Ecology and prevention of Lyme borreliosis

Transcription

1 Ecology and prevention of Lyme borreliosis edited by: Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong Wageningen Academic P u b l i s h e r s Ecology and control of vector-borne diseases Volume 4

2 Ecology and prevention of Lyme borreliosis

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4 Ecology and prevention of Lyme borreliosis Ecology and control of vector-borne diseases Volume 4 edited by: Marieta A.H. Braks Sipke E. van Wieren Willem Takken and Hein Sprong Wageningen Academic P u b l i s h e r s

5 Buy a print copy of this book at EAN: e-ean: ISBN: e-isbn: DOI: / ISSN: First published, 2016 Wageningen Academic Publishers The Netherlands, 2016 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned. Nothing from this publication may be translated, reproduced, stored in a computerised system or published in any form or in any manner, including electronic, mechanical, reprographic or photographic, without prior written permission from the publisher: Wageningen Academic Publishers, P.O. Box 220, 6700 AE Wageningen, the Netherlands, Copyright@WageningenAcademic.com, The individual contributions in this publication and any liabilities arising from them remain the responsibility of the authors. The publisher is not responsible for possible damages, which could be a result of content derived from this publication.

6 Ecology and control of vector-borne diseases In the past century, many advances were made in the control of vector-borne diseases. Malaria disappeared from the northern hemisphere, diseases such as typhus, Bartonella and yellow fever were seriously reduced in prevalence and in many countries effective methods of disease control contributed to a greatly reduced incidence of such diseases. Most of these advances were beneficial to the industrialised world, whereas underdeveloped countries continued to suffer much as before. Indeed, several diseases such as malaria, Rift Valley fever and African sleeping sickness are still highly prevalent in parts of the tropics. New vector-borne diseases such as dengue, chikungunya fever and West Nile fever, have emerged and are invading previously disease-free regions. The discovery of new drugs and vaccines has made great advances and allows for the effective treatment and control of many diseases. In contrast, vector control has lagged behind in development, even though it is realised that effective vector control would allow for an immediate interruption of the transmission of disease, and aid in disease control and eradication. In the last decade new initiatives on vector control have been undertaken, leading to a rapid development of effective and lasting methods of vector control. For example, the Roll Back Malaria control programme of the World Health Organization has led to significant reductions in malaria in many countries. In order to achieve further advances, however, additional tools are required. The development of molecular genetics has provided new insight in vector biology and behaviour, which is being used for developing new strategies of vector control. Advances in geographic information systems allow for precision targeting of interventions. The collective information on new developments in vector ecology and control for vector-borne diseases is scattered over numerous periodicals and electronic databases. This book series intends to bring together this information in sequential volumes arranged around selected themes that are currently of interest. Willem Takken is the senior editor of the series. The editors of Volume 4 are Marieta Braks, Sip van Wieren, Willem Takken and Hein Sprong. The editors of the current volume are well-known experts in the field of Lyme tick biology, tick-borne diseases and ecology. Ecology and prevention of Lyme borreliosis 5

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8 Table of contents 1. Introduction: choosing a One Health approach for the control of Lyme borreliosis 11 Hein Sprong and Marieta A.H. Braks 2. The complexity of patients with (suspected) Lyme borreliosis 19 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius Ecology life cycles Life cycle and ecology of Ixodes ricinus: the roots of public health importance 31 Gábor Földvári 4. Ecology of Borrelia burgdorferi sensu lato 41 Elena Claudia Coipan and Hein Sprong 5. Rodents as hosts for Ixodes ricinus and Borrelia afzelii 63 Gilian van Duijvendijk, Gerrit Gort and Willem Takken 6. The role of large herbivores in Ixodes ricinus and Borrelia burgdorferi s.l. dynamics 75 Sipke E. van Wieren and Tim R. Hofmeester 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe 91 Dieter J.A. Heylen 8. Neglected hosts: the role of lacertid lizards and medium-sized mammals in the ecoepidemiology of Lyme borreliosis 103 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári 9. Emerging tick-borne pathogens: ticking on Pandora s box 127 Setareh Jahfari and Hein Sprong 10. Phenology of Ixodes ricinus and Lyme borreliosis risk 149 Willem Takken Ecology disease ecology How landscapes shape Lyme borreliosis risk 161 Lucy Gilbert 12. The role of host diversity in Borrelia burgdorferi s.l. dynamics 173 Tim R. Hofmeester 13. Greener cities, a wild card for ticks? 187 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock Ecology and prevention of Lyme borreliosis 7

9 14. A resource-based habitat concept for tick-borne diseases 205 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink 15. Modelling the ecological dynamics of tick borne pathogens in a risk assessment perspective 217 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio Risk management hazard control How can forest managers help to reduce the risk for Lyme borreliosis? 233 Kris Verheyen and Sanne C. Ruyts 17. The role of large herbivores in tick-reducing intervention schemes 243 Sipke E. van Wieren 18. Sheep mopping 253 Sipke E. van Wieren 19. Effectiveness and environmental hazards of acaricides applied to large mammals for tick control 265 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr 20. Biological control of the tick Ixodes ricinus by pathogens and invertebrates 279 Ingeborg Klingen and Gilian van Duijvendijk 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future 295 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Risk management exposure control Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis 319 Desiree J. Beaujean and Hein Sprong 23. Prevention of Lyme borreliosis after a tick bite 327 Hein Sprong and Kees (C.C.) van den Wijngaard 24. How an extreme weather spell in winter can influence vector tick abundance and tick-borne disease incidence 335 Hans Dautel, Daniel Kämmer and Olaf Kahl 25. Grasping risk mapping 351 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint 26. From guessing to GIS-ing: empowering land managers 373 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks 8 Ecology and prevention of Lyme borreliosis

10 27. Personal protection for people with occupational risk in the Netherlands 389 Mirjam C.G. de Groot 28. The protection of European dogs against infection with Lyme disease spirochaetes 409 K. Emil Hovius Conclusion Lyme borreliosis prevention strategies: United States versus Europe 429 Lars Eisen and Jeremy S. Gray 30. Concluding remarks 451 Hein Sprong About the editors 453 Contributors 455 Reviewers 460 Ecology and prevention of Lyme borreliosis 9

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12 1. Introduction: choosing a One Health approach for the control of Lyme borreliosis Hein Sprong * and Marieta A.H. Braks National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; hein.sprong@rivm.nl Keywords: nature conservation, One Health, risk assessment, zoonoses Introduction The prevention of disease emergence and preservation of ecosystems are both vital to protect human well-being and health (Bernstein 2014). Diseases arising from the interaction of humans with domesticated and wild animals are a growing international public health concern that is likely to increase with the continuing anthropogenic environmental changes. Approximately 75% of recently emerging infectious diseases affecting humans are diseases of animal origin and approximately 60% of all human pathogens are of zoonotic origin (Jones et al. 2008). Outbreaks of bovine spongiform encephalopathy, West Nile fever, Q fever, avian influenza, severe acute respiratory syndrome, Middle East respiratory syndrome, and Ebola haemorrhagic fever had expensive and multi-sectoral consequences across the world, and caused widespread concern with the general public and decision makers. An adequate response to these kinds of diseases is not straightforward: diagnostic tools and treatment or vaccines are often missing, and conventional measures to eradicate the main sources of infection are nowadays publicly criticised by local media and experts, and have often become socially undesirable (Piesman and Eisen 2008, Sibbald 2003). For example, the widespread application of insecticides might be effective in reducing local vector populations for a short period of time hence reduce disease transmission, their long-term effects on the ecosystem and biodiversity including on non-target arthropods, wildlife as well as humans are often not known. The dichotomy of nature conservation and infectious disease control Perhaps the biggest threat to ecosystems and biodiversity is habitat destruction occurring in terrestrial as well aqueous environments such as the oceans, rivers, and lakes. At the European level, first steps have been made to halt biodiversity loss. Nature legislation at the European Union level forms the backbone of biodiversity policy and the legal basis for our nature protection network. Furthermore, a network of protected areas in Europe, known as Natura 2000, has been built over the last 25 years. Nowadays, these areas comprise one-fifth of the EU s land area ( ec.europa.eu/environment/nature). The European Union aims to connect these natural areas by using green infrastructure to restore the health of ecosystems and allow species to thrive across their entire natural habitat. Expanding and creating ecological networks across Europe is not only beneficial for wildlife, but also for the pathogens they carry, allowing easier maintenance and spread to new areas. The current policy of some European national governments is to create more green spaces in (sub)urban areas to improve human health and well-being, and to mitigate the effects of heat wave, air pollution, flooding and other health risks currently associated with our changing climate (Committee on Climate Change 2014). In addition, green spaces in urban areas can also be utilised as part of conservation strategies for wildlife. It is important to realise, however, that these spaces also provide perfect opportunities for contact between humans and Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 11 DOI / _1, Wageningen Academic Publishers 2016

13 Hein Sprong and Marieta A.H. Braks arthropods and other wildlife, posing risks for acquiring wildlife and vector-borne diseases in urban areas. A prominent case is Lyme borreliosis, which is the most prevalent vector-borne disease affecting humans in temperate regions. In the past decades, Lyme borreliosis has risen from relative obscurity to become a major public health problem, a prime example of an emerging infection (Radolf et al. 2012). Several epidemiological studies describe a two to threefold increase in the incidence of this disease over the last decades, in Europe as well as in the United States (Bacon et al. 2008, Bennet et al. 2006, Smith and Takkinen 2006). The same tick species transmitting the etiologic agents of Lyme borreliosis also serves as vector of pathogens causing tick-borne encephalitis, babesiosis, several forms of rickettsioses and anaplasmoses. The European Centre for Disease Prevention and Control has predicted that the incidence of tick-borne diseases will rise in the near future (Lindgren et al. 2012). The rise in Lyme borreliosis cases and the trailing of related tick-borne infections is in part the consequence of reforestation of land formerly used for agriculture or industry and the increase in deer populations in many countries (Barbour and Fish 1993, Spielman 1994, Sprong et al. 2012). One Health approach How can nature be protected and biodiversity be preserved while the threats of zoonotic diseases are minimised is one of the major paradoxes that our modern society faces today (Keesing et al. 2010). All kinds of governmental agencies, (inter)national organisations and interest groups exist, which either focus on various aspects of nature preservation or on the prevention and control of diseases, but there are hardly any organisations that take responsibility for both these issues simultaneously. The implementation and anticipated effects of cross-sectoral interventions are difficult to predict for strategic and financial planners and decision makers (Aenishaenslin et al. 2013). This complexity calls for transdisciplinary and multi-sectoral approaches in order to achieve effective disease management without harming the environment (Anholt et al. 2012). A One Health approach is needed to develop effective and sustainable management of zoonoses. Such approach recognises the intimate linkages between human, animal and environmental health systems and proposes an international, interdisciplinary, and cross-sectoral approach to disease surveillance, monitoring, prevention, control and mitigation of emerging and re-emerging diseases (Zinsstag et al. 2005). In recent years, the concept of One Health was adopted by many (inter)national organisations as a promising way to improve public health interventions. The next challenge is to bring the One Health concept into practice. One example of a successful implementation of a One Health approach is the integrated humanveterinary risk analysis structure in the Netherlands in The aim of this so-called zoonoses structure is to signal, assess and control (potentially) emerging zoonotic infections that may pose a risk to animal and/or human health in an integrated human-veterinary approach. Its framework consists of consultation structures at several organisational levels: ministries, governmental agencies, national and local animal/human health services. The heart of this structure is the Signalling Forum Zoonoses (SOZ). The task of the SOZ is to signal zoonotic infections in humans and animals. Current zoonotic issues are brought together, discussed and assessed in monthly meetings with representatives from the human and veterinary health domains, and supplemented with other professionals when deemed necessary. In the event of a (potentially) urgent threat, ad hoc meetings are organised. If an assessment identifies a public health threat, the chair of the Response Team Zoonosis is informed, and, based on a risk assessment, subsequent steps within the zoonosis structure will be considered. In order to keep professionals of both the human and 12 Ecology and prevention of Lyme borreliosis

14 1. A One Health approach for controlling Lyme borreliosis veterinary field updated, a monthly review of relevant signals is send out to enlisted professionals. Important signals will actively be addressed in various newsletters (vetinf@ct, inf@ct, labinf@ct) to veterinary and/or public health professionals (Langelaar et al. 2009). The One Health concept does not provide a one fits all solution, but promotes continuous effort and constructive interactions at different levels from organisations in different fields. Thorough knowledge and insights in ecological processes as well as in disease epidemiology are prerequisites to be able to develop sustainable intervention methods and strategies, but also to be able to assess their ecological consequences and effectiveness in disease control. Somehow, the seemingly incompatible values of nature conservation and disease control need to be combined and weighted, or at least put into a common perspective to guide decision making (Table 1). Tools that would facilitate this are needed. Tackling Lyme borreliosis In recent years, efforts to improve the collaboration between the public and veterinary health, has paid off (Wendt et al. 2015). Since the transmission cycles of the pathogens causing Lyme borreliosis occur mostly in nature, involvement of stakeholders of forest and nature management is a logical next level (Braks et al. 2011). An important step would be that policy/decision makers and organisations from both fields, nature conservation and public health, would interact regularly and in a constructive manner with each other. The experience from the Signalling Forum Zoonosis showed that such a multi-sectorial consultation structure would start to create awareness and mutual understanding of challenges within public and veterinary health domain, and could eventually result in the generation of parallel interest and common goals. Tools that would be able to combine the values of nature conservation and disease control are helpful in decision making and planning durable intervention strategies. Examples of these kind of tools are multicriteria decision and cost-benefit analyses, and their first implementations in the control of Lyme borreliosis are available (Aenishaenslin et al. 2013, 2015, Hongoh et al. 2011). Epidemiological measurements to assess infectious diseases are disease incidence, disease burden and cost of illness (Van den Wijngaard et al. 2015). Costs of illness is most commonly measured in monetary terms and is used in the formulation and prioritisation of health care issues. Knowledge on the costs of illness is also required for the calculation of cost-effectiveness of potential intervention strategies, such as vaccination campaigns (Smit and Postma 2016). Management tools to evaluate the values of nature, however, are based on indicators derived from ecosystem services (The QUINTESSENCE Consortium 2016). Ecosystem services can be divided into four broad categories: (1) provisioning, such as the production of food and water; (2) regulating, such as the control of climate and disease; (3) supporting, such as nutrient cycles and crop pollination; and (4) cultural, such as spiritual and recreational benefits. A global initiative, called The Economics of Ecosystems and Biodiversity, is trying to assign ecosystem services to economic values (MacDonald and Corson 2012, Van Wensem 2013). Monetary parameters might eventually be helpful for policy/ decision makers in finding a good balance between nature conservation and the control of wildlife and vectorborne diseases. Another valuable management tool that would facilitate interaction and collaboration between nature management and public health is the generation of local risk maps. Nature value maps are already being used for nature conservation and for landscape management. The nature values of areas can be assessed by their contribution to ecosystem services or to the Natura 2000 (infra) structure. Strictly speaking, any kind of measure of risk describes the probability for a certain Ecology and prevention of Lyme borreliosis 13

15 Hein Sprong and Marieta A.H. Braks hazard to occur combined with the exposure to that hazard. In short, the most basic risk definition is exposure times hazard. Entering a tick s biotope poses a risk for people, because they can acquire a tick bite. The level of the risk depends on the level of exposure by the person (exposure) to the density of infected questing ticks, infected with tick-borne pathogens (hazard). The latter is often referred to as the acarological risk. Control measures to reduce the risk of acquiring Lyme borreliosis should in first instance focus on locations where and time periods in which the risk is highest, either by large hazard or exposure or both. The choice for a certain measure should be based on the nature value of the location in consideration. At high risk locations with high nature values, one might consider to either restrict access of recreants or to warn for tick bites (exposure reduction), while those with lower nature values (such as camp sites) one could focus on lowering the tick density (hazard reduction; Figure 1). Future investigations should also focus on urban areas, where the hazard and nature values might be low, but where exposure is (extremely) high, such as parks and (private) gardens (Mulder et al. 2013, Starostzik 2015). Ideally, full protection against a zoonotic disease, but particularly against Lyme borreliosis, should be achieved at the individual level, for example by vaccination. Efficient personal protection would make other preventive or control measures that would negatively affect the environment redundant. Current research activities focus on the development and implementation of vaccines protecting against Lyme borreliosis and other tick-borne diseases (Sprong et al. 2014, Wressnigg et al. 2013). While vaccines are not available yet, (future) cost-effectiveness studies can investigate, which part of the population should be vaccinated. In some European countries, cost-effectiveness analyses on TBE-vaccination pointed towards national vaccination programmes (Smit and Postma 2015), whereas studies on an off-market Lyme vaccine pointed towards vaccination of high risk groups, such as forest workers (Prybylski 1999). Health education and public health communication focusing on behavioural measures, including avoidance of areas inhabited by ticks, performing routine body checks, using protective clothing, and the application of tick repellents is relatively easy to achieve, but its effectiveness is difficult to measure. The intensified communication with and education of public (toolkits.loketgezondleven.nl) and public health professionals (CBO-guide) since 2003, have not Risk reduction in recreational areas Risk Exposure in nature conservation areas Hazard Figure 1. Schematic representation of the dual aspects of risk reduction of Lyme borreliosis by hazard reduction in recreational areas or exposure reduction in conservation areas. 14 Ecology and prevention of Lyme borreliosis

16 1. A One Health approach for controlling Lyme borreliosis resulted directly in a decline or even a stabilisation of the incidence of Lyme borreliosis in the Netherlands (Hofhuis et al. 2016). The development and implementation of sustainable measures for the prevention of Lyme borreliosis requires thorough insight in the eco-epidemiology of ticks and tick-borne pathogens. The complexity of the eco-epidemiology of Lyme borreliosis results from the interactions of ticks with multiple host species and pathogenic agents, which are modulated by a plethora of abiotic and biotic factors that interact with each other, varying in space and time. Because of this ecological complexity, preventive and control interventions can have environmental, social and economic impacts, aside of the anticipated public health gain. On top of that, the public health impact, of the various tick-borne pathogens and even that of the distinct genospecies of Lyme spirochaetes, is different. The impact of tick-borne pathogens on public health does not only depend on their infection rate in ticks, but also other factors such as their pathogenicity, disease aetiology in humans, the ability to diagnose and treat the corresponding diseases, and their potential to cause persisting symptoms. The complexity of eco-epidemiology makes the prevention and control of Lyme borreliosis a true challenge. Outline of the book A Dutch research programme called Shooting the messenger has focussed on the development of a One Health approach for the prevention of Lyme borreliosis and other tick-borne diseases. The complexity of its eco-epidemiology was unravelled by studying its key elements, human, natural hosts, pathogens, vegetation, and weather separately, but always in combination with either ticks or Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis (Life cycles). The next step was to link the characteristics of tick habitats to Lyme disease risks (Disease ecology). Old, new and futuristic tools for the reduction of acarological risk (Hazard control) and the reduction of tick bites (Exposure control) were investigated. A key aspect of the latter two is the involvement of nature managers: only intervention strategies and tools that are not in conflict with the major aims of land owners will have a chance to be implemented. Ultimately, we hope to inspire policy makers, researchers, property owners, and health and nature organisations to utilise and apply the information provided when deemed necessary and to jointly decrease the disease burden of Lyme borreliosis (Table 1). Table 1. Schematic representation of the ambitions of policy, operation and person in respect to health and nature and translated into an One Health tools. Ambition Health One Health approach Nature policy disease burden reduction interdisciplinary consultation multi criteria decision tools economics of ecosystems and health operational community health services occupational health interdisciplinary consultation knowledge on disease ecology intervention options local risk mapping tools nature conservation biodiversity person healthy living personal preventive measures recreation occupation ecosystem services: provisioning regulating habiting/supporting cultural Ecology and prevention of Lyme borreliosis 15

17 Hein Sprong and Marieta A.H. Braks Additional issues surrounding Lyme borreliosis, not covered in this book Besides its emergence, there are also other public health concerns related to Lyme borreliosis, which are beyond the scope of this book, but undoubtedly require actions of public and medical health professionals. There are uncertainties and controversial opinions about chronic health consequences of treated and untreated Lyme borreliosis (Feder et al. 2007, Hofhuis et al. 2016). A small group of doctors, and a large number of patients, convey that Lyme borreliosis can manifest as chronic illnesses that evades conventional medical tests and treatments. The physicians, who support this theory, choose not to use standard medical guidelines and treat patients with longterm antibiotic therapies that are considered unproven and potentially dangerous by mainstream researchers (Anonymous 2008). Straightforward keys to solve this controversy are not yet available, while the demand for a solution is urgent: a Dutch study calculated that the disease burden associated with chronic symptoms attributed to Lyme borreliosis accounts for roughly 90% of all Lyme borreliosis cases, whereas this category comprises less than 10% of all Lyme borreliosis cases (Van den Wijngaard et al. 2015). Public health relevance Implementation of sustainable control of wildlife- and vector-borne zoonoses requires: consultation structures between nature conservation and disease control organisations; decision making tools that combine the values of ecosystem services and public health; thorough knowledge of disease ecology and epidemiology; sustainable disease control options. References Aenishaenslin C, Gern L, Michel P, Ravel A, Hongoh V, Waaub JP, Milord F and Belanger D (2015) Adaptation and evaluation of a multi-criteria decision analysis model for lyme disease prevention. PLoS ONE 10: e Aenishaenslin C, Hongoh V, Cisse HD, Hoen AG, Samoura K, Michel P, Waaub JP and Belanger D (2013) Multi-criteria decision analysis as an innovative approach to managing zoonoses: results from a study on Lyme disease in Canada. BMC Public Health 13: 897. Anholt RM, Stephen C and Copes R (2012) Strategies for collaboration in the interdisciplinary field of emerging zoonotic diseases. Zoonoses Public Health 59: Anonymous (2008) The chronic debate over Lyme disease. Nat Med 14: Bacon RM, Kugeler KJ and Mead PS (2008) Surveillance for Lyme disease United States, MMWR Surveill Summ 57: 1-9. Barbour AG and Fish D (1993) The biological and social phenomenon of Lyme disease. Science 260: Bennet L, Halling A and Berglund J (2006) Increased incidence of Lyme borreliosis in southern Sweden following mild winters and during warm, humid summers. Eur J Clin Microbiol Infect Dis 25: Bernstein AS (2014) Biological diversity and public health. Annu Rev Public Health 35: Braks M, Van der Giessen J, Kretzschmar M, van Pelt W, Scholte EJ, Reusken C, Zeller H, Van Bortel W and Sprong H (2011) Towards an integrated approach in surveillance of vector-borne diseases in Europe. Parasit Vectors 4: Ecology and prevention of Lyme borreliosis

18 1. A One Health approach for controlling Lyme borreliosis Committee on Climate Change (2014). Managing climate risks to well-being and the economy: ASC progress report Committee on Climate Change, London, UK. Available at: Feder HM, Jr., Johnson BJ, O Connell S, Shapiro ED, Steere AC, Wormser GP, Ad Hoc International Lyme Disease G, Agger WA, Artsob H, Auwaerter P, Dumler JS, Bakken JS, Bockenstedt LK, Green J, Dattwyler RJ, Munoz J, Nadelman RB, Schwartz I, Draper T, McSweegan E, Halperin JJ, Klempner MS, Krause PJ, Mead P, Morshed M, Porwancher R, Radolf JD, Smith RP, Jr., Sood S, Weinstein A, Wong SJ and Zemel L (2007) A critical appraisal of chronic Lyme disease. N Engl J Med 357: Hofhuis A, Bennema S, Harms M, Van Vliet AJ, Takken W, Van den Wijngaard CC and Van Pelt W (2016) Decrease in tick bite consultations and stabilization of early Lyme borreliosis in the Netherlands in 2014 after 15 years of continuous increase. BMC Public Health 16: 425. Hongoh V, Hoen AG, Aenishaenslin C, Waaub JP, Belanger D and Michel P (2011) Spatially explicit multi-criteria decision analysis for managing vector-borne diseases. Int J Health Geogr 10: 70. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL and Daszak P (2008) Global trends in emerging infectious diseases. Nature 451: Keesing F, Belden LK, Daszak P, Dobson A, Harvell CD, Holt RD, Hudson P, Jolles A, Jones KE, Mitchell CE, Myers SS, Bogich T and Ostfeld RS (2010) Impacts of biodiversity on the emergence and transmission of infectious diseases. Nature 468: Langelaar M, Braks M and Van der Giessen J (2009) Emzoo (emerging zoonoses): to an humane veterinary method of threatening zoonoses. Tijdschr Diergeneeskd 134: Lindgren E, Andersson Y, Suk JE, Sudre B and Semenza JC (2012) Public health. Monitoring EU emerging infectious disease risk due to climate change. Science 336: MacDonald KI and Corson C (2012) TEEB begins now : a virtual moment in the production of natural capital. Dev Change 43: Mulder S, Van Vliet AJ, Bron WA, Gassner F and Takken W (2013) High risk of tick bites in Dutch gardens. Vector Borne Zoonotic Dis 13: Piesman J and Eisen L (2008) Prevention of tick-borne diseases. Annu Rev Entomol 53: Prybylski D (1999) The cost-effectiveness of vaccinating against Lyme disease. Emerg Infect Dis 5: Radolf JD, Caimano MJ, Stevenson B and Hu LT (2012) Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10: Sibbald B (2003) Larvicide debate marks start of another West Nile virus summer. CMAJ 168: Smit R and Postma MJ (2015) The burden of tick-borne encephalitis in disability-adjusted life years (DALYs) for Slovenia. PLoS ONE 10: e Smit R and Postma MJ (2016) Vaccines for tick-borne diseases and cost-effectiveness of vaccination: a public health challenge to reduce the diseases burden. Expert Rev Vaccines 15: 5-7. Smith R and Takkinen J (2006) Lyme borreliosis: Europe-wide coordinated surveillance and action needed? Euro Surveill 11: E Spielman A (1994) The emergence of Lyme disease and human babesiosis in a changing environment. Ann N Y Acad Sci 740: Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJ, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Sprong H, Trentelman J, Seemann I, Grubhoffer L, Rego RO, Hajdusek O, Kopacek P, Sima R, Nijhof AM, Anguita J, Winter P, Rotter B, Havlikova S, Klempa B, Schetters TP and Hovius JW (2014) ANTIDotE: anti-tick vaccines to prevent tickborne diseases in Europe. Parasit Vectors 7: 77. Starostzik C (2015) Ticks lurk in the best cared for gardens. [in German] MMW Fortschr Med 157: 24. The QUINTESSENCE Consortium (2016) Networking our way to better ecosystem service provision. Trends Ecol Evol 31: Ecology and prevention of Lyme borreliosis 17

19 Hein Sprong and Marieta A.H. Braks Van den Wijngaard CC, Hofhuis A, Harms MG, Haagsma JA, Wong A, De Wit GA, Havelaar AH, Lugner AK, Suijkerbuijk AW and Van Pelt W (2015) The burden of Lyme borreliosis expressed in disability-adjusted life years. Eur J Public Health 25: Van Wensem J (2013) Use of the ecosystem services concept in landscape management in the Netherlands. Integr Environ Assess Manag 9: Wendt A, Kreienbrock L and Campe A (2015) Zoonotic disease surveillance inventory of systems integrating human and animal disease information. Zoonoses Public Health 62: Wressnigg N, Pollabauer EM, Aichinger G, Portsmouth D, Low-Baselli A, Fritsch S, Livey I, Crowe BA, Schwendinger M, Bruhl P, Pilz A, Dvorak T, Singer J, Firth C, Luft B, Schmitt B, Zeitlinger M, Muller M, Kollaritsch H, Paulke-Korinek M, Esen M, Kremsner PG, Ehrlich HJ and Barrett PN (2013) Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomised, dose-escalation phase 1/2 trial. Lancet Infect Dis 13: Zinsstag J, Schelling E, Wyss K and Mahamat MB (2005) Potential of cooperation between human and animal health to strengthen health systems. Lancet 366: Ecology and prevention of Lyme borreliosis

20 2. The complexity of patients with (suspected) Lyme borreliosis Jeanine Ursinus 1, Jeroen Coumou 1 and Joppe W.R. Hovius 1,2,3* 1 Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; 2 Amsterdam Multidisciplinary Lyme borreliosis Center, Academic Medical Center, University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; 3 Department of Internal Medicine, Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; lyme@amc.uva.nl Abstract The causative agents of Lyme borreliosis (LB) are spirochaetes that belong to the Borrelia burgdorferi sensu lato (s.l.) group which are transmitted by ticks. It is estimated that LB affects 300,000 patients per year in the USA and 65,000 patients in Europe, the latter likely being an enormous underestimation. The incidence of LB has substantially increased in the past two decades. When B. burgdorferi s.l. is transmitted from the tick into the host s skin, it can cause an erythematous skin lesion designated as erythema migrans. Untreated infection can result in dissemination of B. burgdorferi s.l. into distant organs, for example the nervous system, the joints or other skin sites, resulting in Lyme neuroborreliosis, Lyme arthritis and acrodermatitis chronica atrophicans, respectively. The diagnosis of LB is predominantly based on the presence of objective clinical symptoms and the exclusion of other causes, supported by the presence of anti-b. burgdorferi s.l. antibodies in serum. If appropriate, a PCR assay or culture for B. burgdorferi s.l. can be performed to support the diagnosis. In daily practice, it can be challenging to diagnose a B. burgdorferi s.l. infection, especially in patients with nonspecific symptoms and a positive serological test, as well as in patients with persisting symptoms after recommended antibiotic treatment for LB. Although most patients have a good prognosis after antibiotic treatment, approximately 10-20% report nonspecific symptoms with a substantial disease burden. Such persisting symptoms are not thought to be caused by a persisting B. burgdorferi s.l. infection, but rather by a post-infectious disease syndrome. The underlying causes of this syndrome, referred to as Post-Treatment LB syndrome, are still under debate. This chapter provides insight into the pathogenesis of LB, the clinical manifestations and diagnostic challenges in patients with (suspected) LB. Keywords: Borrelia burgdorferi sensu lato, clinical manifestations, epidemiology, Lyme borreliosis, pathogenesis Historic overview of Lyme borreliosis Lyme disease, or Lyme borreliosis (LB), is the most prevalent vector-borne disease in Western Europe and North Eastern USA. The disease is named after Old Lyme, a small village in Connecticut, USA, where researchers from Yale University identified a causal relation between tick bites and arthritis in a group of children (Steere et al. 1977). Five years later, the causative agent, namely spirochaetes belonging to the Borrelia burgdorferi sensu lato (s.l.) group, was identified by Willy Burgdorfer (Burgdorfer et al. 1982). After Burgdorfer s initial discovery of the genospecies B. burgdorferi sensu stricto (s.s.), which is present in the USA as well as Europe and Asia, other B. burgdorferi s.l. genospecies were also identified that are exclusively present in Eurasia such as B. afzelii and B. garinii (Baranton et al. 1992, Canica et al. 1993). B. burgdorferi s.l. genospecies are transmitted by different Ixodes tick species. In the USA, B. burgdorferi s.s. is predominantly transmitted by Ixodes scapularis ticks, whereas the B. burgdorferi s.l. genospecies present in Europe and Asia are transmitted by I. ricinus and I. persulcatus, respectively. In the Netherlands, roughly Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 19 DOI / _2, Wageningen Academic Publishers 2016

21 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius 20% of adult ticks are infected with B. burgdorferi s.l. compared to 10% of nymphs and solely 0.62% of larvae (Rauter and Hartung 2005, Van Duijvendijk et al. 2016). Epidemiology The estimated annual incidence of LB in the USA is approximately 300,000 and in Europe approximately 65,000, the latter likely being an underestimation since multiple factors influence the reliability of estimated annual LB cases (Hubalek 2009, Nelson et al. 2015). For example, in not all European countries LB is a reportable disease. Moreover, LB is significantly underreported by physicians due to diagnostic pitfalls and various case definitions that are being used. Furthermore, epidemiological studies often differentiate between local and disseminated Lyme manifestations and overall LB incidence rates are scarce. Most of the patients in Europe (77-89%) are diagnosed with the first local stage of LB, erythema migrans (EM). In a minority of patients (10-23%), B. burgdorferi s.l. disseminates from the tick bite site to other organs, such as the central nervous system and joints (Berglund et al. 1995, Huppertz et al. 1999). A detailed description of the various disease manifestations will be discussed below. Several studies have shown that in the last two decades, the incidence of LB is on the rise. For example, based on retrospective questionnaire-based studies performed since 1994, the number of consultations at the general practitioner for EM (see below) in the Netherlands has increased from 39 EM cases per 100,000 inhabitants in 1994 up to 134 EM cases per 100,000 inhabitants in 2009 (Hofhuis et al. 2015b, 2006). This increase could partially be attributed to a higher awareness of LB by both physicians and patients. However, Sprong et al. (2012) also showed an increase in the total number of B. burgdorferi s.l.-infected ticks in the Netherlands, as well as an increase in the length of the annual tick questing season, the surface area of tick-suitable habitats and an increase in the feeding and reproductive tick hosts. Pathogenesis of Lyme borreliosis Non-infected larval ticks acquire B. burgdorferi s.l. by taking a blood meal on infected animals, most commonly small rodents. B. burgdorferi s.l. is then transmitted by nymphal and adult ticks to various other hosts, such as birds, large mammals and humans. Therefore, B. burgdorferi s.l. needs to adapt quickly in their metabolism as well as their defence against different host immune responses. In order to survive these different environments, transcriptional changes required for differential gene expression seem to be mainly regulated by a pathway with three major constituents, the so called Rrp2-RpoN-RpoS pathway (Radolf et al. 2012). Activation of this regulatory pathway promotes transcription of genes that are required for infection of the mammalian host. The nymphal blood meal activates this pathway, resulting in the differential expression of B. burgdorferi s.l. outer-surface lipoproteins (Osp s). Osp s are predominantly presented on the outer-surface of B. burgdorferi s.l. and have been found to interact with tick proteins. Within the tick, B. burgdorferi s.l. expresses OspA while residing in the tick. Binding of OspA to the tick receptor for OspA (TROSPA) on tick midgut epithelial cells ensures colonisation and survival of B. burgdorferi s.l. in the tick midgut during the interval between blood-meals (Pal et al. 2004, Yang et al. 2004). During tick feeding, as B. burgdorferi s.l. migrates towards the salivary glands of the tick, the spirochaetes upregulate expression of OspC and downregulate expression of OspA. Subsequently, OspC can bind to the tick salivary gland protein Salp15, which has immunomodulatory properties and thereby protects the spirochaetes from complementdependent (antibody-mediated) killing by the host (Hovius et al. 2008b, Ramamoorthi et al. 2005, Schuijt et al. 2008). Moreover, Salp15 is inoculated into the cutaneous bite site where it suppresses 20 Ecology and prevention of Lyme borreliosis

22 2. The complexity of patients with (suspected) Lyme borreliosis the human immune system by impairment of T cell receptor signalling and inhibited CD 4+ T-cell activation and proliferation (Anguita et al. 2002). Hovius et al. (2008a) showed that Salp15 also suppresses human dendritic cell function and causes an impaired pro-inflammatory cytokine response. For dissemination within the mammalian host, B. burgdorferi s.l. induces multiple host matrix metalloproteinases (MMPs) that degrade extracellular matrix components. B. burgdorferi s.l. can also bind plasminogen to penetrate the collagenous matrix. When the spirochaetes enter the bloodstream, adhesins are expressed that mediate the attachment to specific host tissues (Radolf et al. 2012). During infection in the host, B. burgdorferi s.l. is able to evade the humoral immune response and to protect itself from antibody-mediated killing by recombinant gene expression of the variable major protein-like sequence locus (Fikrig et al. 1998). When B. burgdorferi s.l. successfully enters the human host through the skin and survives the human innate and adaptive immune response, it can cause a multisystemic inflammatory disease affecting a range of tissues, including skin, nervous system, joints or heart and to a lesser extent other organs. Clinical manifestations Stages and treatment LB manifestations can be divided into a localised, early disseminated and late disseminated disease stage. Well-defined case definitions for each of these stages have been described extensively (Stanek et al. 1996, 2011, Steere 2001). Recommended antibiotic treatment regimens for LB depend on the Lyme manifestation as well as the disease duration and are based on the guidelines formulated by the Dutch Institute of Health Care Improvement (CBO) and the Infectious Disease Society of America (IDSA) (CBO 2013, Wormser et al. 2006). Patients with LB are generally treated with doxycycline orally, except for patients with Lyme neuroborreliosis (LNB) or advanced atrioventricular heart block (see below), who should be treated with ceftriaxone intravenously. Early Lyme borreliosis (days to weeks) EM is the sole manifestation of early-localised LB and is characterised by a centrifugally expanding, bluish-red macule with or without central clearing located at the tick bite site (Figure 1A). Typically, the size of an EM is more than 5 cm in diameter and occurs several days to weeks after a tick bite, with a median delay of 14 days. Erythematous skin lesions smaller than 5 cm starting within two days after detachment of the tick, represent most likely hypersensitivity reactions to the tick bite and should disappear within several days. EM is a clinical diagnosis and no further diagnostic testing is necessary. In case of an atypical EM, a serological test can be performed and if necessary be repeated after 6-8 weeks to look for a seroconversion (further explained below). Finally, detection of B. burgdorferi s.l. by culture or PCR in a biopsy of the affected skin when performed under well-controlled circumstances proofs an infection. Another early, while rare, disease manifestation in Europe is Borrelial lymphocytoma, which is a solitary, soft bluish-red painless nodule or plaque with a diameter of up to several centimetres (Figure 1B). Approximately 2-3% of patients with LB present with this manifestation. When an EM does not occur, is not witnessed by the patient or not recognised by the physician and remains untreated, B. burgdorferi s.l. can disseminate through the lymphatic and blood system to other organs and tissues, such as the nervous system (LNB), the joints (Lyme arthritis) or the skin (acrodermatitis chronica atrophicans; ACA). Other organs such as the heart and the eyes can Ecology and prevention of Lyme borreliosis 21

23 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius A B C Figure 1. (A) Erythema migrans. (B) Borrelial lymphocytoma. (C) Acrodermatitis chronica atrophicans (left median and lateral malleolus of the same patient). also be infected by B. burgdorferi s.l., but does occur rarely (Hofhuis et al. 2015a, Mikkila et al. 2000, Raja et al. in press, Wang et al. 1999). Early disseminated Lyme borreliosis (weeks to months) LNB (3-16% of LB patients in Europe) develops in the subacute phase (weeks to months) when B. burgdorferi s.l. disseminates and infiltrates the central nervous centre or peripheral nerves (Rupprecht et al. 2008). Although the spectrum of clinical observations in patients with LNB is diverse, LNB in Europe typically presents as a painful radiculitis, while patients with LNB in the USA most often present with lymphocytic meningitis and cranial nerve palsy, usually the facial nerve. The triad of these symptoms has previously been described as the Garin-Bujadoux- Bannwarth syndrome (Horstrup and Ackermann 1973). The current diagnostic criteria for definite LNB include neurological symptoms suggestive for LNB without evidence for another underlying disease, combined with lymphocytic pleocytosis in cerebrospinal fluid and intrathecal production of specific B. burgdorferi s.l. antibodies. Patients who fulfil only two of the three diagnostic criteria can be categorised as having possible LNB according to the guidelines on LNB by the European Federation of Neurological Societies (Mygland et al. 2010). Although late LNB is rare, encephalomyelitis, encephalopathy and chronic axonal polyneuropathy lasting longer than six months may develop after a long period of latent or untreated B. burgdorferi s.l. infection (Logigian et al. 1990). Lyme arthritis occurs in 5-7% of LB patients in Europe. In the early disseminated phase of B. burgdorferi s.l. infection, the spirochaetes can cause intermittent or long-lasting mono- or oligoarthritis, primarily in large joints and particularly the knee (Berglund et al. 1995, Steere et al. 1977). This manifestation is more common in the USA than in Europe since Lyme arthritis is associated to the genospecies B. burgdorferi s.s. A PCR on B. burgdorferi s.l. DNA or culture of synovial tissue or synovial fluid can support the diagnosis (see below). Persistent arthritis after at least two months of oral antibiotic treatment or one month of intravenous antibiotic therapy occurs in approximately 10% of patients with Lyme arthritis in the USA and can persist months or even several years (Steere et al. 1994). 22 Ecology and prevention of Lyme borreliosis

24 2. The complexity of patients with (suspected) Lyme borreliosis Late Lyme borreliosis (months to years) ACA (1-3% of LB patients in Europe) is a late cutaneous stage of LB, and presents as a slow, progressive skin condition which can develop even up to ten years after the causative tick bite (Asbrink and Hovmark 1985). ACA initially presents as an oedematous bluish-red skin lesion. However, persistent infection eventually leads to irreversible atrophic changes of the skin (Figure 1C). ACA is mainly located on the distal parts of the extremities. It can be challenging to differentiate ACA from vascular conditions such as chronic venous insufficiency, deep vein thrombosis or superficial thrombophlebitis. Therefore, establishing the diagnosis can be delayed for months or years (Mullegger and Glatz 2008). Picken et al. (1998) showed that B. afzelii is the predominant, but not exclusive, causative agent of ACA. Only a few cases of patients with ACA have been described in the USA, most likely due to the absence of B. afzelii in the USA (Steere et al. 1986). ACA can be accompanied by hyperalgesia or paraesthesia as the result of a peripheral neuropathy caused by large fibre axonal damage (Kindstrand et al. 1997). Since the clinical appearance of ACA is not distinctive, the diagnosis must be proven serologically, histopathologically or by B. burgdorferi s.l. culture or PCR. Nearly 100% of patients have specific B. burgdorferi s.l. IgG antibodies, therefore a significantly elevated antibody titre is obligatory for the diagnosis. Although histopathological aspects of ACA are not pathognomonic and depend on the duration of the infection, a typical finding is lymphocytic perivascular and interstitial infiltration with an admixture of plasma cells and histiocytes and the presence of telangiectasia (Brehmer-Andersson et al. 1998). Diagnostic challenges in Lyme borreliosis The diagnosis of LB manifestations is predominantly based on the presence of objective findings or symptoms (see above) in combination with the presence of anti-b. burgdorferi s.l. antibodies in serum or cerebrospinal fluid. Depending on the manifestation, biopsy for PCR or culture can support the diagnosis. Importantly, for LB manifestations other than EM, other diseases must be excluded. For the detection of anti-b. burgdorferi s.l. antibodies in serum, a two-tiered testing algorithm based on a screening enzyme-linked immunosorbent assay (ELISA) followed by a confirmation immunoblot is recommended to enhance specificity (CBO 2013). The screenings assay for B. burgdorferi s.l. antibodies in serum is based on an enzyme-(linked) immunosorbent assay (EIA or ELISA) on B. burgdorferi s.l.-specific recombinant antigens or specific peptides (mainly the C6 peptide). The IgM/IgG immunoblot assay or Western blot is used to differentiate between specific anti-b. burgdorferi s.l. antibodies and antibodies as a result of cross-reactivity. Therefore, a combination of various selected recombinant and native antigens of the three defined humanpathogenic European B. burgdorferi s.l. subspecies are used (Wilske et al. 2007). Overall sensitivity of ELISA and immunoblot in the early phase of infection is low as it can take several weeks before an antibody response can be detected. Therefore, serological examination in patients with short disease duration in case of an atypical EM or other putative LB related symptoms and negative B. burgdorferi s.l. serology results should be repeated after 6 to 8 weeks. A recently published systematic review by Leeflang et al. (2016) showed that the overall sensitivity of ELISAs and immunoblots used in Europe for ACA and Lyme arthritis is ~95%, for EM ~50% and for LNB ~77%. In general, diagnostic reliability of serological tests for LB depends on the pre-test probability and disease duration. Serological testing for LB is limited by false-positive results as 4-8% of the general population has detectable B. burgdorferi s.l. antibodies in their serum. This is mostly due to previous (asymptomatic) infection (Nohlmans et al. 1991). Thus, in patients with nonspecific symptoms such as fatigue, myalgia or arthralgia, the presence of anti-b. burgdorferi s.l. antibodies does not proof active B. burgdorferi s.l. infection. As nonspecific symptoms can accompany B. burgdorferi s.l. infection and estimating the pre-test probability can be difficult and arbitrary, Ecology and prevention of Lyme borreliosis 23

25 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius confirming or excluding LB in clinical practice can be challenging. National and international guidelines therefore do not recommended to perform B. burgdorferi s.l. serological tests in patients with nonspecific symptoms and a low a-priori change of B. burgdorferi s.l. infection. However, this recommendation is not applied in daily clinical practice as it was found that 82% serological requests by general practitioners in the Netherlands were not supported by the CBO guideline and 70% of the requests concerned patients with a low a-priori chance for LB (Coumou et al. 2014b). In addition, the CBO and IDSA guideline state that serological testing is not recommended to confirm the efficacy of antibiotic treatment of a (suspected) B. burgdorferi s.l. infection, since antibodies might remain detectable for years (CBO 2013, Wormser et al. 2006). A minority of patients (10-20%) that have been treated for LB report nonspecific symptoms, most commonly pain, fatigue, neurologic or cognitive disturbances (Cairns and Godwin 2005). This syndrome has been defined as post-lyme disease syndrome or post-treatment LB syndrome (PLDS or PTLBS) for which criteria have been postulated in the IDSA guideline of 2006 (Wormser et al. 2006). Until now, there has been no evidence for a causal relationship between these symptoms and an active B. burgdorferi s.l. infection. However, the disease burden in these patients is substantial. A recent study estimated the total LB disease burden in disability-adjusted life years (DALYs) per 100,000 population in the Netherlands. Interestingly, the disease burden is predominantly due to patients with Lyme-related persisting symptoms (9.09 DALYs per 100,000 population), compared to a more modest disease burden in patients with EM and disseminated manifestations (0.60 and 0.86 DALYS per 100,000 population, respectively) (Van den Wijngaard et al. 2015) (Figure 2). The prevalence of persisting symptoms has been extensively studied in both Europe and the USA. However, the prevalence of persisting symptoms in literature variates considerably and percentages depend on the applied case definition, follow up, geographic location, LB manifestation and delay between onset of symptoms and treatment. A systematic review of Dersch et al. (2015) reported a mean prevalence of residual symptoms in 28% of patients treated for LNB. A recent study by the Dutch National Institute for Public Health estimated an annual incidence of 1000 to 2,500 patients in the Netherlands with persisting symptoms attributed to LB (Hofhuis et al. 2015b). Previous randomised, clinical trials have shown no convincing beneficial effect of long-term antibiotic treatment in patients with persisting symptoms attributed to LB, suggesting that an underlying persisting B. burgdorferi s.l. infection is very unlikely to cause PTLBS (Cameron 2008, Klempner et al. 2001, Krupp et al. 2003). The PLEASE study in the Netherlands, published 3.7% 5.3% 86.2% Erythema migrans Disseminated Lyme manifestations Lyme-related persisting symptoms A 91% B 8.1% 5.7% Figure 2. Relative proportions of the incidence and disease burden of Lyme borreliosis (LB) in 2010 in the Netherlands. (A) Relative proportions of LB diagnoses in 2010 in the Netherlands for erythema migrans (22,000 cases), disseminated LB (1,300 cases) and persisting symptoms attributed to LB (900 cases) (Hofhuis et al. 2015b). (B) Relative proportions of the total LB disease burden estimated in 2010 in the Netherlands expressed in disabilityadjusted life years (DALYs) caused by patients with erythema migrans (0.60 DALYs), disseminated LB (0.86 DALYs) and Lyme-related persisting symptoms (9.09 DALYs) (Van den Wijngaard et al. 2015). 24 Ecology and prevention of Lyme borreliosis

26 2. The complexity of patients with (suspected) Lyme borreliosis by Berende et al. (2016), is the only European study available on this topic and confirms that prolonged antibiotic treatment for persisting symptoms attributed to LB has no effect compared to placebo. The underlying causes of PTLBS have been extensively debated and it is hypothesised that (a combination of) microbiological, immunological, cognitive and behavioural underlying mechanisms play a role. An important approach in the treatment and management of patients with persisting symptoms after treatment for LB is the exclusion of a persisting infection and exclusion of other diseases. Interestingly, a recent retrospectively case series describing 200 LB patients referred to a tertiary multidisciplinary Lyme centre in the Netherlands found that 60% of patients did not have LB. Moreover, 36% of patients in which LB was excluded were diagnosed with another disease (Coumou et al. 2014a). In line with previous findings, only a few patients (3/200) had objective evidence of a probable persisting infection. In conclusion, LB is a multisystemic diseases and the incidence has substantially increased in the past twenty years. Patients with LB can present with early localised, early disseminated and late disseminated disease manifestations affecting skin, central nervous system, joints and to a lesser extent other organs. As described in the CBO guideline, the diagnosis is predominantly based on objective symptoms supported by the presence of anti-b. burgdorferi s.l. antibodies, sometimes combined with biopsy for B. burgdorferi s.l. PCR or culture. Duration, type and route of administration of the antibiotic treatment depends on the disease manifestation and duration. It can be challenging in some patients to either rule out or prove an association between symptoms, serological results and a (persistent) B. burgdorferi s.l. infection. Therefore, future research will hopefully gain more insight into the underlying cause for persisting symptoms, and should focus on a diagnostic test that can distinguish between a past and active B. burgdorferi s.l. infection. An ongoing, prospective, clinical study in the Netherlands, the LymeProspect study for more information visit will hopefully provide more insight into this subject, as the reported symptoms, disabilities and decreased quality of life in these patients call for a thorough evaluation. Public health relevance This chapter provides insight into the pathogenesis of Lyme borreliosis (LB), its clinical manifestations and the diagnostic challenges in patients suspected of LB. LB is a multisystemic disease and the incidence has substantially increased in the past twenty years. The diagnosis of disseminated LB is based on objective clinical symptoms, the exclusion of other causes and is supported by the presence of anti-borrelia burgdorferi s.l. antibodies in serum. It can be challenging in some patients to either rule out or prove an association between symptoms, serological results and a B. burgdorferi s.l. infection. Ecology and prevention of Lyme borreliosis 25

27 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius References Anguita J, Ramamoorthi N, Hovius JW, Das S, Thomas V, Persinski R, Conze D, Askenase PW, Rincon M, Kantor FS and Fikrig E (2002) Salp15, an Ixodes scapularis salivary protein, inhibits CD4+ T cell activation. Immunity 16: Asbrink E and Hovmark A (1985) Successful cultivation of spirochetes from skin lesions of patients with erythema chronicum migrans afzelius and acrodermatitis chronica atrophicans. Acta Pathol Microbiol Immunol Scand B 93: Baranton G, Postic D, Saint Girons I, Boerlin P, Piffaretti JC, Assous M and Grimont PA (1992) Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group vs461 associated with Lyme borreliosis. Int J Syst Bacteriol 42: Berende A, Ter Hofstede HJ, Vos FJ, Van Middendorp H, Vogelaar ML, Tromp M, Van den Hoogen FH, Donders AR, Evers AW and Kullberg BJ (2016) Randomized trial of longer-term therapy for symptoms attributed to Lyme disease. New Engl J Med 374: Berglund J, Eitrem R, Ornstein K, Lindberg A, Ringer A, Elmrud H, Carlsson M, Runehagen A, Svanborg C and Norrby R (1995) An epidemiologic study of Lyme disease in southern Sweden. New Engl J Med 333: Brehmer-Andersson E, Hovmark A and Asbrink E (1998) Acrodermatitis chronica atrophicans: histopathologic findings and clinical correlations in 111 cases. Acta Derm Venereol 78: Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E and Davis JP (1982) Lyme disease-a tick-borne spirochetosis? Science 216: Cairns V and Godwin J (2005) Post-Lyme borreliosis syndrome: a meta-analysis of reported symptoms. Int J Epidemiol 34: Cameron D (2008) Severity of Lyme disease with persistent symptoms. Insights from a double-blind placebo-controlled clinical trial. Minerva Med 99: Canica MM, Nato F, du Merle L, Mazie JC, Baranton G and Postic D (1993) Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand J Infect Dis 25: CBO (2013) Richtlijn Lymeziekte. CBO, Utrecht, the Netherlands. Available at: Coumou J, Herkes EA, Brouwer MC, Van de Beek D, Tas SW, Casteelen G, Van Vugt M, Starink MV, De Vries HJ, De Wever B, Spanjaard L and Hovius JW (2014a) Ticking the right boxes: classification of patients suspected of Lyme borreliosis at an academic referral center in the Netherlands. Clin Microbiol Infec 21: 368.e Coumou J, Hovius JWR and Van Dam AP (2014b) Borrelia burgdorferi sensu lato serology in the netherlands: guidelines versus daily practice. Eur J Clin Microbiol Infect Dis 33: Dersch R, Sommer H, Rauer S and Meerpohl JJ (2015) Prevalence and spectrum of residual symptoms in Lyme neuroborreliosis after pharmacological treatment: a systematic review. J Neurol 263: Fikrig E, Feng W, Aversa J, Schoen RT and Flavell RA (1998) Differential expression of borrelia burgdorferi genes during erythema migrans and Lyme arthritis. J Infect Dis 178: Hofhuis A, Arend SM, Davids CJ, Tukkie R and Van Pelt W (2015a) General practitioner reported incidence of Lyme carditis in the netherlands. Neth Heart J 23: Hofhuis A, Harms M, Bennema S, Van den Wijngaard CC and Van Pelt W (2015b) Physician reported incidence of early and late Lyme borreliosis. Parasit Vectors 8: 161. Hofhuis A, Van der Giessen JW, Borgsteede FH, Wielinga PR, Notermans DW and Van Pelt W (2006) Lyme borreliosis in the Netherlands: strong increase in GP consultations and hospital admissions in past 10 years. Euro Surveill 11: E Horstrup P and Ackermann R (1973) Tick born meningopolyneuritis (Garin-Bujadoux, Bannwarth). [in German] Fortschr Neurol Psychiatr Grenzgeb 41: Hovius JW, Levi M and Fikrig E (2008a) Salivating for knowledge: potential pharmacological agents in tick saliva. PLoS Med 5: e Ecology and prevention of Lyme borreliosis

28 2. The complexity of patients with (suspected) Lyme borreliosis Hovius JW, Schuijt TJ, De Groot KA, Roelofs JJ, Oei GA, Marquart JA, De Beer R, Van t Veer C, Van der Poll T, Ramamoorthi N, Fikrig E and Van Dam AP (2008b) Preferential protection of Borrelia burgdorferi sensu stricto by a Salp15 homologue in ixodes ricinus saliva. J Infect Dis 198: Hubalek Z (2009) Epidemiology of Lyme borreliosis. Curr Probl Dermatol 37: Huppertz HI, Bohme M, Standaert SM, Karch H and Plotkin SA (1999) Incidence of Lyme borreliosis in the wurzburg region of germany. Eur J Clin Microbiol Infect Dis 18: Kindstrand E, Nilsson BY, Hovmark A, Pirskanen R and Asbrink E (1997) Peripheral neuropathy in acrodermatitis chronica atrophicans a late Borrelia manifestation. Acta Neurol Scand 95: Klempner MS, Hu LT, Evans J, Schmid CH, Johnson GM, Trevino RP, Norton D, Levy L, Wall D, McCall J, Kosinski M and Weinstein A (2001) Two controlled trials of antibiotic treatment in patients with persistent symptoms and a history of Lyme disease. New Engl J Med 345: Krupp LB, Hyman LG, Grimson R, Coyle PK, Melville P, Ahnn S, Dattwyler R and Chandler B (2003) Study and treatment of post Lyme disease (stop-ld): a randomized double masked clinical trial. Neurology 60: Leeflang MM, Ang CW, Berkhout J, Bijlmer HA, Van Bortel W, Brandenburg AH, Van Burgel ND, Van Dam AP, Dessau RB, Fingerle V, Hovius JW, Jaulhac B, Meijer B, Van Pelt W, Schellekens JF, Spijker R, Stelma FF, Stanek G, Verduyn- Lunel F, Zeller H and Sprong H (2016) The diagnostic accuracy of serological tests for Lyme borreliosis in europe: a systematic review and meta-analysis. BMC Infect Dis 16: 140. Logigian EL, Kaplan RF and Steere AC (1990) Chronic neurologic manifestations of Lyme disease. New Engl J Med 323: Mikkila HO, Seppala IJ, Viljanen MK, Peltomaa MP and Karma A (2000) The expanding clinical spectrum of ocular Lyme borreliosis. Ophthalmology 107: Mullegger RR and Glatz M (2008) Skin manifestations of Lyme borreliosis: diagnosis and management. Am J Clin Dermatol 9: Mygland A, Ljostad U, Fingerle V, Rupprecht T, Schmutzhard E and Steiner I (2010) EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis. Eur J Neurol 17: 8-16, e Nelson CA, Saha S, Kugeler KJ, Delorey MJ, Shankar MB, Hinckley AF and Mead PS (2015) Incidence of cliniciandiagnosed Lyme disease, United States, Emerg Infect Dis 21: Nohlmans MK, Van den Bogaard AE, Blaauw AA and Van Boven CP (1991) Prevalence of Lyme borreliosis in the Netherlands. Ned Tijdschr Genees 135: Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, Desilva AM, Bao F, Yang X, Pypaert M, Pradhan D, Kantor FS, Telford S, Anderson JF and Fikrig E (2004) TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 119: Picken RN, Strle F, Picken MM, Ruzic-Sabljic E, Maraspin V, Lotric-Furlan S and Cimperman J (1998) Identification of three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) among isolates from acrodermatitis chronica atrophicans lesions. J Invest Dermatol 110: Radolf JD, Caimano MJ, Stevenson B and Hu LT (2012) Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 10: Raja H, Starr MR and Bakri SJ (in press) Ocular manifestations of tick-borne diseases. Surv Ophthalmol DOI: org/ /j.survophthal Ramamoorthi N, Narasimhan S, Pal U, Bao F, Yang XF, Fish D, Anguita J, Norgard MV, Kantor FS, Anderson JF, Koski RA and Fikrig E (2005) The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 436: Rauter C and Hartung T (2005) Prevalence of borrelia burgdorferi sensu lato genospecies in ixodes ricinus ticks in europe: a metaanalysis. Appl Environ Microbiol 71: Rupprecht TA, Koedel U, Fingerle V and Pfister HW (2008) The pathogenesis of Lyme neuroborreliosis: from infection to inflammation. Mol Med 14: Schuijt TJ, Hovius JW, Van Burgel ND, Ramamoorthi N, Fikrig E and Van Dam AP (2008) The tick salivary protein Salp15 inhibits the killing of serum-sensitive Borrelia burgdorferi sensu lato isolates. Infect Immun 76: Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJ, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of borrelia-infected ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Ecology and prevention of Lyme borreliosis 27

29 Jeanine Ursinus, Jeroen Coumou and Joppe W.R. Hovius Stanek G, Fingerle V, Hunfeld KP, Jaulhac B, Kaiser R, Krause A, Kristoferitsch W, O Connell S, Ornstein K, Strle F and Gray J (2011) Lyme borreliosis: clinical case definitions for diagnosis and management in europe. Clin Microbiol Infec 17: Stanek G, O Connell S, Cimmino M, Aberer E, Kristoferitsch W, Granstrom M, Guy E and Gray J (1996) European union concerted action on risk assessment in Lyme borreliosis: clinical case definitions for Lyme borreliosis. Wien Klin Wochenschr 108: Steere AC (2001) Lyme disease. New Engl J Med 345: Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JH 3 rd, Liu NY and Schmid CH (1994) Treatment of Lyme arthritis. Arthritis Rheum 37: Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross MR and Steele FM (1977) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three connecticut communities. Arthritis Rheum 20: Steere AC, Taylor E, Wilson ML, Levine JF and Spielman A (1986) Longitudinal assessment of the clinical and epidemiological features of Lyme disease in a defined population. J Infect Dis 154: Van den Wijngaard CC, Hofhuis A, Harms MG, Haagsma JA, Wong A, De Wit GA, Havelaar AH, Lugner AK, Suijkerbuijk AW and Van Pelt W (2015) The burden of Lyme borreliosis expressed in disability-adjusted life years. Eur J Public Health 25: Van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersoz J, Oei A, Foldvari G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: 97. Wang G, Van Dam AP, Schwartz I and Dankert J (1999) Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin Microbiol Rev 12: Wilske B, Fingerle V and Schulte-Spechtel U (2007) Microbiological and serological diagnosis of Lyme borreliosis. FEMS Immunol Med Microbiol 49: Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, Krause PJ, Bakken JS, Strle F, Stanek G, Bockenstedt L, Fish D, Dumler JS and Nadelman RB (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the infectious diseases society of america. Clin Infect Dis 43: Yang XF, Pal U, Alani SM, Fikrig E and Norgard MV (2004) Essential role for ospa/b in the life cycle of the Lyme disease spirochete. J Exp Med 199: Ecology and prevention of Lyme borreliosis

30 Ecology life cycles

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32 3. Life cycle and ecology of Ixodes ricinus: the roots of public health importance Gábor Földvári Department of Parasitology and Zoology, University of Veterinary Medicine, 2 István str., 1078 Budapest, Hungary; foldvarigabor@gmx.de Abstract Ixodes ricinus is a common arthropod species with high reproduction rate, broad host range and an ability to withstand or circumvent most environmental constraints. These adaptive strategies make it the most common tick species in temperate Europe with a geographical distribution from southern Spain to northern Scandinavia. I. ricinus is undoubtedly one of the most important tick species of the world resulting in a plethora of zoonotic diseases in Europe. Despite the great amount of studies dealing with various aspects of physiology, behaviour, life cycle, ecology and especially its role in pathogen transmission, many parts of the tick s basic biology are obscure and unexplored. This chapter attempts to bring together key elements of I. ricinus life cycle and ecology off and on the host that contribute to the species public health importance with special emphasis on the eco-epidemiology of Lyme borreliosis. Keywords: diapause, ecology, hosts, Ixodes ricinus, life cycle, public health, seasonality Introduction The sheep tick, I. ricinus (Linnaeus, 1758) was named in the 18 th century; however, the first detailed studies on this species were performed only between the 1930s and 1950s (reviewed by Arthur 1963) mostly in relation to sheep rearing on hill farms in UK. These studies resulted in the perhaps misleading common name of sheep tick for I. ricinus (Gray 1991). From the 1960s most studies in parts of Europe and the former Soviet Union on I. ricinus were carried out in relation to its vector role in the epidemiology of tick-borne encephalitis. In the same decade, several new aspects of the ecology of I. ricinus have been explored, particularly with regard to the microclimate and to diapause phenomena (reviewed by Balashov 1972). I. ricinus gained even more attention in 1983 when it was shown to be a vector of the newly described spirochaetes responsible for Lyme borreliosis (Burgdorfer et al. 1983). In the 21 th century I. ricinus is undoubtedly one of the most important tick species of the world resulting in a plethora of zoonotic diseases in Europe (Rizzoli et al. 2014). Despite the great amount of studies dealing with various aspects of physiology, behaviour, life cycle, ecology and especially its role in pathogen transmission, many parts of the tick s basic biology are obscure and unexplored. This chapter attempts to summarise the key elements of I. ricinus life cycle that contribute to the species public health importance with special emphasis on the eco-epidemiology of Lyme borreliosis. Life cycle and ecology Off the host I. ricinus is a common arthropod species. It has a high reproduction rate, broad host range and an ability to withstand or circumvent most environmental constraints. These adaptive strategies make it the most common species in temperate Europe with a geographical distribution from southern Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 31 DOI / _3, Wageningen Academic Publishers 2016

33 Gábor Földvári Spain to northern Scandinavia (Figure 1). Until recently, its southern range was considered to extend to North Africa. However, these records of I. ricinus may apply to a different species, namely the recently described novel species Ixodes inopinatus Estrada-Peña et al. (2014) pointing out that I. ricinus might be absent from north Africa. As most members of the family Ixodidae, I. ricinus spends 90-99% of its life off the host. Therefore, it has gained a unique set of adaptive traits that enables survival in the environment. The most limiting environmental factor for I. ricinus is relative humidity which optimally should not fall below 80% for prolonged periods (Estrada-Peña et al. 2013). This requirement and the necessary presence of vertebrate hosts means that I. ricinus is mainly found in deciduous woodland containing small mammals and deer, but in some areas with sufficient rainfall large populations may occur in open habitats such as meadows and moorland, where the majority probably feed on livestock (Gray 1991). Besides host abundance and diversity, abiotic factors play a key role in shaping the reproduction, development, and consequently the density and distribution of ticks. The most obvious sign of tick activity is the questing behaviour, i.e. searching for hosts. I. ricinus has an ambush strategy for host finding (Sonenshine 1993). This implies climbing the vegetation, clinging to the tips of stems Figure 1. Current known distribution of Ixodes ricinus in Europe at regional administrative level (NUTS3), based on published historical data and confirmed data provided by experts from the respective countries as part of the VectorNet project ( Braks et al. 2016). 32 Ecology and prevention of Lyme borreliosis

34 3. Ixodes ricinus life cycle or branches of the undergrowth and waiting for the direct contact with the host that brush against this vegetation. While questing for the passing host, ticks rest with their forelegs folded, holding themselves with the other legs. Vibrations caused by host movement and odours, body heat and shadows from the hosts excite response from the tick, resulting in extension and rapid waving of the forelegs where subtle receptors of the Haller s organ are located (Sonenshine 1993). This specific waving movement of the forelegs and the Haller s organ make ticks able to smell in stereo (A. Lakos, personal communication) that greatly enables them to locate and attach to the host. During questing, ticks may lose water that they can regain by descending back into the litter zone where the ticks actively reabsorb water vapour from the atmosphere (Rudolph and Knulle 1974). After rehydration, the ticks are ready to climb again onto the vegetation. Tick water balance is influenced by the saturation deficit of water in the air (affecting water loss) and by relative humidity (affecting the possibility of water gain by active water vapour uptake). Ticks can also influence water balance by moving to places with more preferred microclimate when the weather is warm and dry, e.g. to the leaf litter. Besides the energy reserves of the tick (depending mainly on the volume of blood meal from the previous stage), it is its ability to maintain a necessary level of body water that regulates questing behaviour (Estrada-Peña et al. 2013). Despite being affected by many different factors, I. ricinus has an extraordinary long life span lasting for several years. This is only possible with the specific adaptation of all three active stages (larva, nymph and adult; Figure 2) going into an inactive state called diapause. Ticks utilise diapause to anticipate suboptimal environmental conditions. Thus, diapause is different from quiescence and is defined as a neurohormonally-mediated dynamic state of low metabolic activity (Sonenshine and Roe 2014). The key drivers that regulate diapause are weather, microclimate and photoperiod, the day-night relative duration (or its change). Two basic forms can be differentiated: behavioural diapause in unfed ticks leading to the seasonal periodicity of I. ricinus and morphogenetic or Figure 2. Developmental stages of Ixodes ricinus. From left to right and top to bottom: adult female, nymph, adult male, larva (picture by Hans Smid from Van Duijvendijk et al. 2016). Ecology and prevention of Lyme borreliosis 33

35 Gábor Földvári developmental diapause in engorged ticks causing interruption of the development. In both cases the inducing stimulus is the photoperiod, which is perceived by ticks (Belozerov 1982), but temperature has also an important modifying influence (Gray et al. 2016). Diapause thus influences both host finding, feeding, reproduction, oviposition and moulting period before the onset of adverse weather conditions such as winter. The seasonal activity of I. ricinus reflects the subtle responses to environmental factors through the above mechanisms and is based on the biological strategy of every stage in which ticks avoid questing and development when environmental conditions are suboptimal (e.g. dry summer or cold winter) (Gray et al. 2016). This seasonality of larvae and nymphs largely influence disease risk. Rapidly increasing spring temperatures result in higher probability of synchronously co-feeding larvae and nymphs that promotes transmission of tick-borne encephalitis virus (TBEV), but not that of Lyme borreliosis spirochaetes. Co-feeding transmission is due to the special mechanism when pathogens, such as TBE virus, are transmitted from one tick to many other neighbouring ones without the systemic infection of the host (Labuda et al. 1993). In contrast to TBEV transmission, where a quantitative change occurs, in case of LB transmission an interesting qualitative change has been observed as an effect of co-feeding (Pérez et al. 2011). In this study it was reported that at the intraspecific level, Borrelia afzelii isolates obtained from the larvae that were feeding on the rodents simultaneously with nymphs displayed a higher diversity (based on outer surface protein C; ospc groups) than isolates from larvae feeding without nymphs. Thus, nymphs that were feeding simultaneously with larvae contributed to enhance the diversity of ospc groups in larvae through co-feeding transmission (Pérez et al. 2011). On the host On the host I. ricinus acquires blood meal for moulting (immatures) for oviposition (females) or feed facultatively and search for females to fertilise (males). The males of I. ricinus have an adaptive advantage to most of the other tick species with the facultative feeding (Schulze 1943); in contrast to most other ticks, I. ricinus males already possess mature sperms prior to feeding and are able to fertilise females on the vegetation before finding a host (Gray 1987). The human risk of infection by males (although unstudied) may exist, because they also readily attach to and feed on humans (Schulze 1943). As most ticks, I. ricinus is a successful r-strategist. This means that it maximises its reproductive potential (r) by laying several thousands of eggs. In ecology, r-selected species are those that place an emphasis on a high growth rate, and produce many offspring, each of which has a relatively low probability of surviving to adulthood (Pianka 1970). Fully fed, fertilised females can lay up to 4,000 (Honzáková et al. 1975), on the average 2,000-2,500 eggs (Balashov 1972) after dropping off the host. According to laboratory data, larvae feed between 2-5 days, nymphs feed between 2-7 days and females for 6-11 days (Balashov 1972). The duration of feeding depends mainly on tick and host and, to a lesser degree, environmental factors. Because most hosts (mammals and birds) are endothermic, ticks attaching to them have a relatively stable microclimate. This is the reason why larvae of I. ricinus were found to feed for the same time on mice at four different temperatures in the laboratory. However, for obvious metabolic reasons I. ricinus feed longer on hibernating compared to active hedgehogs (Balashov 1972). On the ectothermic reptiles, in contrast, immature I. ricinus feed for several days longer if environmental temperature decreases. The time needed for engorgement also depends on the physiological age of the ticks. Individuals that had been starving for long and those that fed relatively recently (in their previous stage) were observed to attach in smaller proportion and feed for longer than average (Balashov 1972). The same author reported also slower feeding on 34 Ecology and prevention of Lyme borreliosis

36 3. Ixodes ricinus life cycle body areas where blood supply was insufficient. The individual differences in the length of blood feeding certainly have a yet unstudied influence on pathogen transmission. Based on the number of hosts, I. ricinus has perhaps the broadest host range described among ticks. Besides sheep, the sheep tick is known to use over 300 other terrestrial vertebrate hosts (Bowmann and Nuttall 2008), including most of the domesticated animals. Among these, reservoir host are of crucial importance from the eco-epidemiological point of view. These are terrestrial vertebrate species that are the source of infection for I. ricinus. As defined by (Gray et al. 2002) they must also fulfil the following criteria: (1) they must be fed on by infected vector ticks, at least occasionally; (2) they must take up a critical number of infectious agents during an infectious tick bite; (3) they must allow the pathogen to multiply and to survive for some time in at least certain parts of their body; and (4) they must allow the pathogen to find its way into other feeding ticks. Larvae and nymphs very often feed on reservoirs of certain Lyme borreliosis spirochaetes, such as rodents, birds or lizards. The relatively common occurrence of these reservoirs leads to abundant occurrence of infected I. ricinus ticks in habitats that are often used by the public. Since the above mentioned vertebrates are often reservoirs of several human pathogenic infectious agents, I. ricinus feeding on them might also carry several pathogens simultaneously. Thus humans can be co-infected with different zoonotic tick-borne bacteria, e.g. Anaplasma phagocytophilum and Lyme borreliosis spirochaetes that can lead to diagnostic problems for the physicians. Many factors influence host finding, but one of the key determining factor for finding a specific host individual is the questing height of the tick individual. Adults seek hosts higher at the vegetation than nymphs and nymphs higher then larvae. This is at least one of the explanations why adults are usually not found on rodents or lizards, but larvae and nymphs can also access larger hosts (Sonenshine and Roe 2014). For the subadult stages, rodents (mice, voles, dormice, squirrels, etc.) are the most important hosts. Birds and lizards can also harbour a considerable number of larvae and nymphs. Among birds, species foraging mostly on the ground and low shrub vegetation, such as common blackbird (Turdus merula), song thrush (Turdus philomelos), and European robin (Erythacus rubecula) were shown to be frequently infested with I. ricinus (Rizzoli et al. 2014). Most of the common European lizard species such as sand lizards (Lacerta agilis), common wall lizards (Podarcis muralis) and green lizards (Lacerta viridis) have been found to carry larvae and nymphs of I. ricinus (Földvári et al. 2009, Majláthová et al. 2006). Where they are abundant, lizards can be equally important hosts as rodents (Richter and Matuschka 2006) and they usually carry more nymphs than rodents do (Majláthová et al. 2006). As larvae and nymphs often have their blood meal in close vicinity of each other on their hosts, it gives rise to the aforementioned co-feeding transmission (Bowmann and Nuttall 2008, Labuda et al. 1993, Pérez et al. 2011). Medium and large-sized mammals such as red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) wild boar (Sus scrofa), and red foxes (Vulpes vulpes) are the typical hosts for the adults. Deer and fox, but particularly hedgehogs and hares can simultaneously serve as hosts for both immature and adult I. ricinus (Földvári et al. 2011). Consequently, they form important maintenance hosts as the presence of a single species of vertebrate host enables the tick to develop a stable population. This can be a key factor in maintaining ticks in special habitats like islands or urban parks (Rizzoli et al. 2014). Many disease agents such as the spirochaetes causing Lyme borreliosis might be easily transmitted among larvae, nymphs and adults feeding close (in space and time) to one another on these hosts. This is another marked eco-epidemiological benefit of hosts harbouring all three stages from the pathogen s point of view. Ecology and prevention of Lyme borreliosis 35

37 Gábor Földvári Public health consequences From the tick-transmitted pathogen s point of view, survival within I. ricinus is not only beneficial because it is able to effectively maintain and transmit them, but also because of its longevity. Due to various forms of diapause, the life cycle of this tick is 4-6 years in average and can be up to 8 years long (Bowmann and Nuttall 2008, Gray 1991, Kahl et al. 2015). In many cases, I. ricinus can be even considered a reservoir for the pathogenic agents. This long lifespan is related to the exceptionally low metabolic rate of I. ricinus compared to other arthropods (Sonenshine and Roe 2014). The extraordinary wide host range of this tick species is another advantage for many pathogens, especially for those ones that are also ubiquitous in many groups of terrestrial vertebrates, such as Lyme borreliosis spirochaetes. In addition, the high reproduction rate of I. ricinus enables this species to be one of the most prevalent arthropods in the optimal habitats. From public health perspective, several features of I. ricinus life cycle promote survival and transmission of various pathogenic agents (Jahfari and Sprong 2016). During blood feeding, ticks produce analgesic, anti-inflammatory and immune suppressing molecules (Bowmann and Nuttall 2008) that not only help sufficient and undisturbed feeding but it also enhance the efficient transmission of pathogens. The small size of I. ricinus especially that of larvae and nymphs compared to larger species of Dermacentor and Hyalomma, increases the chances not to find them on the human body (Figure 2). Determined by seasonal patterns and the usually aggregated distribution of ticks on a host individual, co-feeding allows pathogens to increase their chance of transmission and form a more diverse population as mentioned before for Lyme disease spirochaetes (Pérez et al. 2011). The colonisation and dissemination of Borrelia burgdorferi spirochaetes in I. ricinus is an intriguing example of co-evolution. It has been first described in case of B. burgdorferi s.s. and I. scapularis that spirochaetes in unfed nymphs are restricted mainly to the tick midgut; most of these spirochaetes express OspA (Schwan and Piesman 2002). When feeding commences, rapid multiplication of the spirochaetes occurs; OspA is downregulated and a proportion of the population now expresses OspC. The downregulation of OspA may allow the spirochaetes to leave the midgut since OspA apparently binds to the tick receptor for outer surface protein A (TROSPA) (Pal et al. 2004). Thus, TROSPA-OspA binding is considered the first and mandatory step of the tick colonisation process. Within the fed tick, a tick salivary gland protein, Salp15, plays an intriguing role in binding to OspC and facilitating transmission and initial survival of the spirochaete in the vertebrate host (Ramamoorthi et al. 2005). Although we have more experimental data about I. scapularis, it has been demonstrated that OspC is also crucial for dissemination of B. afzelii from I. ricinus midgut to the salivary glands, a prerequisite for infection of the vertebrate host (Fingerle et al. 2007). Salp15 was also originally described in I. scapularis but homologues were found in I. ricinus (Hovius et al. 2007). Interestingly, the Lyme borreliosis spirochaetes are able to increase the tick s Salp15 gene expression, which in turn protects B. burgdorferi sensu lato from antibody-mediated killing and facilitates infection of the mammalian host (Murase et al. 2015). In addition, Salp15 has been also shown to inhibit T-cell activation (Ramamoorthi et al. 2005). The event of tick bite and thus the infection with LB spirochaetes can only occur if tick and human activities overlap both in time and in space. Besides the typical natural habitats with high I. ricinus density (e.g. deciduous forests), urban parks (Szekeres et al. 2016) might be also ideal locations for tick-human interactions. However, the highest risk of infestation is usually seen when humans also actively visit tick habitats. Within urban parks (with similar number of visitors in every season) 36 Ecology and prevention of Lyme borreliosis

38 3. Ixodes ricinus life cycle the disease risk may depend more on tick activity patterns alone. At natural sites, on the other hand, where many more people can be expected during summer holidays, the highest risk of infection is probably during summer, the period of maximum human exposure (Sumilo et al. 2008). As pointed out for TBEV eco-epidemiology, socio-economic factors as the annual patterns of human activity may have a great influence on the annual LB incidence as well. Therefore, for a more comprehensive understanding of infection risk (composed of hazard and exposure), an interdisciplinary approach is needed that incorporates not only tick seasonality and ecology but also socio-economic and human behaviour sciences. The different adaptive life cycle traits making I. ricinus the number one tick species of public health concern in Europe are summarised in Figure 3. High reproduction rate Longevity Over 300 hosts Low metabolic rate Behavioural Small size diapause Abundance Ambush strategy Tick bite risk Developmental diapause Public health importance Infection Anti-inflammatory molecules Co-evolution with patogens Immune suppression Analgesia Co-feeding Figure 3. Key features of Ixodes ricinus life cycle that contribute to its public health importance. Ecology and prevention of Lyme borreliosis 37

39 Gábor Földvári Public health relevance Ixodes ricinus is the most widespread tick species in Europe. Besides the abiotic needs of the ticks, vertebrate hosts that maintain both the ticks and Borrelia burgdorferi sensu lato are the key factors in the natural cycle of Lyme borreliosis spirochaetes. Intimate and adaptive interactions between the tick and the host and between spirochaetes and the tick enable stable natural pathogen maintenance and also the effective infection of humans. I. ricinus poses a risk of Lyme borreliosis in many natural and urban habitats in Europe. Acknowledgements The author was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. References Arthur DR (1963) British ticks. Butterworths, London, UK. Balashov YS (1972) Bloodsucking ticks (Ixodoidea) vectors of diseases of man and animals. Entomol Soc Amer Misc Publ J 8: Belozerov V (1982) Diapause and biological rhythms in ticks. In: Obenchain FD and Galun R (eds) Physiology of ticks. Pergamon Press, Oxford, UK, pp Bowmann A and Nuttall P (2008) Ticks biology, disease and control. Cambridge University Press, New York, USA. Braks MAH, Mulder AC, Swart A and Wint W (2016) Grasping risk mapping. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Burgdorfer W, Barbour AG, Hayes SF, Peter O and Aeschlimann A (1983) Erythema chronicum migrans a tickborne spirochetosis. Acta Trop 40: Estrada-Peña A, Gray JS, Kahl O, Lane RS and Nijhof AM (2013) Research on the ecology of ticks and tick-borne pathogens-methodological principles and caveats. Front Cell Infect Microbiol 3: 29. Estrada-Peña A, Nava S and Petney T (2014) Description of all the stages of Ixodes inopinatus n. ssp. (Acari: Ixodidae). Ticks Tick Borne Dis 5: Fingerle V, Goettner G, Gern L, Wilske B and Schulte-Spechtel U (2007) Complementation of a Borrelia afzelii OspC mutant highlights the crucial role of OspC for dissemination of Borrelia afzelii in Ixodes ricinus. Int J Med Microbiol 297: Földvári G, Rigó K, Jablonszky M, Biró N, Majoros G, Molnár V and Tóth M (2011) Ticks and the city: ectoparasites of the Northern white-breasted hedgehog (Erinaceus roumanicus) in an urban park. Ticks Tick Borne Dis 2: Földvári G, Rigó K, Majláthová V, Majláth I, Farkas R and Pet Ko B (2009) Detection of Borrelia burgdorferi sensu lato in lizards and their ticks from Hungary. Vector-Borne Zoonotic Dis 9: Gray JS (1987) Mating and behavioural diapause in Ixodes ricinus L. Exp Appl Acarol 3: Ecology and prevention of Lyme borreliosis

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41 Gábor Földvári Szekeres S, Majláthová V, Majláth I and Földvári G (2016) Neglected hosts: the role of lacertid lizards and medium-sized mammals in the eco-epidemiology of Lyme borreliosis. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersöz J, Oei A, Földvári G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: Ecology and prevention of Lyme borreliosis

42 4. Ecology of Borrelia burgdorferi sensu lato Elena Claudia Coipan 1,2 and Hein Sprong 1,2* 1 National Institute for Public Health and the Environment, Centre for Infectious Disease Control, P.O. Box 1, 3720 BA Bilthoven, the Netherlands; 2 Laboratory of Entomology, Wageningen University & Research, P.O. Box 16, 6700 AA Wageningen, the Netherlands; hein.sprong@rivm.nl Abstract Components of the enzootic cycle of Borrelia burgdorferi s.l. in Europe. The various developmental stages of the ticks feed on various classes of vertebrate hosts. The competence of the vertebrates for the B. burgdorferi s.l. genospecies determines what bacteria will be taken up by the ticks feeding on them. The host preference of the tick stages and the abundance of the hosts determines the prevalence of the bacteria in the next stage of the ticks. For simplicity rodents and insectivores are grouped. Bacteria that have been shown to cause disease in humans are marked by the darkblue frame. Keywords: Borrelia burgdorferi s.l., ecology, Ixodes ricinus, pathogenicity, transmission, vertebrate host Introduction Notwithstanding the causality dilemma of the egg and the hen as to whichever transmitted the first Borrelia burgdorferi s.l. spirochaete the host or the vector, we assume that the enzootic cycle of these spirochaetes begins with competent vertebrate hosts. These can carry, amplify, and transmit the bacteria to the blood-sucking vectors that feed on them i.e. ticks. The ticks that manage to maintain the Borrelia spirochaetes through the moulting process can transmit them further to a next vertebrate they feed on and the transmission cycle of B. burgdorferi s.l. resumes. A Borrelia transmission cycle that has been shown to involve up to 18 different Ixodes and more than 300 vertebrate species. When accounting for densities of some individual host species of up to 1,200 and ticks of up to two million per square kilometre, the simple transmission cycle becomes a process of enormous proportions. When considering also that one of the feeding hosts of the ticks is represented by humans, the simple transmission cycle becomes a complicated public health issue, with an incidence of more than 100,000 Lyme borreliosis cases in Europe alone! B. burgdorferi s.l. is a group of 20 genospecies of spirochaetes, some of which are known the Lyme disease spirochaetes. The disease was named after the town Old Lyme in Connecticut, USA, where it was first diagnosed (Burgdorfer et al. 1982). Later, the causative bacteria were identified as highly motile spirochaetes that are transmitted by hard ticks (Acari: Ixodida) (Burgdorfer et al. 1983). During the last three decades Lyme disease has gained increasingly more interest, being identified as the most common vector-borne human disease in the temperate area of the Northern hemisphere (ECDC 2011). With an intricate enzootic cycle and a genetic complexity to match it, B. burgdorferi s.l. is one of the most puzzling pathogenic microorganisms. This chapter will address the ecology and molecular adaptations of B. burgdorferi s.l. at various scales, from complex to genospecies level, pinpointing the implications for public health and highlighting questions that are still unanswered. Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 41 DOI / _4, Wageningen Academic Publishers 2016

43 Elena Claudia Coipan and Hein Sprong Genomes and genetic diversity in B. burgdorferi s.l. The ecological adaptations of B. burgdorferi s.l. are underpinned by a complex genomic structure and gene expression. The genome of these spirochaetes is highly fragmented, with, next to the chromosome, up to 21 different plasmid types (Casjens et al. 2011, Fraser et al. 1997, Schutzer et al. 2011, 2012). The linear chromosome contains the core house-keeping genes, with a summed length of approximately 950 kbp (Fraser et al. 1997). The plasmids contain the majority of the lipoproteins genes (Barbour 1988, Casjens et al. 2012) that are essential for transmission between vertebrates and ticks and are differentially expressed in the various phases of the enzootic cycle (Schwan and Piesman 2000). The high fragmentation of the genome is considered to be a facilitating element in the multiple niche shifts that a spirochaete has to undergo. However, as complex the genome of B. burgdorferi s.l. may be, it misses many of the essential house-keeping genes, which is what makes it an obligate parasite, having to use the vertebrate host as well as the tick host for survival (Posey and Gherardini 2000, Purser et al. 2003). There is large genetic variation between the genospecies. Recent whole-genome studies have revealed that these genetic differences consist mainly in plasmid content and gene location on the various plasmids (Casjens et al. 2011, Casjens et al. 2012, Schutzer et al. 2011, 2012). The content of the linear plasmids may be shuffled by telomere fusion (Kobryn and Chaconas 2005) but the repertoire of genes remains relatively consistent (Casjens et al. 2012). Beside the differences among the various genospecies in the B. burgdorferi s.l. complex, there are also marked differences within the genospecies. These have been investigated both at chromosomal and plasmid level, and appear to span over house-keeping as well as virulenceencoding genes. Furthermore, chromosomal and plasmid genes have been found in linkage disequilibrium (Bunikis et al. 2004), which is unexpected for genetic elements that are not physically linked. The gold standard for genotyping of B. burgdorferi s.l. nowadays is multilocus sequence typing (MLST), based on eight housekeeping genes on the chromosome, which undergo slow evolution and show nearly neutral variation (Margos et al. 2008, Urwin and Maiden 2003). Furthermore, MLST has revealed geographical structuring of B. burgdorferi s.l. populations (Vitorino et al. 2008, Vollmer et al. 2011). Previous studies have shown that the 5S-23S rdna intergenic spacer (IGS) is also a marker that can discriminate between the genospecies of B. burgdorferi s.l. and detect genetic differentiation between the bacteria of various geographic origins, while having a comparable predictive value of human pathogenic B. burgdorferi s.l. to that of MLST analysis (Coipan et al. 2013a, 2016). Transmission cycle B. burgdorferi s.l. is a vector-borne microorganism it cannot be transmitted between vertebrate hosts in the absence of a tick vector. Ixodes ricinus is the main vector of B. burgdorferi s.l. in Europe (Gern and Humair 2002). In certain habitats Ixodes hexagonus and Ixodes uriae can also transmit B. burgdorferi s.l. (Gern et al. 1997, Olsen et al. 1993), although their importance in the maintenance of the spirochaetes seems to be lower. Early studies have indicated that also Ixodes canisuga and Ixodes frontalis might act as vectors (Estrada-Pena et al. 1995). Recent experimental studies have shown, however, that birdspecialised ticks such as I. frontalis and Ixodes arboricola can get infected with B. burgdorferi s.l. 42 Ecology and prevention of Lyme borreliosis

44 4. Ecology of Borrelia burgdorferi sensu lato bacteria but cannot transmit them to the vertebrate hosts (Heylen et al. 2013b). This highlights the importance of experimental studies for the assessment of the vectorial competence of the various bacterial species. Transovarial transmission is considered to have a negligible contribution to the maintenance of the bacteria in enzootic cycles (Richter et al. 2012, Rollend et al. 2013). However, recently, Van Duivendijk et al. (2016) have shown that 0.62% of the larvae in nature is infected with B. burgdorferi s.l. Considering that the number of larvae questing and on the small rodents is 100 and 50 times, respectively, higher than that of nymphs (Randolph 1998, Van Duijvendijk et al. 2016), larvae could be just as important as nymphs in the maintenance of some B. burgdorferi s.l. genospecies. The presence of the spirochaetes in the larvae could be the result of partial feeding of larvae on a host with a subsequent change of host, but it could also be the result of transovarial transmission. Future studies have to clarify the importance of this transmission route in the maintenance of B. burgdorferi s.l. and the transmission to humans. The main transmission route of these bacteria is the interstadial one, from larvae to nymphs and from nymphs to adult ticks. Larvae of I. ricinus can become infected during a blood meal from an infected host (Piesman and Sinsky 1988) and during a blood meal in the vicinity of an infected nymph feeding on a host, process known as co-feeding (Gern and Rais 1996). The infected engorged larvae then moult into infected nymphs, which can transmit the spirochaetes to new hosts (Radolf et al. 2012). The same process is repeated for the next developmental stage nymph to adult. Thus, the maintenance of the bacteria in enzootic cycles is dependent on all sorts of vertebrates and the ticks they feed. Many small mammals, birds and lizards act as transmission and/or amplification hosts for B. burgdorferi s.l. (Hofmeester et al. 2016). Deer are among the few vertebrates known as incompetent for transmission of B. burgdorferi s.l. The inability of Borrelia to circumvent the innate immune response of ungulates, makes these animals incompetent transmitters of the spirochaetes (Kurtenbach et al. 2002). It has been suggested (Hofmeester et al. 2016) that there are at least two distinct mechanisms behind the maintenance of small mammal-transmitted and bird-transmitted Borrelia spp.: 1. Because small mammals have low nymphal burdens, their infection prevalence with B. burgdorferi s.l. is relatively low. However, because they feed a large proportion of the larvae, even a small infection prevalence of the host species can result in a high density of infected nymphs with small mammal-transmitted Borrelia spp. like Borrelia afzelii. This high density of larvae infected with small mammal-transmitted Borrelia spp. results in a sufficiently-large number of infected nymphs to, in turn, infect small mammals in spite of their low nymphal burdens. Furthermore, the life cycle of I. ricinus takes 2-6 years to complete, with each life cycle stage (larva, nymph and adult) taking one year or even more (Gray 1991). Small rodents, on the other hand, are short-lived, with few adults surviving from one summer to the next in the wild (Ostfeld 1985). Thus, the infected larvae that will moult into infected nymphs can infect a couple of generations of rodents. 2. Bird-transmitted Borrelia spp., like Borrelia garinii and Borrelia valaisiana, on the other hand, seem to be dependent on high infection prevalence of their host species due to relatively high nymphal burdens. Therefore, even with a low larval burden and intermediate host density, sufficient numbers of infected nymphs are produced to infect birds, which completes the maintenance cycle for bird-transmitted Borrelia spp. However, this strategy is probably not only restricted to bird-transmitted Borrelia spp. Borrelia spielmanii is a candidate for a Ecology and prevention of Lyme borreliosis 43

45 Elena Claudia Coipan and Hein Sprong similar maintenance strategy in mammals as it is often found with low prevalence in questing ticks, but with high prevalence in one of its principal hosts, Eliomys quercinus and Erinaceus europaeus (Richter et al. 2004). These differences in maintenance strategies could indicate that less common Borrelia spp., or other tick-borne pathogens with low infection prevalence in questing nymphs, might be maintained by host species with high nymphal or adult burdens (Ostfeld et al. 2014). e.g. one would expect that B. garinii will be more abundant in questing adults than in questing nymphs. Comparative studies on the infection prevalence of the various stages of I. ricinus with B. burgdorferi s.l. genospecies or other tick-borne pathogens could test this hypothesis. These alternative transmission strategies indicate that different B. burgdorferi s.l. genospecies have specialised either on host species that occur in high densities, or on host species that feed large numbers of ticks, with the exception of larger bodied mammalian species such as deer. How do the vertebrate hosts contribute to the maintenance of B. burgdorferi s.l.? Distribution Maintenance of the different Borrelia genospecies in enzootic cycles occurs via direct transmission between various vertebrate hosts and hard ticks (Acari: Ixodida), often in distinct cycles. Tick and host associations shape, thus, the geographical distribution of B. burgdorferi s.l. (Kurtenbach et al. 2006, Vollmer et al. 2011). Most of the B. burgdorferi s.l. genospecies are specialist in terms of the class of vertebrate hosts that they exploit. They can be either mammal-, bird- or reptile-associated (Table 1). However, some of them are generalist, being able to infect two vertebrate classes both mammal and avian hosts (Borrelia bissettii), or all three vertebrate classes (B. burgdorferi s.s.) (Kurtenbach et al. 2006, Newman et al. 2015). At large geographical scale the distribution of the various Borrelia genospecies is primarily driven by the vertebrate host they are adapted to (Kurtenbach et al. 2006, Vollmer et al. 2011), with bird-associated Borrelia having a wider areal than rodent-associated ones. Thus, bird-associated Borrelia, such as B. garinii, Borrelia turdi, and B. valaisiana, are spread over both Europe and Asia. The genospecies that are mammal-associated, such as B. spielmanii, Borrelia yangtze, and Borrelia tanukii, seem to be confined to certain geographic areas (Fukunaga et al. 1996, Margos et al. 2011, 2015, Richter et al. 2004). Exceptions are B. afzelii and Borrelia bavariensis, which are spread across all Eurasia (Margos et al. 2013, Rauter and Hartung 2005). Host specificity could be also the reason that some of the genospecies remain confined to certain geographical areas, where the competent hosts are most abundant. This is the case of Borrelia yangtzensis or B. tanukii in Asia, which are amplified by rodents of the species Suncus murinus and Mus caroli (Kawabata et al. 2013, Margos et al. 2015), and Myodes rufocanus, Myodes smithii and Apodemus speciosus (Masuzawa et al. 1996b), respectively. In areas where the specific vertebrate hosts are absent or less abundant the genospecies cannot persist or, if they do, it is at very low abundance levels. However, many of the genospecies that were once thought to have a relatively limited areal (e.g. B. turdi or B. bavariensis), have been later proved to be widespread (Margos et al. 2013, Norte et al. 2015). In some cases it could be a matter of recent introduction of the genospecies by means of migratory birds (Hasle et al. 2011). For B. bavariensis, recent whole genome studies have shown that the European strains are almost clonal, while in the Asian strains there is a higher genetic diversity. This could be the result of a recent introduction of the genospecies in the European landscape 44 Ecology and prevention of Lyme borreliosis

46 4. Ecology of Borrelia burgdorferi sensu lato Table 1. Genospecies of the Borrelia burgdorferi s.l. complex distribution, hosts and vectors. Borrelia genospecies Continent Vertebrate host Vector tick B. afzelii Europe, Asia rodents, insectivores Ixodes ricinus, I. persulcatus, I. hexagonus B. americana North America birds I. pacificus, I. minor B. andersonii North America birds I. dentatus B. bavariensis Europe, Asia rodents I. ricinus, I. persulcatus B. bissettii North America, Europe rodents 1, birds I. pacificus, I. spinipalpis, I. affinis B. burgdorferi North America, Europe rodents, insectivores, birds, reptiles B. californiensis North America rodents unknown B. carolinensis North America rodents unknown B. garinii Europe, Asia birds I. ricinus, I. persulcatus, I. uriae B. japonica Asia rodents, insectivores I. ovatus B. kurtenbachii North America rodents unknown B. lusitaniae Europe reptiles I. ricinus B. mayonii North America unknown unknown B. sinica Asia rodents I. ovatus B. spielmanii Europe rodents I. ricinus B. tanukii Asia rodents I. tanuki B. turdi Japan birds I. turdus, I. frontalis B. valaisiana Europe, Asia birds I. ricinus, I. columnae B. yangtzensis Asia rodents I. granulatus, I. nipponensis 1 Vertebrate hosts known only for North America. I. ricinus, I. hexagonus, I. scapularis, I. pacificus, I. affinis, I. minor, I. spinipalpis, I. muris by a shift in the vector tick species from only Ixodes persulcatus to also I. ricinus (Gatzmann et al. 2015). It is equally possible that the earlier failure to detect these genospecies was the result of methodological limitations. This kind of questions will probably be answered with the use of phylogeography and population genetics studies. It has been shown that IGS can detect genospecies-specific population subdivisions and population expansion (Coipan et al. 2013a). In a recent study on 1,182 IGS sequences, fixation indices were significantly different from zero for B. afzelii, supporting molecular divergence. That is likely due to isolation by distance of the common ancestor of the B. afzelii samples; an event that occurred at some point of time in the past. In addition to B. afzelii, a statistically significant trace of isolation by distance was detected among B. garinii also. Especially for these two genospecies, the molecular marker IGS possesses a high resolution to differentiate subpopulations within a single genospecies, which are defined according to geographical location (Coipan et al. 2013a). Furthermore, the genetic differentiation between geographical areas was higher for B. afzelii than for B. garinii. Similarly, Vollmer et al. (2011), have shown that MLST profiles can capture the movement of the vertebrate hosts, observing a higher genetic differentiation between distant countries for B. afzelii than for B. garinii. This is consistent with the admixture theory where birds would be able to bridge remote areas, mixing the B. garinii strains, while rodents, with their limited movement range, contribute to keeping the B. afzelii subpopulations separate. At another scale, Vitorino et al. (2008), using MLST, have shown that the fine-scale phylogeographic population Ecology and prevention of Lyme borreliosis 45

47 Elena Claudia Coipan and Hein Sprong structure of Borrelia lusitaniae in Portugal reflects the parapatric population structure of the lizards in the same area. Transmission capacity Host species differ in their transmission capacity for the different genospecies of B. burgdorferi s.l. and consequently, their ability to infect I. ricinus larvae with the bacteria. For example, B. afzelii is mainly transmitted by small mammals, while B. garinii is predominantly transmitted by birds (Hanincova et al. 2003a, 2003b, Heylen et al. 2013a), and even within genospecies, different host species differ in their ability to transmit B. burgdorferi s.l. (Kurtenbach et al. 1994). Both the number of ticks a host can feed and the transmission of B. burgdorferi s.l. could be linked to general host characteristics (Carbone et al. 2005, Lee 2006, Previtali et al. 2012), and could therefore influence both tick burden and reservoir competence/capacity for B. burgdorferi s.l. (Barbour et al. 2015, Huang et al. 2013, Marsot et al. 2013). What makes a Borrelia specific to a certain host type is still an open question. Previous studies have shown that the associations are primarily dependent on the ability of the bacteria to circumvent the innate immune response of the host (Kurtenbach et al. 1998, Ullmann et al. 2003). However, this does not explain the observed association of European B. burgdorferi s.s. with rodents of the Sciuridae family (Humair and Gern 1998, Marsot et al. 2011, Pisanu et al. 2014). The advent of genomic analysis allows the detailed comparison of the genospecies and hints on the potential marked differences are already emerging. For example, the absence of ospb in B. garinii has been suggested to be a result of the host specificity of this genospecies, since the same gene appears to function during Borrelia infections within mammalian hosts (Qiu and Martin 2014). The success of transmission and maintenance of B. burgdorferi s.l. in enzootic cycles depends on the density and abundance of the various vertebrate host species. Hofmeester et al. (2016) have calculated the relative importance of vertebrate species that are abundant in European forests for maintenance of B. burgdorferi s.l. as well as their realised reservoir competence, i.e. the proportion of blood fed larvae that become infected with B. burgdorferi s.l. (LoGiudice et al. 2003). Among small mammals, E. quercinus, Microtus agrestis, and Sorex araneus have the highest realised reservoir capacity. It is, however, Apodemus sylvaticus and Myodes glareolus that have the highest relative importance for infecting larvae with B. burgdorferi s.l.; that is due to their high densities and relatively large larval burdens. The second most important group for B. burgdorferi s.l. maintenance is that of thrushes (Turdus merula and Turdus philomelos), which have intermediate densities and larval burdens, but a very high realised reservoir competence. This indicates that the number of larvae feeding on a host species and its density are more important than the reservoir competence of that host species in determining their contribution to larvae infection. Furthermore, it suggests that the prevalence of the two main B. burgdorferi s.l. genospecies in questing ticks is mainly dependent on the distribution of larvae over rodents and thrushes. Genetic differentiation Genetic differentiation is a precondition for speciation (Avise 2007). Among B. burgdorferi s.l. genospecies, B. garinii is the one that has the largest genetic differentiation, with phylogenetic trees based on MLST housekeeping genes showing long branches (Coipan et al. 2016). One event of speciation within B. garinii could have been B. bavariensis, a genospecies similar to B. garinii. Yet, another ongoing speciation event could be that of some strains of B. garinii group NT29 that are found in rodents, but not in birds (Miyamoto and Masuzawa 2002). 46 Ecology and prevention of Lyme borreliosis

48 4. Ecology of Borrelia burgdorferi sensu lato The host community has been hypothesised to generate the intraspecific genetic diversity of B. burgdorferi s.l. by various mechanisms. One of them is the multiple niche polymorphism balancing selection that implies that various hosts can act as ecological niches for a subset of the strains of a species (Gliddon and Strobeck 1975, Levene 1953). Such host specialisation of the B. burgdorferi s.l. strains has been described especially for B. burgdorferi s.s. in North America, based on the outer surface protein C gene (ospc) (Brisson and Dykhuizen 2004) and MLST (Mechai et al. 2016). Also European studies reported differentiation among the strains of B. afzelii isolated from various rodents, based on ospc and ribosomal protein L2 gene (Jacquot et al. 2014). The second mechanism that could maintain the genetic diversity of B. burgdorferi s.l. at some loci is the presence of negative frequency dependent polymorphisms. This postulates that no strain has a maximum fit within a certain host species but that initial infection of a host triggers an immune response that will be protective against subsequent infections with genetically similar bacterial strains (Barthold 1999, Gromko 1977). Thus, the strain that is most abundant at some point in time will be gradually decreased in frequency by negative selection from the host, favouring another one to become more frequent; a temporal shift in the frequency of the various strains occurs in this manner. This theory has also been supported by the results of some European studies (Durand et al. 2015, Hellgren et al. 2011). The second hypothesis has more ecological support, in the sense that bacterial haplotypes for ospc (one of the strongest elicitor of the vertebrates immune response to B. burgdorferi s.l.) are found to have variable frequencies in different geographical areas while the local assemblage of haplotypes seems to reflect the large-scale assemblage (E.C. Coipan et al. unpublished data). Thus, while in Switzerland the most abundant ospc type in a study by Durand et al. (Durand et al. 2015) was A10, followed at more than 30% difference by A9, in another study by E.C. Coipan et al. (unpublished data) A9 and A10 came in the 3 rd and 4 th positions. This could be the reflection of a negative frequency-dependent selection mechanism, which allows for fluctuations in time of the alleles frequencies and consequently for the shift in frequencies at different geographic locations. Another observation in favour of the negative-frequency dependent selection is the existence of a high degree of linkage disequilibrium between the alleles at loci on the chromosome and plasmids (Bunikis et al. 2004, E.C. Coipan et al. unpublished data). Thus, in spite of the fragmented genome of B. afzelii, and subsequent facility for gene exchange, the horizontal gene transfer is not a pervasive phenomenon in these bacteria. That could be another indication that these spirochaetes have evolved to have equal fitness for both species of the main vertebrate hosts. Given the frequency of double/multiple Borrelia infections observed in the larvae feeding on rodents, there would be plenty of opportunities for lateral gene transfer, should one of the genotypes have an advantage in resisting the host s immune response. This implies that the innate immune response of the various small rodents does not exempt a strong selective pressure among the genotypes of B. afzelii. Coinfection The coinfection with other microorganisms may facilitate or impair the transmission efficiency of the Borrelia. These coinfections seem to not represent an exception but more likely the rule. In a study on questing ticks in the Netherlands, 6.3% (350/5,570) were found infected with more than one pathogen of different genera. A negative significant association was found between B. afzelii and Rickettsia helvetica, as well as between Neoehrlichia mikurensis and R. helvetica. On the other hand, significant positive associations were found between B. afzelii and N. mikurensis and between Borrelia and Babesia spp. These findings, together with a seasonal synchrony of the infection prevalences with these pathogens in questing ticks indicate that B. afzelii, N. mikurensis, Ecology and prevention of Lyme borreliosis 47

49 Elena Claudia Coipan and Hein Sprong and Babesia share the same reservoir hosts, while R. helvetica is maintained in other enzootic cycles, probably with birds (Coipan et al. 2013b, Heylen et al. 2016). Multiple studies have reported coinfection in questing ticks with some of the tick-borne pathogens (Belongia 2002, Burri et al. 2011, Ginsberg 2008, Lommano et al. 2012, Nieto and Foley 2009, Reye et al. 2010). Some others have reported serological evidence of coinfection with spotted fever group rickettsiae and B. burgdorferi s.l. in patients suspected of Lyme neuroborreliosis (Koetsveld et al. 2016). It is possible that the severity of Lyme disease is affected by simultaneous infections with other tick-borne pathogens (Belongia 2002, Swanson et al. 2006). Some of them, such as Anaplasma phagocytophilum, modulate host immunity and increase susceptibility to various second pathogens, including B. burgdorferi s.l. (Holden et al. 2005, Thomas et al. 2001). Others, such as Rickettsia spp., infect endothelial cells, which form the basic layer of the blood brain barrier, rendering this temporarily permeable to B. burgdorferi s.l. (Koetsveld et al. 2016). Thus, coinfection might be partly responsible for the transmission efficiency of B. burgdorferi s.l. between hosts and ticks but also for the variability in clinical manifestations that are usually associated with Lyme borreliosis. How do the ticks contribute to the maintenance of B. burgdorferi s.l.? The bacteria have to adapt to either the vertebrate or invertebrate environment, in a matter of hours. For this, it uses a whole cascade of regulatory mechanisms that promote its activation, detachment, immune evasion, and attachment. The best studied gene expression shift is the down-regulation of outer surface protein A gene (ospa) and up-regulation of ospc (Schwan and Piesman 2000). The up-regulation of ospc is necessary for infecting the host while its downregulation, together with the up-regulation of ospa is responsible for infecting the tick. These latter processes also protect the bacteria in the midgut of the tick from the destructive effects of the host s immune response targeted against ospc (Tsao 2009). Some of the B. burgdorferi s.l. genospecies are vectored by different tick species in different geographical areas. E.g. bird-associated Borrelia have a cycle that involves I. frontalis, Ixodes turdus and Ixodes columnae in Asia (Masuzawa et al. 1996a, Miyamoto and Masuzawa 2002) and I. ricinus and I. persulcatus in Europe (Gern and Humair 2002). Likewise, B. burgdorferi s.s. is transmitted in Europe by I. ricinus and I. hexagonus (Gern and Humair 2002, Toutoungi and Gern 1993) and in North America by Ixodes scapularis and Ixodes pacificus (Piesman 2002). While some Ixodes species transmit multiple B. burgdorferi s.l. genospecies, other tick-borrelia associations seem to be less efficient (Masuzawa et al. 2005). It is possible that the interaction bacterium-tick species contributes to the augmentation of the host spectrum of the bacterium. The tick species present in the areal of a B. burgdorferi s.l. genospecies could promote genetic differentiation of the bacteria and differential transmission efficiencies by various mechanisms. Transmission efficiency One of these relies on the intrinsic properties of the ticks such as the receptors for the spirochaetal proteins. Some of the proteins important for the persistence of the bacteria in ticks are OspA and OspB, their removal leading to the impossibility of the spirochaetes to colonise the tick midgut (Pal et al. 2000, Pal et al. 2004). OspA has been found to bind to the TROSPA protein (tick receptor for OspA) of the midgut of I. scapularis (Pal et al. 2004). Recently, homologues of TROSPA have been found in I. persulcatus (Konnai et al. 2012) and I. ricinus (Figlerowicz et al. 2013). Different receptors 48 Ecology and prevention of Lyme borreliosis

50 4. Ecology of Borrelia burgdorferi sensu lato for OspA could account for different attachment rates of the spirochaetes to the tick midgut and, hence, for their abundance in enzootic cycles. Such a situation could be that of B. burgdorferi s.s. in North America. While in Europe, this is a bacterium that infects only mammals, especially rodents of the Sciuridae family (Humair and Gern 1998, Marsot et al. 2011, Pisanu et al. 2014), in the Nearctic it is the dominant B. burgdorferi s.l. genospecies, thriving in a variety of vertebrate hosts of all classes (mammal, avian, and reptilian) (Piesman 2002). Furthermore, while it is relatively rare in the questing I. ricinus ticks less than 2% (Coipan et al. 2013b, Rauter and Hartung 2005), it is much more frequent in I. scapularis and I. pacificus 25-35% (Kurtenbach et al. 2006). Another tick-protein that plays a role in the transmission of B. burgdorferi s.l. is Salp15. This is a feeding-induced salivary protein that binds to OspC of the spirochaetes, protecting them from antibody-mediated immune responses (Ramamoorthi et al. 2005). Homologues of Salp15 were found recently in I. ricinus (Hovius et al. 2007) and I. persulcatus (Murase et al. 2015). Host range of ticks Both the genetic diversity of B. burgdorferi s.l. and their abundance in enzootic cycles are influenced by the host range of the tick species; whether a tick is a generalist or a specialist will implicitly affect the circulation of the bacteria it carries in enzootic cycles. The issue of tick specialisation to vertebrate hosts is highly controversial. There are studies that describe the majority of the tick species (700 of the extant 800) as host specialists (McCoy et al. 2013). One of the most compelling evidence of host specialisation of a tick species to the host is that of I. uriae, where stronger genetic differentiation was found among tick populations of sympatric host species than among geographically isolated tick populations of the same host species (McCoy et al. 2001). On the other hand there are other studies that suggest that the main determinant of the tick dispersion is the set of abiotic conditions characteristic to a geographic area (Klompen et al. 1996), the proof thereof being that approximately 50% of the investigated tick species had more restricted areal than that of their hosts. Another explanation for observed specialisation is the mere absence of other feeding hosts. For example, more than 70 different tick species have been reported to bite humans (Estrada-Pena and Jongejan 1999). The feeding pattern of ticks could explain why, in most areas in Europe, B. afzelii is the most common genospecies found in questing nymphs (Rauter and Hartung 2005). Hofmeester et al. (2016) found that 89% of the infected larvae analysis had fed on rodents. This should result in a large percentage of B. afzelii-infected nymphs as B. afzelii is transmitted by small mammals (Hanincova et al. 2003a). Thrushes fed only 10% of the infected larvae, which could explain the relatively low percentages of B. garinii and B. valaisiana in questing infected nymphs (Coipan et al. 2013b, Gassner et al. 2011, Ruyts et al. 2016) (Figure 1). It could be that the specialisation of B. burgdorferi s.l. genospecies is partly influenced by the tick feeding behaviour. Evolutionary theory predicts that specialist pathogens are favoured if their hosts are abundant, whereas generalists would do better when the encounters with host species are less predictable (Woolhouse et al. 2001). In this context, the larvae of I. ricinus, that are heterogeneously distributed, have a higher encounter rate with small mammals that are highly abundant and very actively foraging in the leaf litter (Mejlon 1997). Thus, small mammals, occurring in high densities and having relatively large larval burdens, represent the most important host group for feeding I. ricinus larvae. The nymphs, in turn, which have a more homogeneous distribution, have more comparable chances of encountering either a rodent or a bird. Therefore, the nymphs are almost evenly distributed on rodents and birds (Hofmeester et al. 2016). Ecology and prevention of Lyme borreliosis 49

51 Elena Claudia Coipan and Hein Sprong Figure 1. Prevalence of infection with the various Borrelia burgdorferi s.l. genospecies of questing Ixodes ricinus nymphs. All prevalences add up to 100%. Tick density Tick density is yet another factor that promotes the genetic diversity of the bacteria and their transmission efficiency. It is known that the larger the population, the higher the genetic diversity. This is especially true for genetic markers that are neutrally evolving, such as the 5S-23S rdna intergenic spacer (IGS). Preliminary data from a study on B. burgdorferi s.l. in 20 different locations in the Netherlands, suggests that the haplotype diversity of IGS in B. afzelii correlates with the density of ticks infected with this genospecies. Similarly, analysis of MLST data showed that there is a higher haplotype diversity within B. afzelii compared to B. garinii, while the genetic differentiation is, on the contrary higher in the latter. These results are consistent with a larger population size of B. afzelii, which in turn is consistent with a higher density of ticks infected with B. afzelii than of those infected with B. garinii. The higher genetic differentiation within B. garinii reflects, also, the lower contact rate of the ticks with the birds when compared with the mammals, allowing thus for evolution of distinct lineages of B. garinii (Coipan et al. 2016). Which are the implications for public health? All Borrelia genospecies are considered equally hazardous for humans. The study of pathogenicity of the various Borrelia genospecies and genotypes should allow for individual hazard assignment. The combination of hazard and exposure (prevalence in questing ticks) would then allow individual genospecies/genotypes risk assessment. Thus, both ecological and clinical studies are necessary to be able to address the public health issue that is nowadays collectively called Lyme borreliosis. Hazard/acarological risk Although infected larvae and adult ticks can cause LB as well, the infected nymphs are considered as constituting the main source of human infection with B. burgdorferi s.l., simply because their shear abundance. Therefore, the acarological risk of human infection with B. burgdorferi s.l. is defined as the density of infected questing nymphs (Dister et al. 1997, Glass et al. 1994, Glass et al. 1995, Kitron and Kazmierczak 1997, Nicholson and Mather 1996). 50 Ecology and prevention of Lyme borreliosis

52 4. Ecology of Borrelia burgdorferi sensu lato Numerous studies addressed the topic of hazard for B. burgdorferi s.l. infection and the way the vertebrate hosts composition influences this (Brisson et al. 2011, LoGiudice et al. 2003, Ruyts et al. 2016, Tälleklint and Jaenson 1996). One of the most prominent controversies on how the acarological risk varies according to the vertebrate community is that around the dilution effect theory. Its initiators, studying habitats in North America, found that increased biodiversity will lead to an increased abundance of unsuitable transmission vertebrates for B. burgdorferi s.s., with the ensuing dilution (reduction) of the spirochaetal infection in the questing ticks (LoGiudice et al. 2008, LoGiudice et al. 2003, Ostfeld and Keesing 2000). Conversely, other authors have suggested that, on the contrary, biodiversity will only amplify the risk, due to the abundance of hosts that will implicitly lead to an increased abundance of the ticks (Ogden and Tsao 2009, Randolph and Dobson 2012). One of the few vertebrate groups that have been identified as incompetent for B. burgdorferi s.l. amplification or transmission is that of artiodactyls (Jaenson and Tälleklint 1992, Matuschka et al. 1993). The introduction of more such animals in a habitat will result, therefore, in a reduction of B. burgdorferi s.l. infection in ticks. However, these animals feed a very large number of ticks, and especially adult ticks, which, in turn will result in a higher number of questing ticks. Thus, even if the prevalence of infection in ticks is decreased, the overall density of infected ticks might follow the opposite trend. From a meta-analysis study it resulted that the overall mean prevalence of B. burgdorferi s.l. in ticks in Europe is 13.7% (Rauter and Hartung 2005), with a lower average for nymphs (10.1%) comparing to adults (18.6%). In a recent study, on 22 different areas in the Netherlands, Coipan et al. (2013b) found an overall prevalence of 11.8%, but also found that in areas where tick densities were highest, the mean prevalence of Borrelia infection had lower values. The hypothesis of a constant prevalence over the range of questing ticks density was tested and the results indicated a slight negative correlation of the prevalence with the tick density. That implies that the density of ticks infected with B. burgdorferi s.l. decreases as the density of questing ticks increases. Plotting the density of infected questing ticks as an exponential function of the questing ticks densities, however, revealed that over the usual range of questing ticks densities the density of infected ticks is also increasing, and the downward trend might be observed only for questing ticks densities of over 200/100 m 2 (Coipan et al. 2013b). This observation is consistent with the finding made by Randolph that, in Europe the density of Borrelia infected ticks depends much more on the density of all ticks than on the infection prevalence, and that only in areas where the tick density is unusually high ( /100 m 2 ) is the infection prevalence consistently low (Randolph 2001). This hypothesis is also confirmed by a 10 years longitudinal study of density of ticks and their infection prevalence with tick-borne pathogens at Duin en Kruidberg (the Netherlands); there, the density of infected nymphs followed the same trend as the overall density of questing nymphs, while the prevalence of infection with B. burgdorferi s.l. remained constant. It is, thus, obvious that the density of questing nymphs is the main driver of the acarological risk of human exposure to B. burgdorferi s.l. What drives the variations in nymphal density might be mostly rodent abundance and climate and is surely an interesting topic of further research. Furthermore, each of the 20 genospecies of the group has its own vertebrate host spectrum. For example, B. afzelii is mainly transmitted by small mammals, while B. garinii is mainly transmitted by birds (Hanincova et al. 2003a, Hanincova et al. 2003b). Under these circumstances, a decrease of biodiversity of one vertebrate class on the expense of the increase of another would lead to the dilution of one B. burgdorferi s.l. genospecies but to the increase in prevalence of another. It is also generally accepted that with the increase in biodiversity there will also be an increase in the diversity of zoonotic agents (Guernier et al. 2004, Hechinger and Lafferty 2005). Surely, biodiversity might be affect de abundance of several of these pathogens, but the key question for Ecology and prevention of Lyme borreliosis 51

53 Elena Claudia Coipan and Hein Sprong public health therefore lies in the accumulation of all the hazards, weighed by their abundance and (potential) disease burden in humans. Differential pathogenicity The most frequently Borrelia genospecies retrieved from human cases of Lyme borreliosis are B. afzelii, B. garinii, B. burgdorferi s.s., and B. bavariensis (Stanek et al. 2012). The genetic differences between the genospecies seem to affect not only their enzootic associations but also the progress of human infection with Borrelia (Stanek et al. 2012). Mammalassociated Borrelia genospecies, such as B. afzelii, B. bavariensis, and B. spielmanii, are more often isolated from patients than bird-associated Borrelia genospecies (B. garinii and B. valaisiana) (Coipan et al. 2016). Also, it is known that B. afzelii is mostly associated with erythema migrans (EM) and acrodermatitis chronica atrophicans (ACA) (Coipan et al. 2016, Stanek et al. 2012) while B. garinii infections can lead to neurological symptoms the so-called neuroborreliosis (Figure 2). The public health implications of multiple strains and lineages within a genospecies of Borrelia have been investigated in several studies. From a public health perspective, it is important to be able to differentiate between the infectious and non-infectious Borrelia spirochaetes or between the invasive and non-invasive ones. Discriminating between these types could be useful for disease risk assessment and management. Research on B. burgdorferi s.s. in North America has shown that some major sequence types of the ospc and certain sequence types of 16S-23S rrna intergenic spacer are more frequently found in disseminated cases of LB (Dykhuizen et al. 2008, Strle et al. 2011, Wormser et al. 2008). Figure 2. Localisation of human clinical manifestations of Lyme borreliosis (NB = neuroborreliosis, EM = erythema migrans, ACA = acrodermatitis chronica atrophicans, LA = Lyme arthritis) and prevalence of various Borrelia burgdorferi s.l. genospecies in each manifestation. EM does not have a preferential localisation it occurs at the site of the tick bite. Prevalences within a manifestation add up to 100%. 52 Ecology and prevention of Lyme borreliosis

54 4. Ecology of Borrelia burgdorferi sensu lato More recently, Hanincova et al. (2013) have used MLST on eight housekeeping genes on the chromosome, which undergo slow evolution and show nearly neutral variation (Margos et al. 2008, Urwin and Maiden 2003), to investigate these associations. They have shown significant associations between clusters of sequence types (clonal complexes) of B. burgdorferi s.s. and localised or disseminated forms of LB. It seems, thus, that the genetic makeup of the pathogenic spirochaetes is determinant for the symptomatology they cause. In a study comprising European isolates of B. burgdorferi s.l. and tick lysates positive for B. burgdorferi s.l., Coipan et al. (2016) have shown that also within the European genospecies B. afzelii, B. bavariensis, and B. garinii, there are sequence types that are more often associated to human cases of Lyme borreliosis than expected based on their frequency in questing ticks. The two species that were significantly more frequent in human cases than in questing ticks were B. afzelii and B. bavariensis both mammal-associated Borrelia. B. lusitaniae and B. valaisiana were, as expected, negatively associated with LB. The association of B. afzelii with human cases could be due to their ability to cause a long lasting and more prominent EM, as it was shown in previous studies (Van Dam et al. 1993), being therefore, easier to detect. In the case of B. bavariensis, the strikingly low frequency in questing ticks and high frequency in LB patients could be explained by higher infectivity of these bacteria. Remarkably, despite its high incidence in ticks and EM, in terms of disease burden (as measured by disability-adjusted life year), B. afzelii is probably of least concern. Most of the EMs disappear after antibiotic treatment and the relatively rare late manifestations of infections with this bacterium pertain to skin alterations (acrodermatitis chronica atrophicans). On the other hand, the low incidence of infections with B. bavariensis and B. garinii lead more often to severe late clinical manifestations, such as neuroborreliosis, which in terms of disease burden, probability to develop long-term sequella and public health impact, is a (far) more severe disease than erythema migrans. Although, both B. garinii and B. burgdorferi s.s. comprised genotypes that were only isolated from LB patients, there was no significant association of these genospecies with the human cases. One possible explanation is the lower sample size available for these genospecies, comparing with B. afzelii; additional sampling of these genospecies might lead, in future studies, to clarification of the matter of differential infectivity of these spirochaetes. We hypothesise that the reason for which mammal-associated Borrelia are significantly more often retrieved from humans than bird-associated Borrelia is that humans are also mammals and the factors that trigger the specificity of Borrelia for small rodents (e.g. outer surface protein B, as suggested by Vollmer et al. 2013) could be the same ones that are responsible for facilitating the establishment of localised infection with these bacteria in humans. This would make the transmission of the bacteria more facile between vertebrates of the same class (i.e. mammals) than between vertebrates of different classes (i.e. birds and mammals). Previous studies have showed the propensity of some genotypes of B. afzelii and B. burgdorferi s.s. to cause LB (Hanincova et al. 2013, Jungnick et al. 2015). Recent studies indicate that at European scale the genetic diversity of Borrelia in humans is much higher than previously acknowledged, with 68 B. afzelii genotypes (Coipan et al. 2016). Furthermore, the ~450 bp fragment of IGS appears to be as good or an even better predictor for pathogenic Lyme spirochaetes as MLST. MLST is, in exchange, capable of identifying sequence types that were more invasive or persistent than others, being much more often found in late (acrodermatitis chronica atroficans) or disseminated (neuroborreliosis) forms of Lyme borreliosis than expected based on their frequency in EM. The finding that not all genospecies, clusters, or genotypes are equally likely to cause disease in humans Ecology and prevention of Lyme borreliosis 53

55 Elena Claudia Coipan and Hein Sprong suggests that the spirochaetes of B. burgdorferi s.l. have different infectivity properties, not only between but also within the genospecies, and this has direct implications on the epidemiology and risk assessment of human infections with these bacteria. While the genetic make-up of the Lyme borreliosis spirochaetes undoubtfully plays a role in the clinical manifestations observed in humans, it alone cannot fully explain the observed variation in prevalence and severity of the various clinical manifestations. The pathogenesis of chronic Lyme disease seems to be a combination of persistent infection and autoimmunity (Singh and Girschick 2004), as it was shown in the case of chronic joint inflammation (Steere et al. 2001) or Lyme carditis (Raveche et al. 2005). Early recognition/treatment of the disease can prevent irreversible damage done by the (immune reaction to the) infection. Furthermore, the genetic/immunological status of the infected person might be equally important (Bramwell et al. 2014, Schroder et al. 2005). The wide range in outcomes in untreated patients reflects most probably the interplay between spirochaetal virulence and host immune response. Public health relevance Few vertebrate hosts account for maintenance of most ticks and Borellia burgdorferi s.l. in enzootic cycles. The high prevalence of B. afzelii in questing nymphs is caused by the high proportion of larvae that feed on small rodents. There is host specificity of B. burgdorferi s.l. at genospecies level, but probably not at the intra-genospecies level. The various micro-organisms co-infecting questing ticks affect the host s immune response and could alter the course of the infection. The prevalence of B. burgdorferi s.l doesn t necessarily reflect the incidence of human Lyme borreliosis cases: exposure risk disease incidence. References Avise JC (2007) On evolution. Johns Hopkins University Press, Baltimore, MD, USA. Barbour AG (1988) Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. J Clin Microbiol 26: Barbour AG, Bunikis J, Fish D and Hanincová K (2015) Association between body size and reservoir competence of mammals bearing Borrelia burgdorferi at an endemic site in the northeastern United States. Parasites & Vectors 8: 1. Barthold SW (1999) Specificity of infection-induced immunity among Borrelia burgdorferi sensu lato species. Infect Immun 67: Belongia EA (2002) Epidemiology and impact of coinfections acquired from Ixodes ticks. Vector Borne Zoonotic Dis 2: Bramwell KK, Teuscher C and Weis JJ (2014) Forward genetic approaches for elucidation of novel regulators of Lyme arthritis sevserity. Front Cell Infect Microbiol 4: 76. Brisson D, Brinkley C, Humphrey PT, Kemps BD and Ostfeld RS (2011) It takes a community to raise the prevalence of a zoonotic pathogen. Interdiscip Perspect Infect Dis 2011: Ecology and prevention of Lyme borreliosis

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64 5. Rodents as hosts for Ixodes ricinus and Borrelia afzelii Gilian van Duijvendijk 1*, Gerrit Gort 2 and Willem Takken 1 1 Laboratory of Entomology, Wageningen University & Research, P.O. Box 16, 6700 AA, Wageningen, the Netherlands; 2 Mathematical and Statistical Methods, Wageningen University & Research, P.O. Box 16, 6700 AA, Wageningen, the Netherlands; gilian.vanduijvendijk@gmail.com Abstract Ixodes ricinus is the vector of the Lyme borreliosis-causing bacterium Borrelia afzelii in Europe. Larvae of I. ricinus mainly feed on rodents, which are reservoir hosts of B. afzelii. Rodent species vary in their suitability as hosts for ticks and B. afzelii. The effects of rodent species on tick burden, host infection rate and infectivity are reviewed and the effect of rodent species on tick physiology was studied in a field experiment. Wood mice (Apodemus sylvaticus) and bank voles (Myodes glareolus) were trapped in the field and body weight of engorged larvae and flat nymphs was measured. Body weight of engorged larvae and flat nymphs was higher for ticks that fed on wood mice compared to ticks that fed on bank voles. Overall, we conclude that wood mice are better hosts for ticks but that bank voles are better hosts for B. afzelii. The density of a rodent species, which is largely affected by food availability, has, however, the largest effect on determining the contribution of a rodent species to the density of infected nymphs and, therefore, Lyme borreliosis risk. Keywords: bank vole, Borrelia burgdorferi, host, Ixodes ricinus, rodent, tick physiology, wood mouse Introduction The sheep tick (Ixodes ricinus, Ixodida/Ixodidae) is the principal vector of the Lyme borreliosiscausing spirochaete Borrelia burgdorferi sensu lato (s.l.) (Spirochaetales/Spirochaetaceae) in Europe. B. burgdorferi s.l. is transmitted to humans through the bite of an infected tick, usually a nymph (Hofhuis et al. 2013). The density of infected ticks, therefore, positively affects Lyme borreliosis incidence (Diuk-Wasser et al. 2012, Stafford et al. 1998). The density of infected nymphs is calculated by multiplying the density of nymphs with the proportion of infected nymphs (hereafter referred to as the infection rate). So, when one of these parameters increases, the chance of getting bitten by an infected nymph increases. Rodents are commonly used as blood hosts by tick larvae and are the natural reservoir host for Borrelia afzelii, one of the most common B. burgdorferi s.l. genospecies in Europe (Hanincová et al. 2003a, Rauter and Hartung 2005). Rodent reservoir hosts, therefore, contribute strongly to both the density of nymphs and the infection rate of nymphs (Tälleklint and Jaenson 1994, 1995). Most rodents have zero to a few ticks and only a small proportion of the rodent populations feeds the majority of ticks (Perkins et al. 2003, Randolph et al. 1999). An individual rodent can feed over 300 ticks, depending on the geographical location, season and year. The role of rodents as hosts for ticks and B. afzelii and the differences between two common rodent species in the Netherlands are discussed in this chapter. Tick ecology Ixodes ricinus has four developmental stages: egg, larva, nymph and adult. Each of the three motile stages must take a blood meal to complete their development to the next stage. All tick stages climb up the vegetation and wait for a vertebrate host, a behaviour called questing. During questing, evaporative water loss forces questing ticks to return to the litter layer to restore their Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 63 DOI / _5, Wageningen Academic Publishers 2016

65 Gilian van Duijvendijk, Gerrit Gort and Willem Takken moisture content (Lees 1946, Perret et al. 2003). Questing duration and questing height are, therefore, influenced by the microclimate around the tick (Randolph and Storey 1999). Questing immature ticks (larvae and nymphs) are activated by CO 2 from the host and are attracted to host odour (Berret and Voordouw 2015, Van Duijvendijk et al. in press). After encountering a host, the tick stretches its forelegs, holds on to the passing host and searches for a suitable place on the host to bite and feed for about 3 to 7 days (depending on the stage). When the tick is fully engorged, it drops from the host and searches for a place in the moist litter layer to digest its blood meal and moult into the next stage. I. ricinus feeds on a wide variety of vertebrate hosts including: rodents, deer, birds and reptiles (Hofmeester et al. 2016, Keirans et al. 1996). The height at which the ticks quest affects the size of the encountered hosts and differs between the three tick stages. Larvae quest at heights of 0-10 cm, nymphs at cm and adult ticks mainly quest at cm above the ground (Mejlon and Jaenson 1997). As a result, questing larvae mainly encounter small mammals like rodents, which are important blood hosts for larvae (Matuschka et al. 1991). Rodent ecology In Europe, bank voles (Myodes glareolus, Rodentia/Cricetidae), wood mice (Apodemus sylvaticus, Rodentia/Muridae) and yellow-necked mice (Apodemus flavicollis, Rodentia/Muridae) (Figure 1) are the most common rodents in tick habitat and are, therefore, often used as blood hosts by larvae. These rodent species can occur together in the same region due to the different niches they occupy (Khanakah et al. 2006). In the Netherlands, the distribution of the yellow-necked mouse is limited, whereas wood mice and bank voles are widespread throughout the country. The abundance of these rodent species is regulated by different factors, like food availability, predation and vegetation cover (Zwolak et al. 2016) and, therefore, varies temporally and geographically. Food availability is one of the main environmental factors that has a large effect on rodent abundance (Bogdziewicz et al. 2016, Ostfeld et al. 2006). The rodents under study are herbivores feeding on the seeds of a variety of plant species. Large tree species, like oak and beech do not produce seeds every year, but produce their seeds synchronously depending on the weather conditions, called masting (Kelly and Sork 2002). The phenomenon causes fluctuations in rodent densities between years and geographic locations, depending on the presence of mast the species and climatic conditions (Ostfeld et al. 1996, Ostfeld and Keesing 2000). Because rodents are the most important hosts for tick larvae, masting increases the density of ticks (Jones et al. 1998). Figure 1. The three most common rodent species used by larval Ixodes ricinus. (A) Bank vole, (B) wood mouse and (C) yellow necked mouse (copyright: bank vole: Peter Trimming, wood mouse: Spencer Wright, yellow-necked mouse: James Lindsey, 64 Ecology and prevention of Lyme borreliosis

66 5. Rodents, Ixodes ricinus and Borrelia afzelii Rodents as hosts for Borrelia afzelii Lyme borreliosis is caused by spirochaetes from the B. burgdorferi s.l. complex. This complex consists of 19 genospecies, which can cause different clinical symptoms in humans (Nau et al. 2009). The different genospecies are all transmitted by I. ricinus, but their enzootic lifecycles depend on different natural reservoir hosts. In Europe, the most common genospecies is B. afzelii (Herrmann et al. 2013a, Rauter and Hartung 2005), which can cause skin manifestations (Stanek and Strle 2003, Strle and Stanek 2009). Borrelia afzelii circulates between ticks and rodents, whereas B. garinii circulates between ticks and birds (Hanincová et al. 2003a, 2003b, Humair et al. 1999, Kurtenbach et al. 1998, Kybicova et al. 2008). Rodents are born free of Lyme borreliosis spirochaetes (Mather et al. 1991) and can become infected with B. afzelii via the bite of an infected larva or nymph (Radolf et al. 2012, Van Duijvendijk et al. 2016), after which they will remain infected throughout their life (Gern et al. 1994). In contrast to Ixodes trianguliceps, adult I. ricinus ticks rarely feed on rodents and are, therefore, not considered to contribute to the lifecycle of B. afzelii. Therefore, B. afzelii cycles from infected questing larvae or nymphs, to rodents and from rodents to feeding larvae, which subsequently develop into infected questing nymphs (Figure 2). This cycle is influenced by different characteristics of the rodent host, tick vector and Borrelia pathogen (Van Duijvendijk et al. 2015). In general, bank voles are better hosts for B. afzelii than wood mice because they have a higher host infection rate (Gassner et al. 2013, Humair et al. 1999, Kybicova et al. 2008, Tälleklint and Jaenson 1994) and the probability that a feeding larvae acquires B. afzelii from a rodent (infectivity), is also higher for bank voles (Humair et al. 1999, Kurtenbach et al. 1995, Perez et al. 2012, Radzijevskaja et al. 2013). A higher B. afzelii spirochaete load in rodent tissue has a positive effect on its infectivity to feeding I. ricinus larvae (Raberg 2012), but the effect may be counteracted by the blood meal size of the tick. The contribution of a rodent to the density of infected nymphs is not only affected by their suitability as host for B. afzelii, but is also affected by their suitability as host for I. ricinus. The suitability of rodents as host for I. ricinus is discussed in the next paragraph. Host finding Effect of host characteristics, tick charasteristics and Borrelia infections Blood feeding and Borrelia transmission Tick development Host finding Blood feeding and Borrelia transmission Tick mortality Figure 2. Schematic overview of the development from an uninfected Ixodes ricinus larva to a nymph infected with Borrelia afzelii and the transmission process of B. afzelii between rodent and tick (Van Duijvendijk et al. 2015; Dotted lines indicate continuation of questing after a partial blood meal, dashed line indicates co-feeding transmission. Ecology and prevention of Lyme borreliosis 65

67 Gilian van Duijvendijk, Gerrit Gort and Willem Takken Rodents as blood hosts for Ixodes ricinus The contribution of rodents to the density of infected nymphs depends on their tick burden, their B. afzelii infection status, the probability that feeding larvae will acquire spirochaetes, the probability that infected engorged larvae moult into infected nymphs and the longevity of the infected nymphs. When rodent density is increased, for example in the year after a mast year (Clotfelter et al. 2007), the tick larvae that quest for a host have a higher chance of encountering a rodent. As a result, nymphal density is expected to increase the following year (Ostfeld et al. 2006). The average number of ticks feeding per rodent declines with high rodent densities (Brunner and Ostfeld 2008, Schmidt et al. 1999), but the total number of feeding larvae per unit area was still higher due to the high rodent density (Rosa et al. 2007), explaining the positive effect of rodent density the density of nymphs the following year. Rodent species differ in their suitability as hosts for ticks. Several studies have analysed the effects of rodent species on tick burden. In general, mice have a greater larval tick burden than bank voles (Boyard et al. 2008, Gassner et al. 2013, Gray et al. 1999, Humair et al. 1993, Kiffner et al. 2011, Kurtenbach et al. 1995, Nilsson and Lundqvist 1978, Tälleklint and Jaenson 1997), which is expected to be the result of an acquired immunity of bank voles to feeding ticks (Dizij and Kurtenbach 1995). It is difficult to determine the contribution of rodents to feeding immature ticks compared to other vertebrate hosts. Doing so requires knowledge about the abundance and mean tick burden for all of the vertebrate host species in the community. Tick burden data was estimated for a vertebrate community in Europe (Hofmeester et al. 2016, Tälleklint and Jaenson 1997) and the USA (Brisson et al. 2008, Keesing et al. 2009, LoGiudice et al. 2003). One innovative (but difficult) approach is to use host blood meal analysis to estimate the contribution of the different vertebrate hosts to feeding immature ticks (Morán-Cadenas et al. 2007, Pichon et al. 2003, 2005, Scott et al. 2012). In Switzerland, host blood meal analysis showed that rodents feed about 20% of the larval ticks (Morán-Cadenas et al. 2007). The higher tick burden on mice compared to bank voles can be the result of either a preference of the tick for one host species over another, or phenotypic differences (e.g. microhabitat preferences, grooming behaviour, immunological differences) between rodent species. Not much is known about the host preference and selection of I. ricinus (McCoy et al. 2013), whereas host preference under laboratory conditions was shown for I. scapularis and I. pacificus (Shaw et al. 2003, Slowik and Lane 2009). Wood mice and bank voles use different ecological niches. Van Duijvendijk et al. (in press) showed that a B. afzelii infection in rodents can also affect tick burden; it changed the odour of the rodents and made them more attractive to ticks. In addition, these authors also found that B. afzelii infection affects tick physiology; infected nymphs were heavier than uninfected nymphs. The effect of rodent species on the physiology of feeding I. ricinus larvae is largely unknown and was determined in an experiment described below. Experiment: effect of rodent species on tick physiology To determine the effect of a rodent species on the physiology of I. ricinus, we trapped rodents with their naturally attached ticks in their natural environment. Rodents were trapped in a forest near Wageningen, the Netherlands, using 144 live traps baited with grain, carrots and hay. Traps were set in the afternoon and inspected the following morning from May to November at threeweek intervals (10 trapping occasions). Trapped female rodents were released at the trapping site. Trapped male bank voles and male wood mice were taken to the laboratory and housed individually in cages over pans with water. The attached larvae were allowed to complete their 66 Ecology and prevention of Lyme borreliosis

68 5. Rodents, Ixodes ricinus and Borrelia afzelii blood meal and the engorged larvae were collected after they dropped off their host and into the water. Engorged larvae were dried for 2 h on filter paper, weighed to the nearest microgram and housed individually at 20 C and 90% RH to digest their blood meal and moult into flat nymphs. The engorged larvae were checked weekly, flat nymphs were reweighed and stored at -20 C. The effect of rodent species on the body weight of engorged larvae and flat nymphs was analysed using mixed linear models with random effects for individual mice. Larval tick burden and rodent body weight had no effect on the body weight of engorged larvae and flat nymphs and were excluded from the models. The log of the body weight of engorged larvae was related to the log of the body weight of emerged nymphs, testing for differences between rodent species, using a mixed linear model with engorged larval weight as covariate, rodent species as factor and their interaction and random effects for mouse. All analyses were performed with SAS statistical software (SAS Institute Inc., Cary, NC, USA), version 9.3. Forty-three male bank voles and 28 male wood mice were trapped and yielded 509 and 651 engorged larvae, respectively. The mean tick burden of the wood mice (23.3±4.7) was two-fold higher than that of the bank voles (11.8±1.9). The engorged larvae that had fed on wood mice had a higher body weight (0.541±0.009 mg) than the larvae that had fed on bank voles (0.447±0.008 mg, P<0.0001, Figure 3A). The nymphs that emerged from the engorged larvae that had fed on wood mice had a higher body weight (0.247±0.005 mg) compared to the nymphs emerged from the larvae that had fed on bank voles (0.181±0.004 mg, P<0.0001, Figure 3B). In addition, the log body weight of the emerged nymphs was positively related to the log body weight of the engorged larvae for ticks that fed on wood mice (P<0.0001) and on bank voles (P<0.0001, Figure 4). Interestingly, the regression coefficient was higher (P<0.001) for ticks that fed on wood mice (log body weight emerged nymphs = log body weight of engorged larvae) than for ticks that fed on bank voles (Log body weight emerged nymphs = log body weight of engorged larvae). This observation may suggest that the ability of the larval tick A 0.6 *** B 0.6 *** Body weight engorged larvae (mg) Bank vole Wood mouse Body weight emerged nymph (mg) Bank vole Wood mouse Figure 3. (A) Body weight of engorged larvae and (B) body weight of emerged nymphs of Ixodes ricinus that had naturally attached to bank voles or wood mice. Top numbers inside bars represent number of ticks, bottom numbers represent number of rodents, error bars represent standard errors. Significance is illustrated as ***P< Ecology and prevention of Lyme borreliosis 67

69 Gilian van Duijvendijk, Gerrit Gort and Willem Takken 0.5 Body weight emerged nymph (mg) Body weight engorged larva (mg) Figure 4. Relationship between body weight of engorged larvae and body weight of emerged nymphs for ticks that fed on field-collected bank voles (black circles) or wood mice (grey circles) collected from May 2013 till November Each dot represents one tick. Lines represent back transformed linear regression lines for bank voles (black) and wood mice (gray). Significance is illustrated as ***P< to convert rodent blood into nympyhal weight was more efficient for wood mice than bank voles. Because total body weight of the flat nymphs was measured, it is, however, unknown whether the steeper slope for wood mouse blood was caused by: (1) a higher tissue weight; (2) fat storage; or (3) undigested blood in the flat nymphs. Previous work has shown that the body weight of nymphs was positively related to fat reserves and nymphal survival (Crooks and Randolph 2006, Herrmann and Gern 2010, Herrmann et al. 2013b). Further experiments are needed to clarify the relationship between nymphal body weight and nymphal survival under natural conditions and, therefore, the contribution of each rodent species to the density of infected nymphs. Discussion We have shown that rodents are important in maintaining the lifecycles of I. ricinus and B. afzelii. They are important hosts for larvae and a high density of rodents, therefore, increases the probability that questing larvae find a host, resulting in an increase in the density of nymphs in the following year. The most common rodent species in the Netherlands differ in their suitability as hosts for I. ricinus. Bank voles, but not mice, have an innate resistance against feeding ticks and can develop acquired resistance against ticks over successive infestations, resulting in a lower tick attachment success, lower tick feeding success and lower larva-to-nymph moulting success (Dizij and Kurtenbach 1995, Hughes and Randolph 2001). This would increase the relative contribution of wood mice to the density of nymphs compared to the contribution of bank voles. We found that this resistance also affects tick physiology under field conditions. Larvae were able obtain a larger blood meal from wood mice, compared to bank voles, resulting in heavier nymphs. The 68 Ecology and prevention of Lyme borreliosis

70 5. Rodents, Ixodes ricinus and Borrelia afzelii proteins in the larger blood meal during the larval stage are stored as fat in the nymphal body, which is expected to enhance nymph survival (Herrmann and Gern 2015, Randolph and Storey 1999), increasing the density of nymphs. In addition, we also found that the ability of larval ticks to convert rodent blood to nymphal body mass was more efficient for wood mice than bank voles. An effect of host species on tick physiology was also found for I. scapularis (Brunner et al. 2011, Jones et al. 2015). The authors, are, however, unaware of studies that show the effect host species on blood conversion. This topic requires further research to determine what caused this effect. Considering Lyme borreliosis risk, infection prevalence in ticks is an important factor affecting the density of infected nymphs. Rodent infection rate and rodent infectivity also differ between the most common rodent species in the Netherlands. These two factors affect nymphal infection prevalence and are lower in wood mice compared to bank voles (Gassner et al. 2013, Humair et al. 1999, Kurtenbach et al. 1995, Kybicova et al. 2008, Perez et al. 2012, Radzijevskaja et al. 2013, Tälleklint and Jaenson 1994). Infectivity may, however, not always differ between mice and voles (Raberg 2012). At equal rodent densities, wood mice appear to contribute more to the density of nymphs than bank voles due to a higher larval tick burden on wood mice (Humair et al. 1993, Kurtenbach et al. 1995) and survival of the emerged nymphs from wood mice. This is in line with the findings of Perez et al. (2016), who concluded that wood mouse density in spring positively affected the density of nymphs one year later, whereas this was not found for bank vole density. These authors contributed the higher contribution of wood mice to the higher larval tick burdens on wood mice compared to bank voles. The densities of wood mice and bank voles depend on different ecological factors, and the (relative) density of a given rodent species obviously determines the contribution of that rodent species to the total number of feeding larvae per unit area, and hence, on the density of nymphs the following year (Brunner and Ostfeld 2008, Perez et al. 2016). The contribution of these rodent species to the density of infected nymphs, may also interact with each other. When the density of wood mice and bank voles are not equal, the effects may change. A relatively low density of wood mice may increase tick burden, and therefore, acquired resistance and infection rate in bank voles. A low density of bank voles, may, however result in a lower nymphal infection prevalence and, consequently infection rate in wood mice one year later. Rodents are important for I. ricinus larvae and B. afzelii (Hanincová et al. 2003a, Hofmeester et al. 2016, Kurtenbach et al. 2002) and rodent density does, therefore affect the density of infected nymphs one year later (Ostfeld et al. 2001, 2006). Rodents are a challenging target for ecological interventions that aim to reduce Lyme borreliosis incidence. Rodent density varies geographically and annually and due to the high numbers of rodents, intervening in their population size, without affecting overall biodiversity is difficult. Rodent density may be used as a predictor for the density of infected nymphs in the next year. Ecology and prevention of Lyme borreliosis 69

71 Gilian van Duijvendijk, Gerrit Gort and Willem Takken Public health relevance Rodents are important blood hosts for Ixodes ricinus larvae. Rodents are the reservoir host for Borellia afzelii and can transmit the spirochaetes to the larvae that feed on them. Infected larvae moult into infected nymphs, which are responsible for transmitting the spirochaetes to humans. There are large differences between the contributions of the most common rodent species to Lyme borreliosis risk in terms of tick burden, rodent infection rate, infectivity and tick physiology. Rodents may be a good predictor for the density of nymphs, but their use in intervention studies may be challenging due to high spatiotemporal variation in their density. Acknowledgements We thank Marloes van Schaijk for her help with the experiment and Hein Sprong, Sip van Wieren and a referee for their comments on an earlier version of this chapter. References Berret J and Voordouw MJ (2015). Lyme disease bacterium does not affect attraction to rodent odour in the tick vector. Parasit Vectors 8: 249. Bogdziewicz M, Zwolak R and Crone EE (2016) How do vertebrates respond to mast seeding? Oikos 125: Boyard C, Vourc h G and Barnouin J (2008) The relationships between Ixodes ricinus and small mammal species at the woodland-pasture interface. Exp Appl Acarol 44: Brisson D, Dykhuizen DE and Ostfeld RS (2008) Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic. Proc R Soc Biol Sci Ser B 275: Brunner JL, Cheney L, Keesing F, Killilea M, Logiudice K, Previtali A and Ostfeld RS (2011) Molting success of Ixodes scapularis varies among individual blood meal hosts and species. J Med Entomol 48: Brunner JL and Ostfeld RS (2008) Multiple causes of variable tick burdens on small-mammal hosts. Ecology 89: Clotfelter ED, Pedersen AB, Cranford JA, Ram N, Snajdr EA, Nolan Jr V and Ketterson ED (2007) Acorn mast drives longterm dynamics of rodent and songbird populations. Oecologia 154: Crooks E and Randolph SE (2006) Walking by Ixodes ricinus ticks: intrinsic and extrinsic factors determine the attraction of moisture or host odour. J Exp Biol 209: Diuk-Wasser MA, Hoen AG, Cislo P, Brinkerhoff R, Hamer SA, Rowland M, Cortinas R, Vourc h G, Melton F, Hickling GJ, Tsao JI, Bunikis J, Barbour AG, Kitron U, Piesman J and Fish D (2012) Human risk of infection with Borrelia burgdorferi, the lyme disease agent, in Eastern United States. Am J Trop Med Hyg 86: Dizij A and Kurtenbach K (1995) Clethrionomys glareolus, but not Apodemus flavicollis acquires resistance to Ixodes ricinus L., the main European vector of Borrelia burgdorferi. Parasite Immunol 17: Gassner F, Takken W, Plas CL, Kastelein P, Hoetmer AJ, Holdinga M and Van Overbeek LS (2013) Rodent species as natural reservoirs of Borrelia burgdorferi sensu lato in different habitats of Ixodes ricinus in the Netherlands. Ticks Tick Borne Dis 4: Ecology and prevention of Lyme borreliosis

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74 5. Rodents, Ixodes ricinus and Borrelia afzelii Randolph SE, Miklisová D, Lysy J, Rogers DJ and Labuda M (1999) Incidence from coincidence: patterns of tick infestations on rodents facilitate transmission of tick-borne encephalitis virus. Parasitology 118: Randolph SE and Storey K (1999) Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission. J Med Entomol 36: Rauter C and Hartung T (2005) Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis. Appl Environ Microbiol 71: Rosa R, Pugliese A, Ghosh M, Perkins SE and Rizzoli A (2007) Temporal variation of Ixodes ricinus intensity on the rodent host Apodemus flavicollis in relation to local climate and host dynamics. Vector Borne Zoonotic Dis 7: Schmidt KA, Ostfeld RS and Schauber EM (1999) Infestation of Peromyscus leucopus and Tamias striatus by Ixodes scapularis (Acari: Ixodidae) in relation to the abundance of hosts and parasites. J Med Entomol 36: Scott MC, Harmon JR, Tsao JI, Jones CJ and Hickling GJ (2012) Reverse line blot probe design and polymerase chain reaction optimization for bloodmeal analysis of ticks from the eastern United States. J Med Entomol 49: Shaw MT, Keesing F, McGrail R and Ostfeld RS (2003) Factors influencing the distribution of larval blacklegged ticks on rodent hosts. Am J Trop Med Hyg 68: Slowik TJ and Lane RS (2009) Feeding preferences of the immature stages of three western North American ixodid ticks (Acari) for avian, reptilian, or rodent hosts. J Med Entomol 46: Stafford III KC, Cartter ML, Magnarelli LA, Ertel SH and Mshar PA (1998) Temporal correlations between tick abundance and prevalence of ticks infected with Borrelia burgdorferi and increasing incidence of Lyme disease. J Clin Microbiol 36: Stanek G and Strle F (2003) Lyme borreliosis. Lancet 362: Strle F and Stanek G (2009) Clinical manifestations and diagnosis of Lyme borreliosis. Curr Probl Dermatol 37: Tälleklint L and Jaenson TG (1994) Transmission of Borrelia burgdorferi s.l. from mammal reservoirs to the primary vector of lyme borreliosis, Ixodes ricinus (Acari: Ixodidae), in Sweden. J Med Entomol 31: Tälleklint L and Jaenson TG (1995) Is the small mammal (Clethrionomys glareolus) or the tick vector (Ixodes ricinus) the primary overwintering reservoir for the Lyme borreliosis spirochete in Sweden? J Wildl Dis 31: Tälleklint L and Jaenson TG (1997) Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in South-Central Sweden. Exp Appl Acarol 21: Van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersoz J, Oei A, Foldvari G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: 97. Van Duijvendijk G, Sprong H and Takken W (2015) Multi-trophic interactions driving the transmission cycle of Borrelia afzelii between Ixodes ricinus and rodents: a review. Parasit Vectors 8: 643. Van Duijvendijk G, Van Andel W, Fonville M, Gort G, Hovius JW, Sprong H and Takken W (in press). A Borrelia afzelii infection increases larval tick burden on Myodes glareolus (Rodentia: Cricetidae) and nymphal body weight of Ixodes ricinus (Acari: Ixodidae). J Med Entomol DOI: Zwolak R, Bogdziewicz M and Rychlik L (2016) Beech masting modifies the response of rodents to forest management. For Ecol Manage 359: Ecology and prevention of Lyme borreliosis 73

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76 6. The role of large herbivores in Ixodes ricinus and Borrelia burgdorferi s.l. dynamics Sipke E. van Wieren * and Tim R. Hofmeester Resource Ecology Group, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; sip.vanwieren@wur.nl Abstract Large herbivores are the most important reproduction hosts for Ixodes ricinus, and, as such, play a major role in maintaining tick populations. As one individual deer can already feed many females during the tick season, we propose that the relationship between deer density and tick density can best be described by a step function rather than a linear function. At high densities, herbivores may negatively affect tick numbers through their effects on vegetation structure and composition by creating and maintaining a short and open herb layer, reducing the shrub layer and decreasing the thickness of the litter layer. These effects may also have a negative effect on rodent densities. Domestic herbivores as added grazers will likely not have a major added effect on tick numbers but at high density they may have, both through their effects on the vegetation and because they may negatively affect the habitat use of the wild ungulates through competitive interactions. Large herbivores are mainly incompetent, in the sense of not-transmitting the parasite Borrelia burgdorferi s.l. to ticks, but to what extent this will affect the density of infected nymphs in a system is dependent of the host community as a whole and cannot be predicted from the density of the large herbivores alone. Keywords: Borrelia burgdorferi s.l., competence, deer, density, domestic herbivores, grazing effects, Ixodes ricinus, prevalence Introduction Ixodes ricinus in Europe is the main vector for the bacterium Borrelia burgdoferi s.l. which can cause Lyme borreliosis in humans. The number of ticks present or active at any time, and the infestation with parasites, is influenced by many factors. As ticks spend more than 90% of their time in the top soil and in the lower parts of the vegetation, environmental and habitat characteristics like temperature, humidity and presence and thickness of the litter layer are key determinants of survival rate and activity levels. The presence of suitable hosts for the different tick stages is equally important. In this chapter we will elucidate the role of large herbivores in the I. ricinus tick cycle and, to a lesser extent, we will pay attention to the role of large herbivores in the B. burgdorferi s.l. life cycle. The large herbivores we will discuss are mainly the more common and abundant deer species roe deer Capreolus capreolus and red deer Cervus elaphus because they are thought to be the most important hosts for the adult tick stage in western Europe (Hofmeester et al. 2016). Apart from these wild ungulates, we will also discuss the role of some species of domestic herbivores, mainly sheep, as many areas are being grazed by them, either for conservation purposes or in a rangeland setting (Van Wieren and Bakker 2008). In many cases domestic stock is present sympatrically with wild ungulates but in some cases they are the only large herbivores present and as such are important for maintaining tick populations. Insight in the role of large herbivores can also be used to try and find suitable and feasible intervention options to reduce tick abundance (see also chapter sheep mopping). Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 75 DOI / _6, Wageningen Academic Publishers 2016

77 Sipke E. van Wieren and Tim R. Hofmeester Ticks on large herbivores Counting ticks on large furry animals is a tedious job but nevertheless Hofmeester et al. (2016) were able to compile a large database with tick counts from various study areas throughout Europe. In Table 1 an overview is given from the counts on various large herbivores. In most cases prevalence of tick infestation was high and most animals carried ticks. The distribution of the various tick stages on the animals is in general different from the distribution in the habitat. Nymphs and adults prevail and in quite a number of cases more adults have been counted than nymphs. Together with the finding that small and medium sized hosts generally only feed juvenile ticks (Hofmeester et al. 2016), it stresses the fact that large herbivores are the main hosts for adult ticks and, as such, are a key element in sustaining tick populations. They are reproduction hosts. Nevertheless, larvae also feed on large herbivores, sometimes in large numbers, while it is likely that despite intensive searching, larvae are easily overlooked. Not many counts have been done on domestic animals but they carry ticks as well. The results of some studies suggest that sheep and cattle might carry fewer ticks than wild species like deer. In sympatric living wild and domestic species, wild animals were found to be more infested (and with a wider variety), than domestic animals (Domínguez-Peñafiel et al. 2011). But also high densities have been reported. Milne (1943) counted female ticks on 10 groups of hill sheep in England. Prevalence varied from , the mean number of females varied from and some sheep were infested by more than 150 females. In a study of L Hostis et al. (1994), the number of females per cow ranged from The low numbers in some grazer populations can probably partly be explained by the fact that much grazing takes place in more open landscapes, which are generally less tick-infested than woodland habitat (Boyard et al. 2008). In Table 1 mean numbers are given but it is well known that tick numbers on animals vary widely and that the distribution is heavily skewed. Milne (1943) was the first, together with RA Fisher, to discover that, although at lower tick densities the data could well be described by a Poisson distribution, at higher densities a negative binomial distribution was more appropriate. Tälleklint and Jaenson (1997) found heavily skewed distributions of larvae in roe deer and hares. Although we might expect somewhat less skewed distributions of nymphs and females, as larvae have a much more clumped distribution than nymphs or females, very skewed distributions have also been reported for females (L Hostis et al. 1994, Milne 1943). Variation in infestation levels can be thought of to arise from a variety of sources: The (patchy) distribution of ticks in the habitat. The distance travelled by the animal/area covered. The physical and physiological state of the animal. We do not know of any study where all these factors have been taken into account to explain infestation rates. Yet some differences between different classes of animals have been found. Milne (1947) counted more ticks on ewes lower in body condition. He also found a clear effect of body size. The larger ewes had more ticks than rams, who had more ticks than lambs. In one of our studies ticks were counted on 17 roe deer (9 does, 8 bucks), randomly chosen from five different areas. Boxplots of the tick counts are given in Figure Ecology and prevention of Lyme borreliosis

78 6. The role of large herbivores Table 1. Tick, Ixodes ricinus, counts on large herbivores from different areas in Europe (ND = not determined). Area Host species n Prevalence Total n ticks Mean number Reference All stages Larvae Nymphs Adults Norway roe deer ND ND ND ND ND Handeland et al. (2013) Thuringia, Germany roe deer , ND ND ND Heyl and de Mendonca (2011) Southern Hungary roe deer 35 ND Hornok et al. (2012) Gottingen, Germany roe deer , Kiffner et al. (2010) Brandenburg, Germany roe deer 67 ND ND Matuschka et al. (1993) North-Western Spain roe deer , Pato et al. (2013) Sweden roe deer 37 ND 14, Tälleklint and Jaenson (1997) Galicia, Spain roe deer , Vazquez et al. (2011) Germany roe deer , Vor et al. (2010) Byalistok province, Poland roe deer ND 2 ND ND ND Wegner et al. (1997) Norway red deer ND ND ND ND ND Handeland et al. (2013) Southern Hungary red deer 32 ND Hornok et al. (2012) Brandenburg, Germany red deer 147 ND 5, ND Matuschka et al. (1993) the Netherlands red deer 38 ND 2, Pacilly et al. (2014) Byalistok province, Poland red deer ND ND 16 Wegner et al. (1997) Brandenburg, Germany fallow deer 115 ND 7, ND Matuschka et al. (1993) the Netherlands mouflon 22 ND Pacilly et al. (2014) the Netherlands wild boar 9 ND Pacilly et al. (2014) Byalistok province, Poland wild boar ND ND 1 Wegner et al. (1997) Norway moose ND ND ND ND ND Handeland et al. (2013) Sweden moose 7 ND 4, Tälleklint and Jaenson (1994) Southern Hungary goat 110 ND Hornok et al. (2012) Southern Hungary sheep 375 ND Hornok et al. (2012) England sheep Ogden et al. (1997) France cattle , ND ND 11 L Hostis et al. (1994) Ecology and prevention of Lyme borreliosis 77

79 Sipke E. van Wieren and Tim R. Hofmeester Number of females Total number of ticks Number of larvae Number of nymphs Bucks Does Bucks Does Figure 1. Tick counts on roe deer bucks (8) and does (8) from 5 different areas in the Netherlands (2012). Bucks carried significantly more ticks of all stages than does. Male and female ticks were found attached to each other 163 times, 63% of the males were attached to a female and 25% of the females to a male. The sex difference cannot easily be explained. Roe deer bucks and does do not differ much in size. Perhaps the most likely explanation is that males tend to move much more than females and so have more chance of encountering a tick. The effect of herbivore density If large herbivores are so important as reproduction hosts, we can expect tick densities to be very much reduced when large herbivores are absent. We compared tick densities of areas with and without large herbivores (Figure 2). Tick densities in the areas without large herbivores are very low indeed (T.R. Hofmeester unpublished data). Ticks are not completely absent as there will always be some medium-sized host species (e.g. birds, hares, martens) that feed a number of adults and so maintain a tick population at low density. We can also expect a positive relationship between herbivore density and tick density. A number of studies suggest a linear relationship between deer density and tick density (Jensen and Jespersen 2005, Rand et al. 2003, Sprong et al. 2012). The problem with most of these studies is that crude estimates of deer densities were used and that the analyses were covering very large areas (countries). Especially what happens at low deer densities is not clear. We studied the relationship between I. ricinus densities as measured by blanket dragging, and the number of deer active as measured by camera trapping, in twenty forest plots of 1 hectare. At this smaller scale, it is clear that this relationship is non-linear (Figure 2). 78 Ecology and prevention of Lyme borreliosis

80 6. The role of large herbivores A 600 D O Larvae/100 m O O O O O O O O O O O O O O B 200 E O Nymphs/100 m O O O O O O O OO OO O O O C 15 F O Adults/100 m O O OO O O O O O OO O O O Absent Present Deer Deer passage rate (m -1 d -1 ) Figure 2. The density of the sheep tick Ixodes ricinus differed significantly (P<0.01) between 17 forest plots with deer and 3 plots without (A-C), but did not significantly increase with deer activity (D-F), for larvae (A, D), nymphs (B, E) and adults (C, F). Dotted lines represent generalised linear mixed model fit for non-significant models. Modified after T.R. Hofmeester (unpublished data). Van Buskirk and Ostfeld (1995) modelled the relationship between host and tick density. They concluded that low densities of hosts for either adults or juveniles were insufficient to maintain populations of Ixodes. They also found that a change in the density of hosts for adults was accompanied by a change in tick density only at relatively low host densities. Once host densities Ecology and prevention of Lyme borreliosis 79

81 Sipke E. van Wieren and Tim R. Hofmeester became high enough for all adult ticks to find a host, further increase in host density had no effect. There thus seems to be a threshold relationship rather than a linear relationship between Ixodes tick density and deer density, which is supported by our own data (Figure 2; T.R. Hofmeester unpublished data). It seems likely that moderate tick densities can be sustained by already very low deer densities. A simple calculation may elucidate this idea. Assume the presence of 1 deer/km 2. One deer may feed up to 500 adult females at a time (Milne 1943, Piesman et al. 1979, Wilson et al. 1990) so it will certainly do so in a season. One female produces 1,500 larvae (Hoch et al. 2010). If 15% of the larvae feed on a host then the next year the density of nymphs will be 11/100 m 2, a moderately large tick population. We therefore agree with Van Buskirk and Ostfeld (1995) and propose the deer-tick relationship to be more of a threshold relationship than a linear one. Large herbivores and (effects on) habitat Because ticks spend most of their lifetime in the vegetation rather than on a host, habitat requirements also play a key role in determining the number of ticks in an area. The persistence of ticks foremost depends on microclimate with a minimum humidity level as most important lifeline for Ixodes ricinus (Lees and Milne 1951). As ticks do not like to be in direct contact with water they do not occur in very wet environments like marshes but when conditions become too dry, as in many homogeneous coniferous forests or dry heathlands, conditions for tick survival decrease and tick densities become very low (Estrada-Peña 2001, Mulder 2014). Between these extremes there is a large middle ground where ticks can occur. The humidity level is positively affected by vegetation parameters that provide shade like trees, shrubs, a tall dense herb layer and a well-developed litter layer (Dobson et al. 2011, Gray et al. 1998, Sonenshine 1991). The forest litter layer forms a refuge for ticks during unfavourable climatic conditions (Randolph and Storey 1999).The thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities (Gassner et al. 2011). Tick density increased with shrub cover (Steigedal et al. 2013, Tack et al. 2012a, 2012b). Together with temperature, the mentioned habitat characteristics and the composition of the host community determine the long term average tick abundance. Figure 3 gives an impression of tick densities in different habitat types, with and without large herbivores. In all open areas, tick densities were very low, with or without large herbivores. In our case the open areas were short grasslands or dry Calluna heathland with unfavourable conditions for ticks. If conditions are more favourable, when a tall or dense herb layer or shrubs are present, tick densities in open areas can be quite high, even comparable with many a woodland (James et al. 2013). Boyard et al. (2009) found an average nymph density of 27/100 m 2 in rough grasslands with a maximum of 388/100 m 2 ). Without large herbivores, ticks were present in forest habitat and in the ecotone (between the forest and the open areas) but densities were generally very low (Figure 3). The exception was an oak forest with a well-developed understory. 80 Ecology and prevention of Lyme borreliosis

82 6. The role of large herbivores 200 Habitat Ecotone Forest Open Total ticks/100 m None Present Heavily grazed Large herbivores Figure 3. Boxplots of estimated tick densities (nymphs plus adults) from a number of selected habitats in the Netherlands, with and without large herbivores (45 and 31 sites, respectively), and from nine areas that were intensively grazed by sheep (2 sheep/ha). The highest mean tick densities were found in forest habitat with large herbivores (roe deer always present, in some cases other deer species, and in a number of cases additional livestock was present at low densities) but variation was also high. In most cases where comparisons have been made, higher tick densities have been reported in oak forests or mixed/deciduous forests compared to coniferous forests (Estrada-Peña 2001, Gray et al. 1998, James et al. 2013, Steigedal et al. 2013, Tack et al. 2012a, 2012b). Deciduous forests with a well-developed understorey most likely harbour more suitable hosts because they provide food and shelter for animals like rodents and deer (Gassner et al. 2008). Nevertheless coniferous forest with high rainfall and with a thick litter layer may support high tick densities too (Estrada-Peña 2001, Gray et al. 1998). In general, forested habitats with a well-developed understory and a well-developed litter layer have high tick abundances because these habitats provide a suitable microclimate for tick survival and they are most likely to harbour a range of suitable hosts for ticks of all stages. Substantial tick numbers were found in the ecotone habitats, with large herbivores, bordering the studied forests (Figure 3). From a tick-human perspective, ecotones are relevant because they are much used for recreational purposes. People use them as picnic sites, as playing ground, to put up their tent, or to walk the dog, and, as such, the ecotone is a risk habitat. High tick numbers in ecotones can, in part, be explained by ecotones still being reasonable good habitats for ticks because of the shading effects of the nearby forest. But ecotones are also preferred, or at least frequently visited, by host species. Many host species prefer forest as foraging habitat (deer) and/ or as area for cover (e.g. hares, badgers, wood mice). From the forest they may go out into open areas to forage during periods of activity and spend time in the ecotone. Wood mice were more abundant in the forest-pasture ecotone than inside the forest or pastures and were thought to be Ecology and prevention of Lyme borreliosis 81

83 Sipke E. van Wieren and Tim R. Hofmeester the major means of transport of Ixodes larvae from woodland to pasture (Boyard et al. 2008). Hosts may also move ticks from the woodland to adjacent open areas and so maintain tick populations there (Hoch et al. 2010, L Hostis et al. 1995), a typical source-sink situation. Tick densities were low in woodlands heavily grazed by, predominantly, sheep (Figure 3). All these areas were mainly heathland habitat with only small patches of woodland. The low tick numbers were obviously not because of the low density of herbivores because sheep density in these semi-natural reserves was about 2 sheep/ha. The low densities can better be explained by the alteration of the woodland through the effects of sheep grazing leaving an open forest with very little understory and a short grass layer with a thin litter layer on dry sandy soil. It is well known that large grazers can modify vegetation structure by affecting the shrub layer, preventing the development of a tree layer, and by creating a short vegetation of grasses and forbs (Van Wieren and Bakker 2008). These effects are generally observed under semi-natural conditions where domestic herbivores are held in a rangeland setting or applied for conservation purposes to mainly maintain open landscapes. In these situations, densities are higher (sometimes much higher) than the densities of wild herbivores under more natural conditions (Van Wieren and Bakker 2008). The grazing effects (under high grazing intensity) can be expected to generally affect tick populations in a negative way given the habitat requirements of ticks. Steigedal et al. (2013) also found lower tick densities under sheep grazing in Norway and could attribute this partly to a decrease in the shrub layer as the result of sheep grazing. In the Netherlands large grazers like cattle and horses are sometimes applied as complement herbivores to the wild herbivores roe deer, red deer and wild boar in forest-heathland systems. Although the densities here are not as high as in the more semi-natural open landscapes, there still could be effects on tick densities through modifications of the habitat. We compared 20, fenced, forest sites that were complementary (next to deer) grazed by cattle with 20 control sites outside the fence (Moonen 2015). The wild herbivores were also present outside the fence. We found no effects of grazing on I. ricinus nymphal density, herb density or saturation deficit (saturation deficit is a measure of the drying power of the atmosphere that depends on both temperature and relative humidity). We did find an effect of grazing on mean litter layer thickness (β=0.062, SE=0.024, P=0.009; Figure 4B). The litter layer was thinner in the grazed plots. We also found an effect on rodent burrow density (GLMM, β=-0.693, SE=0.215, P=0.0012). Rodent burrow density was shown to be a good proxy for rodent density (Mulder 2014). Rodent density was higher in the ungrazed plots. We also found a positive relationship between rodent burrow density and I. ricinus nymphal density. The relationship between herbivory and rodents is relevant because small rodents are the most important hosts for the juvenile ticks (mainly larvae) and the Borrelia pathogen (Hofmeester et al. 2016). Rodents need cover to protect them from predators and rodent density was significantly correlated with vegetation height (Smit et al. 2001). It is well known that grazing by large herbivores (both domestic and wild) can have negative (indirect) effects on rodent communities (Putman 1986, 1989, Smit et al. 2001, Van Wieren and Bakker 2008). In our study we found no effect of grazing on tick density. Apparently, nor the added grazers or the thinner litter layer, did lead to significant changes in the number of nymphal ticks produced. We did not find any changes in the herb layer. 82 Ecology and prevention of Lyme borreliosis

84 6. The role of large herbivores A 30 B 20 Rodent burrow density/100 m Mean litter layer (cm) 10 * 0 Ungrazed Grazed 0 Ungrazed Grazed Figure 4. (A) Rodent burrow density and (B) mean thickness of the litter layer in 20 grazed and ungrazed forest sites. It can be expected that added grazers may increase tick densities because they are additional reproduction hosts. In most cases, however, this effect will be relatively small when wild herbivores like deer are present as high tick densities are already reached at low deer densities while raised densities have never been observed. Obviously, tick numbers will increase substantially when grazers are applied in areas where no wild herbivores are present. In these cases, the interaction between the density of the grazers and their effects on the vegetation structure will determine tick density. Grazing may have an effect on tick densities through interactions with wild herbivores. It is well known that in many situations domestic grazers compete with other wild herbivores which can lead to local habitat displacement, lower densities or even almost complete disappearance of the wild herbivores (Fritz et al. 1996, Kie et al. 1991, Osborne 1984, Putman 1986, Stewart et al. 2002, Van Wieren and Bakker 2008). Also Steigedal et al. (2013) speculate that the lower tick densities they find with sheep grazing might be partly the result of a displacement of red deer from the sheep-grazed areas. The reason for the lower tick densities, then, might be the fact that domestic animals generally have fewer ticks than deer (Porter et al. 2011). Large herbivores and Borrelia burgdorferi s.l. As large herbivores are important reproduction hosts for ticks the question arises to what extend large herbivores play a role in the Borrelia cycle. Large herbivores can be infected with Borrelia but infection prevalence may vary widely (see Hofmeester et al. 2016). In roe deer it varied from (n=8), in red deer from (n=5), in fallow deer from (n=5), in horses from (n=7), in sheep from (n=4), and in wild boar from (n=5). Cases of Lyme borreliosis have been reported from sheep, cattle and horses (Fridriksdottir et al. 1992, Magnarelli et al. 1988, Parker and White 1992) but the disease is not known to occur in wild ungulates. In Table 2, infection prevalence of various Borrelia species are given that have been found in engorged ticks taken from a number of ungulate species in European systems. The infection prevalence is generally very low. In fact, it has always been found to be much lower than the prevalence in questing ticks in the vegetation (Table 3). This suggests that large herbivores Ecology and prevention of Lyme borreliosis 83

85 Sipke E. van Wieren and Tim R. Hofmeester Table 2. Infection prevalence for various species of Borrelia in engorged ticks taken from large herbivores. 1,2 Host species Pathogen Hosts tested Total ticks tested Infection prevalence All ticks Larvae Nymphs Adults Reference moose B. afzelii moose B. burgdorferi s.s moose B. garinii moose B. burgdorferi s.l ND ND 2 roe deer B. burgdorferi s.l ND ND 3 roe deer B. burgdorferi s.s roe deer B. afzelii roe deer B. garinii roe deer B. burgdorferi s.l ND ND ND 4 roe deer B. afzelii ND ND ND ND 5 roe deer B. burgdorferi s.l ND ND roe deer B. burgdorferi s.s. ND ND ND ND 5 roe deer B. garinii ND ND ND ND 5 roe deer B. valaisiana ND ND ND ND 5 roe deer coinfection B. afzelii ND ND ND ND 5 + B. burgdorferi s.s. roe deer coinfection B. afzelii ND ND ND ND 5 + B. garinii roe deer coinfection B. afzelii ND ND ND ND 5 + B. valaisiana roe deer B. afzelii ND ND roe deer B. burgdorferi s.l. ND ND roe deer B. valaisiana ND ND roe deer coinfection B. afzelii ND ND B. garinii roe deer coinfection B. afzelii + B. valaisiana ND ND roe deer coinfection B. burgdorferi s.s. ND ND B. garinii roe deer B. burgdorferi s.l ND ND red der B. burgdorferi s.l ND ND ND 4 red deer B. burgdorferi s.l ND ND fallow deer B. burgdorferi s.l ND ND ND 4 horse B. afzelii ND ND ND ND 8 sheep B. burgdorferi s.l. ND mouflon B. burgdorferi s.l ND ND ND 4 wild boar B. burgdorferi s.l ND ND References: 1 = Kjelland et al. (2011); 2 = Tälleklint and Jaenson (1994); 3 = Jaenson and Tälleklint (1992); 4 = Matuschka et al. (1993); 5 = Rijpkema et al. (1996); 6 = Schouls et al. (1999); 7 = Wegner et al. (1997); 8 = Ionita et al. (2013); 9 = Ogden et al. (1997). 2 ND = not determined. 84 Ecology and prevention of Lyme borreliosis

86 6. The role of large herbivores Table 3. Summary overview of reported infection prevalence of Borrelia burgdorferi s.l. in engorged ticks (nymphs and/or adults) taken from large herbivores and from questing ticks in the vegetation. Species Prevalence engorged ticks Prevalence questing ticks Reference roe, red, fallow deer Matuschka et al. (1993) goats, cattle Richter and Matuschka (2010) red deer, wild boar Pacilly et al. (2014) sheep this study are not very competent in transmitting the bacterium to the ticks and there is even evidence that the complement of these species might clear the infection from feeding ticks (Kurtenbach et al. 2002). In a series of in vitro experiments, Kurtenbach et al. (2002) also found that competence of domestic sheep, cattle and horses was not total for Borrelia burgdorferi s.l., although it was for some other species of Borrelia. If large herbivores are largely incompetent for transmitting Borrelia to ticks and even may clear the infection from ticks that feed on them, the question can be raised what role herbivores play in the Borrelia s.l. cycle. It is likely that ticks that are added to the system through large herbivores will not be infected. If questing larvae, that have a very low infection prevalence (Van Duijvendijk et al. 2016), will feed on a large herbivore, the nymphs in the next stage will not be infected. Larvae generally get infected when feeding on a competent infected host and these are mainly smaller species, notably rodents. Although large herbivores feed most of the adult ticks and juvenile ticks mainly feed on small species, large herbivores nevertheless sometimes carry many larvae as well. At very high herbivore densities a dilution effect might then become apparent. Very little work has been done to study the effects of varying large herbivore densities on the density of infected nymphs (DIN), the most relevant risk factor for humans. James et al. (2013) report higher infection prevalence in areas with higher deer abundance. They also found higher infection prevalence in mixed/deciduous forests when compared to coniferous forests. In contrast, Mysterud et al. (2016) found a negative correlation between deer density and infection prevalence with B. burgdorferi s.l. in questing nymphs. However, it is not always straightforward how these differences in infection prevalence affect the DIN. Richter and Matuschka (2006) found a negative effect of cattle on B. burgdorferi prevalence within French pastures. In the study mentioned above, where we compared 20 grazed (with cattle) forest sites with 20 ungrazed control sites, we found no effects of grazing on B. burgdorferi prevalence in nymphs and adults of I. ricinus. The value of the results of all of these studies is very limited because the effects of important other hosts have not been taken into account. In particular rodents should be included in any such study. The density of infected nymphs is the result of the interplay between the densities of all relevant hosts, their capacity to feed ticks and their capacity to transmit the bacteria to the ticks (Hofmeester et al. 2016, see also Van Wieren, 2016). It may be clear that large herbivores play a key role in maintaining tick populations in most European systems because they are the most important reproduction hosts. The relationship Ecology and prevention of Lyme borreliosis 85

87 Sipke E. van Wieren and Tim R. Hofmeester between herbivore abundance and tick density and infection prevalence with B. burgdorferi, however, varies between countries. In the past decades tick densities have increased greatly together with an increase in distribution, both latitudinally and altitudinally, due to changes in habitat characteristics and host densities (Medlock et al. 2013). Important drivers for this change are increasing temperatures, an increase in forest cover, changes in forest management, a higher frequency of mast years, range expansion of roe deer, red deer and wild boar, a lower hunting pressure on these species, changes in agricultural use (land abandonment, bush encroachment), agricultural schemes, and increased connectivity between habitats (Apollonio et al. 2010). In many systems a large number of these factors act simultaneously and in conjunction because they all have a positive effect on tick populations. Public health relevance Large herbivores are the most important species for maintaining tick populations. At high density large herbivores can manipulate the vegetation to such an extent that survival conditions for ticks are being compromised but also that exposure of ticks to humans is reduced. The density of infected nymphs, being the greatest risk factor for humans, is not likely to be much affected by varying herbivore density, except when this density is almost zero, or when densities are exceptionally high. References Apollonio M, Andersen R and Putman R (2010) European ungulates and their management in the 21 st century. Cambridge University Press, Cambridge, UK. Boyard C, Vourc h G and Barnouin J (2008) The relationships between Ixodes ricinus and small mammal species at the woodland-pasture interface. Exp Appl Acarol 44: Dobson ADM, Taylor JL and Randolph SE (2011) Tick (Ixodes ricinus) abundance and seasonality at recreational sites in the UK: hazards in relation to fine-scale habitat types revealed by complementary sampling methods. Ticks Tick Borne Dis 2: Domínguez-Peñafiel G, Giménez-Pardo C, Gegúndez MI and Lledó L (2011) Prevalence of ectoparasitic arthropods on wild animals and cattle in the Las Merindades area (Burgos, spain). Parasite 18: Estrada-Peña A (2001) Distribution, abundance, and habitat preferences of Ixodes ricinus (acari: Ixodidae) in northern Spain. J Med Entomol 38: Fridriksdottir V, Nesse LL and Gudding R (1992) Seroepidemiological studies of Borrelia burgdorferi infection in sheep in Norway. J Clin Microbiol 30: Fritz RS, Roche BM, Brunsfeld SJ and Orians CM (1996) Interspecific and temporal variation in herbivore responses to hybrid willows. Oecologia 108: Gassner F, van Vliet AJH, Burgers S, Jacobs F, Verbaarschot P, Hovius EKE, Mulder S, Verhulst NO, van Overbeek LS and Takken W (2011) Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in the Netherlands. Vector Borne Zoonotic Dis 11: Gassner F, Verbaarschot P, Smallegange RC, Spitzen J, Van Wieren SE and Takken W (2008) Variations in Ixodes ricinus density and Borrelia infections associated with cattle introduced into a woodland in the Netherlands. Appl Environ Microbiol 74: Ecology and prevention of Lyme borreliosis

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89 Sipke E. van Wieren and Tim R. Hofmeester Mulder AC (2014) Deer and rabbit density as determinants of tick density on a local scale. MSc thesis, Wageningen University & Research, Wageningen, the Netherlands. Mysterud A, Easterday WR, Stigum VM, Aas AB, Meisingset EL and Viljugrein H (2016) Contrasting emergence of lyme disease across ecosystems. Nat Commun 7: Ogden NH, Nuttall PA and Randolph SE (1997) Natural Lyme disease cycles maintained via sheep by co-feeding ticks. Parasitology 115: Osborne BC (1984) Habitat use by red deer (Cervus elaphus l.) and hill sheep in the west Highlands. J Appl Ecol 21: Pacilly FCA, Benning ME, Jacobs F, Leidekker J, Sprong H, Van Wieren SE and Takken W (2014) Blood feeding on large grazers affects the transmission of Borrelia burgdorferi sensu lato by Ixodes ricinus. Ticks Tick Borne Dis 5: Parker JL and White KK (1992) Lyme borreliosis in cattle and horses: a review of the literature. Cornell Vet 82: Pato FJ, Panadero R, Vázquez L, López CM, Díaz P, Vázquez E, Díez-Baños P, Morrondo P and Fernández G (2013) Seroprevalence of Borrelia burgdorferi sensu lato in roe deer (Capreolus capreolus) from northwestern Spain. J Zoo Wildl Med 44: Piesman J, Spielman A, Etkind P, Ruebush TK and Juranek DD (1979) Role of deer in the epizootiology of Babesia microti in Massachusetts, USA. J Med Entomol 15: Porter R, Norman R and Gilbert L (2011) Controlling tick-borne diseases through domestic animal management: a theoretical approach. Theor Ecol 4: Putman RJ (1986) Competition and coexistence in a multi-species grazing system. Acta Theriol 31: Putman RJ, Edwards PJ, Mann JCE, How RC and Hill SD (1989) Vegetational and faunal changes in an area of heavily grazed woodland following relief of grazing. Biol Conserv 47: Rand PW, Lubelczyk C, Lavigne GR, Elias S, Holman MS, Lacombe EH and Smith RP (2003). Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 40: Randolph SE and Storey K (1999) Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission. J Med Entomol 36: Richter D and Matuschka FR (2006) Modulatory effect of cattle on risk for Lyme disease. Emerging Infect Dis 12: Richter D and Matuschka FR (2010) Elimination of Lyme disease spirochetes from ticks feeding on domestic ruminants. Appl Environ Microbiol 76: Rijpkema SGT, Herbes RG, Verbeek de Kruif N and Schellekens JFP (1996) Detection of four species of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from roe deer (Capreolus capreolus) in the Netherlands. Epidemiol Infect 117: Schouls LM, Van De Pol I, Rijpkema SGT and Schot CS (1999) Detection and identification of ehrlichia, Borrelia burgdorferi sensu lato, and Bartonella species in dutch Ixodes ricinus ticks. J Clin Microbiol 37: Smit R, Bokdam J, den Ouden J, Olff H, Schot-Opschoor H and Schrijvers M (2001) Effects of introduction and exclusion of large herbivores on small rodent communities. Plant Ecol 155: Sonenshine DE (1991) Biology of ticks. Oxford University Press, New York, NY, USA. Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJH, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 1. Steigedal HH, Loe LE, Grøva L and Mysterud A (2013) The effect of sheep (Ovis aries) presence on the abundance of ticks (Ixodes ricinus). Acta Agric Scand A Anim Sci 63: Stewart KM, Bowyer RT, Kie JG, Cimon NJ and Johnson BK (2002) Temporospatial distributions of elk, mule deer, and cattle: resource partitioning and competitive displacement. J Mammal 83: Tack W, Madder M, Baeten L, De Frenne P and Verheyen K (2012a) The abundance of Ixodes ricinus ticks depends on tree species composition and shrub cover. Parasitology 139: Tack W, Madder M, Baeten L, Vanhellemont M, Gruwez R and Verheyen K (2012b) Local habitat and landscape affect Ixodes ricinus tick abundances in forests on poor, sandy soils. For Ecol Manage 265: Ecology and prevention of Lyme borreliosis

90 6. The role of large herbivores Tälleklint L and Jaenson TGT (1994) Transmission of Borrelia burgdorferi s.l. from mammal reservoirs to the primary vector of lyme borreliosis, Ixodes ricinus (Acari: Ixodidae), in Sweden. J Med Entomol 31: Tälleklint L and Jaenson TGT (1997) Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in south-central Sweden. Exp Appl Acarol 21: Van Buskirk J and Ostfeld RS (1995) Controlling Lyme disease by modifying the density and species composition of tick hosts. Ecol Appl 5: Van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersöz J, Oei A, Földvári G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: 97. Van Wieren SE (2016) Sheep mopping. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Wieren SE and Bakker JP (2008) The impact of browsing and grazing herbivores on biodiversity. In: Gordon IJ and Prins HHT (eds.) The ecology of browsing and grazing. Springer, Berlin/Heidelberg Germany, pp Vazquez L, Panadero R, Dacal V, Pato FJ, Lopez C, Diaz P, Arias MS, Fernandez G, Diez-Baos P and Morrondo P (2011) Tick infestation (Acari: Ixodidae) in roe deer (Capreolus capreolus) from northwestern Spain: population dynamics and risk stratification. Exp Appl Acarol 53: Vor T, Kiffner C, Hagedorn P, Niedrig M and Ruhe F (2010) Tick burden on european roe deer (Capreolus capreolus). Exp Appl Acarol 51: Wegner Z, Stańczak J, Racewicz M, Kubica-Biernat B and Kruminis-Łozowska W (1997) The etiological agent of Lyme disease, Borrelia burgdorferi, in ticks (Acari: Ixodidae) from eastern Poland. Zentralbl Bakteriol 286: Wilson ML, Ducey AM, Litwin TS, Gavin TA and Spielman A (1990) Microgeographic distribution of immature Ixodes dammini ticks correlated with that of deer. Med Vet Entomol 4: Ecology and prevention of Lyme borreliosis 89

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92 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe Dieter J.A. Heylen Evolutionary Ecology Group, Department of Biology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium; Abstract More than any other host type, birds are capable to carry ticks and tick-borne diseases over long distances, and introduce infected ticks across geographical barriers into (sub-)urban environments. This chapter discusses the role of songbirds and their Ixodes tick-community in the terrestrial transmission cycles of Borrelia burgdorferi s.l. in Europe, which has been underappreciated for a long time. In addition to the host generalists Ixodes ricinus and Ixodes persulcatus, two abundant Ixodes species have frequently been derived from wild birds with Borrelia infections: Ixodes arboricola (adapted to tree-holes where it infests nestlings, roosting and breeding birds) and Ixodes frontalis (generalistic and ornithophilic, but with a largely unknown ecology). Abiotic constraints and seasonal activity patterns that characterise the ticks biology, in addition to the birds behaviour that relates to tick encounter rates (including foraging height, nesting and roosting habits) are the most important factors for explaining the spatio-temporal variation in Ixodes tick infestations among and within bird species. As the activity patterns of the ornithophilic ticks, I. arboricola and I. frontalis, overlap in time and/or space with I. ricinus and I. persulcatus, they share avian hosts. Consequently, Borrelia spirochaetes of ornithophilic ticks may be bridged via the host generalist ticks to other hosts outside the enzootic bird-tick cycles. Recent experiments have shown, that in addition to I. ricinus (Borrelia garinii, Borrelia valaisiana, Borrelia turdi), I. frontalis (Borrelia turdi) is truly vector-competent. The non-homogeneous distributions of the avian genospecies found in bird-derived I. ricinus larvae suggest differential transmission and amplification of Borrelia genospecies depending on the avian reservoir species. Future studies should further focus on the intra-specific infestation risk factors in birds, the reservoir competence of members in the bird community, and how the different vector-bird niches contribute to the transmission dynamics of the Borrelia genospecies community. Keywords: Borrelia garinii, Borrelia turdi, Borrelia valaisiana, Ixodes arboricola, Ixodes frontalis, Ixodes ricinus, songbird Introduction After several years of controversy, the contribution of songbirds in the terrestrial cycles of Borrelia burgdorferi s.l. has become more than obvious (Humair 2002). Songbirds are now recognised as important hosts for ticks maintaining tick populations (Hudde and Walter 1988, Lundqvist et al. 1998, Ulmanen et al. 1977), and as reservoirs for Lyme disease spirochaetes (Gylfe et al. 2000, Heylen et al. 2016, Humair 2002, Richter et al. 2000). Despite the importance of the songbird-tick cycles, the insight in the processes driving tick-infestations, and in the birds ability to acquire and transmit Borrelia spirochaetes, remains poor and lags behind that of mammal-tick systems (Brunner and Ostfeld 2008, Ginsberg et al. 2005, Heylen et al. 2013a, 2014a, Hughes and Randolph 2001, Humair et al. 1998, Kiffner et al. 2010, Richter et al. 2000, Rosà et al. 2007, Tälleklint and Jaenson 1997,Wikel 1996). Arguably, practical and organisational constraints in capturing and manipulating birds have, among others, slowed down our knowledge acquisition. In the terrestrial Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume 4 91 DOI / _7, Wageningen Academic Publishers 2016

93 Dieter J.A. Heylen ecosystem, mammals have been assumed to contribute most to the Borrelia cycles, because they are generally more abundant than resident birds (Giardina et al. 2000). Recent research indicates that the real contribution of songbirds in infecting and disseminating ticks may be at least as important as that of other host types (Comstedt et al and references herein, Gray et al. 1999, Heylen et al. 2013b). The discovery of bird-associated Borrelia genospecies (including Borrelia garinii and Borrelia valaisiana) (Humair et al. 1998, Kurtenbach et al. 1998) that are pathogenic to humans, and their omni-presence in questing Ixodes ricinus and Ixodes persulcatus ticks have increased our interest in ecological interactions in the bird-tick-borrelia system. Birds act as long-distance carriers of ticks infected with pathogenic agents and can introduce them on their migration and dispersal routes across geographical barriers (Bjöersdorff et al. 2001, Elfving et al. 2010, Hasle et al. 2011, Olsen et al. 1995, Waldenström et al. 2007). They may be particularly important as reservoir hosts in areas such as islands and green patches in urbanised regions that are less accessible to migrating mammals. So far, the majority of the European bird studies have focused on tick infestations at the time of autumn and spring migration, but the role of songbirds in the transmission dynamics when birds are at their breeding or wintering sites, as well as the role of resident birds remain poorly investigated (Dubska et al. 2011, Heylen et al. 2013b, James et al. 2011, Marsot et al. 2012, Norte et al. 2013c). In what follows, I will discuss three themes that (in)directly relate to human Borrelia exposure risks. First, I will delve deeper into the processes that explain inter- and intra-species differences in tick infestation risks among birds. It is obvious that defining the ecological patterns affecting the tick infestations is important for predicting the transmission dynamics of tick-borne diseases between bird and tick populations, and hence the risk of human infections. Second, I will shed light on the vector competence of common bird-associated tick species for the most prevalent B. burgdorferi s.l. genospecies. The third section is dedicated to the capacity of native songbirds to facilitate the transmission of B. burgdorferi s.l. spirochaetes. Tick ecology and infestation processes in the songbird-tick community In European songbirds, at least 10 tick species belonging to the genus Ixodes have been found, all having different habitat requirement and showing contrasting host-specificities (Hillyard 1996). The five most common bird-derived Ixodes ticks are Ixodes lividus, Ixodes arboricola, Ixodes frontalis, I. ricinus and I. persulcatus. I. lividus shows an extreme host specificity, virtually only infesting bank swallows (Riparia riparia L.) inside the sandy borrows the birds excavate at arrival in their breeding location (Ulmanen et al. 1977). Because of the highly specific habitat in which both the host and the tick live outside the reach of humans, of I. ricinus and I. persulcatus this bird and tick species can be considered of low epidemiological importance and will not be discussed further. I. arboricola (the tree-hole tick ) shows an intermediate host-specificity, infesting secondary cavitynesting birds that breed and roost inside its preferred habitat: tree-holes and cavities (Hudde and Walter 1988, Van Oosten et al. 2016). Although I. arboricola can be found often in close proximity to humans in woodland and gardens, it rarely comes into contact with people due to its endophilic ecology. This contrasts with I. frontalis, a generalist ornithophilic tick (Doby 1998) that, because of its partially exophilic ecology, does encounter humans on which the adult female stage has shown to be able to attach (Gilot et al. 1997). Many aspects of I. frontalis ecology remain unresolved. The collection of I. frontalis from understory vegetation, the pronounced questing behaviour on leaf-like substrates (cf. Ixodes brunneus; Goddard 2013) and diurnal detachments from diurnally active birds (cf. I. ricinus; Heylen and Matthysen 2010) all suggest that this tick species tends to be exophilic rather than endophilic, which was previously assumed (Hillyard 1996). Adult 92 Ecology and prevention of Lyme borreliosis

94 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe females can have a strong health impact on its hosts, by causing avian tick paralysis a syndrome characterised by birds showing acute depression or death due to secreted tick toxins (Monks et al. 2006). The most common ticks infesting songbirds are I. ricinus and I. persulcatus. These extreme host generalists are found on a huge variety of terrestrial vertebrates (Hillyard 1996). Similar to I. frontalis, they infest a very broad range of both open- and cavity-nesting birds. The immature developmental stages are particularly susceptible to desiccation (Gray 1991, Perret et al and references herein), and therefore this tick is restricted to the under-storey vegetation and litter layer of forests and semi-open areas. Adult females rarely infest songbirds (but see Norte et al. 2015, Olsen et al. 1995) and are considered to only feed successfully on larger mammals, which contrasts with the adult females of the three strictly ornithophilic tick species. This finding questions whether birds alone can maintain I. ricinus and I. persulcatus populations. As is characteristic in most ectoparasite-host systems, Ixodes ticks are heterogeneously distributed among host individuals, so that a few host individuals carry most of the ticks (Brunner and Ostfeld 2008 and references herein, Comstedt et al. 2006). These aggregated distributions imply that only a small proportion of the host population maintains the tick population and sustains the transmission of tick-borne diseases (Woolhouse et al. 1997). Unravelling the ecological processes that shape this heterogeneity, i.e. evaluating the risk factors for infestation, is therefore essential in understanding the population dynamics of ticks and the epidemiology of diseases they transmit. In free-flying birds, the abundant I. ricinus dominates the infestation pattern during those months when the immature developmental stages actively quest (March-October) (Gray 1991); which include the bird s breeding and post-breeding seasons (Marsot et al. 2012) and migration periods (Comstedt et al. 2006, Hasle et al. 2011). In southern areas, due to higher temperatures the questing period may be extended throughout the winter (Barandika et al. 2011) leading to winter infestations (Norte et al. 2015). The inter-specific variation in infestation is to a large extent determined by the host species vertical space use (Comstedt et al and references herein, Dubska et al. 2011, Marsot et al. 2012). Ground-foraging birds, that search for food inside the vertical distribution of I. ricinus and I. persulcatus (e.g. blackbirds (Turdus merula), song thrushes (Turdus philomelos), European robins (Erithacus rubecula) and Eurasian wren (Troglodytes troglodytes)) are generally more often and more heavily infested than birds that forage in the forest canopy (e.g. blue tits; Cyanistes caeruleus) and nuthatches (Sitta europea)). Crucial information on interspecific differences in behavioural and immunological resistance mechanisms is still lacking, but it is clear, that some birds just feed more I. ricinus ticks and are more abundant than others, hence having a higher potential to contribute to its pathogen transmission cycles (Marsot et al. 2012). Therefore, obtaining reliable estimates for bird abundances (correcting for inter-specific variation in capturability (Marsot et al. 2012) and detection probability) and infestation levels, are all the more essential in addition to information on reservoir and vector competence (see below) to improve our insights in the local involvement of songbird communities to the pathogen-cycles maintained by I. ricinus and I. persulcatus. Both ornithophilic ticks I. frontalis and I. arboricola show a very noticeable winter activity (Doby 1998, Heylen et al. 2014c, Literak et al. 2007, Norte et al. 2015), which contrasts with the phenology of I. ricinus characterised by a spring-feeding and an autumn-feeding cohort, and very little host seeking activity during winter time (Gray 1991). Likely, these poikilothermic ticks use the birds body heat to become activated when they roost inside cavities (I. arboricola infestations) or inside the bushes (I. frontalis infestations). Ixodes ricinus-infested hole-breeding hosts (Parus major and Cyanistes caeruleus) have often been found to be co-infested with immature stages of ornitophilic ticks, in particular during late-autumn and early-spring (Heylen et al. 2014c). And also I. ricinus- Ecology and prevention of Lyme borreliosis 93

95 Dieter J.A. Heylen infested blackbirds show a high tendency to be co-infested with I. frontalis during these moments of the year (Doby 1998). The information about the temporal overlap with I. ricinus is all the more important when pathogen transmission is limited in time due to the acquisition of host resistance after an infection, or in the situation when transmission depends on co-occurring ticks that feed in close proximity on the same host (i.e. co-feeding transmission) (Gern and Rais 1996, Randolph et al. 1996). Thus, even though bird-specific ticks generally do not infest humans and livestock, they often share avian hosts with generalist I. ricinus that do so, via which they can introduce pathogens outside the bird-tick cycles. Therefore, the seasonal feeding activity of ornithophilic ticks can likely have an impact on the transmission of pathogens outside bird-tick transmission cycles. Very little research has been done so far on the risk factors for Ixodes infestations at the intrahost level, except in the hole-breeding great tit (P. major). This bird species shows a tolerance for I. ricinus infestations, rather than acquiring immunological resistance (Heylen et al. 2010). Although tick burdens cause measurable clinical symptoms (reduced haematocrit and increased inflammation levels) in songbirds (Heylen and Matthysen 2008, Norte et al. 2013a), no fitness effects have been found (Heylen et al. 2009). In this host species, ecological risk factors have been identified that to some extent explain the variation in I. ricinus infestation during the breeding season (April-June) (Heylen et al. 2013a). They include extrinsic exposure risk factors, that directly relate to tick abundance and questing activity, as well as intrinsic host characteristics such as the foraging range and intensity (which determines encounter rates) that are associated to the brood conditions. The authors hypothesise that the investments in brood care compete with the energy allocation to behavioural anti-tick resistance mechanisms. Nestlings do not escape Ixodes tick infestations either. The nidicolous I. arboricola prefers the more developed nestlings (highest nutritional value) with the weaker immune system (Heylen and Matthysen 2011), on which they aggregate, and this may eventually create a situation with high potential for cofeeding transmission of tick-borne pathogenic agents. Adult female ticks typically wait until the nestlings grow old, in order to optimise their fitness (Heylen et al. 2012). Consequently, adults can hypothetically infect naïve birds (i.e. nestlings) with a high diversity of tick-borne agents (Heylen et al. 2013b, Hillyard 1996, Lichard and Kozuch 1967, Spitalska et al. 2011) that have been accumulated during the feeding by the larval and nymphal developmental stage. Ixodes ricinus has a high potential to infest nestlings of ground-nesting songbirds and birds that build their nests inside the vertical distribution of I. ricinus (Gallizzi et al. 2008, Roulin et al. 2003), but little systemic research has been done so far. Although I. frontalis has been associated with songbird nests (Hillyard 1996), we still lack information on its infestation patterns in nestlings. Vector-competence of bird-specialised ticks Besides in I. ricinus and I. persulcatus, B. burgdorferi s.l. has been detected in several other European tick species (Gray 1998, Heylen et al. 2013b, Piesman and Gern 2004). The presence of B. burgdorferi s.l. does not mean that the tick is vector-competent, i.e. being able to acquire, maintain, and transmit the micro-organism to a new host. Many tick species may get infected with Borrelia, and contain the bacteria even after moulting to the next instar, but are not able to effectively transmit them. Only few European tick species have shown to actually transmit Borrelia (Gray et al. 2002, Piesman and Gern 2004). As the activity patterns of bird-specialised ticks, I. arboricola and I. frontalis, overlap in time and space with I. ricinus, they frequently share host individuals (e.g. the great tit and the blackbird). Consequently, Borrelia spirochaetes of bird-specialised ticks may be bridged via I. ricinus (the generalist tick) to other hosts outside the enzootic cycles (Piesman and Gern 2004). Screenings of 94 Ecology and prevention of Lyme borreliosis

96 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe bird-derived I. arboricola and I. frontalis ticks from the wild show that both species carry Borrelia spirochaetes (Estrada-Peña et al. 1995, Heylen et al. 2013b, Norte et al. 2013c, Palomar et al. 2012). Two elaborative vector-competence studies revealed that I. arboricola and I. frontalis do not transmit B. garinii and B. valaisiana bacteria to its most common hole-breeding host, the great tit (Heylen et al. 2014b). However, experimental data convincingly shows that I. frontalis is able to transmit B. turdi to blackbirds (D. Heylen et al. unpublished data). The association of B. turdi with bird-derived I. frontalis ticks in Europe is very strong (Estrada-Peña et al. 1995, Heylen et al. 2013b, Norte et al. 2013c, Palomar et al. 2012), which suggests B. turdi is only successfully transmitted by I. frontalis that is maintaining a cryptic cycle in songbirds. Interestingly, in the latter experiment with blackbirds also I. ricinus was able to acquire and effectively transmit B. turdi, supporting the potential occurrence of enzootic B. burgdorferi s.l. cycles in the wild involving songbirds and their ornithophilic ticks, with the generalist I. ricinus acting as bridging vector towards other hosts outside this cycle. We may wonder how I. frontalis ticks impact the Borrelia-cycles, and more importantly, whether they would cause an indirect risk to humans. A way to obtain better insights hereto, is the analysis of Borrelia genotype strains, and investigate whether these are common to both cryptic (I. frontalis) and endemic (I. ricinus) cycles. Although B. valaisiana has often been detected in questing I. ricinus nymphs collected from understory vegetation, there are no records of B. turdi-infections in these ticks, as far as I know. Explanations for this could be that B. turdi shows a low persistence in I. ricinus over the long-term reducing the detection chances in field-derived ticks, that I. ricinus survival is negatively affected by B. turdi reducing the chance to collect infected ticks, or that B. turdi has only recently emerged in Europe and is still too rare to be of epidemiological importance. Borrelia turdi is not known to infect humans, and only recently has been increasingly recorded in Europe. This genospecies, first described in a Japanese study (Fukunaga et al. 1996), may be more common in Eurasia than previously thought, and it is possible that B. turdi has been present since long in Europe and potentially has been overlooked. Under the hypothesis that Borrelia is frequently transmitted via I. frontalis and its host to I. ricinus, another question that can be put forward is how the Borrelia community will be affected by I. frontalis when I. ricinus seasonal activity will be extended because of the milder winters. In this climate change -scenario, there will be more temporal overlap in host-finding activity between I. ricinus individuals (Dautel et al. 2008, Gray 2008) and the winter-active I. frontalis (Doby 1998, Norte et al. 2015), increasing the number of transmission events between ticks species sharing the same host. This scenario would imply a time-based invasion of I. ricinus into the cryptic transmission cycles that will occur during winter time (later autumn and early spring), allowing for bridging pathogenic strains to animals and humans (cf. spatial invasion of I. scapularis on the American continent (Hamer et al. 2011). Although the winter-active I. arboricola is not able to vector B. burgdorferi s.l., similar scenarios as described above could still be expected for other pathogenic agents this tick species shares with I. ricinus (Spitalska et al. 2011). Facilitation of Borrelia transmission by songbirds The competence of host organism to acquire, replicate, and most importantly, to transmit pathogens to naïve organisms is fundamental to understanding the epidemiology of infectious diseases in their natural environment. In several field-studies, bird-derived Borrelia-infected larvae have been collected. This suggests that their hosts facilitated the transmission of Borrelia spirochaetes. Given that B. burgdorferi s.l. spirochaetes are generally not believed to have vertical transmission in Ixodes ticks (Richter et al. 2012, Rollend et al. 2013), the presence in larvae gives indirect proof for their acquisition either via (local) infection in the host, or via co-feeding transmission with an infected nymph. Based on larval infection rates, we can conclude that there is host-dependent structuring in the genospecies composition, in that certain bird species are Ecology and prevention of Lyme borreliosis 95

97 Dieter J.A. Heylen more prone to transmit particular genospecies than others (see Figure 1, which is based on data of the following studies: Comstedt et al. 2006, Dubska et al. 2011, Dubska et al. 2009, Franke et al. 2010, Hanincová et al. 2003, Hasle et al. 2011, Heylen et al. 2013b, Humair et al. 1993, James et al. 2011, Kipp et al. 2006, Kjelland et al. 2010, Lommano et al. 2014, Mannelli et al. 2005, Michalik et al. 2008, Norte et al. 2013c, in press, Olsen et al. 1995, Taragel ova et al. 2008). Blackbirds (T. merula) and song thrushes (T. philomelos) have by far yielded most B. valaisiana- and B. garinii-infected larvae. Both host species show to be relevant for the transmission of the recently discovered B. turdi in Europe (Hasle et al. 2011, Norte et al. 2013b). On the other hand, tree-pipit (Anthus trivialis), great tit (P. major), chaffinch (Fringilla coelebs) and the Eurasian wren (T. troglodytes) tend to yield more B. garinii-infected than B. valaisiana-infected larvae. Among the avian genospecies, B. garinii is responsible for neuroborreliosis and by far the most relevant for human Lyme borreliosis. On the other hand, Borrelia valaisiana is considered as rarely if at all pathogenic for humans (Humair and Gern 2000, Lipsker and Jaulhac 2009). The epidemiological importance and pathogenicity of B. turdi is currently unknown. Interestingly, from several birds, substantial numbers of B. afzelii-infected larvae have been collected, which questions the consensus that B. afzelii is strictly incompatible with avian hosts (Franke et al. 2010). Co-feeding transmission between B. afzelii-infected nymphs and naïve larvae could be one of the pathways resulting in infected bird-derived B. afzelii-larvae, by which the spirochaetes could escape the bird s complement immune response, but this hypothesis needs experimental testing. Another hypothesis would be that the larval ticks have acquired B. afzelii before feeding on the birds, either via trans-ovarial transmission or because the larvae had partially fed on a previous mammalian host (Van Duijvendijk et al. 2016). Also, other mammal- Fringilla coelebs (510; 148; 10) Parus major (413; 322; 17) Troglodytes troglodytes (248; 86; 11) B. garinii B. garinii-b.valaisiana B. valaisiana B. turdi B. afzelii Others Turdus philomelos (563; 262; 14) Turdus merula (1,194; 473; 17) Anthus trivialis (299; 185; 4) Prevalence in tested Ixodes ricinus larvae Figure 1. Distribution of Borrelia burgdorferi s.l. genospecies in infected Ixodes ricinus larvae collected from frequently infested songbird species (number of larvae screened for Borrelia; number of birds; number of studies) that were caught via mist-nets at different locations spread over Europe. Means and standard errors (bars) were based on the overall (pooled) infection rates per bird species per study, because several studies did not report the infection rates of individual birds. All studies (see main text) reported larvae as infected with one Borrelia genospecies, likely referring to the genospecies that was most dominant in the tick. 96 Ecology and prevention of Lyme borreliosis

98 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe associated genospecies have been found in bird-derived ticks (including Borrelia bavariensis and Borrelia spielmanii), and some genotypes possibly have the ability to survive the bird complement such that they can be found in high concentrations in the next developmental stages (Heylen et al. 2013b, 2014b, 2016). The epidemiological significance of this possibly successful transstadial transmission of mammalian pathogens through birds remains to be investigated. Surprisingly little experimental work on the reservoir competence of native European birds has been done so far. In blackbirds, B. valaisiana, B. turdi, and to a lesser extent B. garinii have been found in bird tissues and xenodiagnostic I. ricinus larvae (Humair et al. 1998, Norte et al. 2013b). Repeated exposures of the great tit with unfed nymphs from a Borrelia-endemic area show that this bird species selectively amplifies certain B. garinii genotypes and is less effective in transmitting B. valaisiana (Heylen et al. 2014a, 2016), confirming the conclusions above based on larvae derived from birds captured in the wild. To quantify the infectiousness of the broad diversity of wild birds (of which many abundant ones have seldom been captured and screened for ticks, e.g. pigeons, and corvids) xeno-diagnoses could be executed with Borrelia-naïve Ixodes larvae (Ginsberg et al. 2005). In the next step, the spirochaetes can be tested to whether they effectively infect the next host, after the larvae have moulted into the nymphal stage (Richter et al. 2000). Aside from the interspecific differences mentioned above, also on the intra-specific level, different state-dependent factors could influence the amplification and transmission efficacies, e.g. stress (Gylfe et al. 2000), age and acquired resistance (Dubska et al. 2011) which largely remain unexplored. The contribution of birds to human infections can be substantial, and it is obvious that future studies need an integrated approach, combining observationally and experimentally obtained information. Public health relevance In Europe, birds maintain communities of Ixodes species and Borrelia genospecies. Each combination of bird and tick species forms a specific niche, and consequently affects the local Borrelia genospecies community in a different way. Among the avian Borrelia genospecies, B. garinii is by far the most important for human Lyme borreliosis. Ecological factors that are linked to the vertical space use of avian reservoirs strongly determine the frequency of Borrelia transmission events with the grounddwelling I. ricinus ticks. The ornithophilic Ixodes frontalis is a competent vector for B. turdi, and therefore can affect the Borrelia community in questing I. ricinus ticks, when both ticks share the same avian hosts. Ecology and prevention of Lyme borreliosis 97

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101 Dieter J.A. Heylen Kiffner C, Vor T, Hagedorn P, Niedrig M and Ruhe F (2010) Factors affecting patterns of tick parasitism on forest rodents in tick-borne encephalitis risk areas, Germany. Parasitol Res 108: Kipp S, Goedecke A, Dorn W, Wilske B and Fingerle V (2006) Role of birds in Thuringia, Germany, in the natural cycle of Borrelia burgdorferi sensu lato, the Lyme disease spirochaete. Int J Med Microb 296: Kjelland V, Stuen S, Skarpaas T and Slettan A (2010) Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in southern Norway. Acta Vet Scand 52: 1-6. Kurtenbach K, Peacey M, Rijpkema SGT, Hoodless AN, Nuttall PA and Randolph SE (1998) Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England. Appl Environ Microbiol 64: Lichard M and Kozuch O (1967) Persistence of tick-borne encephalitis virus in nymphs and adults of Ixodes arboricola and its transmission to white mice. Acta Virol 11: 480. Lipsker D and Jaulhac B (2009) Lyme borreliosis: biological and clinical aspects. Karger, Basel, Switzerland. Literak I, Kocianova E, Dusbabek F, Martinu J, Podzemny P and Sychra O (2007) Winter infestation of wild birds by ticks and chiggers (Acari: Ixodidae, Trombiculidae) in the Czech Republic. Parasitol Res 101: Lommano E, Dvorak C, Vallotton L, Jenni L and Gern L (2014) Tick-borne pathogens in ticks collected from breeding and migratory birds in Switzerland. Ticks Tick Borne Dis 5: Lundqvist L, Gray JS and Hillyard PD (1998) Ixodes frontalis on the Baltic island of Gotland, Sweden. Med Vet Entomol 12: Mannelli A, Nebbia P, Tramuta C, Grego E, Tomassone L, Ainardi R, Venturini L, De Meneghi D and Meneguz PG (2005) Borrelia burgdorferi sensu lato infection in larval Ixodes ricinus (Acari: Ixodidae) feeding on blackbirds in northwestern Italy. J Med Entomol 42: Marsot M, Henry P-Y, Vourc h G, Gasqui P, Ferquel E, Laignel J, Grysan M and Chapuis J-L (2012) Which forest bird species are the main hosts of the tick, Ixodes ricinus, the vector of Borrelia burgdorferi sensu lato, during the breeding season? Int J Parasitol 42: Michalik J, Wodecka B, Skoracki M, Sikora B and Stańczak J (2008) Prevalence of avian-associated Borrelia burgdorferi s.l. genospecies in Ixodes ricinus ticks collected from blackbirds (Turdus merula) and song thrushes (T. philomelos). Int J Med Microb 298: Monks D, Fisher M and Forbes NA (2006) Ixodes frontalis and avian tick-related syndrome in the United Kingdom. J Small Anim Pract 47: Norte AC, Araújo PM, da Silva LP, Tenreiro PQ, Ramos JA, Núncio MS, Zé-Zé L and de Carvalho IL (in press) Characterization through multilocus sequence analysis of Borrelia turdi isolates from Portugal. Microb Ecol DOI: org/ /s Norte AC, da Silva LP, Tenreiro PJQ, Felgueiras MS, Araújo PM, Lopes PB, Matos C, Rosa A, Ferreira PJSG, Encarnacão P, Rocha A, Escudero R, Anda P, Núncio MS and Lopes de Carvalho I (2015) Patterns of tick infestation and their Borrelia burgdorferi s.l. infection in wild birds in Portugal. Ticks Tick Borne Dis 6: Norte AC, Lobato DNC, Braga EM, Antonini Y, Lacorte G, Goncalves M, de Carvalho IL, Gern L, Núncio MS and Ramos JA (2013a) Do ticks and Borrelia burgdorferi s.l. constitute a burden to birds? Parasitol Res 112: Norte AC, lopes de Carvalho I, Núncio M, Ramos JA and Gern L (2013b) Blackbirds Turdus merula as competent reservoirs for Borrelia turdi and Borrelia valaisiana in Portugal: evidence from a xenodiagnostic experiment. Environ Microb Rep 5: Norte AC, Ramos JA, Gern L, Núncio MS and Lopes de Carvalho I (2013c) Birds as reservoirs for Borrelia burgdorferi s.l. in Western Europe: circulation of B. turdi and other genospecies in bird-tick cycles in Portugal. Environ Microb 15: Olsen B, Jaenson TGT and Bergström S (1995) Prevalence of Borrelia burgdorferi sensu lato-infected ticks on migrating birds. Appl Environ Microbiol 61: Palomar AM, Santibáñez P, Mazuelas D, Roncero L, Santibáñez S, Portillo A and Oteo JA (2012) Role of birds in dispersal of etiologic agents of tick-borne zoonoses, Spain, Emerg Infect Dis 18: Perret JL, Rais O and Gern L (2004) Influence of climate on the proportion of Ixodes ricinus nymphs and adults questing in a tick population. J Med Entomol 41: Ecology and prevention of Lyme borreliosis

102 7. Ecological interactions between songbirds, ticks, and Borrelia burgdorferi s.l. in Europe Piesman J and Gern L (2004) Lyme borreliosis in Europe and North America. Parasitology 129: S191-S220. Randolph SE, Gern L and Nuttall PA (1996) Co-feeding ticks: epidemiological significance for tick-borne pathogen transmission. Parasitol Today 12: Richter D, Debski A, Hubalek Z and Matuschka FR (2012) Absence of Lyme disease spirochetes in larval Ixodes ricinus ticks. Vector-Borne Zoonotic Dis 12: Richter D, Spielman A, Komar N and Matuschka FR (2000) Competence of American robins as reservoir hosts for Lyme disease spirochetes. Emerg Infect Dis 6: Rollend L, Fish D and Childs JE (2013) Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: a summary of the literature and recent observations. Ticks Tick-Borne Dis 4: Rosà R, Pugliese A, Ghosh M, Perkins SE and Rizzoli A (2007) Temporal variation of Ixodes ricinus intensity on the rodent host Apodemus flavicollis in relation to local climate and host dynamics. Vector-Borne Zoonotic Dis 7: Roulin A, Brinkhof MWG, Bize P, Richner H, Jungi TW, Bavoux C, Boileau N and Burneleau G (2003) Which chick is tasty to parasites? The importance of host immunology vs. parasite life history. J Anim Ecol 72: Spitalska E, Literak I, Kocianova E and Taragel ova V (2011) The importance of Ixodes arboricola in transmission of Rickettsia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato in the Czech Republic, Central Europe. Vector-Borne Zoonotic Dis 11: Tälleklint L and Jaenson TGT (1997) Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in south-central Sweden. Exp Appl Acarol 21: Taragel ova V, Koci J, Hanincova K, Kurtenbach K, Derdakova M, Ogden NH, Literak I, Kocianova E and Labuda M (2008) Blackbirds and song thrushes constitute a key reservoir of Borrelia garinii, the causative agent of borreliosis in Central Europe. Appl Environ Microbiol 74: Ulmanen I, Saikku P, Vikberg P and Sorjonen J (1977) Ixodes lividus (Acari) in Sand Martin Colonies in Fennoscandia. Oikos 28: Van Duijvendijk G, Coipan EC, Wagemakers A, Fonville M, Ersöz J, Oei A, Foldvari G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: 97. Van Oosten RA, Heylen DJA, Elst J, Philtjens S and Matthysen E (2016) An experimental test to compare potential and realised specificity in ticks with different ecologies. Evolut Ecol 30: Waldenström J, Lundkvist A, Falk KI, Garpmo U, Bergstrom S, Lindegren G, Sjostedt A, Mejlon H, Fransson T, Haemig PD and Olsen B (2007) Migrating birds and tickborne encephalitis virus. Emerg Infect Dis 13: Wikel SK (1996) Host immunology of host-ectoparasitic arthropod relationships. CABI, Wallingford, UK, 331 pp. Woolhouse MEJ, Dye C, Etard JF, Smith T, Charlwood JD, Garnett GP, Hagan P, Hii JLK, Ndhlovu PD, Quinnell RJ, Watts CH, Chandiwana SK and Anderson RM (1997) Heterogeneities in the transmission of infectious agents: implications for the design of control programs. Proc Natl Acad Sci U S A 94: Ecology and prevention of Lyme borreliosis 101

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104 8. Neglected hosts: the role of lacertid lizards and medium-sized mammals in the eco-epidemiology of Lyme borreliosis Sándor Szekeres 1*, Viktória Majláthová 2, Igor Majláth 3 and Gábor Földvári 1 1 Department of Parasitology and Zoology, University of Veterinary Medicine, 2 István str Budapest, Hungary; 2 Institute of Parasitology SAS, Hlinkova 3, Košice, Slovakia; 3 University of P.J. Šafárik, Faculty of Science, Institute of Biology and Ecology, Šrobárova 2, Košice, Slovakia; sanyi.szekeres@gmail.com Abstract Borrelia burgdorferi sensu lato (s.l.) is the most abundant tick-borne pathogen of the northern hemisphere, and the causative agent of Lyme borreliosis (LB). LB occurs all over Europe, where conditions are suitable for tick survival and competent reservoir hosts species are abundant. The geographical distribution of B. burgdorferi s.l. genospecies in Europe can differ even over relatively small areas as well as over time period. Several host species contribute in the epidemiology of LB such as rodents, lizards and wild or domestic herbivores and carnivores. Lacertid lizards and medium-sized mammals such us squirrels, hedgehogs and mustelid species can live in close proximity to human dwellings and can have important role in the urban and natural cycle of the B. burgdorferi sensu lato spirochaetes. These vertebrate species can harbour Ixodes ricinus or Ixodes hexagonus the main vector species of B. burgdorferi and some of them could live also in highly urbanised habitats. We also present the collected data about presence of Lyme spirochaetes in tissue and ticks removed from these hosts. In this chapter we would like to focus on the role of these often neglected host species through available data about geographical distribution, ecology and epidemiological studies, and highlight the public health relevance of these hosts in rural and urban environment. Keywords: hedgehog, host species, lacertid lizard, Lyme borreliosis, mustelids, squirrel Introduction People living in urban areas love to be in green for leisure activities or just to enjoy the calmness of nature, therefore, cities and houses are designed with some kind of green areas, like alleys, smaller or bigger city parks and nicely cared front or back gardens. These green areas could serve as suitable habitat for some urban animal species. For example in Budapest, the capital and the biggest city of Hungary, forty-eight different mammal species from bats (Chiroptera) to wild boars (Sus scrofa Linnaeus) have been recorded, since 1990 (Tóth-Ronkay et al. 2015). Some of these urbanised mammal species, such as hedgehogs (Erinaceus spp.) and squirrels (Sciurus spp.), can even reach higher densities in urban/suburban habitats than usually in rural environments (Reeve 1994, Tóth-Ronkay et al. 2015). Borrelia burgdorferi sensu lato (s.l.) is the most abundant tick-borne pathogen of the northern hemisphere, and the causative agent of Lyme borreliosis (LB). LB occurs all over Europe, where conditions are favourable for the tick survival and competent reservoir hosts are abundant. The geographical distribution of B. burgdorferi s.l. genospecies in Europe is heterogeneous even over relatively small areas as well as over the time period (Derdákova and Lenčáková 2005). Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _8, Wageningen Academic Publishers 2016

105 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári The main vectors of these bacteria are the larvae, nymphs and adults of Ixodes ricinus Linnaeus, an exophilic, three-host tick species. Two additional species, Ixodes hexagonus Leach and Ixodes uriae White are also vectors and can maintain B. burgdorferi s.l., without the involvement I. ricinus (Gern and Humair 2002). In addition, Ixodes acuminatus Neumann also contributes to the B. burgdorferi s.l., transmission cycles in nests of the host, the so-called endophilic pathogen cycle (Szekeres et al. 2015). The main blood meal source for the non-adult tick stages are rodents like mice (Muridae), voles (Arvicolinae) and dormice (Gliridae), lizards and birds. Adult ticks usually feed on larger mammals like dogs (Canis lupus familiaris Linnaeus), red foxes (Vulpes vulpes Linnaeus), wild and domestic herbivores and occasionally also on humans. Nymphs and adults pose the highest risk for humans to become infected, but in a recent paper it was shown that also the larval stage seem to play a role (Van Duijvendijk et al. 2016). In urban habitats, the diversity of host species is not as high as in rural habitats (e.g. forest), but in contrast, the few species present are abundant and they serve as hosts for a stable and large tick population increasing the risk of acquiring tick-borne pathogens. Reservoir hosts are proven natural hosts of vector ticks, and ticks may become infected while feeding on these animals (Kahl et al. 2002). Distinct genospecies of B. burgdorferi s.l. are associated with different reservoir hosts (Hanincová et al. 2003a, 2003b, Humair and Gern 1998, 2000, Humair et al. 1995, 1998, 1999, Kurtenbach et al. 1998a, 1998b). According to individual groups of reservoir hosts, specific maintenance cycles are distinguished. In this section, we would like to introduce additional important but often neglected hosts: lizards and medium-sized mammals. Lacertid lizards Lacertidae (order Squamata, suborder Sauria) comprises about 40 genera, and over 180 species of lizards (Ananjeva et al. 2006). The family Lacertidae include small to medium sized and rarely large, agile lizards, with well-developed legs and long tails capable of autotomy. The head plates are fused with the skull bones. The body is covered with granular smooth scales. There is a lack of tendency of losing limbs and thus forming of snake forms (Baruš et al. 1992). Lacertid species occur in Europe, Asia and Africa with the exception of Madagascar. In Europe, the lizards are distributed across the entire continent, on the north, the distribution of sand lizard (Lacerta agilis Linnaeus) reaches the Arctic Circle and the distribution of viviparous lizard exceeds the Arctic Circle (Baruš et al. 1992, Moravec et al. 2015). They occur in a wide range of biotopes of the temperate and tropic zones. These terrestrial animals inhabit rocky, sandy, grassy or scrub habitats. Strictly arboreal species are rare, although some species, for example European green lizard (Lacerta viridis Laurenti) and Balkan green lizard (Lacerta trilineata Bedriaga), may prefer to use trees and shrubs as shelters or hunting places during high temperatures in the season (Baruš et al. 1992, Majláth 2001, Moravec et al. 2015). Lacertids are diurnal animals with monophasic or biphasic activity in hot summer days (Majláth, 2001). They are carnivorous, feed small invertebrates mainly on spiders but also molluscs and annelids, small or juvenile mammals, birds, lizards. Cannibalism is also occurring (Baruš et al. 1992, Majláth 2001, Moravec et al. 2015). Almost all lacertid lizards are oviparous with exception of viviparous lizard (Zootoca vivipara Lichtenstein) in Europe. During the mating season the males of many species are highly territorial with aggressive displays which sometimes leads to fighting. 104 Ecology and prevention of Lyme borreliosis

106 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Males of several species show head coloration that is changing over the breeding season, e.g. blue colour on the throat of European green lizard (Majláth 2001, Moravec et al. 2015). The most common genus in Europe is Lacerta. The 32 species of this genus have tiny dorsal scales, increasing in size on the flanks, broad, smooth, ventral plates, a well-defined scaly collar, and in males of most species the hemipenis has small, sulcal lips. Lizards as hosts for ticks and spirochaetes Besides mammals and birds, lizards also play an important role as hosts for ticks. The whole family Lacertidae host ticks in localities where the habitat is suitable for survival of the ticks. The most common tick in central Europe I. ricinus was found to feed on European green lizard, sand lizard (Rosický 1953), wall lizard and viviparous lizard (Lác et al. 1971). Besides I. ricinus, Haemaphysalis punctata Canestrini & Fanzago, Haemaphysalis inermis Birula, Dermacentor marginatus Sulzer (Nosek 1971, 1972), Haemaphysalis concinna Koch, Dermacentor reticulatus Fabricius (Lác et al. 1971) were found to parasitise on species of the family Lacertidae in Slovakia. Other species of the genera Ixodes, Haemaphysalis, Hyalomma, Amblyomma, and Aponomma were found on lacertids in the world (Chilton et al. 1992, Whitworth et al. 2003). Lizards usually harbour immature ticks, nymphs and larvae (Grulich et al.1957). The seasonal changes in tick infestation depend on activity of ticks as well as that of lizards. The tick infestation peaked in April and May in California (Tälleklint-Eisen and Eisen 1999, Eisen et al. 2001) and in July in Italy (Scali et al. 2001). The infestation is higher in males (Tälleklint-Eisen and Eisen 1999, Eisen et al. 2001, Scali et al. 2001). The front site of lizard s body is the most preferred for tick attachment (Figure 1 and 2). Ticks were found around the forelimbs and under the collar scales (Grulich et al. 1957, Dudek et al. 2016), in areas with smallest scales with relatively wide gaps between them (Dudek et al. 2016). The prevalence of infestation with I. ricinus ranged from Figure 1. Ixodes ricinus ticks on the flank of a Lacerta agilis (photo by Igor Majláth). Ecology and prevention of Lyme borreliosis 105

107 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári Figure 2. Ixodes ricinus larvae around the eye and ear area of a Lacerta viridis (photo by Igor Majláth) to 95.6% (Dudek et al. 2016, Grulich et al.1957, Lác et al. 1971, Majláthová et al. 2006, 2008). Differences in tick infestation were found between lizard species what can be influenced by the behavioural differences and type of preferred habitat of lizards (Lác et al. 1971). The tick load is bigger in forest habitats and on its border with pastures. The infection with B. burgdorferi s.l. was found to have negative impact on tick burden (Eisen et al.2004). The age, gender and size of the lizard also affect the infestation (Scali et al. 2001, Dudek et al. 2016). The presence of ticks on physiological state, growth and reactions of immune system was found to have no influence (Uller et al. 2003). Recent studies suggest associations of Borrelia lusitaniae with lizards. This Borrelia genospecies was isolated from patients in Portugal, thus has also public health relevance (Collares-Pereira et al. 2004, De Carvalho et al. 2008). The reservoir competence of several lizard species was proven recently. Conversely, some lizard species such as southern alligator lizard (Elgaria multicarinata Blainville) and western fence lizard (Sceloporus occidentalis Baird and Girard) have proven borreliacidal activity (Lane and Loye 1989, Wright et al. 1998), therefore the role of different lizards in the circulation of B. burgdorferi s.l. has been underestimated. Borrelia lusitaniae was detected in skin biopsy specimens from tails, toe tips, collar scales and blood samples and in larvae and nymphs of I. ricinus ticks feeding on different lacertid species in Europe (Table 1). B. lusitaniae was isolated from I. ricinus ticks for the first time in Portugal (Núncio et al. 1993) and named by Le Fleche et al. (1997), the type strain PotiB2 was isolated from I. ricinus from Portugal, Belorussia and Czech Republic (Le Fleche et al. 1997). In the Mediterranean area, in Portugal (De Michelis et al. 2000, Lopes de Carvalho et al. 2008, Norte et al. 2013), Tunisia (Younsi et al. 2001, 2005, Zhioua et al. 1999) and Morocco (Gern and Humair 2002, Sarih et al. 2003), B. lusitaniae is the dominant genospecies in I. ricinus, whereas in other geographical areas it has been only sporadically reported with low prevalence in ticks. Its presence in ticks has been shown in the 106 Ecology and prevention of Lyme borreliosis

108 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Table 1. Borrelia burgdorferi s.l. in lacertid lizards in Europe. Source Pathogen Prevalence (positive/tested) Country Reference European green lizard (Lacerta viridis) tissue collar scale B. lusitaniae 18.6% (19/102) Slovakia Majláthová et al. (2006) B. lusitaniae 8% (7/92) Hungary Földvári et al. (2009) toe clip B. lusitaniae 2% (1/47) Hungary Földvári et al. (2009) B. lusitaniae 47.19% (42/89) Slovakia Václav et al. (2011) removed tick I. ricinus B. burgdorferi s.l. 16.6% (77/464) Slovakia Majláthová et al. (2006) B. lusitaniae 12.9% (60/464) Slovakia Majláthová et al. (2006) B. lusitaniae 8% (32/397) Hungary Földvári et al. (2009) B. lusitaniae 10.6% (85/799) Slovakia Václav et al. (2011) B. afzelii 1.3% (6/464) Slovakia Majláthová et al. (2006) B. afzelii 0.02% (1/397) Hungary Földvári et al. (2009) B. garinii 0.4% (2/464) Slovakia Majláthová et al. (2006) B. burgdorferi s.s. 0.6% (3/464) Slovakia Majláthová et al. (2006) B. burgdorferi s.s. 0.02% (2/397) Hungary Földvári et al. (2009) B. valaisiana 0.5% (1/464) Slovakia Majláthová et al. (2006) B. lusitaniae + B. burgdorferi s.s. 1% (5/464) Slovakia Földvári et al. (2009) Sand lizard (Lacerta agilis) tissue collar scale B. lusitaniae 45.5% (5/11) Slovakia Majláthová et al. (2008) B. lusitaniae 1.2% (2/171) Poland Ekner et al. (2011) B. lusitaniae 57.1% (16/28) Romania Majláthová et al. (2008) B. lusitaniae 10% (1/10) Hungary Földvári et al. (2009) removed tick I. ricinus B. lusitaniae 19.7% (30/152) Slovakia Majláthová et al. (2008) B. lusitaniae 3.7% (7/187) Poland Majláthová et al. (2008) B. lusitaniae 13.4% (13/97) Romania Majláthová et al. (2008) B. lusitaniae 26.4% (19/72) Germany Richter and Matuschka (2006) B. lusitaniae 2.8% (8/290) Poland Ekner et al. (2011) B. afzelii 1.3% (2/152) Slovakia Majláthová et al. (2008) B. afzelii 0.9% (2/222) the Netherlands Tijsse-Klasen et al. (2010) B. garinii 0.5% (1/187) Poland Majláthová et al. (2008) B. valaisiana 1.6% (3/187) Poland Majláthová et al. (2008) B. burgdorferi s.s. 0.7% (2/290) Poland Ekner et al. (2011) B. burgdorferi s.s. 0.45% (1/222) the Netherlands Tijsse-Klasen et al. (2010) Ecology and prevention of Lyme borreliosis 107

109 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári Table 1. Continued. Source Pathogen Prevalence (positive/tested) Country Reference Iberian emerald lizard (Lacerta schreiberi) removed tick I. ricinus B. lusitaniae 41.2% (7/17) Portugal Norte et al. (2015) Iberian wall lizard (Podarcis hispanica) tissue B. lusitaniae 3.7% (1/27) Portugal Norte et al. (2015) removed tick I. ricinus B. lusitaniae 1.8% (2/111) Portugal Norte et al. (2015) Large psammodromus (Psammodromus algirus) tissue B. lusitaniae 37.8% (14/37) Tunisia Dsouli et al. (2006) removed tick I. ricinus B. lusitaniae 17% (37/218) Portugal Norte et al. (2015) xenodiagnosis I. ricinus B. lusitaniae 17% (40/229) Tunisia Dsouli et al. (2006) Wall lizard (Podarcis muralis) tissue B. lusitaniae 18.8% (6/32) Italy Amore et al. (2007) removed tick I. ricinus B. lusitaniae 32% (55/172) Germany Richter and Matuschka (2006) B. valaisiana 0.6% (1/172) Germany Richter and Matuschka (2006) B. valaisiana 0.6% (1/172) Germany Richter and Matuschka (2006) B. lusitaniae 23% (104/452) Italy Amore et al. (2007) Madeira wall lizard (Teira dugesii) tissue B. lusitaniae 4.6% (7/151) Madeira Island De Sousa et al. (2012) removed tick I. ricinus B. burgdorferi s.l. 11.8% (25/211) Madeira Island De Sousa et al. (2012) Balkan wall lizard (Podarcis taurica) tissue B. lusitaniae 9.4% (3/32) Hungary Földvári et al. (2009) removed tick I. ricinus B. lusitaniae 2% (1/55) Hungary Földvári et al. (2009) B. burgdorferi s.s. 2% (1/55) Hungary Földvári et al. (2009) 108 Ecology and prevention of Lyme borreliosis

110 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Czech Republic, Moldavia, Ukraine (Postic et al. 1997), Slovakia (Gern et al. 1999, Hanincová et al. 2003a, Majlathová et al. 2006, 2008, Taragelova et al. 2016), Poland (Mizak and Krol., 2000, Wodecka and Skotarczak, 2005), Spain (Barral et al. 2002, Escudero et al. 2000), Switzerland (Jouda et al. 2003, Poupon et al. 2006), Hungary (Földvári et al. 2009), Italy (Amore et al. 2007, Bertolotti et al. 2006), Germany (Richter and Matuschka 2006, Richter et al. 2013, Schwarz et al. 2012), Romania (Majlathová et al. 2008) and Turkey (Guner et al. 2003) (Table 1). B. lusitaniae was the only Borrelia genospecies reported in tissue samples of lacertid lizards. In ticks removed from lizards, single infections of B. burgdorferi s.l., B. burgdorferi s.s., Borrelia afzelii, Borrelia garinii and Borrelia valaisiana and co-infection of B. lusitaniae and B. burgdorferi s.s. were found (Table 1). De Michelis et al. (2000) hypothesised that B. lusitaniae has a narrow ecological niche that involves host genospecies restricted to the Mediterranean Basin, that are highly reservoir competent for this genospecies. However, the recent report on the presence of this genospecies in countries located outside of the Mediterranean Basin, demonstrates that competent reservoirs are present also outside of this well-defined focus. Nevertheless, the fact that B. lusitaniae is by far the dominant species in I. ricinus from southern Europe indicates that the genospecies diversity of B. burgdorferi s.l. decreases towards the southern margin of its European distribution. While B. lusitaniae appears clearly to dominate in southern Europe, data from northern Europe show a dominance of B. afzelii (Jenkins et al. 2001, Junttila et al. 1999, Schouls et al. 1999). The role of lizards in the epidemiology of B. burgdorferi s.l. has been unknown until the last decade. Today it is evident that the contribution of lizard species can be very different, e.g. the southern alligator lizard and the western fence lizard has borreliacidal activity, however other species such as lacertid lizards can and do maintain B. lusitaniae spirochaetes. With controlled laboratory and field experiments, the causes of these differences will hopefully be clarified. Squirrels European red squirrels (Sciurus vulgaris Linnaeus) are common rodent species living in natural forests and city parks in Eurasia. This squirrel species, like most tree squirrels, has sharp, curved claws that help to climb on broad tree trunks and thin branches. The long tail helps the squirrel to balance, when jumping with its strong hind legs from tree to tree and running along branches. The coat of the red squirrels varies from red to greyish or blackish red, the ventral part is always white. These tree squirrels are omnivorous, solitary animals being active during daylight. The size of the territory of the species depends on the nesting and food source trees and also on the sex of the squirrel. The red squirrel is found in both coniferous forest and temperate broadleaf woodlands. Squirrels build dreys out of twigs in a branch-fork, forming a domed structure or use a tree hole or a forsaken woodpecker hole as shelter lined with moss, grass and leaves. In western and southern Europe, they are found in broad-leaved woods where the mixture of tree and shrub species provides a better year-round food source. The main food sources are hazelnuts (Coryllus avellana Linnaeus), walnuts (Juglans spp.), beechnuts (Fagus sylvatica Linnaeus), acorns (Quercus spp.) and younger cones and nuts of pine trees (Pinaceae); the seeds of these plants are rich in vitamins and nutrients. Squirrels supplement their diet with young shoots, leaf and flower buds, tree flowers, bark-growing fungi and insects (Grönwall and Pehrson 1984, Gurnell 1987, Moller 1983, Wauters and Dhondt, 1987, Wauters et al. 1992). Rarely, red squirrels may eat bird eggs or nestlings (Fontaine and Martin 2006). For the harsh winter times these arboreal rodents store excess food in tree holes, underground holes or other proper storage places. Ecology and prevention of Lyme borreliosis 109

111 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári The eastern grey squirrel (S. carolinensis Gmelin) has predominantly grey fur, but it can have a brownish colour and a usual white underside. This invasive species competes with the native red squirrel for resources, such as food and habitat. It was introduced from North America to several locations like South Africa, Australia and also Europe. In Europe, the Eastern grey squirrel was introduced several occasions from the late 19 th century to the British Isles and Italy. In the last century, they have colonised Great Britain except the northern parts of Scotland, and also big territories in Ireland and Italy. In Great Britain, they are considered a pest because of bark stripping and ring barking of trees. Natural predators of the red squirrel are wild cats (Felis silvestris Schreber), pine and stone martens (Martes martes Linnaeus and Martes foina Linnaeus) (Tóth Apáthy 1998), red foxes, stray dogs and cats (Felis catus Linnaeus) and also bird of prey like northern goshawks (Accipiter gentilis Linnaeus) and common buzzards (Buteo buteo Linnaeus) (Bősze 2007). Squirrels as hosts for ticks and spirochaetes Squirrels forage most of the day after food on the ground when they can collect ticks from the leaf litter (Figure 3). The first report about Borrelia infection related with European red squirrel was in 1998 by Humair and Gern (1998) from Switzerland. They found B. burgdorferi s.s., B. afzelii, B. garinii, Borrelia sp. single infection and B. burgdorferi s.s. and B. afzelii co-infection in I. ricinus from a road killed carcass (Humair and Gern 1998). In red squirrel tissue samples all the aforementioned species were present and even single infection of B. valaisiana (Morán Cadenas et al. 2007), coinfection of B. burgdorferi s.s. and B. garinii and triple infection of B. burgdorferi s.s., B. afzelii and B. garinii (Pisanu et al. 2014) (Table 2). Figure 3. Urban red squirrel in Budapest (Pet Zoo, Margaret Island) (photo by Sándor Szekeres). 110 Ecology and prevention of Lyme borreliosis

112 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Table 2. Borrelia burgdorferi s.l. in squirrels in Europe. Source Pathogen Prevalence (positive/tested) Country Reference Eastern grey squirrel (Sciurus carolinensis) tissue B. burgdorferi s.l % (15/106) United Kingdom Craine et al. (1997) removed tick I. ricinus B. burgdorferi s.l. 32% (8/25) 1 United Kingdom Craine et al. (1997) 16.14% (31/192) 1 United Kingdom Craine et al. (1997) B. afzelii 3 nymph in a pool 2 United Kingdom Craine et al. (1997) European red squirrel (Sciurus vulgaris) tissue B. burgdorferi s.s % (2/6) Switzerland Humair and Gern (1998) 8.1% (11/135) 3 Switzerland Morán Cadenas et al. (2007) 11% (30/273) France Pisanu et al. (2014) B. afzelii 5.5% (15/273) France Pisanu et al. (2014) 6.7% (9/135) 3 Switzerland Morán Cadenas et al. (2007) B. garinii 16.66% (1/6) 4 Switzerland Humair and Gern (1998) 0.74% (1/135) 3 Switzerland Morán Cadenas et al. (2007) 1.8% (5/273) France Pisanu et al. (2014) B. valaisiana 0.74% (1/135) 3 Switzerland Morán Cadenas et al. (2007) B. burgdorferi s.l. 1.48% (2/135) 3 Switzerland Morán Cadenas et al. (2007) B. burgdorferi s.s. + B. afzelii 33.33% (2/6) Switzerland Humair and Gern (1998) 4.4% (12/273) France Pisanu et al. (2014) B. burgdorferi s.s. + B. garinii 0.74% (2/273) France Pisanu et al. (2014) B. burgdorferi s.s. + B. garinii + B. afzelii 0.37% (1/273) France Pisanu et al. (2014) removed tick I. ricinus B. burgdorferi s.s. 13.6% (31/227) Switzerland Humair and Gern (1998) B. afzelii 19% (43/227) Switzerland Humair and Gern (1998) B. garinii 1.76% (4/227) Switzerland Humair and Gern (1998) B. burgdorferi s.s. + B. afzelii 4.4% (10/227) Switzerland Humair and Gern (1998) Borrelia sp. 2.2% (2/227) Switzerland Humair and Gern (1998) 1 Xenodiagnostic ticks analysed with PCR (32%) and with IFAT (16.14%). 2 Xenodiagnistic nymph pool (3 individual) from squirrel (code: C). 3 Based on blood meal analysis of questing ticks. 4 Not confirmed: the mentioned data is in an unpublished report. Ecology and prevention of Lyme borreliosis 111

113 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári In tissue samples of grey squirrel, B. burgdorferi s.l. was found. In a xenodiagnostic experiment, Eastern grey squirrel was proved to serve as a reservoir for LB spirochaetes. In a pool from three nymphs from an experimentally used squirrel (Craine et al. 1997) B. afzelii was found (Table 2). Hedgehogs Hedgehogs are common insectivores in Europe. They feed on annelids, insects (larvae, pupae and imagoes as well), snails and slugs, small vertebrates (amphibians, lizards and occasionally young rodents), chicks and eggs of birds (Jackson and Green 2000) and even some berries and fruits (Jones and Norbury 2010, Yalden, 1976). The hedgehog s back contains two large muscles that control the position of the quills. When a hedgehog feels danger they can roll into a ball, hereby the quills on the back protect the tucked head, feet and belly, which are not protected with spines. Hedgehogs are nocturnal animals. In daytime, they are sleeping in a hiding under litter, dead plant material or against base of tree or bush (Reeve and Morris 1985). The foraging behaviour starts after sunset and during this time they can be easily found when moving in the litter. Hedgehogs use a combination of grunts, snuffles and squeals to communicate. Their spine-armour is not adequate defence against some predators that are able to force access to the hedgehog s vulnerable underbelly. Hedgehogs are reported in pellets and gut contents of Eurasian eagle-owl (Bubo bubo Linnaeus) (Penteriani et al. 2002), stoat (Mustela erminea Linnaeus) (King and Moody 1982), European polecat (M. putorius Linnaeus) (Smith et al. 1995), red fox (Doncaster et al. 1990) and also European badger (Meles meles Linnaeus) are predators of hedgehogs (Doncaster and Krebs 1993, Young et al. 2006). Badgers are not just predators but also food competitors of hedgehogs, because their main food source is arthropods and earthworms as well (Ward 1997). Surprisingly, hedgehog remains were found in herring gull pellets (Larus argentatus Pontoppidan) (Camphuysen et al. 2010), most likely from a consumed carcass. In urban habitat, motorised vehicles and dogs pose a large risk to hedgehogs. The majority of the run overs happen in the mating period when the males search intensively for females. Some dogs (including strays) are known to prey upon them when the opportunity arises. There are some interesting reports about hedgehogs using toad (Bufo spp.) venom for increasing their defence (Brodie 1977). These spiny mammals sometimes grab the toad and rub against their spines or apply the toad secretion-saliva mixture to the spines with their tongue. With this chemical support they can increase the impact of spines, the burn or irritation could even last for two weeks. Three hedgehog species live in Europe. The European hedgehog (Erinaceus europaeus Linnaeus) occurs in Western Europe, Scandinavia and the Baltic region. The Northern white-breasted hedgehog (Erinaceus roumanicus Barrett-Hamilton) inhabits from the Eastern part of Europe to the European part of Russia and the Ponto-Mediterranean region. The third species, the Southern white-breasted hedgehog (Erinaceus concolor Martin), is found in Asia Minor and Eastern-Mediterranean. Among the European and Northern white-breasted hedgehogs, there are hybridisation zones; one in north-south direction from Poland to Italy and another in westeast direction in the Baltic-Russian border of the two areas. For the Northern and the Southern white-breasted hedgehog, the Caucasus and the two straits of the Sea of Marmara (Bolfíková and Hulva 2012) form natural barriers. After the last glacial period the ancestors of these hedgehogs recolonised the thawing Europe from Mediterranean refuges (Bolfíková and Hulva 2012) (Figure 4). 112 Ecology and prevention of Lyme borreliosis

114 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Figure 4. Distribution of the three hedgehog species (Erinaceus europaeus (blue), E. roumanicus (red), E. concolor (green), hybridisation zones (purple), and main colonisation routes from the refues after the last ice age in Europe based on Bolfíková and Hulva (2012). Hedgehogs as hosts for ectoparasites and spirochaetes Hedgehogs are appropriate and attractive hosts for several ecto- and endoparasites (Figure 5). First of all, they feed on the typical intermediate host species (e.g. slugs, snails, earthworms, beetles) of different endoparasitic helminths such as roundworms, tapeworms and acanthocephalans. Second, the undergrowth and dry leaf litter dwelling lifestyle is ideal for collecting and maintaining ectoparasites such as ticks and fleas, which are often vectors of several viruses, bacteria and protozoa (some of them have veterinary and public health importance). I. hexagonus, the hedgehog tick, I. ricinus (Földvári et al. 2011, Pfäffle et al. 2011) and Archaeopsylla erinacei Figure 5. All removed ectoparasites (fleas and ticks) from a road-hit Northern white-breasted hedgehog (photo by Sándor Szekeres). Ecology and prevention of Lyme borreliosis 113

115 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári Bouché, the hedgehog flea (Földvári et al. 2011, Gilles et al. 2008, Hornok et al. 2014, Marié et al. 2011, Visser et al. 2001) are common ectoparasites of hedgehogs in Europe. Ixodes acuminatus Neumann and Hyalomma marginatum Koch nymphs were also reported from Northern whitebreasted hedgehog from a city park of Budapest (Földvári et al. 2011). High tick burden can exert negative effect on hedgehog s health. Tick burden can cause tick-induced regenerative anaemia in European hedgehogs by blood loss (Pfäffle et al. 2009). The energy, which is invested into immune responses and regeneration combined with suboptimal environmental factors could lead to secondary infections. Moreover, the spiny armour is ideal for maintaining ectoparasites, because it limits antiparasitic behaviour of hedgehogs (Figure 6 and 7). The summer and winter shelter (hibernaculum) of the hedgehogs play important roles in the life cycle of the nidicolous hedgehog ectoparasites. Eggs and larvae of the hedgehog flea (A. erinacei) develop in the bedding of the nest. Moreover, the non-adult stages of some tick species also live in the nest (e.g. Dermacentor spp.) and there are some species of which all the developmental Figure 6. Engorged Ixodes ricinus female on the ventral part of a road-hit Northern white-breasted hedgehog (photo by Sándor Szekeres). Figure 7. Tick collection from Northern white-breasted hedgehog on Margaret Island, Budapest (photo by Mihály Földvári). 114 Ecology and prevention of Lyme borreliosis

116 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle stages live in the nest (e.g. I. hexagonus) (Morris 1973). The occurrence of I. hexagonus in the urban environment is due to the presence of suitable hosts such as hedgehogs, cats and dogs in gardens and public parks (Gern et al. 1991, 1997). European hedgehogs are reservoir hosts for B. burgdorferi s.l., and take part in the maintenance of several Borrelia species in an enzootic cycle (Gern et al. 1997, Skuballa et al. 2007). In tissue samples of European hedgehogs from Germany, Switzerland and Czech Republic B. afzelii, Borrelia spielmanii, Borrelia bavariensis, B. garinii and B. burgdorferi s.s. have been found (Table 3). In a recent paper B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi s.s. were detected in both tick species commonly found on European hedgehog (Krawczyk et al. 2015). The eastern relative of the aforementioned hedgehog species, the Northern white-breasted hedgehog, had been studied only in the previous decade. Tissue samples were collected from naturally died specimens from an Austrian rehabilitation centre not far from the Hungarian border and B. afzelii and B. bavariensis infection was detected (Skuballa et al. 2012). In addition, in I. ricinus ticks removed from anaesthetised Northern white-breasted hedgehogs, B. afzelii was found. Interestingly, Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis were also found in tissue samples of urban habitat living white-breasted hedgehogs (Földvári et al. 2014). European hedgehogs might also serve as reservoir hosts for the tick-borne pathogen A. phagocytophilum (Silaghi et al. 2012), which causes granulocytic anaplasmosis in humans (Dumler et al. 2005). Unfortunately, we do not have any data about Borrelia infection of the third European hedgehog species. Nevertheless, the area of I. ricinus and E. concolor is overlapping in Turkey, suggesting that this hedgehog species could possibly serve as a suitable host for Borrelia spirochaetes. Mustelids In addition to the easily noticeable urban mammals such as hedgehogs and squirrels, mustelid species form another group of urbanised medium-sized mammals with a more hidden, nocturnal nature (Crooks 2002). Mesocarnivores, like mustelids are generally rather successful in highly fragmented and urbanised landscapes (Crooks 2002). In general, mustelids are carnivores, but some species (e.g. stone martens and European badgers) have considerable amount of fruits in their diet. Stone martens, M. foina is the most abundant mustelid in urban areas, use lofts and abandoned garrets in downtowns, and outbuildings and sheds in suburban regions as hiding places (Figure 8). In central Europe, it is generally regarded as a synanthropic species (Tóth-Ronkay et al. 2015). The food sources of this species are very broad from arthropods, fishes, reptiles and amphibians, small mammals, birds and eggs to fruits and seeds (Lanszki 2003, Lanszki et al. 1999, Tóth-Ronkay et al. 2015). In urban environment, they supplement their diet with garbage and leftover dog and cat food (Tóth et al. 2011). In addition to stone martens, three other mustelids are sporadically reported in urban habitats. The smallest of these species is the least weasel (Mustela nivalis Linnaeus), the medium is the stoat and the biggest is the European badger. In Budapest, there are few sightings of the least weasel in gardens and bushy forest edges in the suburban parts of the city (Tóth-Ronkay et al. 2015). Least weasel has been found in three out of twelve trapping areas with various habitat characteristics (e. g. scrubs, orchards or long grass areas) in built-up areas of Oxford (Dickman 1986). European badgers are also commonly reported in the rural areas near to the cities, where Ecology and prevention of Lyme borreliosis 115

117 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári Table 3. Borrelia burgdorferi s.l. in hedgehogs in Europe. Source Pathogen Prevalence (positive/tested) Country Reference European hedgehog (Erinaceus europeaus) tissue B. spielmanii 1.4% (3/211) Germany Skuballa et al. (2012) B. afzelii 5.68% (12/211) Germany Skuballa et al. (2012) 25% (4/16) Czech Republic Skuballa et al. (2012) 14.3% (1/7) Switzerland Gern et al. (1997) B. bavariensis 0.94% (2/211) Germany Skuballa et al. (2012) B. garinii 42.9% (3/7) Switzerland Gern et al. (1997) B. afzelii + B. bavariensis 2.37% (5/211) Germany Skuballa et al. (2012) 12.5% (2/16) Czech Republic Skuballa et al. (2012) B. afzelii + B. spielmanii 0.94% (2/211) Germany Skuballa et al. (2012) B. bavariensis + B. spielmanii 0.94% (2/211) Germany Skuballa et al. (2012) B. burgdorferi s.s. + B. garinii 14.3% (1/7) Switzerland Gern et al. (1997) B. afzelii + B. bavariensis + B. spielmanii 0.47% (1/211) Germany Skuballa et al. (2012) Borrelia sp. 0.94% (2/211) Germany Skuballa et al. (2012) removed tick I. hexagonus B. burgdorferi s.l. 14% (60/435) the Netherlands Krawczyk et al. (2015) B. afzelii 76% (37/49) the Netherlands Krawczyk et al. (2015) B. bavariensis 6% (3/49) the Netherlands Krawczyk et al. (2015) B. spielmanii 14% (7/49) the Netherlands Krawczyk et al. (2015) B. burgdorferi s.s. 4% (2/49) the Netherlands Krawczyk et al. (2015) I. ricinus B. burgdorferi s.l. 28% (7/25) the Netherlands Krawczyk et al. (2015) serum B. burgdorferi s.l. 1 France Doby et al. (1991) Northern white-breasted hedgehog (Erinaceus roumanicus) tissue B. afzelii 25% (1/4) Austria Skuballa et al. (2012) B. bavariensis 25% (1/4) Austria Skuballa et al. (2012) removed tick I. ricinus B. afzelii 0.4% (4/959) Romania Dumitrache et al. (2013) 1 Serological evidence from one individual: hedgehog titre 1/ Ecology and prevention of Lyme borreliosis

118 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle Figure 8. Urban stone marten (photo by Mária Tóth-Ronkay). the human disturbance such as noise pollution, vehicles and dogs are not frequently presented (Tóth-Ronkay et al. 2015). Mustelids as hosts for ticks and spirochaetes Our knowledge about Borrelia infection in mustelid species is scarce, thus we tried to collect all data about Borrelia infection in these animals (Table 4). The main tick species associated with mustelid species is I. hexagonus (Jaenson et al. 2012, Lorusso et al. 2011), but there are reports about I. ricinus ticks as well (Lorusso et al. 2011). There are no records about Borrelia infection in stone martens. In an article about pathogens and diseases in mustelid species, Borrelia burgdorferi s.l. infection was mentioned from British stoats (McDonald and Lariviere 2001). There is one serological report of B. burgdorferi s.l. infection in one least weasel (Doby et al. 1991). In European badgers, B. afzelii (Gern and Sell 2009, Morán Cadenas et al. 2007) and B. afzelii and B. valaisiana coinfection was found (Gern and Sell 2009). In other not urbanised mustelid species, like marbled polecat (Vormela peregusna Güldenstädt), European mink (Mustela lutreola Linnaeus) and European polecat, Borrelia infections were reported. Borrelia burgdorferi s.s. was found in marbled polecat and in European mink in Romania (Gherman et al. 2012). In Switzerland, analysis of host blood remnants in field collected ticks showed that the European polecat had been the previous host of ticks that were fond infected with Borrelia burgdorferi s.s (Morán Cadenas et al. 2007). Some mustelids live in close proximity around human dwellings. We conclude that, in urban environment these species can serve as host for B. burgdorferi s.l, especially the highly adaptive and synanthropic stone martens, but the role of these medium-sized mammals in B. burgdorferi s.l. cycle needs further examination. Ecology and prevention of Lyme borreliosis 117

119 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári Table 4. Borrelia burgdorferi s.l. in mustelids in Europe. Source Pathogen Prevalence (positive/tested) Country Reference European polecat (Mustela putorius) tissue B. burgdorferi s.s. 1.48% (2/135) 2 Switzerland Morán Cadenas et al. (2007) European mink (Mustela lutreola) tissue B. burgdorferi s.s. 66.6% (2/3) Romania Gherman et al. (2012) Marbled polecat (Vormella peregusna) tissue B. burgdorferi s.s. 50% (1/2) Romania Gherman et al. (2012) Stoat (Mustela erminea) tissue B. burgdorferi s.l. 22.2% (10/45) 3 UK McDonald and Lariviere (2001) Least weasel (Mustela nivalis) serum B. burgdorferi s.l. 1 France Doby et al. (1991) European badger (Meles meles) tissue B. afzelii 24% (2/8) Switzerland Gern and Sell (2009) B. afzelii 0.74% (1/135) 2 Switzerland Morán Cadenas et al. (2007) B afzelii + B valaisiana 12.5% (1/8) Switzerland Gern and Sell (2009) 1 Serological evidence from one individual, least weasel titre 1/50. 2 Based on blood meal analysis of questing ticks. 3 Not confirmed: the mentioned data is in an unpublished report. 118 Ecology and prevention of Lyme borreliosis

120 8. Role of lacertid lizards and medium-sized mammals in Lyme borreliosis cycle In contrast to I. ricinus, I. hexagonus is an endophilic (or nidicolous) tick species living in the nest of the vertebrate host. Therefore, the host range of I. hexagonus is more restricted than that of I. ricinus and it feeds primarily on carnivores such as foxes and mustelids, and on hedgehogs, but also, less frequently on other species such as rodents, hares and rabbits (Arthur 1953, Toutoungi et al. 1991). I. hexagonus has occasionally been collected from Eurasian magpie (Pica pica Linnaeus), common kestrel (Falco tinnunculus Linnaeus) and Eurasian roe deer (Capreolus capreolus Linnaeus) (Hubbard et al. 1998, Toutoungi et al. 1991). Domestic animals such as cats, dogs, horses, goats and cows have also been found to be infested (Arthur 1968, Bernasconi et al. 1997, Földvári and Farkas 2005, Toutoungi et al. 1991). Although less frequently than I. ricinus, I. hexagonus apparently also bite humans (Arthur 1953, Hubbard et al. 1998, Liebisch et al. 1998), thus its epidemiological role in transmitting LB spirochaetes deserves further investigations. Discussion Urbanisation is a phenomenon, within the population shift from rural areas to the urban. In % of the population lived in urban areas, and it is expected that until 2050 this ratio will increase to 60% (UN 2014). Thus when big cities were designed, it was crucial to have relatively big green recreational areas in the concrete jungle. On a smaller scale, our gardens also have a recreational function. Gardens are the places where people can use their imagination and creativity to make a nice and refreshing environment. On the other hand, this diverse patchy habitat provides a huge quantity of food for animals that can adapt to this urban environment. For example, planted shrubs and trees serve a good food source in city gardens. These decorative plants can also serve as shelter for several species. City people love small songbirds, therefore in wintertime provide extra food (sunflower seeds, nuts) to help the survival of these animals, which could be also beneficial for urbanised squirrels and other rodents. In addition, the leftover food from companion animals is a good opportunity, but the main food source insured by the city is the regenerative and inexhaustible food waste. In the European Union more than 88 million tonnes of food is wasted annually (around 173 kg/person/ year; estimated cost: 143 billion euros), which will likely rise (Stenmarck et al. 2016). Conclusion These host species can live in the immediate vicinity of human dwellings, such as lizards in open grassy habitats, hedgehogs and squrrels in urban parks, gardens and cemeteries with trees and shrubs and martens in lofts of inner districts of cities. They can also carry the vectors of B. burgdorferi s.l. and some of them can also maintain the spirochaetes. Thus, these species can serve as a link between the ticks/tick-borne pathogens and companion animals/humans. Some wild animal species (e.g. red foxes, martens) during the centuries have become not only urbanised but also became synanthropic species, and the number of these species might grow in the future. The cities take away bigger and bigger areas from the natural habitats while cities with growing food waste also serve as an inexhaustible and easily obtainable food source. Thus, more and more known and potential vertebrate reservoir species of B. burgdorferi s.l. might find suitable habitat in urban areas. I. ricinus can also be found in parks and green areas in cities. With proper management of these areas, the suitable questing substrate and habitats for exophilic ticks can be minimised without harming other species or reducing minerals and organic material of the habitats. This Ecology and prevention of Lyme borreliosis 119

121 Sándor Szekeres, Viktória Majláthová, Igor Majláth and Gábor Földvári management practices include reducing the undergrowth of shrubs and bushes, collecting the litter after winter (composting and recycling), and cutting the loan short. To reduce the number of nidicolous (endophilic) ticks like I. hexagonus, the use of artificial nesting boxes for squirrels and hedgehogs (especially in gardens), where the bedding of these nest can be sterilised and changed regularly, can be a possible way to reduce the number of ticks. These animals in urban and suburban areas are possible risk factors for humans to get infected from tick-borne pathogens. Nonetheless, with proper usage of repellents and a thorough selfinspection after a walk in risky areas the hazard of infection could be minimised. Public health relevance Lacertid lizards, squirrels, hedgehogs and mustelid species are important hosts of Ixodes ricinus and can carry Borrelia burgdorferi s.l. in close proximity of human dwellings. Some wild animal species become urbanised, synanthropic species and in the future several Borrelia burgdorferi s.l. reservoir species might find suitable habitats in urban areas. Ixodes ricinus and Ixodes hexagonus can be found in urban areas. The number of these ticks with proper management of the questing places can be reduced. Competing interests The authors declare that they have no competing interests. Acknowledgements We would like to thank Mária Tóth-Ronkay and Mihály Földvári for providing us their original photos. The two reviewers provided great help to improve the manuscript. We are indebted to Marieta Braks for the critical reading and language editing. This work was done under the frame of COST action TD1303 EurNegVec. GF was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and an NKB and Research Faculty grants from the Faculty of Veterinary Science, Szent István University. SSz was supported by the Hungarian Eötvös Scholarship and IM was supported by VEGA 1/0417/14 grant. References Amore G, Tomassone L, Grego E, Ragagli C, Bertolotti L, Nebbia P, Rosati S and Mannelli A (2007) Borrelia lusitaniae in immature Ixodes ricinus (Acari: Ixodidae) feeding on common wall lizards in Tuscany, central Italy. J Med Entomol 44: Ananjeva NB, Nikolai Ol, Roman KG, Ilya DS, Sergei RA and Barabanov AV (2006) Atlas of the reptiles of North Eurasia: taxonomic diversity, distribution, conservation status. Coronet Books Incorporated, Philadelphia, PA, USA. Arthur DR (1953) The host relationships of Ixodes hexagonus leach in Britain. Parasitology 43: Arthur DR (1968) British ticks. Butterworths, London, UK, p Ecology and prevention of Lyme borreliosis

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128 9. Emerging tick-borne pathogens: ticking on Pandora s box Setareh Jahfari * and Hein Sprong National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; seta.jahfari@gmail.com Abstract Ixodes ricinus transmits a whole array of causative agents of human and veterinary diseases. In addition, the list of newly or re-emerging tick-borne pathogens transmitted by this tick species has been mounting and is constantly evolving. In the past decades, human exposure to tick bites has risen in most European countries. The public health burden of the pathogens transmitted by ticks seems to be increasing, mostly due to increased exposure to ticks, but also the changing demographics, changing climate and changing landscapes in most European countries. Another reason is the newly emerging pathogens that have been described over the past two decades. Hence, emergence of tick-borne pathogens leads to the development of unknown public health risks. Despite exposure through tick bites, tick-borne diseases other than Lyme borreliosis are rarely diagnosed. Besides Borrelia burgdorferi senso lato genospecies, the Ixodes ricinus tick can transmit other established tick-borne pathogens, like Borrelia miyamotoi, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, several Babesia species, Louping ill virus and tick-borne encephalitis virus. The focus of this review is tick-borne pathogens other than Lyme borreliosis that are an increasing emerging public health concern in Europe, with an emphasis on the Netherlands. Keywords: Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, Candidatus Neoehrlichia mikurensis, Ixodes ricinus, spotted fever Rickettsia, tick-borne encephalitis virus Introduction Since the discovery of Borrelia burgdorferi as the causative agent of Lyme borreliosis in 1982, tick-borne pathogens have been the focus of extensive attention in scientific research, in the fields of veterinary and human medicine. The list of new or re-emerging tick-borne pathogens transmitted by the Ixodes species is growing and constantly evolving (Rizzoli et al. 2014). These developments may be attributed to various factors, including: (1) due to the progresses in the field of molecular biology, new tick-borne pathogens have been discovered; (2) new findings suggest that certain pathogens cause disease in humans although they were previously thought to be restricted to animal diseases or were not considered as causative agents of disease (Tijsse- Klasen et al. 2014); and (3) the geographical spread of tick vectors, hosts and their pathogens into new areas in Europe (Medlock et al. 2013). Furthermore, there are other factors that have resulted in an increase in the number of humans who are bitten by ticks in Europe, namely: (4) changes in land use have enabled the resurgence of large hosts, contributing to a sharp increase in tick densities (Gilbert et al. 2012, Sprong et al. 2012); (5) habitat encroachment and alterations due to recreational activities and tourism in areas with high tick densities (Barbour and Fish 1993); and (6) demographic changes such as an aging population and a higher number of chronically ill people (Gijsen et al. 2014, Kunze 2011). The increase in the number of reported tick bites demonstrates that the risk of acquiring a tick-borne infection is growing (Hofhuis et al. 2015). Hence, these tickborne diseases constitute a novel public health burden of unknown proportions. Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _9, Wageningen Academic Publishers 2016

129 Setareh Jahfari and Hein Sprong Besides the Borrelia burgdorferi senso lato (s.l.) genospecies which causes Lyme borreliosis, the generalist tick species Ixodes ricinus can transmit other established tick-borne pathogens, namely Borrelia miyamotoi, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, several Babesia species, Louping ill virus, and tick-borne encephalitis virus. Besides these established pathogens, there are other bacteria families, like Bartonella, Coxiella, and Francisella that can be transmitted by I. ricinus. Ticks may play a role in the enzootic cycles of these pathogens; still, other transmission routes have a more prevalent role in the transmission to humans (Duron et al. 2015, Eisen and Gage 2012, Hestvik et al. 2015). Furthermore, there are new potential pathogens currently emerging (Moutailler et al. 2016a, Moutailler et al. 2016b). The human infection incidence of these microbes caused by a tick bite is still a matter of debate; none of these pathogens will be covered here. This review focuses on tick-borne pathogens other than Lyme borreliosis that pose an emerging public health concern in Europe, with an emphasis on the Netherlands. Transmission dynamics of tick-borne pathogens in ticks and vertebrate hosts An understanding of the relative risks of (re-)emergence of a tick-borne pathogen requires insight into the transmission dynamics of that pathogen in ticks and vertebrate hosts. As humans are considered accidental hosts of all tick-borne pathogens, these pathogens require one or more vertebrate host species for maintenance in enzootic cycles in nature (Swanson et al. 2006). A greater understanding of the biology of the vector, the host and the vector-borne pathogens therefore provides the best basis for risk assessment in the field of public health and public health policy-making. An interdisciplinary approach offers benefits because it addresses questions regarding tick-borne pathogens in the context of public health. I. ricinus is a three-host tick, meaning that it must find a new host to take one blood meal during each life stage after hatching into larva, nymph, or adult female. Hence, the feeding behaviour of I. ricinus in each life stage has an impact on the risk of tick-borne infection and co-infection for humans. The transmission dynamics are multi-faceted and different for each pathogen. Overall, at least three conditions must be met for transmission cycles to be sustained. Firstly, a vertebrate host must be present that is susceptible to infection with the pathogen. In addition, the host must experience a sufficient level of infection in the blood or skin tissue to enable the pathogen to be transmitted to other ticks during blood feeding. Secondly, it must be possible to maintain a pathogen in the tick for extended periods, including moulting into the next life stage. After moulting, the tick must be able to transmit the infection to another vertebrate host. Thirdly, sufficient numbers of susceptible vertebrate hosts must be present in an area to maintain both the ticks and the pathogens in enzootic cycles. Transmission cycles among ticks and vertebrate hosts can only be preserved when ticks transfer pathogens between susceptible hosts (horizontal transmission), but cannot be sustained when transmission is directed toward dead-end hosts. These dead-end hosts are incapable of experiencing high levels of the pathogen in blood or tissue (tangential transmission). Depending on the micro-organism, the reservoir or amplification host responds differently to infection with a tick-borne pathogen. This has a direct impact on transmission dynamics. For example, Babesia species are parasites of red blood cells and are often associated with relatively mild- or asymptomatic-chronic infections of the reservoir host. These long-term infected animals therefore offer many opportunities for feeding ticks to become infected. On the other hand, most 128 Ecology and prevention of Lyme borreliosis

130 9. Pandora s box viral and bacterial infections are either potentially fatal or can induce an immune response in the vertebrate host. This limits the period during which the pathogen circulates in high numbers. In situations where the time window for other feeding ticks to become infected is limited, the tick itself becomes the crucial link in maintaining the enzootic cycle in nature. When the perpetual route of survival of the pathogen is the tick, ticks can serve as the reservoir for the pathogens, mostly due to the ability of that pathogen to be transmitted transstadially and transovarially. In that case, the vertebrate host acts as the amplifier of the pathogen. Organisms can be transmitted to the next life stage of the tick in different ways: (1) through transstadial transmission, i.e. by transmission between different stages of tick development, from larva to nymph or from nymph to adult; (2) through transovarial transmission, i.e. by transmission of the pathogen between generations, or more precisely, from an adult female to her eggs; or (3) by means of a mechanism called co-feeding, where the micro-organism is transmitted from one tick to another during feeding in close proximity on the same host. All tick-borne pathogens described in this review have their own specific enzootic cycle and drivers (Table 1). Therefore, a customised approach is required for each pathogen when the disease risk is analysed. At each life stage, an I. ricinus bite has an impact on the risk of tick-borne infection and co-infection for humans. For instance, larvae do not play a major role in transmitting Lyme borreliosis to humans, but may transmit other pathogens such as B. miyamotoi. Consequently, the contribution of larvae to the disease burden should be re-evaluated for each individual tick-borne pathogen. Anaplasma phagocytophilum Anaplasma phagocytophilum is a gram-negative obligate intracellular pathogen of the family Anaplasmatacae in the order Rickettsiales. It causes disease in humans and animals by infecting neutrophils, granulocytes and monocytes (Stuen et al. 2013). In 2001, the Anaplasmatacae family underwent extensive reorganisation based on detailed phylogenetic analyses. Bacteria that were known under various designations were all renamed A. phagocytophilum, and synonymised with the organisms formerly known as Ehrlichia phagocytophila, Ehrlichia equi and Cytoecetes phagocytophila (Dumler et al. 2001). A. phagocytophilum has been detected in questing nymphal and adult I. ricinus ticks in studies across Europe, with infection rates ranging from 0.4 to 34% (Stuen et al. 2013) and with lower prevalence rates in nymphs in comparison to adult ticks. In the Netherlands, infection rates in questing nymphs and adults vary between 0 and 11% in questing I. ricinus ticks (Coipan et al. 2013, Jahfari et al. 2014a). Transovarial transmission of A. phagocytophilum has not been proven to occur in Ixodes species. However, there are some indications that transovarial transmission does occur in low frequencies. Since, low rates of A. phagocytophilum in questing larvae have been reported (Jahfari et al. 2014a), and A. phagocytophilum has even been detected in larvae of a breeding colony after two to four generations of moulting (Krucken et al. 2013). Overall, the bacteria are thought to be primarily maintained through enzootic cycles between vector and vertebrate hosts. Various reservoir hosts are implicated to play a role in the maintenance of the endemic life cycle of A. phagocytophilum in nature. The animals identified as reservoir hosts range from domestic and wild ungulates, to small mammals like rodents and insectivores, to birds and lizards (Rizzoli et al. 2014, Stuen et al. 2013). Ecology and prevention of Lyme borreliosis 129

131 Setareh Jahfari and Hein Sprong Table 1. Tick-borne pathogens transmitted by Ixodes ricinus. With the pathogen, reservoir or amplifying host, mode of transmission in the vector, human or veterinary disease, cell tropism, and characteristics. Pathogen Reservoir or amplifying host Transmission in vector Human or veterinary disease Cell tropism Characteristics Anaplasma phagocytophilum ungulates, rodents, insectivores, birds and lizards transstadial and transovarial Babesia divergens cattle, ungulates transstadial and transovarial anaplasmosis, human granulocytic anaplasmosis neutrophils, granulocytes and monocytes obligatory intracellular bacterium babesiosis erythrocytes protozoan parasites Babesia microti rodents, shrews transstadial babesiosis erythrocytes protozoan parasites Babesia venatorum roe deer transstadial and babesiosis erythrocytes protozoan parasites transovarial Borrelia miyamotoi rodents; Myodes glareolus, Microtus arvalis, Apodemus flavicollis, Apodemus sylvaticus Candidatus Neoehrlichia mikurensis rodents; Apodemus agrarius, A. flavicollis, A. sylvaticus, M. glareolus, Microtus agrestis, Mi. arvalis transstadial and transovarial Rickettsia helvetica birds; Parus major transstadial and transovarial hard tick-borne relapsing fever, Borrelia miyamotoi disease extra-cellular blood pathogen transstadial neoehlrichiosis leukocytes and endothelium spirochaete bacterium obligatory intracellular bacterium rickettsiosis endothelium obligatory intracellular bacterium Rickettsia monacensis rickettsiosis obligatory intracellular bacterium tick-borne encephalitis neural tissue Flavivirus tick-borne encephalitis virus (TBEV-Eu) A. flavicollis, A. sylvaticus, M. glareolus, and Mi. arvalis transstadial, transovarial, sexual transmission 130 Ecology and prevention of Lyme borreliosis

132 9. Pandora s box To study the enzootic cycles of A. phagocytophilum in nature, different genetic markers are used. Sequences of the groel gene (part of the heat-shock protein operon) have shown more clearly delineated genetic variants of A. phagocytophilum than have sequences of other genes (Sumner et al. 1997). Using this genetic marker, four different ecotypes of A. phagocytophilum have been described in vertebrates and vectors in Europe, each with their particular enzootic cycle (Jahfari et al. 2014a). Ecotype I is associated with a wide range of hosts, including humans and domesticated animals. Ecotype II is associated with roe deer, ecotype III only with rodents, and ecotype IV with birds. It is hypothesised that each of these ecotypes has its own main vector and therefore its own ecological niche. This notion is supported by experimental findings that point to variations in the susceptibility of different mammalian species to A. phagocytophilum strains (Rikihisa 2011). The generalist I. ricinus tick species is the main vector for ecotype I, the only ecotype that is considered zoonotic. This particular ecotype also showed significant population expansion in genetic tests, implying that expansion occurs in either the vector, the vertebrate host, or in both (Jahfari et al. 2014a). Whether the other ecotypes are pathogenic to humans remains to be determined. In domestic ruminants and horses, clinical manifestations of anaplasmosis have been described since the 1950s (Foggie 1951) and 1960s (Gribble 1969), respectively. However, the first cases of human granulocytic anaplasmosis (HGA) were not reported until 1994 in the USA (Chen et al. 1994). Cases of human infection were later reported worldwide (Dumler et al. 2007). Symptoms are generally mild, flu-like and aspecific. In the Netherlands, the first and strangely enough the only reported human case dates from 1999 (Van Dobbenburgh et al. 1999), despite the widespread presence of A. phagocytophilum in questing I. ricinus ticks. More recently, five other apparently asymptomatic human cases were reported in people exposed to tick bites, some of whom suffered from an erythema migrans (S. Jahfari et al. unpublished data). Furthermore, seropositivity against A. phagocytophilum has been observed in the Netherlands in at-risk groups such as forestry workers (1.3%), febrile patients (3.7%), and potential Lyme borreliosis patients (4%), but not in healthy controls (Groen et al. 2002). Since several A. phagocytophilum strains are reported in nature, some could be more virulent to humans than others. This may well explain the discrepancy in infection rates reported in questing ticks and the exposure levels measured using serological methods in comparison to reported symptomatic human cases. All A. phagocytophilum isolates appear to have serological crossreactivity (Rikihisa 2011). Serological tests are generally group-specific and cannot be used to distinguish between individual strains. HGA is currently thought to be the third most common tick-transmitted disease in Europe (Dumler 2012), and therefore poses a growing public health concern. Since HGA is not a nationally reportable disease in most European countries, an accurate estimate of HGA incidence remains a challenge. Candidatus Neoehrlichia mikurensis Candidatus Neoehrlichia mikurensis is a relatively novel clade in the family of Anaplasmataceae and was first described in wild rats and found in Ixodes ovatus ticks in Japan (Kawahara et al. 2004). This bacterium has retained its Candidatus status, since it has not been cultivated in vitro thus far (Raoult 2014). This obligatory intracellular bacterium was initially detected in several variously designated species of ticks and rodents from Europe and Asia (Beninati et al. 2006, Brouqui et al. 2003, Pan et al. 2003, Rar et al. 2008, Sanogo et al. 2003, Schouls et al. 1999, Shpynov et al. 2006). The Asian strains showed a 99% similarity, based on the 16S rrna component, to the Schotti variant found in I. ricinus that was first described in the Netherlands (Schouls et al. 1999). Since Ecology and prevention of Lyme borreliosis 131

133 Setareh Jahfari and Hein Sprong then, Candidatus Neoehrlichia mikurensis has been found in I. ricinus all over mainland Europe, with infection rates varying from 0.1% to 22% (Silaghi et al. 2016, Wennerås 2015). In the Netherlands, infection rates of Candidatus Neoehrlichia mikurensis in questing I. ricinus nymphs range from 6% to 8%, rising to 11% for questing adult I. ricinus (Jahfari et al. 2012). Transovarial transmission in ticks is thought not to occur (Burri et al. 2014). So far, Candidatus Neoehrlichia mikurensis is thought to depend entirely on vertebrate hosts to maintain its endemic life cycle in nature. However, one study found Candidatus Neoehrlichia mikurensis in questing larvae (Derdáková et al. 2014). Rodents are the assumed amplifying hosts of this bacterium. Experimentally, infected wild rodents have proven to be competent hosts that transmit Candidatus Neoehrlichia mikurensis to laboratory ticks (Burri et al. 2014). In Europe, six rodent species (Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus agrestis and Microtus arvalis) have been shown to be infected with Candidatus Neoehrlichia mikurensis (Silaghi et al. 2016). Prevalence rates in rodents and ticks follow a seasonal pattern (Andersson et al. 2014, Coipan et al. 2013, Silaghi et al. 2012b). Furthermore, infection rates may vary considerably between rodent species. In studies where ticks from humans were tested for Candidatus Neoehrlichia mikurensis in Italy, Germany and the Netherlands, infection rates varied from 4 to 10% (S. Jahfari et al. unpublished data, Brouqui et al. 2003, Otranto et al. 2014, Richter and Matuschka 2011, Tijsse-Klasen et al. 2011). These figures indicate that exposure to Candidatus Neoehrlichia mikurensis is significant and real. The relatively low number of human neoehrlichiosis infections reported is largely due to the virtual absence of routine diagnostic tools, and perhaps a lack of awareness. Since, Candidatus Neoehrlichia mikurensis has not yet been cultivated in the laboratory, serological assays such as whole-cell IFA and ELISA tests are not yet available. In 2010, the first case of human neoehrlichiosis was reported in a patient from Sweden (Welinder- Olsson et al. 2010). In the same year, five other human infections were described in Germany, Switzerland and the Czech Republic (Fehr et al. 2010, Pekova et al. 2011, Von Loewenich et al. 2010), and curiously a report of neoehrlichiosis in an immunocompromised dog (Diniz et al. 2011). The symptoms described in all the human cases were generally non-specific and are usually seen in any other ordinary inflammatory reaction. These reports of human infection suggested that reevaluation was needed regarding the pathogenesis of this bacterium. Most of the previously reported neoehrlichiosis cases occurred in immunocompromised patients who often showed severe clinical manifestations and prolonged disease (Grankvist et al. 2014, Silaghi et al. 2016, Wennerås 2015). Other neoehrlichiosis cases have been reported more recently in immunocompetent patients who had relatively mild symptoms or were even asymptomatic in Poland, China, Sweden and the Netherlands (S. Jahfari et al. unpublished data, Alberdi et al. 2015, Grankvist et al. 2015a, 2015b, Welc-Falęciak et al. 2014). Two of these studies also showed that the DNA of Candidatus Neoehrlichia mikurensis can be detected for several months in the blood of these patients, who nevertheless did not display major clinical manifestations or complications (Grankvist et al. 2015a, Welc-Falęciak et al. 2014). These findings indicate that the true infection rates have been underestimated. 132 Ecology and prevention of Lyme borreliosis

134 9. Pandora s box Borrelia miyamotoi Borrelia miyamotoi belongs to the relapsing fever group of the Borrelia genus and was discovered in Japan in 1995 (Fukunaga and Koreki 1995, Fukunaga et al. 1995). This relapsing fever bacterium is more distantly related to the group of spirochaetes that are the causative agents of Lyme borreliosis (Barbour et al. 2009, Scoles et al. 2001). It is the only member of the relapsing fever family that is transmitted by the hard tick Ixodes species. In Europe, B. miyamotoi infection rates vary between 0.5% and 4% in I. ricinus tick vectors (Wagemakers et al. 2015). In the Netherlands, these figures are the same for questing I. ricinus ticks, and the bacterium is found in all three life stages (Cochez et al. 2015, Fonville et al. 2014, A.J. Wagemakers et al. unpublished data). B. miyamotoi is thought to be transmitted transovarially and transstadially by ticks, and coexists with B. burgdorferi s.l. (Barbour et al. 2009, Richter et al. 2012, Scoles et al. 2001). Experimentally, transovarial transmission has only been shown in I. scapularis (Scoles et al. 2001), but since B. miyamotoi is widely found in questing I. ricinus larvae, it is likely that transovarial transmission does occur (Van Duijvendijk et al. 2016), although probably rarely and inefficiently (Wagemakers et al. 2015). Nonetheless, since the total abundance of larvae is substantial, transovarial transmission probably plays an essential role in maintaining the enzootic cycle (Van Duijvendijk et al. 2016). Interestingly, B. miyamotoi results in a short-term systemic infection in rodents, therefore making rodents excellent but transitory amplifying hosts of this bacterium (Burri et al. 2014). B. miyamotoi was detected in blood or tissue of various rodent species, namely M. glareolus, Mi. arvalis, A. flavicollis and A. sylvaticus (Burri et al. 2014, Cosson et al. 2014, A.J. Wagemakers et al. unpublished data). Vertebrates other than rodents may also become infected: B. miyamotoi DNA was also found in the tissue (spleen or liver) of an European greenfinch, a great tit (A.J. Wagemakers et al. unpublished data), and a hedgehog (S. Jahfari et al. unpublished data). The role of these animals in the transmission cycle is not clear; they could be a transitory or dead-end host. It took more than 15 years after the first discovery of B. miyamotoi before the first human cases were recognised. Mainly due to the non-specific nature of the illness (Molloy et al. 2015), cases may have been confused with viral infections or other tick-borne diseases such as anaplasmosis. Relapsing fever Borreliae infections are characterised by influenza-like illness and one or more relapse episodes of bacteremia and fever. In 2011 a series of cases were reported in Russia (Platonov et al. 2011). After this report, the pathogenicity of this bacterium was reconsidered and in 2013, several case reports from the USA and the Netherlands followed in rapid succession (Chowdri et al. 2013, Gugliotta et al. 2013, Hovius et al. 2013, Krause et al. 2013). Cases were later also reported from Japan (Sato et al. 2014). Additionally, another study revealed that people in the Netherlands are indeed exposed on a wide scale to ticks that carry this pathogen (Fonville et al. 2014). In this study, 3.6% of the ticks that fed on humans tested positive for B. miyamotoi. Furthermore, exposure levels in the Netherlands as measured using serological methods showed that risk groups like forestry workers (10%) had higher seroprevalence levels than the control group (blood donors). Interestingly, higher rates were also found in patients who were suspected to have anaplasmosis (14.6%), presumably due to the portrait symptoms (Jahfari et al. 2014b). This study and other studies confirm substantial levels of B. miyamotoi exposure, infection and disease, and show that patients are not regularly recognised due to factors other than the absence of patients. Ecology and prevention of Lyme borreliosis 133

135 Setareh Jahfari and Hein Sprong Spotted fever group Rickettsiae: Rickettsia helvetica and Rickettsia monacensis In Europe, Rickettsia helvetica and Rickettsia monacensis are the two main Rickettsia bacteria found in I. ricinus. These obligate intracellular bacteria belong to the spotted fever group of the Rickettsiae. R. helvetica was first described in 1979 from a questing I. ricinus tick in Switzerland (Burgdorfer et al. 1979), but it was not until 1993 that it was officially recognised as a spotted fever group Rickettsia (Beati et al. 1993). R. monacensis, on the other hand, is a relatively novel bacterium in this same group. It was first described in 2000 in ticks from Slovakia (Sekeyova et al. 2000), and later also in Germany (Simser et al. 2002). Since their discovery, R. helvetica and R. monacensis have both been reported in questing I. ricinus ticks all over Europe. The infection rates vary from 0.5% to 66% and from 0.5% to 57% for R. helvetica and R. monacensis, respectively (Parola et al. 2013). In the Netherlands, R. helvetica is widespread in the questing tick population, with infection rates ranging from 6% to 66% (Coipan et al. 2013, Sprong et al. 2009, Tijsse-Klasen et al. 2010). In contrast, R. monacensis is reported in only about 0.8% of questing ticks (Coipan et al. 2013). Like all spotted fever Rickettsiae, transmission occurs transstadially and transovarially. Therefore, ticks in nature are usually thought to be the main reservoir and vectors of this group of Rickettsiae (Socolovschi et al. 2009). Although transovarial transmission has not been studied in R. monacensis, it is not thought to differ from the process in other species in the spotted fever group. However, since transovarial transmission rates are less than 100%, vertebrate hosts act as an amplifier of Rickettsiae, playing a vital role in transmission cycles. A study investigating transmission competence found that rodents were not able to transmit R. helvetica or R. monacensis to I. ricinus larvae (Burri et al. 2014). However, various reports of bird-feeding ticks that tested positive for R. helvetica suggest that birds may play a role in the transmission cycle. In a recent study, it was shown that birds are indeed important amplifying host for R. helvetica (Heylen et al. 2016). The amplification hosts of R. monacensis seem to be more elusive. A study where R. monacensis was detected in lizard tissue (7%) and fed I. ricinus ticks (41%) on Madeira Island, Portugal (De Sousa et al. 2012) suggest that lizards may be a potential reservoir or amplification host for R. monacensis. Both Rickettsiae have been reported in a small number of human cases. Infections with Rickettsia helvetica were reported in different cases in Sweden (Nilsson 2009, Nilsson et al. 1999, 2010, 2011). Furthermore, R. helvetica infections were reported in France, Italy and Slovakia (Fournier et al. 2000, 2004, Sekeyova et al. 2012). Rickettsia monacensis infections have been reported in patients with Mediterranean spotted fever-like illness in Spain and Italy (Jado et al. 2007, Madeddu G. 2012). In the Netherlands, R. helvetica or R. monacensis were identified using serological and molecular methods in skin biopsies of erythema migrans patients. Interestingly, co-infections with both bacteria were also found in Lyme neuroborreliosis patients (Koetsveld et al. 2015, Tijsse-Klasen et al. 2013). However, these findings are difficult to interpret for clinical settings. It is not yet clear if co-infection affected the clinical manifestations and the severity of the disease, especially in Lyme borreliosis patients. However, since the medical experts remain sceptical, further evaluation and isolation of the bacterium from clinical samples is required to determine the pathogenicity of R. helvetica. Not only is there a need for more clinical research, but studies of the cellular response and more in-depth molecular studies could perhaps shed more light on the population dynamics and evolution of R. helvetica variants, and provide greater understanding of the pathogenesis and virulence mechanisms. Some R. helvetica strains may be more virulent to humans than others. 134 Ecology and prevention of Lyme borreliosis

136 9. Pandora s box Babesia species In Europe, three intra-erythrocytic protozoan parasites are known to have I. ricinus as their vector: Babesia divergens, Babesia venatorum and Babesia microti. They are classified as apicomplexan parasites of the Piroplasmida suborder and the Babesiidae family. The infection rate of Babesia spp. in questing ticks ranges from 0.9 to 20% (Hildebrandt et al. 2013). The infection rate of Babesia species in questing I. ricinus ticks in the Netherlands is 1.7% (Coipan et al. 2013, Wielinga et al. 2009), and all three Babesia genospecies have been found to occur. The maintenance and persistence of Babesia s within the tick vector is ensured through transstadial and transovarial transmission, with the exception of B. microti where transovarial transmission does not appear to occur in I. ricinus (Chauvin et al. 2009). Cattle have been identified as the main reservoir host of B. divergens. Other ungulates like roe deer, fallow deer, red deer, mouflon and sheep can also be infected with this protozoan parasite (Chauvin et al. 2009). The main reservoir of B. venatorum seems to be roe deer, and small rodents and shrews appear to be the main reservoir of B. microti (Rizzoli et al. 2014). Although babesiosis is mainly known to cause disease in animals, it is a zoonotic disease that can be transferred to humans. However, the main impact of babesiosis is still in the veterinary field, where it causes disease in livestock and in companion animals (Hunfeld et al. 2008). In humans, B. divergens has been reported as the causative agent in most of the cases of babesiosis found in Europe (Hunfeld et al. 2008, Rizzoli et al. 2014). Only four clinical cases caused by B. venatorum have been described in Europe, namely in Austria, Italy and Germany (Blum et al. 2011, Häselbarth et al. 2007, Herwaldt et al. 2003). Only one case of B. microti infection has been reported in Germany (Hildebrandt et al. 2013). All these babesiosis reports concerned immunocompromised patients, mainly patients without a spleen (Hunfeld et al. 2008). This is largely due to the fact that disease manifestations are more severe and often life-threatening in this fraction of the population. In immunocompetent patients, Babesia infection is often mild with influenza-like symptoms or even asymptomatic. The reported seroprevalence rates in Germany (5.4% to 8% for B. microti and 3.6% for B. divergens) suggest that exposure to Babesia is more widespread and that the number of patients is underestimated, probably due to the relatively mild symptoms. Only two cases of human babesiosis have been reported in immunocompetent patients from France, of which one was caused by B. divergens (Martinot et al. 2011). Three cases of B. divergens infection in immunocompetent patients have recently been reported in the Netherlands (S. Jahfari et al. unpublished data). Tick-borne encephalitis viruses In Europe, the first reported case of tick-borne encephalitis (TBE) occurred in 1931 in Austria, when an outbreak of meningitis was reported (Schneider 1931). The causative agent tick-borne encephalitis virus (TBEV) was not isolated until 1937 in the former Soviet Union (Silber and Soloviev 1946). TBEV is a member of the genus Flavivirus and the Flaviviridae family. Most members of this virus genus are arthropod-borne. This group of tick-borne flaviviruses (the TBEV serocomplex) comprises ten other viruses, including Omsk hemorrhagic fever virus, Powassan virus, and Louping ill virus (Calisher and Karabatsos 1988, Theiler and Downs 1973). The virus has a linear positive-stranded RNA genome that consists of a single open reading frame (Demina et al. 2010). TBEV can be divided into three subtypes: Siberian (TBEV-Sib), Far Eastern (TBEV-Fe) and European (TBEV-Eu) (Ecker et al. 1999). Only the TBEV-Eu subtype has I. ricinus as its main vector; the other two subtypes are associated with I. persulcatus. Ecology and prevention of Lyme borreliosis 135

137 Setareh Jahfari and Hein Sprong In endemic areas in Europe, TBEV infection rates vary between 0.1 and 5% in ticks. Infection rates increase during the tick s development from stage to stage. Reported infection rates are usually less than 1% (Süss 2011), with fairly high infection rates (up to 27%) in microfoci (Bormane et al. 2004). Transmission in tick populations occurs transstadially, with co-feeding identified as the most efficient route of infection in naive larvae. Transovarial transmission also occurs, and sexual transmission has even been suggested in ticks (Pettersson et al. 2014, Süss 2011). Various rodent species serve as the main reservoir or amplifying host. They are able to transmit the virus via viremia to feeding ticks and by co-feeding. The rodent species A. flavicollis, A. sylvaticus, M. glareolus and Mi. arvalis are important reservoir hosts for TBEV-Eu (Süss 2011). These species can even maintain the virus in nature through latent persistent infection (Achazi et al. 2011, Tonteri et al. 2011, 2013). Migratory birds have been described as playing an essential role in the geographical distribution of TBEV-infected ticks, thus contributing to new foci. Still, little is currently known about the possible role of birds as TBEV reservoirs (Waldenstrom et al. 2007). However, the prevalence of TBEV-infected bird-feeding I. ricinus is relatively low (Rizzoli et al. 2014). Ungulates that roam freely such as goats, sheep, deer and wild boar are thought not to contribute to the amplification of the virus. These animals are only viremic during a very short period and do not display any clinical symptoms, although they can serve as sentinels for the identification of TBEV foci in serological studies (Klaus et al. 2010, Süss 2011). In the Netherlands some sero-reactivity has been observed in wild animals and horses, although none of these cases could be confirmed through hemagglutination inhibition or serum neutralisation testing for neutralising antibodies (Cleton 2016, Poel et al. 2005). Although the I. ricinus vector of the TBEV- Eu subtype as well as the rodent reservoir are widely present in the Netherlands, it is unclear why TBEV does not thrive in an enzootic cycle. More recently, questing I. ricinus have been reported to be infected with a TBEV-Eu variant on one location in the Netherlands (S. Jahfari et al. unpublished data). This is particularly notable considering recent reports from Belgium, where wild cervids and 2 to 4% of cattle have tested positive for TBEV with neutralising antibodies (Linden et al. 2012, Roelandt et al. 2011, 2014). These findings indicate that TBEV foci are present. According to the European Centre for Disease Prevention and Control, TBEV is currently endemic in 27 European counties (ECDC 2012, Süss 2011). Furthermore, expansion northwards and to higher altitudes has been reported in recent years (Danielová et al. 2010, Dobler et al. 2011). Still, no known autochthonous cases have been reported from Spain, Portugal, the United Kingdom, Ireland, Belgium, or the Netherlands (Süss 2011). The western European TBE subtype often has a biphasic course. The first phase is associated with non-specific flu-like symptoms (e.g. fever, fatigue, myalgia, nausea, or headache). This initial phase is followed by an afebrile asymptomatic interval that could precede the second phase, when the central nervous system is affected (such as meningoencephalitis, myelitis or paralysis) (ECDC 2012, Haglund and Günther 2003). Considering that two-thirds of human TBEV infections are believed to be asymptomatic (Kunze 2012) and that the TBEV-Eu subtype is associated with milder disease (Haglund and Günther 2003), it is possible that human cases are underreported in Europe. Co-infection Since all the previously described agents can coexist in I. ricinus ticks, co-infections in ticks are frequently reported. However, only a small number of systematic and large-scale studies have been conducted to investigate the composition of mixed tick-borne pathogen infection rates (Swanson et al. 2006). Some studies have attempted to determine the infection rates of the entire 136 Ecology and prevention of Lyme borreliosis

138 9. Pandora s box range of pathogens among I. ricinus through reverse line blot analysis and other PCR amplification methods. However, the findings of these studies are difficult to compare due to differences in methodology. The outcomes of such studies are strongly affected by the methods used for tick collection, the sample size, the selection of tested tick life-stages, the selection of tested tickborne pathogens, the DNA extraction methods, and the selection of primers and probes. Less specific PCR primers and probes will potentially yield higher reported infection rates in ticks. Furthermore, without sequence analysis there is little discrimination amongst strain variants that are not associated with human disease. Consequently, in most European countries there is little accurate information about the co-infection rates for all tick-borne pathogens among I. ricinus. According to two studies, co-infection of two or more tick-borne pathogens occurs relatively frequently in questing I. ricinus ticks. For instance, about 6 to 7% of questing ticks are infected with more than one pathogen (Coipan et al. 2013, Lommano et al. 2012). In a more recent study that used a novel molecular platform to test an entire range of tick-borne microbiota in adult ticks, 45% of the ticks were found to be co-infected with at least two microbes (Moutailler et al. 2016b). In the Netherlands, 37 to 38% of all Borrelia-positive ticks are infected with at least one other pathogen of a different genus (Coipan et al. 2013, S. Jahfari et al. unpublished data). Some combinations are found at significantly higher rates than others, probably indicating a common reservoir host. These include co-infections of B. afzelii and Candidatus Neoehrlichia mikurensis or Babesia spp., whereas co-infections of R. helvetica with either B. afzelii or with Candidatus Neoehrlichia mikurensis occurred significantly less frequently (Coipan et al. 2013). These findings suggest that people bitten by ticks run the risk of being exposed to multiple pathogens at once or concomitantly. In other words, human co-infection with tick-borne pathogens can occur following a single bite from a tick infected with multiple pathogens, or following several simultaneous bites from ticks that carry single pathogens. Both these scenarios can potentially result in co-infection of different tick-borne pathogens in humans (Swanson et al. 2006). In one study of patients suffering from an erythema migrans (the first and main symptom of Lyme borreliosis), individuals were shown to be also infected with B. miyamotoi, A. phagocytophilum, Candidatus Neoehrlichia mikurensis and B. divergens (S. Jahfari et al. unpublished data). And as mentioned, R. helvetica or R. monacensis were identified in skin biopsies of erythema migrans patients and co-infections with both bacteria were found in Lyme neuroborreliosis patients (Koetsveld et al. 2015, Tijsse-Klasen et al. 2013). Co-infections may affect the severity of disease and influence clinical outcomes, especially since some tick-borne pathogens like A. phagocytophilum may modulate host immunity (Belongia 2002, Swanson et al. 2006, Thomas et al. 2012). Moreover, mixed infections of different tick-borne pathogens may be partly responsible for the wide variety of reported clinical manifestations of Lyme borreliosis (Krause et al. 1996), such as fever and influenza-like symptoms that are caused by other pathogens but are contributed by physicians and others to the common denominator of Lyme borreliosis. The role of other tick species in pathogen maintenance and transmission to Ixodes ricinus Other tick species maintain their specific set of pathogens in enzootic cycles. However, they may still play a key role in transmitting those pathogens to the generalist feeder I. ricinus via common vertebrate hosts. I. ricinus can be infected by pathogens that are associated with another vector, through feeding on an infected shared host. Because of its generalist behaviour, I. ricinus does not play a key role in the maintenance of the enzootic cycle of these pathogens. Nevertheless, I. ricinus may occasionally transmit these pathogens to humans or animals. Other tick species Ecology and prevention of Lyme borreliosis 137

139 Setareh Jahfari and Hein Sprong like Dermacentor reticulatus and Ixodes hexagonus are also known to occasionally bite humans and companion animals, although less frequently than I. ricinus. D. reticulatus is geographically distributed throughout almost all of North-Western and Central Europe. In the past few decades, D. reticulatus has substantially expanded into regions formerly thought to be unsuitable for the survival and maintenance of this tick species (Rubel et al. 2016). D. reticulatus is a known vector for several pathogens, i.e. Anaplasma marginale, Babesia canis, Babesia caballi, Rickettsia raoultii, Rickettsia slovaca, Theileria equi, Omsk haemorrhagic fever virus, and tick-borne encephalitis virus (Földvári 2016). In the Netherlands, D. reticulatus has tested positive for B. canis and B. caballi (Jongejan et al. 2015). I. hexagonus is a widespread tick species that is mostly associated with hedgehogs and plays a role in the maintenance of several genospecies of the B. burgdorferi s.l. complex (Gern et al. 1997, Skuballa et al. 2012) and the zoonotic variant of A. phagocytophilum (Jahfari et al. 2012, Silaghi et al. 2012a, Skuballa et al. 2010). Remarkably, I. hexagonous and I. ricinus appear to share some pathogens, such as B. bavariensis and A. phagocytophilum. Particularly hedgehogs and their host-specific parasite I. hexagonus play a role in maintaining these pathogens in cryptic cycles, especially in urban areas such as gardens and parks. The relatively small populations of I. ricinus in these areas and their generalist feeding behaviour suggest that they do not play a main role in the maintenance of enzootic cycle of these pathogens. However, when feeding on hedgehogs I. ricinus may still be infected by I. hexagonous-associated pathogens and transmit them to humans (Figure 1). Ixodes hexagonus (N/A) Ixodes ricinus (N/A) Borrelia spielmanii Borrelia bavariensis A. phagocytophilum Ixodes hexagonus (L/N) Ixodes ricinus (L/N) Figure 1. The cryptic cycle of Ixodes hexagonus and its host, the hedgehog, and the pathogens associated with the two in relation to the generalist tick Ixodes ricinus and the risk of exposure to humans (L/N = larva nymph; N/A = nymph/adult). 138 Ecology and prevention of Lyme borreliosis

140 9. Pandora s box Furthermore, the host-specific Ixodes trianguliceps species feeds nidicolously only on rodents and shrews and is a driver of A. phagocytophilum and B. microti (Bown et al. 2006, 2008). When I. ricinus larvae feed on small rodents, they are infected and moult to the next stage. Other factors that influence human exposure to ticks and tick-borne pathogens The transmission cycles of tick-borne pathogens have multifactorial drivers,. Many factors influence vector distribution and pathogen dynamics, such as land use, habitat destruction, degradation and fragmentation. These factors influence host density and host composition in specific areas. In addition, weather factors are important since they affect the intensity and temporal patterns of vector activity throughout the year, leading particularly to increased biting rates in humans. Climate also influences habitat suitability and therefore the survival and reproduction rates of (new) vectors and hosts. In the Netherlands, climate change is one of the many factors that influence vector habitats. Changes in landscape management (e.g. the conversion of agricultural land into habitats suitable for the maintenance of large populations of deer and other ungulates) contribute to a sharp increase in tick densities (Barbour and Fish 1993, Gilbert et al. 2012, Spielman 1994, Sprong et al. 2012). In addition, socio-economic factors such as recreational activities in rural areas with high tick densities have resulted in increased human exposure to ticks. Demographic changes are another important factor, with elderly people making up a substantial portion of the population and improvements in healthcare for chronically ill or immunocompromised patients. In 2013, for instance, 16% of the Dutch population was older than 65 and almost one-third of the entire Dutch population suffered from one or more chronic diseases (Gijsen et al. 2014). Furthermore, public awareness of ticks and the pathogens carried by them also plays a role. Concluding remarks Taking Lyme borreliosis as a proxy for tick exposure, several European countries including the Netherlands have reported marked increases in the incidence of Lyme borreliosis over the past ten to twenty years (De Mik et al. 1997, Hofhuis et al. 2006, 2015, Hubálek 2009). Studies in the Netherlands have shown that more than one-third of questing ticks and more than half of ticks feeding on humans harbour one or more of the tested tick-borne pathogens (Coipan et al. 2013, S. Jahfari et al. unpublished data). This means that the risk of exposure to and infection with a tick-borne pathogen is substantial. It is quite possible that transmissions of other tick-borne pathogens have been common, but have not been distinguished from other general infections, or that infections with these emerging pathogens have all been classified under the common denominator of Lyme borreliosis, simply because most of these pathogens display a wide range of non-specific symptoms in clinical cases. Many clinicians have limited awareness of and experience in recognising or managing tick-borne pathogens other than Lyme borreliosis, let alone co-infections of different tick-borne pathogens. When patients report tick bites, clinicians should consider additional laboratory testing or differential diagnoses for patients that display an intense or persistent array of aspecific, influenzalike symptoms, especially fever, chills, and headache with or without signs of Lyme borreliosis (Swanson et al. 2006). Medical professionals should therefore be aware of and consider the likelihood of infection with tick-borne pathogens other than Lyme spirochaete and co-infections with different tick-borne pathogens when pursuing laboratory testing or selecting therapy for patients with tick-borne diseases (Swanson et al. 2006), especially since protozoan and viral infections are nonresponsive to antibiotics. Furthermore, infections with other pathogens like A. phagocytophilum should be considered when (suspected) Lyme borreliosis patients fail to respond Ecology and prevention of Lyme borreliosis 139

141 Setareh Jahfari and Hein Sprong to β-lactam antimicrobial therapy (Swanson et al. 2006). More specific laboratory tests need to be developed to support clinicians in making a correct diagnosis and selecting appropriate therapy. Most tests currently available are not specific to European tick-borne pathogens. Furthermore, some tick-borne pathogens have not yet been cultivated in the laboratory, meaning that serological assays like whole-cell IFA and ELISA tests are not yet available. A multifactorial issue like tick-borne diseases requires a multidisciplinary and interdisciplinary approach. This means that different approaches will be needed to gain greater insight into the drivers at play in vector and disease dynamics. Clinical studies are needed to gain a better picture of the clinical spectrum of these often newly emerging or re-emerging pathogens. These studies should assess patients suffering from fever following a tick bite. In addition, more relevant laboratory findings and strict case definitions for each individual pathogen are required. Furthermore, surveillance and epidemiological approaches to newly emerging or re-emerging pathogens should be integrated with ecological and biological driving factors. This will allow the relative public health and veterinary burden of each pathogen to be assessed appropriately. Study results will shed light on disease and infection, and will help to identify any risks of outbreaks of zoonotic diseases and important veterinary diseases. Public health relevance Ixodes ricinus transmits many more pathogens other than Lyme spirochaetes. Tick-borne pathogens generally cause self-limiting infections with mild symptoms. Tick-borne infections may cause severe symptoms, particularly in immunecompromised patients. More awareness and better diagnostic modalities for all tick-borne diseases is needed. References Achazi K, Růžek D, Donoso-Mantke O, Schlegel M, Ali HS, Wenk M, Schmidt-Chanasit J, Ohlmeyer L, Rühe F and Vor T (2011) Rodents as sentinels for the prevalence of tick-borne encephalitis virus. Vector-Borne Zoonotic Dis 11: Alberdi P, Ayllon N, Cabezas-Cruz A, Bell-Sakyi L, Zweygarth E, Stuen S and de la Fuente J (2015) Infection of Ixodes spp. tick cells with different anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death. Ticks Tick-Borne Dis 6: Andersson M, Scherman K and Råberg L (2014) Infection dynamics of the tick-borne pathogen Candidatus Neoehrlichia mikurensis and coinfections with Borrelia afzelii in bank voles in southern Sweden. Appl Environ Microb 80: Barbour AG, Bunikis J, Travinsky B, Hoen AG, Diuk-Wasser MA, Fish D and Tsao JI (2009) Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. Am J Trop Med Hyg 81: Barbour AG and Fish D (1993) The biological and social phenomenon of Lyme disease. Science 260: Ecology and prevention of Lyme borreliosis

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150 10. Phenology of Ixodes ricinus and Lyme borreliosis risk Willem Takken Laboratory of Entomology, Wageningen University & Research, P.O. Box 16, 6700 AA Wageningen, the Netherlands; Abstract The phenology of Ixodes ricinus and its consequence for Lyme borreliosis risk are important factors to consider in the prevention of disease. Here, determinants that affect the phenology of I. ricinus are reviewed, and it is discussed to what extent they contribute to the risk of transmission. Ixodes ricinus expresses a strongly seasonal phenology, driven mostly by an endogenous rhythm that is regulated by temperature. Ticks feed on vertebrates, with larvae, nymphs and adults each feeding on specific groups of animals, with rodents the preferred hosts of larvae. Borrelia burgdorferi s.l. parasites, the agents of Lyme borreliosis, use rodents as their main reservoir host, and hence larvae are the tick stage in which infection of ticks occurs. Nymphs, emerging from the dormant larvae the following year, are the infectious stage for humans, and the density of infected nymphs is considered an indicator of Lyme borreliosis risk. Various environmental factors affect development time, feeding success and survival of ticks, where ambient temperature and saturation deficit regulate the rate and probability of successful development. These factors combined, contribute to the variation in Lyme borreliosis risk as reported in western Europe. Although climate change will impact on abiotic and biotic factors regulating the ecology of I. ricinus, this is unlikely to have a large impact on its phenology. Lyme borreliosis risk, however, may be affected due to changes in the diversity and population density of vertebrates on which the ticks depend, causing shifts in tick population density and hence risk of exposure to infection. Keywords: Borrelia burgdorferi sensu lato, climate change, development, environment, Europe, Ixodes ricinus, Lyme borreliosis, phenology, sheep tick, transmission Introduction Phenology is the study of periodic plant and animal life cycle events and how these are influenced by seasonal and inter-annual variations in climate, as well as habitat factors ( org/wiki/phenology). For hard ticks (Acari, Ixodidae), many species of which undergo a period of dormancy, studies on phenology are concentrated on the period when they are active: engaged in mating, host seeking, feeding and egg laying. Studies that assess Lyme borreliosis risk, which is determined mainly by the density of Borrelia burgdorferi s.l. infected nymphs of the sheep tick Ixodes ricinus and their biting behaviour (Hubalek et al. 1996, James et al. 2013) are usually done in periods when ticks are most active and are abundantly present (Estrada-Peña et al. 2011, Mannelli et al. 2012). Recent work has shown, however, that the period in which tick bites can be acquired, and hence B. burgdorferi s.l. transmission to humans can take place, is not limited to these few months, and can be stretched over many months depending on local climate (Gassner et al. 2011). In areas with a short summer season and long periods with cold weather, such as northern Scandinavia (Hvidsten et al. 2015), the tick season is more limited than in areas with longer summers and relatively mild temperatures, such as the Netherlands, Belgium and France (Kilpatrick and Randolph 2012, Paul et al. 2016). The phenology of ticks and their associated B. burgdorferi s.l. infections is therefore an important factor to consider when assessing Lyme borreliosis risk. In this review a short overview is presented in which the main determinants Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _10, Wageningen Academic Publishers 2016

151 Willem Takken driving this phenology are discussed, and how environmental change leads to significant changes in the phenology of Lyme borreliosis risk. The vector Ixodes ricinus consists of a complex of sibling species distributed across the northern hemisphere (Gray et al. 2016). Of these, I. ricinus sensu stricto is the most abundant species in Europe and the principle European vector of Borrelia burgdorferi s.l., the agent of Lyme borreliosis as well as tickborne encephalitis virus, Anaplasma phagocytophilum and several other pathogenic agents (Carpi et al. 2016). Ixodes ricinus has a characteristic phasic annual phenology, with highly active ticks in the spring and in the summer and mostly inactive ticks in the winter, when they are in diapause (Cadenas et al. 2007, Estrada-Peña et al. 2004, Gray 1998) (Figure 1). Because of its central role in the transmission of B. burgdorferi s.l. in Europe, this chapter will focus on I. ricinus. Life cycle Ixodidae have four life stages: egg, larva, nymph and adult (Sonenshine and Roe 2013). Eggs are deposited on moist soil or leaf litter, where they remain for one to several months, depending on the ambient temperature. Newly emerged larvae climb into the stems of grass where they will wait for a suitable host to pass by a process termed questing. Hosts are perceived by odour, body heat and moisture. After attachment, larvae blood feed to repletion, release their mouthparts and drop to the ground to enter diapause, which may last up to 12 months. As the larvae do, newly emerged nymphs climb into the vegetation to engage in questing behaviour. Having attached to a host, nymphs feed to repletion, drop to the ground where they, like larvae, enter diapause during which they morph into adults. Adult ticks, having emerged from the nymphal shell, engage in mating: unlike larvae and nymphs, they walk around the vegetation until they, too, ascend grass or other plants for questing. Mating occurs on the ground or on host, and is, like with Amblyomma and Dermacentor spp. (Sonenshine 2006), presumably regulated by a female-produced mating pheromone (Zemek et al. 2007). In I. ricinus not much is known about the chemical ecology of Mean number of questing ticks collected per 200 m 2 (Log(n+1)) July Nov March July Nov March July Nov L N A March July Nov March July Nov March July Nov March July Nov March July Nov Figure 1. Phenology of Ixodes ricinus in the Netherlands over a 9 year period, expressed as the monthly mean number of questing nymphs collected in 15 locations (adapted from Takken et al. in press; L = larvae; N = nymphs; A = adults). 150 Ecology and prevention of Lyme borreliosis March

152 10. Ixodes ricinus phenology and disease risk mating behaviour. Once females have fed to repletion, they drop to the ground, where eggs are matured and laid over a period of several days. There is a mono-gonotrophic reproductive cycle and females die after egg laying. Availability of hosts All three mobile life stages of I. ricinus feed on vertebrate blood, derived from mammals, birds or reptiles. Larvae are mostly found on small rodents, which forage on the forest floor. They are also found on a variety of other mammals, but most blood meals occur on rodents (Hofmeester et al. 2016, Perez et al. 2012). Unlike larvae, nymphs may feed on a large variety of vertebrates, where birds play an important role as blood host (Hofmeester et al. 2016). Adult I. ricinus depend on large grazers such as roe deer, red deer and wild boar (Pacilly et al. 2014). For a successful life cycle I. ricinus requires, therefore, an abundant population of small rodents (e.g. Myodes glareolus and Apodemus spp.) for larvae and large grazers for adults, while a diverse population of the local vertebrate fauna benefits the survival of the nymphal stages. Environment The geographic distribution of I. ricinus covers a large surface area, from Ireland and Portugal to the Ural mountains in Russia and from northern Scandinavia to the Mediterranean (Estrada- Peña et al. 2006). In general, the species thrives in a temperate climate with abundant rainfall and moderate temperatures. Areas that experience seasonally high saturation deficits are not suitable, and I. ricinus also avoids direct exposure to sunlight, looking for shady areas (Tack et al. 2012,Tagliapietra et al. 2011). Forest zones and adjacent ecotones, with dispersed shrubs and trees, are much preferred above open meadows and heathland (Boeckmann and Joyner 2014, Jaenson et al. 2012, James et al. 2013, Paul et al. 2016). In a countrywide study in the Netherlands, across 24 study sites, I. ricinus was found in a multitude of diverse habitats, which were characterised by being forested or covered with shrubs, and where a diverse fauna was naturally present (Gassner et al. 2011). The current distribution of Lyme borreliosis is thus much determined by the coverage of land surface areas with habitat suitable for I. ricinus. Economic developments and government policies that benefit nature and nature protection are likely to lead to expansion of I. ricinus habitat across Europe, as well as to increased wildlife populations. For example, in the Netherlands the closure of farms has led to an expansion of forested areas and nature protection measures caused an increase in roe deer populations (Sprong et al. 2012). Similar developments are reported from many other European countries, favouring I. ricinus populations and associated B. burgdorferi s.l. prevalence (James et al. 2013, Medlock et al. 2013). Climate Suitable ambient temperatures coupled with saturation deficit are the most important determinants affecting tick development and survival (Gray 1998); hence the importance of vegetation (see above) in which a suitable micro-climate for ticks can be established (Randolph and Storey 1999). Especially larvae are vulnerable to high saturation deficits (SD), and even short periods with high SDs in the summer can have a large impact on larval survival (Knulle and Rudolph 1982, Lees 1946). Such conditions are more often present in open areas lacking shade than in forested zones, and explain the absence of I. ricinus from sunlit habitats. In mountainous areas such as the Alps, ticks can be found up to altitudes of 1,450 m where the limit of distribution Ecology and prevention of Lyme borreliosis 151

153 Willem Takken is largely temperature determined (Danielova et al. 2006, Gern et al. 2008, Medlock et al. 2013). Their seasonal activity at this altitude is more limited than in lowland areas. Further west, in Spain and Portugal, however, I. ricinus has been reported at altitudes of up to 2,000 m, which can be explained by the lower latitude of these countries. Ixodes ricinus is active when the ambient temperature exceeds 5 C, although this can vary between the life stages. At temperatures <5 C, larvae and nymphs are dormant (Gray et al. 2016; see also Seasonal and inter-annual effects ). The present distribution of I. ricinus is to a large extent determined by climate, and climate change as it is being reported at present has already led to shifts in geographic distribution of I. ricinus, moving further north into Scandinavia (Hvidsten et al. 2015, Lindgren et al. 2000, Medlock et al. 2013). Seasonal and inter-annual effects In geographic areas that experience severe winters, all I. ricinus activity is halted when the mean temperature drops below 5 C (Takken et al. in press). Larvae and nymphs enter a seasonal diapause, which is initiated in the autumn and broken in spring (Gray et al. 2016, Randolph et al. 2002, Sonenshine and Roe 2013). In areas with a milder climate, unfed I. ricinus may remain active throughout the year, as this depends on a daytime threshold temperature. Engorged ticks, however, enter a developmental diapause presumably triggered by day length and the duration of which is much determined by the mean temperature to which ticks are exposed. Although the occurrence of diapause in I. ricinus has been well documented, the exact physiology of diapause in this species is still unclear and needs to be further verified (Gray et al. 2016). As 5 C appears the critical threshold temperature below which I. ricinus is inactive, this temperature can be considered an ecological isotherm, which helps to explain the phenology of this species. The seasonal variations in number of questing ticks as shown in Figure 1 are typical for the species, and occur throughout Europe (Gray 1991). In northern Europe ticks become active later in the spring and enter into a state of dormancy by mid-october, while in southern Europe they can be found all year round. In the Netherlands, which enjoys a maritime climate with irregular winter temperatures, I. ricinus usually goes dormant between December and March, but in some winters nymphs and adult ticks can be found questing in January and February. These activities could be linked to relatively and unseasonal mild temperatures (Dautel et al. 2008, Takken et al. in press). Whereas the seasonal phenology of I. ricinus is genetically determined and largely driven by temperature, the inter-annual phenology, although remarkably similar (Figure 1), can be affected by various environmental factors. Drought, for example, can severely affect larval survival and, consequently, population structure and density (Medlock et al. 2008, Perret et al. 2000). Unexpected high mortality of blood hosts, in turn, can affect survival and/or reproduction of I. ricinus. Hence, the inter-annual variation in the phenology of I. ricinus may appear small, it can nevertheless be variable because of the environmental effects on population density, and hence population growth. Borrelia burgdorferi s.l. infections in Ixodes ricinus Lyme borreliosis is caused by members of the B. burgdorferi complex of spirochaetes (Mysterud et al. 2016, Wright et al. 2012). All members are vector-borne, with I. ricinus being the main vector in Europe (Mannelli et al. 2012). At least seven B. burgdorferi genotypes are present in Europe, with different host specificity: Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia bavariensis and Borrelia spielmanii are strongly associated with small rodents; Borrelia garinii and Borrelia 152 Ecology and prevention of Lyme borreliosis

154 10. Ixodes ricinus phenology and disease risk valaisiana are associated with birds, while Borrelia lusitaniae is found mostly in lizards (Kurtenbach et al. 2002, Mannelli et al. 2012). Ticks become infected with Borrelia parasites when taking a blood meal. Once infected, ticks remain infected for life. Most infection studies are done on nymphs, which acquired their infection in the larval stage, although recently Van Duijvendijk et al. (2016) reported infections of B. afzelii and Borrelia miyamotoi in larvae that had not yet taken a blood meal, which suggests a low-level vertical transmission of Borrelia through the parent tick. B. miyamotoi, is not a Lyme disease spirochaete, and is, unlike B. burgdorferi afzelii, known to be vertically transmitted (Heylen et al. 2016). From larvae to nymph to adult, there is an increasing Borrelia prevalence (Jouda et al. 2004, Rauter and Hartung 2005). Natural infection rates in nymphs can vary greatly within and between habitats and geographic zones. The mean B. burgdorferi s.l. prevalence in questing nymphal ticks in a number of European countries was 10.1% (Rauter and Hartung 2005). From a longitudinal study in the Netherlands, covering 11 different locations, B. burgdorferi s.l. prevalence of questing nymphal I. ricinus varied from 7.1 to 26% (Takken et al. in press). The mean B. burgdorferi s.l. prevalence of ticks in these 11 sites increased significantly from early spring to autumn. It is generally accepted that humans acquire Borrelia infections through the bite of a nymph, and the density of infected nymphs is often used as a measure for Lyme borreliosis risk (Eisen and Eisen 2016, Van Duijvendijk et al. 2015). Climate change and tick phenology The main effects of climate change, increased ambient temperatures and rainfall, are expected to affect the length of the tick season as well as tick development rate (Alonso-Carne et al. 2015, Gray et al. 2016). Although climate change is unlikely to affect the seasonal phenology of I. ricinus, increased temperatures and rainfall may lead to changes in Borrelia prevalence, because of the expected shifts in diversity and population densities of vertebrates (birds, mammals and reptiles), which may occur as a result of climate change (Bowler et al. 2015, Maiorano et al. 2013, Plard et al. 2014). Additionally, shifts in distribution of endemic and exotic diseases associated with climate change are likely to impact tick and vertebrate populations as well (Medlock and Leach 2015). It is widely agreed that climate change will most probably lead to the expansion of the range of I. ricinus in Europe (Lindgren et al. 2000, Randolph 2001, Semenza et al. 2012), and hence the expansion of areas of Lyme borreliosis risk. Discussion The phenology of I. ricinus is affected by multiple factors, but regulated by a genetic trait that is driven by temperature (Mannelli et al. 2012). In optimal habitat and climate, I. ricinus becomes active in the spring and achieves peak population densities in July/August, to decline towards autumn with a quiescent (dormant) period in winter. These patterns appear fixed, and can be found along north-south as well as east-west transects throughout Europe. Distribution limits are marked by temperature and saturation deficit, as long as a vegetation type with sufficient cover is present. The first, temperature, is mostly determinant in the north, while the latter (saturation deficit) in the south. There are many environmental variations throughout the natural distribution area of I. ricinus, but as long as the above conditions are met, the species can be found (Estrada- Peña et al. 2006). For Lyme borreliosis to occur, however, B. burgdorferi s.l. spirochaetes also must be present in habitats that overlap the distribution zone of I. ricinus. A large and wide-spread occurrence of various Apodemus spp. and M. glareolus ensure the continued presence of B. burgdorferi genospecies., and the parasites circulate between these animals mostly through the bites of I. ricinus nymphs (Van Duijvendijk et al. 2015). Transovarial transmission of B. burgdorferi s.l. has not been reported, although the recent discovery of B. burgdorferi s.l. infected larvae suggests Ecology and prevention of Lyme borreliosis 153

155 Willem Takken such transmission may be possible (Van Duijvendijk et al. 2016). Nevertheless, the maintenance of B. burgdorferi genospecies in European ecosystems appears to depend much on the association of I. ricinus and small rodents such as Apodemus spp. and M. glareolus (Hofmeester et al. 2016). Populations of I. ricinus are themselves much dependent on the presence of large grazers such as roe deer, red deer and wild boar (Carpi et al. 2008, Medlock et al. 2013). These animals function as blood hosts for adult females and hence contribute much to the reproductive success of I. ricinus. As the large grazers appear incompetent hosts for B. burgdorferi s.l. (Jaenson and Tälleklint 1992, Mannelli et al. 2012, Pacilly et al. 2014), they themselves do not play a role in the circulation of the parasite but provide the basis for a continuing transmission through the production of parasitecompetent vectors. Herein lies the key to reducing Lyme borreliosis risk: the basic reproductive number of the disease depends much on the population density of its vector, which in turn is dependent on small rodents (larvae) and large grazers (adult ticks) (Harrison et al. 2011, Hartemink et al. 2008). Considering the high consistency of I. ricinus phenology, with populations fluctuating between seasons but with high inter-annual regularity (Paul et al. 2016) and the continental-wide prevalence of B. burgdorferi s.l. (Rauter and Hartung 2005), the risk of Lyme borreliosis is highest when populations of questing nymphal ticks are at their maximum, which in most European regions is between June and September. Even though the prevalence in ticks can vary throughout the year (Pawelczyk and Sinski 2004, Takken et al. in press), with highest prevalence recorded in autumn, the higher abundance of ticks during the summer explains the relatively higher transmission risk for Lyme borreliosis. Measures aimed at the prevention of Lyme disease should therefore focus on those periods when people are most likely to be exposed to highest risk. Climate change is expected to lead to an expansion of I. ricinus and Lyme borreliosis risk zones in Europe, shifting the northern boundary of current distribution limits and possibly reducing risks in southern Europe due to longer periods of drought. These processes are unlikely to cause a major shift in tick phenology, however, but Lyme borreliosis risk may be affected due to changes in populations of blood hosts, which may occur as a result of shifts in biodiversity of vertebrates associated with global change. Public health relevance For risk assessment: the seasonal variation in tick density (phenology) explains that highest risk for a tick bite, and hence transmission of Borrelia burgdorferi parasites, is associated with months when ticks are most abundant. For prevention: the phenology of Ixodes ricinus, with its strong seasonal variation, helps to determine when measures for the prevention of tick bites are most needed; in the Netherlands this will be at times when tick populations are most abundant. For these reasons a thorough understanding of the phenology of ticks and associated Borrelia burgdorferi infections is needed for the assessment of temporal distribution of Lyme borreliosis risk and the development of preventive measures. 154 Ecology and prevention of Lyme borreliosis

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158 10. Ixodes ricinus phenology and disease risk Pawelczyk A and Sinski E (2004) Prevalence of Ixodes ricinus infection with Borrelia burgdorferi s.l.: seasonal and annual variations. [in Polish] Wiad Parazytol 50: Perez D, Kneubuhler Y, Rais O and Gern L (2012) Seasonality of Ixodes ricinus ticks on vegetation and on rodents and Borrelia burgdorferi sensu lato genospecies diversity in two Lyme borreliosis-endemic areas in Switzerland. Vector- Borne Zoonotic Dis 12: Perret JL, Guigoz E, Rais O and Gern L (2000) Influence of saturation deficit and temperature on Ixodes ricinus tick questing activity in a Lyme borreliosis-endemic area (Switzerland). Parasitol Res 86: Plard F, Gaillard J-M, Coulson T, Hewison AM, Delorme D, Warnant C and Bonenfant C (2014) Mismatch between birth date and vegetation phenology slows the demography of roe deer. PLoS Biol 12: e Randolph SE (2001) The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme borreliosis in Europe. Philos Trans R Soc Lond B Biol Sci 356: Randolph SE, Green RM, Hoodless AN and Peacey MF (2002) An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus. Int J Parasit 32: Randolph SE and Storey K (1999) Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission. J Med Entomol 36: Rauter C and Hartung T (2005) Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a metaanalysis. Appl Environ Microbiol 71: Semenza JC, Suk JE, Estevez V, Ebi KL and Lindgren E (2012) Mapping climate change vulnerabilities to infectious diseases in Europe. Environ Health Perspect 120: Sonenshine DE (2006) Tick pheromones and their use in tick control. Annu Rev Entomol 51: Sonenshine DE and Roe RM (eds.) (2013) Biology of ticks, Volume 1. Oxford University Press, New York, NY, USA, pp Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJH, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Tack W, Madder M, Baeten L, De Frenne P and Verheyen K (2012) The abundance of Ixodes ricinus ticks depends on tree species composition and shrub cover. Parasitology 139: Tagliapietra V, Rosà R, Arnoldi D, Cagnacci F, Capelli G, Montarsi F, Hauffe HC and Rizzoli A (2011) Saturation deficit and deer density affect questing activity and local abundance of Ixodes ricinus (Acari, Ixodidae) in Italy. Vet Parasitol 183: Takken W, Van Vliet AJH, Verhulst NO, Jacobs F, Gassner F, Hartemink N, Mulder S and Sprong H (in press) Acarological risk of Borrelia burgdorferi sensu lato infections across space and time in the Netherlands. Vect Borne Zoon Dis DOI: Van Duijvendijk G, Coipan C, Wagemakers A, Fonville M, Ersöz J, Oei A, Földvári G, Hovius J, Takken W and Sprong H (2016) Larvae of Ixodes ricinus transmit Borrelia afzelii and B. miyamotoi to vertebrate hosts. Parasit Vectors 9: 97. Van Duijvendijk G, Sprong H and Takken W (2015) Multi-trophic interactions driving the transmission cycle of Borrelia afzelii between Ixodes ricinus and rodents: a review. Parasit Vectors 8: 643. Wright WF, Riedel DJ, Talwani R and Gilliam BL (2012) Diagnosis and management of Lyme disease. Am Fam Physician 85: Zemek R, Bouman EAP and Dusbábek F (2007) The influence of conspecific chemical cues on walking behavior of Ixodes ricinus males. Exp Appl Acarol 41: Ecology and prevention of Lyme borreliosis 157

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162 11. How landscapes shape Lyme borreliosis risk Lucy Gilbert The James Hutton Institute, Macaulay Drive, Craigiebuckler, Aberdeen AB15 8QH, United Kingdom; Abstract Landscapes are topographically, geologically and biotically heterogeneous and the spatial pattern of these factors influences local climates, habitats and vertebrate distribution and abundance over the landscape. The interaction between these factors results in a heterogeneous distribution in tick abundance, Borrelia burgdorferi s.l. prevalence and Lyme borreliosis risk. This chapter examines how the spatial distribution of these factors, and their interactions, help shape Lyme borreliosis hazard over landscapes, and introduces some modelling approaches that integrate these spatial factors and identifies important gaps in our knowledge that need to be filled if we are to better predict disease risk over landscapes. Keywords: Borrelia, deer, habitat, hosts, landscapes, spatial modelling, ticks, woodlands Introduction Tick abundance, Borrelia burgdorferi s.l. prevalence and the risk of Lyme borreliosis are highly heterogeneous: they are not evenly distributed in space or time, but vary dramatically from place to place. What is it about a landscape that determines distribution, abundance and risk, how do these change over time, and what are the implications of landscape management, implemented for other human objectives, on Lyme borreliosis hazard? In this chapter, the term hazard refers to the source of potential pathogen infection, and the density of infected ticks is the most commonly used measure of this. Risk, however, also includes the human element, for example, there is higher risk of infection to the human population if more humans use an area, but human usage does not alter the source (or hazard) of infection. Perhaps the most fundamental aspect of many landscapes is how landscape can determine climate, particularly due to varying altitudes (i.e. landscapes with mountains), but also the angle of slopes and the amount of shade which affects local micro-climate. Since tick activity, like that of most terrestrial arthropods, is limited by cold temperatures as well as by high saturation deficits (a function of temperature and humidity) (Perret et al. 2000, Randolph 2004, Gilbert et al. 2014), the physical form of a landscape can shape the fundamental climate envelope that determines the possible distribution and abundance of ticks (Gilbert 2010, Jouda et al. 2004). Within this climate envelope, however, are layered factors that refine the distribution and abundance of ticks and B. burgdorferi s.l.: habitats and land use, that affect local micro-climates and potential host distributions; and another layer within that, which is the actual densities of each host species, which are influenced by detailed habitat characteristics, ecological interactions within each habitat, and anthropogenic activities and management. Clearly, even in a climate perfect for ticks, if there is no habitat suitable for hosts, there can be no ticks or B. burgdorferi s.l.. Furthermore, within a suitable climate and even with habitat suitable for hosts, if there are no hosts, e.g. due to anthropogenic activities, there can be no ticks; or if there are only tick reproduction hosts such as deer, but no B. burgdorferi s.l. transmission hosts such as rodents or birds, there may be ticks but there is unlikely to be any B. burgdorferi s.l. Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _11, Wageningen Academic Publishers 2016

163 Lucy Gilbert This chapter therefore addresses the key constituents of a landscape that affect ticks, B. burgdorferi s.l. and Lyme borreliosis risk, including climate, habitat, hosts and anthropogenic effects, and how these factors may interact in a landscape to impact Lyme borreliosis hazard. Landscape-driven climate As a rule, average temperatures decline with increasing elevation, known as the environmental lapse rate. This varies depending on humidity but a typical value used, the global mean environmental lapse rate, is a drop of 6.5 C for every 1000 m elevation (Barry and Chorley 1987, p. 56). Climate is also cooler in areas shaded from the sun, such as north-facing slopes in the Northern Hemisphere, again due to the physical shape of a landscape. Tick interstadial development, oviposition rate and egg development rates are slower in cooler temperatures (MacLeod 1935b, Randolph et al. 2002). In addition, several studies have quantified how tick activity is inhibited by cool temperatures as well as with high saturation deficits (the drying power of the air, a function of both temperature and relative humidity; e.g. Perret et al. 2000, 2004, Randolph 2004, Gilbert et al. 2014). Ixodes ricinus ticks are remarkably able to survive in cold temperatures almost down to -15 C, but mortality starts to increase rapidly at temperatures above 30 C, even with high (>80%) humidities (MacLeod 1935a). We therefore expect the shape of the landscape, through affecting climate, to set the outer boundaries dictating the maximum potential distribution for ticks, and therefore also B. burgdorferi s.l. (there can be no B. burgdorferi s.l. and Lyme borreliosis risk where there are no ticks). Further studies show how the effect of temperature on tick development, survival and activity translates into a general decline in tick abundance at higher altitudes in real landscapes (Jouda et al. 2004, Barandika et al. 2006, Cadenas et al. 2007, Burri et al. 2007, Gilbert 2010, Qviller et al. 2013). However, the elevation at which ticks become scarce varies depending on other climatedriving factors such as latitude. For example, in the northern hemisphere the altitudinal limit for ticks is lower in colder northern countries than for warmer southern countries: in western Norway ticks are scarce above approximately 400 m above sea level (Qviller et al. 2013), in NE Scotland the limit is around 550 m (Gilbert 2010; Figure 1), while areas with much warmer summers such Figure 1. Altitude and local climate are closely linked and set the potential outer limits for tick distribution (and therefore Lyme borreliosis risk) across landscapes. In Scotland, pictured here, ticks become scarce above 550 m and are absent at the top of these mountains at 1000 m; the altitudinal limit is higher in countries with warmer climates (photo by Lucy Gilbert). 162 Ecology and prevention of Lyme borreliosis

164 11. How landscapes shape Lyme borreliosis risk as central Europe (northern Italy, Switzerland, Czech Republic) ticks do not become scarce until 1,100-1,500 m (Burri et al. 2007, Cadenas et al. 2007,Daniel et al. 2009, Rizzoli et al. 2002). In addition to latitude, slope aspect affects local climate, such that in the northern hemisphere north facing slopes are cooler than south facing slopes while south facing slopes can have higher saturation deficit (greater drying power), and this can create different tick abundance patterns between north- and south-facing slopes (Cadenas et al. 2007). Further landscape factors that drive climate and thus affect tick abundance and distribution include proximity to the ocean; maritime climates are characterised by cooler and wetter summers and warmer winters which promotes tick survival and activity during a longer season, while continental climates are characterised by hotter drier summers and colder winters, which can concentrate tick seasonal phenology. For example, in Norway distance from the coast is a major influence on both I. ricinus tick abundance and Lyme borreliosis risk, with considerably more ticks and disease risk nearer the coast than inland (Jore et al. 2011). While the effects of temperature on ticks impose a decrease in tick abundance at higher altitudes, any climate-related altitude effects on the prevalence of B. burgdorferi s.l. in ticks is less easy to predict. There is an argument that higher tick densities could be associated with increased B. burgdorferi s.l. transmission because, if more larvae or nymphs are available feed on transmission hosts (birds and rodents), more uninfected ticks will become infected. Therefore, it may follow that because ticks become scarcer at higher altitudes, prevalence of B. burgdorferi s.l. may also decline as there is a lower transmission potential on transmission hosts. However, the evidence that higher densities of ticks produces higher prevalences of B. burgdorferi s.l. is equivocal, with some studies showing a positive correlation between tick density and B. burgdorferi s.l. prevalence (e.g. Rizzoli et al. 2002) and others finding no such effect (e.g. James et al. 2013). One key factor influencing B. burgdorferi s.l. prevalence must be the densities of transmission hosts, so how these are distributed with altitude will play a role. Theoretical and empirical studies on the effect of host composition on B. burgdorferi s.l. transmission are discussed under the section The effect of host distribution over the landscape later in this chapter. Whatever the mechanism, several studies have found a decline in B. burgdorferi s.l. prevalence with increasing altitude (James et al. 2013, Jouda et al. 2004), while other studies have found no clear altitudinal prevalence pattern (Cadenas et al. 2007). However, in terms of Lyme borreliosis risk, the most important parameter for gauging disease hazard is the density of B. burgdorferi s.l.-infected ticks, a function of both B. burgdorferi s.l. prevalence and questing tick density. Due to the general decline in tick density at higher altitudes, as described above, there is likely to be a decline in Lyme borreliosis hazard (density of infected ticks) with increasing altitude. Indeed, those studies that have investigated the density of infected nymphs over altitude have found a negative relationship, confirming that the hazard of an infected tick bite is lower at higher altitudes (e.g. Rizzoli et al. 2002). The effect of habitat distribution over the landscape It should be emphasised that within the altitudes/climates where ticks thrive, i.e. at altitudes well below the threshold for tick survival, habitat and host communities are likely to be stronger drivers of Lyme borreliosis hazard than climate. This is illustrated by a study in NE France where the lowest altitude site (230 m above sea level) had the lowest B. burgdorferi s.l. infection prevalences in questing ticks and the lowest rates of B. burgdorferi s.l. infection in small mammals, in comparison to sites at m above sea level, and this was attributed to differences in habitat patch size (Ferquel et al. 2006). Ecology and prevention of Lyme borreliosis 163

165 Lucy Gilbert Habitats affect both the micro-climate that ticks are exposed to and the host communities that feed ticks and transmit B. burgdorferi s.l.. Habitats that maintain a mild and humid micro-climate of benefit to tick activity and survival tend to be those with enclosed canopies covering the soil, such as tall and dense leafy vegetation or woodlands. Mammals and birds that host ticks and may (or may not) transmit B. burgdorferi s.l. have habitat preferences or even specific habitat requirements. Generally speaking, the main transmission hosts for the various B. burgdorferi s.l. types (rodents and birds in Europe; rodents in North America) are most numerous in woodlands or mixed habitats with shrubs and trees, which are also habitats that create beneficial micro-habitats for ticks. Therefore, the distribution of these types of habitat over a landscape helps determine the potential distribution of tick hosts and B. burgdorferi s.l. transmission hosts and influence tick survival and activity. Indeed, recently collected data show a striking difference between habitats in Lyme borreliosis hazard (the density of B. burgdorferi s.l.-infected ticks), with five times the hazard in unfenced woodland habitats compared to open habitats (grassland and moorland) in Scotland (see Figure 2 for methods and results of this study). Woodland management, however, may help mitigate this increased risk; for example, Scottish woodlands that are fenced to exclude deer benefit from a 96% reduction in tick density compared to unfenced woodlands that allow access to deer (Gilbert et al. 2012). This highlights another important layer of complexity influencing Lyme borreliosis risk on top of climate and habitat: that of anthropogenic management. Within each habitat category, the vegetation characteristics refine the abundance of ticks and B. burgdorferi s.l. further. For example, within the woodland/forest habitat category, James et al. (2013) documented higher B. burgdorferi s.l. hazard in semi-natural mixed/deciduous woodlands Density of infected nymphs (n/ha) Open habitat n=10 sites Woodland n=16 sites Figure 2. Surveys in Royal Deeside, NE Scotland, show five times the hazard of Borrelia burgdorferi s.l. (density of infected Ixodes ricinus nymphs) in unfenced woodland compared to adjacent open habitats (moorland or grassland). This was a result of both higher questing nymph densities (1.8 versus 0.92 nymphs per 10 m 2 cloth lure transect) and higher B. burgdorferi s.l. prevalence in nymphs (2.5 versus 0.1%). DNA extraction of 960 nymphs (range 0 to 110 per site depending on tick densities at the site) was conducted using the DNeasy Blood & Tissue Kit (QIAGEN) and qpcr was performed using the QuantiNova SYBR Green PCR Kit (QIAGEN). Separate genospecies were not assayed. 164 Ecology and prevention of Lyme borreliosis

166 11. How landscapes shape Lyme borreliosis risk than in coniferous forest. Prusinski et al. (2006) found a higher rate of B. burgdorferi s.l. infection in small mammals in New York State woodlands that had smaller trees (with lower canopy height), increased understorey vegetation cover and dense understory shrubs coverage. It is not just habitat type and detailed vegetation characteristics per se that affect the density of infected ticks, but also how the habitats are distributed across the landscape, i.e. habitat patch size, fragmentation and connectivity. Increased forest fragmentation was found to be associated with higher Lyme borreliosis incidence in Maryland (Jackson et al. 2006). However, in NE France Ferquel et al. (2006) found that a small patch of forest surrounded by fields, i.e. fragmented away from other forest areas, had lower B. burgdorferi s.l. prevalence in questing ticks, lower density of infected ticks and lower Lyme borreliosis incidence reported in humans compared with areas of more continuous forest. Perhaps the apparent disparity is due to the definition of fragmentation and how much connectivity there is between fragmented patches. With respect to habitat connectivity, Estrada-Peña (2003) examined the relationships between habitat connectivity and tick abundance in Spain and found that stepping-stone patches that are crucial to connectivity had the highest tick density; ticks were absent in habitat patches that were far separated from the main network. This was attributed to host movements, i.e. how hosts move between patches of habitat using connecting stepping stones rather than small, isolated habitat patches. Of course habitat on its own does not produce ticks or B. burgdorferi s.l.; it is the hosts that reside in the habitat that are the main drivers of the correlations between habitat types and connectivity/ fragmentation and tick abundance or B. burgdorferi s.l. Some hosts occur or move through a variety of habitats, while identical habitats often have different host communities and host densities due to other factors such as human activity (e.g. deer culling). Therefore, we also need to examine how host distribution and movements through the landscape shape the risk of Lyme borreliosis across landscapes. A formal theoretical investigation of the predicted impacts of host movements and habitat fragmentation on tick-borne disease was conducted by Jones et al. (2011). They used mathematical models to predict the impact of host movements from forest onto moorland on the prevalence of the tick-borne louping-ill virus on moorlands, depending on habitat fragmentation and patch size at a landscape scale. The models predicted higher tick-borne disease risk in moorland with increased fragmentation (i.e. more, and smaller, moorland and forest patches), because this increased the frequency of hosts (deer) carrying ticks from forest (which harbours more ticks than moorland) onto moorland. This type of approach is applicable to other tick-borne pathogens, and highlights the importance of habitat fragmentation interacting with host movements as influences on disease risk. The effect of host distribution over the landscape As highlighted above, how hosts are move across the landscape will influence tick abundance, infection prevalence and the density of infected ticks (the Lyme borreliosis hazard). In terms of tick abundance, large mammals such as deer that carry a lot of ticks, especially adult ticks, are generally the most important driving factor: deer are often the main tick reproduction host, feeding adult ticks which then produce the next generation of immature ticks (Gray 1998). As expected, therefore, many studies show positive correlations between deer abundance and questing tick density, both in eastern North America for white-tailed deer, Odocoileus virginianus (Deblinger et al. 1993, Kilpatrick et al. 2014, Rand et al. 2003, 2004, Stafford 1993, Stafford et al. 2003, Werden et al. 2014, Wilson et al. 1990), and in Europe for roe, Capreolus capreolus, red deer, Ecology and prevention of Lyme borreliosis 165

167 Lucy Gilbert Cervus elaphus, and fallow deer, Dama dama (Estrada-Peña et al. 2015, Gilbert 2010, 2013, Gilbert et al. 2012, Gray et al. 1992, James et al. 2013, Qviller et al. 2013, Rizzoli et al. 2002, Ruiz-Fons and Gilbert 2010). However, deer are not competent transmission hosts for B. burgdorferi s.l. (Jaenson and Tälleklint 1992, Telford et al. 1988, for roe deer, Matuschka et al. 1993, but see Oliver et al for whitetailed deer and Kimura et al for Sika deer Cervus nippon). Therefore, the effect of deer density on B. burgdorferi s.l. prevalence is likely to be equivocal and dependent on many factors, not least the species of deer and the relative density of alternative hosts. With respect to deer abundance affecting B. burgdorferi s.l. prevalence in questing ticks, studies have found positive effects (in Europe: James et al. 2013, Rizzoli et al. 2002), negative effects (in Europe: Gray et al. 1992, Millins 2016, Mysterud et al. 2013,) and no effects (in North America: Ostfeld et al. 2006, Werden et al. 2014; in Europe: Pichon et al. 1999). What does this mean for Lyme borreliosis hazard (density of infected ticks) or Lyme borreliosis incidence in humans? Although studies do not agree on the patterns of deer affecting B. burgdorferi s.l. prevalence in questing ticks, many studies do find that higher deer densities produce higher tick populations, so perhaps we might expect the density of infected ticks (i.e. the hazard) or the incidence of Lyme borreliosis to also increase with deer density (or at least have no relationship if increased deer densities reduced prevalence greatly). Indeed, from the few available studies so far that have reported the density of infected ticks, or have reported both tick abundance and B. burgdorferi s.l. prevalence so that relative hazard can be surmised, there are generally positive effects of deer abundance on Lyme borreliosis hazard or incidence (in North America: Garnett et al. 2011, Kilpatrick et al. 2014; in Europe: James et al. 2013, Mysterud et al. 2016, Rizzoli et al. 2002). No studies have yet reported negative effects although some studies from North America have not found any effect of white-tailed deer on Lyme borreliosis hazard or incidence (Jordan et al. 2007, Ostfeld et al. 2006). This area clearly requires further research dedicated to Lyme borreliosis hazard or incidence, covering a full and wide range of deer densities as well as, importantly, densities (or proxies) of transmission hosts (birds and rodents). This is needed because there is likely to be an interaction between deer and transmission host densities in determining B. burgdorferi s.l. prevalence and Lyme borreliosis hazard, since prevalence and hazard are likely to be affected by the proportion of immature ticks that feed on transmission hosts (birds and rodents) compared to non-transmission hosts (such as deer), while tick density is often driven by deer abundance, as they are usually the main tick reproduction hosts (Gray 1998). Such studies are difficult because assessments of bird and rodent densities are extremely time-consuming and notoriously difficult. Therefore, proxies are usually used instead, to estimate very approximate abundance indices or categories. For example, habitat category is sometimes used as a proxy, with semi-natural mixed or deciduous woodlands assumed to have relatively high densities of rodents and birds, in contrast to, for example, commercial conifer plantations or heather moorland that are assumed to relatively low densities of rodents and birds. While such rough proxies can be broadly useful, it must be understood that, importantly, there are several B. burgdorferi s.l. species and each has different host type associations and, therefore, different spatial distributions across landscapes (e.g. Estrada-Peña et al. 2011, James et al. 2014). Furthermore, even within a particular B. burgdorferi s.l. genospecies there are many competent transmission host species and each will have a different importance in the B. burgdorferi s.l. transmission cycle and each also has slightly different habitat requirements. For example, not all bird species are equally important in contributing to the transmission cycle of Borrelia garinii (which is a B. burgdorferi s.l. genospecies associated with birds): James et al. (2011) found that 166 Ecology and prevention of Lyme borreliosis

168 11. How landscapes shape Lyme borreliosis risk ground foragers such as European blackbirds and song thrushes were around ten times more important than other bird species in contributing B. burgdorferi s.l. infection to ticks in Scotland. Therefore, for bird-associated B. burgdorferi s.l. genospecies (B. garinii and B. valaisiana) the relevant habitat proxy needs to be those habitats preferred by European blackbirds and song thrushes. Modelling and mapping B. burgdorferi s.l. hazard over landscapes The distribution over the landscape of multiple factors such as climate parameters, vegetation characteristics and hosts can be combined together using various modelling approaches to predict Lyme borreliosis hazard or risk across landscapes. There are growing numbers of studies taking this approach, and they can be valuable for testing methodological approaches, identifying gaps in the data necessary for more robust modelling and, hopefully, for actually making spatial predictions about areas of higher risk. Some modelling studies first use statistics to determine correlations between empirical tick abundance data or Lyme borreliosis cases as a basis and environmental variables at those locations, then use the correlation algorithm to predict risk over entire landscapes. For example, Rizzoli et al. (2002) and Altobelli et al. (2008) mapped Lyme borreliosis risk in northern Italy using climatic, topographic and biotic data. An alternative approach is to use agent-based models or susceptible-infectious (SI) dynamics models that model the tick or pathogen population dynamics in relation to temperature or hosts (for example); then these are run using temperature values over a landscape to create risk maps. A recent example is Li et al. (2016) who used agent-based models of tick seasonality and B. burgdorferi s.l. according to temperature and hosts to predict Lyme borreliosis risk over Scotland both currently and under climate warming. All models must be used with caution as they are, by definition, simplifications of the real world. A useful recent review paper outlined the tick-borne disease models to date, their practical uses and pitfalls (Norman et al. 2016). It is particularly difficult to validate predictions made for circumstances that fall outside the values used to parameterise the models; for example, predictions into future climates in areas that do not yet experience such temperatures, or predictions into new geographic areas. Model predictions that have been validated using additional data sets can be trusted more than those that have no form of validation. Models that conduct sensitivity analyses can be useful in identifying which parameters cause the largest effect on predicted disease risk, so that future research can focus on these parameters. The accuracy of model predictions depends heavily on the accuracy of the parameter values put into the model, and the process of building disease models can therefore help identify which parameters have detailed and robust empirical data behind them and which parameters are in need of further research. Essential fundamental information that is still unknown includes the transmission efficiency of pathogens between hosts and ticks and even the proportion of ticks fed by each host type is difficult to estimate. This is because of the time and resources needed to gauge the densities of each host species and the number of ticks biting each host species per year, which is required at the same time and at the same sites. From the vector perspective, models have identified that basic biological information is still required such as birth and death rates of ticks in different habitats and different climates. So, while validated disease risk models based on good empirical data can be of practical use to land managers, health professionals and the public, there are still many fundamental gaps in our knowledge that are needed to improve the accuracy of disease risk models. In conclusion, the physical form and land management across landscapes determines the climate, habitat and host distribution, which in turn influences tick abundance, B. burgdorferi s.l. prevalence Ecology and prevention of Lyme borreliosis 167

169 Lucy Gilbert and Lyme borrieliosis hazard over the landscape (Figure 3). Detailed data on these factors allow various modelling techniques to combine these factors to create risk maps over landscapes, for both now and under scenarios of environmental changes. However, landscapes are heterogeneous and complex, and there is still much we don t understand about how host movements in relation to landscape heterogeneity drive disease spread, or how mixtures of host communities drive B. burgdorferi s.l. prevalence or pathogen hazard. A further layer of complexity human behaviour and activity over landscapes adds a further challenge to how we can predict the actual risk of Lyme borreliosis infection over and above the density of infected ticks. Figure 3. The spatial distribution of climate, habitats and hosts, including fragmentation and connectivity, across heterogeneous landscapes influences tick abundance and Lyme borreliosis hazard. In this picture (Lake District National Park, NW England), assuming free roaming deer, we might predict highest hazard in the extensive continuous woodland in the background, and no risk on these mountain tops (800 m above sea level) (photo by Lucy Gilbert). Public health relevance Understanding the pattern of Lyme borreliosis risk over a landscape can help in personal risk assessment and mitigation. Landscape planning such as building new woodlands or expansion of urban areas should take into account this knowledge of disease risk patterns over the landscape. Disease awareness campaigns and control strategies can be targeted more effectively in particularly high risk parts of the landscape. Landscape scale disease risk models can predict changes in risk with environmental changes for various landscape features, aiding policy on future disease risk to the public. 168 Ecology and prevention of Lyme borreliosis

170 11. How landscapes shape Lyme borreliosis risk References Altobelli A, Boemo B, Mignozzi K, Marialisa B, Floris R, Menardi G and Cinco M (2008) Spatial Lyme borreliosis risk assessment in north-eastern Italy. Int J Med Microbiol S1: Barandika JF, Berriatua E, Barral M, Juste RA, Anda P and García-Pérez AL (2006) Risk factors associated with ixodid tick species distributions in the Basque region in Spain. Med Vet Entomol 20: Barry RG and Chorley RJ (1987) Atmosphere, weather and climate. Routledge, London, UK, p.56. Burri C, Cadenas FM, Douet V, Moret J and Gern L (2007) Ixodes ricinus density and infection prevalence of Borrelia burdorferi sensu lato along a north-facing altitudinal gradient in the Rhone valley (Switzerland). Vector Borne Zoonotic Dis 7: Cadenas FM, Rais O, Jouda F, Douet V, Humair P-F, Moret J and Gern L (2007) Phenology of Ixodes ricinus and infection with Borrelia burgdorferi sensu lato along and north- and south-altitudinal gradient on Chaumont mountain, Switzerland. J Med Entomol 44: Daniel M, Materna J, Honig V, Metelka L, Danielová V, Harcarik J, Kliegrová S and Grubhoffer L (2009) Vertical distribution of the tick Ixodes ricinus and tick-borne pathogens in the northern Moravian mountains correlated with climate warming (Jeseniky mountains, Czech Republic). Cent Eur J Public Health 17: Deblinger RD, Wilson ML, Rimmer DW and Spielman A (1993) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following incremental removal of deer. J Med Entomol 30: Estrada-Peña A (2003) The relationships between habitat topology, critical scales of connectivity and tick abundance Ixodes ricinus in a heterogeneous landscape in northern Spain. Ecography 26: Estrada-Peña A, de la Fuente J, Ostfeld RS and Cabezas-Cruz A (2015) Interactions between ticks and transmitted pathogens evolved to minimise competition through nested and coherent networks. Sci Rep 5: Estrada-Peña A, Ortega C, Sánchez N, DeSimone L, Sudre B, Suk JE and Semenza JC (2011) Correlation of Borrelia burgdorferi sensu lato prevalence in questing Ixodes ricinus ticks with specific abiotic traits in the western Palearctic. App Environ Microbiol 77: Ferquel E, Garnier M, Marie J, Bernèd-Bauduin C, Baranton G, Pérez-Eid C and Postic D (2006) Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France. Appl Env Microbiol 72: Garnett JM, Connally NP, Stafford KC and Cartter ML (2011) Evaluation of deer targeted interventions on Lyme disease incidence in Connecticut. Public Health Reports 126: Gilbert L (2010) Altitudinal patterns of tick and host abundance a potential role for climate change in regulating tickborne diseases? Oecologia 162: Gilbert L (2013) Can restoration of afforested peatland regulate pests and disease? J Appl Ecol 50: Gilbert L, Aungier J and Tomkins JL (2014) Climate of origin affects tick (Ixodes ricinus) host-seeking behaviour in response to temperature: implications for resilience to climate change? Ecol Evol 4: Gilbert L, Maffey GL, Ramsay SL and Hester AJ (2012) The effect of deer management on the abundance of Ixodes ricinus in Scotland. Ecol Appl 22: Gray JS, (1998) The ecology of ticks transmitting Lyme borreliosis. Exp Appl Acarol 22: Gray JS, Kahl O, Janetzki C and Stein J (1992) Studies on the ecology of Lyme disease in a deer forest in County Galway, Ireland. J Med Entomol 29: Jackson LE, Hilborn ED, Thomas JC (2006) Towards landscape design guidelines for reducing Lyme disease risk. Int J Epidemiol 35: Jaenson TGT and Tälleklint L (1992) Incompetence of roe deer (Capreolus capreolus) as reservoirs of the Lyme disease spirochete. J Med Entomol 29: James MC, Bowman AS, Forbes KJ, Lewis F, McLeod JE and Gilbert L (2013) Environmental determinants of Ixodes ricinus ticks and the incidence of Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in Scotland. Parasitol 140: James MC, Furness RW, Bowman AS, Forbes KJ and Gilbert L (2011) The importance of passerine birds as tick hosts and in the transmission of Borrelia burgdorferi, the agent of Lyme disease: a case study from Scotland. Ibis 153: Ecology and prevention of Lyme borreliosis 169

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174 12. The role of host diversity in Borrelia burgdorferi s.l. dynamics Tim R. Hofmeester Resource Ecology Group, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; Abstract There has been substantial debate about the influence of vertebrate host diversity on Lyme borreliosis risk. In North America, studies investigating Borrelia burgdorferi s.l. and the Blacklegged tick (Ixodes scapularis) have shown that on a large spatial scale there seems to be a negative correlation between host species diversity and Lyme borreliosis risk. However, studies on this relationship in Europe are lacking. I discuss the work done in North America and translate the findings and assumptions of these studies to the European situation, where the sheep tick (Ixodes ricinus) is the most important vector of B. burgdorferi s.l. The European situation is fundamentally different compared to the North American situation due to the high diversity of B. burgdorferi s.l. genospecies, which are transmitted by different groups of vertebrate species. Disease risk in Europe is hypothesised to increase with vertebrate diversity due to an increase in B. burgdorferi s.l. genospecies diversity. However, it seems that the majority of genospecies in Europe is transmitted by two functional groups of host species, rodents and thrushes, which are present in most vertebrate assemblages. Therefore, it seems plausible that a dilution effect can also occur in Europe. This might result in high risk in urban areas where a few dominant species are very abundant, among which the most important reservoir hosts for B. burgdorferi s.l. in Europe. Keywords: biodiversity, dilution effect, host behaviour, host density, Ixodes ricinus, Ixodes scapularis, urbanisation Introduction The presence and abundance of ticks is determined by the combination of a suitable microclimate and the presence and abundance of vertebrate host species, which is mainly present in forested areas (Randolph 2004). Tick-borne pathogens are transmitted to ticks by these host species and host species differ in their ability to transmit pathogens (Randolph 2009). Therefore, it seems logical that differences in tick-borne disease risk are related to differences in vertebrate assemblage composition. Vertebrates play an important role as (reservoir) hosts for both ticks from the Ixodes ricinus complex and Borrelia burgdorferi s.l. (Coipan and Sprong 2016, Földvári 2016, Heylen 2016, Hofmeester et al. 2016, Szekeres et al. 2016, Van Duijvendijk et al. 2016, Van Wieren and Hofmeester 2016). These host species differ in their quality as hosts for ticks and in their competence in transmitting B. burgdorferi s.l. Ticks can better feed from one host species than the other because of differences in grooming behaviour (Keesing et al. 2009) and immunological responses of hosts to feeding ticks (Dizij and Kurtenbach 1995). At the same time, some host species are better at transmitting B. burgdorferi s.l. to feeding ticks (i.e. have a higher reservoir competence) than other species (Kurtenbach et al. 1994), which differs for the different B. burgdorferi s.l. genospecies (Kurtenbach et al. 1998). Therefore, changes in host composition can change the number of ticks in the environment, the number of ticks infected with B. burgdorferi s.l. and the number of genospecies found in any tick population (Hofmeester et al. 2016). In order to better understand the relationship between vertebrate host composition and Lyme borreliosis risk, I will try to answer the following three questions: (1) Which vertebrate species are Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _12, Wageningen Academic Publishers 2016

175 Tim R. Hofmeester the best hosts for the most important vectors of B. burgdorferi s.l. in North America and Europe?; (2) Which vertebrate species have the highest reservoir competence for B. burgdorferi s.l.?; and (3) How are these species represented in different vertebrate assemblages? To answer these questions, I will first discuss the initial work on the relationship between host composition and Lyme borreliosis risk performed in North America, as most work on this subject has been done there. Afterwards, I will translate the North American findings to the European situation and relate this to the work that has been done in Europe. In the final section I will speculate about future changes in vertebrate distribution and abundance in Europe and the possible effects on Lyme borreliosis risk. It all started in North America Lyme borreliosis was first described in North America, where it was soon discovered that the disease was caused by a spirochaete transmitted by Ixodes spp. ticks (Burgdorfer et al. 1982). The most important vector in North America, the black-legged tick (Ixodes scapularis), was found to mainly parasitise white-footed mice (Peromyscus leucopus) as larva (Piesman and Spielman 1979) and white-tailed deer (Odocoileus virginianus) as adult (Watson and Anderson 1976). Humans are mainly bitten and infected by nymphal black-legged ticks, and therefore, the density of nymphs infected with B. burgdorferi s.l. was proposed as an important measure determining disease risk (Falco and Fish 1989). As a consequence, the white-footed mouse was pinpointed as the most important host determining disease risk for Lyme borreliosis, as the majority of infected nymphs had probably fed from this species (Mather et al. 1989). A theoretical study showed that although deer are important hosts maintaining black-legged tick populations, it is mainly the density of hosts for immature ticks that determines the density of nymphs, B. burgdorferi s.l. nymphal infection prevalence (NIP) and eventually the density of infected nymphs (DIN; Van Buskirk and Ostfeld 1995). This is because, as soon as hosts for adult ticks are present, the number of hosts for the immature stages become limiting. Furthermore, Van Buskirk and Ostfeld (1995) showed that NIP was dependent on the ratio of species with high reservoir competence and species with low reservoir competence, while DIN was mainly influenced by the density of highly competent reservoir hosts. As a consequence of this model, most work on the influence of vertebrate hosts on NIP and DIN for B. burgdorferi s.l. has focused on hosts for immature black-legged ticks and their reservoir competence, especially because white-tailed deer (as hosts for adult black-legged ticks) are present in most forests (Augustine and Jordan 1998). Based on the high reservoir competence for larvae of white-footed mice compared to other host species (Mather et al. 1989), and the fact that white-footed mice are omnipresent, Ostfeld and Keesing (2000a) proposed the dilution effect hypothesis. The dilution effect hypothesis states that disease risk increases with loss of biodiversity and was based on the ecology of Lyme borreliosis in North America (Ostfeld and Keesing 2000a). This hypothesis is based on the assumptions that: (1) white-footed mice are present in all vertebrate assemblages; (2) white-footed mice are the species with the highest reservoir competence; and (3) the distribution of ticks over the different host species is dependent on the relative abundance of these host species. In a species-poor assemblage, the relative proportion of white-footed mice was hypothesised to be highest resulting in a high proportion of the larvae feeding on this host species, and a high NIP and DIN. In contrast, in a species-rich assemblage, the relative proportion of white-footed mice was hypothesised to be lower, resulting in a lower proportion of the larvae feeding on this host species, and a lower NIP and DIN. A large scale study showed that Lyme borreliosis incidence was highest in states 174 Ecology and prevention of Lyme borreliosis

176 12. Host diversity and Borrelia burgdorferi with a low diversity of mammals and reptiles, but a high diversity of birds (Ostfeld and Keesing 2000a). Another early field test of this hypothesis showed that NIP was lower than expected if all larvae would feed on white-footed mice and the eastern chipmunk (Tamias striatus), the two species with the highest reservoir competence (Schmidt and Ostfeld 2001). Schmidt and Ostfeld (2001) calculated that as many as 60% of the larvae had to feed on other host species to obtain the observed NIP, showing that indeed the presence of other host species could reduce tick-borne disease risk. Furthermore, the results of Schmidt and Ostfeld (2001) suggest that although whitefooted mice and eastern chipmunks feed an important proportion of black-legged tick larvae (~40%), a substantial amount of larvae take their blood meal from other host species. Subsequent studies have shown that white-footed mice are indeed the species with the highest reservoir competence in North America (LoGiudice et al. 2003), and they were present in all fragments in a multi-state study investigating the relationship between host species diversity and Lyme borreliosis risk (LoGiudice et al. 2008). Furthermore, white-footed mice were found to be the least effective groomers (Keesing et al. 2009). Therefore, white-footed mice indeed seem to be the most ubiquitous species, which are the best host for both black-legged ticks and at the same time have the highest reservoir competence for B. burgdorferi s.l. However, using genetic techniques, Brisson et al. (2008) showed that only ~25% of black-legged nymphs infected with B. burgdorferi sensu stricto (s.s.) got their infection from white-footed mice, and that the more difficult to study shrews, masked shrew (Sorex cinereus) and short-tailed shrew (Blarina brevicauda) actually infected ~50% of the infected nymphs. Also, they estimated that ~50% of larvae get their blood meal from host species with a low reservoir competence (Brisson et al. 2008). This shows that multiple species of small mammal are important reservoir-competent hosts for B. burgdorferi s.s. in North America, and that other host species can indeed dilute the infection prevalence in questing black-legged ticks. However, it was still the question if adding or loosing host species would really change the distribution of ticks over the different host species, or if it would only mean that a different number of ticks would find a host. Keesing et al. (2009) presented a model in which they included feeding success of larval blacklegged ticks on six vertebrate host species in North America. They started with six host species and subtracted the species according to empirical data on host species presence in fragmented landscapes. The most reservoir competent species, the white-footed mouse and the eastern chipmunk were the last species to be left in the community, as these were generally present even in the most fragmented landscapes (LoGiudice et al. 2008). The modelling results showed that when subtracting less reservoir competent species, only 10% of larvae needed to be redistributed to the other host species for the DIN to increase. This showed that, in theory, only a fraction of the ticks that would otherwise feed on the added species need to be redistributed in order for a dilution effect to occur. In a recent study, Levi et al. (2016) tried to find empirical evidence for the percentage of ticks that redistribute over other hosts by looking at natural variations in white-footed mouse densities and testing for a correlation between mouse densities and the number of black-legged ticks feeding on white-footed mice. They found that both larval and nymphal burden decreased with rodent density and interpreted this as a redistribution of the same number of ticks over more host individuals with increasing density (Levi et al. 2016). However, the same correlation could be caused by changes in behaviour of white-footed mice with increasing density (Hofmeester 2016). These results show that there seems to be a mechanism reducing the encounter rate between white-footed mice and larval and nymphal black-legged ticks which is related to the presence and abundance of different vertebrate species in an assemblage. However, it is still unsure if this is caused by other hosts catching away ticks from the most reservoir competent host species, or if this is caused by competition or predation-induced changes in host behaviour. Ecology and prevention of Lyme borreliosis 175

177 Tim R. Hofmeester Although the generality of the dilution effect hypothesis has been questioned within the framework of Lyme borreliosis (Randolph and Dobson 2012, Wood and Lafferty 2013), recent work in North America showed that the risk of obtaining Lyme borreliosis is correlated with biodiversity. Turney et al. (2014) showed that in the USA, the incidence of Lyme borreliosis was negatively correlated with mammal host species richness and that this correlation was stronger in the most recent time period. I used the same data sources as Turney et al. (2014) to recreate their analysis with the most up to date data. Lyme borreliosis incidence in 2014 in 35 states in the western USA was negatively correlated with mammal host species richness (generalised linear model with negative binomial distribution and log link: β=-0.24, P=0.004; Figure 1). Lyme borreliosis is spreading towards the north in North America, and a study in Canada showed that there was a positive correlation between Peromyscus spp. density and the number of nymphal black-legged ticks infected with B. burgdorferi s.l., while the number of infected nymphs was negatively correlated to small mammal species richness (Werden et al. 2014). However, there is not always a negative correlation between species richness and DIN. In a study comparing speciespoor islands with more species-rich mainland sites, there was no difference in NIP, while DIN was higher in the mainland sites (States et al. 2014). These results show that it is very difficult to infer mechanisms from large-scale studies. For example, it might be that there are more people recreating in areas with low mammal species richness, leading to an increase in Lyme borreliosis incidence. Furthermore, the identity of the species added or subtracted from a community is very important (Randolph and Dobson 2012), which was acknowledged as one of the assumptions for a dilution effect to occur (Ostfeld and Keesing 2000b). Therefore, it is important to study the presence/absence and densities of specific host species at a small spatial scale to better understand the mechanisms behind a possible dilution effect at a larger spatial scale. Several small mammals (eastern chipmunk, masked shrew, short-tailed shrew, and white-footed mouse,) are clearly the most important host species feeding larval black-legged ticks and infecting these ticks with B. burgdorferi s.l. (Brisson et al. 2008, Levi et al. 2016). It is also clear that these 100 USA 500 Europe LB incidence (new cases per 100,000 population) Mammal host species richness Figure 1. Correlation between Lyme borreliosis (LB) incidence and the species richness of mammal hosts in (A) North America and (B) Europe. Solid lines represent the outcomes of two generalised linear models. 176 Ecology and prevention of Lyme borreliosis

178 12. Host diversity and Borrelia burgdorferi species are present everywhere, even in the most fragmented landscapes, and therefore, it seems self-evident that the risk of getting Lyme borreliosis in North America is indeed related to the number of larvae feeding on small mammals. There is, however, mixed evidence for a dilution effect of host species diversity both on a large and small spatial scale. The big question that still remains is: Can all the work done on biodiversity, different vertebrate hosts, black-legged ticks and B. burgdorferi s.l. be translated to the European situation? Translation to Europe There are two important differences between North America and Europe with respect to ticks and B. burgdorferi s.l. First, in Europe, the most important vector for B. burgdorferi s.l. is another tick species, namely the sheep tick (I. ricinus). However, this should not make a very big difference as the sheep tick is closely related to the black-legged tick (Xu et al. 2003) and both species have a similar ecology (Gray 1998). Secondly, in Europe, a distinction is made between the different genospecies of B. burgdorferi s.l., which are transmitted by different groups of host species (Kurtenbach et al. 2002). Therefore, the correlation between mammal species richness and Lyme borreliosis risk (Ostfeld and Keesing 2000a, Turney et al. 2014, Werden et al. 2014) and all findings related to the mechanisms behind this correlation in the North American system (e.g. Keesing et al. 2009, Levi et al. 2016, LoGiudice et al. 2008) cannot be directly translated to the European situation. I will try to pinpoint the important differences and possible similarities between the two systems and the implications for a possible dilution effect and the role of vertebrate hosts in determining B. burgdorferi s.l. infection risk for humans in Europe. The ecology of the sheep tick is very similar to that of the black-legged tick (Gray 1998). Small mammals such as the wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis) and bank vole (Myodes glareolus) seem to be the most important host species feeding the larval stage and deer such as roe deer (Capreolus capreolus), red deer (Cervus elaphus) and fallow deer (Dama dama) seem to be the most important host species feeding the adult stage (Coipan and Sprong 2016, Földvári 2016, Heylen 2016, Hofmeester et al. 2016, Szekeres et al. 2016, Van Duijvendijk et al. 2016, Van Wieren and Hofmeester 2016). Therefore, host species richness might not be well correlated to densities of hosts for the two stages, as a system with roe deer and wood mice might function similarly to a system with three rodent species and two deer species. The most important difference between North America and Europe in terms of hosts for ticks is, that in Europe, the majority of sheep tick nymphs seem to feed on birds (Földvári 2016, Heylen 2016, Hofmeester et al. 2016) making birds more important as hosts for ticks in the European compared to the North American situation. Another major difference between most studies in North America and the European situation is that in Europe differences in ecology between the genospecies of B. burgdorferi s.l. have been acknowledged for some time (Kurtenbach et al. 1998), while most studies in North America do not distinguish between genospecies (e.g. LoGiudice et al. 2003, Werden et al. 2014). It might be that the differentiation between genospecies is not necessary in the north-eastern part of North America as the most abundant genospecies in questing black-legged ticks is B. burgdorferi s.s. (Margos et al. 2012). In Europe, several genospecies have been found in questing sheep ticks, which are associated with different host groups: Borrelia afzelii and Borrelia bavariensis are associated with rodents, Borrelia spielmanii with dormice and hedgehogs, Borrelia lusitaniae with lizards and Borrelia garinii and Borrelia valaisiana with birds (Margos et al. 2012). Due to this host differentiation of B. burgdorferi s.l. in Europe, NIP and DIN of B. burgdorferi s.l. might not be the best measure for Lyme borreliosis risk as different genospecies have different clinical manifestations Ecology and prevention of Lyme borreliosis 177

179 Tim R. Hofmeester (Coipan and Sprong 2016, Coipan et al. 2016). Therefore, it might be better to look at the NIP and DIN for each genospecies separately, and it has been suggested that disease risk might increase with host diversity, as more host species might result in a higher genospecies richness (Ruyts et al. 2016). The most prevalent B. burgdorferi s.l. genospecies in Europe are B. afzelii, B. garinii and B. burgdorferi s.s. (Rauter and Hartung 2005). These genospecies are transmitted by rodents and birds, making the presence and abundance of these species groups the most important determinants of Lyme borreliosis risk (Hofmeester et al. 2016). Furthermore, the two genospecies, B. garinii and B. bavariensis, that cause neuroborreliosis, the most severe clinical manifestation of Lyme borreliosis, are transmitted by birds and rodents (Coipan et al. 2016, Margos et al. 2012). In order for a dilution effect to occur, a distinction could be made between rodent-transmitted Borrelia spp. and birdtransmitted Borrelia spp., and in both cases the assumptions made by Ostfeld and Keesing (2000b) should be met: (1) there is variation in reservoir competence between host species; (2) the most reservoir competent host species are present in vertebrate assemblages with a low host diversity; and (3) ticks feed on hosts relative to the density of the different host species (Figure 2). For rodent-transmitted Borrelia spp. the first two assumptions are met because there are differences between host species in reservoir competence and the species with the highest reservoir competence are all small rodents present in many forests in Europe (Hofmeester et al. 2016). Therefore it is important to know if the number of ticks feeding on each rodent is related to host diversity. As far as I know, there has only been one study on the correlation between host diversity and the number of ticks feeding on rodents in Europe. Krasnov et al. (2007) showed that the number of immature sheep ticks feeding on rodents was negatively correlated with rodent species richness. However, rodent species richness was highly correlated with rodent density, making it A B Figure 2. Schematic representation of the dilution effect hypothesis for vertebrate assemblages and the sheep tick in Europe. (A) A vertebrate assemblage with low species richness in which both black birds and wood mice are present in high density, resulting in a high density of infected nymphs with both rodent-transmitted Borrelia spp. (red spirochaetes) and bird-transmitted Borrelia spp. (green spirochaetes). (B) A vertebrate assemblage with higher species richness in which predators and competitors reduce densities of blackbirds and wood mice, and the relative number of larvae that will feed on these host species, resulting in a lower infection prevalence and a lower density of infected nymphs. 178 Ecology and prevention of Lyme borreliosis

180 12. Host diversity and Borrelia burgdorferi impossible to distinguish between the two parameters. Other studies have also shown that the number of larvae feeding on rodents decreases with rodent density (Hofmeester 2016, Kiffner et al. 2011). Furthermore, a recent study showed that tick burden on rodents decreased with the activity level of predators, such as the European pine marten (Martes martes), polecat (Mustela putorius), and red fox (Vulpes vulpes; Figure 3), in a plot resulting in a reduced DIN for rodent-transmitted tick-borne pathogens (Hofmeester 2016). Hofmeester (2016) suggested that this correlation could be caused by predator-induced changes in rodent behaviour. This mechanism might also explain the negative correlation found between red fox densities and Lyme borreliosis incidence in North America (Levi et al. 2012). This implies that if predator activity or density is correlated with host diversity, a dilution effect for rodent-transmitted Borrelia spp. is plausible. For bird-transmitted Borrelia spp. there is also a substantial difference between birds in reservoir competence where thrushes of the genus Turdus have the highest reservoir competence (Hofmeester et al. 2016). The species with the highest reservoir competence, the blackbird (T. merula), is a very common species in many European countries and is present in most forested areas (Gregory et al. 2007). Furthermore, blackbirds have adapted to live in fragmented areas close to humans, making them an important host species determining Lyme borreliosis disease risk (Gregoire et al. 2002). Again, it is the last assumption that has to be verified to suggest a dilution effect in Europe for bird-transmitted Borrelia spp. Bird species that forage on the ground generally have higher tick burdens than species that forage in the canopy (Marsot et al. 2012). Therefore, it can be expected that changes in the foraging behaviour of blackbirds might change the number of ticks feeding on blackbirds and therefore NIP and DIN for bird-transmitted Borrelia spp. However, to my knowledge the correlation between blackbird tick burdens and differences in host assemblage has not been investigated. The previous examples show that although there has been little work done to test the dilution effect hypothesis in Europe, the possible mechanisms behind an expected dilution effect have partly been studied. These studies show a big similarity between the North American and the European situation in terms of mechanisms. It is the density and number of immature ticks feeding on reservoir-competent species compared to reservoir-incompetent species that determines NIP and DIN. To test if this similarity on a small spatial scale results in a similar pattern on a larger spatial scale, I performed an analysis similar to the one performed by Turney et al. (2014) but for the European situation. I used Lyme borreliosis incidence data as summarised and standardised A B C Figure 3. Predators such as (A) the European pine marten (Martes martes), (B) the polecat (Mustela putorius) and (C) the red fox (Vulpes vulpes) can reduce tick burdens on rodents (photos by Tim Hofmeester). Ecology and prevention of Lyme borreliosis 179

181 Tim R. Hofmeester for Western European countries by Sykes and Makiello (in press) and correlated these with the number of mammalian host species for the sheep tick present in each country. I used the list of host species provided by Anderson and Magnarelli (1993) and the presence/absence of species as described in the database of the IUCN (2014) to determine the mammalian host species richness per country. In Europe, Lyme borreliosis incidence increased with mammal host species richness (generalised linear model with negative binomial distribution and log link: β=0.08, P=0.02; Figure 1). The positive correlation in Europe might be caused by the fact that many of the mammalian host species are mice, voles and shrews, all relatively good reservoirs for rodent-transmitted Borrelia spp. (Hofmeester et al. 2016). These species might be able to fulfil the same ecological role in different habitats or parts of a country causing a rescue effect, as was suggested by Ostfeld and Keesing (2000a). This contrasting result shows that, although mechanisms on a small scale might be very similar, factors influencing Lyme borreliosis incidence on a large scale might be very different between continents. This analysis also indicates that a correlation between mammal host species richness per state or country might not be a good predictor of Lyme borreliosis incidence and other factors such as recreation habits of people might be far more important (Vanwambeke et al. 2010). The work in North America and Europe shows that, on a small spatial scale, it is the specific identity of host species that determines their role in tick-borne pathogen dynamics, and biodiversity is only related to tick-borne pathogen risk if there is a correlation between the presence and abundance of specific host species and biodiversity (Randolph and Dobson 2012). Multiple species are important for both black-legged ticks and sheep ticks as maintenance hosts, reservoir-competent hosts and reservoir-incompetent hosts. Therefore, the study of mechanisms related to specific host species rather than biodiversity is needed to better understand the drivers of acarological hazard in both North America and Europe. In Europe, the most important host species that can be distinguished are several species of deer (as hosts for the adult stage of the sheep tick), several species of small mammal (as hosts for the larval stage and reservoirs for rodent-transmitted Borrelia spp.), and several species of thrush (as hosts for the nymphal stage and reservoirs of bird-transmitted Borrelia spp.). Other species, such as competitors or predators might influence the density or tick burden of these species groups, changing tick-borne pathogen dynamics (Hofmeester 2016, Keesing et al. 2006). Looking into the future Sheep tick populations have expanded and increased in density over the last couple of decades (Medlock et al. 2013). This can be partly explained by an increase in distribution and abundance of several of the most important host species such as bank voles, blackbirds, red deer and roe deer (Apollonio et al. 2010, Gregory et al. 2007, Van Strien et al. 2015). These species have most probably been so successful because they have been able to adapt to a fragmented and human-dominated landscape (Hewison et al. 2001, Michel et al. 2006). In the future, vertebrate assemblages are expected to further change as both small to medium-sized (Proulx et al. 2005) and large (Chapron et al. 2014) carnivores have also started to increase their distribution in Europe. The increase in carnivores and the return of apex predators to some parts of Europe (Chapron et al. 2014) might result in cascading effects on Lyme borreliosis risk by changes in prey behaviour. The three host groups most important for maintaining sheep tick populations, deer, thrushes and rodents, are all prey species. It is well known that prey species can change their behaviour as a response to predators. Rodents reduce their time spent active, moving less and changing their spatial behaviour (Borowski and Owadowska 2010, Haapakoski et al. 2015) and blackbirds 180 Ecology and prevention of Lyme borreliosis

182 12. Host diversity and Borrelia burgdorferi decrease the time spent on the ground as a response to predators (Post and Götmark 2006). Both of these changes in behaviour will lead to a decrease in encounter rate between hosts and immature ticks, leading to a decrease in NIP and DIN for B. burgdorferi s.l. (Hofmeester 2016). Also, deer can change their habitat use in relation to predation risk (Tufto et al. 1996) and predators might also impact deer population densities (Melis et al. 2009). Furthermore, intra-guild predation or competition between predator species will affect tick-borne pathogen dynamics, as was shown in North America, where Lyme borreliosis incidence decreased with red fox density, but increased with coyote (Canis latrans) density (Levi et al. 2012). Therefore, the current comeback of carnivores in Europe might have impacts on Lyme borreliosis risk in the future, but the exact outcome will be hard to predict. Another change in distribution and behaviour of vertebrate hosts for ticks in Europe is the adaptation of species to living in urban areas. More and more bird species have adapted to living in an urbanised world (Møller 2008), and rodents have been thriving close to humans for centuries (Meyer 2003). Although deer might not be present in the centre of urbanised areas, roe deer have formed small populations in city parks (Wang and Schreiber 2001). In the current situation, tick populations in the city seem to be relatively low, and blackbirds living in urban areas have a lower tick burden than blackbirds living in the forest (Gregoire et al. 2002). However, with increasing deer densities in the city as a possible response on the comeback of apex predators, this might change. Furthermore, by moving into the city, ticks might come into contact with novel host species. For example, brown rats (Rattus norvegicus), that are absent in most forests, are an important host for ticks in urban environments (Matuschka et al. 1996). This spillover into new host species is very hard to predict and might result in novel host species with a very high reservoir competence (Morse et al. 2012). Within cities, deer might be present in city parks, while hedgehogs might take over the role of deer as maintenance hosts in gardens (Pfäffle et al. 2013). The release from predation has been suggested as the driver of increased hedgehog abundances in urban areas (Hof et al. 2012, Poel et al. 2015). Both the European hedgehog (Erinaceus europaeus) in western Europe and the Northern white-breasted hedgehog (Erinaceus roumanicus) in eastern Europe have been suggested to be reservoirs for different genospecies of B. burgdorferi s.l. including one of the causative agents of neuroborreliosis, B. bavariensis (Coipan et al. 2016, Skuballa et al. 2012). Hedgehogs are infested with both the sheep tick and the more specialised hedgehog tick (Ixodes hexagonus), which can both transmit B. burgdorferi s.l. (Pfäffle et al. 2011, Skuballa et al. 2012). The hedgehog tick is a nest-dwelling tick, and is therefore less prone to desiccation compared to the sheep tick, which might result in higher numbers of hedgehog ticks on hedgehogs compared to sheep ticks in urban environments (Gern et al. 1997). This might result in the hedgehog tick playing an important role in B. burgdorferi s.l. maintenance in urban areas (Pfäffle et al. 2011). Therefore, this tick species should also be considered in studies investigating the role of different vertebrate species in determining Lyme borreliosis risk in urban environments. The blackbird is an important host species for another neuroborreliosis causing genospecies, B. garinii (Coipan et al. 2016). Blackbirds are present in high densities in urban areas, especially in gardens (Luniak et al. 1990). Generally, the few species that survive in urban areas occur in high densities (McKinney 2006), which suggests that reservoir-competent species, such as blackbirds and hedgehogs, can reach high densities in urban areas. Due to these high densities, the infection prevalence in ticks in cities and city parks might be very high as was found in a city park in Munich (Fingerle et al. 2008). The large amount of time people spent in city parks and gardens further increases the risk of getting Lyme borreliosis in these habitats (Mulder et al. 2013, Rizzoli et al. Ecology and prevention of Lyme borreliosis 181

183 Tim R. Hofmeester 2014). As a result, tick-borne disease risk in urban areas might already be higher than expected, and will most probably increase in the near future. Therefore, studies in urban areas are needed to better understand how urbanisation, adaptation of vertebrate hosts and spillover into novel host species will influence Lyme borreliosis risk. Conclusion There is a negative correlation between mammal host species richness and Lyme borreliosis incidence in North America, while this correlation is positive in Europe. At the same time, studies investigating the mechanisms at a small spatial scale show a large similarity between the North American and European situation. Therefore, it is far more important to study interactions between important host species rather than correlations with species richness or other metrics for biodiversity without studying the underlying mechanisms. Both in North America and in Europe, small mammals are important hosts for larval ticks, while deer are the most important hosts for the adult stage. In Europe, birds and especially thrushes also play an important role in feeding nymphal sheep ticks and infecting them with different genospecies of B. burgdorferi s.l. The most important host species for the sheep tick have increased in distribution and abundance in recent decades and are present in many parts of Europe, also in fragmented landscapes close to humans. Furthermore, these host species are all prey for predators, which might result in differences in tick-borne disease dynamics with changes in predator abundance. Several carnivore species are also increasing their distribution and abundance in Europe, which might lead to changes in tick densities and Lyme borreliosis risk in the future. Many vertebrate species are adapting to an urban landscape, including some of the most important host species for B. burgdorferi s.l. genospecies (B. garinii and B. bavariensis) that cause severe disease in humans. Very little is known about the circulation of these genospecies in urban areas. Therefore, the ecology of tick-borne pathogens in urban environments should be better understood, making this an important topic for future research. Public health relevance Correlations on a large spatial scale between mammal host species richness and Lyme borreliosis incidence are not informative for the underlying mechanisms. In Europe, few species are important for feeding ticks (bank vole, blackbird, red deer, roe deer, wood mouse and yellow-necked mouse) and infecting them with B. burgdorferi s.l. (thrushes and rodents). Deer, thrushes and rodents are omnipresent and reside in fragmented and urban landscapes, close to humans. Predators might change the density or tick burden of these important host groups, changing tick-borne disease dynamics. Gardens are an important habitat in which Lyme borreliosis risk might be larger than expected. 182 Ecology and prevention of Lyme borreliosis

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188 13. Greener cities, a wild card for ticks? Fedor Gassner 1*, Kayleigh M. Hansford 2,3 and Jolyon M. Medlock 2,3 1 Gassner Biological Risk Consultancy, Jachthoeve 22, 3992 NV Houten, the Netherlands; 2 Medical Entomology & Zoonoses Ecology, Emergency Response Department Science & Technology, Health Protection Directorate, Public Health England, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom; 3 NIHR Health Protection Research Unit in Environmental Change and Health, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom; fedor.gassner@gmail.com Abstract Over recent decades urban green areas have been expanded, partly to create more space for nature, but also to counter urban warming and to enhance human wellbeing through access to nature. In parallel, the effects of the risk posed by tick borne disease in rural areas surrounding urbanised areas has been recognised in many regions in Europe. This urban greening, coupled with increasing rural tick and Lyme borreliosis issues may have facilitated, in some areas, circulating urban Lyme borreliosis. One of the main issues with ticks found in urban areas is that communities not previously aware of ticks are now being exposed. This lack of awareness may result in few personal protective measures being practiced, elevated concerns over the risk posed by ticks in urban areas, and heightened alarm raised with local councils. Given that urban areas tend to be intensively managed, this public and professional, including occupational, concern raises questions over all land management actions by local municipalities in urban areas. Whilst some accept that management of ticks in the countryside is a challenge, re-wilding urban areas for wildlife, and inadvertently increasing populations of ticks, may create local concern. Here we describe how environmental and anthropological factors may contribute to the establishment of ticks in urban areas, and discuss preventive strategies for public health management. Keywords: city, ecology, Lyme borreliosis, prevention, tick, urban green space Introduction The tick Ixodes ricinus has long been considered a tick of rural woodland dominated areas (Gray et al. 1998). Much of the research in Europe has focussed on such habitat and it follows therefore that our perception of tick-infested habitats are in the countryside, with our human exposure similarly dictated by us entering tick habitat. However, there is evidence in many parts of Europe that tick habitats may be changing, with numerous reports of ticks in urban areas (Rizzoli et al. 2011). Are ticks becoming an increasing urban issue and what may be the drivers of this change? Let us first consider the possible drivers for change. As part of ongoing assessments of the likely impacts of climate change on vector-borne disease in Europe, concerns have been raised over the possible direct and indirect effects of climate change on vector-borne disease systems (Medlock and Leach 2015). Considering the direct effects, it is widely recognised that changes in temperature and precipitation (IPCC 2013) as well as extreme weather (e.g. flooding, drought) will impact on both vector biology and habitat suitability, as well as the extrinsic incubation of pathogens in vectors and also human behaviour (Jaenson et al. 2009, Kjelland et al. 2010). European governments have been developing adaptation plans to mitigate the effects of heatwave, air pollution, flooding and possible other health risks, and in doing so this is resulting in indirect effects of climate change. One key element of mitigating the urban heat island effect, Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _13, Wageningen Academic Publishers 2016

189 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock where the urban infrastructure and human activity cause cities to be several degrees Celsius warmer than surrounding areas, involves the provision of urban greenspace, to essentially keep cities cooler. Green space and green corridors also improve the public s access to the countryside, which in turn improves human well-being, as well as providing additional habitat for wildlife and hence improved biodiversity. There are therefore many benefits to greener cities. Coupled to this, are also increases in the size of towns and cities, with a natural extension of urban areas into greenspace, bringing natural habitats into closer proximity to residential housing (C. Millins et al. unpublished data). In some instances, a possible consequence of greener towns and cities has been the incursion of ticks, via their animal hosts, potentially increasing the exposure of residents to tick bites, and the risks of tick-borne diseases such as Lyme borreliosis (LB). Ticks and LB have been long associated with visits to the forest and other natural areas. Are we now seeing an expansion of ticks and LB risk in urban green space? The habitat of ticks and Lyme borreliosis LB occurs throughout temperate areas north of the equator and is considered the most prevalent vector borne disease in Europe and North America. It is considered to be linked to the distribution of its main Ixodes tick vector, which can be found in a range of habitats such as woodland, rough grassland, moorland and even gardens and urban parks. The ecology of LB, Borrelia species and ticks is surprisingly similar throughout its range. Borreliae are picked up from a great variety of small-sized vertebrate hosts (Coipan and Sprong 2016, Heylen 2016, Hofmeester 2016, Szekeres et al. 2016, Van Duijvendijk et al. 2016b). The most important tick species in the transmission of Borrelia spirochaetes to humans are I. ricinus in Europe, Ixodes persulcatus in northern and eastern Europe and Asia, and Ixodes scapularis as well as Ixodes pacificus in the United States. Despite their distinct geographical distribution, their life cycles and habitat requirements are rather similar. Tick ecology The current and expected future increase in urban greenspace has the potential to allow the incursion of ticks into urban areas, via their animal hosts, potentially increasing the exposure of residents to tick bites, and the risks of LB. For I. ricinus to survive and establish in such spaces it essentially needs the same ecological and environmental requirements that can be found in the countryside; these are: 1. A variety of vertebrate hosts for three blood meals and for their dispersal (Coipan and Sprong 2016, Heylen 2016, Szekeresa et al. 2016, Van Duijvendijk et al. 2016, Van Wieren and Hofmeester 2016); 2. Moist soil and litter layer or moss or dense vegetation that affords protection and recovery from the drying effect of warm temperatures and low aboveground air humidity (typically I. ricinus desiccate at air humidities below approximately 80%, depending on air temperature) (Perret et al. 2004, Randolph and Storey 1999). Understanding these basic requirements helps to understand why ticks may or may not survive or establish in certain urban areas. Urban areas provide a mosaic of tick habitats, made up of woodland, hedges and grasslands managed as meadows for flowers and insects. In some cases, the expansion of towns has brought established woodland, and hence more traditional tick habitat, closer to the urban environment, with some cities actually engulfing woodland within its limits (K.M. Hansford et al. unpublished data). Ticks also require an assemblage of hosts, many of 188 Ecology and prevention of Lyme borreliosis

190 13. Urban ecology which can be found in urban areas. The larvae normally require small mammals such as mice and voles, the nymphs tend to be found on birds such as blackbird, as well as on squirrel, with adults on larger animals such as deer. Deer species such as roe deer (Capreolus capreolus) are increasing in numbers in many parts of Europe, but scientific evidence on increase of deer presence in urban areas is hard to find. Many anecdotal examples of deer species entering urbanised green areas exist however (F. Gassner and J.M. Medlock, personal observations). Dogs and cats can also be hosts for the adult tick stages, but it is unclear to what extent they contribute to tick survival and establishment in urban areas. Ticks in the I. ricinus species complex strictly depend on vertebrate hosts for blood meals during their two to six year lifespan. Larvae usually feed on smaller vertebrates for a few days and then retreat to the litter layer to moult to nymphs. Nymphs tend to feed on a wider range of hosts, including smaller vertebrates such as rodents and larger vertebrates such as deer. Adult females require mainly larger vertebrates such as deer for their final blood meal, which is used to produce approximately 2,000 eggs (Sonenshine 1991). Borrelia bacteria can be picked up from competent hosts during all three blood meals, and once infected, ticks remain infected transstadially. Transovarial transmission of Borrelia is probably negligible, but recent research shows it cannot be excluded (Van Duijvendijk et al. 2016a). One of the distinctive traits of ticks in the I. ricinus species complex is that they quest for a host, waiting on vegetation for a host to brush past, and such hosts can sometimes include humans. Other ticks species, such as Ixodes arboricola and Ixodes hexagonus are common but generally not found questing in the vegetation, but can transmit Borrelia (Gern et al. 1997, Heylen et al. 2014). These tick species are much more associated with their associated host s nests, making human encounters less likely. Although humans are probably rarely bitten by these other tick species (Estrada-Peña and Jongejan 1999, Jaenson et al. 1994), it is assumed that these can play a role in maintaining Borrelia species circulation between hosts (Heylen et al. 2013). To understand tick biology in relation to urban habitats, we have visualised where ticks can generally be found and their route to biting humans in Figure 1. The figure illustrates where ticks reside at any given time: either in a natural habitat (resting, (developmental) diapause, or questing), or on-host. Routes of transmission of ticks between habitats and host types remain Ticks on pets Ticks in nonurban natural areas Tick bites on humans Ticks in urban areas Ticks on wild animals Figure 1. The hypothetical sources of Ixodes ricinus ticks biting humans. Line weight depicts the relative role of the route in the total number of tick bites among humans. Dashed lines indicate that routes are not well described in literature. Ecology and prevention of Lyme borreliosis 189

191 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock partially unknown, as illustrated by the dashed lines in Figure 1, especially those routes between urbanised areas and pets. Tick bites acquired in natural (non-urban) areas such as woodlands are well described, as well as tick bites in urban areas. Ticks that bite humans and originate from wild animals (i.e. all free roaming animals that are not kept as companion animal) are probably rare in the general human population, but a common phenomenon among people (e.g. hunters) that handle recently killed game such as deer. The role of pets in human tick bites and transportation of ticks will be described later in this chapter. Ticks and tick-borne diseases Over recent decades, incidence of tick borne diseases, mainly LB and tick borne encephalitis have increased (ECDC 2012, Rizzoli et al. 2011). There are many, sometimes complex and interrelated, drivers of these trends, involving socio-economical, climatic, epidemiological and ecological changes (Randolph 2008, Sprong et al. 2012). For LB specifically, the most striking development is not only expressed in terms of increasing incidence, but also the effect of rising global temperature on the expansion of ticks to higher latitudes and altitudes in Europe as well as Northern America and Canada (Jaenson et al. 2012, Medlock et al. 2013, Ogden et al. 2006). The expansion of tick populations and Borrelia species in ticks and host animals in more residential and urban settings have been studied only recently. Such studies are providing evidence of differences in tick abundance within greenspace in urban areas, with more natural, wooded areas considered to be more important than managed parks (Corrain et al. 2012, Pangrácová et al. 2013). The importance of adjacent woodland habitat to urban parks and gardens have also been highlighted, with higher tick abundance being found in such spaces (Koci et al. 2007, Maetzel et al. 2005). Varying Borrelia prevalence rates are reported, with some suggesting urban greenspace habitat type is significant (Junttila et al. 1999), but no differences are reported by others (Plch and Basta 1999). It appears that like rural habitats, tick abundance and pathogen prevalence rates are equally variable in urban habitats and that they will likely be influenced by the same ecological and anthropological factors. A changing urban landscape: green into city, city into green Intra-urban developments One of the key traits of most urban areas is that they tend to expand and develop. In view of a changing climate, notably expressed by the warming up of large areas of the earth and more extreme weather events with extreme rainfall and prolonged periods of heat (IPCC 2013), cities need to adapt. Apart from adaptation to climate related events, humans living in cities are facing threats to their wellbeing by traffic and industrial emissions, excessive heat development in concrete-dominated environments, noise, and urban pests. Human wellbeing can be improved by improving nature access to green areas in or around urban areas. These phenomena can be largely countered by making use of ecosystem services : by making clever use of natural traits of blue and green development to buffer excessive rainfall, to capture fine dust and to improve urban living climate for humans and by providing a cool place for rest and recreation (Hansen and Pauleit 2014). Improving or restoring green areas in urban environments may offer solutions, but may also introduce new issues that may threaten human health: including arthropod disease vectors that 190 Ecology and prevention of Lyme borreliosis

192 13. Urban ecology may thrive in urban greenspace. In view of the hazard of climate change and global urbanisation, we may underestimate the potential effects of making cities greener (Lohmus and Balbus 2015). Over recent decades several urban planning activities have been undertaken that involve improvement and interconnection of existing urban green areas, or by establishing new green areas. Although many of these new urban green areas may be currently unsuitable for ticks or tick hosts to survive and establish, in time such areas may become suitable. For example, if areas such as fields, scrubby areas or construction sites are left without maintenance and human disturbance, tick host presence and abundance can increase and a litter layer may develop after a few years to sustain local tick populations. This development may be more likely under economic recession, leading to less maintenance, differences in human use of green areas, and by a reduction in pest control efforts. Not all urban areas will represent the same degree of risk in terms of tick-borne disease transmission. It is possible that a changing urban microclimate could also impact tick phenology and activity, in contrast to rural populations and that this could have impact on tick bite incidence and disease transmission (Buczek et al. 2014, Pfäffle et al. 2013). Similarly interannual abundances of ticks have been shown to vary between urban parks in the Czech republic (Basta et al. 1999, Plch and Basta 1999, Zakovska et al. 2007). Since fauna differs between urban and forest areas, genospecies dominance may also differ, with some reporting dominance of Borrelia valaisiana and Borrelia afzelii (Maetzel et al. 2005, Pangrácová et al. 2013, Reye et al. 2013) and others reporting Borrelia garinii (Pejchalová et al. 2007, Schicht et al. 2011). However, this genospecies heterogeneity is also the case in rural areas (Cekanac et al. 2010, Coipan and Sprong 2016, Hofmeester 2016, Jaenson et al. 2009, Junttila et al. 1999, Lommano et al. 2012, Sprong et al. 2012). Extra-urban developments Apart from the development within cities mentioned above, geographical expansion of cities is, by definition, directed towards rural areas. Rural areas often contain typical habitats that are suitable for established tick populations (Maetzel et al. 2005, Paul et al. 2016, Vourc h et al. 2016). This may lead to an integration of (sub)urban areas into habitat already supporting established tick populations. Along with expanding urban areas, human populations within such cities are expanding as well. This will increase the recreation pressure on greenspace areas within the city boundaries, and also outside cities. Modes of transport Although I. ricinus and closely related ticks are motile, they walk only on hosts or up and down vegetation and hardly disperse horizontally. While active dispersal is considered to be less than approximately 5 metres during the tick s lifetime (Carroll and Schmidtmann 1996, Healy and Bourke 2008, Lane et al. 2009), these ticks are transported over large distances only when attached to a host. Other dispersal mechanisms, such as with soil or plant material or even directly by humans, are largely hypothetical or anecdotal. Ecology and prevention of Lyme borreliosis 191

193 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock Dispersal by natural hosts Stage-dependent feeding times ranges from three days for larvae to up to 14 days for adults (Földvári 2016, Sonenshine 1991). With such a long attachment on the host, host-mediated dispersal can occur over very long distances, and may differ per tick life-stage. Dispersal through hosts is also determined by host associated home-range of the host and the migration distance. Home-ranges of vertebrates are usually considerably smaller than migration distances. For example, some migratory bird species migrate over thousands of kilometres, whereas once settled, their home-range, often territory associated, may be as short as a few hundred metres. In addition, the time the host resides in such habitats determines the chance of drop-off and survival of ticks. For example, compared to a tick-infested bird flying over a small urban park, the chance of tick survival and establishment of a tick-infested bird that rests, forages, builds a nest or dies (Figure 2A and 2B) in the park is much higher. Factors that influence host migration are numerous. Except for the case of birds, physical boundaries largely determine the connectivity between habitats. The ability of host species to detect and reach isolated habitats in the urban environment is crucial for tick dispersal. In addition, the urban habitat has specific characteristics with respect to availability of food, water, suitable micro-climate, mates, nesting material, nesting sites, and the level of competition, predation and disturbance compared to natural habitats. Hosts that are able to adapt to these factors will be most successful. General traits of these hosts are that they have an easy mode of dispersal, and are generalists in terms of food and habitat requirements. Rats are omnivorous and can readily disperse using their running, climbing and swimming and therefore have been associated with tick survival and establishment in urban habitats (Matuschka et al. 1996, 1997). Connectivity between urban habitats and rural natural habitats affect the host dispersal. Another factor of pushing animals into urban areas is the situation outside the city boundaries. The level of disturbance and competition in natural habitats can strongly determine the tendency of host species to disperse into urban areas. A recent example of this is fallow deer that leave their original (tick infested) habitat in a Dutch coastal dune forest due to very high interspecific competition for territory and food in a relatively small dune area, and subsequently invade surrounding villages. A B Figure 2. (A) Tick-infested bird (here a magpie Pica pica, in this image no ticks are visible) may release ticks locally at the place of its death (photo by Fedor Gassner). (B) Greenfinch (Chloris chloris) with viable engorged ticks (photo by Robert Cheke). Live birds may introduce and propagate ticks in a larger area during their lifetime. Both birds are common visitors of urban environments. 192 Ecology and prevention of Lyme borreliosis

194 13. Urban ecology Bird-associated ticks and Borrelia genospecies may readily disperse between habitats by movement of I. ricinus and more host-specific ticks such as I. arboricola (Heylen 2016, Heylen et al. 2013). Interestingly, many Borrelia genospecies have specific host-associations, which are mediated by the host immune system (Kurtenbach et al. 2002). Moreover, the bird associated Borrelia, B. garinii, is associated with neurological symptoms, while the mice-associated B. afzelii is associated with skin manifestations of LB, and the more generalist Borrelia burgdorferi sensu stricto with arthritis (Stanek and Reiter 2011). The hosts specificity may have led to differentiation of Borrelia species based on host availability posing differential spatial risk for LB in humans. Dispersal through companion animals Apart from stray dogs and cats, and released other companion animals such as rodents, turtles, rabbits, birds, etc., companion animals have all their habitat requirements in or around the owner s premises. Dispersal of companion animals is usually restricted to the owner s home location and immediate surrounding area, and the daily trip(s) that the owner may undertake with their pet. The significance of companion animals in tick dispersal is not frequently studied. Ticks do seem common on pets, even in an urban setting (Krol et al. 2015, Nijhof et al. 2007). The diversity of ticks on host is far greater than can be expected from the dominance of I. ricinus and closely related Asian and American counterparts in the vegetation (Krol et al. 2015, Nijhof et al. 2007). Indirect associations between pets and tick bites in humans have been shown in several studies. For example, a Norwegian study on risk factors for tick bites showed a significant correlation with ownership of domesticated animals, along with more straightforward risk factors such as time spent outdoors or activities such as hunting (Hjetland et al. 2013). Similar results are described in a country-wide study in German children, where having companion animals was significantly correlated with seropositivity for Borrelia species (Dehnert et al. 2012). How companion animals may potentially contribute to tick bites in humans, apart from stimulating the owner to walk in green areas, is shown in Figure 3. Companion animals associated ticks may hypothetically lead to either direct tick bites, or indirect tick bites through establishment of a tick population in the Engorged Garden In-house Moults / lays eggs Dries out in 5 days (excluding exotic spp.) Establishment depends on habitat Tick on pet Survives Dies through grooming or tick prevention methods (Partially) unengorged Garden In-house Directly on human Searches new host Dries out in 5 days (excluding exotic spp.) Survives actively host-searching 5 days Establishment depends on habitat Figure 3. Pet-associated Ixodes ricinus tick introduction and establishment into (peri)domestic settings. Ovals refers to the location of ticks, rectangles refer to the tick s status and large arrows refer to action. Black filling indicates tick death, red indicates hazard for humans. Ecology and prevention of Lyme borreliosis 193

195 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock peridomestic zone (garden). However, it remains largely unclear how much this contributes to the number of human ticks bites and LB cases. It can also be reasoned that companion animals could reduce tick survival in an enclosed habitat where pets are the prime food source for ticks if they are treated with tick-killing substances. Habitat types that may be(come) suitable for ticks Within the city boundaries, many different greenspace habitats could be(come) suitable for tick survival and establishment such areas as parks, public, business and peridomestic gardens, zoos, theme-parks, and play grounds. These areas are not equally visited by humans and therefore will not pose the same risk in terms of tick-borne disease transmission. The focus of tick bite prevention should be on those areas where contact between humans and ticks is at its highest, or where vulnerable citizens who may not be sufficiently able to perform tick checks (elderly, children, disabled) are using the greenspace. Land use types with hardly any human presence that may act as amplifying place or green corridor for ticks and tick hosts are for example vegetation covered infrastructure (e.g. roofs, sound barriers or facades of buildings), fallow land, water sides, yards and road as well as railroad verges. The more connected two fragmented tick habitats are, the more likely the tick population will survive, with rodents and birds probably providing the necessary link between populations. It may be expected therefore that urban areas with well-connected habitats, would probably support more ticks due to important tick hosts accessing these areas via green corridors. Also, urban greenspace on the margins of towns and cities, with direct connectivity to the countryside may also be significantly more suitable for tick survival. Suitability of urban environment Urban habitats are usually not considered highly suitable for ticks because of its buildings, concrete structures, tarmac and other hard ground surfaces or even sandy areas. Development of urban greenspace can create a more moist and cooler environment. Notably these moist conditions can promote tick survival. Moreover, increasing tree cover can also increase litter layers, which are favourable for ticks. Increasing plant diversity does not increase tick establishment chance per se. For example, grassy meadows with annual flowers are often too dry for tick survival, and lack the top soil composition and cover that is essential for ticks during resting and transstadial development In colder geographical areas, ticks may benefit from the fact that urban areas act as heat islands, with on average temperatures that are 5 C higher than surrounding rural areas. Although higher temperatures may also cause stronger desiccating conditions, they enable a longer activity season for ticks compared to the countryside, and speed up tick movement and transstadial development. The urban environment may give rise to another composition of the Borrelia species populations. For example, Borrelia infected ticks are more resistant to desiccating conditions in the laboratory (Gassner et al. 2011, Herrmann and Gern 2015). Urban infrastructure and limited habitat size, but enough food for generalist species, may lead to high exposure of (reservoir) hosts to ticks in small sized tick-suitable habitats. Related to this, 194 Ecology and prevention of Lyme borreliosis

196 13. Urban ecology is the general lack of biodiversity among tick hosts in urban areas. In many urban areas, large tick hosts such as deer are lacking. Deer are known to amplify tick populations by feeding large numbers of adult female ticks, but also to buffer Borrelia infection due to the incompetence of deer to transmit Borrelia. It has even been suggested that deer-feeding ticks are cleared from the Borrelia infection they acquired during a blood meal in the previous life-stage (Pacilly et al. 2014). In an urban setting, reservoir-competent hosts such as mice, rats, squirrels, blackbirds and hedgehogs are dominant over buffering species such as deer. Here tick densities may be not as high as in deer-inhabited areas, but the infection rate in ticks in urban areas may exceed 30%, even if considerable numbers of ticks are tested (reviewed in Rizzoli et al. 2011). Host composition in urban areas can be strongly affected by human behaviour. For example, discarded food in urban parks can support tick hosts, such as rats, stray cats and omnivorous birds. Moreover, releasing (exotic) animals (e.g. the grey squirrel), and letting pets roam free may also contribute to the chance of a tick finding a host. Finally it should be noted that tick species other than I. ricinus may thrive under urban hot and dry conditions. These include those tick species that originate from warmer and drier climatic regions. The main examples associated with human bites and potency to transmit several tick/ borne pathogens are Hyalomma marginatum, Rhiphicephalus sangineus and the soft tick Argas reflexus (Estrada-Peña and Jongejan 1999). To date, establishment of such species in urban areas seem to be rare. The urban population at risk: are they more vulnerable? Besides relatively low tick densities in urban areas, the exposure of humans to these ticks might be high. However, exposure is not often sufficiently quantified (Eisen and Eisen in press). It is well known that high densities of (infected) ticks in a publically accessible site pose a risk to the public (Jaenson et al. 2009, Soleng and Kjelland 2013). Furthermore, as stated earlier there is a wealth of evidence in the literature for the dilution effect of large mammals in the circulation of Lyme borreliae in ticks. Deer and cattle are not considered to infect ticks with Borrelia species. Although increasing tick densities, cows and deer may actually decrease infection rates of ticks. Therefore in the absence of large mammals in urban areas, the lower tick densities might be offset by the increasing Borrelia prevalence (Coipan et al. 2013, James et al. 2013, Tack et al. 2011). Coupled with the high human exposure, an urban tick habitat can present a new challenging risk to public health. Other indicators of human risk are LB prevalence in humans (Skotarczak 2014, Smith et al. 2012), tick abundance on dogs (Jennett et al. 2013), the presence of reservoir hosts and degree of habitat fragmentation (Estrada-Peña 2009, Rizzoli et al. 2014) or home proximity of humans to woodland or favourable tick habitat (Mavin et al. 2009). This makes an assessment of human risk, particularly in an urban setting, a complex interaction of many factors, but it should not be assumed that urban greenspace and the peri-urban fringes present no or lesser risk that more natural habitats in the countryside. What we do know that tick-borne disease risk is associated with the presence of tick vectors, the presence of suitable tick habitats, and high human contact; all of which are present in urban areas (Junttila et al. 1999). Humans may be less well prepared for tick bites in urban areas than they are in rural settings. For example, Bayles et al (2013) describe that knowledge and prevention of tick-borne diseases can vary between urban and rural areas in the United States (Bayles et al. 2013). Moreover, persons such as the elderly, disabled or young children who are less able to perform tick checks and normally do not venture out into woodland areas may be exposed to ticks in urban areas such as parks. Ecology and prevention of Lyme borreliosis 195

197 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock Available interventions in the urban setting Prevention of LB would benefit from information on the hazard and exposure to ticks and Borrelia species. What are tick habitats, how many (infected) active ticks are present? It is also important to identify the major groups at risk, and what behaviour is associated with exposure to infected ticks: all humans that come into direct contact with tick suitable habitats and with tick hosts, either deliberately through their profession or type of recreation, or unintended. Once risk groups and risk activities are identified, epidemiological research can focus on monitoring exposure to ticks and tick-borne pathogens such as Borrelia species. Two strategies of preventive measures can be implemented: hazard control and exposure control. Hazard control involves management of the tick habitat, tick hosts and ticks themselves and of vaccination strategies. Exposure control involves increasing self-effectiveness of humans, communication on tick activity and presence, personal protection methods and protection of pets against ticks and LB. In the next section, we will briefly discuss some current relevant examples for each strategy which are described in detail elsewhere in this book. No single method will suffice to prevent LB. Depending on the specific situation, an integrated approach that combines different strategies will be most effective. Tick habitat targeted strategies Few evidence based methods exist that support habitat targeted control of ticks in Europe. Examples from the United States include burning of the top vegetation, creating physical boundaries between (potentially) infested vegetation and areas where people are active, such as lawns. Also mowing the lawn or removing overhanging vegetation is suggested (Stafford 2007). Most methods focus on removing the humidity providing elements of the vegetation, such as tall grass, or other dense undergrowth, or leaf litter and humus, to desiccate ticks. Removing overhanging vegetation can also reduce the contact between questing ticks and passers-by. Minimising the use of plants that may attract tick amplifying hosts such as deer is also suggested (Stafford 2007). Although very labour intensive in nature areas due to the large scale of such areas, urban green areas are more manageable due to their smaller size and presence of nearby infrastructure such as roads. Also, nature conservation regulation that can prevent habitat intervention methods in natural areas are less likely to hamper habitat management in urban settings. Tick host targeted strategies Since ticks completely depend on vertebrate hosts for blood, the presence of host species can be altered by placing fences, limiting food or shelter and other methods. If host species are regarded as pest species themselves, for example rats (Himsworth et al. 2014), they can be either captured and released elsewhere, or exterminated. Removal of hosts may also have unwanted side effects, for example when a non Borrelia reservoir hosts is removed, and already present ticks switch to a reservoir competent host, which may in turn lead to increased infection prevalence (Perkins et al. 2006, Rand et al. 2004). Indeed removal of hosts may also be conflicting with one of the objectives of development of urban green areas: the improvement of biodiversity. 196 Ecology and prevention of Lyme borreliosis

198 13. Urban ecology Tick hosts may also be treated with pesticides that either repel ticks, or kill ticks that come into contact with the host. Such approaches may be relatively easy for pets, but very challenging to apply to natural free roaming hosts. Various methods to apply acaricides to deer and mice are available, but not commonly applied due to high maintenance costs and insufficient tick reduction (Stafford 2007). Vaccination provides another tool, and ongoing research on LB vaccination of reservoir hosts has been under development in the past decade, but with no large scale application to date. However, the ideal method to prevent tick bites and pathogen transmission in hosts may eventually be antitick vaccines, which are objectives of current and future research (Sprong et al. 2014). Tick targeted strategies Targeting ticks directly can be achieved by chemical (products of natural or synthetic origin) or biological control methods. In the United States, pesticide spraying is widely applied in peridomestic settings. Spraying may reduce (but not eradicate) ticks on a single property for some weeks, but has recently been proven to have insufficient effect in preventing tick bites and LB in humans living in the treated areas (Hinckley et al. 2016). Moreover, most chemical substances also kill numerous non-target organisms such as mites, spiders and insects, which are beneficial or even protected species in many ecosystems. Biological control of ticks has been widely researched, for example using a parasitoid wasp, various entomopathogenic fungi, various tick-eating birds and nematodes (see review Samish et al. 2008, Klingen and Van Duijvendijk 2016). Unfortunately no biological control strategy has proven widely applicable, i.e. affordable specific and effective, to date. Human targeted strategies The most widely applied method of LB prevention involves strategies aimed at humans, such as personal protection products, behaviour, tick checks and early removal of ticks and vaccination. These strategies propagated by governmental institutions for the general population, and by occupational health professionals for highly exposed outdoor workers (De Groot 2016). An array of personal protection products and strategies can be applied. In order of increased effectiveness, the most common methods are: covering the skin with clothes and tucking trouser pants into the socks; application of various repellent or acaricidal products to the skin or clothes, and wearing permethrin-impregnated clothes. Behavioural strategies are avoiding contact with vegetation and leaf litter in tick-infested areas, awareness of indirect tick contact through pets and clothes, checking the body for ticks after possible exposure, early removal of ticks and alertness for tick-borne disease symptoms. Other behavioural adaptation in the urban setting may be prevention of littering potential food for tick hosts, and for example feeding birds only outside the tick activity season (Stafford 2007). Vaccination is available for tick-borne encephalitis, but not for LB prevention. The aforementioned studies on anti-tick vaccines can also be very useful for future application in humans (Klouwens et al. 2016). Ecology and prevention of Lyme borreliosis 197

199 Fedor Gassner, Kayleigh M. Hansford and Jolyon M. Medlock Since ticks can survive several days inside clothes and laundering at 40 C (Carroll 2003, Jennett and Wall 2012), a recent study provides useful methods to eliminate ticks that remain in clothing after exposure to tick infested areas or animals. These methods involve either washing at approximately 60 C, or briefly tumble-dry dry clothes at 60 C (Nelson et al. 2015). There are several methods available for improving self-efficacy of humans to prevent LB (Connally et al. 2009, Mowbray et al. 2014). Evaluation of the effect of such methods are rare however (Beaujean et al. 2016a). Recently, a flyer and an information film for the general public were evaluated in the Netherlands. Both tools were effective in increasing knowledge, self-efficacy and intention to adopt protective strategies, but no actual behavioural change could be observed in the general population that was studied (Beaujean et al. 2016b). Choosing which prevention method may be tailor made depending on the target group and the setting. Of course, web-based interventions such as web-sites, on-line videos, social media and apps offer great dispersal potential, but also may not reach certain risk groups such as the elderly. It is also unclear whether strategies such as tick warning signs near tick infested habitats are effective in preventing LB. Governmental action Governmental institutions can develop, evaluate and disperse public information tools and provide guidelines for medical staff and occupational health managers. Also, they can initiate, direct or fund research and improve national and international cooperation on LB prevention, diagnostics and treatment. Concluding remarks In conclusion, we should begin to accept that ticks are becoming part of urban fauna in some areas of Europe. Our challenge now is how do we manage this situation, and what advice can we give to the public about reducing their exposure to ticks, and how can local authorities be advised to mitigate tick hot-spots. Crucially, the way in which we manage greenspace for nature, and increase access to the public for improved well-being will be critical for the public s exposure to Borrelia-infected ticks. Advice that may be relevant for avoiding tick areas in the countryside may be more challenging in urban areas. Public health relevance Lyme borreliosis may be acquired in some urban areas. There are multiple factors that need to be considered when assessing the risk of urban Lyme borreliosis. Future planning of urban greenspace should take tick-borne disease risk into account, to help prevent tick-human encounters. Intervention methods need to be developed and evaluated for urban use. Future studies are needed to further understand and mitigate the risk of urban Lyme borreliosis. 198 Ecology and prevention of Lyme borreliosis

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206 14. A resource-based habitat concept for tick-borne diseases Sophie O. Vanwambeke 1*, Sen Li 2 and Nienke A. Hartemink 3 1 Université catholique de Louvain, Earth & Life Institute, Georges Lemaître Centre for Earth and Climate Research, Place Pasteur 3, 1348 Louvain-la-Neuve, Belgium; 2 Environmental Change Institute, University of Oxford, South Parks Road, Oxford OX1 3QY, United Kingdom; 3 Theoretical Ecology group, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, P.O. Box 94248, 1090 GE Amsterdam, the Netherlands; sophie.vanwambeke@uclouvain.be Abstract Because tick and tick-borne disease distributions are so tightly linked to the environment, a robust conceptual background is necessary to build useful empirical and process-based models and to interpret results coherently with pathogen, vector and host ecology. This is especially pressing when considering that tick-borne pathogen circulation is a complex ecological system that has been studied in a great diversity of ways, producing results that may appear challenging to synthesise. We propose that a resource-based habitat concept can provide helpful leads to collect data, elaborate models and interpret and assemble upcoming and existing results. Two elements in the existing knowledge and its gaps, and in the current ecological context encourage us to explore such a conceptual framework. First, it is rare in the current practice to focus on the ecology of the pathogen, whereas this may be a key element in understanding the role of biodiversity in pathogen circulation. Second, the role of non-classical tick habitats (e.g. forest encroached areas and meadows) is getting more and more often highlighted, indicating that summarising tick habitat, or pathogen habitat, by a single vegetation class is not suitable. We revisit two simulation models (a cellular automata and an agent-based model) that focus on infected ticks and on tick population dynamics. In these models, the potential of a resource-based habitat concept can be highlighted. We then discuss how to implement this concept for studying the ecology of tickborne pathogens. Keywords: biological resources, environmental factors, functional habitats, movement ecology, tick-borne pathogens Introduction Tick distribution and tick-borne disease transmission is closely tied to the environment (e.g. Medlock et al. 2013, Randolph 2001). This relates to the fact that ticks, the pathogens they may host, and the organisms they feed from all have specific habitat requirements. Identifying these requirements allows identifying and predicting the riskiest environments and, potentially, modifying these conditions to make them less suitable for pathogen circulation and transmission. Studies investigating associations between environmental factors and elements of tick-borne disease transmission systems have been carried out in many different ways: examining different groups of environmental factors, using different entry points into the system (i.e. the variable to be explained or predicted by a quantitative model), or different methodologies. Environmental factors highlighted for ticks, their hosts, and their pathogens range from regional or continentalscale environmental factors to the micro-scale. At the regional or continental scale, environmental factors considered mostly aim at identifying the climatic envelope of ticks: where are climatic conditions suitable for ticks to survive and develop. These factors are mostly considered at a coarse scale and are monitored using remote sensing or interpolated weather station data such Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _14, Wageningen Academic Publishers 2016

207 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink as WorldClim ( (Estrada-Peña et al 2006, 2016). At the landscape scale, focus usually shifts to the fine or mid-resolution habitat characteristics mostly related to vegetation. Land cover maps are often used and can be processed to describe various elements of the landscape such as composition (how much of a land cover) and configuration (how it is arranged) (Dobson et al. 2011b, Halos et al. 2010, Vanwambeke et al. 2010). Finally, some studies also focus on fine scale characteristics of the environment that can cover weather conditions (instantaneous or short-time scale temperature, relative humidity), fine scale vegetation and soil conditions, such as presence of ground vegetation or litter (Gassner et al. 2011, Randolph and Storey 1999, Tack et al. 2012). Few studies combine scales of studies (De Keukeleire et al. 2015, Jore et al. 2014, Li et al. 2012b). Studies may also use different entry points, that is, look at different dependent variables or model outputs. Often used entry points are the presence and abundance of (infected) ticks, usually measured through flagging (Barandika et al. 2006, Boyard et al. 2011, Richter and Matuschka 2011). Other entry points related to the vector include counting ticks on hosts (Carpi et al. 2008, Claerebout et al. 2013), and reports by veterinarians (Jore et al. 2011). Other used or potential entry points include infected hosts (Juřicová and Hubálek 2009), tick bites (in a survey or in citizen or at-risk population reports) (De Keukeleire et al. 2015, Hugli et al. 2009), and human disease cases (Linard et al. 2007, Zeimes et al. 2014). The latter, using human case reports, is an attractive option as it relies on the most systematic, spatially exhaustive and temporally deep datasets available, whether the disease considered is reportable or not. However, it adds the important filter of reporting and exposure. Domestic animals serology libraries offer an interesting alternative with temporal depth (Jore et al. 2014), but an exposure filter persists for most domestic animals populations. The consequence of this diversity of measurements of the intensity of tick-borne disease transmission is that we are effectively looking at the process through keyholes, none of which allow us to get an extensive view of the phenomenon, either because it is practically difficult to get a large number of consistent measures, or because the measure can only report on a part of the system. This is why studies on tick-borne disease transmission are often considered to only assess the emerged part of an iceberg (Randolph and Šumilo, 2007). Methods encountered include both empirical studies (most of the studies cited above), or processbased models that attempt to model the effect of host, host habitat, tick habitat or weather and climate variables on tick emergence and population dynamics (Dobson et al. 2011a, Hancock et al. 2011, Hoch et al. 2010, Ogden et al. 2005). While a wealth of results has come out of such studies, our understanding of how the observed associations relate to the ecology of tick-borne disease transmission remains proportionally poor, and can be confusing to those less acquainted with ecological modelling. Results may appear contradictory, while they may simply reflect different local conditions, or a focus on a different part of the system. A striking example of this concerns habitats described in some areas as of intermediate suitability, whereas in some regions they may represent the best habitat available and therefore the most significant one (for an example on hantavirus, see Zeimes et al. 2015). Because some such habitats are related to intense human exposure to tick bites, they are of high relevance from a public health perspective. Also, the choice of a scale of study is often suggested as more advantageous than another, whereas they are complementary, as is the case in most ecological processes (Forman 1995). In this paper, we identify and discuss two specific challenges that are currently poorly addressed in most empirical studies, such as those cited above, or the less numerous process-based studies. 206 Ecology and prevention of Lyme borreliosis

208 14. A resource-based habitat concept for tick-borne diseases First, few studies include the ecology of the pathogen in their focus. This is a key element for modelling tick-borne disease transmission, particularly considering the role of biodiversity on the circulation of pathogens. This is beginning to be conceptually formalised but it is still poorly represented in models, with many studies producing apparently idiosyncratic results. Second, more and more studies are pointing towards the role of non-classic environments for ticks, primarily non-forest habitats (Hartemink and Takken 2016, Rizzoli et al. 2014, Špitalská et al. 2016, Uspensky 2014). Several reasons can be highlighted for their possible role: they are suitable habitat for tick hosts; transitional environments (e.g. bushy areas, forest encroached areas, grasslands) are a major feature of landscapes in some regions, or one that is expanding (Estel et al. 2015, Meyfroidt and Lambin 2011, Navarro and Pereira 2015); or these areas are highly suitable for human exposure to infected ticks. It is becoming clear that there is not just one type of vegetation or land cover that is linked to tick-borne pathogen (TBP) transmission. This highlights the need for a new perspective on the role of the landscape in shaping the risk of TBP transmission. Focusing only on forests as risk areas is clearly not sufficient, we need to look at other vegetation and land cover types as well, provided that the conditions in those areas are suitable for TBP transmission. Assessing whether this is the case, requires a framework that incorporates the link between landscape factors and the functional ecology of the actors involved in TBP transmission (ticks, hosts and pathogens). We propose that a habitat concept focusing on ecological functions and their associated resources for all species involved not only for the vector or for the hosts, but also for the pathogen as drawn from conservation ecology, can bring a robust and useful perspective on these questions and help consolidate both existing knowledge and future studies. In our conceptual model, the organism of focus is the pathogen considered. After presenting the concept and its adaptation to tick-borne pathogens transmission systems, we revisit existing models in the light of the resource-based habitat concept and discuss a few lines for implementation of the concept to better understand the ecology of these diseases. The perspective of functional ecology: the resource based-habitat concept In a functional ecology perspective, we adopt a focus on the organism and its ecological requirements, rather than approaching it from the angle of habitat characteristics or classes such as vegetation types. Initially proposed in the field of conservation biology (Dennis et al. 2003, 2006), this approach has been conceptually adapted to deal with vector-borne diseases (Hartemink et al. 2015) and illustrated with the case of bluetongue virus. At various life stages, an organism needs to be able to carry out a certain number of functions (e.g. feeding, mating, diapausing). These functions can be associated to specific resources found in the environment, which can be either a consumable (e.g. host plant for an herbivorous insect) or utility (e.g. suitable micro-climate). The resource-based habitat concept (RBHC), in line with the classical ecological niche concept, considers in a bottom-up way the ecological resources needed by the organism of interest (here, the pathogen) to complete its life cycle, from which a functional habitat arises. In order to determine whether a habitat is suitable for an organism, the RBHC explicitly takes into account the movement capacity of animals and the distance between the different resources needed during all life stages of the organism. This means that a habitat may not correspond to a single land cover or landscape patch type, but rather to a combination of different land cover types or landscape elements, that offer all required resources within a range than can be covered by the individual. Ecology and prevention of Lyme borreliosis 207

209 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink A resource-based habitat concept for tick-borne diseases The primary purpose of RBHC is to set up a conceptual framework, which can subsequently be used in various ways to support modelling (Hartemink et al. 2015). Here we present the structure of the concept as adapted to TBP, generally, illustrated with Borrelia spp. The concept is illustrated in Figure 1. In this general model, we focus on fine-scale environmental features and ecological interactions, and assume climatic conditions favourable to pathogen, vectors, and hosts. Pathogen level Three essential functions are identified for vector-borne pathogens: replication in the host, replication in the vector and transmission. This can be identified for any TBP. Two resources are thus necessary to complete the life cycle: reservoir hosts and vectors. Their functional habitat needs to be considered as well. In the case of Ixodes-borne diseases, many reservoir hosts need to be considered, also depending on which pathogen is considered. For Borrelia burgdorferi genospecies of public health relevance, the role of rodents can be highlighted, especially Apodemus spp., particularly as vectors of genospecies Borrelia afzelii, and of birds (Borrelia garinii, Borrelia valaisiana) (Lommano et al. 2014, Mannelli et al. 2012). Replication in host Transmission Replication in vector Pathogen Reservoir host Vector, mostly nymphs and larvae Suitable thermal conditions Reservoir hosts Feeding Food resource for host Resting, shelter Reproduction Watering Vector: e.g. Ixodes ricinus ticks Questing/host finding Vegetation-increasingly high with life stages: grassy, bushy vegetation Feeding Resting/diapausing* Resource for host resting Resource for mating/nesting Water Non competent hosts Feeding Food resource for host Resting, shelter Reproduction Watering Resource for host resting Resource for mating/nesting Water Food (other than blood) e.g. nectar Shelter from extreme temperatures or dessication: litter layer, moist soil Function Resource *: diapause: developmental or behavioural Mating Ovipositing Blood feeding Resources for mating Breeding site (suitable for egg laying): litter layer, moist soil Host (reservoir or not), increasingly large with life stages Suitable thermal conditions Figure 1. Application of the resource-based habitat to tick-borne pathogens. Identification of the functions (light grey boxes) and resources (grey boxes) is first done at the level of the pathogen and then at the level of both the vector and hosts. A box for non-competent hosts is added to account for their role in tick reproduction and population dynamics. 208 Ecology and prevention of Lyme borreliosis

210 14. A resource-based habitat concept for tick-borne diseases Vector level Vectors are a necessary resource for the vector-borne pathogen functions of transmission and replication in the vector, and vectors have their own functions and resources that need to be accounted for, which include hosts. For Ixodes vectors, a number of functions will be the same for all motile life-stages (larvae, nymph, adult) but correspond to different resources (Figure 1), with older life-stages generally feeding on gradually larger hosts, and questing higher on the vegetation. Larvae quest on low, grassy vegetation and feed primarily on small mammals and birds among which many reservoir host species are found, while adults quest also on bushes and on larger animals, such as deer, often as a result considered as key for reproduction. Each stage takes a single blood meal over three to seven days. When not questing or attached to a host, for oviposition, for eggs to moult into larvae, ticks require a protective layer of litter, such as what can be found on the ground of deciduous forest. Tick movement is extremely restricted and mostly vertical along the vegetation. Ticks can however end up far from their place of hatching as their feeding hosts can move considerable distances host movement capacity is therefore the main component for tick spread. Host level While their functions and resources may not differ greatly, or not differ in relation to this divide, we need to consider two types of tick hosts: those competent for supporting pathogen functions (that are thus a pathogen resource as well as a tick resource) and those that are not competent (i.e. in which no systemic infection/pathogen replication is observed) and that only support tick functions. As some tick species, in particular those considered here in priority (Ixodes spp.), are opportunistic feeders, it is not feasible to list here all possible host species and their associated resources but when looking at Borrelia burgdorferi we can make a general distinction between reservoir hosts (including many rodent species) and non-competent hosts (that include e.g. red deer, roe deer). It is necessary and useful to assess which areas of the landscape, including in the sense of a combination of spatially explicit land cover or vegetation types, contain the set of resources for possible hosts. This will lead to consider specifically host movement capacity, permitting or not to access the necessary combination of resources for survival. Likely, it is a combination of land covers that holds the resources making up a host s habitat, also considering that resources will be needed in sufficient quantities. This brief outline focuses on the general concept and did not outline precisely the functions and resources of specific species concerned here, which would be beyond our scope. Our main purpose is to present the concept and highlight its potential when studying TBP. Also, considering the resource needs of the vector(s) as well as of the hosts sheds light on why such diverse habitats have been identified as favourable for tick-borne disease transmission in the general sense of the term. Cellular automata and agent-based models to implement RBHC We here revisit two simulation models that have implicitly integrated RBHC principles as outlined above and examine their set-up and results in the light of the RBHC. Ecology and prevention of Lyme borreliosis 209

211 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink Landscape fragmentation and transitional habitats Li et al. (2012a) elaborated a cellular automata model to assess the role of landscape fragmentation on tick abundance and infection prevalence (for a schematic overview of the model see Figure 2). This model explicitly represented the movements and habitat preferences of two types of host species. A generic small reservoir host, calibrated following bank voles Myodes glareolus, is assumed to move small distances and stay in the forest, whereas the other host type, a generic large reproduction host (i.e. that provides blood meals but does not become infected), calibrated following roe deer Capreolus capreolus, moves across larger distances and used forests (mostly for shelter) as well as grassland (for foraging). The composition and configuration of the highly suitable tick and host habitat (assumed to be forest) was modified in the different simulations: the proportion of forest highly suitable habitat, that is resource-rich habitat varied between 20 and 80% and forest patch ( block ) size varied between 1 and 100 ha. Non-forest areas were nonvegetated (low or no resources), or grassland (medium level of resources for hosts). Depending on whether the areas surrounding the forest were grassland or non-vegetated areas, the outcomes in terms of the effect of fragmentation of the forest patches were radically different. When the forest is surrounded by grassland, the model predicts the density of infected nymphs to be highest in large patches of forest, whereas if the surrounding areas consist of non-vegetated areas (unsuitable habitat for deer), the density of infected nymphs was highest in the scenarios with few and small patches of forest. These seemingly contrasting outcomes are a direct result of the underlying assumptions of the model. In case of forest surrounded by grassland, deer will move into the grassland for foraging and also drop off ticks there. Ticks have a lower survival in grassland compared to forest (due to an increased desiccation risk). More grassland thus means more deer movements into grassland, more ticks being dropped off in grassland and lower survival Weekly survival Eggs Larvae Nymphs Adults drop, egg laying & egg hatching Questing larvae Feeding larvae Larvae drop & development Weekly survival Questing nymphs Feeding nymphs Nymphs drop & development Weekly survival Questing adults Attach on Attach on Attach on Weekly random movement (forest) Small mammal/ reservoir host (H) Weekly random movement Reproduction host (R) Adults (female & male) Feeding adults Figure 2. General structure of the simulation models revisited (modified from Li et al. 2012a, 2014, where details of the models can be found). 210 Ecology and prevention of Lyme borreliosis

212 14. A resource-based habitat concept for tick-borne diseases for ticks (grassland basically acts as a sink for ticks), which hampers TBP transmission. In contrast, if the forest is surrounded by unsuitable areas for deer, the deer will stay in the forest, as will the ticks, and the interaction between ticks, small hosts and deer intensifies as the area of forest (patches) becomes smaller, which increases TBP transmission. This shows that the assumptions made in such a simulation model heavily affect the simulation results, and a robust ecological base is therefore absolutely necessary. The cellular automata model presented in Li et al. (2012a) thus implicitly incorporates several aspects of the RBHC approach, such as the focus on the movement capacities and use of the landscape of the different hosts and the results underline in a striking manner that tick dynamics are heavily impacted by how hosts of various types may effectively use the landscape. However, now that we can use the RBHC framework to provide a checklist of the relevant actors (i.e. ticks, pathogens and hosts), resources and mechanisms, it becomes clear that the biological realism of this and other models can still be improved. For example, including more specific resources, possibly associated to vegetation types more easily addressed by mapping and land management, would allow better nuancing of scenarios and results. Rather than dividing the area in just forest and grassland, or forest and non-vegetated areas, it would be useful to include the ecological functions of the border zones, the so-called ecotones, with their own specific resources. Also, the amount of resources needed per individual need to be taken into account. The results of the cellular automata model may have been different if the resource availability had been modelled explicitly, since resources may not be available in sufficient amounts in very small forest patches as those assumed in the model. Making such small forest patches unusable by deer would thus improve the biological realism of the model. Another straightforward next step is to increase the host diversity in the model, as long as information on resource/landscape use, movement capacity and interaction with ticks are known. Feeding host control: impact of deer management on ticks Li et al. (2014) used an agent-based simulation to examine the consequences of deer management, through scenarios modifying mortality or movement, on the spatial dynamics of Ixodes ricinus (Li et al. 2014). The main focus concerned a realistic representation of deer movement, and scenarios based on existing or foreseen practices related to game or disease risk management. Ticks were also included in the model, including a detailed life cycle and attachment on (a generic) small mammal, and (generic) deer. Pathogen transmission was not considered. Deer movements were calibrated based on existing knowledge concerning roe deer and red deer movements and included two types of movements: home ranging (short distance movement for e.g. foraging) and displacement (long range movements to other, e.g. less populated, habitats). These two types of movement correspond to specific sets of rule that include several steps ( phases ) and are constrained by movement capacity which is longer in displacement than in home ranging. Animals moved around an artificial landscape made out of three patches of woodland and a matrix of grassland. Four scenarios were considered: (1) reducing local deer density through hunting (across the landscape or in a local woodland patch); (2) controlling deer grazing intensity in grassland (applied by reducing movement capacity and time spent in the grassland matrix in home ranging); (3) translocation and reintroduction of deer; and (4) controlling human disturbances and deer displacement (reducing displacement caused by human disturbances). This therefore, from a RBHC point of view, considers tick resources as well as deer resources and movement capacity to access diverse resources. Ecology and prevention of Lyme borreliosis 211

213 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink This multi-host, multi-land cover model underlines the need to consider host movement capacity in order to understand tick population dynamics at the landscape level, and that the range of movement, combined with the landscape fragmentation, modifies the outcome of scenarios. The results indicated that the effect of locally controlling deer population on tick population differs according to the movement capacity of deer: if it allows deer to reach patches where control was applied, the effect on tick population is greatly reduced. The second scenario tested the effect of a decrease of the use of grassland by deer. This reduced tick abundance in grassland, but increased it in woodland, where ticks may thus feed more often on reservoir species. The third scenario indicated that suppressing deer in an area does not suppress ticks, which can still feed on other hosts. The suppression of deer may result in an increase in small mammal densities, and once deer are reintroduced, density levels can reach higher level than at the start of the simulation. The decrease in deer displacement applied in the fourth scenario indicated resulted in a decrease in overall and maximum tick density, which may relate to the model set up for use of grassland, where tick mortality is higher, and to movement rules (choice of a destination cell). Still, it does point at relevant public health implication of the establishment of quiet zones (areas excluding human disturbances) in isolated areas as movement capacity and landscape structure, as presented above, modify the effect, in terms of tick abundance and locations, of such processes. Results were consistent with other studies, both empirical and of simulation (see Li et al. 2014). A full RBHC perspective as presented above would need to have the pathogen as its main organism of focus. This is feasible as the pathogen s resources (host and vector) are effectively present in the model. Concerning host and vector, several resources are included (e.g. grassland as a key element for feeding; hosts for ticks). However, the results do highlight that movement capacity, as well as landscape structure (are landscape features, such as woodlands, accessible?) do affect the outcome of a change in pathogen resource abundance. The results raise the issue of substitutions between resources or else the need to carefully assess the potential of various animals or landscape features to serve as resources. A model set up such as this one indeed allows testing the effect of substitution or the lack thereof. In the context of dynamic landscapes that are subjected to managements by diverse public health authorities, this is an important issue, and one that a conceptual framework such as RBHC can usefully address. Discussion: challenges and perspectives for RBHC in understanding and management of disease risk At the most basic level, RBHC underlines two major ideas. First, that, often, a single vegetation type cannot represent the entire habitat, whatever the species concerned. Any host, vector, pathogen species we may consider in the context of vector-borne and zoonotic diseases, including tickborne diseases, has a range of functions that requires specific resources in order to be fulfilled. Second, as Figure 1 makes very clear, the habitat of a pathogen, does not equate to the habitat of the vector. Far from approaching the issue of environmental suitability for TBP in an overly complicated and detailed fashion, the structure that RBHC encourages to build offers keys to understand a complex system. Operational constraints clearly remain, such as those generated by the information content of existing environmental databases or what is currently possible to monitor when it comes to vectors, hosts or the TBP themselves. This should not preclude the use of a RBHC concept, and may help in directing data collection in a resource tight context. It does not require mapping resources specifically, but primarily encourages the identification of any vegetation type or landscape feature that could provide them, rather than the element providing it mostly. Then, 212 Ecology and prevention of Lyme borreliosis

214 14. A resource-based habitat concept for tick-borne diseases examining the distribution of these features through the lens of host movement completes the picture. Such operationalisation of the concept have been demonstrated previously for insects (Kalarus et al. 2013, Turlure et al. 2009, 2010). Across a continent such as Europe, the suitability of vegetation types can vary for the transmission of zoonotic pathogens, with some vegetation types classically assessed as unfavourable in some areas that are found suitable in others. This was found for hantavirus by Zeimes et al. (2015). Medlock et al. (2008) found that grass/dwarf heath likely offers better protection from desiccation for ticks than the nearby deciduous forest. Considering resources available rather than vegetation types allows making such interpretations in diverse environments. In existing models, RBHC offers additional perspectives on results that may appear unexpected, and in areas where the primary habitat of I. ricinus ticks, deciduous forest, is poorly represented (Vanwambeke et al. 2016). A major challenge, but also a major opportunity, for the application of RBHC to TBP is the opportunistic feeding behaviour of ticks, which may feed on reservoir or non-competent hosts. Models are currently unlikely, in relation to parameterisation and computing, to accommodate a representation of the full palette of hosts that ticks feed on. However, the use of scenarios that offer more or fewer reservoir hosts, on which ticks would therefore feed more or less often, opens interesting opportunities. Such models have been elaborated in the past but in a non-spatial way (Levi et al. 2016, Ogden and Tsao 2009). The results by Li et al. (2012a, 2014) suggest the very core of the RBHC: availability and accessibility of the resources do modify these outcomes, and including them may help solve some of the current questions. Also, as indicated by that model, the time spent in an area (e.g. grassland) does affect the importance each will play in providing resources and in distribution vectors across the landscape. Some research also has indicated that hosts may affect vector resources (other than blood meal) by e.g. keeping the vegetation low or by keeping key reproduction hosts away, this lowering tick abundance (Steigedal et al. 2013). Understanding the details of a pathogen s habitat bears relevance also when considering humans, exposure and prevention. Two pathways can be highlighted for this. First, as outlined in Hartemink et al. (2015) concerning blue tongue virus transmission, key resources in the system can be identified that lend themselves to removal or displacement, making them inaccessible and therefore unusable. Second, the concept can be easily extended to include the resources required for human ecological functions that would lead to exposure to tick bites. Like other ecological resources, recreational resources (or related to other exposure activities) can be mapped in relation to other resources of the TBP and their accessibility through host movement. Those resources (e.g. signposted walks, recreational equipment) could potentially be placed in areas where infected tick abundance is lower, information is focused and/or control applied. The use of RBHC in formalised models such as those reviewed here allows exploring the effect of such interventions on exposure and on risk. Conclusion A great wealth of knowledge has been accumulated thanks to decades of research and modelling of the suitability of diverse landscapes for tick-borne disease transmission. Because many datasets represent keyholes that only offer a limited insight into what are complex ecological systems, the coherence, beyond the most obvious results related to primary habitats, can be difficult to see. The RBHC approach offers a promising perspective to consolidate existing knowledge, to guide further data acquisition and modelling. Beyond the scientific interest of the concept, its potential use for understanding the risk of zoonotic tick-borne diseases for humans and for exploring options for prevention and control is significant. Ecology and prevention of Lyme borreliosis 213

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217 Sophie O. Vanwambeke, Sen Li and Nienke A. Hartemink Meyfroidt P and Lambin EF (2011) Global forest transition: prospects for an end to deforestation. Annu Rev Environ Resource 36: Navarro LM and Pereira HM (2015) Rewilding abandoned landscapes in Europe. In: Pereira HM and Navarro LM (eds.) Rewilding European landscapes. Springer International Publishing, New York, USA. Ogden NH and Tsao JI (2009) Biodiversity and Lyme disease: dilution or amplification? Epidemics 1: Ogden NH, Bigras-Poulin M, O Callaghan CJ, Barker IK, Lindsay LR, Maarouf A, Smoyer-Tomic KE, Waltner-Toews D and Charron D (2005) A dynamic population model to investigate effects of climate on geographic range and seasonality of the tick Ixodes scapularis. Int J Parasitol 35: Randolph SE (2001) The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme borreliosis in Europe. Philos Trans R Soc B Biol Sci 356: Randolph SE and Storey K (1999) Impact of microclimate on immature tick-rodent host interactions (Acari: Ixodidae): implications for parasite transmission. J Med Entomol 36: Randolph SE and Šumilo D (2007) Tick-borne encephalitis in Europe: dynamics of changing risk. In: Takken W and Knols BGJ (eds.) Emerging pests and vector-borne disease in Europe. Ecology and Control of Vector-borne diseases, Volume 1. Wageningen Academic Publishers, Wageningen, the Netherlands. Richter D and Matuschka FR (2011). Differential risk for Lyme disease along hiking trail. Emerg Infect Dis 17, Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubalek Z, Foldvari G, Plantard O, Vayssier-Taussat M, Bonnet S, Špitalská E and Kazimírová M. (2014) Ixodes ricinus and its transmitted pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health. Front Public Health 2: Špitalská E, Stanko M, Mošanský L, Kraljik J, Miklisová D, Mahríková L, Bona M and Kazimírová M (2016) Seasonal analysis of Rickettsia species in ticks in an agricultural site of Slovakia. Exp Appl Acarol 68: Steigedal HH, Loe LE, Grøva L and Mysterud A (2013) The effect of sheep (Ovis aries) presence on the abundance of ticks (Ixodes ricinus). Acta Agric Scand Sect Anim Sci 63: Tack W, Madder M, Baeten L, Vanhellemont M, Gruwez R and Verheyen K (2012) Local habitat and landscape affect Ixodes ricinus tick abundances in forests on poor, sandy soils. For Ecol Manag 265: Turlure C, Choutt J, Baguette M and Van Dyck H (2009) Microclimatic buffering and resource-based habitat in a glacial relict butterfly: significance for conservation under climate change. Glob Change Biol 16: Turlure C, Choutt J, Van Dyck H, Baguette M and Schtickzelle N (2010) Functional habitat area as a reliable proxy for population size: case study using two butterfly species of conservation concern. J Insect Conserv 14: Uspensky I (2014) Tick pests and vectors (Acari: Ixodoidea) in European towns: introduction, persistence and management. Ticks Tick-Borne Dis 5: Vanwambeke SO, Šumilo D, Bormane A, Lambin EF and Randolph SE (2010) Landscape predictors of tick-borne encephalitis in Latvia: land cover, land use, and land ownership. Vector-Borne Zoonotic Dis 10: Vanwambeke SO, Van Doninck J, Artois J, Davidson RK, Meyfroidt P and Jore S (2016) Forest classes and tree cover gradient: tick habitat in encroached areas of southern Norway. Exp Appl Acarol 68: Zeimes CB, Olsson GE, Hjertqvist M and Vanwambeke SO (2014) Shaping zoonosis risk: landscape ecology vs. landscape attractiveness for people, the case of tick-borne encephalitis in Sweden. Parasit Vectors 7: 370. Zeimes CB, Quoilin S, Henttonen H, Lyytikäinen O, Vapalahti O, Reynes J-M, Reusken C, Swart AN, Vainio K, Hjertqvist M and Vanwambeke SO (2015) Landscape and regional environmental analysis of the spatial distribution of hantavirus human cases in Europe. Front Public Health 3: Ecology and prevention of Lyme borreliosis

218 15. Modelling the ecological dynamics of tick borne pathogens in a risk assessment perspective Alessandro Mannelli 1*, Agustin Estrada-Peña 2 and Donal Bisanzio 3 1 University of Turin, Dipartimento di Scienze Veterinarie Università degli Studi di Torino, Largo P. Braccini, 2, Grugliasco (Torino), Italy; 2 University of Zaragoza, Department of Animal Health, Faculty of Veterinary Medicine, Miguel Servet, 177, Zaragoza, Spain; 3 Big Data Institute, Nuffield Department of Medicine, University of Oxford, c/o Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom; alessandro.mannelli@unito.it Abstract Modelling the ecological dynamics of tick borne pathogens (TBP) must be based upon the integration of information from multiple disciplines. As an example, including genetic variability of Borrelia burgdorferi s.l., into next generation matrix models suggested that conclusions from previous approaches, based upon quantitative relationships among interacting ticks and hosts, should be reconsidered. Individual-based models, such as those based on networks, by taking into account the aggregation of ticks among hosts, show that the epidemic threshold (a critical value of transmission probability, under which the TBP eventually dies out) is lower than expected based upon other methods. In a similar approach, food web models can be used to evaluate the relative importance of vertebrates in supporting the persistence of the ticks and TBP. Models can be used in the main phases of risk assessment of expanding home range of TBP: release assessment from endemic areas (potential sources of hazards for other areas), dispersal of TBP to previously free areas (exposure assessment); consequence assessment in newly invaded areas. Surveillance of introduction, and of trends of TBP following the invasion of a new area, can benefit from modelling, by investigating the relationships between indicators, such as, for example, acarological risk, and prevalence of antibodies in sentinel animals. Keywords: ecology, epidemiology, modelling, network analysis, risk assessment Introduction The emergence of tick borne pathogens (TBP) in Europe is associated with complex interactions between environmental factors, and societal changes affecting land use (Medlock and Jameson 2010). Understanding mechanisms leading to changes in the abundance and distribution of ticks and TBP is an important objective of ecological research, which can provide scientific evidence for prediction, surveillance, and prevention of infections (Braks et al. 2014, Medlock et al. 2013, Rizzoli et al. 2014, Sprong et al. 2012). Modelling gained a prominent place in different phases of ecological research. The approach can provide the conceptual framework to identify objectives, to guide study design, to integrate and summarise results from different disciplines, and to interpret the observed patterns. Since modelbuilding usually requires a careful review of the available information on key parameters, gaps in knowledge can be identified, and models may serve as guides for further research (Heesterbeek et al. 2015, Randolph and Craine 1995). Conditions affecting the persistence and the intensity of transmission of pathogens by means of the estimation of the basic reproductive number of the infection, R 0, have been the main objective Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _15, Wageningen Academic Publishers 2016

219 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio of ecological modelling of TBP. Conversely, the role of these models in estimating the risk of infection of humans and susceptible animals has been less frequently explored (Li et al. 2012, 2016, Wang et al. 2012). In this chapter, we carry out a synthetic review of models of the transmission dynamics of TBP, with emphasis on Borrelia burgdorferi sensu lato (s.l.), the agents of Lyme borreliosis, which is the most commonly reported tick borne zoonosis in Europe. A secondary aim is the identification of the role of models in risk assessment, with a focus on the link between ecological research and public health. A broad classification of models In the ecology of TBP, modelling methods can be classified into two broad categories: mechanistic (or process-driven) models, and correlative models. Furthermore, a third category, mechanistic niche models, can be identified, where features of mechanistic and correlative models are integrated. Mechanistic models are attempts to represent (at least in part) the biological mechanisms leading to an outcome of interest, such as persistence of a TBP. Examples of mechanistic models include the simulation of the transmission of TBP s, and the calculation of the basic reproduction number, R 0, under different assumptions on tick-host dynamics and on routes of transmission. The model s key parameters, such as, for example, tick-host encounter rates, are usually based upon previous knowledge, or upon ad hoc observations (Rosà and Pugliese 2007). In correlative models, the association between selected predictors (i.e. weather, landscape features) and the outcome of interest (i.e. presence/absence, or counts of the vector) are estimated, for example, by a regression analysis, which is carried out on field observations. Based upon the estimated regression parameters, the probability of presence of an organism (a vector) can be calculated. Less frequently, tick counts are used as the model s outcome (Boehnke et al. 2015). In correlative models, no attempt is made to represent the processes underlying the estimated associations. For instance, the probability of the presence of tick in an area may be because its low mortality, or its fast development, but these processes are not explicitly included in correlative models, which are based upon empirical observations. Accordingly, they can be defined as phenomenological models (Ostfeld and Brunner 2015). Usefulness of correlative models depends upon the proper selection of predictors, and on the careful interpretation of results, which need to be based on sound hypotheses on the underlying biological mechanism (although these are not explicitly considered in the estimation process itself). Examples of correlative models include species distribution models, and environmental niche model, which have been used to predict the geographic range of ticks and TBP. Potentials, and caveats of correlative models were recently reviewed (Estrada-Peña et al. 2014a, 2016). In mechanistic niche models, the geographic distribution of ticks and TBP s can be predicted based upon previous knowledge of their environmental requirements (mechanistic component), and on available data on the current geographic distribution of those favourable conditions. Therefore, these models are not solely based on the statistical correlation between tick presence or abundance, and, for example, habitat humidity and temperature (or their satellite-derived proxies). Instead, prediction is based on the knowledge of the physiological mechanisms relating tick survival and development to those environmental characteristics (Estrada-Peña and Estrada- Sánchez 2014, Kearney and Porter 2009). 218 Ecology and prevention of Lyme borreliosis

220 15. Modelling tick borne pathogens Modelling the transmission of TBP: dealing with complexity Mechanistic models have been widely used in the dynamics of directly transmitted diseases (Lloyd-Smith et al. 2009). The complexity of the transmission of TBP, however, needs specific modelling efforts. (Alexander et al. 2012, Estrada-Peña et al. 2014b, Gilbert 2016, Heesterbeek et al. 2015, Hollingsworth et al. 2015). As an example of how complexity affects models outcomes, the effects of varying density and composition of the populations of vertebrate hosts for Ixodes ricinus might have different and, sometimes, counter-intuitive effects on the transmission of TBP. Field studies carried out at locations in North America and in Europe, supported the conclusion that biodiversity has a negative effect on the intensity of transmission of B. burgdorferi s.l. and of tick borne encephalitis virus (TBEV) (Bolzoni et al. 2012, Cagnacci et al. 2012, Keesing et al. 2006, LoGiudice et al. 2003). The hypothesised mechanism, which was also supported by mechanistic models, is based upon ticks feeding on hosts, which are characterised by limited, or no reservoir competence for the agents, therefore leading to a dilution of the transmission. However, other studies provided contrasting evidence, and models showed that, under certain circumstances, host diversity might support or even amplify transmission. It is the case of non-competent hosts that contribute to transmission by vector augmentation (Ogden and Tsao 2009, Swei et al. 2011). Furthermore, even secondary reservoir hosts may sustain transmission, when primary reservoir hosts population are at low levels (Mannelli et al. 2012a, Randolph and Dobson 2012). We will further elaborate about these effects in the section food web models. Recent advances in mechanistic models of TBP Among recently developed mechanistic models for transmission, next generation matrix models (NGMM) have been used to estimate R 0, by integrating the complexity of the system under study. Hartemink et al. (2008) used NGMM to explore in detail the role of different transmission routes of B. burgdorferi s.l. and TBEV: systemic infection of vertebrate reservoir hosts and sufficient circulation of the pathogen in its blood to allow infection of feeding ticks, as well as transmission by co-occurrence of feeding (co-feeding) ticks and transovarial transmission. They provided sound and clear support for the major role of systemic vertebrate transmission for B. burgdorferi s.l., and of transmission among co-feeding ticks for TBEV. NGMM were also used to show that the key components of transmission were significantly affected by the variability across six strains of Borrelia afzelii (Tonetti et al. 2015). Based upon results of this study, previously built models that focused only on quantitative relationships among hosts and vectors should be revisited, and their conclusions reconsidered. This is in line with our realised knowledge about the variability of host-pathogen relationships, which are susceptible to change along the gradient of genetic variability of the association. Furthermore, relationships between vertebrate cross immunity and B. burgdorferi s.l. genetic variability would also be suitable to be explored by modelling (Durand et al. 2015, Jacquet et al. 2015), confirming the importance of the integration of modelling with laboratory investigations. Individual based models Individual based models, where individuals are the units of analysis and simulation, allow to take into account the heterogeneity of hosts regarding their role in the transmission of TBP. Ferreri et al. (2014), as an example, showed that, due to the aggregated distribution (following a power law) of I. ricinus immature on a limited fraction of the rodent population, the transmission of TBEV was possible even during the season of low abundance of hosts. Ecology and prevention of Lyme borreliosis 219

221 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio The frequency and structure of contacts among individuals are the specific focus of network and food web models. In the ecology of TBP, vertebrate hosts and ticks can be represented as nodes in a graph, whereas agent transmission is represented by link, or edges among nodes (Godfrey 2013, Pastor-Satorras and Vespignani 2001). Since transmission may occur only between nodes of different types (such as vertebrate hosts, or ticks), but not between two individuals of the same type, such graphs are defined as bipartite. Even transmission via co-feeding, in fact, requires that two ticks feed on a vertebrate host. The application of a bipartite network model, by Bisanzio et al. (2010), showed that, due to the aggregation of ticks among hosts, B. burgdorferi s.l. can be maintained even in case of very low levels of transmission between ticks and hosts. To more effectively represent the distribution of ticks on hosts, as a key factor in non-systemic transmission of TBEV (via co-feeding), Ferreri et al. (2016) applied dynamical, star network models, where hosts are represented by a central node, and ticks by peripheral nodes which are connected with the central node by edges called rays. During each step of the numerical simulation, a constellation of star graphs is generated, and the infection is spread among rays of the stars, following methods described by Bisanzio et al. (2010). Connections among nodes are allowed to change with season, improving the model s realism. Such an approach would be suitable for application to other TBP and to different transmission route. Network approaches may provide helpful frameworks for understanding structural patterns and the functional and complementary roles of different animal species in ecosystems (Bersier et al. 2008, Clauset et al. 2008). The only report on the structure of TBP has been recently addressed (Estrada-Peña et al. 2015). These studies feed on massive data sets for assessing ecological, epidemiological, and evolutionary patterns among vertebrate hosts, vectors, and transmitted pathogens. Like networks, food web models consist of nodes and links that represent a given system in terms of its components (nodes) and the relations between those components (edges). In the same way that food webs are descriptions of who eats whom in an ecosystem, the method may be translated to describe who is a parasite of whom. To represent the network, each node symbolises a species, and the resulting edge between two nodes represents a relationship. These models are not aimed to predict, but to capture the structure and the dynamics of a system. Of importance in these models are the measures of centrality (Martınez-Lopez et al. 2009), from which the semantics of the system emanate. The purpose is to understand the relative importance of vertebrates in supporting the persistence of the ticks and pathogens in a network of clusters defined by their species composition. Results and ongoing studies show that these approaches to TBP s are adequate to capture the complex co-evolving forces that shape the relationships between species. Most important, it is now evident that in the case of B. burgdorferi s.l., the genetic diversity of the hosts feeding the ticks would be responsible for the maintenance of the system, and that each species of host would have very different roles in the circulation of the pathogens. That circulation capacity would be even a local effect, with a wide variability according to the local fauna of vertebrates. As stated, these interpretations are only aimed to address the complete picture of the epidemiology a TBP, and to obtain conclusions from the many reports available, which commonly have a local or regional focus. Previous studies on Anaplasma phagocyotphilum (De la Fuente et al. 2015), however, suggest that there is a wide field of research for the application of food web models to the epidemiology of TBP. 220 Ecology and prevention of Lyme borreliosis

222 15. Modelling tick borne pathogens Role of models in risk assessment of geographic range expansion of TBP s Risk assessment is a systematic process, aimed to providing answers to specific risk questions, based upon the best scientific evidence (Brookes et al. 2015, MacDiarmid and Pharo 2003). The introduction in free territories of pathogens of humans and animals is a frequent application of risk assessment, and examples include tick borne zoonoses (Gale et al. 2010, Hoek et al. 2012). In this section, we will attempt to identify the role of ecological models in the main phases of a risk assessment, to provide answer to key questions related to the geographic range expansion of I. ricinus and of B. burgdorferi s.l., and to the increasing trends in transmission intensity which are observed in Europe (Figure 1). The following, general risk questions would be relevant: What is the risk of introduction and establishment of I. ricinus and of B. burgdorferi s.l. in a previously free area (or in an area where these agents were present at a low level of transmission) from pre-existent endemic areas? Once established in a new area, will the agents undergo increasing trends, or will decrease, possibly resulting in extinction? Introduction of an agent of infectious or parasitic disease in a hitherto free area can be represented through a general conceptual model or risk pathway. In addition to producing a Risk pathway Role of models Release assessment Release of TBP in endemic areas Evaluation of the intensity of transmission of tick borne pathogens (TBP) in geographic areas potentially serving as sources of TBP for free areas. Application of correlative, and mechanistic niche models to predict intensity of transmission across the current range of TBP. Exposure assessment Exposure of free areas to TBP Evaluation of dispersal of TBP from sources, to hitherto free areas, by network analysis, cellular automata, integrated with GIS and spatial analysis. Application of phylogenetic analysis to phylogeography. Consequence assessment Consequence of TBP in free areas Evaluation of establishment and persistence of TBP in previously free areas, surveillance and analysis of trends, evaluation of indicators in surveillance. Integration of mechanistic, and correlative models. Figure 1. Role of models in the main phases of a risk pathway for the geographic range expansion of tick borne pathogens (TBP) in a risk assessment approach. Ecology and prevention of Lyme borreliosis 221

223 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio graphical representation, occurrence of events, which are part of the risk pathway can be assigned probabilities, whose combinations result in a probabilistic model (EFSA 2015). Different steps can be identified, and terminology needs to be adapted, when applying the risk assessment approach to a specific field of application (De Vos et al. 2011, EFSA 2012). To favour the identification of the role of models in assessing the risk of the geographic expansion of tick borne pathogens to hitherto free areas, we distinguish three main phases: release assessment, exposure assessment, and consequence assessment. Release assessment from endemic areas In our approach, this is the evaluation of the capability of endemic areas to serve as sources of I. ricinus and B. burgdorferi s.l. for other areas. It can be assumed that the probability of movements of the agents from an endemic area is positively associated with: The intensity of transmission within the area, resulting in abundant infected vectors and reservoir hosts, including different B. burgdorferi s.l. genospecies, which can be associated with different vertebrate reservoir hosts, characterised by variable dispersal capability. Movements of vertebrate hosts from the endemic area, as the responsible of dispersal of both vectors and infectious agents; in fact, pathogens can be carried by ticks attached to hosts, or by infected, reservoir hosts. In general, models could be used to estimate the intensity of transmission across the current geographic range of the agents. Correlative models could be used to predict abundance of infected vectors and vertebrate hosts, based on the collection of data at sampled locations, and on the subsequent generalisation to wider areas. Mechanistic niche models, on the other hand, could be used to identify those endemic areas where environmental condition are most suitable for the vector s physiological requirements, and where transmission might be most intense. Modelling dispersal of TBP among geographic areas (exposure assessment) Although this phase is, in some extent, overlapping with the previous phase of release assessment, we consider it separately to focus on the application of methods, such as, for example, network analysis, which are specifically suited to investigate dispersal of pathogens among geographic areas. Long distance dispersal of I. ricinus and B. burgdorferi s.l. is mostly associated with movements of migratory birds, whereas short distance dispersal is associated with movements of resident animals (De la Fuente et al. 2015, Diuk-Wasser et al. 2010, Ogden et al. 2008, 2013). Modelling can be applied to the dispersal of agents outside endemic area, based upon information on: (1) geographical locations of endemic (source), and free (target) areas; and (2) routes, intensity, and time pattern of dispersal of hosts from endemic areas and to free areas. A correlative models, based upon survival analysis, was used to predict the northward spread of Ixodes scapularis in Canada (Leighton et al. 2012). Time to establishment to a new location was the model s outcome, whereas environmental suitability, and available data on tick dispersal were the predictors. The effect of temperature as a major component of environmental suitability suggested the importance of climate warming in range expansion. Network modelling would be particularly appropriate to model dispersal of I. ricinus and B. burgdorferi s.l.: geographic areas, which can be identified by GIS and spatial analysis, are 222 Ecology and prevention of Lyme borreliosis

224 15. Modelling tick borne pathogens represented as nodes, whereas dispersal routes are represented as link among nodes (Figure 2). The use of graph theory and networks to investigate animal movements was recently reviewed (Jacoby and Freeman 2016, Jacoby et al. 2012). Network analysis was used by Fenner et al. (2011) to investigate ticks and other parasites spread in lizard populations. Geographic areas can serve as primary sources of agents for other areas, but they can also serve as intermediate nodes, connecting two or more nodes (Bodin and Saura 2010). The distribution of links among nodes, including distances, as well as the intensity of dispersal are the focus of network analysis. Frequency and distribution of links among nodes can be summarised by computing centrality indices (Martınez-Lopez et al. 2009).These are useful to identify territories which have high connections with other territories and, thus, might play an important role in the dispersal of TBP among geographic areas (Ortiz-Pelaez et al. 2006). Dispersal of I. ricinus and B. burgdorferi s.l. can be quantitatively simulated by dynamic networks, where the seasonality of the dispersal can be included. Consequently, the structure of the network changes through time due to variations in the attributes of links and nodes. Simulating the dispersal of agents through dynamic networks may lead to results which were not predicted by centrality measures. Home range of wild animal hosts, as well as direction, intensity, and seasonality of their dispersal are fundamental data which are needed to build dynamic network models of dispersal. Their Forest Patch Connection Figure 2. Graph representation of hypothetical animal movement data, in the province of Pisa, Tuscany, Italy, where abundance of Ixodes ricinus was positively associated with normalised difference vegetation index (NDVI) and deciduous woods (Bisanzio et al. 2008). Deciduous wood patches are represented as nodes, whereas edges represent potential animal movement routes. To model dispersal of I. ricinus and Borrelia burgdorferi s.l., further information should be used, on actual movements of several animal species, and on physical barriers, such as urban areas, highways, major rivers. Ecology and prevention of Lyme borreliosis 223

225 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio limited availability, and difficulties which might be encountered in collecting these data are major obstacles in the use of this approach. Once available, these data would be integrated with other information on habitat, which would be more easily gathered by, for example, remote sensing. Tools for data management and analysis, GIS and spatial analysis, and software for network analysis should be applied, by integrating proper analytical capability, with the best knowledge of the biological processes to be modelled. Proper data collection, together with the development of such a comprehensive set of tools and competences can be suggested as priorities to face the current emergence of vector borne zoonoses. Spread pattern of TBP greatly benefits from the integration of network modelling and phylogeographic analysis of the agents (Weidmann et al. 2013). Consequence assessment in previously free areas Mechanistic models can be used to provide answers to questions on whether the agents, once introduced by dispersal from endemic areas, or from other areas serving as intermediate passages, may persist in a newly invaded area. More specifically, models aimed to the calculation of the basic reproduction number, R 0, can be used to assess what will happen if a pathogen is introduced in a naïve population. Correlative models, as well as mechanistic niche models could provide information on the effects of environmental factors affecting the successful establishment of I. ricinus and B. burgdorferi s.l. in new areas and, therefore, should be integrated with mechanistic models. Transmission intensity of the agents may, subsequently, undergo an increasing trend, or remain at low levels, with the possibility of extinction. In this way, network modelling of dispersal may be integrated with models of successful establishment and trends. Suitability of new areas may be the object of mechanistic niche models. Areas with suitable conditions for the establishment of the transmission cycle of B. burgdorferi s.l. may be distributed in patches (Estrada-Peña 2009). The effects of landscape fragmentation on the spread of I. ricinus and B. burgdorferi s.l. across the landscape has been explored by the application of models based upon cellular automata (Li et al. 2012). By including both the transmission dynamics of the agent, as well as movements across different habitat types, the model combined all phases of the risk assessment process, and it was useful to make predictions on relevant risk parameters, such as density of nymphs, nymph infection prevalence (NIP), and density of infected nymphs (DIN). By providing suggestions for landscape management, the model showed contrasting effects of limiting host movements by fencing. Modelling may be applied to investigate potential interactions between the transmission dynamics of newly introduced, and existing TBP, including competition between established, and invading tick species (Kershenbaum et al. 2012, Martello et al. 2014, Vaumourin et al. 2013). Moreover, one TBP can be favoured over another by changes in the composition of vertebrate hosts, which can be due to hunting or predation (Levi et al. 2012). Given the clinical consequences of infections by multiple, tick borne agents in people, simultaneous transmission of several agents, by the same tick species, also deserves a modelling effort (Coipan et al. 2013). The density of host-seeking, infected I. ricinus nymphs (DIN), and NIP (Jouda et al. 2004, Mather et al. 1996) can be combined to obtain the acarological risk, which is an estimate of the probability of encountering at least one infected I. ricinus in a defined land unit (Mannelli et al. 2003). Mechanistic models could be specifically geared to provide estimates of such parameters. In this way, TBP s 224 Ecology and prevention of Lyme borreliosis

226 15. Modelling tick borne pathogens ecology would be better integrated in risk assessment, and would be more directly relevant for public health (Li et al. 2012, 2016, Wang et al. 2012). Based upon the combination of release, exposure, and consequence assessment, predictions can be generated of the range expansion of TBP. Subsequent surveillance would allow to validate prediction and to investigate on trends of TBP in the newly invaded areas. Role of modelling in surveillance of TBP Surveillance of zoonoses can be conceptually represented in surveillance pyramids, allowing the visualisation of targets for data collection (such as ticks and hosts), in different contexts (Braks et al. 2014). Based upon a pyramid for Lyme borreliosis, targets may include B. burgdorferi s.l.- infected, questing ticks, and the acarological risk, or competent reservoir hosts. Alternatively, other indicators can be used, such as the detection of antibodies against B. burgdorferi s.l. in animals (wild ungulates or domestic animals) which are not competent reservoirs for the agents, but that can be exposed to infectious tick bites (Mead et al. 2011, Wagner and Erb 2012). In these cases, models can be applied to the relationships between prevalence of antibodies in sentinel animals in a certain study area, and the acarological risk. A compartmental model was built to simulate trends in the prevalence of antibodies against B. burgdorferi s.l. in dogs, following trends in acarological risk (Mannelli et al. 2012b). Key parameters, in a set of differential equations, were: b, the probability of tick bite on a susceptible dog; b, when multiplied by the acarological risk, determines the transition of dogs from susceptible to exposed; c, the rate of development of a detectable antibody response in exposed dogs which can become serologically positive; a, the rate of disappearance of detectable antibody response. The range of acarological risk values, where changes in prevalence of seropositive dogs were greatest, were obtained for different values of b, thus providing support for sampling in surveillance and for the interpretation of results. Moreover, such as model can be useful to identify potential factors, which might affect the above key parameters and, therefore, which must be taken into account in surveillance. For example, the frequentation, by dogs, of areas suitable to B. burgdorferi s.l., and treatments with tick repellents may affect b, and their effects could be included in modelling. The development of detectable antibody response, and the rate of antibody disappearance following an infectious tick bite, as well as sensitivity and specificity of diagnostic tests, are currently not completely known and, therefore, limit the use of animal serology in surveillance, and should be the objects of further research. Conclusion Models are useful in the ecology of TBP as well as in risk assessment. Best results can be obtained by integrating in-depth knowledge of the biological systems under study, with the most advanced modelling techniques. The complexity of the ecological dynamics of TBP, leading to contrasting results of models relaying on different assumptions and field observations, justifies a cautious approach to prediction by modelling. Indeed, the identification of gaps in knowledge and of research priorities is one of the most important objectives of models. This is of particular interest in risk assessment, where modelling can be used to investigate the chain of events leading to the outcomes of interest, such as the risk of range expansion of TBP, and its consequences. Here, Ecology and prevention of Lyme borreliosis 225

227 Alessandro Mannelli, Agustin Estrada-Peña and Donal Bisanzio the conceptual framework and the comprehensive view, which are provided by models, would enhance the usefulness of ecology as a guide for policy in public health. Public health relevance The expansion of the geographic range of ticks and tick borne pathogens is a threat to public health across Europe. Modelling the ecological dynamics of these agents would be most useful for the prevention of diseases in people and animals, if integrated within a risk assessment framework. Based upon results of a transparent and science-based risk assessment, proper surveillance can be designed, and disease prevention measures can be adopted. Acknowledgements We would like to thank Mario Giacobini, University of Torino, for useful discussion on dynamic network modelling, and Giuseppe Ru, Istituto Zooprifilattico Sperimentale di Piemonte Liguria e Valle d Aosta, for suggestions on definitions in risk assessment. References Alexander KA, Lewis BL, Marathe M, Eubank S and Blackburn JK (2012) Modeling of wildlife-associated zoonoses: applications and caveats. Vector-Borne Zoonotic Dis 12: Bersier LF, Banašek-Richter C, Cattin MF and Sep N (2008) Quantitative descriptors of food-web matrices. Ecology 83: Bisanzio D, Amore G, Ragagli C, Tomassone L, Bertolotti L and Mannelli A (2008) Temporal variations in the usefulness of normalized difference vegetation index as a predictor for Ixodes ricinus (Acari: Ixodidae) in a Borrelia lusitaniae focus in Tuscany, central Italy. J Med Entomol 45: Bisanzio D, Bertolotti L, Tomassone L, Amore G, Ragagli C, Mannelli A, Giacobini M and Provero P (2010) Modeling the spread of vector-borne diseases on bipartite networks. PLoS ONE 5: e Bodin O and Saura S (2010) Ranking individual habitat patches as connectivity providers: integrating network analysis and patch removal experiments. Ecol Modell 221: Boehnke D, Brugger K, Pfäffle M, Sebastian P, Norra S, Petney T, Oehme R, Littwin N, Lebl K, Raith J, Walter M, Gebhardt R and Rubel F (2015) Estimating Ixodes ricinus densities on the landscape scale. Int J Health Geogr 14: Bolzoni L, Rosà R, Cagnacci F and Rizzoli a (2012) Effect of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. II: population and infection models. Int J Parasitol 42: Braks M, Medlock JM, Hubalek Z, Hjertqvist M, Perrin Y, Lancelot R, Duchyene E, Hendrickx G, Stroo A, Heyman P and Sprong H (2014) Vector-borne disease intelligence: strategies to deal with disease burden and threats. Front Public Health 2: Brookes VJ, Hernández-Jover M, Black PF and Ward MP (2015) Preparedness for emerging infectious diseases: pathways from anticipation to action. Epidemiol Infect 143: Cagnacci F, Bolzoni L, Rosà R, Carpi G, Hauffe HC, Valent M, Tagliapietra V, Kazimirova M, Koci J, Stanko M, Lukan M, Henttonen H and Rizzoli a (2012) Effects of deer density on tick infestation of rodents and the hazard of tick-borne encephalitis. I: empirical assessment. Int J Parasitol 42: Ecology and prevention of Lyme borreliosis

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234 16. How can forest managers help to reduce the risk for Lyme borreliosis? Kris Verheyen * and Sanne C. Ruyts Forest & Nature Lab, Ghent University, Geraardsbergsesteenweg 267, 9090 Melle-Gontrode, Belgium; kris.verheyen@ugent.be Abstract In the northern temperate zone, the highest Ixodid tick densities are found in forests. However, tick densities and Borrelia prevalence strongly vary from one forest location to another. Multiple drivers underlie this variability. In this chapter we provide more information on the way forest managers can influence three important risk determining factors, notably forest stand composition and structure, visitor rates and the probability that visitors come in contact with questing ticks. Using a simple probabilistic model, we demonstrate that guidance of visitor flows in forests combined with a trail management that focuses on reducing the contact probability with questing ticks appear to be the most effective risk reducing measures. Densities of infected nymphs vary between forest types, but the differences are too small to significantly affect Lyme borreliosis risk. Altering the forest composition through changing the dominant tree species or the forest structure should therefore not be the primary focus of risk management. However, forest type also affects the Borrelia genospecies community composition and a forest type-dependent prevalence of more or less pathogenic genospecies deserves more attention in risk assessments. Keywords: forest composition, forest management, Ixodes ricinus, recreation management, risk modelling Introduction In the northern temperate zone, the highest Ixodid tick densities are found in forests due to the favourable microclimate and high abundances of hosts (Gray et al. 1998, Lindstrom et al. 2003). Nevertheless, tick densities and Borrelia prevalence strongly vary from one forest location to another (e.g. Gray et al. 1998, Jaenson et al. 2009, Ruyts et al. 2016). Multiple drivers underlie this variability (see Ostfeld 2011 for an overview). Some drivers, such as macroclimatic conditions, cannot be influenced by forest management, but others, such as forest composition and structure, can. Next to tick densities and Borrelia prevalence, the number of visitors in a forest is another important factor determining the risk for Lyme borreliosis, especially in densely populated urban and peri-urban regions. Forest recreation differs strongly in space and time (Hill and Courtney 2006) and this variability in visitor flows to and in a forest area can to a large extent be explained by management decisions. Finally, the likelihood of getting in contact with questing ticks within a forest is strongly impacted by the vegetation height on and along paths (cf. Mejlon and Jaenson 1997); another factor under control of forest managers. Hence, it is clear that forest management can play an important role in reducing the risk for Lyme borreliosis by acting upon multiple important risk determining factors. In this chapter we start out by providing more information on the way forest managers can influence three risk determining factors that can, to a certain extent, be controlled by forest management, notably forest stand composition and structure, visitor rates and the probability that visitors come in contact with questing ticks. We will show that forest stand composition and Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _16, Wageningen Academic Publishers 2016

235 Kris Verheyen and Sanne C. Ruyts structure can have strong direct and indirect impacts on tick densities and Borrelia prevalence and that the probability to come in contact with questing ticks is likely to be strongly determined by the vegetation height on and along paths. Next, we will present a simple model that allows quantifying the relative importance of the above-mentioned drivers on the probability that forest visitors make contact with Borrelia infected ticks. We will end the chapter with some concluding remarks. This chapter is mainly based on data and insights obtained in the Kempen, a region in northeastern Belgium, where we have done extensive research on the multiple relationships between forest stand composition and structure on the one hand and tick abundance, host community composition and Borrelia prevalence on the other (Tack 2013, Ruyts et al. 2016). Forest covers roughly 20% of the land area in this region and is principally composed of even-aged monoculture pine stands (mainly Pinus sylvestris and Pinus nigra). However, due to changing forest management objectives and due to the gradual aging of the forest stands, which have mostly been established on former heathlands from the second half of the 19 th century onwards, the pine stands are now gradually transformed into more diverse, multi-layered stands admixed with oak (Quercus robur or Quercus petraea), birch (Betula pendula or Betula pubescens) and several other, mainly broadleaved, tree and shrub species. Although part of the results presented in this chapter principally apply to the forest types mentioned above, we think that they can be generalised to other regions where strongly contrasting forest types differing in habitat suitability for ticks and their hosts occur. Impact of forest management on risk determining factors In order to decrease the risk for Lyme borreliosis, actions to reduce tick densities and Borrelia prevalence in ticks, to guide visitor flows in the forest and to decrease the probability that visitors come in contact with questing ticks should be considered. Below we will highlight measures a forest manager can take to either directly or indirectly influence these risk determining factors. Changing forest structure and composition Guiding forests towards a desired structure and composition is the essence of forestry and foresters can rely on knowledge and insights which have accumulated over centuries to achieve the targeted forest stand type (Den Ouden et al. 2010). Below we will show the impact that forest stand structure and composition may have on tick densities and Borrelia prevalence. Impact on tick densities Since nymphal ticks are small, abundant and may carry a Borrelia infection (Barbour and Fish 1993), nymphs are the most important tick stage in the light of human health risk and thus also the most studied stage. As mentioned above, due to suitable microclimatic conditions and available host species, nymphal tick densities are usually highest in forests (Gray et al. 1998). Moreover, in different regions in Europe, structure-rich deciduous forests and mixed forest dominated by deciduous tree species are found to harbour more ticks compared to young, homogenous and/ or coniferous forests (Estrada-Peña 2001, Lindstrom et al. 2003). This pattern was confirmed in the Kempen, where most nymphs were found in deciduous forests dominated by oaks with a welldeveloped shrub layer, and fewer in homogenous pine forests without a shrub layer (Figure 1A). The tick density patterns, not unexpectedly, also coincided with differences in roe deer density between these forest types (Ruyts et al. 2016, Tack 2013). Changing the forest composition through 234 Ecology and prevention of Lyme borreliosis

236 16. Forest management A 60 Density of nymphs (#/100 m²) B 20 Nymphal infection prevalence (%) C 10.0 Density of infected nymphs (#/100 m²) pine shrub (n=20) pine+shrub (n=32) oak shrub (n=19) oak+shrub (n=22) Forest type Figure 1. (A) The density of Ixodes ricinus nymphs, (B) nymphal infection prevalence and (C) density of infected nymphs in oak and pine stands, with or without shrub layer. n is the number of forest stands studied per forest type (modified after Ruyts et al. 2016). Averages over all investigated forest stands are shown ± standard deviation. The density of nymphs is calculated as the number (#) of nymphs, caught using the flagging technique, per 100 m 2, and prevalence as the percentage of nymphs infected with Borrelia burgdorferi sensu lato. Density of infected nymphs is the product of the density of nymphs and prevalence, and is expressed as numbers infected per 100 m 2. Ecology and prevention of Lyme borreliosis 235

237 Kris Verheyen and Sanne C. Ruyts changing the dominant tree species or the forest structure thus potentially changes the density of host species and ticks. Impact on Borrelia prevalence An area with a high density of ticks does not directly imply a high Lyme borreliosis risk. To interpret the human health risk, or the risk of Lyme borreliosis for a visitor of a forest, Borrelia infection of the ticks should be quantified. Indeed, the density of infected nymphs is one of the most powerful risk predictors (Begon 2008, Ogden and Tsao 2009, Wood and Lafferty 2013). In the Kempen, nymphal infection prevalence in oak and pine forests is the same (±15%; Figure 1B). The presence of a shrub layer has no effect on prevalence either. From this it follows that the density of infected nymphs is mainly determined by the density of nymphs, and is thus lowest in the pine forests without a well-developed shrub layer (Figure 1C). The prevalence of Borrelia and the density of infected nymphs are not the only important indicators of disease risk. The Borrelia burgdorferi sensu lato species complex consists of multiple pathogenic genospecies that cause different disease symptoms, and are associated with different host species (see Kurtenbach et al and Stanek et al and references therein). The diversity of hosts for (larval) ticks is assumed to be higher in structure-rich oak forest than in homogenous (non-indigenous) pine forests (Alexander et al. 2006, Brockerhoff et al. 2008, Carnus et al. 2006, Du Bus De Warnaffe and Deconchat 2008, Kennedy and Southwood 1984, Laiolo 2002). The diversity of the Borrelia genospecies that are found in the nymphs is likewise highest in oak forest. Borrelia afzelii appears to be the most prevalent genospecies in nymphs from the Kempen, occurring in 73.8% of the infected nymphs. It dominates the genospecies community in the pine forests (83.9% of all Borrelia occurrences), but less so in the oak forests (62.4%). In oak forests, the genospecies Borrelia garinii and B. burgdorferi sensu stricto appear to be more prevalent than in pine forests (Ruyts et al. 2016). This high genospecies diversity in oak forests has important health implications because the latter two genospecies are associated with more severe disease manifestations than B. afzelii. The chance of getting infected by a more pathogenic Borrelia genospecies thus seems to be higher in diversified forest communities than in homogenous, less diversified communities such as pine forests, since the host community diversity is most probably higher. This finding applies to the Kempen in particular, but the insights can probably be generalised to other regions in Europe. In general, the most common genospecies in questing nymphs in Europe are B. afzelii and B. garinii, but the relative prevalence of the different genospecies varies between regions (Rauter and Hartung 2005). Directing visitor flows Managers can have an important impact on the visitor flows in forests and can ascertain that zones with high densities of Borrelia-infected ticks are visited less frequently. In Figure 2 different instruments that can be used to direct visitor flows are depicted. These instruments can be ranked along two axes, one characterising the nature of the instrument (physical versus social) and the other characterising the perceived freedom of itinerary choice given to the visitors. In general, visitors prefer instruments with a high perceived freedom of choice. Points of attractions, such as parking lots and bird observation towers, are examples of physical instruments with a high perceived freedom of choice, whereas marked out routes that visitors have to follow are examples of physical instruments with a low freedom of choice. Intermediate on the freedom of choice axis are guiding measures, such as the presence of well-maintained trails versus trails of much poorer quality. A social measure that generates a high perceived freedom of choice is the provision of public information on Lyme borreliosis risk, e.g. via info panels and a website. The 236 Ecology and prevention of Lyme borreliosis

238 16. Forest management Nature of instrument High perceived freedom of choice Low perceived freedom of choice Physical Points of attraction (e.g. parking lot, observation tower) Guidance (e.g. variation in path pavement quality) Marking (e.g. signposted routes that need to be followed) Social Education (e.g. info panels) Legal regulation of trail access Figure 2. Overview of the instruments managers can apply to guide visitor flows. The instruments are ranked along two axes, one characterising the nature of the instrument (physical vs social) and the other characterising the perceived freedom of choice given to the visitors. Graph adapted from Van Marwijk et al. (2010). legal regulation of trail access is an example of a social instrument with a low perceived freedom of choice. In practice, however, a combination of instruments is generally most appropriate to steer visitor flows. Although not yet supported by data, concentrating visitor flows on certain trails could generate an additional risk-reducing effect. For instance, Arnberger et al. (2011) found indications that roe and red deer avoided areas with heavily used trails in the UNESCO biosphere reserve Untere Lobau in Vienna, Austria. Deer are important hosts for ticks and close correlations have been found between the number of (roe) deer beds and local tick densities (Tack et al. 2012). Hence, it is not unlikely that tick densities will decrease in areas that are too disturbed for deer. Reducing the contact probability with questing ticks Ixodes ricinus ticks find their hosts by climbing the vegetation and adopting a sit-and-wait tactic (see e.g. Tomkins et al. 2014), the so-called questing behaviour. It has been shown that the greatest numbers of nymphal and adult ticks can be found at height intervals of 50 to 70 cm (Mejlon and Jaenson 1997). To our knowledge, little research has been done on the factors determining questing success, i.e. the probability that ticks effectively attach to a host. However, the questing behaviour is in principle very similar to epizoochorous dispersal of plant seeds attaching to passing dispersal vectors. Couvreur et al. (2008) studied this process in detail by building a dispersal model parameterised using an extensive set of field data on epizoochorous dispersal by large herbivores. Model validation showed that seed accessibility, which was mainly determined by vegetation height, was a key factor to explain variability in seed load on the animals. Hence, we assume that questing success will strongly depend on vegetation height as well and that Lyme borreliosis risk will strongly decrease when the vegetation is kept low, i.e. well below 50 cm. Managers can obviously strongly influence vegetation height by more or less frequently mowing the vegetation along trails. Depending on the site conditions, at least several mowing sessions per year are needed to keep the vegetation continuously below 50 cm. Quantifying variability in Lyme borreliosis risk The Lyme borreliosis risk R, defined here as the probability of a person making contact with at least one Borrelia-infected tick along a 100 m forest trail, can be expressed as: R = (v c) [1 exp (-p DT) ] Ecology and prevention of Lyme borreliosis 237

239 Kris Verheyen and Sanne C. Ruyts Where: v = the probability of at least one visitor passage per hour; c = the contact probability with questing ticks; p = the prevalence of B. burgdorferi sl.; DT = the density of I. ricinus nymphs and adults along a 1 m wide and 100 m long forest trail section; p DT = DIT = the density of infected ticks. This formula was taken from Mannelli et al. (2003), but was extended with a factor v, the probability of visitor passage, and a factor c, the vegetation contact probability. As outlined above, all terms in this equation can to a certain extent be influenced by forest management. Figure 3 depicts the outcomes of the risk calculations for situations with low (v c = 0.1), over intermediate (v c = 0.5) to high (v c = 1.0) combined probabilities of visitor passage and questing tick contact. Couvreur et al. (2008) arbitrarily set the seed accessibility factor mentioned-above to 1.0 for plants with exposed seeds on a stem >30 cm, whereas the factor was set to 0.1 for other plants. Here, we followed the same reasoning and used a value for c of 1.0 and 0.1 when the vegetation was >50 cm and <50 cm, respectively. Values for v can vary widely: along heavily used trails in well-visited forests this value can easily reach 1.0, whereas in more remote areas the value can be <<0.01. The parameter for values p and DT are based on Tack (2013) and Ruyts et al. (2016), with p and DT ranging between 0 and 0.15 and 0 and 40 ticks per 100 m long trail section, respectively. The calculations demonstrate that the Lyme borreliosis risk steeply rises with increasing DIT and that a plateau is reached at DIT-values between two and four. Beyond DIT-values of two to four the risk simply equals the product of the visitor passage and contact probabilities. In the Kempen region DIT-values larger than four are fairly common: Ruyts et al. (2016) found a mean density of infected nymphs of 5.9 per 100 m 2 in transects laid out within forest stands. Furthermore, DITvalues are expected to increase in the future due to ongoing transformation of the former pine plantations towards more mixed, multi-layered forest stands (see Figure 1). Therefore, the main message that can be taken from this analysis is that appropriate recreation management seems to 1.0 v c = 0.1 v c = 0.5 v c = 1 Contact probability Density of infected ticks (number/100 m 2 ) Figure 3. The probability of making contact with at least one infected tick along a 100 m forest trail section (y-axis) as a function of different densities of infected ticks (p DT = DIT; x-axis) and for different combined visitor passage and questing tick contact probabilities (v c; coloured lines). 238 Ecology and prevention of Lyme borreliosis

240 16. Forest management be the most effective measure to reduce the risk. Managers could opt for a zonation of the forest and make sure that the contact probability is as low as possible in areas where the visitor flows are high (e.g. in the vicinity of parking lots). This can, for instance, be achieved by establishing wide paths that are frequently mown or even paved. In more remote zones where visitor flows are much lower, managers could opt not to invest in wide paths and/or frequent mowing activities and still achieve the same level of risk, as illustrated by the calculations in Table 1. The risk-values along the two path types are similar, but the much higher visitor flows in path type II are compensated by a reduced contact probability due to the higher mower frequency. The latter obviously comes at a higher cost, so managers will have to carefully think how to optimally allocate their available budget to reduce the risk and importantly to also achieve the other management goals (e.g. wood production and biodiversity conservation), that are at play. Finally, it should be noted that temporal zonation is a valid option too. Tick densities are typically highest between April and September (see and therefore managers could choose to temporarily close certain high risk zones for recreation. Table 1. Overview of the recreation and management characteristics of two path types exhibiting a similar Lyme borreliosis risk of 0.1, calculated according to formula 1 and assuming a DIT of 5. Path type I: low visitor frequency low mowing frequency Path type II: high visitor frequency high mowing frequency Visitor probability (v) Contact probability (c) Mowing frequency (year -1 ) 1 5 Mowing cost (100/m/year) The mowing costs are taken from Van Raffe and De Jong (2008) and imply the manual mowing of a 1 m wide strip with a brush cutter at two sides of the path over a distance of 100 m. Concluding remarks Lyme borreliosis risk is determined by multiple, interacting factors and many of them can be influenced by forest management. However, our analyses point out that some management measures are more effective than others. Reversing the ongoing trend towards more species-rich and structurally diverse forests may not be the most effective strategy. Even in structurally poor monocultures, densities of infected nymphs and adults are often still too high to achieve low Lyme borreliosis risk-values (Figure 1 and 3). However, the higher prevalence of more pathogenic genospecies in more diversified forests is a new element that may need to be taken into account in this discussion. An additional argument, though, not to reverse the trend towards more diverse forests is that more mixed forests are better able to deliver a whole suite of other ecosystem services compared to monocultures (Van der Plas et al. 2016). Smart recreation management, on the other hand, can be effective (Figure 3): instruments exist to direct visitor flows to low-risk zones and managers can make sure that the contact probability with questing ticks is low in these zones. Less visited zones can be subjected to a less intensive management. Ecology and prevention of Lyme borreliosis 239

241 Kris Verheyen and Sanne C. Ruyts However, the inferences made here are to a large extent based on theory and several assumptions had to be made. Among others, the factors determining questing success and the associated contact probability c warrant further investigation. Spatio-temporally explicit data on tick densities, Borrelia prevalence, host community composition and visitor flows in forests to validate the predictions made here would be highly welcome too. Public health relevance Forest management can potentially impact several factors determining the risk for Lyme borreliosis. Guidance of visitor flows in forests combined with a trail management that focuses on reducing the contact probability with questing ticks appear to be the most effective risk reducing measures. Densities of infected nymphs vary between forest types, but the differences are too small to significantly affect Lyme borreliosis risk. Forest type also affects the Borrelia genospecies community composition and a forest type-dependent prevalence of more pathogenic genospecies deserves more attention in future risk assessments. Acknowledgements S.C. Ruyts was supported by the Agency for Innovation by Science and Technology (IWT). References Alexander K, Butler J. and Green T. (2006) The value of different tree and shrub species to wildlife. Brit Wildl 18: Arnberger A, Eder R, Taczanowska K, Tomek H, Frey-Roos F, Muralt G, Nopp-Mayr U and Zohmann, M (2011) Recreation impacts on wildlife in the UNESCO biosphere reserve Untere Lobau in Vienna. In: Conference proceedings of Public recreation and landscape protection hand in hand?, Brno, Czech Republic, 4-6 May 2011, pp Availabe at: Barbour A and Fish D (1993) The biological and social phenomenon of Lyme disease. Science 260: Begon M (2008) Effects of host diversity on disease dynamics. In: Ostfeld RS, Keesin F and Eviner V (eds.) Infectious disease ecology: effects of ecosystems on disease and of disease on ecosystems. Princeton University Press, Princeton, NJ, USA, pp: Brockerhoff EG, Jactel H, Parrotta JA, Quine CP and Sayer J (2008) Plantation forests and biodiversity: oxymoron or opportunity? Biodivers Conserv 17: Carnus J-M, Parrotta J, Brockerhoff E, Arbez M, Jactel H, Kremer A, Lamb D, O Hara K and Walters B (2006) Planted forests and biodiversity. J Forest 104: Couvreur M, Verheyen K, Vellend M, Lamoot I, Cosyns E, Hoffmann M and Hermy M (2008) Epizoochory by large herbivores: merging data with models. Basic Appl Ecol 9: Den Ouden J, Muys B, Mohren F and Verheyen K (2010) Bosecolgie en bosbeheer. Uitgeverij Acco, Leuven, Belgium. Du Bus De Warnaffe G and Deconchat M (2008) Impact of four silicultural systems on birds in the Belgian Ardenne: implication for biodiversity in plantation forests. Biodivers Conserv 17: Ecology and prevention of Lyme borreliosis

242 16. Forest management Estrada-Peña A (2001) Distribution, abundance, and habitat preferences of Ixodes ricinus (Acari: Ixodidae) in northern Spain. J Med Entomol 38: Gray JS, Kahl O, Robertson JN, Daniel M, Estrada-Peña A, Gettinby G, Jaenson TGT, Jensen P, Jongejan F, Korenberg E, Kurtenbach K and Zeman P (1998) Lyme borreliosis habitat assessment. Zentralbl Bakteriol 287: Hill GW and Courtney, PR (2006) Demand analysis projections for recreational visits to countryside woodlands in Great Britain. Forestry 79: Jaenson T, Eisen L, Comstedt P, Mejlon H, Lindgren E, Bergström S and Olsen B (2009) Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden. Med Vet Entomol 23: Kennedy CEJ and Southwood TRE (1984) The number of species of insects associated with British trees: a re-analysis. J Anim Ecol 53: Kurtenbach K, De Michelis S, Etti S, Schäfer SM, Sewell H-S, Brade V and Kraiczy P (2002) Host association of Borrelia burgdorferi sensu lato the key role of host complement. Trends Microbiol 10: Laiolo P (2002) Effects of habitat structure, floral composition and diversity on a forest bird community in north-western Italy. Folia Zoologica 51: Lindstrom A, Jaenson TGT, Lindström A and Jaenson TGT (2003) Distribution of the common tick, Ixodes ricinus (Acari: Ixodidae), in different vegetation types in southern Sweden. J Med Entomol 40: Mannelli A, Boggiatto G, Grego E, Cinco M, Murgia R, Stefanelli S, De Meneghi D and Rosati S (2003) Acarological risk of exposure to agents of tick-borne zoonoses in the first recognized Italian focus of Lyme borreliosis. Epidemiol Infect 131: Mejlon HA and Jaenson TGT (1997) Questing behaviour of Ixodes ricinus ticks (Acari: Ixodidae). Exp Appl Acarol 21: Ogden NH and Tsao JI (2009). Biodiversity and Lyme disease: dilution or amplification? Epidemics 1: Ostfeld R (2011) Lyme disease: the ecology of a complex system. Oxford University Press, New York, NY, USA. Rauter C and Hartung T (2005) Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe : a metaanalysis. Appl Environ Microbiol 71: Ruyts SC, Ampoorter EVY, Coipan EC, Baeten L, Heylen D, Sprong H, Matthysen E and Verheyen K (2016) Diversifying forest communities may change Lyme disease risk: extra dimension to the dilution effect in Europe. Parasitology 143: Stanek G, Wormser GP, Gray J and Strle F (2012) Lyme borreliosis. Lancet 379: Tack W (2013) Impact of forest conversion on the abundance of Ixodes ricinus ticks. PhD thesis, Ghent University, Ghent, Belgium. Tack W, Madder M, Baeten L, De Frenne P and Verheyen K (2012) The abundance of Ixodes ricinus ticks depends on tree species composition and shrub cover. Parasitology 139: Tomkins JL, Aungier J, Hazel W and Gilbert L (2014) Towards an evolutionary understanding of questing behaviour in the tick Ixodes ricinus. PLoS ONE 9: e Van der Plas F, Manning P, Allan E, Scherer-Lorenzen M, Verheyen K, Wirth C, Zavala MA, Hector A, Ampoorter E, Baeten L, Barbaro L, Bauhus J, Benavides R, Benneter A, Berthold F, Bonal D, Bouriaud O, Bruelheide H, Bussotti F, Carnol M, Castagneyrol B, Charbonnier Y, Coomes D, Coppi A, Bastias CC, Muhie Dawud S, De Wandeler H, Domisch T, Finér L, Gessler A, Granier A, Grossiord C, Guyot V, Hättenschwiler S, Jactel H, Jaroszewicz B, Joly F-X, Jucker T, Koricheva J, Milligan H, Müller S, Muys B, Nguyen D, Pollastrini M, Raulund-Rasmussen K, Selvi F, Stenlid J, Valladares F, Vesterdal L, Zielínski D and Fischer M (2016) Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests. Nature Comm 7: Van Marwijk RBM, De Vreese R and Van Herzele A (2010) Maatregelen voor recreatie. In: Den Ouden J, Muys B, Mohren GMJ and Verheyen K (eds.) Bosecologie en bosbeheer. ACCO Uitgeverij, Leuven, Belgium, pp Van Raffe JK and De Jong JJ (2008) Normenboek natuur, bos en landschap 2006: tijd-en kostennormen voor inrichting en beheer van natuurterreinen, bossen en landschapselementen. Alterra, Wageningen, the Netherlands. Wood CL and Lafferty KD (2013) Biodiversity and disease: a synthesis of ecological perspectives on Lyme disease transmission. Trends Ecol Evol 28: Ecology and prevention of Lyme borreliosis 241

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244 17. The role of large herbivores in tick-reducing intervention schemes Sipke E. van Wieren Resource Ecology Group, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; Abstract Of all the stages of the tick Ixodes ricinus, adults are the stage with the lowest numbers in any tick population. The majority of the adult ticks feed on large ungulates like deer, who are generally also in low numbers compared to other important tick hosts like rodents. To reduce tick populations, lowering wild ungulate densities to close to zero or using acaricide applications on them are not really feasible options for large areas in many situations. Since large reductions of wild ungulates in any system will likely conflict with other management objectives, fencing to exclude wild ungulates can be applied in relatively small areas. Many semi-natural systems are being grazed by domestic herbivores. When these herbivores are treated with an acaricide during the tick season for a number of years, tick numbers can be substantially reduced. Short-term applications with treated domestic animals like sheep in small areas are also possible. Keywords: acaricide, deer, fencing, Ixodes ricinus, sheep, tick-intervention scheme, ticks Introduction Of all the stages of the tick Ixodes ricinus, adults are the stage with the lowest numbers in any tick population. The majority of the adult ticks feed on large ungulates like deer, which are generally also in low numbers compared to other important tick hosts like rodents. If one wants to reduce tick populations by intervening in the tick cycle, it makes sense to see to what extent large herbivores can be targeted for this task. Much work has been done already, both with wild ungulates and with domestic animals. Wild herbivores Reducing numbers In case ungulates are important as reproduction hosts for ticks (Gray 1998), it can be expected that lowering ungulate density will lead to a lower density of questing ticks in the vegetation. Indeed this has been found a number of times: eastern North America for white-tailed deer Odocoileus virginianus (Deblinger et al. 1993, Kilpatrick et al. 2014, Rand et al. 2003, 2004, Stafford 1993, Stafford et al. 2003, Werden et al. 2014, Wilson et al. 1990). Europe for roe Capreolus capreolus, red deer Cervus elaphus and fallow deer Dama dama (Estrada-Peña et al. 2015, Gilbert et al. 2012, Gray et al. 1992, James et al. 2013, Qviller et al. 2013, Rizzoli et al. 2002, Ruiz-Fons and Gilbert 2010). The key question here is to what level ungulate density should be reduced to get an acceptable or required tick density. Theoretically, a reduction to zero would give the best results (see below). A number of studies suggest a linear relationship between deer density and tick density (Jensen and Jespersen 2005, Rand et al. 2003, Sprong et al. 2012). However, in most of these studies crude estimates of deer densities were used and analyses were sometimes covering very large areas (countries). Kugeler et al. (2015) summarised a number of studies that looked at a reduction of deer on tick density (Ixodes scapularis). The largest reduction occurred when deer densities were Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _17, Wageningen Academic Publishers 2016

245 Sipke E. van Wieren brought to zero, but studies with reductions close to elimination yielded mixed results. Deer densities at the end of those studies were generally still quite high for West European standards (2.3, 10,11,18, 18 and 25 deer/km 2 ). A complicating factor when reducing deer density is a rise in tick burden per deer, which may counter balance the effect of the fewer deer (Kugeler et al. 2015). It seems that tick reduction is only substantial when deer numbers are sharply reduced (Stafford et al. 2003, Van Buskirk and Ostfeld 1995, Van Wieren and Hofmeester 2016, Wilson et al. 1988). The relationship between the densities of ungulates and ticks is more likely to be a threshold relationship than a linear one. In practice, a wish to reduce deer density will likely meet with various complicating factors. In itself, it is already a difficult task to reduce the larger part of the deer population, but it might also be in conflict with the interests of hunters, the management objectives for the area or the public opinion. Fencing Fencing to reduce the ungulate density to zero might be an alternative strategy to reduce tick density. In early spring 2013, four 1 ha exclosures were created in a coniferous forest with roe deer as the only ungulate species and the development of the tick population after two years of exclusion was compared with control sites. A strong decrease in nymphal density was observed in the exclosures (β=-0.41, SE=0.08, P<0.001; Figure 1). Also the control plots showed a decrease in nymphal density, but we think this can be explained by a strong decrease in roe deer numbers in recent years in the area. Many other studies have shown that tick densities can be reduced substantially, in some cases up to 90%, by fencing (Gilbert et al. 2012, Ruiz-Fons and Gilbert 2010, Stafford 1993, Wilson et al. 1988). When many alternative hosts are present, results can be less pronounced (Ginsberg et al. 2002). In the first year after creating exclosures, adult ticks present are likely to quest longer in the exclosure, because then they are questing for a host which is not there, suggesting a higher adult density. Nymphal density (number/100 m 2 ) Deer Year *** Exclosure Control Exclosure Control Figure 1. Questing nymphal density after two years of deer exclusion in four 1 ha exlosures. 244 Ecology and prevention of Lyme borreliosis

246 17. Herbivores and intervention schemes How big does an exclosure need to be to be effective? Small exclosures might suffer from an edge effect whereby rodents bring in engorged larvae. In these situations, exclosures act as sinks whereby a questing nymphal population can be maintained. In our small exclosures, we did not find an edge effect. Both nymphal density (β=0.30, SE=0.41, P=0.46) and adult density (β=-1.01, SE=0.62, P=0.13) did not differ significantly between the edge and the core of the exclosures. This is in line with Stafford (1993), who also did not find an effect in abundance of I. scapularis nymphs within 45 m from the fence in continuous woodland. In a meta-analysis Perkins et al. (2006) found that in exclosures smaller than 2.5 ha, the density of questing ticks in deer exclosures was higher than in controls. Perkins et al. (2006), however, did make use of results obtained from different studies involving different tick species, which differ in host preferences and ecology (Gilbert et al. 2012). Also the number of years after deer exclusion, ranging from one year to eight years, was not taken into account in the analysis. The findings of Perkins et al. (2006) are in contrast with those of Gilbert et al. (2012), who found large effects in very small exclosures of 0.2 and 0.25 ha, and with those of our study (exclosures of around 1 ha). Fencing is very effective to reduce local tick densities. Although erecting and maintaining fences is quite costly, it is a feasible intervention option for relatively small areas. For larger areas, it meets similar complications as mentioned above with the reduction options. Fences can be very ugly and not very appreciated by the public (and most managers) and they can have negative impacts on other species, such as badgers and foxes. At present more attractive new types of high-strength, less visible black or green vinyl fences are available (Piesman 2006) but one can also think of a sunken fence, which consists of a ditch with a low fence (Figure 2). Use of acaricides The best and probably only known method to use acaricides on wild herbivores (white-tailed deer) is the so called 4-Poster approach. Here, deer are attracted to corn at a bait station, and they apply themselves with acaricides from four paint rollers when feeding. 4-Poster devices, with 10% permethrin as acaricide, are now commercially available. The use of 4-Poster devices has led to reductions up to 70% in nymphal tick populations in 4 years (Stafford et al. 2009). A 10 year model simulation indicated that 70-90% of the deer in a targeted area need to be treated to achieve a large reduction in the density of infected nymphs (Stafford et al. 2009). To treat such a high percentage of deer, rather large numbers of 4-Poster devices are needed. In one study, 24 of these devices were used on 6 km 2. Tick densities at the onset of the treatments were low in the experiments (Hoen et al. 2009). Figure 2. A sunken fence to exclude red deer in Doneraile, Ireland (photo by Rory Harrington). Ecology and prevention of Lyme borreliosis 245

247 Sipke E. van Wieren Various problems can be anticipated when implementing the 4-Poster system, especially in many parts in Europe. In the first place, the system is expensive. Many devices are needed, even for a medium sized area, and they need regular maintenance. And because the devices are developed for white-tailed deer, they need to be adjusted to make them suitable for the smaller red deer and roe deer in Europe. Further, roe deer, the most common host species for the adult ticks in Europe, lives solitary, and therefore even more devices are probably needed than for group-living whitetailed deer or red deer, even at lower density. In addition, roe deer is known to avoid artificial or strange objects and probably will not stick its head in the device, defeating its purpose. The fact that in many, if not most, habitats in Europe three species of ungulates, roe deer, red deer and wild boar, are present poses an insurmountable challenge for applying the 4-Poster device in most areas in Europe. However, this method might have something to offer, if any, in small specifically target areas like green suburbs, small islands and recreational sites. The use of acaricides on host animals in tick control measures needs special attention. The great advantage is that it is effective, and also protects the animals themselves against tick bites. However, there are serious downsides as well (for details see also Van Wieren et al. 2016). Some chemicals, like the synthetic pyrethroids, are not completely devoid of toxic effects on vertebrates, particularly when used in combination with other biocides (Ostfeld et al. 2006). As acaricides are also insecticides (better: insecticides are generally also acaricides), most if not all the commercial insecticides are lethal to many invertebrates, including pollinators and predators on arthropod pests (Schauber et al. 1997). These potential negative effects on biodiversity are of particular concern in conservation areas (Piesman and Eisen 2008), but they have rarely been studied (Schulze et al. 2001). Finally, repeated insecticide applications can cause the development of insecticide resistance (Roush 1993, Van Wieren et al. 2016). Domestic herbivores Domestic herbivores can be found grazing in many landscapes. The rationale behind these grazing approaches can be secondary production in a rangeland setting like hill grazing or grazing in rural coastal areas, or for conservation purposes like grazing of heathlands or other semi-natural habitats (Van Wieren and Bakker 2008). In many cases, these goals are combined. Recently, domestic herbivores (cattle and horses) have also been introduced in more forested areas as part of a new rewilding strategy in conservation. Most of the domestic grazing occurs in more open landscapes, which are not the most tick-infested areas. In general, domestic grazers are not the sole ungulate species in an area and roe deer or red deer are almost always present as well. Options without acaricides In case domestic grazers are introduced in an area without any other ungulate species present, they are likely to cause a boost in tick numbers. In case they are added to already present wild species, effects may be different. We compared 20 fenced forest sites that were complementary (in addition to deer) grazed by cattle with 20 control sites outside the fence where deer were present but no cattle. In this study, we found no effects of grazing on I. ricinus nymphal density. Domestic animals usually have fewer ticks than deer (Van Wieren and Hofmeester 2016). As a consequence the addition of domestic grazers to systems where deer are already present in not too low numbers will likely not lead to a strong increase in tick numbers. 246 Ecology and prevention of Lyme borreliosis

248 17. Herbivores and intervention schemes When grazing intensity increases, things may change. In many cases, grazing is applied to control vegetation, for instance to maintain a short herb layer, to control shrub development or to prevent forest regeneration (Van Wieren and Bakker 2008; Figure 3). Intensive grazing can also reduce the thickness of the litter layer. All these effects have a negative effect on survival conditions for ticks: less shade, dryer conditions, more direct sunlight, less soil moisture (Medlock et al. 2008, 2012). These effects will be less prominent in wetter mild climates where humidity is very high all year. The grazing effects mentioned also negatively affect small rodents, which are important hosts for the juvenile ticks. It is therefore not surprising that we found very low tick densities in forest habitats that are heavily grazed by sheep (2 sheep/ha; see Figure 3 in Van Wieren and Hofmeester 2016). The presence of domestic herbivores may also affect the wild species. Competition between wild and domestic ungulates has frequently been described (Van Wieren and Bakker 2008). Also in tick research papers anecdotal evidence suggests a negative interaction between deer and sheep where deer tend to avoid areas where sheep are present (Porter et al. 2011). In cases where deer are outcompeted by domestic grazers, it can be expected that tick density may decrease, because of the lower number of ticks that feed on livestock (Steigedal et al. 2013). Options with acaricides It is much more easy to treat domestic animals with acaracides than wild ones. With seasonal grazing, domestic animals can be treated when they are put out in the field, which is effective for 5-6 weeks, or, and better, they may be treated a few times during the tick season. In some rangeland systems, regular dipping of cattle is normal procedure and it can be expected that this will have effects on tick populations. In a long-term study, Keesing et al. (2013) indeed found that treated cattle reduced nymphs and adult ticks in the environment enormously in a multi wildlife-cattle species system in Kenya. Larvae were still being produced because much large game was present. Figure 3. Highland cattle creating a very short herb layer in dune vegetation. Ecology and prevention of Lyme borreliosis 247

249 Sipke E. van Wieren In Europe, good results have been achieved with sheep, generally in sheep-deer systems. In the uplands of England and the moorlands of Scotland, I. ricinus and the tick-borne louping ill virus cause major economic losses in both sheep farming and moorland shoots of red grouse Lagopus lagopus scoticus (Newborn and Baines 2012). When sheep were treated four times between March and October, and vaccinated against louping ill, tick burdens on grouse chicks were reduced with 90% and louping ill seroprevalence also decreased (Laurenson et al. 2007). In upland UK, sheep coexist with red deer and it can be expected that the effect of treating sheep is predominantly dependent on deer density. Porter et al. (2011) modelled the relationship between treated sheep density against the density of deer. The model showed that increasing the density of treated sheep for a given deer density led to a decrease in the tick population. The model also predicted that using acaricide-treated sheep can be an effective method to reduce the tick population providing there are few deer (<6 per square kilometre). The model was very sensitive to deer density and, at high deer numbers, the sheep mops became ineffective. In a situation where sheep were treated once (with a 5 weeks efficacy) and where deer were also present, Steigedal et al. (2013) found significant but rather small effects on tick densities. The interpretation of the relationships found between deer densities and tick densities in various studies in Europe and North America is confounded by the enormous variation in deer densities reported. In the summary overview of the deer reduction studies by Kugeler et al. (2015), starting deer densities ranged from deer/km 2. In the European context these are very high densities. At deer densities of 2-4 deer/km 2, high nymphal densities of >40/100 m 2 can regularly be found. This again underlines the idea that the relationship between deer density and tick density is a step-function. In our pilot study with treated sheep grazing mixed forest-heathlands, we also found a strong reduction in density of questing nymphal ticks after already four months (Klouwens et al. 2016). Using treated domestic herbivores can thus be an effective method to reduce tick numbers. Also here the method has to comply with other management objectives and, likely, with interests of other stakeholders. Also the use of acaricides is subject to similar debate as described above. Short-term application In the chapter Sheep mopping (Van Wieren 2016) we describe how sheep can be used to reduce tick numbers in small areas during short bouts of intensive use. With untreated sheep, the method is less effective than when treated sheep are used. This method is suitable for recreational areas, rides, forest trails, ecotones, camping grounds, parks, suburbs, and the like. For this, a flock of sheep is needed that can flexibly be moved from one place to the other and that is professionally herded. When the mopped areas simultaneously are being grazed, the effect will even be greater because of the grazing effects on the vegetation. Apart from negative effects on tick survival conditions, the lowering of the vegetation height will also reduce the exposure of humans to ticks. It has been observed that the presence of a flock of sheep on many a recreational site has an added value as a tourist attraction. In conclusion, there are various options to use large herbivores in tick-intervention schemes. 248 Ecology and prevention of Lyme borreliosis

250 17. Herbivores and intervention schemes When wild herbivores are present options include reducing densities through culling or fencing. As it is not likely that large areas will be fenced because of other conflicting interests, this method is only suitable for relatively small areas. When domestic herbivores are or can be present, and the density of wild herbivores is low, treating livestock will reduce tick numbers in the system. Other conflicts of interests and the use-of-acaricide-issue need to be resolved. Short bouts of the presence of treated (or untreated) sheep in small targeted areas can also lead to short-term reductions of questing tick numbers. Most of the described methods are only suitable for relatively small areas. In large areas of suitable tick habitat, and with large herbivores present, it is more difficult for tick populations to be controlled use these techniques. Public health relevance Large herbivores are potentially suitable target species for tick-intervention schemes because they are the main reproduction hosts for ticks. Reducing ungulate densities to close to zero or applying acaricide treatments on them are not feasible options for large areas in many situations. Fencing out is the most effective method to get a substantial reduction in tick numbers. This method has most potential for small areas where humans run a high risk of being bitten by ticks, like recreational sites and camping grounds. References Deblinger RD, Wilson ML, Rimmer DW and Spielman A (1993) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following incremental removal of deer. J Med Entomol 30: Estrada-Peña A, De la Fuente J, Ostfeld RS and Cabezas-Cruz A (2015) Interactions between tick and transmitted pathogens evolved to minimise competition through nested and coherent networks. Sci Rep 5: Gilbert L, Maffey GL, Ramsay SL and Hester AJ (2012) The effect of deer management on the abundance of Ixodes ricinus in Scotland. Ecol Appl 22: Ginsberg HS, Butler M and Zhioua E (2002) Effect of deer exclusion by fencing on abundance of Amblyomma americanum (Acari: Ixodidae) on Fire Island, New York, USA. J Vector Ecol 27: Gray JS (1998) The ecology of ticks transmitting lyme borreliosis. Exp Appl Acarol 22: Gray JS, Kahl O, Janetzki C and Stein J (1992) Studies on the ecology of lyme-disease in a deer forest in county Galway, Ireland. J Med Entomol 29: Hoen AG, Rollend LG, Papero MA, Carroll JF, Daniels TJ, Mather TN, Schulze TL, Stafford KC and Fish D (2009) Effects of tick control by acaricide self-treatment of white-tailed deer on host-seeking tick infection prevalence and entomologic risk for Ixodes scapularis-borne pathogens. Vector-Borne Zoonotic Dis 9: James MC, Bowman AS, Forbes KJ, Lewis F, McLeod JE and Gilbert L (2013) Environmental determinants of Ixodes ricinus ticks and the incidence of Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in Scotland. Parasitology 140: Jensen PM and Jespersen JB (2005) Five decades of tick-man interaction in Denmark an analysis. Exp Appl Acarol 35: Keesing F, Allan BF, Young TP and Ostfeld RS (2013) Effects of wildlife and cattle on tick abundance in Central Kenya. Ecol Appl 23: Ecology and prevention of Lyme borreliosis 249

251 Sipke E. van Wieren Kilpatrick HJ, Labonte AM and Stafford KC, III (2014) The relationship between deer density, tick abundance, and human cases of lyme disease in a residential community. J Med Entomol 51: Klouwens MJ, Trentelman JJ and Hovius JWR (2016) Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Kugeler KJ, Jordan RA, Schulze TL, Griffith KS and Mead PS (2015) Will culling white-tailed deer prevent lyme disease? Zoonoses Public Health 63: Laurenson MK, McKendrick IJ, Reid HW, Challenor R and Mathewson GK (2007) Prevalence, spatial distribution and the effect of control measures on louping-ill virus in the forest of bowland, Lancashire. Epidemiol Infect 135: Medlock JM, Pietzsch ME, Rice NVP, Jones L, Kerrod E, Avenell D, Los S, Ratcliffe N, Leach S and Butt T (2008) Investigation of ecological and environmental determinants for the presence of questing Ixodes ricinus (Acari: Ixodidae) on Gower, South Wales. J Med Entomol 45: Medlock JM, Shuttleworth H, Copley V, Hansford KM and Leach S (2012) Woodland biodiversity management as a tool for reducing human exposure to Ixodes ricinus ticks: a preliminary study in an English woodland. J Vector Ecol 37: 1-9. Newborn D and Baines D (2012) Enhanced control of sheep ticks in upland sheep flocks: repercussions for red grouse co-hosts. Med Vet Entomol 26: Ostfeld RS, Price A, Hornbostel VL, Benjamin MA and Keesing F (2006) Controlling ticks and tick-borne zoonoses with biological and chemical agents. Bioscience 56: Perkins SE, Cattadori IM, Tagliapietra V, Rizzoli AP and Hudson PJ (2006) Localized deer absence leads to tick amplification. Ecology 87: Piesman J (2006) Strategies for reducing the risk of Lyme borreliosis in North America. Int J Med Microbiol 296: Piesman J and Eisen L (2008) Prevention of tick-borne diseases. Annu Rev Entomol 53: Porter R, Norman R and Gilbert L (2011) Controlling tick-borne diseases through domestic animal management: a theoretical approach. Theor Ecol 4: Qviller L, Risnes-Olsen N, Baerum KM, Meisingset EL, Loe LE, Ytrehus B, Viljugrein H and Mysterud A (2013) Landscape level variation in tick abundance relative to seasonal migration in red deer. PLoS ONE 8: e Rand PW, Lubelczyk C, Holman MS, Lacombe EH and Smith RP (2004) Abundance of Ixodes scapularis (Acari: Ixodidae) after the complete removal of deer from an isolated offshore island, endemic for Lyme disease. J Med Entomol 41: Rand PW, Lubelczyk C, Lavigne GR, Elias S, Holman MS, Lacombe EH and Smith RP (2003) Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 40: Rizzoli A, Merler S, Furlanello C and Gench C (2002) Geographical information systems and bootstrap aggregation (bagging) of tree-based classifiers for lyme disease risk prediction in Trentino, Italian Alps. J Med Entomol 39: Roush RT (1993) Occurrence, genetics and management of insecticide resistance. Parasitol Today 9: Ruiz-Fons F and Gilbert L (2010) The role of deer as vehicles to move ticks, Ixodes ricinus, between contrasting habitats. Int J Parasitol 40: Schauber EM, Edge WD and Wolff JO (1997) Insecticide effects on small mammals: influence of vegetation structure and diet. Ecol Appl 7: Schulze TL, Jordan RA, Hung RW, Taylor RC, Markowski D and Chomsky MS (2001) Efficacy of granular deltamethrin against Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs. J Med Entomol 38: Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJ, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Stafford KC (1993) Reduced abundance of Ixodes scapularis (Acari: Ixodidae) with exclusion of deer by electric fencing. J Med Entomol 30: Ecology and prevention of Lyme borreliosis

252 17. Herbivores and intervention schemes Stafford KC, Denicola AJ and Kilpatrick HJ (2003) Reduced abundance of Ixodes scapularis (Acari: Ixodidae) and the tick parasitoid Ixodiphagus hookeri (hymenoptera: Encyrtidae) with reduction of white-tailed deer. J Med Entomol 40: Stafford KC, III, Denicola AJ, Pound JM, Miller JA and George JE (2009) Topical treatment of white-tailed deer with an acaricide for the control of Ixodes scapularis (Acari: Ixodidae) in a connecticut Lyme borreliosis hyperendemic community. Vector-Borne Zoonotic Dis 9: Steigedal HH, Loe LE, Grøva L and Mysterud A (2013) The effect of sheep (Ovis aries) presence on the abundance of ticks (Ixodes ricinus). Acta Agric Scand A Anim Sci 63: Van Buskirk J and Ostfeld RS (1995) Controlling Lyme disease by modifying the density and species composition of tick hosts. Ecol Appl 5: Van Wieren SE (2016) Sheep mopping. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Wieren SE and Bakker JP (2008) The impact of browsing and grazing herbivores on biodiversity. In: Gordon IJ and Prins HHT (eds.) The ecology of browsing and grazing. Springer, Berlin, Heidelberg, Germany, pp Van Wieren SE and Hofmeester TR (2016) The role of large herbivores in Ixodes ricinus and Borrelia burgdorferi s.l. dynamics. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Wieren SE, Braks MAH and Lahr J (2016) Effectiveness and environmental hazards of acaricides applied to large mammals for tick control. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Werden L, Barker IK, Bowman J, Gonzales EK, Leighton PA, Lindsay LR and Jardine CM (2014) Geography, deer, and host biodiversity shape the pattern of Lyme disease emergence in the thousand islands archipelago of Ontario, Canada. PLoS ONE 9: e Wilson ML, Ducey AM, Litwin TS, Gavin TA and Spielman A (1990) Microgeographic distribution of immature Ixodes dammini ticks correlated with that of deer. Med Vet Entomol 4: Wilson ML, Telford SR, Piesman J and Spielman A (1988) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following elimination of deer. J Med Entomol 25: Ecology and prevention of Lyme borreliosis 251

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254 18. Sheep mopping Sipke E. van Wieren Resource Ecology Group, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; Abstract Adult female ticks mainly feed on ungulates like deer, which makes these large mammals potential suitable targets for tick-intervention schemes. As wild vertebrate species are not easy to work with, experiments have been done with domestic herbivores, mainly sheep. In this study, we describe short-term experiments with flocks of sheep to reduce tick densities. In the first experiment, we targeted small (ca. 1 ha) campsites and forest plots for two days, using sheep. In the second experiment, forest paths were systematically walked for one or two days, using sheep treated with an acaricide. In the third, acaricide treated sheep grazed a forest-heathland for a whole season (May-September). In all experiments, the density of questing nymphs decreased with >60% shortly after the experiments. The effect was generally of short duration (few weeks to few months). Sheep mopping can thus be a useful tool to effectively reduce questing tick numbers for a short period of time in small areas. Suitable places are forest paths, picnic places, recreational playing grounds and tick-infested camping grounds. Keywords: acaricide, intervention, Ixodes ricinus, sheep mopping Introduction In the search for intervention methods to reduce the number of ticks in an area one can think of large mammals as a potential useful mopping tool. Large mammals are the main host for the adult female ticks, a crucial link in the reproductive part of the tick cycle. Further, when looking at the population dynamics of the ticks, the number of adults is less than 1% of the number of larvae which makes them more realistic targets than either larvae or nymphs. The latter also having a large variety of different hosts. Also, the density of large mammals is much lower than that of all other hosts. Targeting the adult females on large mammals can thus potentially be an effective way to intervene in the tick cycle. In many rangelands in the world it is common practice to treat livestock with acaricides to protect animals against tick bites, and thus against tick-borne diseases, in tick-infested areas. Although this practice is meant to protect livestock, a side effect could be that the tick population itself decreases. Indeed such effects have been found, even in areas where other large wild, and untreated, ungulates were present (Keesing et al. 2013, Newborn and Baines 2012). The acaricides used not only work as repellent but also kill the ticks that are coming into contact with the acaricide. The question is if this technique can be applied in areas where people run a high risk of getting tick bites, such as recreational areas like forest paths or trails or woody picnic places or small sheltered camp-sites (Figure 1). These areas may have high tick densities because of the presence of deer and other hosts. Although there are possibilities to apply acaricides on wild ungulates, and some good results have been obtained experimentally, the method is not readily applicable and also not very cost- Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _18, Wageningen Academic Publishers 2016

255 Sipke E. van Wieren Figure 1. Sheep mopping might be a feasible option for reducing ticks on tick-infested forest paths and camp sites. effective (Van Wieren et al. 2016). As these areas normally are not grazed by livestock, grazing livestock must be brought in the area. A treatment should be of limited timespan to avoid unwanted grazing effects and other inconveniences. Nevertheless it is worth trying to look at the effect of a short treatment on tick abundance and to find out how long this effect might last. Here we report the results of three intervention experiments with sheep. In experiment one, five forest plots and four small camp-sites are grazed with a flock of sheep, without acaricides applied, for two days. In experiment two, sheep with acaricides applied were grazing eight forest paths during two days. In experiment three a pilot study was carried out with acaricide-treated sheep grazing on two heathlands for 12 weeks during the summer months. 254 Ecology and prevention of Lyme borreliosis

256 18. Sheep mopping Experiments with sheep without acaricides In 2012 we selected nine plots of 1-2 ha for the experiments, five forest plots and four campsites. The forest plots were of mixed forest type with sessile oak, Quercus robur, and scots pine Pinus sylvestris as the dominant tree species and with bramble, Rubus sp., ferns and some grass (predominantly Deschampsia flexuosa, Molinia caerulea and Agrostis sp.) in the herb layer. Roe deer, Capreolus capreolus, were present in all plots, as well as bedding sites. The camp-sites can best be described as decidious woodland (mainly sessile oak) with small tent-sites (ca m 2 ). Roe deer were present and all camp-sites suffered from high tick densities. Sheep mopping consisted of walking slowly and systematically (to cover the whole of the plot) with a flock of 130 herded sheep through a plot (Figure 2). Figure 2. Plots were slowly and repeatedly walked by a closely herded flock of sheep. Ecology and prevention of Lyme borreliosis 255

257 Sipke E. van Wieren This was done two or three times a day for two days. Special attention had to be given to the length of the treatment because if the treatment would last for more than two days, the engorged larvae and nymphs might drop off again. Because this would happen anyway, the sheep were moved to a short grassland after the treatment for at least ten days so the ticks could drop off. After this period the sheep could be used again for another treatment. The forest plots were so grazed two times (between 22 July and 14 August, and between 20 August and 3 September), the camp-sites were grazed once (between 16 June and 12 July). After each treatment 25 sheep were inspected for ticks. Half of the body parts head, groins, udder and arm pits was inspected. This method gives a good impression of the number of ticks on a sheep (Milne 1943). Just before and a few days after the treatment m 2 transects were dragged in a plot with a cotton cloth of 1 m 2. Only nymphs and adults were counted. Parallel to the treated plots, also seven control plots of about 1 ha were dragged for ticks. These plots were situated close to the treatment plots and measurements were done on the same day. To analyse the data of 2012, the treated and untreated plots were compared before and after each of the two acaricide applications. The data were analysed using linear mixed models (LMM) with plot-id as subject, sampling moment as the repeated fixed factor, and two different dependent variables: the number of adult ticks and the number of nymphs. The dependent variables were lntransformed to meet the assumptions of normality of the residuals. Location was used as random factors, and treatment, sampling moment and the interaction between treatment and sampling moment as fixed factors. The best fitting model was selected by choosing the covariance matrix with the lowest Aikaike s information criterion (AIC) index. Additionally, either the restricted maximum likelihood or maximum likelihood was used, depending on the lowest AIC score. Using the post hoc Sidak test, significances between the treated and the untreated plots were tested. Results In Figure 3 the results of the treatments on the density of questing ticks is given. There was a significant and large (ca 70%) reduction in the number of nymphs after the first mopping treatment (Figure 3A). At the start of the second treatment the questing nymph population apparently had not yet recovered and the second treatment, again, led to a reduction in nymph density but the difference was not significant, possibly due to large variation in the data. Although the density of adults was much lower after the first treatment, the effect was not significant because the numbers in the control plots were also much lower. Also the second treatment did not have any effect. The density of questing adults was very low. Because we did not treat the sheep with an acaricide, we were able to study the effects of the sheep on the ticks in a different way than by comparing questing ticks before and after a treatment. We could estimate the number of ticks on the sheep to make a calculation how many ticks had left the area with the sheep. We could also compare the two methods. A summary of the average number of ticks counted on the sheep is given in Table 1. On average 9.8 (±3.8) ticks were counted per sheep, mainly nymphs and females. The mean number of males on a sheep was <0.2. The ticks were mainly found in the axillae (arm-pits) and on the head. The greatest variation was found in the females (min. 0, max. 24 per sheep). 256 Ecology and prevention of Lyme borreliosis

258 18. Sheep mopping A Number of nymphs/100 m ** * Pre 1 st tr. Post 1 st tr. Pre 2 nd tr. Post 2 nd tr. * B Number of adults/100 m Untreated Treated Pre 1 st tr. Post 1 st tr. Pre 2 nd tr. Post 2 nd tr. Figure 3. Density of (A) questing nymphs and (B) adults, before and after the first (all plots) and second (only forest plots) treatment in plots grazed by sheep (treated), and in ungrazed control plots (untreated). Table 1. The average number of ticks found per body part and per sheep. Results of 14 inspections of sheep per inspection. Body part Larvae Nymphs Females Males Total Head Ears Axillae Groin Total sheep For each location and after each mopping treatment, we calculated how many ticks had disappeared with the sheep, based on the estimate of ticks counted on the sheep after a mopping event (Table 2). On average 536 nymphs (range ) and 385 females (range ) disappeared per ha. After the second treatment quite a few more ticks were mopped than after the first treatment in the forest plots. This shows that still many ticks were present at the start of the second treatment. Figure 4 and Figure 5 give the relationship between the density of questing nymphs and females in the field before a treatment, and the number that have been removed with the sheep. While expecting a positive relationship in both nymphs and adults, there is no clear relationship in nymphs (Figure 4). Probably there is an overabundance of nymphs questing in the vegetation and perhaps sheep are not the most preferred hosts for nymphs. The discrepancy between the change of density of ticks in the vegetation and the estimates based on the number found on the sheep is very large. Three to seven times more nymphs have been counted in the field. With the females we see a totally different pattern. Here we do find the expected positive relationship but much more striking is that many more adult females (three to four times more) Ecology and prevention of Lyme borreliosis 257

259 Sipke E. van Wieren Table 2. The total number of nymphs and females that disappeared with the sheep per ha during the different treatments in the plots. Location Habitat Size (ha) After 1 st treatment After 2 nd treatment Total Number of nymphs disappeared with sheep/ha Nymphs/ha Females/ha Total/ha Nymphs/ha Females/ha Total/ha Total/ha Blauwe Bos forest ,096 1,841 Kiekenberg2 forest ,129 Kiekenberg3 forest Schurega forest , ,040 2,600 Tolhekbos forest , ,452 2,310 Dronten campsite ,609 Lochem campsite Ootmarsum campsite Winterswijk campsite , ,000 4,000 6,000 8,000 Number of questing nymphs in vegetation/ha Figure 4. Relationship between the number of questing nymphs in the vegetation before mopping and the number of nymphs that disappeared with the sheep after a mopping event. Number of females disappeared with sheep/ha R 2 = Number of questing females in vegetation/ha Figure 5. Relationship between the number of questing females in the vegetation before mopping and the number of females that were picked up by the sheep during mopping. 258 Ecology and prevention of Lyme borreliosis

260 18. Sheep mopping have disappeared with the sheep than have been counted in the field. Apparently sheep are very efficient in attracting females. This efficiency has been reported before (Milne 1943, Randolph and Steele 1985) and, perhaps, explains the name sheep tick for Ixodes ricinus. This result also shows that the drag-method is a convenient but biased method, and certainly not well applicable in counting females. Experiments with sheep with acaricide applied (2014) In 2014, experiments were carried out with acaricide-treated sheep with a focus on forest paths and their verges (Figure 6). While these habitats pose high risk for man and animals (dogs) for acquiring tick bites, there are not many options for intervention methods. The sheep were treated with Butox 7.5 pour on (Intervet bv, Boxmeer, the Netherlands), with the active ingredient deltamethrin, a chemical which has been proven to be very effective against ticks (Mehlhorn et al. 2011). Deltamethrin is effective for about six weeks. In total eight forest paths situated in three large forest areas and three camp-sites were mopped. Each path was mopped over a length of 200 m, while another part of the same path was used as control. Mopping consisted of (very) slowly walking the transect with a flock of 60 sheep twice a day. Each path was mopped two times, once in June and once early September. Shortly before and a few days after mopping, questing tick density was estimated with flagging m 2 on each transect. To analyse the 2014 data, LMM were again applied with similar procedure as for the 2012 data. For the 2014 data, pair was added as additional random factor because the mopped and the unmopped plots were paired. Figure 6. An additional effect of walking the sheep along road verges is that there can be some grazing too. The lowered vegetation might have a reducing effect on the exposure to questing ticks. Ecology and prevention of Lyme borreliosis 259

261 Sipke E. van Wieren Results Both the density of questing nymphs and that of questing females decreased substantially and significantly after the first treatment (Figure 7). The density of nymphs decreased from 40 to 15 per 100 m 2 and that of females from 10 to 2 per 100 m 2. At the start of the second treatment the density of ticks was still quite low but that was also the case with the controls so no conclusion can be made how long the effect of the first treatment lasted. Nevertheless in both experiments the density of questing ticks remained low throughout the summer months after the first treatment. The second treatment did not have a signifant effect on the nymphs but it did have a significant effect on the females. Experiment with treated grazing sheep In 2015, a pilot experiment was carried out with acaricide-treated sheep, grazing two small heathlands of about 15 ha, each including a forest patch of about 2 ha. Roe deer were abundant in both areas. The grazing session lasted from mid-may until the end of September m 2 transects were dragged in the woodland patches five times, once before the grazing and four times during the summer season. Larvae were not counted. The sheep were treated with Butox 7.5 pour on (Intervet bv). This was repeated three times during the treatment period. Both areas were grazed by 25 sheep, one area was also grazed by eight Scottish Highland cattle who were also acaricide-treated. Close to each grazed area, an ungrazed forest plot was chosen of about 2 ha as a control. Tick draggings were all carried out on the same day (11 May (pre-grazing), 8 June, 9 July, 5 Aug, 5 Sep). Results At the start of the experiment, all plots had similar densities of ticks (Figure 8). After two months (July), a reduction in tick density was observed in the two treated plots. A Number of nymphs/100 m ** Pre 1 st tr. Post 1 st tr. Pre 2 nd tr. Post 2 nd tr. B Number of females/100 m Untreated Treated ** * Pre 1 st tr. Post 1 st tr. Pre 2 nd tr. Post 2 nd tr. Figure 7. Number of (A) questing nymphs and (B) questing females, before and after the first and the second mopping event on the mopped (treated) and control (untreated) forest paths (2014). 260 Ecology and prevention of Lyme borreliosis

262 18. Sheep mopping Total nymphs and adults/100 m Start treatment C1 C2 T1 T2 May June July Aug Sep Figure 8. Density of questing ticks (nymphs and adults) during the summer in two forest plots grazed by acaricidetreated sheep (T1 and T2) and two ungrazed forest plots (C1 and C2). Tick densities remained at the same level in the following months. Tick density in one plot seemed to be more reduced than in the other. The former plot was also used by the Highland cattle and it was observed (by means of cameratraps) that the cattle used this plot regularly as a resting site. Especially on these bedding places tick numbers declined strongly, stressing the point that contact is needed to kill the ticks but also that more contact leads to a greater effect. Discussion The results of the experiments show that significant and large reductions of tick populations can be obtained with sheep mopping, already after a few days (Figure 8). Apparently, sheep efficiently pick up ticks, especially females, the most important target of the tick population when it comes to an effective intervention. Randolph and Steele (1985) also found that sheep are more efficient than blanket dragging in picking up ticks. All three methods tested in the three experiments have their pros and cons. The short-term experiment with sheep not treated with acarcide showed that good tick reductions can be obtained within a few days in small forest plots and on small camp-sites. How long the effect lasts is not known precisely, but in most cases tick numbers were still low at the start of the second mopping a few weeks or a few months later in the season. People who camped on the mopped camp-sites also noticed and reported that they were less troubled by ticks during the summer. A return frequency of about four weeks is probably needed to be of help for a whole summer season. In this experiment, the treatments should not last longer than two days, otherwise the attached ticks would start to drop off again. After a mopping session, then, the sheep have to go in quarantine for 10 days to let the ticks drop off in a safe place. This makes this method a quite laborious and not very cost-effective one, even though it is the preferred method from an environmental, and public health point of view. Ecology and prevention of Lyme borreliosis 261

263 Sipke E. van Wieren With acaricide-treated sheep the above disadvantages do not apply and sheep can be used for at least 6 weeks on end. The second experiment mainly focused on walking paths and trails in forests and camp-sites. Also here large and significant reductions in tick numbers were obtained after a flock of sheep had walked a certain path length a few times on one day. This method seems to be more efficient and cost-effective than the one described above. It also has the additional advantage that the sheep graze the verges, which can be a management goal. By reducing the height of the herb layer, the habitat becomes less attractive for ticks which reduces the contact rate of humans with ticks. The downside of using treated sheep is the introduction of biocides in the environment. Deltamethrin for instance is not only effective against ticks but it also kills other arthropods. However, compared to residual acaricidal treatment of the environment, non-target arthropods only come in contact with acaricides when in contact with the sheep. In areas with more permanent grazing, treating the grazers with an acaricide on a regular basis can also reduce tick populations. In our pilot experiment, tick densities were significantly reduced already after a few months. Reduction was most prominent in places regularly used for resting. Large effects of treated livestock on ticks have also been reported by other workers (Keesing et al. 2013, Newborn and Baines 2012). The downside of these long treatments is that the potential negative effects of the biocide can (and most likely will) be much more pronounced. When considering the use of biocides for tick control the pros and cons have to be weighed between on the one hand the benefits for human and animal health and on the other the negative side effects on the environment and biodiversity. In almost all situations, grazing livestock form additional hosts to the already present large wild mammals. As a consequence sheep mopping will not eradicate a tick population and has to be repeated and sustained for a long time to be effective over time. Nevertheless it shows potential, even in large areas. In a savanna area, with much other wildlife, Keesing et al. (2013) found a strong negative effect on tick densities with treated cattle after more than five years. (Porter et al. 2011) in a modeling study, found that the use of acaricide-treated sheep was only effective in areas with rather low deer density (<6 deer/km 2 ). In conclusion, tick populations can be effectively reduced (especially females) by sheep mopping. The strongest effects can be expected with acaricide-treated livestock, when grazing areas permanently, when animals are regularly treated with an acaride during the tick season, and when the density of wild grazers is not too high (this study, Keesing et al. 2013, Newborn and Baines 2012, Porter et al. 2011). Short-term mopping treatments are suitable for forest paths and trails, picnic places, recreational playing grounds and in tick-infested camping grounds. As said, the use of acaricides is not without risk. They may be toxic for the animals or in the environment, ticks may become resistant. The potential benefits always need to be weighed against the various costs involved before mopping is being considered (Van Wieren et al. 2016). 262 Ecology and prevention of Lyme borreliosis

264 18. Sheep mopping Public health relevance Sheep mopping can be used to reduce tick numbers, at least for a few months, in places that poses risks for people to contract tick bites. A discussion is needed about the pros and cons of using acaricides in tickintervention schemes. Acknowledgements We thank Staatsbosbeheer (Forest Service), the municipality Aa and Hunze, and the society NTKC for permission to use their terrains for our research. Thanks also to Herman van Oeveren, Irene Mouthaan and Frans Jacobs for indispensable help with tick dragging. Without the creative and flexible cooperation of Henry Hoiting (Natuurlijk Beheer) and shepherd Hilde Groen this study would not have been possible. References Keesing F, Allan BF, Young TP and Ostfeld RS (2013) Effects of wildlife and cattle on tick abundance in central Kenya. Ecol Appl 23: Mehlhorn H, Schumacher B, Jatzlau A, Abdel-Ghaffar F, Al-Rasheid KAS, Klimpel S and Pohle H (2011) Efficacy of deltamethrin (Butox 7.5 pour on) against nymphs and adults of ticks (Ixodes ricinus, Rhipicephalus sanguineus) in treated hair of cattle and sheep. Parasitol Res 108: Milne A (1943) The comparison of sheep-tick populations (Ixodes ricinus L.). Ann Appl Biol 30: Newborn D and Baines D (2012) Enhanced control of sheep ticks in upland sheep flocks: repercussions for red grouse co-hosts. Med Vet Entomol 26: Porter R, Norman R and Gilbert L (2011) Controlling tick-borne diseases through domestic animal management: a theoretical approach. Theor Ecol 4: Randolph SE and Steele GM (1985) An experimental evaluation of conventional control measures against the sheep tick, Ixodes ricinus (L.) (Acari: Ixodidae). II. The dynamics of the tick-host interaction. Bull Entomol Res 75: Van Wieren SE, Braks MAH and Lahr J (2016) Effectiveness and environmental hazards of acaricides applied to large mammals for tick control. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Ecology and prevention of Lyme borreliosis 263

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266 19. Effectiveness and environmental hazards of acaricides applied to large mammals for tick control Sipke E. van Wieren 1*, Marieta A.H. Braks 2 and Joost Lahr 3 1 Resource Ecology Group, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; 2 National Institute for Public Health and the Environment, Centre for Infectious Disease Control, P.O. Box 1, 3720 MA Bilthoven, the Netherlands; 3 Wageningen Environmental Research, Wageningen University & Research, P.O. Box 47, 6700 AA Wageningen, the Netherlands; sip.vanwieren@wur.nl Abstract Ticks are important vectors of a large number of pathogenic organisms. In the Netherlands, Ixodes ricinus is the most abundant tick species and the main vector for several Borrelia species that may cause Lyme borreliosis. Many chemicals have been developed for tick control. In this chapter, a few commonly used acaricides are discussed with the main aim to assess whether they could be both effective and environmentally safe enough for tick control in the field through application on large mammals. This method is currently still at the experimental stage and only limitedly applied. The focus was on amitraz, permethrin, flumethrin, deltamethrin and ivermectin. After a qualitative comparison of the pros and cons, it was concluded that the pyrethroids flumethrin and deltamethrin are potentially the most useful, despite their high toxicity to various other animals in the environment. Both compounds act as contact-acaricides and are not easily leached in the environment. Environmental hazards can therefore be minimised if they are applied correctly and thoughtful, and contamination of the aquatic environment is avoided. Nevertheless, all synthetic acaricides have a number of serious downsides and, when considering using them in field situations, the benefits always need to be weighed against all costs involved. More environmentally-friendly alternatives are being developed of which vaccines against ticks seem most promising. Keywords: acaricides, amitraz, deltamethrin, flumethrin, ivermectin, large mammals, tick control Introduction Ticks are important vectors of a large number of pathogenic organisms like protozoans, bacteria and viruses. Many of these pathogens can seriously affect livestock, pets and humans. In the Netherlands, the hard tick Ixodes ricinus is the main vector for a number of human pathogens including several Borrelia species that may cause Lyme borreliosis (Coipan et al. 2013, Jahfari and Sprong 2016). Ticks themselves can also have great impact. Ticks are considered to be a major limiting factor of the cattle husbandry in many parts of the world, especially in developing countries in the tropical and subtropical region (Kiss et al. 2012). For more than hundred years, attempts have been made to control ticks. However, very few substances have specifically been developed against ticks and tick control generally relies on the use of chemical pesticides, developed against insects, which also have acaricidal effects. As mentioned in Van Wieren (2016) and Klouwens et al. (2016), large herbivores, both wild and domestic may be used for tick control by applying acaricides to the skin of these animals. In this chapter, we will review what is known about both the effectiveness and the environmental hazards of a few common acaricides that can be applied to large herbivores to control (reduce) Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _19, Wageningen Academic Publishers 2016

267 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr the number of I. ricinus in the field. It is important that the chemical will have a strong killing and not only a repellent effect on ticks. But this capacity may in turn lead to potential adverse environmental impact because of possible effects on non-targeted fauna. This chapter does not represent an in-depth review of all the literature about the selected substances. Only some key literature has been consulted and linked to the potential use of the substances for tick control using large mammals. The review also focusses on the environmental hazards only, i.e. on the intrinsic properties of the compounds that make them more or less environmentally harmful, not on the environmental risk, i.e. not on quantification of the probability of harmful effects at certain dosages of use. At the end of the chapter, the development of resistance to chemical acaricides and the available alternatives to the use of chemicals are briefly discussed. Finally, we present a qualitative assessment of effectiveness and environmental effects of the selected acaricides. Selection of compounds The chemicals used in the treatment of ectoparasites of veterinary importance act either systemically, following uptake of the compound by the parasite, or externally by direct contact (Taylor 2001). Systemically acting chemicals may be given parenterally (by subcutaneous or intramuscular injection) or applied topically to the skin from where the active ingredient is absorbed percutaneously and taken up into the blood circulation of the host. Depending on the nature of the compound used, the following effects may be obtained, alone or in combination: (1) disruption of contact between the ectoparasite and the host; (2) prevention of feeding; (3) death of the ectoparasite; and (4) interference with egg fertility and subsequent development of life-cycle stages of the ectoparasite in the case of ticks (George et al. 2004). Any ectoparasiticide applied on or given to a vertebrate animal is considered a veterinary drug. It must undergo a registration process as veterinary medicine through health agencies in each country to obtain their marketing authorisation. The chemicals that act on arthropods that are, however, used in their environment, even when being the same active ingredients, are not regulated as veterinary drugs but as pesticides in the broader sense (Beugnet and Franc 2012). Presently a number of acaricides is used for tick control through the application to large mammals. The acaricide is applied to the mammals by dipping them in a solution of the acaricide or by topical application in the so-called four poster method (Daniels et al. 2009) or as pour-on. The former method is widely applied in the tropics against Boophilus ticks. The four poster method is implemented in the USA. In the Netherlands, anthelmintic pour on s are widely used off label against tick infestations of sheep. Tick control is still at the experimental stage in Europe and therefore final dose rates have not yet been established (e.g. Gilbert 2016, Laurenson et al. 2007, Newborn and Baines 2012). An overview of a number of commonly used chemicals on large herbivores against ectoparasites, which are arthropods that live on the outside of the host permanently (lice, fleas, and some mites) or temporarily (ticks), is given in Table 1 (modified from the review by Taylor 2001). These substances are all neurotoxins, exerting their effect on the nervous system of ectoparasites. The environmental properties of a few, commonly used, individual active ingredients (a.i.) and groups of a.i. from Table 1 will be discussed in more detail in the following sections. The five 266 Ecology and prevention of Lyme borreliosis

268 19. Acaricides Table 1. Some ectoparasiticide groups of active ingredients which are commonly used for tick control on large mammals (modified from Taylor 2001). Chemical group Active ingredient Registered for veterinary use in the Netherlands 1 organophosphates fenthion no diazinon yes propetamphos no coumaphos no chlorfenvinphos no amidines amitraz yes synthetic pyrethroids flumethrin yes cypermethrin yes permethrin yes deltamethrin yes avermectins ivermectin yes 1 individual substances reviewed are each registered in the Netherlands for veterinary use in a considerable number of commercial products. The topics addressed per group/substance are globally: acaricidal/insecticidal properties; use as parasiticidal and for tick control; environmental behaviour; potential environmental toxicity. Organophosphates Organophosphates are synthetic chemicals that belong to the group of organic esters of phosphoric acid. While this chemical group has been known since the 19 th century, its lethal effect on many arthropods was not. Organophosphates inhibit the enzyme acetylcholinesterase that breaks down the neurotransmitter acetylcholine in the synapses between nerve cells. Several extremely toxic compounds were discovered around 1930 and were used in toxic gases for military purposes. The organophosphates with lethal effects on arthropods (insecticides, acaricides) were discovered in the 1950s. Very soon, they were widely used against agricultural and urban pests as well as against veterinary ectoparasites. Organophosphates formed the answer to organochlorines such as DDT of which its environmental risks became evident in the early 1960s (George et al. 2008). Many organophosphates have a broad spectrum of activity, i.e. they act against a large number of different ectoparasites. They are effective against both adult and immature stages of arthropods. Most organophosphates act by contact with the arthropod, and a few are also systemic. Usually they do not accumulate in the food chain, because they are either metabolised by microorganisms in the soil or otherwise broken down to less toxic molecules in the soil (George et al. 2008). The use of the majority of organophosphates has been abandoned in quite a few countries, because of the following issues (George et al. 2008). In the first place, organophosphates affect Ecology and prevention of Lyme borreliosis 267

269 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr non-target species. Many organophosphates if not all, are very toxic to birds that for example feed on dead ticks from treated livestock. Some organophosphates are also toxic for other vertebrates, like fish and mammals (and people that handle them). Almost all of them are highly toxic to aquatic invertebrates. Secondly, the development of resistance in arthropods to organophosphates has eliminated or minimised their efficacy in Australia, much of Africa and parts of Latin America (Kunz and Kemp 1994). By cross-resistance, the effectivity of other compounds was lowered. Consequently, these older generation pesticides have largely, but certainly not completely, been replaced by more recent safer alternatives, such as synthetic pyrethroids. Amitraz Amitraz is a formamidine (Figure 1) that acts selectively towards Acari (mites and ticks) and has been used to control ticks since the late 1960s. The acaricidal activity of amitraz is due to its antagonistic effect on octopamine receptors of the nerve cells in the brain. Parasites become hyperexcited, paralysed and eventually die (Beugnet and Franc 2012). Amitraz is commercially available as a spray or dip for use in domestic livestock. In cattle, for example, amitraz has been widely used in dips, sprays or pour-on formulations for the control of single-host and multi-host tick species (Taylor 2001). Dermal absorption of topically administered amitraz is quite low. Treated animals may ingest amitraz through licking and grooming. Amitraz is rapidly broken down to metabolites in the liver. This occurs rather fast in ruminants and pigs, but much slower in horses, which may explain why they do not tolerate amitraz. Excretion is achieved through the kidneys: 24 hours after treatment >60% of the administered dose is already excreted (Junquera 2015). Apart from the acaricidal properties, amitraz has a detachment effect; in case they are not directly killed, ticks leave the host before completing or even before initiating their blood meal. Amitraz has also a repellent effect that keeps many ticks away from treated animals. While amitraz was widely replaced with synthetic pyrethroids in the 1980s, recently amitraz has experienced a strong comeback because of increasing resistance of ticks to synthetic pyrethroids (George et al. 2008). The environmental properties of amitraz seem to be little studied. Some information was found at Ecotoxnet ( In soil containing oxygen amitraz is broken down rapidly; the half-life is less than one day. The substance is moderately toxic to fish, but highly toxic to Daphnia water fleas. It is relatively non-toxic to bees. Figure 1. The chemical structure of amitraz. 268 Ecology and prevention of Lyme borreliosis

270 19. Acaricides Pyrethroids Pyrethroids are synthetic organic compounds. They are similar to the natural pyrethrins extracted from the flower heads of chrysanthemum species (Chrysanthemum cinerariifolium, Dalmatian pyrethrum). They are lipophilic molecules that once in the body generally undergo rapid absorption, distribution and excretion. Pyrethroids act on the sodium channel proteins in nerve cells resulting in paralysis and eventually death (Davies et al. 2007). Pyrethroids are known for their strong knock-down effect. An arthropod stops all movement and behaves as if dead. This knock-down effect can be reversible and after a few seconds the arthropod may wake up and enter a second phase, which involves hyperexcitation due to action on peripheral nerves, with rapid, brief, and inconsistent movements, which can lead to death (Beugnet and Franc 2012). We have observed similar effects in the tick I. ricinus (personal observations). The natural pyrethrins are generally available as shampoos, sprays and powders. Pyrethrins are unstable and therefore have a poor residual activity. A second generation of photostable synthetic pyrethroids emerged in the 1970s. They show residual activity (several weeks) on the skin and in the environment. Important second-generation pyrethroids are permethrin, deltamethrin, and flumethrin (Beugnet and Franc 2012). They are available in many countries as pour-on, spot-on, spray and dip formulations. Three of the more commonly used synthetic pyrethroids, permethrin, flumethrin and deltamethrin, will be discussed in more detail. Permethrin Permethrin has a high affinity for soil and sediment, making it generally immobile in soil. At acidic and neutral ph s, permethrin is relatively stable, but will hydrolyse slowly under alkaline conditions. Permethrin is moderately susceptible to degradation via photolysis in water and soil (Laskowski 2002). It also has a strong tendency to bind to soil and sediment. As such, permethrin is not likely to leach through soil or move in the aqueous phase (ground water). However, sedimentbound permethrin residues can be transported into surface waters along with sediment during heavy runoff events (Imgrund 2003). Permethrin, like many of the synthetic pyrethroids, presents a relatively low toxicological hazard to birds and mammals, but is extremely toxic to some fish and aquatic arthropods (Coats et al. 1989). Permethrin is also classified as highly toxic to honey bees. Permethrin s high octanol-water partition coefficient suggests that it may have a tendency to bioaccumulate into living organisms (Ney 1990) but also is rapidly metabolised (ICPS 1990). Flumethrin Flumethrin, an a-cyano-substituted pyrethroid (Figure 2), was designed for application on cattle as a pour-on formulation, but there is also an emulsifiable concentrate formulation that can be applied as a dip or spray. The active ingredient in the pour-on has a remarkable capacity for spreading rapidly on the skin and hair from points of application along the dorsal line of an animal to all areas of the body. The residual flumethrin for the control of both one-host and multi-host tick species on cattle is effective at low concentrations compared to other pyrethroids (George et al. 2004). Mehlhorn et Ecology and prevention of Lyme borreliosis 269

271 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr Cl Cl H 3 C H 3 C O O CN O F Figure 2. The chemical structure of flumethrin. al. (2012) tested the efficacy of flumethrin and residual effect. Their tests showed that flumethrin is able to kill both tropical ticks of cattle, and ticks of Central Europe, with a residual effect for 4-5 weeks in the case of I. ricinus and Dermacentor reticulatus. Flumethrin is volatile and therefore also seems to have a repellent effect towards flying insects and ticks (Beugnet and Franc 2012, Taylor 2001). Topically administered flumethrin remains mostly on the hair-coat of the treated animals and is very poorly absorbed through the skin. Treated animals can ingest flumethrin through licking or grooming that is largely excreted unchanged through the faeces. The absorbed flumethrin is quickly metabolised in the liver to inactive metabolites that are excreted through urine. As a general rule, livestock (cattle, sheep, goats) tolerate flumethrin and most other synthetic pyrethroids very well. The toxicity is about 1000 higher to insects and other arthropods than to mammals. However, its toxicity to mammals can be increased in case of sustained skin or inhalation exposure, or after direct contact with open wounds (Junquera 2015). Flumethrin is quite resistant to photodegradation, i.e. it breaks down rather slowly even when exposed to sunlight. Like all pyrethroids, flumethrin is extremely toxic to fish and aquatic invertebrates. Disposal of residues in water courses should be absolutely avoided. Flumethrin is not toxic to birds. Flumethrin is almost insoluble in water and tends to bind to soil particles. Therefore, groundwater contamination is unlikely to occur. Persistence in water depends on ph and temperature. Soil bacteria contribute to the biodegradation of flumethrin. Flumethrin does not bioaccumulate. Correct use on livestock is unlikely to result in any significant environmental pollution (Junquera 2015). Deltamethrin Deltamethrin is a synthetic pyrethroid insecticide (Figure 3) that kills insects on contact and through digestion. It quickly paralyses the insect s nervous system giving a rapid disabling effect on the feeding behaviour and a quick knock-down effect. 270 Ecology and prevention of Lyme borreliosis

272 19. Acaricides O Br Br H 3 C CH 3 O O CN Figure 3. The chemical structure of deltamethrin. Many products with deltamethrin remain effective for a long period. Even when cattle and sheep were wetted twice a week, deltamethrin remained effective for 28 days (Schmahl et al. 2010). While many typical insecticides have been successfully tested for their activity against a broad spectrum of insects, their activity against ticks was only occasionally a topic of investigation (Schmahl et al. 2010). Mehlhorn et al. (2011) studied the acaricidal effects of deltamethrin (Butox 7.5 pour on; Intervet bv, Boxmeer, the Netherlands) on hair (head, leg, etc.) from cattle and sheep on a number of tick species after various periods after treatment. When sheep received 10 ml and cattle 30 ml per head, strong acaricidal effects were seen. Along the whole body of sheep, the acaricidal effect was noted for the whole period of 28 days with respect to adults and nymphs of I. ricinus, while reduced acaricidal effects of deltamethrin were seen for another tick species, Rhipicephalus sanguineus, beginning at day 21 after treatment. In cattle, the full acaricidal effect was seen for 21 days in I. ricinus stages and for 14 days in R. sanguineus, while the acaricidal efficacy was reduced after these periods of full action. Besides these acaricidal effects, repellent effects were also noted. Full repellency for both species was seen during the first 14 days in sheep and cattle against both tick species, while the repellency was later reduced, especially in contact with hair from the legs of the animals. The authors conclude that deltamethrin brings acaricidal as well as repellent effects against ticks. We also used deltamethrin (in the form of Butox 7.5 pour on) in our sheep mopping studies (Van Wieren 2016) and also found strong effects through a significant reduction of the number of ticks in the field immediately after treatment. Deltamethrin can be used for all life stages of ticks and is safely applicable on animals of all age groups. It is recommended to use it at intervals of 3-4 weeks. Like the other pyrethroids, it has similar effects on the environment in that it is highly toxic for aquatic organisms. It is rapidly adsorbed by sediment in water and, in soil, and is degraded within 1-2 weeks. Ivermectin Ivermectin is a macrocyclic lactone and an avermectin (Figure 4), a group of broad-spectrum agents with the ability to kill endo- and ectoparasites (Omura 2008). Due to its potent anthelmintic and insecticidal activity, ivermectin has been predominantly used worldwide as a veterinary drug in livestock (sheep, swine, horses, some cattle) and pets to protect against a broad variety of parasites (Rath et al. 2016). The mode of action of avermectins has been studied, but has still not been completely elucidated. Ivermectin is known to act on g-aminobutyric acid (GABA) neurotransmission at two or more sites Ecology and prevention of Lyme borreliosis 271

273 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr O HO O O O O O O O H O O OH B 1a O H OH O HO O O O O O O O H O O OH B 1b O H OH Figure 4. The chemical structure of ivermectin B1a and B1b. It is the mixture of 22,23-dihydro-avermectin B1a (at least 90%) and 22,23-dihydro-avermectin B1b (less than 10%). in nematodes blocking interneuronal stimulation of excitatory motor neurones and thus leading to a flaccid paralysis (Taylor 2001). Macrocyclic lactones can be given orally, parenterally or topically (as either pour-ons or spot-ons). In cattle, for example, available products can be given orally (ivermectin is available in a controlled release device), by injection, or topically using pour-on formulations. Following administration, macrocyclic lactones are stored in fat tissue, from where they are slowly released, metabolised and excreted in the gut. Ivermectin has been shown to be absorbed systemically following oral, subcutaneous or dermal administration, but it is absorbed to a greater degree, and has a longer half-life, when given subcutaneously or dermally (Taylor 2001). Excretion of the unaltered molecule is mainly via the faeces with less than 2% excreted in the urine of ruminants. In cattle, the reduced absorption and bioavailability of ivermectin when given orally, may be due to its metabolism in the rumen. The affinity of these compounds to fat explains their persistence in the body and the extended periods of protection afforded against some species of internal and external parasites (Taylor 2001). The efficacy of ivermectin against I. ricinus has received limited attention. The most detailed study is that of Taylor and Kenny (1990), who compared treated and untreated calves. Treatment was carried out with an intraruminal bolus that slowly released ivermectin. They found no significant differences between treatment groups for mortality of female ticks. However, ivermectin treatment significantly reduced the numbers and weights of engorged female ticks, females laying eggs, and the number of larvae produced from blood of the treated calves was reduced to 3% of that of the control group. The effects on nymphs, although similar, were less consistent than on adults. The effects of the prolonged ivermectin treatment lasted at least for 36 days. In another study on the 272 Ecology and prevention of Lyme borreliosis

274 19. Acaricides effective duration of ivermectin against Ixoididae, it was concluded that cattle would have to be treated with ivermectin subcutaneously at intervals of not more than 31 days apart to ensure that no viable ticks could reach repletion and detach from the host (Davey et al. 2010). Taylor and Kenny (1990) conclude that while the effects of a single such treatment of cattle on a particular farm would not produce a dramatic reduction in tick-borne disease, repeated applications over several years during periods of maximum tick activity would presumably result in a gradual reduction in the population of I. ricinus and of the incidence of babesiosis, tick-borne fever and Borrelia burgdorferi infections. Ivermectin apparently has no great direct killing effect on I. ricinus, but detachment from the host is efficient while viability is reduced which may lead to a reduction of ticks in the environment. Metabolisation of ivermectin by cattle is limited and 70-90% of the applied dose is excreted in the faeces. Here, it keeps its insecticidal activity and the excreted ivermectin can remain in the dung and the soil for years (Edwards et al. 2001). This long lasting activity can have deleterious effects on a large number of invertebrate species attracted to the dung. In some cases, this results in retardation of dung decomposition (Floate et al. 2005). Negative effects have been demonstrated on the survival and reproduction of fly larvae, adult dung beetles, collembolans, and other arthropods, and plankton (summarised by e.g. Liebig et al. 2010, Rath et al. 2016). Repeated treatment may increase the local extinction probability of dung fauna, more in particular dung beetles (Lumaret and Errouissi 2002). Secondary effects can be expected on the numerous species that forage on dung fauna, like meadow birds and their chicks (McCracken 1993). Resistance The development of resistance to insecticides and acaricides constantly reduces the number of chemicals to choose from. Particularly in tropical and subtropical regions of the world, ticks cause significant problems in livestock resulting in major economic loss. Other chemical acaricides, therefore, are being extensively used for tick control with the consequence that resistance to all major classes of acaricides in several countries has subsequently developed (George et al. 2004). Several studies confirm the resistance of ticks in Mexico to organophosphates, synthetic pyrethroids (in the 1990s), amitraz (in 2001) and recently (2008) the first case of resistance to fipronil was reported in Northern States of Mexico (Perez-Cogollo et al. 2010). Rhipicephalus microplus has been shown to have become resistant to ivermectin (Rodriguez-Vivas et al. 2014). Alternatives Plant-based acaricides The insecticidal and acaricidal properties of plant extracts have already been studied since the 1950s, but applications of plant extracts are still rare. Nevertheless, there is a renewed interest in the use of plant extracts, because of the high cost of developing new drugs and vaccines, development of drug resistance and concerns over drug residues associated with the continuous use of these chemicals (Abbas et al. 2014). The application of botanicals to livestock in order to control the ectoparasites of veterinary importance is particularly widespread in the developing countries. At least twenty-one plant species have been identified with an acaricidal activity and many plant-essential oils are extensively being tested to establish the efficacy of their parasiticidal activity, their mechanism of action and their target parasite species (Abbas et al. 2014, Kiss et al. 2012). Ecology and prevention of Lyme borreliosis 273

275 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr As far as current studies show, botanical acaricides are not suitable if a 100% control of ticks is desired in a short time. Even if some field studies suggest that the effects of plant extracts are comparable to that of chemical acaricides, the time needed to attain the same effect is usually longer. The persistence of most phytoacaricides is usually lower too, due to their high degree of biodegradation, but on the other hand, this is the exact trait that makes them more eco-friendly and attractive as replacements of synthetic chemicals in the first place (Kiss et al. 2012). It is suggested that a thoughtful integration of botanical products with synthetic acaricides may be expected to produce better effects in terms of controlling ticks and delaying the resistance than the use of acaricides alone (Abbas et al. 2014). Vaccination The fact that control of tick infestations is possible and feasible through immunisation of hosts with selected tick antigens was demonstrated with the development of commercial vaccines that reduced R. microplus infestations on cattle (Kiss et al. 2012, Merino et al. 2011). These vaccines contain the recombinant R. microplus BM86 gut antigen. They reduce the number of engorging female ticks, their weight and reproductive capacity. Effects of vaccination are predominantly seen in the late stages of the life cycle, that is in adults and post-engorgement survival and egg laying. This means that the first generation of ticks infecting vaccinated cattle would show some diminution of numbers, typically no more than 50% at best. The greatest effect of vaccination, however, would be seen not until the second and subsequent generations when the impact of vaccination on reproductive performance was translated into reduced numbers of larvae (Kiss et al. 2012). One of the major limitations in anti-tick vaccine development was the lack of the discovery of suitable antigen targets. With advances in characterisation of tick genomes, and other technologies allowing for a rapid, systematic and comprehensive approach to tick vaccine discovery, this constraint has largely been overcome (De la Fuente and Kocan 2006). Numerous trials have demonstrated the advantages of tick control by vaccination, by being a non-contaminating, sustainable and cheap technology, potentially applicable to a wide variety of hosts (Kiss et al. 2012). Nevertheless, there is enormous variation in the efficacy of vaccines. Efficacy varies between different tick species and may even vary within a species when applied in different geographical regions (Abbas et al. 2014). Extensive screening is therefore always needed before a vaccine can be used against a certain tick species in a region. A vaccine against I. ricinus has not been developed yet. Discovery of protective antigens, along with proper vaccine formulations, field trial evaluation and commercialisation, are all major components required for development of tick vaccines. These new tick vaccines will probably play a key role in future integrated tick control strategies (Kiss et al. 2012, Klouwens et al. 2016). Evaluation and discussion In Table 2 some key features of the above discussed acaricides are summarised and evaluated qualitatively. These can be used to judge their usefulness for the main goal, to reduce tick densities of I. ricinus in field situations with minimal environmental side-effects. Least suitable for tick control is ivermectin. While ivermectin will give fair protection to the host, the reported negative effects on the environment are an issue. It has low direct killing potential, but ticks may be killed indirectly because of fitness effects and ticks detach quickly. Also the 274 Ecology and prevention of Lyme borreliosis

276 19. Acaricides Table 2. Some key aspects of the four discussed acaricides with respect to their usefulness to reduce tick numbers. Effect Acaricide Amitraz Permethrin Flumethrin Deltamethrin Ivermectin Kill ticks Detach ticks Repel ticks Environmental hazard Residual effect (weeks) >4 >4 1 Depending on species and application method this period varies. The periods shown are generally reported for pour-products for cattle and sheep. 2 If applied properly. killing power of amitraz is weak, but amitraz is mainly effective in protecting the host through detachment and repellency. Amitraz is unstable and broken down quickly by microbial and chemical degradation and does not seem to be very toxic to the environment. Although amitraz is effective and used frequently in protecting the host from ticks, it is probably less effective in reducing the tick density in the field because of its strong repellent and not killing effects. The most effective candidates for tick reduction in the field are the two synthetic pyrethroids, flumethrin and deltamethrin. Both have great killing power and are effective for many weeks. A drawback is their toxic effects in the environment, especially on aquatic organisms. Nevertheless, when applied correctly and with care in a terrestrial environment, the toxic effects may be limited. By ensuring that the treated animals and the substance applied do not get in contact with waterbodies, most negative effects can be avoided. The evaluation above represents a first rough assessment of the usefulness of a few selected acaricides for tick control through application to large mammals. However there are still several issues when one is trying to select an agent for tick control that need more research and action. A short list: With a high tick infestation, frequent application is needed. This may be constrained by cost and time considerations. The development of resistance is an ongoing and increasing problem. Much more knowledge is needed on the fate and (toxic) effects of the acaricides and their metabolites on non-target organisms in the environment. Does accumulation take place? Can contamination of surface waters effectively be avoided? Are there no effects in the terrestrial environment? Although a product may be effective against ticks, it is not always licensed for tick control. This may vary between countries. In summary, the implementation of an intervention using, even very effective, acaricides in field situations is a task in which the costs and benefits need to be weighed carefully. The applications of acaricides on large mammalian hosts can indeed lead to a (sometimes strong) reduction in tick densities, but never to eradication (which increases the probability of resistance development). Ecology and prevention of Lyme borreliosis 275

277 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr The relation between reduction of tick density and reduction in health risk for humans is still object of study (Van Wieren and Hofmeester 2016, Van Wieren 2016). Public health relevance Very effective acaricides are available for potentially reducing tick densities in the field through application on large mammalian hosts. The most useful seem to be the synthetic pyrethroids: permethrin, deltamethrin and flumethrin. Because of the many potential environmental side-effects, a proper cost-benefit analysis should be made when considering tick control through the application of acaricides to large mammals. The public health benefits for tick control need to be carefully weighed against the environmental consequences of such use. The many environmental issues with acaricides call for the search for alternative ways of tick control. Vaccine development seems to be the most promising alternative. References Abbas RZ, Zaman MA, Colwell DD, Gilleard J and Iqbal Z (2014) Acaricide resistance in cattle ticks and approaches to its management: the state of play. Vet Parasitol 203: Beugnet F and Franc M (2012) Insecticide and acaricide molecules and/or combinations to prevent pet infestation by ectoparasites. Trends Parasitol 28: Coats JR, Symonik DM, Bradbury SP, Dyer SD, Timson LK and Atchison GJ (1989) Toxicology of synthetic pyrethroids in aquatic organisms an overview. Environ Toxicol Chem 8: Coipan EC, Jahfari S, Fonville M, Maassen C, van der Giessen J, Takken W, Takumi K and Sprong H (2013) Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus. Front Cell Infect Microbiol 3. Davey RB, Pound JM, Miller JA and Klavons JA (2010) Therapeutic and persistent efficacy of a long-acting (la) formulation of ivermectin against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and sera concentration through time in treated cattle. Vet Parasitol 169: Daniels TJ,Falco RC, Mchugh EE, Vellozzi J, Boccia T, Denicola AJ, Pound JM, Miller JA, George JE and Fish D (2009) Acaricidal treatment of white-tailed deer to control Ixodes scapularis (Acari: Ixodidae) in a New York Lyme diseaseendemic community. Vector-Borne Zoonotic Dis 9: Davies TGE, Field LM, Usherwood PNR and Williamson MS (2007) DDT, pyrethrins, pyrethroids and insect sodium channels. IUBMB Life 59: De la Fuente J and Kocan KM (2006) Strategies for development of vaccines for control of ixodid tick species. Parasit Immunol 28: Edwards CA, Atiyeh RM and Römbke J. (2001) Environmental impact of avermectins. Rev Environ Contam Toxicol 171: Floate KD, Wardhaugh KG, Boxall ABA and Sherratt TN (2005) Fecal residues of veterinary parasiticides: nontarget effects in the pasture environment. Ann Rev Entomol 50: George JE, Pound JM and Davey RB (2004) Chemical control of ticks on cattle and the resistance of these parasites to acaricides. Parasitology 129: S353-S Ecology and prevention of Lyme borreliosis

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279 Sipke E. van Wieren, Marieta A.H. Braks and Joost Lahr Rodriguez-Vivas IR, Perez-Cogollo CL, Rosado-Aguilar AJ, Ojeda-Chi MM, Trinidad-Martinez I, Miller RJ, Li AY, De Leon AP, Guerrero F and Klafke G (2014) Rhipicephalus (boophilus) microplus resistant to acaricides and ivermectin in cattle farms of mexico. Rev Bras Parasitol Vet 23: Schmahl G, Al-Rasheid KAS, Abdel-Ghaffar F, Klimpel S and Mehlhorn H (2010) The efficacy of neem seed extracts (Tresan, MiteStop ) on a broad spectrum of pests and parasites. Parasitol Res 107: Taylor MA (2001) Recent developments in ectoparasiticides. Vet J 161: Taylor SM and Kenny J (1990) An ivermectin sustained-release bolus in cattle its effects on the tick Ixodes-ricinus. Med Vet Entomol 4: Van Wieren SE (2016) Sheep mopping. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Wieren SE and Hofmeester TR (2016) The role of large herbivores in Ixodes ricinus and Borrelia burgdorferi s.l. dynamics. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Ecology and prevention of Lyme borreliosis

280 20. Biological control of the tick Ixodes ricinus by pathogens and invertebrates Ingeborg Klingen 1* and Gilian van Duijvendijk 2 1 Norwegian Institute of Bioeconomy Research (NIBIO), P.O. Box 115, 1431 Ås, Norway; 2 Laboratory of Entomology, Wageningen University & Research, P.O. Box 16, 6700 AA, Wageningen, the Netherlands; ingeborg.klingen@nibio.no Abstract In this chapter we will focus on the tick Ixodes ricinus, with its main geographical distribution in Europe. It is known to transmit a variety of pathogens, among them Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Tick population control is one of the measures to reduce the incidence of tick-borne diseases. Due to non-target effects of chemical acaricides, acquired resistance against chemical acaricides and increased regulations, there is a demand for sustainable control measures that may be used in integrated vector management (IVM) of ticks. This chapter describes and evaluates the present knowledge on biological control of I. ricinus as an alternative to the use of chemical acaricides. Biological control makes use of living organisms (e.g. fungi, bacteria, nematodes, invertebrate predators, parasitoids) to suppress a pest population. The natural occurrence of these organisms in I. ricinus and the use of these organisms as biological control agents against I. ricinus are reviewed. Entomopathogenic fungi (Beauveria and Metarhizium spp.) are the most commonly used biocontrol agents against ticks. A variety of nematode species are also shown to be effective against different tick species, but the knowledge on the operational use of invertebrate predators and parasitoids to control ticks is limited. We conclude that there are several candidates for the biological control of ticks, but that the knowledge on the natural occurrence and efficacy of these to control I. ricinus populations is very limited. There is, therefore, a need of more studies on naturally occurring enemies of I. ricinus to be able to suggest possible biocontrol candidates. These candidates should be tested in controlled laboratory and field studies with the aim to develop elegant, precise and effective biocontrol strategies for the control of I. ricinus that may be used alone or in combination with other control strategies in IVM. Keywords: biological control, fungus, Ixodes ricinus, nematode, parasitoid, predators, integrated vector management (IVM) Introduction Many pathogens of man and animals are transmitted by ticks. In Europe, the deer tick, Ixodes ricinus (Ixodida/Ixodidae), is known to transmit a variety of pathogens, among them Borrelia burgdorferi sensu lato (Spirochaetales/Spirochaetaceae), the causative agent of Lyme borreliosis (disease) (Jongejan and Uilenberg 2004, Mannelli et al. 2012). Ticks belong to the phylum Arthropoda, which is the largest group in the order Acarina (Sonenshine 1991), they belong to the superorder Parasitiformes, the order Ixodida, and the superfamily Ixodoidea (Keirans 2009). They are divided into two groups: soft bodied ticks (Argasidae) and hard bodied species (Ixodidae). The ixodidae family encompasses the Protista and just one tick genus, Ixodes, which currently includes 244 recognised species (Durden and Beati 2014). Hard ticks, in which I. ricinus is included, feed for extended periods of time on their hosts (Apanaskevich and Oliver Jr. 2014). I. ricinus is an exophilic species found in open or semi open biotopes, most often on the vegetation litter or herbaceous shrubs (Vassallo et al. 2000). The geographical distribution of I. ricinus includes Europe from Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _20, Wageningen Academic Publishers 2016

281 Ingeborg Klingen and Gilian van Duijvendijk Portugal to Russia and from North Africa to Scandinavia but no other parts of the world (Gray et al. 1992, Jore et al. 2011, Kurtenbach et al. 2001, Rich et al. 1995, Yukari and Umur 2002, tinyurl.com/zfn6u4r). In this chapter, we will focus on the main geographical distribution area for I. ricinus, which is Europe. Climate change (i.e. higher winter temperature), changes in land use (i.e. bush encroachment) and an increase in the wildlife host population are factors expected to increase the population of ticks (Sprong et al. 2012). I. ricinus is extending its distribution further north and to higher altitudes (Jore et al. 2011, Lindgren et al. 2000, Medlock et al. 2013), giving rise to concern that challenges with Lyme borreliosis will increase in Europe. Preventive measures to reduce the incidence of Lyme borreliosis include tick population control, which can be achieved by: (1) the clearing of vegetation; (2) draining land; (3) reduction of host animals including wildlife; and (4) acaricide treatment on and off humans, pets, livestock and wildlife hosts (Sonenshine 1991, various chapters in this book). Public concern, however, on the negative effects of chemical pesticides and problems with acquired resistance to chemical pesticides in tick populations (Van Wieren et al. 2016) have led to a demand for a more effective and sustainable control measure of ticks and tick-borne diseases. Biological control is one of these measures, representing relevant alternatives to chemical I. ricinus control and will be the focus of this chapter. Chemical acaricides and their possible limitations in Ixodes ricinus control Considering tick population control, chemical pesticides (acaricides) are the most commonly used method (Guerrero et al. 2014, Schulze et al. 1987, 1991, 2000). However, the use of synthetic chemical acaricides is becoming more and more questionable because they do not only affect ticks, but can also have a negative effect on the populations of non-target organisms, including small vertebrates (Schauber et al. 1997) and invertebrate natural enemies (Ostfeld et al. 2006). Chemical acaricides applied on the host to kill feeding ticks reduces exposure to non-target hosts and reduces the amount of chemicals needed and hence leakage of acaricides to soil and water. In practice, on-host application of chemical pesticides is mostly done to control ticks feeding on livestock and pets (Beugnet and Franc 2012, Drummond et al. 1973, George et al. 2004, 2008, Sonenshine 1991, Van Wieren et al. 2016). Application of acaricides on wildlife hosts has also been conducted, however, and the application of fipronil on white footed mice (Peromyscus leucopus, Rodentia/Cricetidae) in USA resulted in a decrease in I. scapularis (Ixodida/Ixodidae) larval and nymphal tick burden (Dolan et al. 2004). To apply acaricides on wildlife hosts to control ticks is questionable and out of scope in many countries since the recreation area in question might be huge. Further, it seems not feasible for the control of I. ricinus because this tick species feeds on a large variety of wild vertebrates. Another disadvantage of using chemical pesticides to control ticks is that there is an increased incidence of acaricide resistance in ticks (George et al. 2004, Guerrero et al. 2014, Thullner et al. 2007). This was for example found in the cattle tick (Rhipicephalus microplus, Ixodida/Ixodidae) in USA (Texas), Latin America (Mexico, Brazil, Costa Rica, Argentina), Australia, New Caledonia and South Africa (Beugnet and Chardonnet 1995, Li et al. 2004, Lovis et al. 2013, Martins 2001, Pohl et al. 2012, Thullner et al. 2007). To our knowledge there are no studies on acaricide resistance in I. ricinus populations and this, therefore, needs to be further investigated. Further, legal restricitions in acaricide residues in meat for consumption limits the use of chemicals on livestock (Koschorreck et al. 2002). 280 Ecology and prevention of Lyme borreliosis

282 20. Biocontrol of Ixodes ricinus Biological control of ticks Considering that I. ricinus spends over 90% of its life cycle off the host (Needham and Teel 1991) and that about 80% of this is spent in the litter resting (unfed, all stages), copulating or laying eggs (fed females) (Perret et al. 2004) (Figure 1), efforts to discover effective biocontrol methods to control ticks in the litter seem warranted. Livestock on-host application of biocontrol agents against other tick species with other life cycles is promising (Fernandes and Bittencourt 2008, Maniania et al. 2007) and it might also be of interest to look closer into this for I. ricinus. This might be especially relevant for I. ricinus on fenced livestock. Biological control is defined as the use of living organisms (e.g. fungi, bacteria, nematodes, predators, parasitoids) to suppress the population density or impact of a specific pest organism (Eilenberg et al. 2001). The different biocontrol methods that may be used on ticks and other pests are described by Eilenberg et al. (2001) and include conservation biological control, inundative- or inoculative biological control or classical biological control. A B C D Figure 1. (A) Unfed Ixodes ricinus in/on litter (80% of time). (B) Unfed I. ricinus climbing grass to hunt hosts. (C) Fed I. ricinus female (big and grey) copulating with I. ricinus male (small and black) (D) Fed I. ricinus female extending its egg laying organ preparing to lay eggs (photos by Karin Westrum, NIBIO, Ecology and prevention of Lyme borreliosis 281

283 Ingeborg Klingen and Gilian van Duijvendijk Natural occurring enemies of I. ricinus among pathogens and invertebrates Many authors have suggested that different biotic and abiotic factors affect the activity and distribution of I. ricinus (e.g. Qviller et al. (2014) and references therein) but few have studied the prevalence and effect of natural enemies of I. ricinus on questing tick density patterns. Fungal pathogens, nematodes, predatory mites and parasitoids are, however, thought to be tick killers (Chandler et al. 2000, Samish and Rehacek 1999, Samish et al. 2008) and will be discussed here. Fungi In nature, 20 species of fungus have been found to be associated with ticks (Samish et al. 2008). Entomopathogenic fungi in the order Hypocreales are abundant in the soil and litter habitats where I. ricinus reside and it is therefore possible that hypocrealean fungi may contribute to natural control. However, only few studies quantify natural fungal infection of ticks. Further, the infection rates reported vary markedly in different studies, probably as a result of both natural variation and differences in the sensitivity of methods used (Ginsberg, 2014). In addition, entomopathogenic fungi in this order also grow saprophytically and dead ticks with fungal outgrowth does not necessarily imply that the tick is killed by the fungus growing on it. To our knowledge, only very few studies are conducted on the natural occurrence of entomopathogenic fungi on I. ricinus. These are mentioned below. Kalsbeek et al. (1995) showed that I. ricinus collected from vegetation, small rodents and deer in Denmark were found naturally infected with the following species within the fungal group Hypocreales: Beauveria bassiana (Hypocreales/Clavicipitaceae), Beauveria brongniartii (Hypocreales/Clavicipitaceae), Paecilomyces farinosus (Eurotiales/Trichocomaceae), Paecilomyces fumosoroseus (Eurotiales/Trichocomaceae), Verticillium lecanii (Lecanicillium lecanii) (Hypocreales/Clavicipitaceae) and Verticillium aranearum (Hypocreales/Clavicipitaceae). Fungal infections were most common on engorged females (22%) and flat females (10%), while infections in males, nymphs and larvae did not exceed 1%. In the Czech Republic, engorged I. ricinus females collected during summer were reported to be infected by Beauveria spp., but also by more opportunistic fungal species such as Aspergillus (Eurotiales/Trichocomaceae), Fusarium (Hypocreales/Nectriaceae) and Mucor spp. (Mucorales/Mucoraceae) (Samsinakova et al. 1974). In a pilot study we conducted in Norway to identify naturally occurring beneficial fungi and nematodes of I. ricinus in sheep grazing areas collected by flagging (382 ticks in 2011, 730 ticks in 2012) and on sheep (112 ticks in 2011) we could not report of any I. ricinus infected with fungi. I. ricinus in litter and sheep resting places should probably be investigated further to get the whole picture. Other studies in Europe indicate that fungal infections may cause the death of up to 50% of Dermacentor (Ixodida/Ixodidae), Ixodes and other ticks (Samish and Rehacek 1999). Methods used in these studies need to be analysed, however, to be able to evaluate whether the infection levels found were not artifacts. Nematodes Nematodes are widespread among insects, but have only rarely been found in ticks. The only nematodes that were found in I. ricinus in nature are mermethids (Lipa et al. 1997). In the Norwegian pilot study mentioned above, we could not report of any I. ricinus infected with nematodes. Other nematode species (Steinernematidae and Heterorhabditidae) are able to kill ticks in the laboratory, but cannot complete their lifecycle in ticks (see below). The effect of nematodes on tick population control under natural conditions, therefore, seems to be limited. 282 Ecology and prevention of Lyme borreliosis

284 20. Biocontrol of Ixodes ricinus Predatory mites Many generalist invertebrate and vertebrate predators will eat ticks on an opportunistic basis. However, few predators appear to specialise on ticks (Ginsberg 2014). Even though predatory mites are suggested to be important tick killers, the prevalence and diversity of predatory mites in tick habitats has barely been evaluated (Venancio et al. 2016). Predatory mites are prevalent in the soil and litter, especially those of the cohort Gamasina of the order Mesostigmata (Lindquist et al. 2009). These have been reported as important predators of nematodes, springtails, mites and insect larvae (Calvo et al. 2011, Castilho et al. 2015, Koehler 1999, Ruf and Beck 2005) and might also be important predators of ticks. Venancio et al. (2016), therefore, determined the groups of gamasine co-occurring with I. ricinus in sheep grazing areas in Western Norway. A total of 2,900 gamasines of 12 families was collected. The most numerous families were Parasitidae (46.9%) and Veigaiidae (25.7%), whereas the most diverse families were Laelapidae, Macrochelidae, Parasitidae and Zerconidae. Parasitoids Few parasitoids are specific to ticks and only the genus Ixodiphagus (Hymenoptera/Encyrtidae), which includes seven species, all of which are tick parasites, is known to specialise on ticks (Ginsberg, 2014). The most widespread species is Ixodiphagus hookeri (synn. Hunreellus hookeri, Ixodiphagus caucurtei), which has been recorded from Asia, Africa, North America and Europe. Most species of Ixodiphagus are host-generalists (Samish et al. 2008). Different biological control methods of ticks Classical biological control of ticks Classical biological control is defined as the intentional introduction of an exotic, usually coevolved, biological control agent for permanent establishment and long-term pest control (Eilenberg et al. 2001). The advantages of classical biological control are that it may be a long lasting and a relatively inexpensive solution (Hajek 2004). The disadvantage is that there may be environmental risks of introducing an exotic species and this risk needs to be evaluated extensively before the biocontrol agent is introduced (Van Lenteren et al. 2003). Classical biological control is probably not relevant for I. ricinus without knowing more about the evolutionary relationship and geographical origin of Ixodes species. It is suggested that the genus Ixodes has two main linages, the Aurtalasian Ixodes and the other Ixodes (Barker and Murell, 2008, Durden and Beati, 2014). Many natural enemies are, however, not species and often not genus specific and, even though it might be risky to introduce generalists in conservation biological control programmes, it might be interesting to search for I. ricinus biocontrol candidates in exotic areas. Conservation biological control of ticks Conservation biological control is defined as modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests (Eilenberg et al. 2001). Habitat management is one way to increase population densities of natural occurring enemies, as has been successfully used to control agricultural pest species (Landis et al. 2000). For the control of ticks, however, modification of the environment to enhance natural enemies has to our knowledge been limited. The reintroduction of yellow-billed oxpeckers (Buphagus africanus, Passeriformes/Sturnidae) that feed on ticks in Zimbabwe in 1975 appears Ecology and prevention of Lyme borreliosis 283

285 Ingeborg Klingen and Gilian van Duijvendijk to be one successful example of conservation biological control of ticks (Grobler 1979). There are several examples of habitat management that are used to control tick populations directly (Schulze et al. 1995, Tack et al. 2013). Inoculation and inundation biological control of ticks Inoculation biological control is defined as the intentional release of a living organism as a biological control agent with the expectation that it will multiply and control the pest for an extended period, but not permanently. Further, inundation biological control is defined as the use of living organisms to control pests when control is achieved exclusively by the released organisms themselves (Eilenberg et al. 2001). Among promising inundative or inoculative (mass release) biocontrol agents, fungi, predatory mites, nematodes and parasitoids are mentioned (Chandler et al. 2000, Ginsberg 2014, Maniania, et al. 2007, Samish and Rehacek 1999) and will be discussed here. Fungi Entomopathogenic fungi seem to be the most promising candidate for biological control of ticks (Ment et al. 2010). Further, spores of the entomopathogenic fungi germinate and penetrate the cuticle of the host and do not need to be eaten like entomopathogenic bacteria and viruses. This is an advantage when controlling ticks since they only feed on blood from their hosts. The most common fungal pathogens studied for inundative- and inoculative biological control of arthropods belong to fungi in the order Hypocreales and include the genera Metarhizium (Hypocreales/ Clavicipitaceae), Beauveria, Isaria (=Paecilomyces), Hirsutella (Hypocreales/Ophiocordycipitaceae), Lecanicillium (=Verticillium), Tolypocladium (Hypocreales/Ophiocordycipitaceae), and Normuraea (Hypocreales/Ophiocordycipitaceae) (Maniania et al. 2007). In vitro studies and field trials have demonstrated that these entomopathogenic fungi not only may cause mortality of ticks, but also reduce subsequent generations due to effects on their reproductive efficacy (Fernandes and Bittencourt 2008, Maniania et al. 2007, Rot et al. 2013, Samish et al. 2014). Several laboratory studies have confirmed the potential for fungi (especially Metarhizium anisopliae and B. bassiana) to control ticks but field studies are still limited (Fernandes and Bittencourt 2008, Kaaya et al. 2011, Manianai et al. 2007). Further, both laboratory and field studies have been conducted mainly with other tick species than I. ricinus (Hartelt et al. 2008, Lonc et al. 2014). There is, therefore, a need for more studies on the effect of fungi for the control of I. ricinus. A few studies have, however, been published and some preliminary studies conducted. Below we will mention these. A laboratory experiment on the killing capacity of M. anisopliae (five strains), B. bassiana (three strains) and Paesilomyces fumosoroseus (two strains) showed that the highest mortality by two M. anisopliae isolates causing 80% mortality in unfed nymphs after 30 and 50 days respectively (Hartelt et al. 2008). Beauveria bassiana isolated from I. ricinus in Moldova and P. farinosus isolated from Ixodes persulcatus (Ixodida/Ixodidae) (St. Petersburg Region, Russia) were tested in the laboratory for their activity against male and female I. ricinus and I. persulcatus (Alekseev 2011). Ticks submerged into a suspension of fungal conidia showed a low level of mortality (15.9±1.6%) while one-day contact of fungal treated males with non-treated females enhanced the fungal action on both sexes (max. 61.9, mean 34±9.2%). In Austria, work conducted by Strasser et al. (2007) on applying M. brunneum (the BIPESCO 5 isolate) on hot spots in the vegetation in order to reduce the number of I. ricinus particularly in areas used for recreation had promising results ( gnabczy). Another field experiment that was recently conducted in Norway in the TICKLESS project ( suggests that Metarhizium brunneum (the BIPESCO 5 isolate) also has promising results against I. ricinus applied in sheep pastures (I. Klingen et al. unpublished data) (Figure 2). 284 Ecology and prevention of Lyme borreliosis

286 20. Biocontrol of Ixodes ricinus A B C Figure 2. (A) Application of the entomopathogenic fungus Metarhizium brunneum (the BIPESCO 5 isolate) against Ixodes ricinus in sheep pasture in Norway (photo by Natasha Iwanicki NIBIO/ESALQ-USP). (B) Flagging I. ricinus to estimate the population number after fungal treatment (photo by Natasha Iwanicki NIBIO/ESALQ-USP). (C) I. ricinus engodged female and male infected and killed by M. brunneum (photo by Karin Westrum NIBIO, M. brunneum strain F52 (029056) is approved as a biocontrol agent (Tick-EX EC and Tick-EX G) against ticks in the USA ( and the Agrifutur strain BIPESCO 5 (= KVL 275 / V 275 / Ma 43) is under development for I. ricinus control in Europe. Development of appropriate formulation- and application methods are critical for the practical application and implementation of fungal pathogens against ticks and many different formulation- and application methods have been tested on different tick species (Fernandes and Bittencourt 2008, Maniania et al. 2007, Strasser et al. 2007). To know which formulation and application method is the best to use, there is a need to have deep knowledge about the life cycle of the tick species in question. Further, it is important to consider whether the intention is to treat the tick off or on the host and if on-host, on which hosts. For our tick, I. ricinus, only off-host fungal application has been tested until now. This is probably mainly because I. ricinus spend most of its time (90%) off the host in the vegetation (Needham and Teel 1991). For the fungus to kill I. ricinus it needs quite some time (not immediate response as for effective synthetic acaricides). On-host application might, therefore, not protect the individual host but it might help in reducing the I. ricinus population since less females will engorge and lay eggs. Ecology and prevention of Lyme borreliosis 285

287 Ingeborg Klingen and Gilian van Duijvendijk Studies presenting on-host fungal application against other tick species are, however, promising (Fernandes and Bittencourt 2008, Maniania et al. 2007, Rot et al. 2013) and it might be of interest to look closer into this for I. ricinus as well. On-host application of entomopathogenic fungi is probably most relevant to control I. ricinus feeding on livestock, pets and wildlife hosts. Application of entomopathogenic fungi on wildlife natural hosts has been conducted and laboratory treatment of the nesting material of the white footed mouse (P. leucopus) with M. anisopliae could increase the mortality (75%) of I. scapularis larvae engorging on white footed mice compared to the control nests (35%). Field trials caused only modest, localised reductions in nymphal I. scapularis (Hornbostel et al. 2005). The off-host fungal application strategies mentioned above that were tested for I. ricinus were hot-spot applications and broad applications in the tick habitat, but also more precise off-host application methods are now under development. One of them is the use of pheromone/carbon dioxide traps baited with fungi (Maranga et al. 2006). On-host fungal application on wildlife to control ticks and treatment of large pasture and recreation areas far away from human infrastructure might be questionable and out of scope. Hot-spot fungal application on- and off-host in pasture and recreation areas close to human infrastructure might, however, be of interest. When developing and choosing appropriate fungal isolates, formulation- and application methods it is also important to consider the susceptibility of the tick species in question. Tick mortality due to fungal pathogens varies considerably with the developmental stage and species of tick, as well as environmental conditions (Gindin et al. 2002, Maniania et al. 2007, Ment et al. 2010, Samish et al. 2001). Further, stimulation of the germination of conidia in the ticks environment can both increase and decrease the fungus anti-tick efficacy (Ment et al. 2010). Based on in vitro studies on the effect of fungi against eggs, larvae, nymphs, adults and engorged females of several tick species in South America larva seem to be the most susceptible stage (Fernandes and Bittencourt 2008). Gindin et al. (2002), report that eggs of three different tick species (Boophilus annulatus (Ixodida/Ixodidae), Hyalomma excavatum (Ixodida/Ixodidae) and Rhipicephalus sanguineus (Ixodida/Ixodidae) were the most susceptible stage to fungal infection. Which I. ricinus stage that is most susceptible is still not clear since only few studies have been conducted, but from the few that have been conducted it is suggested that unfed nymphs might be the most susceptible (Hartelt et al. 2008). Nematodes Nematodes are another potential inundation biological control agent against ticks due to their broad host range, high virulence, host-seeking capability, and the possibility to produce nematodes in large amounts (Grewal et al. 2005). Several studies have confirmed that entomopathogenic nematodes within the families of Steinernematidae and Heterorhabditidae are virulent for ticks (Samish and Glazer 2001). When using entomopathogenic nematodes as a biological control agent, the infective juvenile stage is generally used. After encountering a susceptible host species, these infective juveniles penetrate through natural openings of the host body and enter into the haemocoel of the host before they release gram-negative gamma proteobacteria (Xenorhabdus sp. (Enterobacteriales/Enterobacteriaceae) and Photorhabdus sp. (Enterobacteriales/Enterobacteriaceae)) that live in symbioses with the nematodes (Forst et al. 1997, Taylor et al. 2012). After these bacteria are released into the hemolymph, they colonise and kill the host. One infective juvenile nematode can be enough to kill a tick (Glazer et al. 2001). To complete their lifecycle, the nematodes need to develop and reproduce inside the killed host, and released into the environment after the host bursts open. While the nematodes are able to kill ticks, they are unable to reproduce and complete their lifecycle in ticks (Hill 1998, Kocan et al. 286 Ecology and prevention of Lyme borreliosis

288 20. Biocontrol of Ixodes ricinus 1998, Mauleon et al. 1993). The ability of steinernematid and heterorhabditid nematodes to kill ticks was first shown in engorged Rhipicephalus annulatus (Samish and Glazer 1991, 1992). Since then, several entomopathogenic nematode species have been tested for the control of a variety of tick species. For Ixodid ticks, 16 species from six different genera were susceptible to nematodes, whereas for Aragsid ticks, two out of three species from two genera were susceptible to nematodes (Samish and Glazer 2001). The ability of nematodes to kill ticks varies between nematode and tick species (Samish and Glazer 2001). The number of experiments conducted on I. ricinus is, however, very limited. Hartelt et al. (2008) investigated the effects of three nematode species (Steinernema carpocapsae (Rhabditida/Steinernematidae), Steinernema feltiae, and Heterorhabditis bacteriophora (Rhabditida/Heterorhabditidae)) on the survival of nymphs (engorged and unengorged) and females (unengorged) of I. ricinus in the laboratory. These authors found that all three nematode species affected I. ricinus survival and the highest mortality (40% in 13 d) was observed in unfed females treated with S. carpocapsae. Different developmental stages of the tick species also vary in their susceptibility to nematodes. In general, eggs are resistant to nematodes, larvae and nymphs are least susceptible, and adult ticks are most susceptible and the engorged female ticks are more susceptible than unengorged females (Hill 1998, Kaaya et al. 2000, Samish et al. 1999a). Engorged larvae and engorged nymphs were, however, not more susceptible than the unengorged stages (Zhioua et al. 1995). Even though many laboratory studies on the efficacy of nematodes against tick have been conducted, the number of (semi-)field trials is limited. Some have been conducted, however, and Samish et al. (1999b) and Alekseev et al. (2006) filled buckets with soil mixed with nematodes in a greenhouse and found that the nematodes were able to kill almost all engorged female ticks. The success, however, differed between tick species, nematode species and nematode concentration in the soil. The application of nematode suspensions on cattle infested with semi-fed R. annulatus females, when the environmental conditions for the nematodes were optimal, resulted in fewer ticks (El-Sadawy and Abdel-Shafy 2007). Environmental conditions like soil type, ph, soil aeration, temperature, moisture, and relative humidity can affect the survival and pathogenicity of released nematodes and the effects differ between nematode species (Alekseev et al. 2006, Glazer and Navon 1990, Kung et al. 1990, 1991). Alekseev et al. (2006) found many infective juveniles of S. carpocapsae and S. riobrave in the upper 6 cm of the soil layer whereas the heterorhabditid nematodes were almost absent from the top layer. The location of nematodes in the soil is affected by the environmental conditions, like humidity, due to their sensitivity to dehydration (Smits 1996). The abiotic conditions are rather stable in the litter, which is the place where the most susceptible stage of I. ricinus (engorged females) spend their time while digesting their blood meal and lay their eggs. Even though a variety of nematode species appear to be suitable as biological control agents against a variety of tick species in the lab, the number of field trials and the number of experiments on I. ricinus are still limited and practical use of entomopathogenic nematodes against feeding or questing ticks is rare. Predatory mites Information on mites, as predators of ticks is very limited, but since mites are known to feed on a large variety of prey and are used commercially to control various arthropod pests this group may contain important candidates for inoculative and inundative biological control (Samish and Rehacek 1999, Venancio et al. 2016). Most predatory mites commercialised around the world for the control of edaphic organisms are gamasines of the family Laelapidae. Four species of this family have been used commercially for the control of soil pests such as fungus gnats (Sciaridae), thrips (Thysanoptera) and mites of the cohort Astigmatina of the order Sarcoptiformes (Gerson et Ecology and prevention of Lyme borreliosis 287

289 Ingeborg Klingen and Gilian van Duijvendijk al. 2003, Moreira and De Moraes 2015). Predatory mites have, to our knowledge, not been used for tick control so far. Only a few laboratory studies have evaluated the possible predation on ticks by mites of the suborder Prostigmata of the order Trombidiformes and of the cohort Astigmata, as summarised by Samish & Rehacek (1999) and Samish and Alekseev (2001). Apparently, nothing has been published about the possible relation between ticks and Gamasina. This does not necessarily mean that these two groups do not interact. Studies about the effect of soil Gamasina on other organisms have referred mainly to arthropods of agricultural importance and, only recently, on important mesostigmatid parasites of laying hens (Lesna et al. 2009, 2012) Parasitoids The majority of current agricultural biological control agents are parasitic Hymenoptera. However, the only species that has been used for tick control is I. hookeri (Ginsberg 2014). Ixodiphagus hookeri is a parasitoid wasp that parasitises I. ricinus (Tijsse-Klasen et al. 2011). Increasing the natural population of these wasps to control the tick population is complicated. In the USA, an increased deer density resulted in an increased density of wasps (Stafford et al. 2003), but did also result in more ticks. It was modelled that releasing 300,000 lab-reared wasps per square kilometre into in the field could result in a lower tick density (Knipling and Steelman 2000). Ginsberg (2014) suggests that well-target release of large numbers of wasps can effectively control ticks in limited areas. Concluding remarks and future perspectives Even though there might be several good candidates for the biological control of I. ricinus in its geographical distribution area, which is mainly Europe, a very limited number of studies has been conducted to investigate these possibilities further. Using results from other tick species with other life cycles will not enable us to develop good biocontrol agents and strategies for I. ricinus, but should serve as an important base of information. Our suggestion would, therefore, be to conduct more studies on naturally occurring enemies of I. ricinus in its geographical distribution area to be able to suggest possible biocontrol candidates. Further, these candidates should be tested in controlled laboratory and field studies with the aim to develop elegant, precise and effective biocontrol strategies for the control of I. ricinus. A focus should be placed on controlling I. ricinus by means of biological control methods in hot spots on and off-host in pasture and recreation areas close to human infrastructure. Further, one should consider focusing on the natural enemy groups that have shown the most promising results against ticks until now. Both conservation, inoculation and inundation biological control should be considered. Combining biological control agents with chemical ecology or other smart combinations as presented in studies above should be emphasised. Further, it is of outmost importance to remember that biological control is only one part of integrated vector management and should be combined with other strategies such as gazing of animals, management of vegetation and acaricide resistance management. 288 Ecology and prevention of Lyme borreliosis

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296 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future Michelle J. Klouwens 1,2,3, Jos J. Trentelman 1,3 and Joppe W.R. Hovius 1,2,3* 1 Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; 2 Department of Internal Medicine, Division of Infectious Diseases, Academic Medical Center (AMC), University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; 3 Amsterdam Multidisciplinary Lyme borreliosis Center (AMLC), Academic Medical Center (AMC), University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, the Netherlands; lyme@amc.uva.nl Abstract Ticks are ectoparasites that transmit various pathogens with great impact on human and animal health throughout the world. In the USA, Europe and Asia, Ixodes ticks are the medically most important vectors for human tick-borne pathogens. After the tick hypostome penetrates the skin, ticks secrete saliva that facilitates feeding and paves the way for pathogen transmission. Tick saliva contains molecules interfering with host defence mechanisms, such as the coagulation system and the immune responses, in order to try to prevent rejection. Despite the tick s countermeasures, repeated tick infestations can lead to natural immunity that partially protects against tick feeding and pathogen transmission, a phenomenon known as tick immunity. Two commercial veterinary anti-tick vaccines have been developed against another tick species, Boophilus microplus, showing the feasibility of mimicking acquired tick-immunity through vaccination. However, the protective veterinary antigen Bm86 does not seem to protect against Ixodes ticks, but other interesting Ixodes vaccine candidates, have been described. These antigens are able to partially protect against Ixodes challenge, and some also against pathogen transmission. The ideal vaccine candidate, one that is expressed in all tick stages, protects against multiple tick species, provides long lasting immunity and blocks pathogen transmission has not been identified yet. Also, the application of such antigens in vaccines for domesticated animals, wildlife, let alone humans, remains a challenge. Therefore, a better understanding of the molecular interactions between Ixodes ticks, hosts and pathogens will help in the identification of new antigens to prevent tick-borne diseases in both humans and animals. In this review we describe the known molecular mechanisms underlying the interaction between ticks and their hosts, the mechanisms by which pathogens exploit these interactions, and selected Ixodes proteins that have been investigated as vaccine candidates to prevent tick feeding and/or transmission of pathogens from the tick to host. Keywords: anti-tick vaccine, Borrelia, Ixodidae, salivary gland proteins, tick-host-pathogen interactions, tick-immunity, tick feeding Introduction: the ideal anti-tick vaccine Many factors influence the efficacy and usefulness of an anti-tick vaccine. An anti-tick vaccine candidate should firstly be antigenic and preferably associated with a vital function of the tick (Nuttall et al. 2006). Ideally, anti-tick vaccines should also provide long-lasting immunity, either through selection of highly immunogenic concealed antigens, or through natural boosters of exposed antigens. In addition the used antigen, or a combination of antigens, should be expressed during different stages of the tick s life cycle, since different tick stages could then be targeted (Almazan et al. 2012). Furthermore, the ability to produce a cross-protective immune response Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _21, Wageningen Academic Publishers 2016

297 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius against more than one tick species, and the ability to block transmission of multiple tick-borne pathogens, would be desirable. In finding the right vaccine candidate, one should also take into account the pluripotency and redundancy of tick salivary proteins; different members of different tick protein families target the same host immune function or the same protein targets different aspects of the host immune response (Chmelar et al. 2016b). Finally, combining tick antigens with pathogen derived antigens, forming multivalent combination vaccines, could aid in vaccine efficacy (Schuijt et al. 2011b). Unfortunately, such an ideal vaccine does not exist to date. Nonetheless, there are veterinary anti-tick vaccines available and many promising candidates for future anti-tick vaccines have been identified in experimental settings. In this chapter we will give an overview of some important interactions between the host and the tick, and the tick vector and the pathogen. In addition, we will review previous and ongoing research on the search for the ideal anti-tick vaccine, with the main focus on Ixodes ticks and the commercially available anti- Rhipicephalus microplus vaccine. This chapter will help to understand the rationale behind antitick vaccines and provides insights into their potential. Depending on its characteristics, a future anti-tick vaccine could be suitable for vaccination of wild life, livestock or companion animals, and eventually also for humans. Tick-host-pathogen interactions Ticks are divided into two major groups: soft ticks and hard ticks (Ixodidae), which differ in their life cycle and blood feeding strategies. Both groups face common host-anti-tick responses, such as haemostasis and acute inflammation. However, hard ticks must also counteract chronic responses and specific humoral and cellular immunity, because of the prolonged time of blood feeding and repetitive feeding on previously tick-exposed animals (Chmelar et al. 2016a). The most important Ixodidae in the USA, Europe and Asia are Ixodes scapularis (USA), Ixodes pacificus (USA), Ixodes ricinus (Europe), and Ixodes persulcatus (Asia and Europe). They transmit a variety of pathogens with medical importance, which include, but are not limited to, Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Anaplasma phagocytophilum, Rickettsia species, Babesia species and tick-borne encephalitis virus (TBEV). Hard ticks require a blood meal to complete development and reproduction, and have three blood feeding stages: larvae, nymphs and adults. Each developmental stage feeds only once and may require several days to complete their blood meal. To acquire a blood meal, ticks insert their specialised mouthparts through the host skin and anchor themselves by producing a cement-like substance (Kazimirova and Stibraniova 2013). These cement proteins not only enable attachment, but also seal the area around the mouthparts at the wound site. During the initial probing and attachment process, capillaries and small blood vessels are injured and form a haemorrhagic pool at the tick-bite site (Kazimirova and Stibraniova 2013). Importantly, during their blood meal, ticks introduce saliva, together with possible pathogens, into the skin of the host. Normally a host would react to damage of the skin and the presence of a tick by activation of the coagulation cascade, vasoconstriction and inflammatory responses aiming to disrupt the tick feeding and start wound healing. However, ticks succeed in blood feeding due to several molecules in tick saliva responsible for anticoagulation, vasodilatory, anti-inflammatory and immunomodulatory effects that are essential for effective attachment and engorgement (Hovius et al. 2008a). The balance between the host immune response and the tick s countermeasures determines the duration of attachment and therefore also the effectiveness of pathogen transmission. 296 Ecology and prevention of Lyme borreliosis

298 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future Haemostasis To overcome the haemostatic response of the host, ticks have developed a broad array of anti-haemostatic agents that counteract coagulation, enhance fibrinolysis and inhibit platelet aggregation in the host (Maritz-Olivier et al. 2007). To antagonise vasoconstrictors released at the tissue injury site of the host, vasodilators are produced by ticks and secreted in the feeding pool. These include lipid derivatives, such as prostacyclin and prostaglandins (Ribeiro et al. 1992), and more recently identified proteins with additional functions such as tick histamine release factor (thrf) in I. scapularis saliva (Dai et al. 2010), as well as serpin (IRS-2), a serine protease inhibitor in I. ricinus saliva (Chmelar et al. 2012). Serine proteases are key players in host haemostasis and are specifically targeted by several serine protease inhibitors present in tick saliva (Kotal et al. 2015). The serine proteinase α-thrombin is one of the important enzymes in haemostasis as it activates several clotting factors and cleaves fibrinogen to form fibrin. In addition, it also activates circulating blood platelets. Interestingly, at least 17 tick thrombin inhibitors have been identified (Maritz- Olivier et al. 2007). Other tick anticoagulants are inhibitors of coagulation factor X and thrombin or both. There are a number of factor X inhibitor families, such as Salp14 and tissue factor pathway inhibitors (e.g. Ixolaris and Penthalaris) that function through inhibition of factor VIIa (Maritz- Olivier et al. 2007, Narasimhan et al. 2002). Our group characterised the anticoagulant potential of the tick salivary protein TIX-5 (tick inhibitor of factor Xa toward factor V) which dose-dependently postpones activation of the coagulation system by specifically preventing activation of factor V through Factor Xa. In addition, ticks are capable of accelerating fibrinolysis and degrading fibrin clots, for instance by the presence of a metalloproteases, such as I. scapularis MP1 in the saliva of I. scapularis (Francischetti et al. 2003). Following vascular damage of the capillaries and small blood vessels by the tick bite, several host-derived agonists such as adenosine diphosphate (ADP), thrombin and collagen that bind to specific platelet membrane receptors, can activate platelets. The tick can target this platelet activation and aggregation at different stages. For example, ticks can inhibit the activation of platelets by producing substances that either remove or compete with agonists and they block binding of fibrinogen to activated platelets, as reviewed by others (Maritz-Olivier et al. 2007). Complement The complement system is part of the innate immune system and consists of a complex network of proteins, which can be activated in a cascade-like manner. The central reaction of all three complement pathways (classic, alternative and lectin) is the conversion of complement component 3 (C3) to C3a and C3b. Several anti-complement molecules in tick saliva have been identified to date (Kotal et al. 2015), such as Isac-1 (Valenzuela et al. 2000) and Salp20 from I. scapularis (Tyson et al. 2007) and IRAC I and II from I. ricinus (Daix et al. 2007). These molecules specifically inhibit formation of C3 convertase by blocking binding of complement factor B to complement C3b. In addition, our group has shown that the tick salivary lectin pathway inhibitor (TSLPI) present in I. scapularis and I. ricinus saliva interfered with the lectin pathway and impaired complement lectin pathway-dependent neutrophil phagocytosis and chemotaxis (Schuijt et al. 2011a, Wagemakers et al. 2016). Innate and adaptive immunity Local modulation of cutaneous immune responses at the tick bite site occurs immediately after tick attachment. Keratinocytes, endothelial cells and various resident leukocytes, such as mast cells, dendritic cells and macrophages come in contact with the tick saliva and the tick Ecology and prevention of Lyme borreliosis 297

299 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius hypostome and are therefore activated (Kotal et al. 2015). In addition, influx of leukocytes, for instance neutrophils, augments the inflammatory response. Recruitment of different leukocyte populations during the inflammation response is triggered by IL-8, tumour necrosis factor (TNF), and IL-1β. However, ticks have evolved strategies to manipulate the host cytokine response. For instance, they evade the host immune response by producing proteins that selectively bind and neutralise the chemokines that normally recruit cells of the innate immune system. Deruaz et al. (2008) have identified chemokine binding proteins, termed Evasins. Also, Hajnicka et al. (2001) reported anti-il-8 activity from hard tick saliva impairing neutrophil function. Other inhibitory activities from tick saliva have been described against pro-inflammatory cytokines such as IL-2, CCL2, CCL3, CCL5 and CCL11 (Hajnicka et al. 2005). Langhansova et al. reported in 2015 that I. ricinus saliva increases the production of three chemokines, CCL1, CCL2 and CXCL2. This upregulation reflects Th2 polarisation of the host immunity due to the effect of tick saliva (Ferreira and Silva 1998, Langhansova et al. 2015, Schoeler et al. 1999). The initial inflammatory responses will shape the adaptive immune responses. Of specific interest in that regard are dendritic cells, including Langerhans cells, which are essential in initiating adaptive immune responses in naive hosts. They recognise antigens by TLRs and C-type lectins and process and present antigens to lymphocytes (Wikel and Alarcon-Chaidez 2001). Tick saliva interferes with multiple dendritic cell functions and signalling pathways, impairing the dendritic cell driven immune response on various levels. Tick saliva directly modulates cytokine production and inhibits phagocytosis by dendritic cells and also impairs effects on the expression of co-stimulatory molecules and polarisation of T cells (Mason et al. 2014). Our group has investigated the interaction of the immunomodulatory Salivary gland protein 15 (Salp15) with human dendritic cells (Hovius et al. 2008c). Salp15 is induced and secreted into the host during I. scapularis tick feeding (Anguita et al. 2002). We showed that Salp15 in Ixodes saliva is responsible for reducing pro-inflammatory cytokine production by dendritic cells. Moreover, Salp15 and tick saliva suppress the T cell stimulatory role of dendritic cells. Salp15 interacts with the C-type lectin DC-SIGN on dendritic cells, which leads to activation of the kinases Raf-1 and mitogen-activated protein kinase (MEK). This activation inhibits TLR-induced proinflammatory cytokine production (Hovius et al. 2008b). Recently, we have described an ex vivo human skin model in which the effect of tick saliva on dendritic cell activation and migration can be further studied (Mason et al. 2016). B lymphocytes play a major role in antimicrobial immunity. After entry of a pathogen, circulating antibodies eliminate most of the pathogens by neutralisation and enhancing opsonisation. Interaction between T and B cells is dependent on the presentation of antigens by the major histocompatibility complex class II molecules of B cells. Hannier et al. (2014) describe the inhibition of host B cells by I. ricinus saliva by preventing IL-10 and tumour-necrosis factor-a (TNF-a) production. This B cell inhibitory activation is likely to play a role in enhancing tick-vectored pathogen transmission. This was further investigated, and subsequently Hannier and colleagues reported the responsible agent for inhibiting B cell proliferation; B cell inhibitory protein (BIP). In nature, ticks usually attach to hosts that have been pre-exposed to ticks. It has been hypothesised that local downregulation of IgE production and consequently the inflammatory response around the feeding lesion may be due to BIP (Hannier et al. 2004). Ticks have also evolved ways to inhibit and alter the production of T lymphocyte cytokines. Tick saliva polarises the host immune response towards a Th2 type profile, which is characterised by the down-regulation of pro-inflammatory Th1 cytokines, such as IL-2, IFN-gamma, and increased production of the Th2 cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 (Gillespie et al. 2000, Wikel and Alarcon-Chaidez 2001). Several T cell inhibitory proteins have been identified in ticks (Kazimirova and Stibraniova 2013). Iris is a protein detected in the saliva of I. ricinus that is produced during 298 Ecology and prevention of Lyme borreliosis

300 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future the feeding process and also suppresses T cell proliferation. In addition, it induces a Th2 immune response and inhibits production of IL-6 and TNF-alpha (Kazimirova and Stibraniova 2013, Leboulle et al. 2002). Finally, Ixodes ticks inhibit activation of naive CD4 positive T cells by the salivary protein Salp15. Anguita et al. (2002) showed that Salp15 inhibits IL-2 production upon T cell receptor engagement by repressing calcium fluxes. Anguita s group showed that the C-terminal part of Salp15 binds to the CD4 co-receptor and prevents the activation of the Src kinase Lck upon TCR engagement, resulting in defective F-actin polymerisation followed by a reduced formation of lipid rafts. These events are essential in the cascade that leads to IL-2 production and T-cell proliferation (Garg et al. 2006). This inhibitory effect may affect the differentiation of naive CD4 positive T cells into immune effector cells. Indeed, Paveglio et al. (2007) demonstrated that recombinant Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. Exploitation of tick salivary glands for pathogen transmission The common route of pathogen migration within a vector is ingestion via blood, migration from the gut lumen to the haemocoel and finally the penetration of the salivary glands, from which they can be transmitted to the host. Salivary gland proteins and saliva are therefore thought to play a major role in pathogen transmission to the vertebrate host (Kazimirova and Stibraniova 2013). By modulating host responses at the site of their attachment by their salivary molecules, ticks create an environment, which is beneficial for their own feeding process as well as for transmission of the microorganisms that they carry. For example in Lyme borreliosis, spirochaetes are transmitted to the host with tick saliva containing various molecules that modulate T cells (Iris, Salp15), complement (ISAC, Salp20, TSLPI) and B cell activities (BIP), only to mention a few. In an unfed tick, spirochaetes express outer surface protein A (and B), which bind to the tick receptor for OspA (TROSPA), located in the tick gut (Neelakanta et al. 2007, Pal et al. 2004a). During engorgement of the tick, OspA is downregulated by the spirochaete and outer surface protein C (OspC) is upregulated (Pal et al. 2004b). OspC is a lipoprotein that facilitates the migration of B. burgdorferi from the Ixodes tick s gut to their tick salivary glands and is crucially important for early mammalian infection (Grimm et al. 2004, Pal et al. 2004b). In the salivary glands OspC binds to Salp15, which protects the spirochaetes from antibody-mediated killing and facilitates their transmission into the host (Ramamoorthi et al. 2005). Thus, the pleiotropic salivary protein Salp15 aids Borrelia to infect the mammalian host. Interestingly, Salp15 is more abundantly expressed in Borrelia-infected ticks, suggesting a co-evolutionary process. Anti-tick vaccine research Naturally acquired resistance to tick infestation: tick immunity As the tick introduces different saliva proteins associated with tick attachment, feeding and immunomodulation into the host, these also serve as antigens for the host to develop a successful protective immunological response; so-called naturally acquired tick resistance. Naturally acquired tick resistance, also referred to as tick immunity, occurs after repeated tick infestations and can lead to the reduction of tick feeding success. It affects tick feeding in multiple ways and causes reduced number of ticks attached, number of ticks fed to repletion, post-engorgement tick weights, and molting or oviposition rates. Already during the first attempts to establish laboratory tick colonies, acquired resistance to tick-bites was observed in laboratory animals that were used to feed the ticks on. The first report of this phenomenon was published in 1939 by William Trager (Trager 1939), describing the effect of repeated tick infestations on guinea pigs. Tick immunity Ecology and prevention of Lyme borreliosis 299

301 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius has since been described for laboratory animals as mice, rabbits and guinea pigs and in cattle (Bowessidjaou et al. 1977, Mbow et al. 1994, Ribeiro 1989, Wada et al. 2010), and is the rationale behind anti-tick vaccines. Interestingly, acquired resistance to tick-bite in mice is rare and much less profound than in other laboratory animals. Tick immunity is established by complex interactions of all the different mediators of the immune system as reviewed above and antigen-presenting cells, T-lymphocytes, eosinophils, mast cells, basophils, cytokines, complement, antibodies and cytokines play a central role (Brossard and Wikel 1997, 2004, Wikel 1996). Tick infestation leads to IgG production against salivary gland proteins and is boosted upon re-infestation (Bowessidjaou et al. 1977, Lawrie and Nuttall 2001). Although IgG titres do not necessarily correlate with the degree of observed tick immunity, transfer of serum from tick immune to naive animals conferred protection to subsequent tick infestations, confirming the protective role of humoral factors (Brossard and Girardin 1979, Roberts and Kerr 1976). Complement also plays a role; C3 is deposited near the tick-bite site and depletion of complement reduced tick immunity (Wikel and Allen 1978). Next to the humoral components, transfer of lymphocytes isolated from the lymph node of tick immune animals resulted in protection against tick infestation in naive guinea pigs (Wikel and Allen 1976). T-cells appear to be involved in the increased cutaneous response associated with tick immunity (Girardin and Brossard 1989). The cutaneous response at the tick bite site is characterised by an influx of basophils and eosinophils. Both influx and degranulation of these cells were elevated at the tick bite site in repeated tick infestations (Brossard and Fivaz 1982, Brossard and Wikel 2004, Monteiro and Bechara 2008). Basophils may play an important role in vertebrate hosts as depletion of basophils reversed tick immunity in mice against another tick species, Haemaphysalis longicornis. As a result, basophils are thought to play a role in the Th2 polarisation by tick feeding (Wada et al. 2010, Wikel 2013). Tick immunity also has implications for tick-borne pathogen transmission through activation of the immune system at the tick bite site. Indeed, activation of the immune system by antigens in tick saliva is likely to create an unfavourable environment for transmitted pathogens. Furthermore, since most of these pathogens require active tick feeding for some time before transmission occurs, Borrelia transmission starts approximately 24 hours after tick attachment (Crippa et al. 2002, Gern et al. 1996), tick rejection might take place before transmission can occur. Indeed, it has been demonstrated that tick immune animals are partially protected against Borrelia infection (Nazario et al. 1998, Wikel et al. 1997). In addition, immunity against tick saliva proteins expressed 24 hours after tick feeding was able to protect animals against Borrelia infection, also passive transfer of 24 hour tick immune serum reduced Borrelia loads in mice challenged with B. burgdorferi-infected I. scapularis nymphs compared to controls (Narasimhan et al. 2007). Interestingly, a study among 1498 residents of Block Island, Rhode Island (a Lyme-disease endemic area) showed that prior exposure to tick bites reduces the change of developing Lyme disease, and repeated tick infestation in humans is associated with increased inflammatory cell influx at the tick bite site (Burke et al. 2005, Krause et al. 2009). The history of anti-tick vaccine research and the discovery of Bm86 Ever since the phenomenon of tick immunity has been described, efforts have been made to identify the antigens involved. It was shown that vaccination with tick homogenates were able to induce protection against tick challenge. Vaccination of guinea pigs and cattle with midguts and reproductive organs of Dermacentor andersoni fed for 5 days also resulted in a decreased engorgement weight, reduced egg mass and loss of offspring. This effect was higher when all internal organs of the tick were used (Allen and Humphreys 1979). Also a single vaccination with salivary gland extracts of partially engorged Amblyomma americanum female adults resulted in 300 Ecology and prevention of Lyme borreliosis

302 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future tick immunity; reduction was observed in tick numbers and in tick weights (Brown et al. 1984). However for the development of a safe and usable anti-tick vaccine well-defined antigens are required. In 1989 a tick antigen was discovered: Bm86, a 89 kda gut protein from the cattle tick Rhipicephalus (Boophilus) microplus. It was discovered by a series of fractionation using the extract of 988 gram of ticks, representing almost 50,000 ticks (Willadsen et al. 1989). Bm86 is expressed in every life stage from eggs to engorged adult tick (Willadsen 2004). Vaccination with the isolated antigen and subsequent R. microplus challenge in an experimental set-up showed 65% reduction in tick numbers, 33% reduction of tick weight and 65% reduction of egg mass resulting in a combined overall efficacy of 92% (Willadsen et al. 1989). The function of this protein is still unknown, but it is situated with a glycosylphosphatidyl inositol anchor on the microvilli of the midgut. Bm86 is not naturally exposed to the immune system (Kemp et al. 1989, Richardson et al. 1993) and thus represents an example of a concealed antigen. It has been shown that the effect of Bm86 vaccination is IgG and complement mediated and that protection is correlated to IgG titres (De la Fuente et al. 1998, Kemp et al. 1989). The development of recombinant protein techniques made it possible to produce single antigens, and on an industrial scale, paving the way for the development of Bm86 as a commercial anti-tick vaccine. Existing recombinant anti-tick vaccines Thus far the only tick antigen to be commercialised as an anti-tick vaccine is Bm86. Two veterinary vaccines have been developed based on the Bm86 antigen produced in yeast: Gavac (Hebertech, Havana, Cuba) and TickGard (Merck Animal Health, Madison, NJ, USA) (De la Fuente et al. 2007b, Jonsson et al. 2000). Currently, only Gavac is available. It has been shown that these vaccines reduce tick numbers up to 74% and reduce tick fertility, combining the overall efficacy of up to 91%. As a result, local tick pressure on farms can be greatly reduced (De la Fuente et al. 1999, 2007b, Jonsson et al. 2000). Vaccine efficacy varies depending on the strain of R. microplus that is abundant in a certain region. In contrast to Australian and Cuban R. microplus tick strains, it appears that some Mexican, Brazilian and Columbian R. microplus tick strains show lower overall efficacy, and the Argentinian R. microplus strain A even appears to be resistant to Bm86 vaccination (De la Fuente et al. 2000, García-García et al. 2000). Despite the success of the Bm86 vaccine, Bm86 based vaccines are not able to replace acaricides due to a lack of a knock-out effect as observed with acaricides. However, experience in the field has shown that vaccination with Bm86 greatly reduced acaricide treatments. This is important due to the rise of acaricide resistance and their detrimental effects on the environment. Indeed, the tightly regulated tick control programme of the Cuban government resulted in a reduction of acaricide treatment of 87% (De la Fuente et al. 2007b, Valle et al. 2004), which is comparable to a recent study in Venezuela (Suarez et al. 2016). Vaccination also resulted in reduction of tickborne diseases, such as Bovine babesiosis and Bovine anaplasmosis (De la Fuente et al. 1998, 2007a). Importantly, vaccination with Bm86 also resulted in increased cattle productivity (Jonsson et al. 2000), which has important economic consequences. These different effects combined make Bm86 vaccination a highly cost-effective tool against tick infestation and the resulting disease burden. Vaccination with TickGard is able to induce cross-reactive antibodies with Bm86 homologues in other tick species. The observed cross-reactivity resulted in varying efficacy across different tick species tick species; Rhipicephalus (Boophilus) annulatus, Rhipicephalus (Boophilus) decoloratus, Ecology and prevention of Lyme borreliosis 301

303 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Rhipicephalus sanguineus, Hyalomma anatolicum anatolicum and Hyalomma dromedarii (De Vos et al. 2001, Perez-Perez et al. 2010). For other tick species, Amblyomma cajennense, Amblyomma variegatum and Rhipicephalus appendiculatus no cross-reactive protection was observed (De Vos et al. 2001, Odongo et al. 2007). Strangely, the efficacy of Bm86 vaccination has a 100% efficacy on R. annulatus, which is higher than the reported efficacy in a homologous challenge with R. microplus. This might be explained by physiological characteristics as lower blood engorgement and less protease activity (Popara et al. 2013). Unfortunately, it is difficult to extrapolate the experiences with the Bm86 vaccines against R. microplus ticks to a vaccine targeting Ixodes ticks. R. microplus is, in contrast to I. ricinus and I. scapularis, a one host tick; it only feeds on cattle, has a short life cycle and does not drop off to molt and find a new host after a blood meal. Efficacy studies on Bm86- vaccinated cows challenge with R. microplus larval ticks and measure parameters associated with tick immunity on the engorged adult females that drop off. As a result, the measured protection is the sum of the effect on 3 tick stages and two molting periods. For R. microplus, it has been shown that Bm86 vaccination results in the damage and subsequent reduced engorgement weight in adult female ticks (Vargas et al. 2010), but unfortunately the relative effect of Bm86 vaccination on the immature life stages of R. microplus is not well known. Bm86 homologues were identified in Ixodes ticks. Bm86 has two homologues in I. ricinus; Ir86-1 and Ir-86-2, which are also present in I. scapularis; Is86-1 and Is86-2 (Nijhof et al. 2010). However, antibodies against Ir86-1 and Ir86-2 were not able to protect rabbits against I. ricinus challenge; neither the number of attached ticks nor tick weights were reduced (Coumou et al. 2015). As a result, Ixodes homologues of the successful Bm86 antigen are apparently not good candidates for an anti-tick vaccine targeting Ixodes ticks. Experimental approaches/potential vaccine candidates There is a growing list of tick proteins that have been identified and evaluated as potential vaccine candidates (Table 1). In literature two groups of possible candidate vaccine antigens are described. The first group consists of the exposed antigens, which are secreted in tick saliva during attachment and feeding on a host. These antigens elicit an immune response at the tickfeeding site. Exposed antigens are likely to be less immunogenic as a result of prolonged exposure to the host immune system (Bishop et al. 2002). Moreover, the redundancy in some tick salivary immunomodulators might also reduce their immunogenicity by lowering the amount of each individual antigen in tick salivary secretions (Chmelar et al. 2016b). Salivary gland proteins There has been extensive research on the question how tick salivary gland gene expression is contributing to host homeostasis and pathogen transmission. The diversity of tick salivary compounds has been established in several transcriptomic studies and more recently by next generation sequencing studies (Chmelar et al. 2008, Cramaro et al. 2015, Kotsyfakis et al. 2015, Lewis et al. 2015, Schwarz et al. 2014). Ribeiro and colleagues showed in 2006 that there were 20 genes at least two fold more abundantly expressed than expected in the salivary glands of adult I. scapularis females after attachment. The most represented protein families were Kunitz domain containing proteins, Salp15, lipocalins, metalloproteases and several proteins of unknown function (Chmelar et al. 2016b), which has been confirmed in a recently published study (Gulia- Nuss et al. 2016). Lewis et al. characterised immunogenic I. scapularis salivary proteins present after 24 hours of feeding, which appeared to be involved in the feeding process before most pathogens could be transmitted (Lewis et al. 2015). 302 Ecology and prevention of Lyme borreliosis

304 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future Table 1. Tick vaccine candidates, discovery method, function and protection. Vaccine candidate Function Discovery method Tick stage during discovery Salp15 inhibits activation of murine CD4 positive T cells and human dendritic cells and binds to Borrelia burgdorferi OspC protecting the spirochaete from antibodymediated and complement dependent killing salivary gland cdna expression library Salp25D anti-oxidant protein salivary gland cdna expression library 64P/TRP tick cement cone immunoblot analysis engorged Ixodes scapularis nymphs engorged I. scapularis nymphs adult Rhipicephalus appendiculatus ticks FER2 binding and transport of iron isolated and cloned from cdna libraries thrf tick histamine release factor (thrf) in I. scapularis saliva Salp20 I. scapularis complement inhibitor Isac-1 I. scapularis complement inhibitor 2-dimensional fluorescence difference gel electrophoresis (DIGE) salivary gland cdna expression library random screening of a salivary gland cdna library by sequencing unfed adult I. ricinus ticks and larvae of Ornithodoros moubata B. burgdorferi-infected, and uninfected, I. scapularis salivary glands extracts engorged I. scapularis nymphs partially engorged female I. scapularis ticks feeding for 3-4 days on a rabbit Antibody/ screening used during discovery Phenotype 1 References tick-immune rabbit serum impaired B. burgdorferi transmission Das et al. (2001), Anguita et al. (2002), Ramamoorthi et al. (2005), Dai et al. (2009) tick-immune rabbit serum tick-immune guinea pig serum cdnas encoding ferritin impaired B. burgdorferi transmission impaired Ixodes ricinus tick feeding and increased tick mortality, as well as decreased TBEV transmission Das et al. (2001), Narasimhan et al. (2007) Shapiro et al. (1989), Trimnell et al. (2002), Havlikova et al. (2009) impaired tick feeding Kopacek et al. (2003), Hajdusek et al. (2010) none decreased tick feeding, impaired B. burgdorferi transmission Dai et al. (2010) tick-immune rabbit serum inhibits murine alternative pathway activity none impaired B. burgdorferi transmission Das et al. (2001) Valenzuela et al. (2000) Ecology and prevention of Lyme borreliosis 303

305 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Table 1. Continued. Vaccine candidate Function Discovery method Tick stage during discovery IRAC-I I. ricinus complement inhibitor isolation from transcriptome of I. ricinus salivary glands IRAC-II I. ricinus complement inhibitor isolation from TSLPI tick salivary lectin pathway inhibitor, impairs neutrophil phagocytosis and chemotaxis Iris I. ricinus suppress T cell proliferation, induces a Th2 immune response, inhibits production of IL-6 and TNFalpha transcriptome of I. ricinus salivary glands immunoscreening of I. scapularis salivary gland yeast surface display library analysis subtractive library from salivary glands Tix-5 anticoagulant immunoscreening I. scapularis salivary gland yeast surface display library Subolesin signal transduction cdna expression library immunisation and analysis of expressed sequenced tags I. ricinus salivary gland of engorged adult female ticks I. ricinus salivary gland of engorged adult female ticks I. scapularis salivary gland unfed and 5 day fed female I. ricinus ticks adult I. scapularis salivary glands I. scapularis cell line (IDE8) derived from tick embryos Antibody/ screening used during discovery Phenotype 1 References none transmembrane IRAC leads to antibody response, marginally impaired tick feeding Daix et al. (2007) none see IRAC-I Daix et al. (2007) tick immune rabbit sera protects both B. garinii and B. burgdorferi s.s. from killing by the human complement system Schuijt et al. (2011) none impaired tick feeding Leboulle et al. (2002), Prevot et al. (2006) tick immune rabbit sera impaired tick feeding Schuijt et al. (2013) tick-immunised mouse sera impaired tick feeding, impaired B. burgdorferi, Anaplasma marginale and Anaplasma phagocytophilum transmission, no effect on TBEV transmission Almazan et al. (2003), De la Fuente et al. (2006), Canales et al. (2009), Havlikova et al. (2013) 304 Ecology and prevention of Lyme borreliosis

306 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future Table 1. Continued. Vaccine candidate Function Discovery method Tick stage during discovery Antibody/ screening used during discovery Phenotype 1 References CDK10 cyclin-dependent kinases participate in cell cycle control Ir86-1 and Ir86-2 Bm86 homologue, gut protein I scapularis TROSPA gut-protein, binding partner of the Borrelia protein OspA ISDLP gut-protein, binds to Borrelia and involved in Borrelia migration Ixofin3D gut-protein, binds to Borrelia and involved in Borrelia migration bioinformatic search for CDK homologues bioinformatic search for Bm86 homologues screening of a I. scapularis cdna library probing of I. scapularis midgut yeast surface display library probing of I. scapularis midgut yeast surface display library Salp14 factor Xa inhibitor I. scapularis salivary gland cdna expression library Sialostatin L2 specifically inhibits cathepsin L activity in cytotoxic T lymphocytes I. scapularis salivary gland cdna expression library and sialome R. microplus none impaired Ixodes feeding Gomes et al. (2015) I. ricinus none no effect on weight/ attachment I. scapularis Borrelia protein OspA impaired B. burgdorferi acquisition, no significant effect on tick feeding in R. microplus ticks I. scapularis midgut Borrelia proteins impaired B. burgdorferi transmission upon silencing, no effect on tick feeding I. scapularis midgut Borrelia proteins impaired B. burgdorferi I. scapularis salivary gland I. scapularis salivary glands from fed nymphs and (un)fed adults tick immune rabbit sera transmission upon active vaccination, no effect on tick feeding Nijhof et al. (2010), Coumou et al. (2014) Pal et al. (2004), Merino et al. (2013) Coumou et al. (2016) Narasimhan et al. (2014) impaired tick feeding Das et al. (2001), Narasimhan et al. (2004) none increased rejection and lower post-engorgement weights of I. scapularis Kotsyfakis et al. (2008), Valenzuela et al. (2002) 1 This column depicts whether vaccination or silencing experiments led to decreased tick feeding and/or pathogen transmission. Ecology and prevention of Lyme borreliosis 305

307 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Many of the abovementioned salivary proteins see also the section on tick-host-pathogen interactions have been studied as potential vaccine candidates. For example vaccination studies with Iris, a salivary gland protein detected in I. ricinus female nymphs and adults report impaired tick feeding on vaccinated rabbits (Prevot et al. 2006). Also Salp15 has been studied as a vaccine candidate. When mice were immunised with Salp15, they were largely protected against tickborne Borrelia, especially when this was combined with low-dose of Borrelia OspC antibodies (Dai et al. 2009), making Salp15 a potent and interesting vaccine candidate. Salp15 is part of a larger family of proteins, which is present in other Ixodes species as well (Hojgaard et al. 2009, Hovius et al. 2007b, 2008c, Schwalie and Schultz 2009). Interestingly, Salp15 Iric-1, the Salp15 homologue in I. ricinus, the European vector for B. burgdorferi s.l., was shown to protect B. burgdorferi sensu stricto, B. garinii and B. afzelii from antibody killing in vitro (Hovius et al. 2008c). Sialostatin L2 is another secreted tick salivary immunomodulatory protein, which does not raise an antibody response upon repeated tick-exposure, but can lead to decreased feeding ability of I. scapularis nymphs when used as an antigen in active immunisation (Kotsyfakis et al. 2008). Dai et al. also described the salivary protein thrf, which is a conserved multiple-function protein that provokes the release of histamine by both IgE dependent and IgE-independent mechanisms from mammalian basophils and mast cells. Histamine increases vascular permeability and is also a mediator of the itch response and promotes the recruitment of pro-inflammatory cells to the tick bite site. Ixodes ticks encode several histamine binding proteins to counteract the effect of histamine early in feeding. Tick HRF is upregulated during rapid engorgement feeding and by modulation of vascular permeability and increasing blood flow to the tick bite site it facilitates rapid engorgement, but it might also facilitate B. burgdorferi transmission (Dai et al. 2010). Immunisation with the recombinant protein in mice drastically decreased tick feeding as well as Borrelia transmission. As such, blocking thrf might offer a strategy to help developing vaccines that block tick feeding and transmission of tick-borne pathogens (Dai et al. 2010). As described, TSLPI, a Tick Salivary Lectin Path Inhibitor, has been identified in I. scapularis. TSLPI appears to be part of a larger protein family conserved among different Ixodes species. It is a salivary protein facilitating B. burgdorferi s.s. transmission and acquisition by inhibiting the host lectin complement pathway through interference with mannose binding lectin activity. Our group identified a TSLPI orthologue in I. ricinus and has shown that TSLPI is upregulated during I. ricinus tick feeding and that it also inhibits the lectin pathway and protects both B. garinii and B. burgdorferi s.s. from killing by the human complement system. There is homology between TSLPI and several other Ixodes saliva proteins, which suggests that antibodies against TSLPI might also be directed against several of its paralogs. This mechanism might add to the effectiveness of TSLPI as the basis for an anti-tick vaccine candidate (Wagemakers et al. 2016). Tick anti-haemostatics A limitation in this group as potential vaccine targets is that little effort has been made to ensure that tick anti-haemostatics differ from their host counterparts to limit any possible side effects. Maritz-Olivier and colleagues describe five orthologous families that have been identified as possible vaccine candidates within the tick anti-haemostatic antigens: BPTI-kunitz thrombin inhibitors, ixolaris, pethalaris, Salp14 and BmTIs/RsTIs (Maritz-Olivier et al. 2007). Vaccination with Salp14 and BmTIs resulted in 50-70% and 72.8% efficacy to interference with tick feeding and development, respectively (Maritz-Olivier et al. 2007, Narasimhan et al. 2004). TIX-5, the anticoagulant salivary protein described above has been investigated in vaccination studies as well. Rabbits immunised with recombinant TIX-5 produced an antibody response that recognised native TIX-5 in salivary gland extract of I scapularis adults. It was also demonstrated that adult 306 Ecology and prevention of Lyme borreliosis

308 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future tick engorgement weights after feeding on rtix-5-immune rabbits were dramatically reduced compared to control rabbits. TIX-5 or its homologs could be used, possibly in combination with other tick proteins, in efforts to develop vaccines that render tick immunity or tick resistance (Schuijt et al. 2011b). Structural components Because Ixodes tick species use cement to attach to their host during feeding, these antigens may be potential anti-tick vaccine candidates against different tick stages and species. For example, tick cement cone protein 64P is a structural protein described by Trimnell et al. (2002). This protein has homology to mammalian skin proteins, possibly to escape host rejection during tick infestation, but again not a desirable capacity for a vaccine candidate (Nuttall et al. 2006). Truncated versions of the protein (64TRPs) showed significant adult and nymphal mortality in guinea pigs. A potent humoral immune response, boosted by tick challenge, has been demonstrated experimentally using 64TRPs (Trimnell et al. 2005). Concealed antigens The second group are concealed antigens that do normally not come into contact with the hosts immune system (Nuttall et al. 2006, Willadsen 2004). Although concealed antigens do not induce an immune response upon tick infestation, they are immunogenic when prepared as extracts or recombinant proteins and inoculated artificially into a host (Nuttall et al. 2006). These antigens rely on vaccine-induced antibodies to be effective and repeated vaccination may be necessary to produce sufficient levels of antibodies. As can be seen below, concealed antigens are a heterogeneous group of proteins. They are either excreted or intracellular, and are not limited to a particular tick compartment. Ferritins Ticks possess two forms of iron-storage proteins (ferritins), a secreted form (FER2) and an intracellular form (FER1). FER2 functions as an iron transporter in the tick haemolymph and is expressed during all tick stages. Hajdusek et al. (2010) described silencing FER2, which had adverse effects on blood acquisition and reproduction of the tick. Vaccine studies in rabbits with recombinant proteins of FER2 from I. ricinus (IrFER2) showed an overall vaccine efficacy of 98% (Hajdusek et al. 2010). Since all ticks must contain large amounts of iron during blood feeding and vaccination against ferritin reduced tick feeding in R. microplus, R. annulatus and H. longicornis (Galay et al. 2014, Hajdusek et al. 2010), one could hypothesise that ferritins are perhaps conserved across tick species (Parizi et al. 2012), making them suitable targets for a cross-reactive vaccine. Gut proteins For I. scapularis several different gut proteins have been described that are involved in tick immunity and/or pathogen transmission. One Ixodes gut protein involved in pathogen colonisation is TROSPA. This protein is located at intercellular spaces and luminal surface of the gut (Pal et al. 2004a). It is expressed through all life stages, but expression decreases with each following life stage. Feeding reduces TROSPA expression, whereas TROSPA expression is increased upon B. burgdorferi infection. Interestingly, anti-trospa antibodies reduce B. burgdorferi acquisition by I. scapularis, but do not appear to damage the ticks gut wall. As TROSPA vaccination also did not Ecology and prevention of Lyme borreliosis 307

309 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius significantly affected tick feeding in R. microplus ticks (Merino et al. 2013), it appears only to affect pathogen-tick interactions essential for pathogen transmission. By using a yeast surface display to identify I. scapulars gut proteins binding to Borrelia antigens, our group discovered two new Ixodes gut proteins: ISDLP and ixofin3d (Coumou et al. 2016, Narasimhan et al. 2014). ISDLP is a dystroglycan-like membrane bound gut protein upregulated upon both feeding and infection. Silencing by RNA interference (RNAi) of ISLDP reduced Borrelia in the salivary glands of the tick and reduced Borrelia loads in the skin 7 days after tick challenge. However, this phenotype was not recapitulated by active vaccination. In both RNAI and vaccination experiments no effect was found on tick parameters (Coumou et al. 2016). The other identified gut protein, ixofin3d, is expressed at the gut surface and upregulated upon Borrelia infection and, to a greater extend, upon feeding of the infected tick. Although active vaccination with ixofin3d did not have an effect on tick parameters, Borrelia loads were significantly reduced in the skin 1 week after tick feeding (Narasimhan et al. 2014). Interestingly, interference with the three above-described Ixodes gut proteins or the Ixodes Bm86 homologues by vaccination or RNAi did not influence tick feeding as compared to Bm86. It is not clear whether this difference is caused by the characteristics of the targeted proteins, or if it is a result of special differentiation in gut morphology/physiology between the two different tick species. Nonetheless, targeting Ixodes gut proteins did reduce Borrelia transmission, and reveals their potential as candidates for anti-tick vaccines. Regulatory proteins Subolesin is a tick regulatory protein involved in signal transduction in several cellular pathways. It could serve as a promising vaccine candidate, as it provides cross-vector protection; against mosquitos, sandflies and ticks (Canales et al. 2009). Subolesin was protective against all tick developmental stages when used in cdna and/or recombinant protein immunisation experiments (De la Fuente et al. 2006). In addition, vaccination against Subolesin has been shown to reduce Borrelia transmission in mice, but was not protective against TBEV (Bensaci et al. 2012, Havlikova et al. 2013). Cyclin-dependent kinases Cyclin-dependent kinases (CDKs) are proteins that participate in cell cycle control in eukaryotes. Gomes et al. (2015) determined the potential to use CDKs as anti-tick vaccine targets by immunising hamsters with recombinant CDK10 followed by tick challenge. They demonstrate significantly reduced tick feeding and reproduction, suggesting cell cycle proteins as possible new targets for anti-tick vaccine. Targeting both ticks and tick-borne pathogens Ixodes species, distributed worldwide, vector several human pathogens such as Borrelia, Anaplasma, Coxiella, Francisella, Rickettsia and Babesia species. The most prevalent tick-borne disease in humans in Europe and the USA is Lyme borreliosis, which is caused by B. burgdorferi s.l. There is currently no human vaccine protecting against Lyme borreliosis on the market. However, there are several B. burgdorferi outer surface proteins that induce protective responses. From these outer surface proteins, OspA has been most intensively studied. It has shown to result in different degrees of immunity when used in animal vaccination models of Lyme disease. It is the only 308 Ecology and prevention of Lyme borreliosis

310 21. Anti-tick vaccines to prevent tick-borne diseases: an overview and a glance at the future Borrelia antigen that resulted in a FDA approved vaccine, which was available from 1998 until 2002 (Steere et al. 1998). One of the reasons leading to the vaccine s withdrawal appear to be potential vaccine safety concerns coming from the hypothesis that suggested that the vaccinogen, outer surface protein A, might serve as an autoantigen and hence was arthritogenic (Abbott 2006). Of special interest, the mechanism by which for instance I. scapularis TROSPA and Salp15 interact with B. burgdorferi could serve as a model to determine tick-pathogen combined vaccination strategies (Hovius et al. 2007a, Schuijt et al. 2011b). Indeed, the presence of anti-salp15 antibodies also enhanced the protective capacity of both OspA and OspC antibodies (Dai et al. 2009), which corroborates the hypothesis that a tick antigen can be used to complement the protective effect of a Borrelia antigen vaccine (Schuijt et al. 2011b). Although a number of antigens show significant effects, few are highly efficacious on their own (Willadsen 2004). This also implies that a multiantigen vaccine has potential greater efficacy. Likewise, a mixture of multiple Borrelia antigens could be used to enhance the protective effect of a vaccine. Concluding remarks Ixodes ticks transmit numerous pathogens, including bacteria, protozoa and viruses. Anti-tick vaccines could potentially prevent transmission of these pathogens from the tick to the host. Commercial anti-tick vaccines based on Bm86, and directed against the cattle tick R. microplus, have shown that vaccination against ticks can be applied successfully in the field. However, caution must be taken when extrapolating the success story of this anti-tick vaccine to other tick species, such as Ixodes ticks. Firstly, it has been shown by Coumou et al. (2015) that vaccination with the I. ricinus homologue of Bm86 does not affect tick feeding. Secondly, as R. microplus is a one-host tick larvae are used to challenge cows and the main read-out is the fully engorged adult female tick the reduction in tick numbers measured and subsequently reported is a cumulative effect on all three life stages of the tick. Ixodes ticks on the other hand change hosts during their life cycle and as such, a vaccine targeting Ixodes ticks should efficiently block tick attachment and/or feeding during one blood meal on one host. Nonetheless, vaccination against Ixodes ticks seems possible and realistic. The molecules discussed here are only a selection of the tick proteins that have been characterised and appear to have potential as vaccine candidates. In addition, some of the proteins might even have therapeutic potential as immunosuppressive or anticoagulant agents (Hovius et al. 2008a). However, the identification and characterisation of novel Ixodes vaccine candidates to prevent tick feeding and pathogen transmission, as well as the application of these antigens in vaccines for domesticated animals, wildlife, let alone humans, remain a challenge. A European initiative, ANTIDotE, is currently investigating exactly this in more detail. It has become clear from our review that the host has many defense mechanisms that try to thwart tick feeding and pathogen transmission by the tick. Yet, the tick, by introducing functionally active tick salivary proteins into the host skin tries to evade these detrimental responses. Nonetheless, some animal species can develop immune responses against these tick protein, which impairs tick feeding and pathogen transmission. To date, several tick-proteins have been identified that have an important role in the attachment and feeding of the tick and/or transmission of pathogens. A future approach focusing on combinations of both vector and pathogen proteins may lead to new prevention strategies. Ecology and prevention of Lyme borreliosis 309

311 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Public health relevance Reduction of Lyme borreliosis incidence and associated disease burden may be possible by: reduction of the prevalence of ticks and Borrelia through vaccination of animals and/or wild life hosts; prevention of tick feeding and pathogen transmission through vaccination of humans. Acknowledgements This chapter has been written in the context of the ANTIDotE project. The ANTIDotE project has received funding from the European Union s Seventh Programme for research, technological development and demonstration under grant agreement No References Abbott A (2006) Lyme disease: uphill struggle. Nature 439: Allen JR and Humphreys SJ (1979) Immunisation of guinea pigs and cattle against ticks. Nature 280: Almazan C, Moreno-Cantu O, Moreno-Cid JA, Galindo RC, Canales M, Villar M and De la Fuente J (2012) Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens. Vaccine 30: Anguita J, Ramamoorthi N, Hovius JW, Das S, Thomas V, Persinski R, Conze D, Askenase PW, Rincon M, Kantor FS and Fikrig E (2002) Salp15, an Ixodes scapularis salivary protein, inhibits CD 4+ T cell activation. Immunity 16: Bensaci M, Bhattacharya D, Clark R and Hu LT (2012) Oral vaccination with vaccinia virus expressing the tick antigen subolesin inhibits tick feeding and transmission of Borrelia burgdorferi. Vaccine 30: Bishop R, Lambson B, Wells C, Pandit P, Osaso J, Nkonge C, Morzaria S, Musoke A and Nene V (2002) A cement protein of the tick Rhipicephalus appendiculatus, located in the secretory e cell granules of the type III Salivary gland acini, induces strong antibody responses in cattle. Int J Parasitol 32: Bowessidjaou J, Brossard M and Aeschlimann A (1977) Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus l. Experientia 33: Brossard M and Fivaz V (1982) Ixodes ricinus l.: mast cells, basophils and eosinophils in the sequence of cellular events in the skin of infested or re-infested rabbits. Parasitology 85: Brossard M and Girardin P (1979) Passive transfer of resistance in rabbits infested with adult Ixodes ricinus l: humoral factors influence feeding and egg laying. Experientia 35: Brossard M and Wikel SK (1997) Immunology of interactions between ticks and hosts. Med Vet Entomol 11: Brossard M and Wikel SK (2004) Tick immunobiology. Parasitology 129: S161-S176. Brown SJ, Shapiro SZ and Askenase PW (1984) Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with amblyomma americanum-derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies. J Immunol 133: Burke GS, Wikel SK, Spielman A, Telford SR, McKay K, Krause PJ and Tick-borne Infection Study Group (2005) Hypersensitivity to ticks and Lyme disease risk. Emerg Infect Dis 11: Canales M, Naranjo V, Almazan C, Molina R, Tsuruta SA, Szabo MP, Manzano-Roman R, Perez de la Lastra JM, Kocan KM, Jimenez MI, Lucientes J, Villar M and De la Fuente J (2009) Conservation and immunogenicity of the mosquito ortholog of the tick-protective antigen, subolesin. Parasitol Res 105: Ecology and prevention of Lyme borreliosis

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315 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Monteiro GE and Bechara GH (2008) Cutaneous basophilia in the resistance of goats to Amblyomma cajennense nymphs after repeated infestations. Ann NY Acad Sci 1149: Narasimhan S, Coumou J, Schuijt TJ, Boder E, Hovius JW and Fikrig E (2014) A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission. PLoS Pathog 10: e Narasimhan S, DePonte K, Marcantonio N, Liang X, Royce TE, Nelson KF, Booth CJ, Koski B, Anderson JF, Kantor F and Fikrig E (2007) Immunity against Ixodes scapularis salivary proteins expressed within 24 hours of attachment thwarts tick feeding and impairs Borrelia transmission. PLoS ONE 2: e451. Narasimhan S, Koski RA, Beaulieu B, Anderson JF, Ramamoorthi N, Kantor F, Cappello M and Fikrig E (2002) A novel family of anticoagulants from the saliva of Ixodes scapularis. Insect Mol Biol 11: Narasimhan S, Montgomery RR, DePonte K, Tschudi C, Marcantonio N, Anderson JF, Sauer JR, Cappello M, Kantor FS and Fikrig E (2004) Disruption of Ixodes scapularis anticoagulation by using RNA interference. Proc Natl Ac Sci USA 101: Nazario S, Das S, de Silva AM, Deponte K, Marcantonio N, Anderson JF, Fish D, Fikrig E and Kantor FS (1998) Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity. Am J Trop Med Hyg 58: Neelakanta G, Li X, Pal U, Liu X, Beck DS, DePonte K, Fish D, Kantor FS and Fikrig E (2007) Outer surface protein b is critical for Borrelia burgdorferi adherence and survival within ixodes ticks. PLoS Pathog 3: e33. Nijhof AM, Balk JA, Postigo M, Rhebergen AM, Taoufik A and Jongejan F (2010) Bm86 homologues and novel ataq proteins with multiple epidermal growth factor (egf)-like domains from hard and soft ticks. Int J Parasitol 40: Nuttall PA, Trimnell AR, Kazimirova M and Labuda M (2006) Exposed and concealed antigens as vaccine targets for controlling ticks and tick-borne diseases. Parasit Immunol 28: Odongo D, Kamau L, Skilton R, Mwaura S, Nitsch C, Musoke A, Taracha E, Daubenberger C and Bishop R (2007) Vaccination of cattle with tickgard induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus. Vaccine 25: Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, Desilva AM, Bao F, Yang X, Pypaert M, Pradhan D, Kantor FS, Telford S, Anderson JF and Fikrig E (2004a) TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi. Cell 119: Pal U, Yang X, Chen M, Bockenstedt LK, Anderson JF, Flavell RA, Norgard MV and Fikrig E (2004b) Ospc facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J Clin Invest 113: Parizi LF, Githaka NW, Logullo C, Konnai S, Masuda A, Ohashi K and da Silva Vaz I, Jr. (2012) The quest for a universal vaccine against ticks: cross-immunity insights. Vet J 194: Paveglio SA, Allard J, Mayette J, Whittaker LA, Juncadella I, Anguita J and Poynter ME (2007) The tick salivary protein, Salp15, inhibits the development of experimental asthma. J Immunol 178: Perez-Perez D, Bechara GH, Machado RZ, Andrade GM, del Vecchio REM, Pedroso MS, Hernandez MV and Farnos O (2010) Efficacy of the Bm86 antigen against immature instars and adults of the dog tick Rhipicephalus sanguineus (latreille, 1806) (Acari: Ixodidae). Vet Parasitol 167: Popara M, Villar M, Mateos-Hernandez L, Fernandez de Mera IG, Marina A, Del Valle M, Almazan C, Domingos A and De la Fuente J (2013) Lesser protein degradation machinery correlates with higher Bm86 tick vaccine efficacy in Rhipicephalus annulatus when compared to Rhipicephalus microplus. Vaccine 31: Prevot PP, Adam B, Boudjeltia KZ, Brossard M, Lins L, Cauchie P, Brasseur R, Vanhaeverbeek M, Vanhamme L and Godfroid E (2006) Anti-hemostatic effects of a serpin from the saliva of the tick Ixodes ricinus. J Biol Chem 281: Ramamoorthi N, Narasimhan S, Pal U, Bao F, Yang XF, Fish D, Anguita J, Norgard MV, Kantor FS, Anderson JF, Koski RA and Fikrig E (2005) The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 436: Ribeiro JM (1989) Role of saliva in tick/host interactions. Exp Appl Acarol 7: Ribeiro JM, Evans PM, MacSwain JL and Sauer J (1992) Amblyomma americanum: characterization of salivary prostaglandins E2 and F2 alpha by RP-HPLC/bioassay and gas chromatography-mass spectrometry. Exp Parasitol 74: Richardson MA, Smith DRJ, Kemp DH and Tellam RL (1993) Native and baculovirus-expressed forms of the immunoprotective protein Bm86 from Boophilus microplus are anchored to the cell membrane by a glycosylphosphatidyl inositol linkage. Insect Mol Biol 1: Ecology and prevention of Lyme borreliosis

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317 Michelle J. Klouwens, Jos J. Trentelman and Joppe W.R. Hovius Wikel SK and Allen JR (1976) Acquired resistance to ticks. I. Passive transfer of resistance. Immunology 30: Wikel SK and Allen JR (1978) Acquired resistance to ticks. III. Cobra venom factor and the resistance response. Immunology 32: Wikel SK, Ramachandra RN, Bergman DK, Burkot TR and Piesman J (1997) Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infect Immun 65: Willadsen P (2004) Anti-tick vaccines. Parasitology 129: S367-S387. Willadsen P, Riding GA, McKenna RV, Kemp DH, Tellam RL, Nielsen JN, Lahnstein J, Cobon GS and Gough JM (1989) Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus. J Immunol 143: Ecology and prevention of Lyme borreliosis

318 Risk management exposure control

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320 22. Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis Desiree J. Beaujean * and Hein Sprong National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; desiree.beaujean@rivm.nl Abstract Despite improvements in prevention, diagnosis and treatment, Lyme borreliosis (LB) is still the most common tick-borne infection in Europe and North America. As long as there are no effective measures to control tick populations in nature and there is no vaccine, we have to primarily rely on information provision and communication to prevent tick bites and LB. Tick and LB prevention programmes are aimed at increasing the public s knowledge of ticks, tick bites and LB, influencing people s perception, and stressing the positive effect of preventive measures. Remarkably, the number of studies aiming at the improvement of education and communication interventions to prevent tick bites and LB is very limited. Recently, four newly developed interventions, a movie, a leaflet, an online educational video game and a mobile app were developed, implemented and evaluated on social-cognitive variables associated with the intention to take measures to prevent ticks and LB and the preventive behaviour of the general public and schoolchildren in the Netherlands. The effects of these tools were comparable: people appreciated the tools, they learn short-term from it and some of the tools motivate them to check their skin on ticks more frequently. Since none of the tools was able to reach a long-term effect, it is important to keep going the attention for education continuously. Keywords: health promotion, intervention tools, prevention Introduction The risk of acquiring Lyme borreliosis (LB) is intimately linked to tick abundance and exposure. High risk is not only associated with residency in rural areas, but also with occupation (e.g. forestry work) and with certain leisure activities (e.g. hiking, camping, and berry picking). Furthermore, the relatively low abundance of ticks in some urban areas (e.g. gardens and city parks) poses a substantial risk as well, because of the elevated exposure rate (Mulder et al. 2013). A silver bullet for the prevention of LB, such as a efficacious vaccine, is currently not available (Schuijt et al. 2011), and prevention predominantly relies on the education of individuals and communities on how to prevent exposure to ticks (and therefore tick bites) and to decrease the risk of LB in case of a tick bite. In theory, individual or community measures could form very effective preventive methods (Clark and Hu 2008, Gould et al. 2008, Vazquez et al. 2008). For example, in order to decrease the risk of tick bites and Borrelia transmission, people living in or visiting tick-infested areas are advised to avoid tick habitats, to wear long, light-coloured trousers (and to tuck them into their socks) and to use insect repellent that contains permethrin (on clothes) or N,N-diethyl-meta-toluamide (DEET) on clothes or directly on skin. After visiting or working in such areas, taking a shower is recommended and a thorough check for ticks should be performed, including careful inspection of the neck, armpits, groin and clothing. Any attached tick should be removed immediately, preferably with tweezers, by seizing and pulling steadily on the mouth parts, without twisting, and Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _22, Wageningen Academic Publishers 2016

321 Desiree J. Beaujean and Hein Sprong the attachment site should subsequently be disinfected, to prevent cutaneous infection (Pitches 2006). Since ticks do not have a high probability of transmitting Borrelia until hours after they begin to feed on their host, immediate removal of ticks is one of the most effective ways of avoiding Borrelia infection, and consequently LB. For three months after the bite, the site of the bite should be monitored for signs of EM and a person s overall health should be monitored for other possible symptoms. All these measures are set down in national and international Lyme prevention guidelines (Box 1). In the Netherlands, there is the multidisciplinary practice guideline on LB by the former Quality Institute for Health Care (CBO), aimed at health professionals involved in the diagnosis, treatment and care of patients suffering from LB. Based on the CBO guideline, there is a guideline for public health care workers produced by the RIVM s National Coordination Center for Communicable Disease Control (LCI) (CBO 2013, LCI 2016). One of the conclusions of the national expert consultation was that efforts concerning education on ticks and LB should be intensified and centrally coordinated by RIVM, as this is currently the most straightforward approach to prevent LB (Hovius et al. 2014) The need for health education on prevention of tick bites and Lyme borreliosis The preventive measures to reduce the risk of tick exposure or checking the body for ticks are well-established but, in general, the compliance from the general public is poor. Research in areas where LB is endemic has demonstrated that despite adequate knowledge about its symptoms and transmission, many people do not act in a way to reduce their risk of infection (Corapi et al. 2007, Beaujean et al. 2013a, 2013b). Daltroy et al. (2007) and Malouin et al. (2003) demonstrated that public education on ticks and LB decreases the chance to contract. Furthermore, little evidence-based research on the effectiveness of educational and communicational intervention methodologies is being performed in recent years, (Beaujean et al. 2016a, Mowbray et al. 2012). Here, we shortly describe four innovative intervention tools to improve the compliance to existing preventive measures (Box 1). The content of these four tools was the same: information about how ticks look like, their preferred habitat and how to check and remove ticks correctly after being outdoors. These tools were developed and evaluated during a four-year PhD-trajectory in a collaboration between the RIVM s National Coordination Center for Communicable Disease Control and the departments of Health Promotion and Health Services Research from the School for Public Health and Primary Care (CAPHRI), Maastricht, the Netherlands. Leaflet and movie In order to inform the Dutch population about preventive measures concerning ticks and LB, a novel leaflet and a movie was developed (available at: An elaborate evidence-based study evaluated the effect of these two educational products and showed that both the leaflet and the movie are valued as effective educational products to enhance the public s knowledge of ticks and LB, self-efficacy, and the intention to take preventive measures (Beaujean et al. 2016b). Although the respective respondents rated the leaflet and the movie in equal measure, the latter group experienced that the five-minute long movie was too lengthy. A long-term effect could not be demonstrated for either the leaflet or the movie. With the acquired effectiveness, measures and feedback from the respondents, these tools can be improved even further, and its implementation can be adapted based on the anticipated effects (Beaujean et al. 2016b). Since 94% of the general public in the Netherlands has Internet access and 56.7% uses 320 Ecology and prevention of Lyme borreliosis

322 22. Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis Box 1. Selection of measures to prevent tick bites and Lyme borreliosis (LB) according to the Dutch guidelines of CBO and LCI. Avoiding tick areas During the tick season (March to November), stay on the beaten paths and avoid contact with dense vegetation, wetlands with high grass, areas with leaf litter under trees, and shrubbery. In the Netherlands, ticks are highly prevalent in wetlands on the ends of blades of grass, in bushy areas with high grass (up to 1.5 meter) and leaf litter. The density of questing ticks varies per season even per day, and also between different geographic locations. If one avoids contact with the vegetation, then one can assume that there is little chance of contact with ticks. There are no studies showing that avoiding certain areas prevents infection. A longitudinal study indicated that in the Netherlands the density of questing ticks is greatest in dune areas, followed by forest areas, (city)parks and moorlands (Wielinga et al. 2006). Wearing protective clothing During the tick season, wear protective clothing (long pants with pant legs into socks, closed shoes, and long sleeved tops). Ticks are more visible on light-coloured clothing. In case of potential occupational exposure to ticks, the employer is strongly advised to provide protective clothing to employees. A prospective study among a rural community in North- Western California in the USA showed that wearing protective clothes during outdoors activities led to less cases of LB (Lane et al. 1992). Another case-control study demonstrated that persons who wore protective clothing during outdoors activities were less likely to get LB than persons who did not wear protective clothing (Vazquez et al. 2008). A study among soldiers showed that seropositivity against anti-tick proteins was higher among soldiers who did not wear pant legs in socks than among those who did (Schwartz et al. 1996). A randomised cohort study demonstrated that Ixodes ricinus is more attracted to lightcoloured clothes than to dark-coloured clothes (Stjernberg and Berglund 2005). Using insect repellent During the tick season, spray or grease your clothes or skin with a DEET-containing repellent. Wear permethrin impregnated clothing in case of occupational exposure to ticks. Permethrin is a synthetic pyrethroid and is deadly to ticks. In the Netherlands, it is not permitted to sell permethrin as a separate product, but it is allowed to pre-treat clothes with it. Permethrin-treated clothes preserve their protective function through several washings. Removing ticks promptly Remove ticks promptly. Borrelia transmission does not occur immediately after tick attachment: it first need to migrate from the intestines of the tick to its salivary glands before it is transmitted to the host. Animal studies have shown that transmission of Borrelia burgdorferi becomes apparent after 24 hours following attachment, sometimes sooner (Piesman et al. 1987, Teece and Crawford 2002). Removing infected ticks more than 24 hours after attachment therefore means an increased risk of Borrelia bacteria transmission. There is no scientific evidence suggesting there is an increased risk of LB when the tick s head remains in the skin and only the tick s body is removed. Ecology and prevention of Lyme borreliosis 321

323 Desiree J. Beaujean and Hein Sprong the Internet to search for information on health, the online leaflet and movie are examples of accessible interventions for the entire general public (Statline 2016). A more targeted approach Rather than developing tools for the general public, improvement of the effectiveness might be achieved when at-risk groups for tick bites and LB within the general public are specifically addressed with educational tools that fit their perceptions and demands. Selection of such at-risk groups, knowledge on their profiles and an insight into their motives allow health organisations to fine-tune their communication strategies to the characteristics and to make the education more effective (see below). In a recent qualitative study among visitors to two municipal health services travel health clinics (one in a high-endemic area and one in a low-endemic area) and parents with young children, four groups at risk of being bitten by ticks and developing LB among the general Dutch population were identified (Beaujean et al. 2013c). These groups were as follows: (1) outdoor people that check for tick bites; (2) outdoor people that do not check for tick bites; (3) parents that check their children for tick bites; and (4) parents that do not check their children for tick bites. Previous experience with ticks or LB was the main denominator between the groups (Beaujean et al. 2013c). Mobile app In order to educate the Dutch public on the risks of ticks and to motivate them to self-check when they return from being outdoors, and if applicable to remove a tick as soon as possible, an application was devised for a mobile phone, named Tekenbeet (Dutch for tick bite, Figure 1). The content of the application was based on the results of several pilot studies (Van Velsen et al. 2015). One study focussed on obtaining an inventory of the wishes and requirements that endusers have in relation to a mobile phone application regarding measures to prevent ticks, tick bites and LB. A focus group discussion with stakeholders and in-depth interviews with the end-users learned us that the mobile app should firstly motivate users to self-check for ticks after a visit to a high-risk area. The app should work in the absence of internet (forest areas) and contain a tick radar, to allow users to see where the ticks are most prevalent and when ticks are most active. Furthermore, stakeholders wanted a reminder function to be available, which alerts the user to self-check after a visit to nature and also allows the option to record a tick bite. The mobile app Tekenbeet is an example of an intervention mainly aimed at members of the high risk groups, since mainly these people are willing to download such a particular app on their mobile phone. Data provided by Google Analytics showed that the app was downloaded nearly 50,000 times in the 20 months following the launch in April A survey evaluating the app showed that the mean satisfaction of the app was 7.44 (on a scale of 1 to 10), and 90.9% of the respondents indicated that they would recommend the app to others. The tick radar and the tick diary were the most viewed sections of the app. On average, when compared to those who had not downloaded the app (n=311), respondents who had downloaded the app (n=243) scored higher on knowledge and intention to take preventive measures (D.J. Beaujean et al. unpublished data). Thus, the Tekenbeet app turned out to be used often, well appreciated, and effective to increase users knowledge of ticks and prevention of tick bites. It is a practical addition to the existing public health products, and enhances the self-efficacy to take preventive measures (checking for and removal of ticks). Furthermore, the app increases the intention for someone to take preventive measures, although these effects are not sustained long-term. However, one of the advantages of mobile technology is that it can be used anywhere when required, which reduces the need for a 322 Ecology and prevention of Lyme borreliosis

324 22. Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis A. Main screen containing the following buttons: Tick info; Tick radar; Tick checks; Tick removal; Lyme disease; Tick alarm; FAQ; Tick diary. B. Tick information screen about the appearance of ticks, their habitat, and how they can cause Lyme disease. C. Tick radar screen showing the current activity of ticks (nymphs) in the Netherlands and a ten day forecast. Light green implies a small risk for risk for tick bites and white a minimal risk. Figure 1. Screenshots of the mobile app Tekenbeet (Dutch for Tickbite ) a mobile application to have always and everywhere access to important information on ticks, tick bites, LB and preventive measures. (A) shows the main screen that is used to navigate within the app, (B) and (C) show the screens with tick information and the tick radar screen, respectively. This app can be downloaded at the App store (ios) and the Google Play Store (Android). long-term effect. The use of applications for mobile phones is popular. The Tekenbeet app fits this trend and offers a modern alternative to more traditional media for information provision such as a leaflet or information on a website. Serious game Epidemiological studies indicate that children are an at-risk group for tick bites and LB (Stanek et al. 2012). An online game was developed specifically aimed to educate schoolchildren in a playful manner (Figure 2). In a large study involving 887 schoolchildren, the effectiveness of the game was compared to the effectiveness of a leaflet detailing ticks and LB. The pupils were divided, per class, into three intervention groups: game (22.4%), leaflet (35.6%) or control (41.9%). Prior to and immediately after the intervention, the children completed a short questionnaire. Knowledge of ticks and LB increased in all groups. The children who had played the game and those in the control group, were checked for ticks more often by their parents/carers when compared to the children in the leaflet group. This indicates the presence of a mere measurement effect, an exposure effect. Since the game did not achieve a significantly better effect than the leaflet or no intervention, we would anticipate the game to have a supplementary role in addition to other information provision products used for public health interventions aimed at children (Beaujean et Ecology and prevention of Lyme borreliosis 323

325 Desiree J. Beaujean and Hein Sprong Figure 2. A screenshot of the serious game Teek control (teek is the Dutch word for tick) where children can learn in what kind of habitats ticks are active in their own environment, how they can recognise ticks and what they can do to prevent LB. The game is available at: al. in press). It would be useful to integrate the online serious game Teekcontrol in an educational school programme on ticks and LB. Recommendations for practice, policy and research Practice In the Netherlands, a central organisation, the National Coordination Center for Communicable Disease Control, actively involves all kinds of organisations, such as the municipal Health Services, general practitioners, national nature organisations, Scouting Netherlands and the Dutch Foundation for Lyme patients in the promotion of a mix of interventions to reach as many as possible people in prevention programmes on ticks and LB. By integrating the game Teekcontrol in a school education programme on ticks and LB, both children and their parents can be reached. Online intervention tools, like online games, movies and apps, need advertisement since these are no tangible products like a leaflet or a poster, you easily, by accident, get in touch with. Therefore, we recommend also to use online social media (Twitter, Facebook, Instagram, etc.), as well as traditional media (newspapers, radio, television) to draw repeatedly attention to these online intervention tools, and to reach as many at-risk groups as possible. Policy Firstly, there is a need of more scientific research into education and communicational interventions (Beaujean et al. 2016a). The old adage prevention is better than cure does not only hold true of LB, and it is of pivotal importance to gain more knowledge and insights on how to achieve this through education and risk communication. The development and evaluation of the tools developed during the PhD-trajectory described in this chapter clearly how that there is a benefit of pre- and post-intervention research to develop effective evidence based intervention tools. 324 Ecology and prevention of Lyme borreliosis

326 22. Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis Therefore, (structural) funding the development of evidence-based prevention programmes based on the principals of intervention mapping is strongly encouraged. Finally, guideline developers should focus on measures which are practical and achievable for individuals, and not on measures that cannot be performed by an individual, like applying pesticides and reducing tick habitat. Practice guidelines should focus primarily on education on avoiding tick areas, wearing protective clothing, using insect repellent and removing ticks promptly. Research Since the number of studies on the topic of education and communication interventions to prevent tick bites and LB is so limited (Beaujean et al. 2016a), research into educational and communicational interventions is needed. The more effective the education and communicational tools on ticks and LB will be, the less diagnostics and therapy will be needed. The development of new interventions tools should focus on measures that the public is likely to adopt and/or fit with their perceptions like checking the body for ticks and removing ticks as quickly as possible. It would be interesting to investigate the barriers to take other preventive measures like using insect repellent skin products or wearing protective clothing. Maybe it will give rise to new opportunities in the field of preventive behaviour. Public health relevance An ounce of prevention is worth a pound of cure (Benjamin Franklin). Knowledge of the profiles and insight in the motives of at-risk groups allow health organisations to fine-tune their communication strategies to make the education more effective. A mobile app ( Tekenbeet ) is a very attractive and suitable tool to reach a very large audience and has the ability to provide adequate information on the right place and time. References Beaujean DJ, Bults M, Van Steenbergen JE and Voeten HA (2013a) Study on public perceptions and protective behaviors regarding Lyme disease among the general public in the Netherlands: implications for prevention programs. BMC Public Health 13: 225. Beaujean DJ, Crutzen R, Gassner F, Ameling C, Wong A, Van Steenbergen JE and Ruwaard D (2016b) Comparing the effect of a leaflet and a movie in preventing tick bites and Lyme disease in the Netherlands. BMC Public Health 10: 495. Beaujean DJ, Crutzen R, Kengen C, Van Steenbergen J and Ruwaard D (2016a) Increase in ticks and Lyme Borreliosis, yet research into its prevention on the wane. Vector Borne Zoonotic Dis 16: Beaujean DJ, Gassner F, Wong A, Van Steenbergen JE, Crutzen R and Ruwaard D (2013b) Determinants and protective behaviours regarding tick bites among school children in the Netherlands: a cross-sectional study. BMC Public Health 9: Ecology and prevention of Lyme borreliosis 325

327 Desiree J. Beaujean and Hein Sprong Beaujean DJ, Gassner F, Wong A, Van Steenbergen JE, Crutzen R and Ruwaard D (in press) Education on tick bite and Lyme borreliosis prevention, aimed at schoolchildren in the Netherlands: comparing the effects of an online educational video game versus a leaflet or no intervention. BMC Public Health DOI: Beaujean DJ, Van Velsen L, van Gemert-Pijnen JE, Maat A, Van Steenbergen JE and Crutzen R (2013c) Using risk group profiles as a lightweight qualitative approach for intervention development: an example of prevention of tick bites and Lyme disease. JMIR Res Protoc 2: e45. CBO (2013) Richtlijn Lymeziekte. CBO, Utrecht, the Netherlands. Available at: Clark RP and Hu LT (2008) Prevention of Lyme disease and other tickborne infections. Infect Dis Clin North Am 22: Corapi KM, White MI and Phillips CB (2007) Strategies for primary and secondary prevention of Lyme disease. Nat Clin Pract 3: Daltroy LH, Phillips C, Lew R, Wright E, Shadick NA and Liang MH (2007) A controlled trial of a novel primary prevention program for Lyme disease and other tick-borne illnesses. Health Educ Behav 3: Gould LH, Nelson RS, Griffith KS, Hayes EB, Piesman J, Mead PS and Cartter ML (2008) Knowledge, attitudes, and behaviours regarding Lyme disease prevention among Connecticut residents, Vector Borne Zoonotic Dis 8: Hovius JW and Sprong H (2014) Combatting Lyme disease. Ned Tijdschr Geneeskd 158:A7986. Lane RS, Manweiler SA, Stubbs HA, Lennette ET, Madigan JE and Lavoie PE (1992) Risk factors for Lyme disease in a small rural community in northern California. Am J Epidemiol 136: LCI (2013) LCI-richtlijn Lymeziekte. LCI, Bilthoven, the Netherlands. Available at: Malouin R, Winch P, Leontsini E, Glass G, Simon D, Hayes EB and Schwartz BS (2003) Longitudinal evaluation of an educational intervention for preventing tick bites in an area with endemic Lyme disease in Baltimore County, Maryland. Am J Epidemiol 157: Mowbray F, Amlôt R and Rubin GJ (2012) Ticking all the boxes? A systematic review of education and communication interventions to prevent tick-borne disease. Vector Borne Zoonotic Dis 12: Mulder S, Van Vliet AJ, Bron WA, Gassner F and Takken W (2013) High risk of tick bites in Dutch gardens. Vector Borne Zoonotic Dis 13: Piesman J, Mather TN, Sinsky RJ and Spielman A (1987) Duration of tick attachment and Borrelia burgdorferi transmission. J Clin Microbiol 25: Pitches DW (2006) Removal of ticks: a review of the literature. Euro Surveill 11: E Schuijt TJ, Hovius JW, van der Poll T, Van Dam AP and Fikrig E (2011) Lyme borreliosis vaccination: the facts, the challenge, the future. Trends Parasitol 27: Schwartz BS, Sanchez JL, Sanders ML and DeFraites RF (1996) Tick avoidance behaviors associated with a decreased risk of anti-tick salivary gland protein antibody seropositivity in military personnel exposed to Amblyomma americanum in Arkansas. Am J Trop Med Hyg 55: Stanek G, Wormser GP, Gray J and Strle F (2012) Lyme borreliosis. Lancet 379: Statline (2016) Internet; toegang, gebruik en faciliteiten. Centraal Bureau voor de Statistiek, The Hague, the Neterlands. Available at: Stjernberg L and Berglund J (2005) Detecting ticks on light versus dark clothing. Scand J Infect Dis 37: Teece S and Crawford I (2002) Towards evidence based emergency medicine: best BETs from the Manchester Royal Infirmary. How to remove a tick. Emerg Med J 19: Van Velsen L, Beaujean DJ, Wentzel J, Van Steenbergen JE and Van Gemert-Pijnen JE (2015) Developing requirements for a mobile app to support citizens in dealing with ticks and tick bites via end-user profiling. Health Informatics J 21: Vazquez M, Muehlenbein C, Cartter M, Hayes EB, Ertel S and Shapiro ED (2008) Effectiveness of personal protective measures to prevent Lyme disease. Emerg Infect Dis 14: Wielinga PR, Gaasenbeek C, Fonville M, De Boer A, De Vries A, Dimmers W, Akkerhuis Op Jagers G, Schouls LM, Borgsteede F and Van der Giessen JW (2006) Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in the Netherlands. Appl Environ Microbiol 72: Ecology and prevention of Lyme borreliosis

328 23. Prevention of Lyme borreliosis after a tick bite Hein Sprong * and Kees (C.C.) van den Wijngaard National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; hein.sprong@rivm.nl Abstract The probability of developing Lyme borreliosis after a tick bite is around 2%, and 0.2% for severe manifestations. Most tick bites thus do not lead to any disease burden due to Lyme borreliosis. Furthermore, the high incidence of localised Lyme borreliosis only gives limited rise to the disease burden in patients that are successfully treated with antibiotics. In contrast, the relatively small incidences of both disseminated Lyme borreliosis and persisting symptoms attributed to Lyme borreliosis are causing a relatively high disease burden, and are considered to be a major public health concern. The high disease burden in these categories might already be reduced by improving the diagnosis and treatment of earlier stages of Lyme borreliosis. Conventional measures to prevent tick bites, such as wearing protective clothing, using repellants, checking for and removing ticks, are simple ways to prevent the development of all stages of Lyme borreliosis. In addition, advising people with a skin rash after a tick bite to visit a general practitioner can help to prevent the development of later stages of Lyme borreliosis. Novel measures, such as point of care testing and post-exposure prophylaxis have not yet been proven to contribute to the prevention of the disease. Keywords: disease burden, disease incidence, prophylaxis, tick bite Introduction Lyme borreliosis is a human bacterial disease, caused by an infection with, or by the immune response to, pathogenic members of the Borrelia burgdorferi sensu lato complex (Coumou et al. 2015). It is important to realise that being infected with a pathogen is not the same as having a disease; there is no disease when there are no symptoms. Consequently there is no Lyme borreliosis in the absence of clinical manifestations. A tick bite, or the mere proof of an infection with B. burgdorferi s.l., is insufficient to be designated as Lyme borreliosis, as symptoms might never develop. However, at these stages, the development of Lyme borreliosis can be stopped by a vaccine or an antibiotic post-exposure prophylaxis (Nadelman et al. 2001, Wressnigg et al. 2013). This book largely focuses on the old adage prevention is better than cure, but where does prevention of Lyme borreliosis end, and disease curation begin? And are all possible preventive measures justified? In this chapter, we will try to explore the shifting borders between prevention and cure by looking at the epidemiological situation of Lyme borreliosis in the Netherlands. For epidemiological purposes, Lyme borreliosis is classified in three consecutive stages (Figure 1). The earliest stage is called localised Lyme borreliosis. The most common clinical manifestation of localised Lyme borreliosis is an erythema migrans, an expanding skin lesion occurring after several days or weeks at the site of the tick bite. Other concomitant symptoms in this early stage of disease can be malaise and flu-like symptoms. An erythema migrans eventually resolves, even without antibiotic treatment (Stanek et al. 2012). Still, medical guidelines strongly advise antibiotic treatment of erythema migrans, as it commonly prevents the development of late and more severe disease stages. Estimates indicate that around 25% of Lyme borreliosis patients do never see an erythema migrans or did not receive treatment for it (Berglund et al. 1995, Krupka et al. Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _23, Wageningen Academic Publishers 2016

329 Hein Sprong and Kees (C.C.) van den Wijngaard Recovery Recovery Ticks Harmless Recovery Disseminated LB Persisting symptoms after LB Exposure Tick bite Infection Localised LB Persisting symptoms attributed to LB Recovery? Figure 1. Outcome tree with possible health outcomes of Lyme borreliosis (LB). Tick bites are generally acquired when working or recreating (Exposure) in tick-suitable habitats (Hazard). After a tick bite, followed by infection with Borrelia burgdorferi sensu lato, three symptomatic outcome categories can be distinguished: (1) Localised LB, with EM being the most common manifestation; (2) (early and late) disseminated LB, with more serious manifestations affecting skin (multiple EM, acrodermatitis chronica atrophicans, (early and late) neuroborreliosis, Lyme arthritis, Lyme carditis; and (3) persisting symptoms attributed to LB, where the etiology of the disease manifestations is not always clear. In many, but not all, cases of LB, symptoms typically resolve during or after completion of a course of antibiotic treatment. 2011). If the infection remains unnoticed and untreated in this early localised stage, B. burgdorferi s.l. can either disappear (self-limiting infection) or it can spread to other tissues and organs. The second, so called disseminated Lyme borreliosis displays more severe manifestations that can involve a patient s nervous system (neuroborreliosis), joints (Lyme arthritis), skin (acrodermatitis chronica atrophicans), and in rare cases heart (Lyme carditis), and lymph nodes (Borrelial lymphocytoma) (Stanek et al. 2012). In a Dutch study based on a general practitioners (GP) survey, 95% of reported LB had an erythema migrans, 0.4% borrelial lymphocytoma, 0.9% ACA, 2% Lyme neuroborreliosis, 2% Lyme arthritis, 0.1% Lyme carditis, and 0.1% ocular manifestations (A. Hofhuis unpublished data). In a German study that registered a case series of patients with Lyme borreliosis comparable proportions were observed: 89% had an erythema migrans, 5% had arthritis, 3% had early neurological manifestations, 2% had borrelial lymphocytoma, 1% had acrodermatitis chronica atrophicans, and less than 1% had cardiac manifestations. None of the patients had late neurological Lyme borreliosis (Huppertz et al. 1999). The characteristic manifestations of Lyme borreliosis typically resolve during or after completion of a course of antibiotic treatment. Any accompanying non-specific symptoms also usually resolve, but some patients report long-term ( 6 months) persistence of fatigue, musculoskeletal pain, or difficulties with concentration (Stanek et al. 2012). The latter complications of Lyme borreliosis result in the third stage, namely persisting symptoms attributed to Lyme borreliosis. Patients with persisting and non-characteristic symptoms, like musculoskeletal pain, neurocognitive symptoms and fatigue, which may last up to six months or longer, and have been diagnosed or are suspected with Lyme borreliosis are assigned to this category. The cause of these medical complaints are attributed to an active or endured B. burgdorferi s.l. infection. It is known that some patients with disseminated Lyme borreliosis report persisting symptoms, even after repeated antibiotic therapy (Cairns and Godwin 2005, Cerar et al. 2010, Stanek et al. 2011, Wormser et al. 2006). 328 Ecology and prevention of Lyme borreliosis

330 23. After tick bite measures In the Netherlands, periodic retrospective surveys among all GP have shown an almost fourfold rise in GP consultations for erythema migrans during the past twenty years from 39 per 100,000 inhabitants in 1994 up to 140 per 100,000 inhabitants in 2014 (Hofhuis et al. 2015b). The most straightforward explanation for this rise is the increased exposure to infected ticks (Sprong et al. 2012). Based on a population survey, approximately 1.1 million tick bites were noticed in the Dutch population in 2007 (Hofhuis et al. 2015b). For every sixty tick bites in the general population in 2007, one consultation for erythema migrans at the general practitioner was observed, resulting in a risk of approximately 2% for acquiring an erythema migrans after a tick bite. Two prospective studies on the transmission of B. burgdorferi s.l. to humans after tick bites, estimated that the risk of a B. burgdorferi s.l. infection was 5.1%, whereas the risk of developing an erythema migrans after tick bites was estimated at 2.1 to 2.6% (Hofhuis et al. 2013, Wilhelmsson et al. 2016). In another cross-sectional retrospective study in 2010, the annual incidence rate for erythema migrans was calculated to be 132 per 100,000 inhabitants, whereas the incidence of disseminated Lyme borreliosis was 7.7 per 100,000 inhabitants. Based on these incidence rates, the risk of acquiring disseminated Lyme borreliosis after a tick bite is between 0.1 and 0.2%. Generally, disseminated Lyme borreliosis can be treated well with antibiotics. In the same study, the estimated incidence of persisting symptoms attributed to Lyme borreliosis was 5.5 GP reports per 100,000 inhabitants (Hofhuis et al. 2015a). The risk of acquiring persisting symptoms attributed to Lyme borreliosis after tick bite is around 0.1%. For policy and decision making, it can be helpful to compare the public health impact of diseases with each other. In theory, the burden of any disease can be expressed in disability-adjusted life years (DALYs), a summary measure of disease burden that aggregates the impact of mortality and morbidity in one figure. A recent study estimated the total disease burden of Lyme borreliosis for the Netherlands in 2010 to be DALYs per 100,000 (Van den Wijngaard et al. 2015). Comparing to 32 other infectious diseases in the Netherlands, Lyme borreliosis ranked 12 th in disease burden. The highest burden is caused by pneumococcal disease, which gives rise to 57 DALYs per 100,000 inhabitants. Lyme borreliosis is preceded by hepatitis C, and followed by norovirus and Salmonella with a somewhat lower burden (Van den Wijngaard et al. 2015). A striking finding is that the persisting symptoms attributed to Lyme borreliosis account for almost 90% of the disease burden by Lyme borreliosis, whereas this category comprises less than 5% of all Lyme borreliosis cases (Van den Wijngaard et al. 2015) (Table 1). From a public health perspective, preventive measures of effective treatment of earlier stages of Lyme borreliosis to reduce the incidence of this category of patients yield the highest effect. Improved diagnosis and more insight in the aetiology of this stage of the disease are necessary, before better treatment or preventive measures can be formulated. After tick bite measures Currently, the control of Lyme borreliosis is predominantly based on public campaigns to increase self-effectiveness by checking for and removing ticks after visiting a risk area and prevention of tick bites by wearing protective clothing or using repellants. Furthermore, people with a skin rash after a tick bite, the erythema migrans, are strongly advised to visit a general practitioner. Photo examples of the skin rash are presented to the public and medical doctors through www. tekenradar.nl, a web platform developed by RIVM and Wageningen University & Research to give timely and accurate information on ticks, tick activity, Lyme borreliosis and preventive measures Ecology and prevention of Lyme borreliosis 329

331 Hein Sprong and Kees (C.C.) van den Wijngaard Table 1. Incidence versus disease burden of the three different stages of Lyme borreliosis (LB). Disease Incidence (/100,000) Incidence (% of total) DALY DALY (/100,000) 1 (% of total) 1 Erythema migrans (localised LB) % % Disseminated LB 7.7 5% % Persisting symptoms attributed to LB 5.5 4% % Total % % 1 DALYs = disability-adjusted life years. for the public. Such information is also available in a freely available software application for smartphones, called Tekenbeet, which was developed by the RIVM. This kind of measures are considered to be cost-efficient methods to prevent an individual from contracting the more severe and disabling forms of Lyme borreliosis. In 2014, a first possible sign of effect by these public education campaigns was observed when the number of tick bite consultations decreased, whereas the number of erythema migrans diagnoses showed a first sign of possible stabilisation (Beaujean and Sprong 2016). Despite the low probability of infection after a tick bite for an individual, hundreds of patients with disseminated Lyme borreliosis and patients with long-term persisting symptoms occur due to the large annual number of tick bites in the population. This is a typical example of a numbers game, and as a result the demand of more preventive measures after a tick bite is increasing. Postexposure prophylaxis after tick-attachment may be an effective way to prevent the development of any stage of Lyme borreliosis. In a randomised trial performed in the USA, the prescription of a single-dose of doxycycline was shown to prevent the development of erythema migrans after a tick bite (Nadelman et al. 2001). Whether a similar single-dose, post-exposure prophylaxis would be effective in Europe, where other Borrelia genospecies occur in ticks than in the USA, is currently under investigation. From a public health perspective, the major disadvantage of providing antibiotic treatment to all people that detect a tick bite is that around 40 to 50 healthy persons have to be treated with antibiotics to prevent erythema migrans formation of a single infected case, a manifestation that is generally cured effectively with a similar treatment. Furthermore, more than 300 healthy persons need to be treated with antibiotics for preventing a singular case disseminated Lyme borreliosis. Although most antibiotics are inexpensive and are commonly used to treat all kinds of infections, they have a range of possible side-effects, such as photosensitivity and gastrointestinal side effects, and noteworthy contraindications, especially amongst pregnant women and young children (Smith and Leyden 2005). In theory, the number needed to treat (NNT) can be reduced if one would be able to predict the development of Lyme borreliosis, for example on the basis on the Borrelia-infection and attachment time or stage of engorgement of the tick (Hofhuis et al. 2013, Tijsse-Klasen et al. 2011). Such a test-algorithm requires a high sensitivity to be able to treat all persons at risk for Lyme borreliosis, and moderate specificity to be able to reduce the NNT. The infection status of the engorged tick alone appears to be a poor predictor of the development of Lyme borreliosis (Hofhuis et al. 2013, Huegli et al. 2011). Approximately 20% of the ticks biting humans is infected 330 Ecology and prevention of Lyme borreliosis

332 23. After tick bite measures with B. burgdorferi s.l. (Hofhuis et al. 2013, Tijsse-Klasen et al. 2011), whereas only 2.1 to 2.6% of humans bitten by ticks developed Lyme borreliosis. There are several explanations for this. First of all, not all members of B. burgdorferi s.l. group are pathogenic for humans. Although a few case reports exist in the literature Borrelia valaisiana and Borrelia lusitaniae are considered to be non-pathogenic. Second, the tick attachment time was too short for the transmission of the pathogens to humans. In general, ticks attached for a few hours do not transmit Borrelia (Piesman et al. 1987, 1991). Thirdly, a pathogenic Borrelia was transmitted but only caused a self-limited infection, without the presentation of overt symptoms. More importantly, in several prospective studies after tick bites, some people developed Lyme borreliosis, whereas their tick was tested negative for B. burgdorferi s.l. (Hofhuis et al. 2013, Huegli et al. 2011, Tijsse-Klasen et al. 2011) Further understanding is needed on the development of Borrelia infection after the bite of a tick that tested negative for Borrelia. Possible explanations for development of Borrelia infection after the bite of a Borrelia-negative tick, may be that these ticks contained Borrelia species that are not detected by our assays, or that all Borrelia bacteria are transmitted during attachment (Huegli et al. 2011). Self-tests for the detection of B. burgdorferi s.l. in ticks are commercially available, and claim to have a high accuracy. Their accuracy-statements may or may not reflect the ability to detect the Lyme spirochaete in the tick (Sprong et al. 2013), but the ability of such a test to predict the development of Lyme borreliosis is often not investigated, because they can only be evaluated in a prospective medical study setting (Sprong et al. 2013). As far as we know, only one commercially available self-test for the detection of B. burgdorferi s.l. in ticks was evaluated for its ability to predict erythema migrans formation (Sprong et al. 2013). This point of care test gave negative results on all the 127 ticks tested from 122 individuals, 14 of whom reported erythema migrans at follow-up. Therefore, the estimated sensitivity of the self-test for prediction of erythema migrans formation was 0%, making this self-test not suitable for reducing the number needed to treat in a post-exposure prophylaxis setting as it already missed all the obvious early Lyme borreliosis cases. A test (algorithm) to effectively reduce the NNT in post-exposure prophylaxis is currently not available. Periodic occupational health examinations are a valuable tool to monitor the development of some occupational-related diseases, such as work-related hearing impairment. A relatively new development is that some employers of high-risk workers, e.g. foresters and gardeners, offer a serological screening for antibodies against B. burgdorferi sl. during the occupational health checks. Serological screening of persons with a high risk of exposure to ticks but without overt clinical symptoms is considered to be useless from a medical point of view: irrespective of the outcome of the serological test in healthy persons, there is no reason for a preventive or curative antibiotic treatment. The prevalence of antibodies against B. burgdorferi s.l. in the (general) Dutch population is already 5%, and more than 20% in healthy forest workers (Hauser et al. 1998). The assessment of whether or not somebody has early or disseminated Lyme borreliosis should primarily be done on the basis of clinical symptoms, in combination with an anamnesis. Serological laboratory tests are primarily meant as supportive tools for the clinical diagnosis. These serological tests are also applied for epidemiological studies to provide information on the exposure to and infection with pathogens in populations and for the identification of (risk)groups. Ecology and prevention of Lyme borreliosis 331

333 Hein Sprong and Kees (C.C.) van den Wijngaard Conclusion Tick bites occur very frequently, at least in the Dutch population. The prevention of tick bites is favourable as it not only circumvents the development of Lyme borreliosis, but also of other tick-borne diseases (Jahfari and Sprong 2016). Currently, there is not a singular method that will completely prevent tick bites when visiting tick-suitable habitats. Public health authorities, nature reserve owners, as well as organisations organising activities in green areas, should create awareness and share knowledge of the presence and activity of ticks to any visitor of nature areas. This knowledge will stimulate the adequate implementation of protective measures, the control of checks for tick bites. Basic knowledge on when and how to check for tick bites, and being able to recognise the first symptoms of Lyme borreliosis, the expanding rash on the site of the tick bite, will help to prevent the development of later stages of Lyme borreliosis. Improvement of the diagnosis and treatment of localised and disseminated Lyme borreliosis will also help to reduce the incidence and the relatively high disease burden of the later stages of Lyme borreliosis. When looking at what is already commercially available, there is a need for other, simple yet effective tools for the public to prevent tick bites and Lyme borreliosis. In the future, a vaccine against Lyme borreliosis or even ticks might be a solution? Public health relevance The risk of acquiring severe manifestations of Lyme borreliosis after a tick bite is around 0.2%. Improving diagnosis and treatment of early and disseminated Lyme borreliosis can reduce the disease burden of Lyme borreliosis. The three best-practice advises for the general public are: 1. check for tick bites after visiting a risk area; 2. prevent tick bites by wearing protective clothing or using repellants; 3. visit a general practitioner in case of disease symptoms, such as a rash, that develop after a tick bite. References Beaujean DJ and Sprong H (2016) Evidence-based health promotion programmes and tools to prevent tick bites and Lyme borreliosis. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Berglund J, Eitrem R, Ornstein K, Lindberg A, Ringer A, Elmrud H, Carlsson M, Runehagen A, Svanborg C and Norrby R (1995) An epidemiologic study of Lyme disease in southern Sweden. N Engl J Med 333: Cairns V and Godwin J (2005) Post-Lyme borreliosis syndrome: a meta-analysis of reported symptoms. Int J Epidemiol 34: Cerar D, Cerar T, Ruzic-Sabljic E, Wormser GP and Strle F (2010) Subjective symptoms after treatment of early Lyme disease. Am J Med 123: Ecology and prevention of Lyme borreliosis

334 23. After tick bite measures Coumou J, Herkes EA, Brouwer MC, Van de Beek D, Tas SW, Casteelen G, Van Vugt M, Starink MV, De Vries HJ, De Wever B, Spanjaard L and Hovius JW (2015) Ticking the right boxes: classification of patients suspected of Lyme borreliosis at an academic referral center in the Netherlands. Clin Microbiol Infect 21: 368e e320. Hauser U, Krahl H, Peters H, Fingerle V and Wilske B (1998) Impact of strain heterogeneity on Lyme disease serology in Europe: comparison of enzyme-linked immunosorbent assays using different species of Borrelia burgdorferi sensu lato. J Clin Microbiol 36: Hofhuis A, Bennema S, Harms M, Van Vliet AJ, Takken W, Van den Wijngaard CC and Van Pelt W (2016) Decrease in tick bite consultations and stabilization of early Lyme borreliosis in the Netherlands in 2014 after 15 years of continuous increase. BMC Public Health 16: 425. Hofhuis A, Harms M, Bennema S, Van den Wijngaard CC and Van Pelt W (2015a) Physician reported incidence of early and late Lyme borreliosis. Parasit Vectors 8: 161. Hofhuis A, Harms M, van den Wijngaard C, Sprong H and van Pelt W (2015b) Continuing increase of tick bites and Lyme disease between 1994 and Ticks Tick Borne Dis 6: Hofhuis A, Herremans T, Notermans DW, Sprong H, Fonville M, van der Giessen JW and van Pelt W (2013) A prospective study among patients presenting at the general practitioner with a tick bite or erythema migrans in the Netherlands. PLoS ONE 8: e Huegli D, Moret J, Rais O, Moosmann Y, Erard P, Malinverni R and Gern L (2011) Prospective study on the incidence of infection by Borrelia burgdorferi sensu lato after a tick bite in a highly endemic area of Switzerland. Ticks Tick Borne Dis 2: Huppertz HI, Bohme M, Standaert SM, Karch H and Plotkin SA (1999) Incidence of Lyme borreliosis in the Wurzburg region of Germany. Eur J Clin Microbiol Infect Dis 18: Jahfari S and Sprong H (2016) Emerging tick-borne pathogens: ticking on Pandora s box. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Krupka M, Zachova K, Weigl E and Raska M (2011) Prevention of lyme disease: promising research or sisyphean task? Arch Immunol Ther Exp 59: Nadelman RB, Nowakowski J, Fish D, Falco RC, Freeman K, McKenna D, Welch P, Marcus R, Aguero-Rosenfeld ME, Dennis DT, Wormser GP and Tick Bite Study G (2001) Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med 345: Piesman J, Mather TN, Sinsky RJ and Spielman A (1987) Duration of tick attachment and Borrelia burgdorferi transmission. J Clin Microbiol 25: Piesman J, Maupin GO, Campos EG and Happ CM (1991) Duration of adult female Ixodes dammini attachment and transmission of Borrelia burgdorferi, with description of a needle aspiration isolation method. J Infect Dis 163: Smith K and Leyden JJ (2005) Safety of doxycycline and minocycline: a systematic review. Clin Ther 27: Sprong H, Docters van Leeuwen A, Fonville M, Harms M, Van Vliet AJ, Van Pelt W, Ferreira JA and Van den Wijngaard CC (2013) Sensitivity of a point of care tick-test for the development of Lyme borreliosis. Parasit Vectors 6: 338. Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJ, Van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Stanek G, Fingerle V, Hunfeld KP, Jaulhac B, Kaiser R, Krause A, Kristoferitsch W, O Connell S, Ornstein K, Strle F and Gray J (2011) Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clin Microbiol Infect 17: Stanek G, Wormser GP, Gray J and Strle F (2012) Lyme borreliosis. Lancet 379: Tijsse-Klasen E, Jacobs JJ, Swart A, Fonville M, Reimerink JH, Brandenburg AH, van der Giessen JW, Hofhuis A and Sprong H (2011) Small risk of developing symptomatic tick-borne diseases following a tick bite in the Netherlands. Parasit Vectors 4: 17. Ecology and prevention of Lyme borreliosis 333

335 Hein Sprong and Kees (C.C.) van den Wijngaard Van den Wijngaard CC, Hofhuis A, Harms MG, Haagsma JA, Wong A, de Wit GA, Havelaar AH, Lugner AK, Suijkerbuijk AW and van Pelt W (2015) The burden of Lyme borreliosis expressed in disability-adjusted life years. Eur J Public Health 25: Wilhelmsson P, Fryland L, Lindblom P, Sjowall J, Ahlm C, Berglund J, Haglund M, Henningsson AJ, Nolskog P, Nordberg M, Nyberg C, Ornstein K, Nyman D, Ekerfelt C, Forsberg P and Lindgren PE (2016) A prospective study on the incidence of Borrelia burgdorferi sensu lato infection after a tick bite in Sweden and on the Aland Islands, Finland ( ). Ticks Tick Borne Dis 7: Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, Krause PJ, Bakken JS, Strle F, Stanek G, Bockenstedt L, Fish D, Dumler JS and Nadelman RB (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic Anaplasmosis, and Babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis 43: Wressnigg N, Pollabauer EM, Aichinger G, Portsmouth D, Low-Baselli A, Fritsch S, Livey I, Crowe BA, Schwendinger M, Bruhl P, Pilz A, Dvorak T, Singer J, Firth C, Luft B, Schmitt B, Zeitlinger M, Muller M, Kollaritsch H, Paulke-Korinek M, Esen M, Kremsner PG, Ehrlich HJ and Barrett PN (2013) Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomised, dose-escalation phase 1/2 trial. Lancet Infect Dis 13: Ecology and prevention of Lyme borreliosis

336 24. How an extreme weather spell in winter can influence vector tick abundance and tick-borne disease incidence Hans Dautel 1#, Daniel Kämmer 1 and Olaf Kahl 1*# tick-radar GmbH, Haderslebener Street 9, Berlin, Germany; olaf.kahl@berlin.de; #these authors contributed equally to the chapter Abstract Natural conditions are always complex with a multitude of different biotic and abiotic factors acting on organisms all the year round. Extreme weather spells may offer the rare opportunity to learn about the influence of certain weather factors on populations, in this case Ixodes ricinus ticks. There was an extreme cold spell in Germany from late January to mid-february 2012 with near-ground temperatures constantly below 0 C for at least 15 days and often dropping to a range of -15 to -20 C or even lower at night. In the tick season after this cold spell, the mean host-seeking activity of I. ricinus nymphs was significantly lower at 4 out of 4 (field-plot method) and 3 out of 4 (flag method) German forest locations, respectively, when compared to that in It seems highly probable that the continuously low temperatures prevailing during that cold spell had a detrimental effect on many I. ricinus nymphs. The decline of I. ricinus host-seeking activity was especially distinct in the absence of any snow cover during the cold spell providing strong evidence that the snow cover acted as an effective buffer, protecting I. ricinus ticks from low temperatures in the subjacent leaf litter. There was also a significant correlation between the local minimum snow depth during the cold spell and the decrease in the local numbers of tickborne encephalitis cases in different southern German administrative districts in 2012 compared with This provides additional indirect evidence that the decrease in tick abundance was particularly strong in those areas with limited snow depth during the cold spell. Keywords: extreme weather, Ixodes ricinus, low temperatures, tick abundance, tick-borne encephalitis incidence, overwinter survival Introduction The hard tick Ixodes ricinus (Linnaeus 1758) (Acari, Ixodidae) is the main European vector of the causative agents of Lyme borreliosis and tick-borne encephalitis (TBE). Its distribution covers an area extending from Scandinavia in the north to southern Europe and from Ireland and the Iberian Peninsula in the west to western Russia and the Caspian Sea in the east (Gray 1991). The success of this species in all these different types of climate (mainly Cfb, Cfc, Csa, Csb, Dfb, and Dfc after the Köppen and Geiger classification (Rubel and Kottek, 2010)), identifies I. ricinus as an ecologically flexible species with a large distribution area. However, the recent description of Ixodes inopinatus as a new species and close relative of I. ricinus occurring mainly in Mediterranean countries (Estrada-Peña et al. 2014), raises some serious questions about the true distribution of I. ricinus in this region. As a 3-host tick with potentially long phases between the blood meals, I. ricinus spends approximately 99% of its 4-6 years life cycle (in central Europe) off the host. During these extended free-living phases, all life stages of the tick have to cope with changing weather conditions in all seasons. Special environmental challenges for this hygrophilic tick are warm and dry periods in the summer, which lead to temporarily reduced tick activity (H. Dautel and O. Kahl unpublished data, Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _24, Wageningen Academic Publishers 2016

337 Hans Dautel, Daniel Kämmer and Olaf Kahl Kahl and Knülle 1988, Morán Cadenas et al. 2007, Perret et al. 2000), and very low temperatures in the winter (Gray 1981). There are various abiotic and biotic factors affecting tick abundance in the field, rendering it very difficult to assess short- and long-term influences of a single factor. Extreme weather events may offer rare chances to learn about the influence of certain weather factors (e.g. temperature) on tick abundance (cf. Gray 2008). The extreme cold spell from late January to mid-february 2012 in whole Germany (and in other parts of Europe as well; com/zcear28) appears to have been such an occasion (see Figure 1 for weather data during that period at the four locations under study). In the following, we present and discuss our findings on I. ricinus in Germany in the year 2012 as an example of how an extreme weather period can affect I. ricinus abundance. Materials and methods Weather data All weather data were provided by MeteoGroup Deutschland GmbH (Berlin, Germany) or the German Weather Service (Deutscher Wetterdienst, Offenbach, Germany). Tick data As part of a multi-year field investigation, we determined the seasonal host-seeking activity (questing) of I. ricinus nymphs at four different forest locations in Germany (Berlin, N E, flagging in 2011: N E; Bielefeld, N 8.48 E; Giessen, N 8.65 E; Regensburg, N E) in the years 2011 to We used two different methods to determine the level of I. ricinus questing, flagging and the newly-developed field-plot method (Dautel et al. 2008). Flagging is the classic method to collect questing exophilic ixodid ticks and to determine their seasonal course of questing (Sonenshine 1993). We flagged approximately m 2 every week at each of the four locations in all seasons as far as the weather permitted. Flagging was not possible when the substrate was wet. At each occasion, a different, randomly chosen route was flagged in an area of totalling approximately 1-4 ha at each location to avoid any unwanted influence of frequent flagging on the local abundance of ticks. For each site and year, we calculated the mean number of ticks collected per 100 m 2, as well as the corresponding standard error and 95% confidence interval of the mean (see Table 1 for the number of field trips per year). Differences between years were evaluated by an ANOVA followed by Tukey s honest significant difference (HSD) test for each site (Sachs and Hedderich 2006). The so-called field-plot method was developed by us to monitor I. ricinus questing under quasinatural conditions (Dautel et al. 2008). Briefly, field-collected I. ricinus larvae were fed on Mongolian gerbils in the laboratory in early summer and in the autumn, and subsequently released onto field plots (Figure 2) where they moulted to the nymphal stage in the leaf litter in the subsequent summer. Questing of these unfed nymphs was monitored at short intervals (3 times a week from 1 st March to 31 st October and once a week from 1 st November to 28 th /29 th February). For each site, we took the average relative nymphal activity in a given calendar year in relation to the number of fully engorged, previously released larvae usually n=960. For each site and year we calculated the mean percentage of active ticks as well as the corresponding standard error and 95% confidence interval of the mean. Differences between years were evaluated by an ANOVA followed by an HSD 336 Ecology and prevention of Lyme borreliosis

338 24. Weather and Ixodes ricinus in Germany in the year 2012 A 0 T max T min Snow 10 Temperature ( C) Snow depth (cm) B 0 10 Temperature ( C) Snow depth (cm) C 0 10 Temperature ( C) Snow depth (cm) D 0 10 Temperature ( C) Snow depth (cm) Observation day 0 Figure 1. Course of temperature (daily temperature minima and maxima 2 m above the ground) and snow depth at four different locations in Germany during a cold spell from late January to mid-february (A) Berlin, (B) Bielefeld, (C) Giessen, and (D) Regensburg. Data for every location were taken from a nearby weather station (MeteoGroup Deutschland GmbH, Berlin, Germany). Each curve covers the full period during which the local temperature did not surpass 0 C (x-axis, day 1: 27 th January 2012). Ecology and prevention of Lyme borreliosis 337

339 Hans Dautel, Daniel Kämmer and Olaf Kahl Table 1. Number of field trips (field plot observations vs flag samplings for active Ixodes ricinus nymphs) per year at four German tick locations (cf. Figure 3-6). Location Year Number of field trips field-plot method flagging Berlin Bielefeld Giessen Regensburg Figure 2. A field plot showing the outer metal barrier, the inner layer of leaf litter and the 6 8 white wooden sticks serving as vantage points for active ticks. Engorged Ixodes ricinus larvae or nymphs were released in the leaf litter and, after moulting, became active as unfed nymphs or adults. 338 Ecology and prevention of Lyme borreliosis

340 24. Weather and Ixodes ricinus in Germany in the year 2012 test for each site. In the present study, emphasis is on the overall level of nymphal tick activity in a given calendar year rather than on the seasonal course of nymphal tick questing. Tick-borne disease data TBE is a notifiable disease throughout Germany, and a reliable reporting system has been developed. In a regression analysis, we correlated snow depth during the cold spell from late January to mid- February 2012 with regional TBE incidence 2012 vs We took only administrative districts with at least 15 TBE cases per year (average from 2001 to 2013; Infekt/SurvStat/survstat_node.html) to minimise the bias, and compared the number of TBE cases in 2011 with that in 2012 for each of the administrative districts (in German: Regierungsbezirke) Freiburg, Karlsruhe and Stuttgart in the state Baden-Wuerttemberg and for Lower Franconia, Central Franconia, Lower Bavaria, Upper Bavaria, and Upper Palatinate in Bavaria. We then plotted the results in relation to local minimum snow depth during the cold spell (data from MeteoGroup; based on daily mean snow depth). Results Tick abundance in 2012 Figure 1 shows the daily temperature minima and maxima as well as snow depth at the four investigated locations during the extreme cold spell from late January to mid-february Daily maximum temperatures were continuously below 0 C for days between 27 th January and 14 th February Night temperatures were mostly below -10 C, often below -15 C, and sometimes even below -20 C. The minimum temperatures close to the ground were usually even 2-3 C lower than the minimum temperatures given in Figure 1 (data from MeteoGroup, not shown). There was almost no snow cover in the western locations Bielefeld and Giessen during that period (Figure 1B and 1C). In the eastern locations, Berlin and Regensburg, however, there was permanent snow cover during the whole cold spell, albeit only 1-2 cm deep in Berlin in the first week (Figure 1A and 1D). The remaining winter 2011/2012 was comparatively mild at all locations always distinctly milder than the corresponding long-term mean and did not constitute a special physiological challenge for I. ricinus ticks (Table 2). From 2011 to 2012, we noticed a highly significant decrease of I. ricinus nymphal questing in the field plots at all investigated locations (Figures 3A-6A) and at 3 out of 4 locations by flagging Table 2. Monthly mean temperatures at the four German tick locations in the winter 2011/2012. In brackets the long-term mean temperatures ( ). All data were taken from nearby weather stations (Deutscher Wetterdienst, Offenbach, Germany). Location December 2011 ( C) January 2012 ( C) February 2012 ( C) Berlin 4.3 (0.7) 1.6 (-0.9) -2.1 (0.2) Bielefeld 5.3 (2.6) 3.2 (1.4) -0.7 (2.0) Giessen 4.1 (0.9) 2.4 (-0.2) -1.6 (0.6) Regensburg 2.8 (-0.6) 1.3 (-2.1) -3.8 (-0.4) Ecology and prevention of Lyme borreliosis 339

341 Hans Dautel, Daniel Kämmer and Olaf Kahl A Questing nymphs (%) a b Year b Mean SE 0.95 CI c B Flagged nymphs/100 m a a Year b b Figure 3. (A) Annual mean percentage of Ixodes ricinus nymphs found questing in field plots at the location Berlin and (B) annual mean number of I. ricinus nymphs flagged in the same area in the years 2011 to 2014 (SE = standard error; CI = confidence interval). Differences between years are marked by letters (a, b, c). See Table 1 for the number of field trips. A 14 Mean SE 0.95 CI B 60 Questing nymphs (%) a c a Flagged nymphs/100 m a 0-2 b Year 10 0 bc b Year c Figure 4. (A) Annual mean percentage of Ixodes ricinus nymphs found questing in field plots at the location Bielefeld and (B) annual mean number of I. ricinus nymphs flagged in the same area in the years 2011 to 2014 (SE = standard error; CI = confidence interval). Differences between years are marked by letters (a, b, c). See Table 1 for the number of field trips. 340 Ecology and prevention of Lyme borreliosis

342 24. Weather and Ixodes ricinus in Germany in the year 2012 A Questing nymphs (%) a b Year c Mean SE 0.95 CI d B Flagged nymphs/100 m a b Year a b Figure 5. (A) Annual mean percentage of Ixodes ricinus nymphs found questing in field plots at the location Giessen and (B) annual mean number of I. ricinus nymphs flagged in the same area in the years 2011 to 2014 (SE = standard error; CI = confidence interval). Differences between years are marked by letters (a, b, c). See Table 1 for the number of field trips. A Questing nymphs (%) a b c Year Mean SE 0.95 CI B 28 b Flagged nymphs/100 m a b ab Year a Figure 6. (A) Annual mean percentage of Ixodes ricinus nymphs found questing in field plots at the location Regensburg and (B) annual mean number of I. ricinus nymphs flagged in the same area in the years 2011 to 2014 (SE = standard error; CI = confidence interval. Differences between years are marked by letters (a, b, c). See Table 1 for the number of field trips. Ecology and prevention of Lyme borreliosis 341

343 Hans Dautel, Daniel Kämmer and Olaf Kahl (Figures 3B-6B). Extraordinarily low mean relative nymphal tick activities of <1% were determined in the field plots at the two western locations, Bielefeld and Giessen, in 2012 (Figures 4A and 5A). Also the annual maximum of nymphal tick activity was distinctly lower and the duration of the seasonal activity was distinctly shorter at those two locations in 2012 than in the years 2011, 2013, and 2014 (Table 3). The decrease of nymphal questing in 2012 in relation to 2011 was also distinct but less pronounced in the field plots at the two eastern locations, Regensburg (Figure 6A, Table 3) and Berlin (Figure 3A, Table 3). In the years 2013 and 2014, nymphal tick activity in the field plots significantly increased again at both western locations but remained rather low in Berlin in 2013 and even decreased in Regensburg from 2012 to The latter was in contrast to the level of adult tick activity in Berlin, although the overall annual trend of adult I. ricinus activity was similar to that of the nymphs in the field plots at all locations (data not shown). Although the duration of the annual nymphal I. ricinus questing period was comparatively short at the locations Bielefeld and Berlin in 2012 when compared to the results of the other three years (Table 3), this was not evident for the location Regensburg. Because the level of nymphal questing was <1% at the location Giessen throughout the whole of 2012, the duration of tick activity there, following our definition (only values 1% were taken as real tick activity), amounted to zero days in that year (Table 3). Furthermore, flagging of questing I. ricinus nymphs in the field yielded the lowest overall tick activity in 2012 when compared to that in 2011, 2013, and 2014 (Figures 3B-6B) even though the level of activity was also rather low in Bielefeld, Giessen, and Regensburg in The decrease of tick activity from 2011 to 2012 was significant at the locations Bielefeld, Giessen, and Regensburg and distinct but not significant in Berlin. The latter may have been caused by the forced change Table 3. Selected data of Ixodes ricinus nymphal questing (relative activity) in field plots at four different locations in Germany (Berlin, Bielefeld, Giessen, Regensburg) in the years Location Year Annual maximum of tick activity (% of released ticks) Annual duration of tick activity (time span in days from first to last day with 1% activity) Berlin Bielefeld Giessen Regensburg Ecology and prevention of Lyme borreliosis

344 24. Weather and Ixodes ricinus in Germany in the year 2012 of the flagging area from 2011 to 2012 (though the Berlin field-plot area was not changed). The mean annual density of active nymphs was lower than 10/100 m 2 at all the locations in The increase of the mean annual density of active nymphs from 2012 to 2013 was The numbers of collected I. ricinus adults in the field were too low to give reliable results, so those results are not presented here. An unusually low abundance of questing I. ricinus nymphs in 2012 was confirmed by other tick workers in various parts of Germany (U. Mackenstedt and G. Dobler personal communication). Moreover, our working group and that of U. Mackenstedt found that I. ricinus collected in 2012 were often not very viable, and many of them died quickly when taken to the laboratory, a very unusual finding. TBE and Lyme borreliosis incidences in 2012 The nymphal stage of I. ricinus is evidently responsible for most human tick bites (Maiwald et al. 1998, Robertson et al. 2000, Wilhelmsson et al. 2013), making it the most important life stage transmitting infectious agents to humans. Therefore, nymphal abundance plays an important role in tick-borne disease incidence, although other factors, e.g. human behaviour (exposure), may also be important (Randolph et al. 2008). Thus, a low abundance of I. ricinus nymphs as in 2012 might result in a comparatively low number of human tick bites, which in turn might result in a relatively low incidence of tick-borne disease. Indeed, the number of reported cases of both TBE and Lyme borreliosis in Germany reached a distinct minimum in 2012 (Figure 7 and 8). A total of 195 reported TBE cases for the whole of Germany was a historical low figure (mean value : 310 cases), and the same is true for Lyme borreliosis in 2012 with 3,143 cases in eastern Germany (the Lyme borreliosis statistics up to 2012 covered only eastern Germany; mean value : 4,922 cases) Germany Selected districts Number of TBE cases Year Figure 7. Annual numbers of the reported human tick-borne encephalitis (TBE) cases in whole Germany vs in selected administrative districts of Germany ( Ecology and prevention of Lyme borreliosis 343

345 Hans Dautel, Daniel Kämmer and Olaf Kahl 7,000 6,000 Number of LB cases 5,000 4,000 3,000 2, Figure 8. Annual numbers of the reported human Lyme borreliosis (LB) cases in the German federal states of Brandenburg, Mecklenburg/West Pomerania, Saxony, Saxony-Anhalt, and Thuringia ( Content/Infekt/SurvStat/survstat_node.html). Year Probable cause of the strong decrease of Ixodes ricinus abundance from 2011 to 2012 The question is which extrinsic factor(s) caused the dramatic decrease in tick abundance in many parts of Germany in the same year. It seems very doubtful that any biotic factor can be consistently effective over such a large area in one and the same year, but weather may well have such a largescale uniform influence. The decrease of tick activity in 2012 when compared to that in 2011 was most distinct at the locations Bielefeld and Giessen where there was almost no snow cover during the cold spell mentioned above. It was less distinct in Berlin and Regensburg where there was at least some snow cover. As snow cover acts as a temperature buffer, it might have protected the ticks in the subjacent leaf litter against the cold (Jore et al. 2014, cf. Medlock et al. 2013). The lower lethal temperature, i.e. the temperature at which 50% of the ticks do not survive a 24-hour exposure, was found to be C in unfed I. ricinus nymphs (Dautel and Knülle 1997). Temperatures below that value (and also slightly above; cf. Gray 1981) might be potentially detrimental to I. ricinus nymphs, especially if they occur repeatedly. In the leaf litter not covered by any snow in Bielefeld and Giessen and only weakly covered at the location Berlin for days (Figure 1), microclimatic temperatures might have been low enough for many I. ricinus nymphs and adults to be damaged or even killed. Unfortunately, there are no comparable tick activity data from other locations in central Europe for 2011 and 2012 to test the hypothesis that a cover of snow protects I. ricinus ticks from low temperatures during extreme cold spells. As an alternative, we tried to correlate snow depth during the cold spell in early 2012 with the extent of decrease of TBE incidence from 2011 to 2012 for different administrative districts in southern Germany, being aware that the relationship between tick abundance and TBE incidence is indirect and also other factors may be influential. In a comparable approach, Diuk-Wasser et al. (2012) took the density of host-seeking I. scapularis Say, 1821, nymphs infected with Borrelia burgdorferi sensu stricto as an indicator for the local risk of humans to acquire an infection with the causative agent of Lyme disease in the eastern United States. Because the infection rate of questing I. ricinus nymphs with the TBE virus in endemic 344 Ecology and prevention of Lyme borreliosis

346 24. Weather and Ixodes ricinus in Germany in the year 2012 areas is almost always <1%, i.e. very constant, it appears reasonable to take the abundance of all questing I. ricinus nymphs as an indicator of infected nymphal tick density here. As shown in Figure 7, 64.4% of all the annual TBE cases in Germany from 2001 to 2015 occurred in the selected 7 administrative districts. The regression analysis was performed with the decrease of TBE case numbers from 2011 to 2012 as the dependent parameter and minimum snow depth during the cold spell as an independent parameter. In the first attempt, we included the administrative district Stuttgart, but later regarded this as an outlier, far outside the 95% confidence interval. Figure 9 shows the regression without this district. The correlation between minimal snow depth during that cold spell and the amount of the local decrease of TBE incidence from 2011 to 2012 is highly significant, and the correlation coefficient is very high as well. This result indirectly supports the hypothesis that the amount of snow cover is important for I. ricinus survival during cold periods and might have a strong protective effect on overwintering ticks especially during extreme cold spells. Concluding remarks The occurrence of a day long extreme cold spell throughout Germany between late January and mid-february 2012 during an ongoing field investigation provided an unusual opportunity to investigate the effect of extreme cold on I. ricinus survival as determined by questing activity before and after the cold spell. This was done by two different methods, the classic flagging method and the newly developed field-plot method at four locations in Germany. Circumstantial evidence suggests that the prevailing daily minimum temperatures killed many I. ricinus ticks and/or affected the fitness of many survivors. The effects on questing tick abundance were most 8 7 r 2 =0.9772; r= ; P= * Stuttgart Minimal snow depth (cm) * Reduction of TBE cases from 2011 to 2012 (%) Figure 9. Decrease of tick-borne encephalitis (TBE) case numbers from 2011 to 2012 for each of the south German administrative districts Freiburg, Karlsruhe, Lower Franconia, Central Franconia, Lower Bavaria, Upper Bavaria and Upper Palatinate as a function of the minimal snow depth during an extreme cold spell from late January to mid-february The administrative district Stuttgart was taken as an outlier and excluded from the analysis (marked by a star) (source of snow-depth data: MeteoGroup Deutschland GmbH, Berlin, Germany). Ecology and prevention of Lyme borreliosis 345

347 Hans Dautel, Daniel Kämmer and Olaf Kahl severe in the western locations Bielefeld and Giessen, where there was virtually no snow cover during the cold spell. In contrast, the effect of the low temperatures on the ticks was negligible at the (coldest) location Regensburg where there was a continuous cover of snow during the cold spell, even if it was only 4-8 cm deep. The tick situation at the location Berlin 2012 versus 2011 was intermediate and so was also the snow cover during that cold period (snow depth 0-5 cm). Jore et al. (2014) working in southern Norway close to the northern border of the distribution area of I. ricinus took the seroprevalence of sheep against Anaplasma phagocytophilum as an indicator of the level of exposure of sheep to I. ricinus, i.e. to its local presence or absence, since this tick species is the only known local vector of that bacterium. They observed that the yearly number of days with black frost (i.e. temperatures <0 C without any snow cover) and the duration of snow cover were among the six variables explaining most of the variation. They concluded: Snow cover affects the survival of ticks during winter (Gray and Male, 1981), as it increases ground surface temperatures (Isaksen et al. 2011). The significance of the number of days with black frost might reflect that absence of snow cover during the winter is far more crucial for I. ricinus than the actual exposure temperature. This might be an important point when building climate models to I. ricinus abundance. There are some more papers stating that snow cover is important for the overwinter survival of I. ricinus in areas with cold winters (e.g. Medlock et al. 2013), but to our best knowledge further experimental evidence for this does not exist. However, Lindsay et al. (1995) concluded that overwinter survival of adult I. scapularis was better at the northern than at the southern sites in Ontario, southern Canada, because of a greater and more continuous snow cover in the north during the winter months. Lindgren et al. (2000) who did not consider the influence of snow cover found that the northward shift of the northern distribution limit of I. ricinus in Sweden from the early 1980s to the mid-1990s was related to fewer days with temperatures lower than -12 C. These findings support the view that very low temperatures such as in Germany in January/February 2012 might be dangerous for I. ricinus. We were surprised that I. ricinus field abundance had mostly returned by 2013 to similar levels as observed in 2011, before the cold spell in 2012 (Figures 3B-6B). Daniel et al. (2015) found that extreme weather events such as extended warm and dry conditions during the tick season impact I. ricinus questing, but similar to the findings in the present study, tick numbers recovered quickly in subsequent years. We have no well-founded explanation of this phenomenon, but it appears impossible that individual nymphs skipped their activity in 2012 and delayed their host-seeking activity to It is notable that the very low I. ricinus abundance in 2012 coincided with a very low TBE incidence in Germany in the same year. Our spatial regression analysis correlating snow depth during the cold spell with the decrease of the local TBE incidence from 2011 to 2012 in different southern German administrative districts resulted in high statistical significance as well as a very high correlation coefficient. The results presented here on tick abundance in 2012 strongly suggest that the extreme cold spell in Germany from late January to mid-february killed many I. ricinus and damaged many survivors especially in those extensive areas where black frost occurred during that period. It seems that the much reduced I. ricinus abundance in these areas led to a decreased density of TBE virus- 346 Ecology and prevention of Lyme borreliosis

348 24. Weather and Ixodes ricinus in Germany in the year 2012 infected ticks in endemic areas with the result that the number of human TBE cases reached a historical trough in It might be much more difficult in other years to find out which factors may have caused changes in I. ricinus abundance and TBE incidence, respectively. Jaenson et al. (2012) gave an instructive example how two successive cold and snowy winters in Sweden might have caused an increase in TBE incidence because many roe deer died and, as a result, I. ricinus immatures probably fed more frequently on abundant bank voles (Myodes glareolus) and other small mammals, which are reservoir hosts of the TBE virus. It is a standard statement in papers on the ecology of I. ricinus that this tick might benefit from milder winters in the future in central and northern Europe. This might be true for areas close to the most northern I. ricinus distribution provided sufficient snow still occurs during the whole winter (Jaenson et al. 2009). But if in the course of climate warming as a side effect the number of winter days with severe black frost increases, this could in fact increase tick mortality in parts of central Europe, also in locations at higher altitudes. This means that milder mean temperatures of winters per se are not necessarily predictive for subsequent I. ricinus performance and that it is usually insufficient to take monthly mean temperatures to explain complex ecological processes. Since our study suggests that winter survival can be a key ecological factor determining the abundance of I. ricinus in central and northern Europe, this topic requires further research in a climate-change setting. Public health relevance Weather conditions affect the seasonal pattern of Ixodes ricinus activity and abundance and therefore also the number of cases of tick-borne disease such as TBE. Weather effects on I. ricinus activity and abundance can be very local. Extremely cold weather conditions combined with a lack of snow cover had a negative effect on I. ricinus abundance, resulting in a lower number of TBE cases in the following season. Acknowledgements The study was supported by the German Federal Environmental Agency (projects FKZ and FKZ ). We appreciate the excellent technical assistance by all the co-workers who carried out the tick flagging and made tick observations at the field plots. We are grateful to Jeremy Gray who read a late version of the manuscript. References Daniel M, Malý M, Danielová V, Kříž B and Nuttall P (2015) Abiotic predictors and annual seasonal dynamics of Ixodes ricinus, the major disease vector of Central Europe. Parasit Vectors 8: 478. Dautel H and Knülle W (1997) Cold hardiness, supercooling ability and causes of low-temperature mortality in the soft tick, Argas reflexus, and the hard tick, Ixodes ricinus (Acari: Ixodoidea) from Central Europe. J Insect Physiol 43: Ecology and prevention of Lyme borreliosis 347

349 Hans Dautel, Daniel Kämmer and Olaf Kahl Dautel H, Dippel C, Kämmer D, Werkhausen A and Kahl O (2008) Winter activity of Ixodes ricinus in a Berlin forest. Int J Med Microbiol 298: Diuk-Wasser MA, Hoen AG, Cislo P, Brinkerhoff R, Hamer SA, Rowland M, Cortinas R, Vourc h G, Melton F, Hickling GJ, Tsao JI, Bunikis J, Barbour AG, Kitron U, Piesman J and Fish D (2012) Human risk of infection with Borrelia burgdorferi, the Lyme disease agent, in Eastern United States. Am J Trop Med Hyg 86: Estrada-Peña A, Nava S and Petney T (2014) Description of all the stages of Ixodes inopinatus n. sp. (Acari: Ixodidae). Ticks Tick Borne Dis 6: Gray JS (1981) The fecundity of Ixodes ricinus L. (Acarina: Ixodidae) and the mortality of its developmental stages under field conditions. Bull Entomol Res 71: Gray JS (1991) The development and seasonal activity of the tick Ixodes ricinus: a vector of Lyme borreliosis. Rev Med Vet Entomol 79: Gray JS (2008) Ixodes ricinus seasonal activity: implications of global warming indicated by revisiting tick and weather data. Internat J Med Microbiol 298 S1: Gray DM and Male DH (1981) Snow and climate. In: Male DH (ed.) Handbook of snow: principles, processes, management and use. Pergamon, Toronto, Canada. Isaksen K, Odegard RS, Etzelmüller B, Hilbich C, Hauck C, Farbrot H, Eiken T, Hygen HO and Hipp TF (2011) Degrading mountain permafrost in southern Norway: spatial and temporal variability of mean ground temperatures, Permafrost Periglac Process 22: Jaenson TGT, Eisen L, Comstedt P, Mejlon HA, Lindgren E, Bergström S, Olsén B. (2009) Risk indicators for Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden. Med Vet Entomol 23: Jaenson TGT, Hjertqvist M, Bergström T, Lundkvist A (2012) Why is tick-borne encephalitis increasing? A review of the key factors causing the increasing incidence of human TBE in Sweden. Parasit Vectors 5: 184. Jore S, Vanwambeke SO, Viljugrein H, Isaksen K, Kristoffersen AB, Woldehiwet Z, Johansen B, Brun E, Brun-Hansen H, Westermann S, Larsen I-L, Ytrehus B and Hofshagen M (2014) Climate and environmental change drives Ixodes ricinus geographical expansion at the northern range margin. Parasit Vectors 7: 11. Kahl O and Knülle W (1988) Wirtssuchaktivität der Schildzecke Ixodes ricinus (Acari: Ixodidae) und ihre Durchseuchung mit Lyme-Spirochäten und dem Frühsommer-Meningoenzephalitis (FSME)-Virus in Berlin (West). Mitt Dtsch Ges Allg Angew Entomol 6: Lindgren E, Tälleklint L and Polfeldt T (2000) Impact of climatic change on the northern latitude limit and population density of the disease transmitting European tick Ixodes ricinus. Environ Health Perspect 108: Lindsay LR, Barker IK, Surgeoner GA, McEwen SA, Gillespie TJ and Robinson JT (1995) Survival and development of Ixodes scapularis (Acari: Ixodidae) under various climatic conditions in Ontario, Canada. J Med Entomol 32: Maiwald M, Oehme R, March O, Petney TN, Kimmig P, Naser K, Zappe HA, Hassler D and von Knebel Doeberitz M (1998) Transmission risk of Borrelia burgdorferi sensu lato from Ixodes ricinus ticks to humans in southwest Germany. Epidemiol Infect 121: Medlock JM, Hansford KM, Bormane A, Derdakova M, Estrada-Peña A, George JC, Golovljova I, Jaenson TG, Jensen JK, Jensen PM, Kazimirova M, Oteo JA, Papa A, Pfister K, Plantard O, Randolph SE, Rizzoli A, Santos-Silva MM, Sprong H, Vial L, Hendrickx G, Zeller H and Van Bortel W (2013) Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe. Parasit Vectors 6: 1. Morán Cadenas F, Rais O, Jouda F, Douet V, Humair P-F, Moret J and Gern L (2007) Phenology of Ixodes ricinus and infection with Borrelia burgdorferi sensu lato along a north- and south-facing altitudinal gradient on Chaumont Mountain, Switzerland. J Med Entomol 44: Perret J-L, Guigoz E, Rais O and Gern L (2000) Influence of saturation deficit and temperature on Ixodes ricinus tick questing activity in a Lyme borreliosis-endemic area (Switzerland). Parasitol Res 86: Randolph SE, Asokliene L, Avsic-Zupanc T, Bormane A, Burri C, Gern L, Golovljova I, Hubalek Z, Knap N, Kondrusik M, Kupca A, Pejcoch M, Vasilenko V and Zygutiene M (2008) Variable spikes in tick-borne encephalitis incidence in 2006 independent of variable tick abundance but related to weather. Parasit Vectors 1: 44. Robertson JN, Gray JS and Stewart P (2000) Tick bite and Lyme borreliosis risk at a recreational site in England. Eur J Epidemiol 16: Ecology and prevention of Lyme borreliosis

350 24. Weather and Ixodes ricinus in Germany in the year 2012 Rubel F and Kottek M (2010) Observed and projected climate shifts depicted by world maps of the Köppen- Geiger climate classification. Meteorol Z 19: Sachs L and Hedderich J (2006) Angewandte Statistik (12 th Ed.). Springer Verlag, Berlin, Heidelberg, Germany, p Sonenshine DE (1993) Biology of ticks. Oxford University Press, Oxford, UK, pp Wilhelmsson P, Lindblom P, Fryland L, Nyman D, Jaenson TG, Forsberg P and Lindgren PE (2013) Ixodes ricinus ticks removed from humans in northern Europe: seasonal pattern of infestation, attachment sites and duration of feeding. Parasit Vectors 6: 362. Ecology and prevention of Lyme borreliosis 349

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352 25. Grasping risk mapping Marieta A.H. Braks 1*, Annemieke C. Mulder 1, Arno Swart 1 and William Wint 2 1 National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; 2 Environmental Research Group Oxford, Department of Zoology, University of Oxford, South Parks Road, OX1 3PS, United Kingdom; marieta.braks@rivm.nl Abstract A picture is worth a thousand words, and so many Lyme borreliosis risk maps have been produced and published in the scientific and grey literature or on websites. The focus of these is usually on the technical and scientific aspects, and facilitating interpretation by their users is frequently overlooked. Designing the maps is made difficult by the complexities of the disease system, the often black box methods used to produce the models from which the maps are derived, and the fact that many of the maps are of risk indicators rather than direct risk measures used by the public health community. In the current chapter, we explore the various research questions, data and tools that underlie available risk maps. We investigate how risk maps fit in a risk analysis framework of Lyme borreliosis, including risk assessment, risk management and risk communication, exemplified with risk maps at Pan-European, national and local scale. Keywords: exposure, hazard, risk maps, risk analyses, scale Introduction Risk maps used in public health are visual representations of intricate geographic data that provide a quick overview of the health risks. Ample examples of Lyme borreliosis risk maps for Europe and the United States have emerged, usually depicting the spatial distribution of (infected) ticks or (less often) disease incidence. The users often interpret these intuitively, rather than objectively understanding the full scope of the model output, which is often derived from spatial modelling techniques (Mannelli et al. 2016, Vanwambeke et al. 2016a). The intuitive aspect of a risk map is both its strength and its weakness. In this chapter, we aim to provide some guidance on the objective interpretation of risk maps. First, we examine two topics that largely determine the features of a risk map, namely: (1) the underlying research question (what kind of risk metric is mapped and for whom and why?); and (2) availability of data and tools (how can available data be mapped usefully?). Second, we review different kinds of risk maps available for Lyme borreliosis, with special attention to those concerning the Netherlands. It is not our objective to review the various spatial modelling techniques (Ozdenerol 2015), but rather to evaluate the utility of the output maps for risk analysis of Lyme borreliosis. In conclusion, we provide guidance for the stakeholders (i.e. risk map consumers) and for researchers (i.e. map developers) confronted with these issues. Research question While most people have strong intuitive ideas about what risk entails, defining, interpreting and subsequently dealing with risks in evidence-based policy making appears to be rather an intricate task. Because risk can mean different things to different people or in different circumstances, risk cannot be captured in a single definition. An important source of confusion in the interpretation is Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _25, Wageningen Academic Publishers 2016

353 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint the probabilistic nature of risk. Risk is an uncertain, generally adverse, consequence of an event or activity with respect to something that human beings value. Strictly speaking, any kind of measure of risk describes the probability for a certain hazard to occur combined with the exposure to that hazard. The most basic risk definition is exposure times hazard. In public health terms, risk has been defined by medical epidemiologists and health organisations as the probability of disease developing in an individual within a specified time interval (Sedda et al. 2014). For many infectious diseases, particularly the vector borne ones including Lyme borreliosis (Rousseau 2015), environmental conditions are a strong predictor of risk. Maps have become increasingly important in assessing the spatial distribution of diseases and are now powerful tools in public health risk analyses (Kraemer et al. 2016). In its most basic form, risk analysis is a framework for the assessment and subsequent management and/or control of the danger posed by an identified disease threat, and includes communication to stakeholders (Sedda et al. 2014). In general terms, risk mapping fits this framework as follows: 1. Risk assessment: a. Descriptive mapping of risks to localise the problem areas or hotspots. b. Predictive mapping using spatial models to produce more detailed and complete maps than those only using the records available, and to provide insight into the complex ecological mechanisms of diseases and to identify associated risk factors, enabling projections in space and time. 2. Risk management: a. Risk mapping to identify areas where intervention is most needed or most cost-effective. b. Risk mapping to help to analyse and/or evaluate interventions or precautions that can reduce the risk 3. Risk communication: a. Risk mapping to inform stakeholders in advance about where and when risks are highest (or lowest), or where and what interventions are useful. One of the preconditions for effective prevention of tick borne diseases including Lyme borreliosis is the determination of both the dynamics of the vector Ixodes ricinus in time and space and the risk of people being bitten by a tick (Daniel et al. 2006). The associated risk is often defined using the following entomological risk metrics: density of nymphs (DON), infection prevalence of Borrelia burdorferi spp. in nymphs (NIP), and the density of infected nymphs calculated by multiplying the DON with NIP (Salkeld et al. 2015). These entomological disease risk measures are therefore actually hazard proxies. A hazard may not lead to a risk. If someone is aware of a hazard, they can do something about it, (e.g. wearing protective clothing against ticks) and it stops being a risk if the precautions taken are effective. A risk in a particular place may vary over time. For example, tick activity depends on temperature so if it is cold, there may be plenty of ticks but they will be inactive. Alternatively, a place may have a high risk of tick borne encephalitis (TBE), but not to people who have been vaccinated. The translation or interpretation from risk maps based on single metrics to risks for an individual is therefore not straightforward because link between risk and hazard is governed by a great number of modifying factors. Due to the various uses a risk maps can have in risk governance and the complexity of Lyme borreliosis, the metrics used for risk are not uniquely defined (Sedda et al. 2014) and may well be constrained by pragmatic concerns such as availability of reliable data (discussed below). Maps can show risk in a variety of ways: e.g. the likelihood of disease introduction, spread, or 352 Ecology and prevention of Lyme borreliosis

354 25. Grasping risk mapping transmission; the number of human cases; and host/vector occurrence or infection status. There are also other elements of a risk map which are of importance, such as its spatial extent and level of detail (resolution); and whether it depicts change or is a static snapshot. A risk-map may also be developed to evaluate risk-factors rather than to predict (or project) risk. Furthermore risk maps may incorporate some measure of uncertainty to show how reliable the map is estimated to be. For both the stakeholders asking for risk maps and for the map developers responding to these requests, it is essential to define and agree on the purpose of the risk map. Within this process, it is important to clearly identify the targeted audience and to carefully consider to whom the risk of interest is posed and for which unit of space and time. Data and tools available It is the availability of data and tools that largely define the technical content of a risk map. For data on the occurrence of Lyme borreliosis, information can be derived at different levels in the human population. For the forecasting, early detection, prevention and control, data on the presence and density of (infected) ticks and (infected) reservoir hosts is required (Table 1) (Braks et al. 2011, 2014a). The data of the different subgroups can be represented as different layers in a surveillance pyramid. The absolute numbers of each layer is affected by the spatial scale considered (global, continental, national or local) but also by the temporal scale (per decade, year, month or for the ticks even per day) (Figure 1). Though an end-user will hardly ever (be able to) investigate the quality of the data that drive the mapping process, it is important to be aware of its quality when using the resulting map: the old adage garbage in, garbage out definitely applies. The data associated with each layer can be acquired through either passive or active collection. Passive activities involve mandatory reporting of cases by laboratories and clinicians, but also by voluntary reporting of events such as erythema migrans or tick bites in citizen science projects (Mulder et al. 2013). Active systems involve searching for evidence of disease (Hofhuis et al. 2015) or proxies through historical (e.g. museum or literature) records, routine, or project-based data collection (Braks et al. 2011). Active field surveillance provides the most accurate and timely information, but is also expensive. Passive activities are less expensive as they rely on reports from third parties. They often provide longer time-series, but the quantity of mandatory data is often limited (see below), and the quality of volunteered data more heterogeneous and less reliable (Garcia-Martí et al. in press). In summary, observational data from various sources can contribute to risk mapping as long as its quantity and quality are understood when constructing and interpreting the map. Data from either passive or active collection can never supply the full picture, since these are a limited set of samples from populations, taken at a specific time and place. Extrapolation of tick presence or abundance to areas that have not been sampled is desirable, but also very challenging. Ticks, the pathogens they carry, and their hosts, have a complex ecology, affected by various biotic and abiotic factors as shown in the preceding chapters of this book. In addition, the complexity of spatial prediction increases further with each ascending layer of the surveillance pyramid (Figure 1): the landscape configuration is known to influence the spatial spread of Lyme borreliosis not only by affecting tick demography, but also human exposure to infected ticks (Barrios et al. 2013, Garcia-Martí et al. in press). Predictive mapping techniques using spatial models have been developed to provide insight into the complex ecological mechanisms of diseases or to identify associated risk factors, Ecology and prevention of Lyme borreliosis 353

355 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint Table 1. Descriptive maps. Area Spatial aspects Dependent variable 1 Reference T E P D World distribution of records reported Lyme disease activity, infected ticks, infected animals, and seropositive human samples Ozdenerol (2015) Europe distribution of records tick distribution (P/A) Mead et al. (2015) USA distribution of records reported Lyme disease cases Mead et al. (2015) Russia distribution of records morbidity and incidence of Lyme borreliosis Malkhazova et al. (2014) Texas USA spatial clustering of cases reported Lyme disease cases Szonyi et al. (2015) Ontario, CA distribution of records tick collection (P/A) and pathogen prevalence Clow et al. (2016) Romania distribution of records tick collection (P/A) and pathogen prevalence Kalmar et al. (2013) States, USA change of spatial clustering of cases reported Lyme disease cases Lantos et al. (2015) S. Bohemia, CZ distribution of records tick collection (P/A) and pathogen prevalence Honig et al. (2015) Southern Canada change of spatial clustering of cases tick distribution (P/A) and sequences/haplotypes of Ogden et al. (2014) tick and pathogen Germany distribution of records tick collection (P/A and species) Rubel et al. (2014) South Norway distribution of records tick collection (P/A) Vanwambeke et al. (2016b) 2 Scotland distribution of records tick collection (P/A) and pathogen prevalence James et al. (2013) 2 the Netherlands distribution of records voluntary tick bite reports/100,000 per province the Netherlands distribution of records GP reports on tick bites/100,000 Hofhuis et al. (2015) the Netherlands distribution of records GP reports on EM/100,000 Hofhuis et al. (2015) the Netherlands distribution of records tick collection (P/A) and pathogen prevalence Coipan et al. (2013) Belgium distribution of records tick collection (abundance and species) Obsomer et al. (2013) Temporal aspects USA change in distribution and abundance 2 decades of tick distribution Eisen et al. (2016) Great Britain change in distribution and abundance historical data on tick density and on deer sighting Scharlemann et al. (2008) Sweden change in distribution tick report (P/A) in questionnaire Lindgren et al. (2000) Sweden change in distribution and abundance historical data on tick density Jaenson et al. (2009) Sweden change in distribution and abundance historical data on tick density Jaenson et al. (2012) South Norway change in distribution of records Anaplasma phagocytophilum antibodies in sheep as Jore et al. (2014) 2 indicators for tick presence 1 P/A = presence/absence; EM = erythema migrans; GP = general practitioner; T = tick; E = exposure; P = pathogen; D = disease. 2 These papers include spatial models, but the maps presented do not include modelled data. 354 Ecology and prevention of Lyme borreliosis

356 25. Grasping risk mapping Risky physiology Chronic Disseminated Risky behaviour Erythema migrans Tick bites People exposed in tick habitats Risky landscapes Infected ( questing ) ticks Wildlife (pathogen host and tick reproduction host) scale Temporal Risky climates Spatial scale Figure 1. Surveillance pyramid for Lyme borreliosis, with decreasing numbers towards the top of the pyramid. In addition to these relative numbers, the absolute numbers of each layer is affected by the spatial scale considered (global, continental, national or local) but also by the temporal scale (per decade, year, month or for the ticks even per day). The drivers of the layers are categorised in four colours. enabling predictions and projections in space and time. There are two main spatial risk-modelling approaches: (1) empirical-statistical models that quantify the correlation between observational data and a number of environmental variables using statistical methods; and (2) mechanistic or process-based models that quantify these relationships based on proven causality, in which the biological and epidemiological aspects of the process are known and taken into account (see also Mannelli et al. 2016, Vanwambeke et al. 2016a). Multiple criteria decision analyses (MCDA) are included in the latter category, even though they are not proper process-based models, as they are based on sequential application of weighted filters. Process-based models need to use quantified relationships between factors and the target variable. For either methodology, explanatory data can be categorised according various dimensions, including data subject (human, vector, host, environmental), data nature (biotic, abiotic), data characteristic (counts, measurements, proxies), data property (continuous, categorical), data collection method (ground truthing, land use maps, aerial photos, satellite imaging, model output), spatial resolution (from point data up to 20 km 2 remote sensing data), data aggregation level (georeferenced points, NUTS 1 level, national level), temporal scale (static, monthly, yearly, unstructured) and data scale (micro, local, regional, national, continental, global). As a result, a plethora of model outputs have emerged from the wide variety of studies using these associations to estimate and map risk. Until fairly recently, most of the time and energy needed for mapping was directed to the collection of environmental data, construction of explanatory spatial layers and laborious spatial analyses. 1 The Nomenclature of Territorial Units for Statistics, NUTS abbreviated to the French Nomenclature des Unités Statistiques Territoriales, a coherent system for the classification of the territory of the European Union with a view to the compilation of regional statistics. Ecology and prevention of Lyme borreliosis 355

357 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint Desktop GIS applications, along with much increased computing power and storage capacity, and the explosion of open source tools and utilities have enabled virtually anybody to produce intuitively appealing maps. Appealing, however, may not mean accurate, and so uncertainty and sensitivity analyses are essential to assess the accuracy and robustness of a study (Barrios et al. 2013). It is also important to realise that model outputs can be statistically significant in technical terms, but may not be biologically significant or relevant to policy needs. For example, a policy maker may need to know best and worst case scenarios, which cannot be extracted from a map that shows average risk. Estrada-Peña et al. (2016) acknowledged that the best methods for modelling the impact of environmental variables on the tick-pathogen cycles are mechanistic. However, the lack of detailed data about the processes involved means that correlative distribution modelling is extensively used instead. They also highlighted the better performance of climatic covariates that are obtained from remotely sensed information compared to interpolated explanatory variables derived from ground measurements. The latter are often flawed with internal issues affecting modelling performance. Further, mapping exercises alone were considered to be not enough to understand the factors underlying risk. A multidisciplinary approach is needed to derive conclusions about the ecological drivers from such models. Here, however, we focus only on the mapped spatial modelling outputs. Ideally, the interpretation of spatial model outputs leads to biologically meaningful conclusions that can be validated in the real world in an iterative manner. The challenge has therefore shifted from making maps into making sensible maps (Estrada-Peña et al. 2016). Lyme borreliosis risk maps Numerous Lyme borreliosis risk maps can be found in peer reviewed and grey literature. An overview of descriptive and explanatory/predictive maps of several kinds of risk metric for Lyme borreliosis is shown in Table 1, in decreasing order of scale. This table was compiled on the basis of examples rather than an exhaustive literature search, since inclusion of a map in a paper cannot be always be deduced from titles or keywords. In the following, we look at their utility within the risk analyses framework and aim to illustrate the issues raised in the preceding rather theoretical disquisition, and provide direction into improving the effectiveness of risk maps. Risk assessment The majority of studies has been concerned with the risk assessment part of risk analysis that includes predictive mapping and the identification of vulnerable groups (Table 1 and 2). In these studies, the associations between environmental factors and elements of tick-borne disease transmission systems are investigated in many different ways (Vanwambeke et al. 2016a). Recently, this has resulted in an explosion of works claiming to have solved problems raised in previous or other approaches. They did this by addressing important, but mostly methodological, issues (Estrada-Peña et al. 2015). Scale Looking at the available Lyme borreliosis risk maps (Table 1), the first thing that is noticeable is the range of scales, from world (Estrada-Peña et al. 2015) to recreational area (Dobson et al. 2011). This results from the fact that environmental factors highlighted for ticks, their hosts, and 356 Ecology and prevention of Lyme borreliosis

358 25. Grasping risk mapping Table 2. Explanatory/predictive maps. Area Map output Dependent variable 1 Independent variable 2 Modelling 3 Reference T E P D H EAB EB In space Western Palearctic prediction of tick infection rate Europe tick habitat suitability (between 0-1) Canada northern geographic limit of tick North Central USA Central Eastern USA tick habitat suitability (4 categories) tick habitat suitability over years (between 0-1) Texas, USA spatial concordance between tick habitat suitability and disease incidence Minnosota, USA California county, USA habitat suitability and increase in distribution tick abundance and pathogen species prevalence temperature, connectivity, vegetation stress tick (P/A) climate, greenness, masking tick survival and seasonality temperature limits, degree days tick abundance soil, forest moisture, rodents, land cover, elevation, climate tick abundance ground temperature, greenness tick presence and pathogen species prevalence and disease cases elevation, temperature, relative humidity, land cover/use, soil texture tick distribution records land cover, rainfall, temperatures tick habitat suitability tick (P/A) tree species, greenness, slope, elevation, aspect, solar insolation precipitation Ireland habitat suitability tick presence and abundance forest composition and fragmentation, hosts (deer and sheep), climatic (temperature and rainfall) ESM Estrada-Peña et al. (2011) ESM com/z79ptpa MM Ogden et al. (2004) ESM Guerra et al. (2002) ESM Estrada-Peña (2001) MM Atkinson et al. (2014) ESM Johnson et al. (2016) ESM Eisen et al. (2006a) ESM, MM/ MCDA Rousseau (2015) Ecology and prevention of Lyme borreliosis 357

359 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint Table 2. Continued. Area Map output Dependent variable 1 Independent variable 2 Modelling 3 Reference T E P D H EAB EB California county, USA the Netherlands projected acaralogical risk tick habitat suitability (between 0-1) Wales habitat suitability (4 categories) Southern Germany Nature reserve S Germany Amsterdam, the Netherlands estimating tick density on landscape scale tick abundance and disease records forest cover ESM Eisen et al. (2006b) tick (P/A) tick bite consultation, deer densities, greenness, temperature proportion of 10 m cloth drags with Borrelia positive ticks aspect, soil, geology, slope, land cover tick abundance sea level, land cover, air temperature, relative humidity saturation deficit distribution of ticks tick abundance air temperature, relative humidity, soil water content, vegetation type spatial prediction tick occurrences Forest, France human risk of infection with Borrelia Recreational sites, UK predicting seasonal tick abundance (10 categories) tick abundance vegetation structure, distance to water or build up tick abundance and pathogen species prevalence (DIN, DON, IR) tick abundance in space and time temperature, humidity, roe deer, wild boar, chipmunks, tree composition, ground cover, vegetation, temperature, relative humidity ESM Swart et al. (2014) ESM Medlock et al. (2008) ESM Boehnke et al. (2015) ESM Schwarz et al. (2009) ESM Gao (2015) ESM Vourc h et al. (2016) MM/MCDA Dobson et al. (2011) 358 Ecology and prevention of Lyme borreliosis

360 25. Grasping risk mapping Table 2. Continued. Area Map output Dependent variable 1 Independent variable 2 Modelling 3 Reference T E P D H EAB EB In time Eastern USA climate-based tick habitat suitability three future periods States USA, Mexico tick habitat suitability with projections on climate scenarios Europe changes in tick habitat suitability with projections on climate scenarios Europe tick habitat suitability with projections on climate scenarios tick abundance (activity) infected and noninfected ticks (P/A) France seasonal tick activity tick abundance (activity) temperature, precipitation, deciduous forest cover ESM Brownstein et al. (2005) temperature, precipitation ESM Feria-Arroyo et al. (2014) tick activity and density temperature, precipitation, NVDI tick (from Britain) solar radiation, precipitation, temperature, GARP ESM, MM Estrada-Peña and Venzal (2006) ESM Boeckmann and Joyner (2014) temperature, terrain MM Beugnet et al. (2009) 1 P/A = presence/absence; DIN = density of infected nymphs; DON = density of nymphs; IR = infection rate; T = tick; E = exposure; P = pathogen; D = disease. 2 NVDI = normalised difference vegetation index; GARP = genetic algorithm for rule set production; H = host; Env AB = environmental abiotic variable; Env B = environmental biotic variable. 3 ESM = emperical/statistical modelling; MM = mathematical modelling; MCDA = multiple criteria decision analyses. Ecology and prevention of Lyme borreliosis 359

361 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint their pathogens are considered to act differently at different scales, from global (>10,000 km), continental-regional (200-10,000 km), landscape ( km), local (1-10 km), site (1-1,000 m) to even micro scale (<1 m) (Boehnke et al. 2015, Estrada-Peña et al. 2014, Pearson and Dawson 2003). Factors can act at more than one scale and sometimes act differently at different scales (Pearson and Dawson 2003). In addition, factors acting at the same scale can act differently in different parts of the same study area. This was elegantly shown for the epidemiology of a non-tick-borne zoonotic pathogen, Puumala virus transmitted by bank voles by Zeimes et al. (2014). They showed that the effect of one variable on disease at large scale can result in three different local response scenario s (Zeimes et al. 2014). These scaling phenomena complicate modelling efforts and the interpretation of modelling outputs (Estrada-Peña et al. 2016). Pearson and Dawson (2003) proposed a useful general framework for addressing the environmentspecies relationships using a hierarchy of factors operating at different scales, which was surprisingly generically applicable: Thus, at the continental scale, climate can be considered the dominant factor, whilst at more local scales factors including topography and land-cover type become increasingly important. Further down the hierarchy, if conditions at higher levels are satisfied, factors including biotic interactions and microclimate may become significant. Thus, the distribution of a species in Europe may be primarily defined by climatic tolerances if the data resolution is 50 km 2, whereas as the resolution is downscaled, to perhaps 5 km 2, land-cover type may become the dominant control over species presence. Similarly, as the resolution is downscaled to less than 1 km 2, biotic interactions may become important. A primary driver of disease risk, of course, is contact rate between a disease carrier and a susceptible target. It is essentially a numbers game driven by the numbers of pathogens reaching susceptible organisms. This is determined partly by what affects an organism s distribution and abundance. A general ecological principle is that climatic factors tend to be more important determinants at the edge of a species range, while biotic interactions like competition, predation and parasitism tend to be more significant in core areas. This pattern is a little blurred if an animal needs a specific host or prey (as opposed to being generalist), because the factors that limit the host/prey may override those that affect the animal in question. Besides environmental factors, Estrada-Peña et al. (2014) introduced three additional levels in their hierarchy of spatial scales in zoonotic systems, namely individual, cell and molecular scale, recognising that disease risk entails more than simply environmental factors. The drivers of pathogen transmission act at the individual scale, referring to the differences between the condition and behaviour of individual animal hosts or humans. The drivers for infection act at the cellular scale, on the immune status of a (sensitive) host or infection route and for the outcome of an infection act also on the molecular scale on resistance to certain genotypes (Estrada-Peña et al. 2014). In the surveillance pyramid of Lyme borreliosis, the framework of spatial hierarchy is simplified and indicated by the risky climate, risky landscape, risky behaviour and risky physiology (Figure 1). Mapping different types of risk Within risk assessment, risk maps can be either descriptive or explanatory. In the former, available records are simply drawn on a map. Risk maps concerning disease incidence are usually descriptive, 360 Ecology and prevention of Lyme borreliosis

362 25. Grasping risk mapping mapping occurrence of Lyme cases at national scale or coarser (Table 1 and 2). The majority of risk maps are explanatory and are derived from works aiming to find factors that play important roles in determining occurrence of tick (risky climate) and, to a lesser degree, the density of (infected) ticks (risky landscape). The latter risk maps address the hazard part of the risk equation, omitting the exposure part. In theory, combining the hazard map with a data layer describing the exposure would result in a proper risk map. In practice, information on exposure is more often deduced from both risk and hazard maps. For example, a model developed in a study predicting tick presence by environmental risk mapping for Lyme borreliosis in the Netherlands, had good predictive power for tick presence in the Netherlands. In addition, the tick-bite incidence per municipality correlated significantly with the modelled average probability of tick presence(swart et al. 2014). In principle, information on the exposure could be subsequently derived by subtracting the latter from the former. In a study on TBE (not included in Table 2) using georeferenced disease records, predictive models were developed on the basis of independent variables describing either hazard or exposure or both (Zeimes et al. 2014). Only explanatory variables within a 2 km buffer zone around TBE records were included in the analysis. Hazard was described using data on species, forest and land cover. Exposure was described using data on accessibility and scenic beauty, which are proxies for number of people entering the risky habitat. On a smaller scale, the identification of factors determining risky behaviour within a risky habitat becomes important. There seems to be insufficient understanding of the factors that determine the risk of acquiring tick bites (exposure) (Hofhuis et al. 2015). Actually there are (few) studies that have identified risky behaviour, but always in risky habitats (Dobson et al. 2011, Garcia-Martí et al. in press, Lane et al. 2004, Quine et al. 2011). The behaviour itself is largely driven by factors acting at the individual scale and not at the site or landscape scale. As a consequence, the risky behaviour, when stripped from the risky landscape, lacks an environmental spatial component and can therefore not be mapped. It is therefore more accurate to say that there is insufficient information to produce an exposure layer for these risky landscapes. However, in cases where certain behaviours are associated with particular landscapes e.g. forestry workers spending time in forests, or tourists walking less when it is raining, there is still an implied environmental component. Risk management Besides identification of explanatory variables of disease risk, the mapped results of these modelling exercises are mainly used to inform the wider community (see also Risk communication ) and help decision makers produce public health strategies for preparedness, adaptation or response (Estrada-Peña et al. 2016). In risk management, risk maps at landscape and coarser scale mainly serve to raise awareness in the public or governments on the spatial distributions of risky areas, possibly with projections in time using climate scenarios and risky landscapes at low resolution (read: coarse scale) (Table 1). For the Netherlands, various national (risk) maps exist (Garcia-Martí et al. in press, Hofhuis et al. 2006, 2015, Swart et al. 2014). The ultimate goal of risk management strategies is the reduction of disease burden by decreasing the Lyme borreliosis risk. Ideally, the explanatory variables identified in the spatial modelling provide clues for entry points to risk management. In recent years, a multitude of explanatory variables have been identified as covariates influencing the complex tick-pathogen ecology. Some covariates are proven to be directly related to actual biological drivers or constraints, while others Ecology and prevention of Lyme borreliosis 361

363 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint cannot be readily interpreted (Estrada-Peña et al. 2016). Covariates may also be wrongly identified (e.g. due to confounding, or chance), or missed. As addressed throughout this book, Lyme borreliosis risk can be reduced by either reducing exposure or hazard or both. As environmental factors act at different scales, their influence on risk reduction do likewise. Of course, some factors such as climate, weather and soil type cannot be manipulated. Other environmental factors such as forest composition and land-use can be manipulated, but it may not be desirable to do so; the benefit of the prevention of Lyme borreliosis may not outweigh other (policy) goals such as nature conservation. Prevention and intervention measures cannot be implemented based on these risk maps unless they provide sufficiently high resolution detail to target focused cost-effective measures. An exception to this rule is the so called online tick radar that projects tick activity on the basis of weather forecasts. It is actually a forecast on whether the weather is suitable for the tick to be active, if they are present (tick occurrence or abundance are not included in this tick radar; The type of precaution available to the public includes avoiding high-risk habitats, wearing protective clothing, application of repellents, checking the skin for ticks, and prompt removal of attached ticks by using fine forceps, when deemed necessary. Eisen and Gray (2016), however, expressed their concern about the practicality of some of these actions, for example, avoidance of high-risk habitats when these are often not clearly defined. For personal risk management, the location of high risk-habitats needs to be known on a finer than landscape scale. In reality while modellers produce maps with spatial distribution of accurate risk outputs of mathematical modelling, the public may want a simple description of the risk: presence or absence of ticks (Braks et al. 2014b). Tourist agencies may prefer to avoid the term risk and present the information in terms of the precautions needed to reduce risk. For many land managers of natural areas, however, their whole terrain would categorise as risky habitat. For the development of effective, feasible and desirable risk management measures for the general public, these managers need to able to focus on locations with the highest risk, defined by the density of (infected) ticks as well as the exposure of the visitors (see below). Risk communication A plethora of recent studies on spatial modelling of Lyme borreliosis is available and the number of papers including a map as visual representation of the model output is increasing. While maps can be effective tools for risk communication, Estrada-Peña et al. (2016) warn that model outputs may not be very effective: mapping exercises should be a secondary aim in the study of the distribution of health threatening arthropods. Hazard maps showing some kind of measure of tick activity, occurrence or density are most intuitive and effective. Maps of national incidence of tick bites or erythema migrans developed for public health purposes are unlikely to be interpreted correctly by untrained individuals. Differences in incidence of reported tick bites between areas on the maps might be due to difference in risky landscapes (many ticks), or risky behaviour (many visitors to habitats with ticks) or reporting bias. The risk for an individual might not, therefore, differ between the areas. These maps are informative for public health agencies informing physicians and recording epidemiological changes. 362 Ecology and prevention of Lyme borreliosis

364 25. Grasping risk mapping As mentioned above, it is important to know whether the risk maps used by individuals can be used to define what preventive personal measures may be necessary. The use of maps in risk communication therefore requires the translation of the modelling output into comprehensive concepts tuned to the targeted user of the map. Examples In the following section, examples of risk maps at Pan-European, national and local scales are described and evaluated in the context of risk analysis. Pan-European risk map The first example we discuss is a map at the scale of Europe as a whole (Figure 2). It was produced for an EFSA/ECDC funded project on disease vectors: VectorNET. The project records tick presence at NUTS level shown in the inset, which has gaps that need to be filled to provide a complete continental map. This was done using standard spatial modelling methods applying calculated relationships between ticks and environment to the admin level data, adapted to include known absences which were defined as unsuitable habitats by expert opinion. These maps help to identify where the ticks are (or not) at quite detailed levels (the outputs are at a resolution of 1 kilometre) so can be summarised back to NUTS level as shown in the bottom of the picture. The map does NOT depict the risk of Lyme, but only where the tick is likely to be found. The ticks may not be infected with the pathogen, they may not be common or very active, or no people may go to an area where ticks are shown. Further, the map is a snapshot, and shows no seasonal detail. There are also technical issues. Though the methods are generally reliable, their outputs depend on the accuracy of the inputs and there is a temptation to look at the value for a single 1 km 2, while looking at groups or pixels is more appropriate for a map that shows probability. It is, however, always easy to find fault. Many of these issues can be addressed by ensuring the input data is good enough, by combining the probability values with other information that determines for example, tick activity or contact with susceptible persons. At the end of the day, there can be no cases of Lyme borreliosis without the tick. In more pragmatic terms, this is the only information that is available in a consistent way for the whole continent, allowing different regions or countries to be compared. As such, though maps like these may best be viewed as a first step, they are clearly useful to national and international public health planners to target resources away from areas with little or no risk towards those where action is most likely to be needed. National risk map As an example of a map at the national level, we briefly discuss the risk-map for tick presence of probability in the Netherlands (Swart et al. 2014) (Figure 3). As for the Pan-European map discussed above, the methodology is based on using absence-presence data on tick occurrence, and correlating this with spatial covariates and produces a kilometre resolution output. In this case, the technique involves a classification algorithm (NLDA), having the desirable property that the absences and presences do not have to be converted, but can be used as-is. As alluded to before, better guidelines and insights are needed for proper handling of spatial scales in riskmapping exercises. For the temporal aspects, a Fourier analysis of the time-dependent covariates was performed, which enables the analyses to identify seasonal influences. This is particularly Ecology and prevention of Lyme borreliosis 363

365 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint Figure 2. Pan-European risk map depicting (A) modelled probability of the vector Ixodes ricinus and (B) the predicted percentage of the area to presence of I. ricinus. 364 Ecology and prevention of Lyme borreliosis

366 25. Grasping risk mapping A Prediction B Figure 3. Hazard map of tick presence in the Netherlands. The colour indicates the probability of presence. Note the virtual absence of intermediate shades, indicating good discriminatory power of the method. In the leftmost panel, pixels with low prediction certainty have been omitted. Areas well known for tick suitability feature prominently, such as the dunes and Hoge Veluwe (Swart et al. 2014). appropriate for maps based on climate and natural factors (being typically cyclic), where additionally the phenomenon to be modelled defines the risk associated with the typical state of the environment, as opposed to peak/anomalous events. Two modes of validation were attempted. Firstly, the output in the form of probability of tick presence, was correlated with an independent data set records of tick bites obtained from general practitioners. A good agreement was observed, in the sense that more tick bites correlated significantly with more calculated hazard. Secondly, the risk-map was compared with a map based on expert elicitation. Experts were asked to quantify tick suitability by giving ranges of tick densities for several land-use types. It was found that inclusion of experts knowledge in the form of a Bayesian prior probabilities did not significantly alter the outcome, which led the authors to conclude that the experts knowledge as represented by the answers to the questionnaire, had roughly the same information content as the risk-map based on climatic and environmental factors. The authors did not perform any analysis on variable importance, so although it can be concluded that the map gives a reliable overview of tick presence at a coarse scale, it does not yield any insight in the drivers. Local risk map The last example that we briefly discuss here, but more extensively in the next chapter, is that of the risk map developed for land managers for estimating the risk for acquiring a tick bite for visitors of nature areas at high resolution (Figure 4). This project focused on the dunes along the North Sea Coast, near Castricum, the Netherlands. Waterworks and natural administrator of dunes along the North Sea Coast (PWN) like to know, which areas within their nature reserve pose a high (or low) risks for visitors. The model was built using ArcGIS based on the definition of risk as hazard times exposure. For both the hazard and the exposure part of the final risk map, the most important parameters were identified based on literature and expert knowledge formed the base of the structure and the final definition of risk categories of the MCDA model. High resolution data of PWN was used for parameterisation of the model which resulted in high resolution rick maps (3 3 m). Ecology and prevention of Lyme borreliosis 365

367 Marieta A.H. Braks, Annemieke C. Mulder, Arno Swart and William Wint N kilometers Final risk category Figure 4. Zoomed section of the risk map of Noordhollandse Duinreservaat, including exposure and hazard parameters. Seven levels of risk from lowest risk (1) to highest risk (7) have been identified (for details see Mulder et al. 2016). The model helped to provide high resolution (local scale) maps of the natural areas showing the risk (7 levels) of visitors getting bitten by a tick. Basing the model on (at least partly) on data that are provided by the stakeholder (the reserve owner) largely improved the interpretation and acceptance of the output map by the stakeholders. The high resolution of local maps increases usefulness at the required scale. Like the first of these three examples, the map does not depict the risk of acquiring Lyme borreliosis, but only the average risk of being bitten by a tick, which only poses a risk if it is active and infected. The actual risk is determined by ecological parameters (hazard) such as vegetation type and exposure parameters, such as accessibility. These three examples serve a purpose in risk analyses framework, each on their own merits. All of them resulted from predictive mapping using spatial models enabling projections in space, which fits in risk assessment. Their difference in scale, however, determines the extent (area covered) and the resolution (amount of detail) and therefore also its utility in risk management. While the risk maps of coarser resolution (Pan-European and national) can assist in identifying areas where intervention is most needed, the details of the local maps are required to implement cost-effective measures in the field. The fact that no biological parameters that drive the risk are identified in the first two examples hampers implementation and evaluation of targeted interventions that can reduce the risk. A more insidious issue is that the thresholds chosen for legend categories and even the colours used for each band can radically influence the story the map tells. However, like any map, the visual aspects of the Pan-European, national, and local maps 366 Ecology and prevention of Lyme borreliosis

368 25. Grasping risk mapping are strong communication tools to inform international health authority, national health planners, and land managers respectively. Conclusion In this chapter, we have provided an overview of what is involved in producing risk maps for Lyme borreliosis in the broader context of risk analysis: what is mapped, how the maps are produced, what they mean, how useful they are and how they can best be interpreted by the users. We show that it is important for map providers and users to agree on the purpose and content of the maps, to ensure that the map conveys the intended message. Accurate maps rely on effective modelling methods based on reliable data advances in information technology, availability of global environmental datasets, and improvements in methods available has meant that the challenge is not to produce maps, but to produce sensible ones that are useful to policy makers and academia alike. This is complicated by a number of factors particularly the scale of the maps and the different disease drivers that act at different scales. The scale of the map also affects whether the maps can be used to determine which risk management strategy to use in general the finer the scale the more effective is the guidance provided. Three examples of risk maps at different scales are described, and their usefulness in the context of risk analysis, management and communication are considered. In particular, most of the models provide information on hazard rather than risk (i.e. hazard times exposure). Once again, scale affects utility, but it is the link between environment and risk (proxy) that drives all of them. It is thus essential to understand the biology of the disease in order to correctly interpret the risk maps. In summary, we have sought to demonstrate that several mapping approaches have their place in risk governance, but they should be interpreted with a thorough understanding of the processes involved. It is however essential that model outputs are translated into comprehensive concepts that the users are familiar with and can intuitively interpret. This is involves a degree of translation of both technical output and user requirements, which means that users and producers of these risk maps need to communicate from the very start of the map generation process. Public health relevance Maps can show risk in a variety of ways. Risk maps can serve different purposes in risk analyses frameworks. Risk maps should be interpreted with a thorough understanding of the limitations involved. Most risk maps provide information on hazard rather than risk (i.e. hazard times exposure). Ecology and prevention of Lyme borreliosis 367

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374 26. From guessing to GIS-ing: empowering land managers Annemieke C. Mulder 1, Marianne Snabilie 2 and Marieta A.H. Braks 1* 1 National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; 2 PWN Het Noord-Hollands Duinreservaat, P.O. Box 2113, 1990 AC Velserbroek, the Netherlands; marieta.braks@rivm.nl Abstract For the land managers of natural areas, knowledge of the location of the highest risk for visitors is required for the implementation of management measures that modify either the density of infected ticks (hazard) or the level of contact of the visitor with these ticks (exposure). We explore the possibilities to develop a simple, but informative reliable model to create local risk maps for land managers. In addition, we aim to disclose the most important parameters, based on literature study and expert opinion, in defining the risk on acquiring a tick bite from the deer tick Ixodes ricinus. We want to predict risk levels within the area of the land managers using these most important parameters and their knowledge of the area. By involving their knowledge, land managers are more likely to embrace the information risk maps can give them on, for example, vegetation structures, grazed pastures and touristic attractions. We developed a generally applicable model in ArcGIS to create the risk maps and based the model on the most important parameters (both hazard and exposure) explaining the risk of acquiring a tick bite for visitors of natural areas. We based the first part of the model on parameters explaining the hazard: vegetation type, absence/presence of wild ungulates, absence/presence of sandy soil and absence/presence of grazing leading to a hazard risk map with three risk levels (low, medium and high tick densities). In the next step, we included the exposure parameters in the model: accessibility, recreational quality, temporary exposure, and stationary exposure (crossroads and benches). This led to seven risk levels in total. We only validated the hazard part of the risk map in the field for a dune natural area: Het Noord-Hollands Duinreservaat. This pilot study presents a novel approach to disclose expert information to land managers to assist them with implementing management measures to prevent that visitors acquire tick bites. We view the results as promising and hope they will inspire additional investigation into the validation of the model and translation into a decision key for cost-effective and risk-based management. Keywords: hazard, ArcGIS tool, decision key, exposure, management measures, risk map, tick, tick bite Introduction Risk of acquiring a tick bite Entering a tick s biotope poses a risk for people, because they can acquire a tick bite. The level of risk depends on the density of infected ticks, called hazard and the level of exposure by the person, in short exposure (Estrada-Peña and Jongejan 1999, Parola and Raoult 2001). Personal risk can be extrapolated to risk to public health by also considering the number of people exposed. To reduce the risk of people acquiring a tick bite, a reduction in either the hazard and/or the exposure is necessary as appears from the following formula (Dobson et al. 2011, Zeimes et al. 2014): Risk = hazard exposure Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _26, Wageningen Academic Publishers 2016

375 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks Where, hazard = number of questing ticks infection rate (density of infected ticks) and exposure = exposure of humans to ticks. Usually, the hazard includes both the number of questing ticks and the infection rate, which together determine the density of infected ticks. Within a geographical area such as the Netherlands, the infection rate is usually less variable than tick density (Coipan et al. 2013, Sprong et al. 2009) and therefore variation in hazard is mainly due to the variation in the density of ticks, in specific, the number of questing nymphs. For practical reasons, the focus within the hazard part of this study is on number of questing nymphs; infection rates of ticks are not taken into account. Since the hazard and the exposure show large variation in time and space within natural areas, the location of the highest risk for visitors is useful to know for the managers of those areas. The temporal aspects are important as well, because the activity of ticks is largely dependent on the season and the weather conditions (Dautel et al. 2016, Takken 2016). The same holds for the amount and type of outdoor activity of people, which affects the exposure level. Despite the importance of temporal aspects, we focus on mapping the spatial aspects of risk to aid the implementation of management measures. Management measures can aim to reduce either the hazard, the exposure or a combination of both. Firstly, hazard reduction may be achieved by making a particular location unattractive for ticks or their primary hosts, such as birds, rodents and deer (Kjelland et al. 2010, Stafford 2004). Hosts can also be physically excluded from an area (Van Wieren and Hofmeester 2016). Another possibility is to increase the attractiveness of an area for rabbits; presence of rabbits appears to be negatively related with tick presence in the Netherlands (Mulder 2015). However, only laboratory studies exist of this relationship (Bowessidjaou et al. 1977, Prevot et al. 2007). Secondly, concerning exposure reduction, management measures can be focussed on changing the accessibility of areas to people or the behaviour of people. Land managers can decide to exclude visitors to areas with high tick densities (high hazard). Signs can also be placed in areas of high risk (high hazard or high exposure or both) or picnic spots can be relocated to an area with a lower hazard (Adalsteinsson et al. 2016). Researchers are generally interested in unravelling the complex ecology of ticks and their pathogens and produce accurate risk outputs of mathematical modelling. The provided results often do not provide answers to practical questions; managers often want a straightforward description of the risk. As a result, pragmatic models to assist land managers in their task are still lacking. Tick densities can differ within a few meters depending on several ecological parameters. To be able to implement management measures linked to the risk for a visitor of acquiring a tick bite, land managers need to know where this risk is highest. This means that location-based information on tick density is necessary. Land managers often have a database containing maps of their terrain containing vegetation structures, grazed pastures, and touristic attractions. Such spatial information, called Geo-Information, is essential to categorise risk levels within their terrain. In the current paper, we summarise the results of our pilot study. We have explored the possibilities to combine various input datasets in ArcGIS in a reliable model. This model creates risk maps for land managers, who can use these maps for the implementation of management measures to reduce the risk of a visitor for acquiring a tick bite. The following questions were addressed: 374 Ecology and prevention of Lyme borreliosis

376 26. From guessing to GIS-ing: empowering land managers Which parameters, available at the level of natural area, can be used to predict density of I. ricinus nymphs reliably and can be used as an input variable for the model? (hazard). What is the overall accuracy of the hazard risk map? (validation) Which parameters, available at the level of natural areas, reflect the level of exposure of people to ticks and can be used as an input parameter for the model? (exposure) How can the different parameters be used to create a reliable model? (ArcGIS model) Methodology Study area Provinciaal Waterleidingbedrijf Noord-Holland (PWN) is an organisation that manages and distributes water for the inhabitants of Noord-Holland, in the Netherlands. The PWN manages 7,300 hectare of dune areas. The Province of Noord-Holland is commissioning the PWN. One of those areas is the Noord-Hollands Duinreservaat, one of the largest natural areas of the Netherlands. The area is 20 km long and 2.5 km wide covering 5,300 hectare. It includes dunes, beaches, and dune edge forests. Model hazard To create the hazard risk map, we defined the most important parameters explaining the variation in tick densities within an area. We did this by literature review and expert opinions. The latter, we obtained by interviewing various experts. A maximum of four input parameters was set to create a hazard risk map that is feasible, reliable, and applicable for all natural areas in the Netherlands. It forced us to focus on the most important parameters of tick density to be able to make a good prediction of the hazard. To make both a practical and reliable model, the hazard risk map shows no absolute number of nymphs per 100 m 2, but just three risk levels. Table 1 lists the parameters that were included for the creation of the hazard map. This table also includes the literature source and the source of the data. Elaborate descriptions of the parameters and the build-up of the model in the model builder of ArcGIS 10.2 are available on request. Experts for Vbornet (ECDC funded project) already assessed various habitat types occurring in Europe for the suitability of those habitat types for ticks. They divided the habitat types in three categories: very suitable (high), suitable (medium) or not suitable (low) (W. Wint personal communication). For the Netherlands, we created a decision rule for the three levels of the hazard risk map based on both literature that is available on the relationship between nymphal density and vegetation type and expert opinion. For example, areas with a high hazard risk level are forests, while areas with a low hazard risk level are, for example, short grass (Coipan et al. 2013, Dobson et al. 2011, Gassner et al. 2011, Sprong et al. 2009). For all forest types considered as high hazard, we listed the number of nymphs found and deducted a hazard risk category. In total, we defined the hazard risk level for 43 different vegetation classes listed in the Dutch Index Nature and Landscape. Ideally, all the vegetation maps of natural areas in the Netherlands have the same vegetation classification. However, the land managers use often (slightly) different vegetation classifications to depict specific details in their nature area maps. Therefore, also in the current study, it was required to do an additional small translation step before assigning the hazard categories to the vegetation layer of PWN map. The second parameter of the hazard part of the model consisted of georeferenced data on presence of wild ungulates in the PWN terrain. The presence of wild ungulates is considered a driver for ticks when present. Therefore, the hazard risk level of locations that are suitable for ticks and have wild ungulates increased (Figure 1, Table 1). The third parameter was soil type. Here, opposed to clay, Ecology and prevention of Lyme borreliosis 375

377 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks Table 1. Hazard and exposure parameters used in the ArcGIS tool and decision key, including the supporting literature, resolution and units of the dataset, threshold and risk category and the source from which the datasets can be collected. Reference Resolution Unit Threshold Risk 2 Source Exposure Hazard 1 Parameter manager of the areas (PWN)-Index Natuur en Landschap, Vegetatie van Nederland vegetation type reference to table later in results high (shapefile) Vegetation type Coipan et al. 2013, Dobson et al. 2011, Estrada-Peña and Jongejan 1999, Gassner et al. 2011, Lindström and Jaenson 2003, Medlock et al. 2013, Medlock et al. 2008, Medlock et al. 2012, Randolph 2004, Sprong et al. 2009, Swart et al roe deer database (Royal Dutch Hunting Association), Manager of the areas (PWN) low high number of roe no roe deer deer/km 2 roe deer high (shapefile) Dobson et al. 2011, Gray 1998, Harrison et al. 2010, Hoch et al. 2010, Jensen et al. 2000, Li et al. 2012, Medlock et al. 2013, Medlock et al. 2008, Medlock et al. 2012, Perkins et al. 2006, Rand et al. 2003, Randolph 2004, Randolph et al. 2002, Swart et al Wild ungulate density RIVM high low low high soil type forest + sand forest sand grass + sand grass sand Soil type Medlock et al. 2008, expert opinion high (shapefile) manager of the areas (PWN) low medium medium high no grazing grazing grazing + wildlife wildlife presence/absence grazing Grazing Hofmeester 2014 high (shapefile) manager of the areas (PWN) no risk = 0 risk = risk hazard layer no paths paths with buffer of 50 m presence/absence paths Accessibility Zeimes et al high (shapefile) 376 Ecology and prevention of Lyme borreliosis

378 26. From guessing to GIS-ing: empowering land managers Table 1. Continued. Reference Resolution Unit Threshold Risk 2 Source Exposure Hazard 1 Parameter Stichting Recreatie, Kennis en Innovatie Centrum (2002) class: 8, 9, 10 class: 5, 6, 7 class: 1, 2, 3, 4 recreative quality (1-10) for the rural areas of the netherlands high (only pdf) Recreative quality Goossen et al. 1997, Recreatie 2002, Zeimes et al manager of the areas (PWN) type of path walking cycling mountain bike bridle way regional road service road public road parking railway access road beach entrance stairs Expert opinion high (shapefile) Temporary exposure manager of the areas (PWN) +1 0 crossroads no crossroads presence/absence crossroads high (shapefile) Bennet et al. 2006, Zeimes et al. 2014, expert opinion manager of the areas (PWN) +1 0 benches (buffer of 5 m) presence/absence benches high (point data) Bennet et al. 2006, Zeimes et al. 2014, expert opinion Stationary exposure crossroads Stationary exposure benches manager of the areas (PWN) +1 0 no benches present absent presence/absence playground high (point data) Bennet et al. 2006, Padgett and Bonilla 2011, Zeimes et al. 2014, expert opinion Stationary exposure playground 1 Precondition: ticks should be active to cause a risk to people. Therefore, the risk map only is valid if the temperature >7 C and the relative humidity is >80% according to the KNMI (Greenfield 2011, Randolph 2004). 2 Risk: if +1 is given in this column, this means that the risk category shifts to a higher risk category e.g. from 1 (low) to 2 (medium) or from 2 (medium) to 3 (high), if -1 is given in this column, this means that the risk category shifts to a lower risk category e.g. from 3 to 2 or from 2 to 1. If 0 is given in this column, the calculated risk that is already there based on the previous layers does not change. So if it was 3 it stays 3. Ecology and prevention of Lyme borreliosis 377

379 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks A >80% canopy closure Risk category High = 3 (>50 nymphs/100 m 2 ) Medium = 2 (5-50 nymphs/100 m 2 ) Low = 1 (<5 nymphs/100 m 2 ) B 60-80% canopy closure Figure 1. Examples of decision tree: for forest with (A) >80% canopy and (B) 60-80% canopy closure. Note that upon adding each layer, the hazard risk category may be: (1) raised; (2) lowered; or (3) kept the same, depending on the previous layer. sandy soil is considered to promote leaf litter build up, providing a good microclimate for survival of ticks. Grazing is the fourth and last parameter that is included in the determination of the risk category in which a specific area within the natural area belongs when looking at the hazard risk map. Areas where grazers are present have a higher nymph density than areas without ungulates (wild or domesticated). However, areas where both grazers and wild ungulates are present have a lower nymph density than areas that contain only wild ungulates (Table 1). In summary, upon adding each parameter, it is possible that the hazard risk level raises, lowers or stays the same. This depends on the previous layer. All decision diagrams are available on request. 378 Ecology and prevention of Lyme borreliosis

380 26. From guessing to GIS-ing: empowering land managers The resolution of the different GIS layers, which represent the different parameters, is as high as the vegetation type layers available (often 3 3 m). Since nymph densities have been down scaled to three categories, it is possible that there is no differentiation in the assigned tick classes within one natural area. However, by including exposure of people to nymphs (see section Model hazard and exposure ), differentiation of risk levels within natural areas nearly always emerged. Based on these outcomes, we made a first estimate of the range of the three ranges for the three hazard levels. The procedure described above resulted in the following hazard categories: Low: ticks are absent or present at a low density. Decision key: 0-5 nymphs/100 m 2. Medium: ticks occur in medium density. Decision key: 6-50 nymphs/100 m 2. High: ticks occur in high density. Decision key: >50 nymphs/100 m 2. Validation fieldwork We acquired the independent validation dataset in Het Noord-Hollands Duinreservaat during two field-sampling days, using VECMAP. VECMAP is a GIS application for both the determination of the sampling locations and the fieldwork planning. Based on two days of sampling with in total 11 teams of two people and an area of 5 30 km, the maximum number of locations visited was 100 locations. The hazard risk map of Het Noord-Hollands Duinreservaat, created using the model described above, formed the input for the determination of the sampling locations. This map contains three risk categories as described earlier. Ranges in nymph densities (number/100 m 2 ) are the basis of those risk categories. VECMAP has special tools for the determination of the sampling locations within a map. It contains several strata taking the difference in surface covered per hazard risk class into account. VECMAP provides a GPS location for which a link to google maps is added to reach the correct location in the field. Some fieldworkers used the radar application of VECMAP to find the sampling location, in case the application did not provide the GPS location in natural areas. We provided paper maps as backup plan in case the application failed. Validation data analysis We validated the hazard risk map created with the first part of the model by means of the collected field data. For each collection site, a risk category (now still only hazard) was assigned on the bases of the number of nymphs found per 100 m 2 per location. To assess the percentage of explained variation of the hazard part of the risk map, we created an error matrix to determine the overall accuracy (Story and Congalton 1986). Model hazard and exposure Personal risk can be extrapolated to risk to public health by also considering the number of people exposed. Exposure was defined as the chance that natural area visitors will get in contact with the hazard (Zeimes et al. 2014). We used a maximum of six exposure parameters as input for the model. The choice for the parameters was based on literature, on expert knowledge and on the availability of georeferenced data. Table 1 shows the chosen parameters. We included the exposure parameters in the model by following the same strategy as for the hazard parameters. Elaborate descriptions of the parameters are available on request. The importance or priority of the parameters determining exposure according to literature and the consulted experts determined the order. Ecology and prevention of Lyme borreliosis 379

381 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks Final risk map We created the final risk map by sequential addition of the hazard layers and subsequently the exposure layers in the model. Based on expert opinion and literature, a decision tree was developed to determine the risk category of each pixel in each sequential step. The layer that is most important in determining tick density, vegetation type, formed the basis of the final risk map. Results Model hazard The parameters used to define hazards are provided in Table 1. Figure 1 shows an example of the decision tree of the hazard model. Model validation hazard risk map We collected tick data at 70 of the 100 planned locations during the 2-day field study. The overall accuracy of the original predicted data with these observed data was 54.3% (not shown). After consultation with the land manager, we corrected the information on the presence of wild ungulates since this was necessary. After the correction, the overall accuracy increased to 62.9%. Table 2 shows the final error matrix of the distribution of the predicted and observed risk categories of the locations, for which we collected tick data in the field. The contribution of the sequential addition of layer to the overall accuracy was as followed; the hazard map with only the first layer (vegetation) had an accuracy of only 38.6%. By adding the second layer (presence of wild ungulates) it increased to 52.9%. Adding the third layer (soil type) did not change it (as only one soil type was present in the whole study area). The four layers together resulted in the final overall accuracy of 62.9%. Most misclassifications occurred in the locations with predicted low risk; 15 of the 32 predicted low risk locations were medium risk. Seven of the 26 predicted medium risk locations belonged, in reality, to the low risk category. Few extreme miss-classifications have occurred (e.g. predicted as low while observed to be high). Figure 2 shows a histogram of the number of locations per interval of nymphal density. Table 2. Error matrix of the predicted and observed risk categories of 70 sampling locations in the field. The number of location with correctly predicted risk categories are indicated in bold. Risk observed Risk predicted Low Medium High SUM Accuracy user Low % Medium % High % Sum Accuracy producer 88.9% 51.5% 57.9% 62.9% 380 Ecology and prevention of Lyme borreliosis

382 26. From guessing to GIS-ing: empowering land managers Locations predicted as: High risk Medium risk Low risk Number of infections Observed nymph density (number of nymphs/100 m 2 ) Figure 2. Histogram of the locations categorised according observed nymphal density (N=70) during the validation process in the field, Provinciaal Waterleidingbedrijf Noord-Holland (PWN). The colour indicates the predicted risk category of each location. Risk category low, medium and high are defined by 0-5, 6-50, >50 nymphs/100 m 2, respectively. Model hazard and exposure To reduce the complexity of combining different parameters, resulting in the correct risk category, only six exposure parameters were used as input for the model. The choice of those parameters was partly based on the literature, but mostly on expert knowledge. In addition, the availability of GIS data played an important role for inclusion. Without such data, it is impossible to build the tool in ArcGIS. Since the tool should be applicable for natural areas all over the Netherlands, datasets that cover the whole country are preferred. Table 1 shows the chosen parameters. Data, necessary for the inclusion of all those parameters, is often not available and certainly not on national scale. Elaborate descriptions of the exposure parameters are provided on request. Risk maps The output maps of the model-builder are shown in Figure 3. The hazard map (Figure 3A) was the first output and was build up from four input layers determining tick suitability. Subsequently, we added one of the exposure parameters, accessibility, to the hazard map (Figure 3B). After adding all exposure layers to the hazard map the final risk map was created (Figure 3C). A small part of the south of the area is not included in the final risk map, because no data was available on the location of the benches. The high risk categories 5 and 6 cover small areas (often only areas of 5 m around benches or around cross roads), which are only visible when zooming in (Figure 3D). Discussion Our main objective was to develop a model that is able to disclose information related to Lyme borreliosis risk, which land managers can use to identify risk areas within their terrain. Such inventory of risk areas is required according the Dutch branch agreements, described in the Health and Safety catalogue of the green sector that includes forestry and nature conservation, and professional gardening and landscaping (De Groot 2016). Besides these occupational health Ecology and prevention of Lyme borreliosis 381

383 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks A B C D Figure 3. Output maps. (A) Hazard risk map of the whole Provinciaal Waterleidingbedrijf Noord-Holland (PWN) terrain. (B) Hazard risk map including the parameter accessibility of the whole PWN terrain. (C) Final risk map of northern part of PWN terrain (hazard and exposure). (D) Zoomed portion of final risk map. policies, land managers are concerned with the risk for a visitor to a quire a tick bite within their own natural area. It is also necessary to look into different risk levels within other natural areas and to compare them to be able to implement focussed management measures to reduce this risk. We aimed to develop a tool that, when ready, can produce reliable maps, which require minimal calibration to the local situation. To be reliable, it needs to provide useful information per location based on data that is easily available to the land manager. Ideally, for each of the input layers, maps or otherwise georeferenced information are available of the chosen parameters that cover the whole of the Netherlands. This would improve the general applicability of the tool for different natural areas in the Netherlands. In this way, a minimum of adaptations would be necessary when 382 Ecology and prevention of Lyme borreliosis

384 26. From guessing to GIS-ing: empowering land managers applying the tool for other natural areas than the one used for this study. During the development phase of this tool, however, thorough validation in the field was essential. The validation was performed only for the hazard part of the final risk map. As appeared within this study, exposure is barely considered in studies on risk mapping with regard to ticks (Zeimes et al. 2014). Methods for gathering and validating the data are highly desirable, but still expensive and labour intensive. The validation showed that the overall accuracy of the hazard risk map is 62.9%. This rather low accuracy of the modelled hazard risk map may relate to the developed model as well as the validation process. First, the complexity of tick ecology is reduced to a hazard proxy based on four parameters. Aiming at a reliable model for land managers, the inclusion of more than four parameters was not desirable, as it would increase the complexity of the final estimation of the risk categories (Randolph 2004). The selection of the parameters was based on literature and expert opinion in hopes to select parameters that drive tick density. The problem is that most studies follow a different methodology and sometimes focus on areas outside the Netherlands, which at least might be resolved using a resource-based habitat concept approach (Vanwambeke et al. 2016). Most misclassifications occurred in the locations with a low risk prediction, but that appeared to be medium risk. The input parameters of many of these locations indicated them as open forest without wild ungulates but with grazing. In the decision tree, these locations were assessed as low risk. From the current result, the decision tree of open forest should be the same as forest with a more closed canopy. Furthermore, not every parameter has enough literature support to be sure it is a correct predictive parameter to add for risk. Next to the fact that possible errors in build-up of the various decision trees, the problem of low accuracy might arise from the ranges of the nymphal density for the three hazard categories. A possibility is to change the range of the low risk category to zero nymphs/100 m 2. This increased the accuracy from 62.9 to 94.2%. However, we used broad nymphal ranges for the assignment of risk categories to the different parameters in the decision tree (see Figure 1), so to just change the ranges in the validation process would be raising the accuracy rather artificial. We need to look into this more carefully. Besides issues related to the model build, some problems we encountered had a more technical nature. The location software of pilot version VECMAP was not accurate enough, which led to the fact that some people could not find their location correctly. It is possible that people sampled in the neighbourhood of their location, but not at the exact location. Probably, fieldworkers did the sampling in another vegetation type, leading to a different risk category when comparing the fieldwork data with the predicted risk categories. With regard to the choice of the different parameters, several authors discussed vegetation types within their studies, but they discuss vegetation structure as well. According to Dobson et al. (2011) and Sprong et al. (2009), the latter is more important than the used vegetation type. However, no map exists of this for natural areas in the Netherlands. Randolph (2004) indicates that the normalised difference vegetation index (NDVI) can be used as well as it is shown to be correlated with tick mortality rates. This index reflects moisture availability on the ground, which is a major determinant of the tick population performance at a certain location. Although satellite images are available for the NDVI extraction, this parameter is not chosen, because the purpose is to use maps provided by the land managers themselves and the NDVI is not available at a very high resolution for most locations. Ecology and prevention of Lyme borreliosis 383

385 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks According to Swart et al. (2014) and Greenfield (2011), soil moisture is also an important explanatory parameter that is considered to vary in both time and space. In the current project, we focus on the spatial and not the temporal aspects of risk. But, because there are no maps of soil moisture for natural areas specifically and only a soil map for whole of the Netherlands is available, we used the absence or presence of sand as proxy for soil moisture. However, this differs between forested areas and open areas, due to the built up litter layer in forests with sandy soils (T.R. Hofmeester personal communication). The presence or absence of grazing is also included in the model, because it is an important explanatory parameter according to the experts based on small number of studies. There are only a few studies about grazing. So, it is not clear what the exact effects of grazing are on the number of active nymphs in particular situations. However, the addition of the grazing parameter to the hazard risk map increased the accuracy from 52.9 to 62.9%. From the preliminary results, we conclude that vegetation class and presence/absence of wild ungulates are the most important parameters and that grazing is the least important one. In our study, we assumed that forest use leads to exposure, and therefore considered relevant too. Zeimes et al. (2015) used many parameters determining exposure in forests. Examples are the number of tick-borne encephalitis (TBE) cases in 20 km, length of roads in forest, distance to watercourse, proportion of forest, mean height of trees and accessibility. They determined the presence (number or percentage) of the different parameters within the 2 km radius around TBE cases. We considered the use of several of them, but our approach does not allow for a standardised surface area. The parameters we did use depended on expert opinion. Besides identifying the main parameters that drive tick density, data of these parameters needs to be readily available within nature organisations to serve as input layer. The maps that we used as input layers date from several years ago. For example, the vegetation map is from 2006 and it is possible that some things have changed during the past nine years and gave rise to some deviation between predicted and observed hazard risk categories. This possibly opens a door to involving the land managers: they are in the position to update that data and hence the risk map. As mentioned above, our main objective was to develop a model that discloses information related to Lyme borreliosis risk that land managers can use to identify risk areas on their terrain. To disclose the information build into the model, the model developed can be used to build a decision key app. By answering four questions relating the four hazard input parameters, land managers can assess the hazard risk of a specific location on site. Conclusion Depending on expectations, the result of the current study may be considered disappointing as well as encouraging. One could quickly conclude that decades of European tick research did not lead to an accurate prediction of rather broad hazard categories on regional scale. However, those studies did not focus on making risk predictions. The current study is a first attempt to produce a risk map without using local tick data as input. The results of this study show that it is possible to build a tool in ArcGIS by means of several smaller submodels in which we combined several ArcGIS tools. This current study resulted in a risk map of the terrain of the PWN Het Noord-Hollands Duinreservaat. Although the result of the validation of the hazard part of the risk map showed that the hazard risk map is not perfect yet, the model can easily be adapted to the changes. For example when new studies on parameters influencing the hazard parameters become available or in case there are changes needed after we do the validation of the exposure part of the risk map. 384 Ecology and prevention of Lyme borreliosis

386 26. From guessing to GIS-ing: empowering land managers We consider the model for creating risk maps together with the decision key app as a spatial explicit decision support system in the future based on considerations of the needs of people on the ground. Public health relevance Study shows proof of principle for a tool to build risk maps for land managers to identify high risk locations in their terrain. The actual risk (7 levels) is determined by ecological parameters (hazard) such as vegetation type and exposure parameters, such as accessibility. The fact that the model data are provided by the land managers means that the map is immediately comprehensible. The high resolution of the map enables the focussed implementations of risk management measures. Accessory files Available on request: marieta.braks@rivm.nl. Acknowledgements The project was financed by Dutch Ministry Public Health, Welfare and Sports. We like to express our great thank PWN for allowing access to their property and data files for the research. Jeroen van Leuken is thanked for his help with the GIS. The two supervisors of Wageningen University & Research, F. de Boer and A. Bergsma, and consulted experts, S. van Wieren, T. Hofmeester and F. Gassner. The help of the members of the field teams, H. Sprong, A. Krawczyk, M. Fonville, S. Jahfari, H. Yilmaz, C. Coipan, A. Docters van Leeuwen, K. Dikmen, G. van Duijvendijk, T. Hofmeester, S. Moonen, and I. Heijmenberg is also highly appreciated. References Adalsteinsson SA, D Amico V, Shriver WG, Brisson D and Buler JJ (2016) Scale-dependent effects of nonnative plant invasion on host-seeking tick abundance. Ecosphere 7: e Bennet L, Halling A and Berglund J (2006) Increased incidence of Lyme borreliosis in southern Sweden following mild winters and during warm, humid summers. Eur J Clin Microbiol Infect Dis 25: Bowessidjaou J, Brossard M and Aeschlimann A (1977) Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus L. Experientia 33: Coipan EC, Jahfari S, Fonville M, Maassen CB, Van der Giessen J, Takken W, Takumi K and Sprong H (2013) Spatiotemporal dynamics of emerging pathogens in questing Ixodes ricinus. Front Cell Infect Microbiol 3: 36. Dautel H, Kämmer D and Kahl O (2016). How an extreme weather spell in winter can influence vector tick abundance and tick-borne disease incidence. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Ecology and prevention of Lyme borreliosis 385

387 Annemieke C. Mulder, Marianne Snabilie and Marieta A.H. Braks De Groot MCG Personal protection for people with occupational risk in the Netherlands. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Dobson AD, Taylor JL and Randolph SE (2011) Tick (Ixodes ricinus) abundance and seasonality at recreational sites in the UK: hazards in relation to fine-scale habitat types revealed by complementary sampling methods. Ticks Tick Borne Dis 2: Estrada-Peña A and Jongejan F (1999) Ticks feeding on humans: a review of records on human-biting Ixodoidea with special reference to pathogen transmission. Exp Appl Acarol 23: Gassner F, Van Vliet AJ, Burgers SL, Jacobs F, Verbaarschot P, Hovius EK, Mulder S, Verhulst NO, van Overbeek LS and Takken W (2011) Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in the Netherlands. Vector Borne Zoonotic Dis 11: Goossen C, Langers F and Lous J (1997) Indicatoren voor recreatieve kwaliteiten in het landelijk gebied. DLO-Staring Centrum, Wageningen, the Netherlands. Gray JS (1998) Review: the ecology of ticks transmitting Lyme borreliosis. Exp Appl Acarol 22: Greenfield BPJ (2011) Environmental parameters affecting tick (Ixodes ricinus) distribution during the summer season in Richmond Park, London. Biosci Horizons 4: Harrison A, Scantlebury M and Montgomery W (2010) Body mass and sex biased parasitism in wood mice Apodemus sylvaticus. Oikos 119: Hoch T, Monnet Y and Agoulon A (2010) Influence of host migration between woodland and pasture on the population dynamics of the tick Ixodes ricinus: a modelling approach. Ecol Modell 221: Hofmeester TR (2014) Waar zitten de teken? De Levende Natuur 115: 4. Jensen PM, Hansen H and Frandsen F (2000) Spatial risk assessment for Lyme borreliosis in Denmark. Scand J Infect Dis 32: Kjelland V, Stuen S, Skarpaas T and Slettan A (2010) Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in Southern Norway. Acta Vet Scand 52: 59. Li S, Hartemink N, Speybroeck N and Vanwambeke SO (2012) Consequences of landscape fragmentation on Lyme disease risk: a cellular automata approach. PLoS ONE 7: e Lindström A and Jaenson TG (2003) Distribution of the common tick, Ixodes ricinus (Acari: Ixodidae), in different vegetation types in southern Sweden. J Med Entomol 40: Medlock J, Hansford KM, Bormane A, Derdakova M, Estrada-Peña A, George J-C, Golovljova I, Jaenson TG, Jensen J-K and Jensen PM (2013) Driving forces for changes in geographical distribution of Ixodes ricinus ticks in Europe. Parasit Vectors 6: 1. Medlock J, Pietzsch M, Rice N, Jones L, Kerrod E, Avenell D, Los S, Ratcliffe N, Leach S and Butt T (2008) Investigation of ecological and environmental determinants for the presence of questing Ixodes ricinus (Acari: Ixodidae) on Gower, South Wales. J Med Entomol 45: Medlock J, Shuttleworth H, Copley V, Hansford K and Leach S (2012) Woodland biodiversity management as a tool for reducing human exposure to Ixodes ricinus ticks: a preliminary study in an English woodland. J Vector Ecol 37: Mulder AC (2015) Deer and rabbit density as determinants of tick density on a local scale a study focussing on explaining differences in nymph densities of the tick species Ixodes ricinus in de Loonse en Drunense duinen, the Netherlands. MSc thesis, Wageningen University & Research, Wageningen, the Netherlands. Padgett KA and Bonilla DL (2011) Novel exposure sites for nymphal Ixodes pacificus within picnic areas. Ticks Tick-Borne Dis 2: Parola P and Raoult D (2001) Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis 32: Perkins SE, Cattadori IM, Tagliapietra V, Rizzoli AP and Hudson PJ (2006) Localized deer absence leads to tick amplification. Ecology 87: Prevot PP, Couvreur B, Denis V, Brossard M, Vanhamme L and Godfroid E (2007) Protective immunity against Ixodes ricinus induced by a salivary serpin. Vaccine 25: Ecology and prevention of Lyme borreliosis

388 26. From guessing to GIS-ing: empowering land managers Rand PW, Lubelczyk C, Lavigne GR, Elias S, Holman MS, Lacombe EH and Smith Jr RP (2003) Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 40: Randolph SE (2004) Tick ecology: processes and patterns behind the epidemiological risk posed by ixodid ticks as vectors. Parasitology 129: S37-S65. Randolph SE, Green R, Hoodless A and Peacey M (2002) An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus. Int J Parasitol 32: Stichting Recreatie, Kennis en Innovatie Centrum (2002) Wandelen en toegankelijkheid bedreigingen en knelpunten. Stichting Recreatie, Kennis en Innovatie Centrum, the-hague, the Netherlands. Sprong H, Wielinga PR, Fonville M, Reusken C, Brandenburg AH, Borgsteede F, Gaasenbeek C and Van der Giessen JW (2009) Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry flea-borne Rickettsia species. Parasit Vectors 2: 41. Stafford KC (2004) Tick management handbook. The Connecticut Agricultural Experimental Station, Connecticut General Assembly, New Haven, CT, USA, 66 pp. Story M and Congalton RG (1986) Accuracy assessment a user s perspective. Photogramm Eng Remote Sensing 52: Swart A, Ibanez-Justicia A, Buijs J, van Wieren SE, Hofmeester TR, Sprong H and Takumi K (2014) Predicting tick presence by environmental risk mapping. Front Public Health 2: 238. Takken W (2016) Phenology of Ixodes ricinus and Lyme borreliosis risk. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Vanwambeke SO, Li S and Hartemink N (2016) A resource-based habitat concept for tick-borne diseases. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Van Wieren SE and Hofmeester TR (2016) The role of large herbivores in Ixodes ricinus and Borrelia burgdorferi s.l. dynamics. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Zeimes CB, Olsson GE, Hjertqvist M and Vanwambeke SO (2014) Shaping zoonosis risk: landscape ecology vs. landscape attractiveness for people, the case of tick-borne encephalitis in Sweden. Parasit Vectors 7: 370. Zeimes CB, Quoilin S, Henttonen H, Lyytikainen O, Vapalahti O, Reynes JM, Reusken C, Swart AN, Vainio K, Hjertqvist M and Vanwambeke SO (2015) Landscape and regional environmental analysis of the spatial distribution of hantavirus human cases in Europe. Front Public Health 3: 54. Ecology and prevention of Lyme borreliosis 387

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390 27. Personal protection for people with occupational risk in the Netherlands Mirjam C.G. de Groot STIGAS, Occupational Health and Safety Center for Dutch Agriculture and Forestry, Stationsweg 1, 3445 AA Woerden, the Netherlands; Abstract Persons working in the green sector (forestry, gardening and landscaping) in the Netherlands have, for years now, received a great deal of information on the prevention of tick bites and Lyme disease. Prevention is a fixed point on the agenda of employers and employees. In spite of the fact that employees are by now well aware of the potential risks, tick bites and Lyme disease still occur. Employers must carry out an effective risk inventory and evaluation of the risks and base their policy for a safe and healthy workplace accordingly. This includes preparing an action plan, providing information, distributing the necessary resources, monitoring the working agreements entered into, and regularly evaluating and updating the measures taken. Stigas, the foundation for occupational health in agriculture, forestry, gardening and landscaping, supports employers and employees in their efforts to ensure that workplaces are safe and healthy. Stigas develops information material about tick bites. Stigas also carries out practical research, including research into the effectiveness of protective clothing. In this regard, Stigas works closely together with other interested organisations and knowledge institutes. Persons working in the green sector need to know, in particular, which total package of measures in relation to their daily work provides optimum protection against tick bites. The environment and the type of work activities carried out have a great impact on the risk and therefore on the preventive measures that need to be taken. If the appropriate knowledge is available, employers can make a good assessment of the risk in a specific work situation and prepare an appropriate action plan accordingly. Keywords: Lyme disease, occupational risk, permethrin-impregnated clothing, personal protection, methodical tick check, preventing tick bites Introduction An increase was reported, between 1994 and 2014, in the number of tick bites and instances of Lyme disease (Hofhuis et al. 2010, 2013). It is striking that, since 2014, the number of consultations with family doctors for tick bites has decreased from 93,000 patients in 2009 to 82,000 patients in This decrease may be due, in part, to increased awareness and knowledge regarding the prevention of tick bites, achieved via information activities such as organising workshops, distributing leaflets and tick removal instruments, and the annual, national Week of the tick. During this week, many newspapers contain information about the prevention of tick bites. A great many films from Dutch National Institute for Public Health and the Environment (RIVM) are also shown on television during this period. Under the Working Conditions Act, employers must carry out a risk inventory and assessment, including a description of high-risk areas and activities, and then use this to prepare an action plan for a healthy and safe workplace. The employer is obligated to take preventive measures, including those described in the multidisciplinary guideline, Work and Lyme disease, from RIVM and the Dutch National Society of Occupational Medicine (NVAB) (Gassner et al. 2014). Clear sector-specific agreements have also been made. These have been made by the social partners in the forestry, gardening and landscaping sectors and can be consulted in Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _27, Wageningen Academic Publishers 2016

391 Mirjam C.G. de Groot the occupational health and safety catalogue for forestry, gardening and landscaping. Although the increase in the number of family doctor consultations for tick bites seems to be flattening out, the number of patients per year remains high (24,000), and the annual disease burden is large (see also Ursinus et al. 2016). The risk of tick-borne encephalitis has also surfaced recently. It is therefore in the interest of persons working in the green sector to monitor the most recent developments with regard to ticks and personal protection. This helps them to protect themselves as effectively as possible against this increasingly frequent occupational disease. The following sections contain additional information about the research carried out in the Netherlands in the forestry sector (2009) and the gardening ( ) (De Groot et al. 2009, 2010) into tick bites, preventive measures and personal protective equipment such as clothing that covers the body, impregnated clothing, tick-repellent agents on the skin or clothing, checking for ticks on or in the skin and on the clothing, and other equipment that can be used. A brief description is then given of an inventory study into the work-related disease burden from Lyme borreliosis, carried out by RIVM (Hofhuis et al. 2013). A brief description is also presented of a field trial, carried out in the forestry and landscaping sector, with different protective trousers against tick bites and their effectiveness (De Groot and Bokdam 2013). A description is provided of the measures that can be taken by employers and employees themselves. In 2015, a system was developed for inspecting the skin and clothing for tick bites. In addition, in 2015, at the request of various interested parties, a working group was established to formulate standards for tickrepellent clothing impregnated with permethrin. The last section presents the conclusions and recommendations for employers and employees, policymakers, and researchers. Present situation in the green sector General situation International studies make it clear that the incidence of tick bites and Lyme borreliosis in the green sectors is two to three times higher than in the general working population. In the green sectors, 25% of the employees are estimated to come into contact with ticks each year. In the Netherlands, Lyme disease is one of the most prevalent zoonoses in green areas. A green working environment is defined here as an environment in the vicinity of trees and/or bushes and/or grassy or herbaceous vegetation, such as forests, dunes, parks, and gardens. Approximately 2% of all tick bites result in the early stage of Lyme disease (Hofhuis et al. 2013). Each year, 24,000 persons in the Netherlands are diagnosed with erythema migrans (EM). In 2013, RIVM carried out a study into the number of consultations with company doctors for work-related disease burden from Lyme borreliosis in the Netherlands. For 2010, the study reported 173 diagnoses of a work-related EM, 100 diagnoses of disseminated Lyme disease, and 163 diagnoses of persistent symptoms due to work-related Lyme disease. This study provided new information about the seriousness and duration of the disease, sickness related absenteeism, and the amount of care needed. The study calculated that the resulting costs for 2010 amounted to 3.9 million. For the employees, this involves a large loss of healthy years (Hofhuis et al. 2013, Ursinus et al. 2016, Van den Wijngaard et al. 2015). State of affairs for employees in the forestry, landscaping and gardening sector In 2009/2010, Stigas carried out a field study involving persons employed in the forestry, landscaping and the gardening sectors. The study consisted of a questionnaire with 53 questions about the work situation, the work activities, preventive measures, tick bites, Lyme disease related 390 Ecology and prevention of Lyme borreliosis

392 27. Personal protection for people with occupational risk in the Netherlands symptoms, and how tick bites and the symptoms mentioned were dealt with. The study report presented recommendations for the sector. In the forestry sector, the occupational health and safety coordinators of the organisations responsible for managing the areas in question, including the National Forest Service in the Netherlands and the Society for the Preservation of Nature in the Netherlands as well as the Provincial Organizations for Nature and Landscape Management and the Landscape Management organisations, were asked to cooperate with the study and the questionnaire. Contractors in the forestry sector and private owners of forested areas were also asked to cooperate with the study. In the gardening and landscaping sector, requests were sent to landscaping companies, tree supply companies, and landscaping companies in the state-subsidised (social) sector asking if employees wished to participate in this study. 1,017 employees from the forestry and landscaping sector participated in this study. This amounted to 60% of the employees that were approached (n=1,700). 798 employees in the gardening sector participated in the study. This amounted to 36% of the employees that were approached (n=2,217). The results are summarised in Table 1. 81% of the 1,017 employees in the forestry and landscaping sector who participated in the inventory study indicated that they had been bitten by ticks one or more times while working. 36% received treatment with antibiotics. In 2009, 67% of the employees had a pair of tweezers or tick remover, and 91% had removed a tick in a timely fashion (in accordance with the 2009 standard). 32% of the employees in the gardening sector (n=798) have been bitten by a tick. 69% have a pair of tweezers or tick remover. 80% removed the ticks in accordance with the 2009 standard. Table 1. Results of the questionnaire based study ( ) in the Netherlands. Gardening (n=798) Forestry and landscaping (n=1,017) The company has a protocol in place (registration, reporting, and 16% 37% screening forms) A form is present for employees for the registration of tick bites 19% 30% Employee reports tick bites to supervisor 63% 41% Employee has a tick remover at the place of work 69% 67% Employee has been bitten by a tick at least once in the past while 32% 81% working Employee suffers from tick bites now during present work 28% 78% activities Which part of the body is most susceptible to tick bites? 38% groin area 62% groin area Employee has at some point in the past been affected by a circular 14% 28% red mark on the skin Employee removes ticks within 20 hours after finishing work 80% 91% Employee has at some point in the past been treated for Lyme 15% 36% disease after a tick bite Contact with animals (n=58 gardening, n=163 landscaping and forestry) and frequent tick bites 40% 17% Ecology and prevention of Lyme borreliosis 391

393 Mirjam C.G. de Groot Legislation and its practical implementation Working Conditions Act The Dutch Working Conditions Act requires the employer to provide a healthy and safe workplace. The obligation to provide a safe workplace also means that employees must be protected, insofar as possible, against risks while carrying out their work, including protection against Lyme disease. In the Working Conditions Act, the term employees also includes volunteers, temp workers, and trainees. The Working Conditions Act imposes obligations on employers and employees. The multidisciplinary guideline, Work and Lyme disease, describes how organisations can comply with these obligations, such as providing information, risk inventory and assessment, and taking preventive measures (Gassner et al. 2014). This guideline is intended to provide a uniform framework for occupational health and safety and optimise interdisciplinary cooperation between all stakeholders in the area of occupational health and safety (Gassner et al. 2015). Occupational disease In the Netherlands, Lyme disease has been classified as an occupational disease since As Lyme disease is not an infectious disease subject to reporting requirements under the Public Health Act, and work-related cases of Lyme disease have not been reported in a structured fashion. 54 reports of Lyme disease contracted during work have been registered with the Dutch National Center for Occupational Diseases (NCvB) over the last five years. This says little about the actual number of work-related cases as many cases are not reported. Risk inventory and evaluation All Dutch based companies in which work is carried out within the framework of an employeremployee relationship must carry out a risk inventory and evaluation (RIandE). An RIandE is a useful tool when it comes to making decisions and formulating policy with regard to risk communication and risk management. Borrelia burgdorferi sensu lato is classified in risk category 2 of biological agents. A checklist can be of assistance in estimating the occupational exposure to tick bites and Lyme disease. Questions can include: is work carried out in a high-risk area and in a high-risk period? RIandE indicates the nature of the work activities and risk of tick bites. If the RIandE makes it clear that the work activities are associated with an increased risk of tick bites and Lyme disease is present, then the multidisciplinary guideline and the recommendations from the RIandE and the occupational health and safety catalogue must be complied with. A strategy based on good working hygiene is followed with regard to risk management. First, a source-based approach is considered, after which technical and organisational measures are implemented, and then personal protective measures are considered. Management measures for Lyme disease can focus on: ecological factors (dealing with ticks and tick hosts or modifications to the habitat of the tick); behavioural factors (such as avoiding high-risk areas, appropriate clothing, checking for ticks, and removing ticks correctly and in a timely fashion); medical factors (preventive antibiotics (Sprong and Van den Wijngaard 2016) and the timely diagnosis and treatment of symptoms of Lyme disease). 392 Ecology and prevention of Lyme borreliosis

394 27. Personal protection for people with occupational risk in the Netherlands Extra attention has to be paid to employees subjected to higher risks and to special groups. Employees who carry out work or spend time in nature areas and deviate from roads and paths in doing so run a higher risk of being bitten by ticks. Special groups are: Employees in the green sector who have difficulty with reading and writing. These include, for example, employees in the state-subsidised (social) employment sector. It is important to take into account the capacity of the employee to understand the information material and to recognise and remove ticks. Pregnant women. Pregnant women are advised to avoid high-risk areas, as antibiotics are not a good option in case of infection. In order to prevent the transfer of the Borrelia bacteria from mother to child, during the pregnancy, the work must be organised in such a manner as to minimise the exposure to ticks. Occupational health and safety catalogue The occupational health and safety catalogue is an online catalogue of occupational risks and measures to be taken to control these risks ( It serves employers as a practical tool in setting up healthy and safe operational practices. The solutions and recommendations were prepared in collaboration between Stigas and employer and employee organisations and were published after approval by the Inspectorate of the Dutch Ministry of Social Affairs and Employment (SZW). An occupational health and safety catalogue is available for the gardening sector and for the forestry sector. The prevention of tick bites and Lyme disease is also dealt with in these catalogues. The occupational health and safety catalogue contains two important agreements (Box 1). The catalogue also contains detailed information on preventive measures (see Box 2 for a summary). Box 1. Agreements entered for the gardening and landscaping sector in the occupational health and safety catalogue. What is the desired situation?: Employees are not bitten by ticks so that Lyme disease cannot be transmitted. Employees and supervisors are aware of the risk, avoid high-risk behaviour, and therefore carry out checks after working in green areas so that ticks can be removed as quickly as possible. Which measures must I take for that purpose?: Prepare a good risk inventory and evaluation, including a description of high-risk areas and high-risk activities. Whenever possible, plan the work activities in periods of lower risk. Provide information to the employees, on an annual basis, about the risks of ticks in the work environment and update them on the most recent developments. Provide protective clothing and insect-repellent agents and tick removers. Employees report tick bites. Register cases of employees being bitten by ticks for each company. Ecology and prevention of Lyme borreliosis 393

395 Mirjam C.G. de Groot Box 2. General preventive measures for the gardening and landscaping sector. It is advisable to avoid working in high-risk areas or to carry out the work in periods of decreased tick activity. If that is not possible, organisational measures must be considered. Possible measures include: Modifying a (walking) route to the place of work. Limiting the number of employees exposed. Limiting the duration of the exposure. The registration of tick bites within a company. Proper selection of a location for lunch and/or toilet facilities. Systematically checking the skin and clothing for ticks after visiting a green environment so that ticks can be removed as soon as possible. Leaving work clothing behind at the company site so that they can be washed or dried at 60 C on the company premises. Providing resources that make it easier to discover ticks on the skin and clothing. One way of doing so is to provide changing rooms with (double) mirrors and good lighting. A fine-pointed pair of tweezers or other tick removal instrument can also be provided, together with appropriate instructions for using it. Finally, personal protective materials, such as clothing that covers the skin, can also be used. However, this does not provide complete protection against ticks. In case of workrelated exposure to ticks, it is advisable to use permethrin-impregnated clothing. Clothing impregnated with permethrin provides a level of protection against tick bites of at least 90%. The use of tick-repellent agents such as DEET or citriodiol on the skin is advisable only in case of incidental and short-term stay in a green environment. Information The annual thematic Week of the tick has been on the calendar in the Netherlands since This is a collaborative and focused initiative aimed at informing employees. The Week of the tick in early spring is already a household name in the Netherlands. The Week of the tick is so effective, because more than 20 participating organisations work together to spread the same message. Information is provided on the prevention of tick bites and Lyme disease, based on the most recent data available. Each organisation provides information to its own specific target groups. For example, RIVM has developed information materials for the general public and physicians. Stigas provides information to employees in the agricultural and green sectors. The website is a collective initiative on the part of the participating organisations. During the theme week and the period surrounding it, the website shows the latest news and items of interest for everyone who deals with ticks during work or leisure time. An article is also prepared for the editors of the many magazines and newspapers in the Netherlands. This article contains the following information: What are ticks and where are they found? What does a tick look like? The importance of carrying out adequate checks. What should one do when bitten by a tick? When should one visit the family doctor? Why is adequate information so important? 394 Ecology and prevention of Lyme borreliosis

396 27. Personal protection for people with occupational risk in the Netherlands Consulting hours focusing on ticks for employees working in green surroundings. What can employers do? What can employees do themselves? An overview of information meetings and information materials at under toolkit for ticks, and Special PowerPoint presentations are available from Stigas for the forestry, landscaping and gardening sector. Systematically and regularly checking for tick bites To prevent the acquisition of Lyme disease, one should regularly check yourself for tick bites and immediately remove any ticks that have attached themselves to the skin. One should always do this after visiting a green area, such as the forest, the dunes, or a park or after working in the garden. Ticks prefer warm areas such as the head, neck, armpits, arms, groin, buttocks, and hollow of the knee. A mirror is useful for inspecting areas that are difficult to see (Figure 1). It is important for employees to learn how to systematically inspect their body. In concrete terms, this means: after being in a green environment, always carry out a tick check as soon as possible, and also do so after finishing work; always do so in the same manner; check all clothing and entire body. By always carrying out the check in the same manner, one is less likely to skip some areas and will discover as many ticks as possible that may be present. The nymphs in particular are very small, the size of a poppy seed, and are difficult to find. In order to carry out the check without any interruption, it is important for the employee to be able to withdraw to a quiet location with adequate space and lots of light. Figure 1. Use of mirror for tick check. Ecology and prevention of Lyme borreliosis 395

397 Mirjam C.G. de Groot That sooner a tick is removed, the smaller the risk is that the Borrelia bacterium will be transmitted. In order to do this appropriately and correctly, it is important for employees to have a personal tick remover. There are a great many different tick removers available. There are tick spoons, tick scoops, tick crowbars, tick lassos, tick hooks, tick cards with a notch, and tick removers with a magnifying glass or a lamp. There are also tick removers for a key chain and of course the pointed set of tweezers. To correctly remove an attached tick, a fine-tipped tweezers should be used to grasp it as close to the surface of the skin as possible and then remove it, in one smooth motion. Which tick remover is the best depends on many factors: Is one experienced in removing ticks or still inexperienced? Is it a small nymph or a fully engorged tick? Does one have to remove the tick oneself or can someone else do it? Is the tick attached in a location one can easily get or a difficult location such as a navel? Find a tool that is appropriate for you, and always read the instructions carefully. A flyer (Figure 2) and video have also been made about this method ( What can employers and employees themselves do? What can employers do? It is important for employers to be familiar with the work environment, the high-risk areas (Gassner et al. 2016, Haverkort 2013, Mulder et al. 2016) and the high-risk work activities. It is important for employers to regularly update the RIandE. The RIandE contains a chapter on biological agents, which contains more information on ticks and tick bites and how to deal with them. Employers must provide information to the employees or set up a toolbox meeting about the risks of ticks in the work environment and the relevant protective measures. The employer must also specify which measures will be taken by the organisation, whereby extra attention should be paid to employees who are at increased risk and special groups. Many employers do this in the month in which the Week of the tick is scheduled to take place. A great deal of updated information is then available. A special leaflet has been prepared, which is easy to read and understand, for employees who are less proficient in the Dutch language. It is important to make agreements on how to prevent tick bites and to set these down on paper. A registration system has been set up to ensure effective registration in the sector. A registration system at the level of the individual company and the overall sector can provide information on the basis of which the employer can adjust his policy. The parties concerned can then also collaborate on the most effective management strategy. Concrete details of what the employer can do are presented in Box 3. What can employees do The information and advice provided to employees includes the following: regularly attend information meetings at your company. Learn everything about ticks, and make sure that you can recognise ticks and remove them correctly. Wear work clothing or protective clothing in accordance with the agreements made. Wear close-fitting, body-covering clothing with long sleeves and long pants. Clothing should preferably have a smooth surface and be of a light colour. Wear high work shoes or boots that protect your ankles, or tuck your pant legs into your socks. Or wear pants with an extra inner sleeve fitted inside the bottom of each pant leg, in which case the inner sleeve is tucked inside the sock. Wear a cap with a neck flap or a broad-brimmed hat. Exposed areas of skin can be rubbed in with an insect-repellent agent that contains DEET for example. Always read the instructions on the packaging carefully. At the end of the workday, 396 Ecology and prevention of Lyme borreliosis

398 27. Personal protection for people with occupational risk in the Netherlands Methodical tick checking If you work outdoors, you run a higher risk of being bitten by a tick. So, after work, you should always check yourself for ticks. Use your hands and, if necessary, a brush or a sticky clothes roller. Make sure there s enough light. First check your clothing. From front to back, top to bottom, and left to right. Pay particular attention to: seams, rolled up trouser bottoms, rolled up sleeves, under your belt and watch, the edge of your underwear, socks and the inside of your shoes. And check your hat or cap. or Once you ve checked the outside of your clothing, check the inside. Take off your clothes and check the inside of your coat, top layers and underwear. Put the clothing straight into a washing machine or dryer, not in a laundry basket with other clothing. Or put your clothing in an organic laundry bag and tie the bag firmly. Do not hang your coat on a coat stand as ticks can walk onto other coats and jackets. The same applies to shoes and hats. Check the front and back of your body; from top to toe, from left to right. Pay particular attention to: behind your ears, the hairline and neck, armpits, navel, the crook of your elbow, between your fingers, the crotch, the groin, your bottom and crack, behind the knees, between the toes, under the feet and round the ankles. Women: check under and between your breasts. Men: check under your scrotum. Check the back of your body using a hand mirror and fulllength mirror. If you see a tick, remove it, preferably using sharp tweezers. Wrap the tick in sticky tape or a bag and throw it away. Once you ve checked your clothing and body, have a shower. This can wash away any unattached ticks you ve not spotted. Note any tick bites in your diary and on Watch the video on Figure 2. Flyer with information on methodical tick checking (Stigas, Ecology and prevention of Lyme borreliosis 397

399 Mirjam C.G. de Groot Box 3. What can the employer do? Identify and locate risks: Prepare a good risk inventory and evaluation, including a description of high-risk areas and high-risk activities. Provide updated information. Then prepare a clear action plan. Make a checklist that specifies who in your company is responsible for what when it comes to the prevention of tick bites. For example, formulate a tick protocol. Register cases of employees being bitten by ticks for each company. This will provide more information on where the risk of tick bites is the greatest. A B C D Figure 3. Examples of personal prevention measures: (A) anti-tick gamaschen (photo by Mirjam de Groot), (B) rubber boots (photo by Mirjam de Groot), (C) inner sleeve fitted inside the pant leg below the knee (photo by Mirjam de Groot), (D) repellent (photo by Margriet Montizaan). 398 Ecology and prevention of Lyme borreliosis

400 27. Personal protection for people with occupational risk in the Netherlands Make clear agreements: Make sure that everyone wears appropriate clothing: shirts with long sleeves and fulllength trousers. Clothing should preferably have a smooth surface and be of a light colour. Socks should also preferably be of a light colour. At the end of the day, have your employees check their clothing and bodies for ticks. Explain, with the help of information materials such as videos or leaflets, how employees should check their clothing and bodies in a systematic fashion. Make sure the employees remove any ticks quickly and effectively and register the date. Take organisational measures: Avoid high-risk work locations whenever possible, and deploy as few people as possible in any such locations. Whenever possible, plan the work activities outside of high-risk periods. Provide protective clothing and tick-repellent agents. Work clothing should preferably be washed on the company premises. Whenever possible, allow your employees to eat their lunch in a mobile trailer or similar facility and not on the ground. Check to make sure that appropriate clothing is being worn. Provide training and instruction: Provide information to the employees about the risks of ticks in the work environment. Employees should be able to recognise ticks, prevent tick bites, and remove ticks correctly. Have them note down the date of the tick bite. It is important to comply with the clothing regulations. Use films, PowerPoint presentations, leaflets, and stickers to supplement the information provided. Employees who are at increased risk and special groups: Pay extra attention to special groups. Employees in the green sector who have difficulty with reading and writing. These include, for example, employees in the state-subsidised (social) employment sector. It is important to take into account the capacity of the employee to understand the information material and to recognise and remove ticks. A leaflet has been developed in the Netherlands especially for this purpose that is written in clear and easy to understand language. Pregnant employees should pay extra attention to checking their clothing and skin. Pregnant women are advised to avoid high-risk areas, as antibiotics are not a good option in case of infection. You should therefore have pregnant women do types of work whereby the risk of contracting Lyme disease is eliminated. Ecology and prevention of Lyme borreliosis 399

401 Mirjam C.G. de Groot check your clothing and body for the presence of ticks (Figure 2). When working in a high-risk area, it is advisable to also carry out interim checks, for example during the break. When checking places that are difficult to access, ask for assistance and use a magnifying glass and extra light to be able to inspect the skin correctly. Use a good tick remover and remove the tick as soon as possible via the correct method. Note down the date and location of the bite in your diary. If you notice any symptoms, consult your family doctor. In case of persistent symptoms, also consult the company doctor. What more can you as an employee do?: Whenever possible, avoid thick vegetation, layers of leaf litter, shaded grassy areas, and bushy areas. Do not walk through forested areas if it is not necessary. Do not take your lunch on the grass if it is a high-risk area. When relevant: before working in a green area, use a leaf blower to clear out leaves from the vegetation. Take extra care when working with or in the vicinity of animals. Also be extra careful when moving cadavers, as these may have lots of ticks on them. Carry out tick checks yourself, but also have others carry out a check for you in places that you cannot easily see yourself. Also carry out a tick check at regular intervals while working, and shake down your work clothing regularly in the course of the day. Also clean your car seat on a regular basis. If a working dog (shepherd dog, police dog, or hunting dog) accompanies you on the back seat, then regularly clean the car. Personal protective equipment Personal protective equipment acts as a barrier to prevent tick bites. This can work in various ways, for example wearing protective clothing or clothing that has been impregnated with insecticide, applying tick-repellent agents directly on the bare skin or on clothing, or using a tick remover. Wherever necessary, appropriate personal protective equipment against tick bites is made available by the employer free of charge and also used by the employee. The employer ensures that they are also used and sets this down in the company regulations. The employer provides the users with information and instructions. There are a great many publications that describe the effectiveness of various types of personal protective equipment. However, this section will also contain some practical pieces of advice based on the experience of employees in the sector or simply on common sense. A distinction is made between clothing that simply covers the skin and protective clothing. By wearing clothing that covers the skin and tucking pant legs into socks, it becomes more difficult for ticks to reach bare skin from the vegetation. It is important for employees to wear appropriate work clothing: long-sleeved shirts and long pants. Clothing should preferably have a smooth surface and be of a light colour which enables spotting ticks easily. Preferably, one should also wear light-coloured socks and high work shoes or boots (protecting the ankles). Pants are also available with an inner sleeve fitted inside the pant leg below the knee, in which case the inner sleeve is tucked inside the sock so that the tick ends up blocked between the outer pant leg and inner sleeve. Anti-tick gamaschen, also referred to as gaiters, are a recent development (Figure 3). The main benefit of these permethrin-impregnated protective coverings for the lower leg is that they can be worn over the pant legs. There is therefore no direct contact between the permethrin and the skin, and one can still continue to use their own (company-issued) pants. This is important 400 Ecology and prevention of Lyme borreliosis

402 27. Personal protection for people with occupational risk in the Netherlands for many companies who issue their own company work clothing to employees. These lower leg protectors are easy to be taken in the car or on the bicycle and also are easy to keep clean. There are many types of impregnated work pants available on the market, whereby the protective agents have been applied via washing, dipping, and spraying. Industrial permethrin-impregnated work pants are an example of protective clothing. Permethrin belongs to the group of synthetic pyrethroids and has a neurotoxic effect on insects. At present, employers and employees are uncertain about the long-term effectiveness of impregnated clothing as a protective measure. The protection provided normally decreases with washing, and the decrease is not visible in the clothing. The impregnated clothing is recognisable via an anti-tick label. The use of clothing that has gone through many washing cycles can form a risk and give an unjustified feeling of safety. It is therefore important to be able, in one way or another, to provide an indication of how much protection is still provided by the clothing. Another type of protective clothing is clothing that has been carefully sprayed with the insect- and tick-repellent agent DEET. More information about DEET is given in Box 4. At present, there is no clear overview available of work clothing that provides effective protection. There is also no clear standard that pants must comply with. Efforts are being made, at the Dutch and European level, to determine which standards tick-repellent clothing must comply with in future. However, there is a WHO guideline. Employers as well as employees would very much like to minimise skin contact with permethrin by partly treating the fabric in a targeted manner or by making use of a double layer of inner and outer fabric. There are also practical questions as to whether safety clothing, such as the saw pants that employees are required to wear, can be treated with an anti-tick agent. Effectiveness of protective clothing In 2011, for a period of six months, 53 agriculture and forestry workers kept a complete registration of the protective work clothing they wore. In this field trial, the operational term used was pants wearing days. After a wearing day, the employee registered his experience with the pants. The possibilities were: wearing permethrin-impregnated pants; wearing regular working pants that had been sprayed with DEET; wearing regular working pants. The results of this field trial showed that wearing permethrin-impregnated pants significantly reduced the number of tick bites in comparison to regular working pants. The pants sprayed with DEET were not significantly more effective against tick bites in comparison to the regular work pants. This may be due to the fact that spraying the pants with DEET at the start of the workday is a procedure that takes quite some time and needs to be carried out carefully, which may not always have been the case. Another possibility is that the effectiveness of the active ingredient diminishes in the course of the day. The permethrin-impregnated pants were worn for 1,463 workdays, and the same type of pants without permethrin were worn for 1,565 workdays. The number of tick bites per unit of time in this field trial was expressed as tick bites per 100 workdays, leading to the following results: 1.8 tick bites per 100 workdays when wearing the permethrin pants; 4.7 tick bites per 100 workdays when wearing the regular work pants without permethrin. Ecology and prevention of Lyme borreliosis 401

403 Mirjam C.G. de Groot Box 4. Insect-repellent agent containing DEET for use on the skin and on clothing. Diethyltoluamide, abbreviated as DEET, is an active ingredient that affects the nervous system of insects. As a result, insects become effectively disoriented and do not bite. What do employees need to know if DEET is used on the skin?: The product should be sparingly and carefully applied to exposed skin. The product should be evenly distributed over the skin. The product should not be applied to the eyes, the lips, or damaged skin (wounds). The product should not be applied in places where, due to the bending of joints, the skin is normally bent very frequently, so it should not be applied in the hollow behind the knee and behind the elbow. The most important side-effect is potential irritation of the skin. After finishing work, you should wash the skin in question with water and soap. When using it on clothing: Carefully apply the product to the outside of the clothing at the location of the wrists, ankles, shoulders, and pants pockets (read the instruction leaflet carefully and use the product accordingly). After using the clothing and before washing it, store it in a closed plastic bag. The percentage of DEET in the insect- and tick-repellent product determines the length of time it remains effective, for example for Care Plus DEET anti-insect spray 40%, the effectiveness against ticks lasts four hours. In that case, you must take the bottle of DEET with you in your car or backpack to treat your clothing again after four hours. After spraying your clothing, wash your hands and also be careful to ensure that DEET does not come into contact with the frame of your glasses or other plastic materials. Some individuals who work outdoors in nature find this DEET procedure rather difficult. How should you use DEET in combination with sunblock?: Sunblock must be applied to the skin 30 minutes before exposure to the sun. After the sunblock has penetrated into the skin, the DEET product can be applied 10 minutes before the skin is exposed to the sun. Care Plus insect- and tick-repellent DEET products should not be applied in full sunlight. NB: the protection factor of a sunblock decreases after a DEET product has been applied over it. It is therefore advisable to choose a sunblock with a higher protection factor. The number of ticks found when regular work pants without permethrin were worn was significantly higher than the number found when permethrin-impregnated work pants were worn. This was true of the number of ticks found on the pants as well as on the skin. Wearing permethrin-impregnated pants reduces the risk of being bitten by a tick by more than 50% in comparison to wearing similar pants without permethrin. The same is true of the number of ticks found on the pants. A number of employees indicated that they would have preferred to also have the option of wearing an impregnated shirt and impregnated socks during this field trial. This could have provided even better protection against ticks. On average, the persons wearing the impregnated pants gave the pants a rating of 8.3 on a scale of 10. Wearing comfort is also very important during work. 402 Ecology and prevention of Lyme borreliosis

404 27. Personal protection for people with occupational risk in the Netherlands Based on various research activities and studies with human subjects, including German and Dutch military personnel (Vaughn and Meshnick 2011, Valstar and Hardij 2013), we can conclude that using a combination of DEET spray and lotion plus permethrin clothing can be effective in preventing tick bites. Safety of protective clothing Various institutes have carried out evaluations of the potential health risks associated with wearing permethrin-impregnated clothing for long periods of time. Based on the data presently available, we may conclude that the resulting health risks, in the short-term as well as long-term, are negligible. However, as this does result in the absorption of permethrin into the body, employers are advised to provide this permethrin clothing only to persons who frequently work in forests and agricultural areas and in high-risk areas. Military personnel exposed to permethrin have reported symptoms such as a tingling sensation of the skin, itchiness and skin irritation, redness, swelling, and skin rash. Many studies have focused on exposure via the skin. However, some exposure via inhalation of dust particles cannot be completely ruled out. Studies carried out until now have not shown any clear relationship between exposure to permethrin and the occurrence of cancer. Most types of cancer occur only after an exposure of 15 to 20 years. However, one can conclude from studies that, should permethrin turn out to have carcinogenic properties, the effect will be very small. In the EU, permethrin is not classified as a carcinogenic compound. At present, there is insufficient data available. The most recent study carried out is the one done by the German accident insurance company (DGUV 2013) into the effect of permethrin-impregnated pants on the health of 171 male forest workers. The study focused not only on the protection provided by these pants against ticks but also on the exposure of the body to permethrin. This study resulted in the following conclusions: Employees who wore saw pants/safety pants had better protection against tick bites than employees who wore the regular work clothing. Permethrin-impregnated saw pants provided little extra protection against ticks than nonimpregnated saw pants. Regular work pants impregnated with permethrin provided significantly greater protection against tick bites than regular work pants that were not impregnated. However, the protection provided was not 100%, and it was therefore still important to carry out a tick check after the work. No allergic reactions to permethrin were noticed, but the risk of allergic reactions is theoretically present. The acceptable daily intake established by the WHO ( g/kg/day) was not exceeded. With regard to the potential carcinogenic properties of permethrin, the study was careful not to draw any definitive conclusions due to the lack of data on the specific long-term exposure under forest workers. The absorption of permethrin into the body is influenced by the climate, by how much exertion the work requires, by the number of hours that the pants are worn, and the brand of the pants. The study recommends using these pants only in cases where there is a high risk of tick bites and not as the standard pants for all employees under all circumstances in forests and nature areas. Ecology and prevention of Lyme borreliosis 403

405 Mirjam C.G. de Groot Standards for tick-repellent clothing In January 2016, a NEN project group, composed of experts and clothing producers, started formulating a Dutch standard, which can then be further developed at the European and international level. The standard deals with the choice, use, and maintenance of the tick-repellent clothing. In addition, a test method will also be described. When the first draft of the standard becomes available, this document will be published (target date is during the Week of the tick in April 2017), and interested parties will be able to submit their comments on the content. During the development of the standard, various interests groups, such as the National Green Lyme Working Group, will be regularly updated on the progress being made. In addition, a broad group of interested parties is involved via the existing network for the development of standards for personal protective equipment. The goal is to provide employers and employees with more information about protective clothing impregnated with permethrin in the Netherlands. The market for this type of clothing is rapidly growing, but there are still no adequate quality guarantees to safeguard the level of protection provided. NEN is collaborating with this project for establishing a Dutch standard. NEN takes care of document management, publication of the standard, promotion via an independent quality mark, and the associated register. The register is accessible to everyone. The logo of the quality mark ensures that it can be easily recognised in the market. Which protection method is to be preferred under which circumstances? Table 2 gives an overview of potential protection methods depending on work activities. Under field conditions in the forestry sector, working pants impregnated with permethrin will not provide sufficient protection for employees working close to the ground. In such situations, the tick can climb on board on the arms or at chest height. This risk is also present if someone is carrying branches (at hip height). Upper-body clothing that has been impregnated or sprayed can then serve as an important supplementary measure. Conclusion We know that ticks are present in forests and wooded areas, but little is yet known about public green spaces, which is where gardeners and landscapers work. Examples of these include parks and gardens. We do know that ticks are present if there are thick layers of ground litter or thin layers of litter with ground covering plants. Little is yet known about the consequences of more natural management practices for green spaces in urban areas and the increase in the number of ticks. At present, there is still little detailed, periodic, geographic information available on tick density. The tick density is important for a risk evaluation (Mulder et al. 2016). However, employers can consult the online tick radar of Wageningen University & Research (Wageningen, the Netherlands) to see what the present and predicted level of tick activity is. This information can be taken into account when planning work activities in a high-risk area. Employers and employees are still not sufficiently familiar with good management measures. In case the RIandE indicates that work activities are associated with an increased risk of tick bites and Lyme disease, then the multidisciplinary guideline and the recommendations from the RIandE and the occupational health and safety catalogue must be complied with. Employers have sufficient information materials such as the RIVM toolkit and the website to encourage healthy work behaviour at the work site. An important part of such behaviour is the structural and systematic tick check on clothing and skin, even when protective or impregnated 404 Ecology and prevention of Lyme borreliosis

406 27. Personal protection for people with occupational risk in the Netherlands Table 2. Protection options against ticks for different types of work activities in the Netherlands. 1 Work activities Protective measures Long pants with the socks rolled up over the pant legs Pants with an Impregnated extra inner pants sleeve inside the legs and the inner sleeves tucked inside the socks Spraying repellent onto exposed skin Working under conditions of high risk for lengthy periods and/or frequently: warmer than 4 C and relative humidity above 80% high grass, bushes known high-risk areas Working at risk: a few hours per day and/or a few days per year Working bent over with high risk of ticks + outer clothing with long sleeves + spray outer + also spray clothing with a outer clothing repellent with a repellent Work carried out in the middle of the path Working on mobile machines Outdoors: colder than 4 C and less than 80% relative humidity 1 = applicable. clothing are worn. Good discipline and motivation are needed for this. Employers should pay attention to workers who are at increased risk and to special groups such as pregnant women and employees with a physical or mental handicap or employees who do not have a good command of the language. It should be clear that effective prevention is possible only with a total package of measures. The proper implementation of these measures must be monitored and supervised. Workers have the opportunity to go to the occupational physician for tick consultations. Occupational physicians are obliged to report occupational diseases. Employers and employees have questions about the long-term health effects of wearing clothing impregnated with permethrin. At present, there are no known negative health effects of longterm use (20-30 years). There are many types of impregnated work pants available on the market, whereby the protective agents have been applied via washing, dipping, and spraying. The protection provided decreases with washing, but this decrease is not visible in the clothing. There is a need, on the part of employers, employees, and clothing purchasers, for a clear overview of safe work clothing that provides effective protection and that meets the requirements for working safely and healthy. Ecology and prevention of Lyme borreliosis 405

407 Mirjam C.G. de Groot Public health relevance Research is needed to also be able to provide adequate information to gardeners and landscapers in the future as well as practical tips on how to reduce the number of tick bites during work activities in gardens and parks. Updated and detailed geographical information is needed about tick density in order to be able to prepare a good risk evaluation. Employers and employees are asking for more information about management measures. There is a need for a Dutch standard for tick-repellent clothing. If impregnated clothing is worn, it would be advisable to further minimise skin contact with permethrin via partial treatment or by making use of a double layer of inner and outer fabric. It is important for employers to supervise the implementation of the measures agreed upon. It is important for employees to do a methodical tick checking during several moments of the day. Additional research is needed into the health effects of the long-term use of DEET and clothing impregnated with permethrin. Acknowledgements My thanks goes out to Brechtje Bokdam, Annemarie van den Hoven, Heleen Mees and Ad de Rooij for their input to an earlier version of the chapter. References De Groot M and Bokdam B (2013). Tekenwerende werkkleding praktijkproef. Commissie Arbeidsomstandigheden van het Bosschap, Driebergen, the Netherlands. De Groot M, Van Houten E, De Rooij A and Van der Zwan A (2010). Teken, tekenbeten en de ziekte van lyme in de hoveniers- en groenvoorzieningssector. Stigas, Woerden, the Netherlands. De Groot M, Reinders E, De Rooij A and Van der Zwan A (2009). Teken, tekenbeten en de ziekte van lyme in de sector bos en natuur. Commissie Arbeidsomstandigheden van het Bosschap, Driebergen, the Netherlands. Deutsche Gesetzliche Unfallversicherung (DGUV) (2013) Biological monitoring and evaluation of potential hazards arising from the use of permethrin treated protective clothing for employees in the forestry sector. DGUV, Sankt Augustin, Germany. Available at: Gassner F, Bruinvels DJ, Appels A, Van Balen J, Bouwmans J, Van der Burg A, Houba R, Hovius JW, De Lange MJ, Maas JJM, Niessen ML, De Rooij A, Schilpzand J, Steenbergen JM and Veders R (2014). Multidisciplinaire richtlijn Arbeid en lymeziekte. National Institute of Public Health and the Environment (RIVM) and Netherlands Society of Occupational Medicine (NVAB), the Hague, the Netherlands. 406 Ecology and prevention of Lyme borreliosis

408 27. Personal protection for people with occupational risk in the Netherlands Gassner F, Hofhuis A and Bastiaanssen M (2015). Nieuwe richtlijn voor preventie van en omgaan met tekenbeten en lymeziekte tijdens het werk. Infectieziekte Bulletin 26: Available at: Gassner F, Lier A, Pronk W and Brandwagt D (2016). Teken in de bebouwde kom in de regio Utrecht. GGD, Utrecht, the Netherlands. Availabe at: Haverkort F (2013) Aanpak van het risico op de ziekte van Lyme door aanpassingen in natuurbeheer, een handboek voor natuurbeheerders. MSc thesis, National Institute of Public Health and the Environment (RIVM), the Hague, the Netherlands. Hofhuis A, Harms MG, Van der Giessen JWB, Sprong H, Notermans D and Van Pelt W (2010) Ziekte van Lyme in Nederland Infectieziekten Bulletin 21: Hofhuis A, Herremans T, Notermans DW, Sprong H, Fonville M, Van der Giessen JW and Van Pelt V (2013) A prospective study among patients presenting at the general practitioner with a tick bite or erythema migrans in the Netherlands. PLoS ONE 8: e Hofhuis A, Meunier N and Van den Wijngaard C (2013) Arbeidsgerelateerde ziektelast door Lyme-borreliosis in Nederland. Cib/RIVM, on behalf of Ministry of Social Affairs, the Hague, the Netherlands. Mulder AC, Snabilie M and Braks MAH (2016) From guessing to GIS-ing: empowering land managers. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Sprong H and Van den Wijngaard CC (2016) Prevention of Lyme borreliosis after a tick bite. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Ursinus J, Coumou J and Hovius JWR (2016) The complexity of patients with (suspected) Lyme borreliosis. In: Braks MAH, Van Wieren SE, Takken W and Sprong H (eds.) Ecology and prevention of Lyme borreliosis. Ecology and Control of Vector-borne diseases, Volume 4. Wageningen Academic Publishers, Wageningen, the Netherlands, pp Valstar MW and Hardij A (2013) Risicobeoordeling permetrine langdurig dragen van geïmpregneerde kleding. Defensie Gezondheidszorg Organisatie, Coördinatiecentrum Expertise Arbeidsomstandigheden en Gezondheid, Doorn, the Netherlands. Van den Wijngaard CC, Hofhuis A, Harms MG, Haagsma JA, Wong A, De Wit GA, Havelaar AH, Lugner AK, Suijkerbuijk AW and Van Pelt W (2015) The burden of Lyme borreliosis expressed in disability-adjusted life years. Eur J Public Health 25: Vaughn MF and Meshnick SR (2011). Pilot study assessing the effectiveness of long-lasting permethrin-impregnated clothing for the prevention of tick bites. Vector Borne Zoonotic Dis 11: Ecology and prevention of Lyme borreliosis 407

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410 28. The protection of European dogs against infection with Lyme disease spirochaetes K. Emil Hovius Amphipoda BV Ecovet, Heike 9, 5508 PA Veldhoven, the Netherlands; Abstract In this chapter the natural, innate immunity of dogs, the added protection offered by tick repellents and acaricidal products, and the activated immunity provided by vaccines against Lyme disease spirochaetes are discussed. The dog s innate immune defence to spirochaetes is incomplete. Only one Borrelia species is killed by serum complement. This means that feral dogs may have a role to play as a reservoir in the ecology of Lyme disease spirochaetes. However, only a minority (5%) of dogs develop Lyme disease symptoms, the reason for which is probably breed related. Thus added protection seems appropriate. Recently acaricidal products have improved in efficacy and in ease of application. Client compliance with the application regime may greatly influence the outcome and care must be taken, at time of reapplication, to prevent ticks from having an opportunity to reinfest. Vaccination with the recently improved newest vaccines which target the European Borrelia species and its related strains can ensure a yearlong active immunity. It is anticipated that the new vaccines will exert nearly 100% protection if they are applied in the way the manufacturer s instructions recommend and even more surely with an extra booster. Experience with the new vaccines has not yet resulted in published field trials. More knowledge of the effects of vaccination is needed in Europe; knowledge which could also benefit the development of human vaccine production. Keywords: acaricides, anti-osp A, anti-osp C, canine borreliosis, One Health, serum resistance, vaccines Introduction The dog is the one household animal that is most often let free to traverse its favoured playing/ hunting grounds, these are usually parks or wooded areas that are not far from the neighbourhood where the dog lives in proximity with its owners. Companion animals in general are daily in close contact with humans and, as such, may put their owners at risk of tick bites from tick infested areas. For these reasons, the protection of dogs against ticks is a rewardable precondition against the risk, not only for the dog itself, but for its owners too. The dog is possibly a competent reservoir host for Lyme spirochaetes and it is not unlikely that feral dogs still roaming in some parts of Europe are playing a local role in Lyme disease ecology. Therefore, it is relevant to explore the dogs own defence against ticks, Borrelia infections and disease. Perhaps, in the near future, vaccination against ticks could be a realistic option (Klouwens et al. 2016). Anti-Borrelia vaccines are already on the market and millions of dogs have been vaccinated across North America and Europe. For dog breeds that are sensitive to the development of Lyme disease after infection and are at risk of tick bite, protection via vaccination may be the ideal choice. The experience gained using these vaccinations may also be useful when vaccination for humans is eventually introduced. Other, more direct, measures should not be omitted and will be discussed in this chapter. Tick avoidance is possible but will restrict the dog s free movement. Therefore repellents and acaricides which can be applied directly to the dog s skin, or orally administered are most frequently used. The role of carefully applied antibiotics in veterinary medicine, taking the suspicion of arthropod borne Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _28, Wageningen Academic Publishers 2016

411 K. Emil Hovius infection into account for a dog with appropriate symptoms, cannot be omitted in the discussion on protective measures. With all the evaluations of risk it is logical that the comparison of city dogs versus outdoor dogs is accounted for (Mather et al. 1988). Dogs are more at risk than their owners and there are no signs that having a dog in the household puts the human occupants at any greater risk for acquiring Lyme disease (Eng et al. 1988, Goossens et al. 2001). Risk of tick borne diseases in dogs Many mammals and birds, including pet animals, are host species for ticks. The dog accompanies man into woods and other tick habitats, and is thereby put at far greater risk of contracting tickborne infections than man himself. Horses may be exposed if pastured in the grassland to wood interface. Free roaming cats can be exposed to ticks, but do not easily develop borreliosis, and their role in the ecology of ticks and Borrelia needs investigation. Dogs are frequently infected, and re-infected, by the spirochaetes, and in some cases (around 5%) develop disease (Hovius 1990, 2005, Hovius and Houwers 2007, Hovius et al. 2000, Kornblatt et al. 1985, Littman 2003, Magnarelli et al. 1985, 1987, Straubinger 2000a, Summers et al. 2005). The dog, therefore, has been selected as the companion animal for which any possible protective measures against Borrelia infection may be appropriately illustrated. Infection and symptomatic manifestations of an array of other tick-borne agents have also been described in dogs. The most important of these other tick-borne agents are the (bacterial and protozoan) genera (Neo)ehrlichia, Anaplasma, Rickettsia (including the recently discovered Neorickettsia), and Babesia moreover dogs are also prone to viral tickborne encephalitis. Some protective measures against Borrelia infections, those preventing tick infestations, for instance, will also offer protection against the other tick borne disease agents. However, not all infectious agents are harboured by the same tick species, Ehrlichia canis in Europe reside in the brown dog tick Rhipicephalus sanguineus, for example, while Babesia canis infecting dogs is transmitted by Dermacentor reticulatus and R. sanguineus. The two Ixodes species, I. ricinus in western Europe and I. persulcatus in the East, transmit several Borrelia species as well as Anaplasma phagocytophilum that propagates in dog granulocytes. Anaplasma platys, which infects dog thrombocytes, is mainly transmitted by R. sanguineus. In some European countries, antibodies against Anaplasma phagocytophilum may be far more prevalent in dog serum than antibodies directed Borrelia and the other tick borne agents and canine anaplasmosis is regularly diagnosed especially in Sweden (Egenvall 1997, Krupka et al. 2007, Kybicova et al. 2009). Preventive measures against tick infestations will also reduce the acquisition of the other disease agents. Symptoms and diagnosis of borreliosis in dogs In dogs the symptoms of disease caused by the array of tick borne zoonotic infectious agents can be similar in appearance especially when the first manifestations of disease develop. In the later stages of disease, symptoms may more clearly define one particular agents but for a diagnosis to be made serological and molecular laboratory investigations usually need to be made at several time intervals after infection. Moreover, coinfection or successive infections can occur which may hamper the interpretation of clinical and laboratory investigations. Thus it appears that, in clinical appearance, symptomology, and diagnostic workup, the dog is as complicated as the human Lyme disease patient. This also concerns borreliosis caused by a single Borrelia infection for which there are no pathognomonic symptoms, such as erythema migrans in man. However, unexplained fever and lameness may be seen as not specific but typical for canine borreliosis (Kornblatt et al. 1985, Littman et al. 2006). Chronic manifestations in the dog may be more easily observable than in man, due to the shorter lifetime of the dog. Also the apparent differences in breed susceptibility seem to indicate a genetic disposition more easily than is observable in man (Gerber et al. 2007, 410 Ecology and prevention of Lyme borreliosis

412 28. Protection of pets Lindenmayer et al. 1991) (Figure 1). Therefore, lately it has been suggested that the dog might not only be used as a sentinel for the infection in humans but could also serve as a model for the pathogenesis of disease in man (Elsner et al. 2015). Re-infection is more easily determined in dogs than in humans, if the appropriate time frames and particular antibodies are studied (Hovius et al. 1999a). These reinfections (probably with more than one Borrelia species; see below) do not usually lead to disease. Apparently, some of the seroconverting antibodies, along with other reactions of the adaptive immune-system clear the spirochaetes. When attributable symptoms (not specific) occur this is accompanied by rising and high titres, which usually leads to a diagnosis when several serum samples are taken throughout one tick season (Hovius et al. 2000). To date, four Borrelia species have been detected in dog tissues and tissue tropism for the three pathogenic species seems to be apparent. However, after limited investigation, it seems that this tropism in the dog is not directed in completely the same way as it is in humans. However, in dogs as in man, Borrelia burgdorferi sensu stricto is mostly detected in tissues which are rich in collagen such as skin and peripheral joint tissues (Straubinger 2000b, Summers et al. 2005, Van Dam et al. 1993). Borrelia garinii is frequently observed in man in the tissue of the nervous system. In the dog it was mainly detected in liver tissue (Hovius et al. 1999b, Van Dam et al. 1993). Borrelia afzelii and Borrelia valaisiana were only found in liver tissue in dogs (Hovius et al. 1999b).The dog s liver may function as a sink in which spirochaetes are collected and killed by liver macrophages, as Sambri has shown in rat livers (Sambri et al. 1996). In a Dutch study on tick infested seropositive dogs, liver samples containing Borrelia DNA were mainly found in symptomatic dogs, putatively diagnosed for Lyme disease. Most of the positive samples contained DNA of more than one Borrelia species and nearly always included B. garinii DNA. Healthy dog livers much less frequently contained Borrelia DNA (Hovius et al. 1999b). Figure 1. The Bernese mountain dog, is frequently found to be seropositive for the Borrelia IR6 antigen, measured with the C6 SNAP test (by Idexx, see text). It may also show episodes of fever and lameness. The diagnosis of an episode of active Lyme disease, is complicated by possible osteological problems or immunological aberrations that may occur in this breed. In general a thorough diagnostic workup with exclusion of differential diagnosis and multiple serological tests is needed to confirm or discard the diagnosis of Lyme disease (copyright photo: Tijdschrift voor Diergeneeskunde 2007). Ecology and prevention of Lyme borreliosis 411

413 K. Emil Hovius The dog is imperfectly protected against Borrelia by its innate immunity Borrelia species circumvent the innate immunity of their hosts by expressing host specific factor H binding proteins that inhibit factor H regulatory proteins in their function as first step in the cleavage of the C3b complement factor (Bhide et al. 2009, Van Burgel et al. 2010). The differing complement resistance of Borrelia species determines the evolutionary coupled reservoir competence of certain animals (mammals, birds, reptiles) (Kurtenbach et al. 1998, 2002). In this chapter it is interesting to explore the known putative reservoir function of canids (and other mammals) for the different Borrelia species, derived from complement mediated killing experiments, (Bhide et al. 2005, Hovius et al. 2000,Van Dam et al. 1997) or factor H binding experiments (Bhide et al. 2009, Van Burgel et al. 2010) (Table 1). Several Borrelia species are resistant to dog serum such as B. burgdorferi s.s, B afzelii, Borrelia bavariensis (formerly B. garinii serotype 4-Pb1) and B. valaisiana. While most B. garinii strains are not resistant and thus killed by the dog and mammalian complement system, in line with B. garinii being hosted by birds. Remarkably, B. afzelii and B. valaisiana are killed by wolf complement and not by dog complement. A factor H binding protein for dog complement was detected for B. afzelii and B. valaisiana, thus preventing bactericidal activity by dog serum (Bhide et al. 2009). In line with these results 4 Borrelia species were found in organ tissues of dogs (Hovius et al. 1999b). It is hypothesised that evolutionary time has been too short for dog and man to develop innate immunity against the 4 species (Skotarczak 2014). B. afzelii is the most frequently occurring species in ticks. The Western European wolf Canis lupus lupus opposes this threat with an evolutionary developed defence strategy of the complement system. The dog Canis lupus familiaris seems to have lost this ability in the domestication process. B. burgdorferi s.s. appears to have made transatlantic colonisation vice versa and some strains, such as B31 used in the assays, have North-American origins (Castillo-Ramirez et al. 2016). Therefore, it is not surprising that the European wolf and its domestic pedigree is maladapted to withstand certain non-european B. burgdorferi s.s. strains (Bhide et al. 2009). This implies that dog and the wolf are a putative reservoir for B. burgdorferi s.s.. The Beagle breed has been experimentally determined to be a competent reservoir animal for B. burgdorferi s.s., which is passed to ticks (Mather et al. 1994). Table 1 also shows the capacities of feline predators to fence off different Borrelia species. The reservoir function of the cat has not been investigated. But, if the cat were to transmit Borrelia to ticks like the Beagle dog does, the feral cats, collecting and releasing ticks in the woods, could contribute to the ecology of the Borrelia species which have serum resistance. Hoofed mammals are dead end hosts for all the Borrelia species and strains investigated and have no reservoir function. The ecological impact of these animals is discussed in earlier chapters of this book. Man is similarly maladapted to withstand the different Borrelia species as is the dog and in this sense the dog may be an animal model for Lyme disease. Do dogs have a role in the transmission cycle of some Borrelia species? The possibility of vectors and their transmissible disease agents being distributed in the wild by wild canines and felids to their domesticated or feral counterparts has been reviewed by Otranto (Otranto et al. 2015). Wolfs are expanding and roam together with feral dogs and dog hybrids in certain parts of Europe. These animals can function as reservoirs for Borrelia and maintain infections in the region that may be picked up by their tame counterparts. The Beagle dog breed has been determined to be a competent reservoir animal for B. burgdorferi s.s., it acquires and delivers the spirochaetes to ticks (Mather et al. 1994). The wolf, however, is a dead end host for more Borrelia species than the dog, see Table 1. Feral dogs and the wolf in the European ecosystem may differ in their reservoir function to propagate different Borrelia species (Bhide et al. 2005).The preliminary bactericidal assays have to be repeated with several dog breeds, with wolves from 412 Ecology and prevention of Lyme borreliosis

414 28. Protection of pets Table 1. Resistance (yes/no) of the most occurring West European Borrelia species and strains to serum of several mammalian species including dog and man. Borrelia garinii is heterogeneous and distinguished by the Osp A serotype as determined by Wilske et al. (1995, 1996). 1,2 Species (OspA serotype) Strain Horse Cow Lynx Cat Wolf Dog Man B. burgdorferi s.s. B31 yes: 2 yes: 1, 5 (serotype 1) SKT2 no: 4 no: 3, 4 yes/no: 3 yes: 3; yes: 3 yes: 3, 4 yes: 3, 4 no: 4 B. afzelii SKT4-5 no: 4 no: 3, 4 yes/no: 3 yes: 3, 4 no: 3 yes: 3,4 yes: 3, 4 (serotype 2) pko yes: 2 yes: 1, 2 B. garinii (serotype 3) Rio 2 no: 4 no: 3, 4 yes: 3 yes: 3; no: 4 yes: 3 yes: 3; no: 4 yes/no: 3; no: 4 B. garinii G117 no: 4 no: 4 no: 4 no: 4 no: 4 (serotype 5) PV6 no: 3 no: 3 yes/no: 3 no: 3 no: 3 no: 3 A87S no: 2 no: 1 B. garinii SKT3 no: 4 no: 3, 4 yes: 3 yes: 3; no: 3 no: 3, 4 no: 3, 4 (serotype 6) no: 4 VSBP no: 1, 5 B. garinii T25 no: 4 no: 4 no: 4 no: 4 yes: 4 (serotype 7) B. garinii (serotype 8) CLI no: 4 no: 3,4 yes: 3 yes: 3; no: 4 yes: 3 yes: 3; no: 4 no: 3,4 B. bavariensis (B. garinii serotype 4) PBi no: 4; yes: 5 no: 3, 4, 5 yes: 3 yes: 3, no: 4, 5 yes: 3 yes: 3, 5; no: 4 yes: 1, 4, 5; yes/no: 3 B. valaisiana (not serotyped) VS116 no: 4 no: 3, 4 yes: 3 yes: 3; no: 4 no: 3 yes: 4; yes/no: 3 yes: 1, 4; yes/no: 3 1 yes = serum does not kill the Borrelia strain; no = serum kills the Borrelia strain. 2 Numbers represent the authors (in the references) that have employed different assays: 1, 2, 3 explored the bactericidal activity of serum against live spirochaetes (yes, is less than 40% killed; yes/no is between 40 and 60% killed; no is 60% or more spirochaetes killed); authors 4 and 5 determined the factor H binding protein on the spirochaetes (yes is detected; no is not detected). References: 1 = Van Dam 1997; 2 = Hovius 2000; 3 = Bhide 2005; 4 = Bhide, 2009; 5 = Van Burgel different countries and with several strains per Borrelia species. However, it may be tentatively expected that the dog will be a reservoir animal for B. burgdorferi s.s. B. afzelii, B. bavariensis and B. valaisiana and the European wolf for B. burgdorferi s.s. and B. bavariensis. Therefore, after expected reintroductions, the wolf may have a direct influence on tick cycles and the ecology of Borrelia systems, besides possibly influencing these cycles by changing the spectrum of reservoir biodiversity. Spanish wolves were found to be seropositive (14.7%), but the infecting species was not determined (Sobrino and Gortazar 2008). In the urbanised western countries of Europe, the influence of feral dogs in the patchy woody ecosystems can be neglected. When dogs are walked by their owners through these environments they contract many ticks, which drop off elsewhere. In gardens, large enough to contain some species of mice, rats, squirrels, hedgehogs and birds, a local cycle of tick and Borrelia may develop Ecology and prevention of Lyme borreliosis 413

415 K. Emil Hovius which could pose an extra threat of tick infestation and Borrelia transmission to the owners (Bhide et al. 2004). But a greater infection rate in dog owners than in non-dog owners has never been documented (Eng et al. 1988, Little et al. 2010). However, the possibility exists that unattached ticks in the dog s coat can walk onto the owner s skin (personal observation). For the above reasons, the protection of dogs against ticks is a rewardable precaution. Tick avoidance is possible but will restrict the dog s free movement. It is, therefore mostly repellents and acaricides that are applied to the dog s skin, or orally administered. In future it may be possible to vaccinate against ticks (Klouwens et al. 2016). Anti-Borrelia vaccines are available for dog breeds sensitive to the development of Lyme disease. Added protection of dogs Protection by avoidance strategies Not roaming the woods and avoiding shrubby areas will destroy the dog s pleasure during field walks. Expecting dog owners to be able to judge the tick favourable habitats is unrealistic, so appropriate warning signs could be placed that owners and dogs enter at risk of contracting ticks. Apart from instructing people how best to avoid tick contact, advice for dog handling in this sense should also be put on the signs. When the tick season is at its most intense and the weather is favourable for questing ticks, with moderate to high temperatures and very high relative humidity, it is particularly important to avoid the undergrowth of woods and thickets. At such times the dog is best kept on a lead. Many people live in the countryside and for these dogs and for working dogs (guard dogs, herding dogs, hunting dogs) the avoidance of tick habitat is impossible. After field work, just as in man, these dogs should be checked for ticks immediately when returning home or to the pen. Particular attention should be paid to checking the areas above the eye, behind the ears, the front of the neck, around the front legs (axil and shoulder) and in the groin and the ticks removed with a special instrument or a pair of pliers (Figure 2). The result of these inspections depends on the texture and colour of the dog s coat and will never be perfect. It is therefore wise to include other preventive measures. It has been shown that in an area which has about 1 in 10 ticks infected, the first contact with an infected tick occurs on the first or second walk. This single attachment of an infected tick in all cases leads to the transmission of Borrelia to the dog (Leschnik et al. 2010). There may be 3 strategies to prevent this transmission. 1. Preventing ticks from clinging to the dog s coat by applying effective repellents. Or, if the repellents do not succeed in preventing attachment and fixation to the skin, by applying acaricides which have immediate efficacy. 2. If the first strategy does not work, ticks can be killed when attached, preferably in the very first 12 hours of attachment when (in the majority of ticks) the Borrelia have not yet reached the salivary gland to be transported by the saliva to the vertebrate host (Cook 2015, Piesman et al. 1987). 3. If the acaricides do not work properly because of the composition of the dog s skin, for instance, Borrelia infection may then be prevented by vaccination. Protection by prevention of ticks clinging onto the coat: repellents With regard to ticks, the repellent effect cannot be viewed the same as with flying vectors where the repellent prevents the host from being landed on. It is hard to discourage ticks from grabbing onto the fur or skin of the host when they come in accidental contact with it, and repellency is defined as the reluctance to attach (insert the mouthparts) or leave altogether (Halos et al. 2012). 414 Ecology and prevention of Lyme borreliosis

416 28. Protection of pets Figure 2. Shown is a dogs ear with on the pinna attached adult ticks at several stages of engorgement. All three developmental stadia of Ixodes ricinus ticks may infest dogs. Deeper in the ear nymphal tick are attached, also engorged. The larval stage may also be found on dogs, especially with high numbers on the ears, when the dog has passed a recent hatched egg cluster. Repellent ointments used by man to fend off mosquitos and ticks do not work sufficiently for furred animals and should not be applied; plant products such as citronella and lemon eucalyptus oil are equally ineffective. Also the synthetic made DEET (N,N-diethyl-m-toluamide) should not be used and can even be toxic for dogs (Schoenig et al. 1999). The only substance having a repellent effect on several tick species is permethrin, and this too is not absolute. Permethrin is one of the synthetic pyrethroidic substances (natural product: pyrethrin, derived from the plant Tanacetum cinerariifolium) whose actions are enhanced with certain solvents like piperonyl butoxide. The liquid, contained per dose in small pipettes (for instance Pulvex spot on; MSD, New York, NY, USA), is applied topically onto the skin between the shoulder blades, so that the dog cannot lick the site if it itches. From the application site, the substance diffuses through the fat layer and exerts its acaricidal effect killing ticks evenly from nose to tail (Lussenhop et al. 2011). Other synthetic pyrethroids that are applied in acaricidal products are deltamethrin (also used in powders and shampoos) and flumethrin, usually used in combination with other products (see also Van Wieren et al. 2016). Protection by inhibiting or killing ticks shortly after attachment: acaricides Amitraz, a strong acaricidal, is a product widely used and applied onto the skin of farm animals (Van Wieren et al. 2016) but can have adverse effects when applied all over the skin of dogs. Also in small concentrations when combined with other products and topically applied it may show unwanted effects. More recently the acaricidal properties of substances like fipronil (a fenylpyrazolon) and imidacloprid (a nicotine derivate) have been utilised with no adverse effects. These substances can be combined with permethrin to enhance the bactericidal effects and are marketed as Frontect (Merial, Lyon, France), Effitix (Virbac, Carros, France) and Advantix (Bayer, Leverkusen, Germany), all of which have been shown to have almost 100% efficacy in experimental settings (Bonneau et al. 2015, Dumont et al. 2015, Otranto et al. 2005). Under natural conditions these combinations Ecology and prevention of Lyme borreliosis 415

417 K. Emil Hovius showed that they were able to prevent the transmission of arthropod transmissible infections in I. ricinus and other tick species (Jongejan et al. 2015, Otranto et al. 2008, 2010). Topical applications are also employed by way of impregnated collars which slowly release the acaricidals and have the advantage of a half year activity. Seresto collars (Bayer), a combination of imidacloprid and flumethrin, and Scalibor collars (MSD), impregnated with deltamethrin, both appear to have enduring action and are approximately 98% effective with one application providing months of protection (Brianti et al. 2010, Stanneck et al. 2012, Van den Bos and Curtis 2002). The effect of these collars in preventing the transmission of tick borne disease agents by killing the vector before transmission occurs, has been shown for E. canis and its vector R. sanguineus (Stanneck and Fourie 2013). Just recently Simparica (Zoetis, Madison, NJ, USA) and Bravecto (MSD) chewable tablets were licensed on the European market. These tablets are given to dogs every 2 months when R. sanguineus is the target or may be given every 3 months when the action is directed at Ixodes or Dermacentor (Williams et al. 2015). The pharmaceutical compounds Sarolaner and Fluranaler belong to isoxazoline organic chemicals. These compounds, as others, exert their action by the selective inhibition of arthropod GABA-gated chloride nervous system channels (Gassel et al. 2014). By blocking these channels the arthropod nervous system is inhibited which results in the death of the insect or tick (McTier et al. 2016). The vertebrate chloride nervous system channels are much less sensitive to these products and are safe for dogs up to several times the prescribed dose (Walther et al. 2014a). The product did not show any adverse effects when administered together with an anthelmintic or the deltamethrin collar (Walther et al. 2014b). The most advantageous property of these drugs is the speed of their action in killing ticks before transmission of disease agents occurs. When applied: after 2 days 100%, and after 12 weeks 98% of ticks are killed within 12 hours (Wengenmayer et al. 2014). Prevention of transmission of B. canis by D. reticulatus has also been shown (Taenzler et al. 2015). Protection by vaccination directed at killing attached ticks Theoretically, the quick killing of a tick could be accomplished by vaccinating with tick proteins functional in the salivary gland or the tick gut. For cattle the standard practice is vaccinating with the Bm86 gut lining protein of Rhipicephalus microplus, (Klouwens et al. 2016). A homolog of this protein from I. ricinus did not work in a laboratory setting (Coumou et al. 2014). In the future we may probably see vaccination of dogs with tick proteins. Protection by vaccination directed at killing Borrelia just before or after infection Antigenic shifts of Borrelia in the vector-host interface and the possibilities for vaccination In nature, Borrelia infection is established with help of tick salivary proteins (Ramamoorthi et al. 2005). The different salivary proteins exert different immunomodulatory actions, enabling the tick to survive the host s immune system (Hovius et al. 2008). Moreover, the Ixodes scapularis protein Salp 15, enables B. burgdorferi s.s. to surpass the immune system by cloaking outer surface protein C (Osp C) (Ramamoorthi et al. 2005). Osp C is thought to be essential for the transfer from the midgut to the salivary glands of the tick and subsequently to the vertebrate host (Pal et al. 2004b). If Salp 15 is inhibited experimentally or if anti-salp 15 has developed after some initial infections, the second blockade against infection becomes apparent when bactericidal antibodies against Osp C, destroy the spirochaete in a complement dependent process (Callister 1999). Osp 416 Ecology and prevention of Lyme borreliosis

418 28. Protection of pets C is expressed on the spirochaete outer membrane after the tick has attached to the host. The temperature and the first vertebrate blood ingestion induces a change of expression (switch) of the outer service proteins. The spirochaete reside in the tick gut connected with Outer Surface Proteins A (Osp A) to TRospA ligand protein (Pal et al. 2004a). A Vaccine directed at Osp A has the remarkable effect of preventing the switch from Osp A to Osp C by killing the spirochaetes in the gut of the tick and thus preventing entrance into the vertebrate host (Coughlin et al. 1995). Anti-Osp A induced by Osp A, that does not seem to have a function when in the vertebrate host, may be detected only in prolonged infection when it may prevent reinfections in the way described above. In some naturally infected dogs Osp A was detected (Gauthier and Mansfield 1999, Hovius et al. 2000). It may be hypothesised that this is an eventual reaction of the body against reinfections. The vaccination with Osp A then seems a natural phenomenon, artificially introduced earlier in the infective process. Upon vaccination with whole cell lysates of spirochaete (bacterins), the most prevalent antibody induced is anti-osp A (Barthold et al. 1995, Gauthier and Mansfield 1999). Other antibodies may be produced after several revaccinations but anti-osp A remains prevalent in high titers. Thus, vaccination with a bacterin induces the same protection after vaccination as with a (recombinant) Osp A vaccine. The high anti-osp A titre makes the differentiation of serum of vaccinated or naturally infected dogs possible (Gauthier and Mansfield 1999). Some recent experiments with vaccinated and naturally infected dogs yielded some re-evaluation on this clear differentiation. The time effect on the intensities of certain antibodies needs to be taken into account to make the distinction (Leschnik et al. 2010). Vaccination of puppies with Osp C proteins or with whole cell lysates containing Osp C induce high levels of bactericidal Osp C antibodies killing the spirochaetes upon entrance with the first infective tick bite (Callister 1999). Anti-Osp C in natural infection is detected as one of the first induced antibodies, which may be explained by the requirement of Osp C for entering the vertebrate host (see above). A dog survey in the Netherlands among naturally infected dogs revealed anti-osp C bactericidal antibodies against three different Borrelia species (Hovius et al. 2000). The implications of this finding for vaccine development need further investigation since the Osp C protein is very variable and regionally fixed (see below). History of vaccination in dogs The Osp A vaccine strategy was developed at first in human vaccination schemes (De Silva et al. 1996), but the manufacturer has withdrawn Lymerix (recombinant Osp A) from the market because it was not generally accepted by the public (Embers and Narasimhan 2013). Several vaccines for dogs have been marketed and vaccine development has recently expanded to a third stage with new vaccines employing different geno-species and antigens exerting more crossprotection (Table 2). The first vaccines contain whole cell lysates of the spirochaete, so-called bacterins, whilst the second stage contains recombinant Osp A and Osp C as dominant antigen. The newest recently developed third stage vaccines are: (1) bacterins composed of a broad array of cross-protective antigens where several Borrelia geno-species are employed together; and (2) recombinant Osp A combined with chimeric Osp C which combines the most essential epitopes in one molecule. These third stage vaccines have only been on the market a few months or years and no field trials are yet available. It is expected that more cross-protection will be gained by vaccinating with these new vaccines. Ecology and prevention of Lyme borreliosis 417

419 K. Emil Hovius Table 2. Vaccines employed for protection of dogs against the Lyme disease Borrelia. First stage vaccines (1) have been employed for decades. The second stage (2) of development appeared somewhat later on the market. And the recent developed vaccines (3) were just licensed and the effect in the field with clinical trials have not yet been determined or published. The first and second stage contain only one strain of Borrelia burgdorferi s.s. (Bb s.s.). The last developed vaccines contain strains of several species in combination including Bb s.s., Borrelia garinii (Bg) and Borrelia afzelii (Ba) or only Bb s.s. with a combination of several Osp C serotypes in one so called chimeric molecule. The references describing experiments or clinical trials with the respective vaccines are discussed in the text. Name vaccin Company Fabrication B. species employed Specific antigens Most induced antibodies Used in Stage Reference lymevax Zoetis lysate and adjuvans Bb s.s. 2 strains mulitple antigens anti-ospa, anti-ospc America, Europe Duramun Lyme Boehringer lysate and adjuvans Bb s.s. multiple antigens anti-ospc America, Europe Merilym Merial lysate and adjuvans Bb s.s. multiple antigens anti-ospa, anti-ospc, anti-66kda Nobivac Lyme Recombitek Lyme America, Europe MSD 2 lysates, adjuvans Bb s.s. 2 strains OspA bivalent OspC anti-ospa, anti-ospc America, Europe 1 Chu et al. (1992), Gauthier and Mansfield (1999), Levy (2010), Levy et al. (1993, 2002), Topfer and Straubinger (2007) 1 Levy (2010) 1 Leschnik et al. (2010), Topfer and Straubinger (2007) 2 LaFleur et al. (2009, 2010) Merial recombinant Bb s.s. OspA anti-ospa America 2 Conlon et al. (2000), Eschner and Mugnai (2015), Levy et al. (2005), Topfer and Straubinger (2007) Biocan B Bioveta 2 lysates and adjuvans Bb s.s.; Bg OspA and multiple anti-flagellin (41 kda) anti 45 and 58 kda Europe 3.1 Topfer and Straubinger (2007) Merilym3 Merial 3 lysates and adjuvans Bb s.s.; Bg; Ba multiple antigens anti-ospa and others Europe 3.1 information by Merial Vanguard rc Zoetis recombinants Bb s.s. OspA chimeric OspC anti-ospa, anti-ospc America 3.2 Earnhart and Marconi(2007) Lyme 418 Ecology and prevention of Lyme borreliosis

420 28. Protection of pets First and second stage vaccines and the bacterin vaccine including B. garinii were comparatively investigated by Töpfer and Straubinger (2007) (Table 2). Ten seronegative dogs per vaccine, of all age groups and differing breeds, were vaccinated with the 5 vaccines using a double vaccination 3 weeks apart. For comparison, another 50 dogs were vaccinated with the same 5 vaccines 3 times at 2 and 3 week intervals and so received a double booster. The dogs, which were normal household dogs, were walked by their owners in Saxony, Germany and blood was taken for serological purposes every month. Titres of an automated whole cell Elisa reached their highest values for all dogs at 45 days after primo vaccination. But the 3 times vaccinated dogs peaked at about a 10% higher value. Thereafter titres more than halved in the course of one year and rose to the maximum level in the 20 days after revaccination on day 365. The gain of the second booster is the prolongation of higher titers during the one year decline to half the maximum value. Depending on which vaccine was used, the second booster resulted in between 0 to 30% difference in titre height. All the vaccines that were derived from B. burgdorferi s.s. performed with markedly lower titres when antibodies were tested against heterologous antigens (B. garinii and B. afzelii). Not so the European fabricated B burgdorferi s.s. and B garinii bacterin, which performed even better when tested with homologous B garinii. The differences between the heights of titers for the sera of dogs which had received one or two boosters became more pronounced in an ELISA that employs only Osp A as antigen. In Western blots of vaccinated dogs only antibodies against Osp A were apparent which made the distinction with the infected dogs that developed a whole array of antibodies against different protein weights in the Western blots at kda 39, 41 (flagellin), and 23 (Osp C). The Osp A antibody in vaccinated dogs is bactericidal in contrast with the serum of experimentally infected dogs without Osp A and will protect dogs from infection and disease in an experimental setting (Chang et al. 1995, Straubinger et al. 1995). Of the 100 vaccinated dogs only one showed signs of infection which means that, in 99% of dogs, infection was prevented by the bactericidal action of the vaccine induced antibodies. The same investigation with one of the vaccines (Merilyme-Merial) was performed in Austria and in this research all the vaccinated dogs remained uninfected while unvaccinated dogs became infected (Leschnik et al. 2010). Borrelia detected in the ticks infecting the dogs were B. gariniii and B. afzelii, not B. burgdorferi s.s., which is the species used in the vaccine, thus good cross protection was achieved. This cross protection was not foreseen from the lowered heterologous antibody titres in the trial performed by Töpfer and Straubinger (2007) (see above). In the Austrian trial more bands were in seen in Western blots of vaccinated dogs such as strong reacting Osp C, and even the p41flagellin B. garinii band reacted strongly which explains the apparent cross protection. In the USA, Recombitek Osp A by Merial is able to protect 12 week old, mixed breed dogs in a laboratory setting. Vaccinated dogs had negative skin biopsies and ticks could not acquire spirochaetes from them. In contrast to the tick infected non vaccinated dogs that had positive skin biopsies and could transmit the spirochaetes to ticks (xenodiagnoses) (Conlon et al. 2000). Interestingly the efficacy of the same Osp A vaccine Recombitek Lyme by Merial could be evaluated in a veterinary practice In Maine USA, where a thorough registration was kept of the 6,202 dogs which were vaccinated over a five year period. The 2 booster protocol recommended by Töpfer and Straubinger (2007) was adopted by the practice; the dogs received an initial injection, a booster after 3 weeks and a second booster after 6 months and subsequent vaccinations were then given annually. Of the 4,551 dogs which fully complied with the protocol, 1.01% became infected in the five year period apparently by being bitten naturally while walking with their owners. Surprisingly, of the 1,294 dogs that did not fully comply with the protocol, some 21.6% became infected during the 5 year period. Infection was measured as a positive reaction in the 4DX SNAP Ecology and prevention of Lyme borreliosis 419

421 K. Emil Hovius test (Idexx) measuring the C6 antibody. C6 is the synthetic counterpart of the IR6 antigen used in the SNAP test that is derived from the conserved part of the variable VlsE protein that changes antigenatically during the course of an active infection (Philipp et al. 2001). The C6 antibody is never induced by vaccination (Liang et al. 2000). The preventable fraction was calculated on a yearly basis and in the last study year (2013) was 98.08% which meant that a non-compliant dog was 52 times more likely to become infected than a fully compliant dog (Eschner and Mugnai 2015). In accordance, a smaller trial employing 60 dogs vaccinated with a single booster resulted in 25% infections (Levy et al. 2005). The vaccine by MSD, Nobivac Lyme, deliberately employs special strains one expressing Osp A combined with a second one which only expresses Osp C containing an epitope that is recognised by B. afzelii and B. garinii (LaFleur et al. 2009). The vaccine prevented Lyme disease development in laboratory dogs for a duration of one year but did not entirely prevent early infection in these dogs (LaFleur et al. 2010). The addition of the second strain not expressing Osp A but only Osp C has recently been shown to induce high bactericidal activity. But used singly (without the Osp A strain included) the vaccine reduced infectivity but in itself was not able to prevent establishment of spirochaetes in the joints of vaccinated laboratory dogs (LaFleur et al. 2015). This may be explained by the extreme variability of the Osp C strains in Lyme spirochaetes (Seinost et al. 1999). The Osp A sero-types, formerly determined by Wilske, broadly coincide with the now recognised Borrelia species (Table 1) (Wilske et al. 1996). Each of the Osp A sero-types includes several Osp C serotypes (Wilske et al. 1995). Broadly around 30 of these Osp C serotypes exist which appear to be geographically located and to have developed by recombination and not by selective pressure on mutations (Earnhart and Marconi 2007). The Osp C type specific epitopes induce bactericidal antibodies that do not cross-react with the other Osp C types. Importantly, Osp C types that are found in dogs are different from the ones found in man, which has to be accounted for when constructing vaccines for dogs (Rhodes et al. 2013). Recently such a third stage vaccine was licensed in the USA (VanguardcrLyme by Zoetis) based on the B. burgdorferi s.s. serotype with one recombinant Osp A protein combined with one chimeric Osp C protein including 7 type epitopes most commonly found in dogs. The first vaccine marketed, under the name of LymeVax and now distributed by Zooetis has been described by Chu, tested under experimental conditions and its cross protection for American strains of B. burgdorferi s.s. determined (Chu et al. 1992). In its current form, it contains two American Borrelia strains, which induces anti Osp A and anti Osp C antibodies. The LymeVax vaccine is widely used in the USA and the trials were performed under clinical conditions in veterinary practices in the USA where large numbers of vaccinated dogs (up to thousands of dogs) were compared with non-vaccinated dogs. Tens of millions of dogs have been vaccinated giving ample opportunity for the efficacy and safety of the vaccines marketed to be studied. Under field conditions the preventable fraction (or disease) was higher (88%) when seronegative dogs were vaccinated compared to seropositive vaccinated dogs (58%). It is important to test for antibodies in serum prior to vaccination since, in contrast to the experimental studies, vaccinated dogs have also been diagnosed as having Lyme disease (1% against 4.7% of non-vaccinated dogs) (Levy et al. 1993). The use of an in House Snap test determining C6 antibodies can easily identify dogs that have been infected before vaccination (Levy et al. 2002), as antibodies against the C6 antigen are not produced as a reaction to vaccination (Liang et al. 2000). On the other hand a reaction to the outer surface proteins Osp A and Osp C has been noted after vaccination with this vaccine (Levy 2010). 420 Ecology and prevention of Lyme borreliosis

422 28. Protection of pets Protection by clearance after infection, use of antibiotics Antibiotics have been used therapeutically in animals with Lyme disease after which improvement of the condition can be noted and antibody decline in serum is monitored usually with the quantitated C6 ELISA to confirm clearance of the spirochaete (Philipp et al. 2001). The argument as to whether antibiotics could be used in (heavily) tick infested humans to clear the spirochaetes before dissemination is disputed (Nadelman et al. 2001, Shapiro 2001). It is not recommended for dogs as new ticks can infest a few days later. However, in veterinary practice, antibiotics are easily administered to treat a fever of unknown origin which may have stopped further dissemination and in some cases may have prevented disease (Wagner et al. 2015). The tighter regulations governing antibiotic prescriptions will stimulate research on the diagnostic aspects of fevers of an unknown origin in general and Lyme borreliosis in particular. And at the same time, restricting antibiotic application, is an encouragement for the alternative employment of protective measures. Public health significance The dog may function as a sentinel for its owners, warning them for the risk of tick bite. Owners may check their dogs on a daily basis and when ticks are detected the chance of having acquired ticks themselves should not be neglected. In this way, owners will also gain knowledge about the whereabouts of tick habitats and may avoid these places. Ticks may be introduced to their owners gardens raising the risk for tick bite close at home. The dog appears to be a good animal model to study Lyme disease. It responds comparably as the human patient to an infection of B. burgdorferi sensu lato. Re-infection and recurrence of disease may be more easily observed in the dog than in man because of the relatively shorter live span. Some breeds may be more prone to the disease revealing a putative genetic predisposition, when further studied may reveal genetically coupled pathogenic mechanisms. Experiences with the different vaccines employed for dogs may pave the way to similar vaccines for man. Veterinarians as well as physicians should adapt the one-health way of thinking and communicate structurally, directed and stimulated by governmental facilities, for maximum benefit of the knowledge gained in dog studies. Public health relevance The susceptibility for the different Borrelia species is similar, which provides the opportunity in comparing human and canine clinics and diagnosis. Lifetime clinically monitoring of dogs and breed predilections provide insight in age and genetic related pathogenic mechanisms. The accelerating vaccine development for dogs, and the experience gained in the application, will provide valuable knowledge for human vaccine development. Ecology and prevention of Lyme borreliosis 421

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430 29. Lyme borreliosis prevention strategies: United States versus Europe Lars Eisen 1* and Jeremy S. Gray 2 1 Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 3156 Rampart Road, Fort Collins, CO 80521, USA; 2 UCD School of Biology and Environmental Science, University College Dublin, Belfield, Dublin 4, Republic of Ireland; evp4@cdc.gov Abstract In recent decades, vector ticks have spread and proliferated, and Lyme borreliosis emerged as a major health problem both in Europe and the United States. In the United States, Lyme borreliosis is a reportable disease and public health agencies and private industry have invested heavily in developing counter-measures, including education, a human vaccine (no longer available), tick repellents and permethrin-treated clothing to prevent tick-bites, and acaricides to suppress hostseeking ticks or target ticks on rodents or deer. Homeowners in the United States have the options of using over-the-counter acaricidal products or to use the services of licensed pest control operators to protect their properties. Similar options are available in Europe, but application of acaricides in peridomestic environments is rarely if ever carried out and licensed pest control companies do not offer this service. The main measure in use in Europe is still the dissemination of information on prevention of tick-bites and guidelines on whether to consult a physician. Current use of existing prevention and control measures have not proven adequate to stem the rising tide of Lyme borreliosis in either the United States or Europe. The evidence base for personal protective measures and environmentally-based tick/pathogen control methods to reduce, or fail to reduce, Lyme borreliosis cases needs to be strengthened to clarify the value of employing such measures, singly or in combination. The re-emergence of a human vaccine remains a critical need to reduce Lyme borreliosis in North America and Eurasia. Keywords: Ixodes ricinus, Ixodes scapularis, Lyme borreliosis, prevention Introduction In the last three decades, Lyme borreliosis has emerged as a major health problem across Europe and in parts of the United States. We describe the emergence and spread of vector ticks and Lyme borreliosis in the United States and Europe, and contrast the actions taken by public health agencies, academic researchers, and private industry in these two geographical regions to control vector ticks and prevent Lyme borreliosis. Emergence and spread of vector ticks and Lyme borreliosis United States The blacklegged tick, Ixodes scapularis Say (Figure 1), is the most important vector of the Lyme borreliosis spirochaete, Borrelia burgdorferi sensu stricto (hereafter called B. burgdorferi), in the United States (Burgdorfer et al. 1982, Eisen et al. 2016). This tick species is widely distributed in the eastern United States, from the Atlantic Coast to the edge of the Great Plains (Figure 2). The immature tick stages feed commonly on rodents, shrews and birds that serve as reservoirs for Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _29, Wageningen Academic Publishers 2016

431 Lars Eisen and Jeremy S. Gray B. burgdorferi, including the white-footed mouse, Peromyscus leucopus (Rafinesque) (LoGiudice et al. 2003). The white-tailed deer, Odocoileus virginianus (Zimmerman), is the preferred host of the adult stage (Piesman and Gern 2004). Presumably in large part due to deforestation and near elimination of white-tailed deer from much of the eastern United States during preceding centuries, the geographic distribution of I. scapularis was spatially restricted and highly focal in the early 20 th century. Reforestation combined with spread and increasing abundance of white-tailed deer are considered key factors in the rise and dramatic spread of I. scapularis in mid-atlantic, northeastern, and north-central states during the latter part of the 20 th century (Dennis et al. 1998, Spielman 1994). The tick has continued to expand its range in these regions in the 21 st century, with two previously distinct foci in the Northeast and Upper Midwest now merging in the Ohio River Valley to form a large single contiguous focus stretching from the Atlantic Coast to the edge of the Great Plains (Eisen et al. 2016). Similar expansions have not occurred either for I. scapularis in the southeastern United States or for the primary vector of B. burgdorferi in the far west, Ixodes pacificus Cooley and Kohls (Eisen et al. 2016). Geographic expansion and increasing abundance of I. scapularis have contributed to increases for both the geographic extent of Lyme borreliosis endemic areas and counties with high incidence of Lyme borreliosis in mid-atlantic, northeastern and north-central states (Kugeler et al. 2015, Mead 2015) (Figure 3). After Lyme borreliosis (Lyme disease) became nationally notifiable in the United States in 1991, the number of annually reported cases rose steadily from less than 10,000 in until they exceeded 20,000 for the first time in 2002 and 25,000 for the first time in 2007 (Bacon et al. 2008, Mead 2015). Following a modification of the national surveillance case definition in 2008, the number of reported confirmed cases alone exceeded 22,000 in all years up to 2013, and combined reports of confirmed and probable cases exceeded 30,000 in all years (Mead 2015) (Figure 4). Moreover, recent reports based on indirect information sources indicate that the true numbers of annual Lyme borreliosis cases in the United States are 10-fold higher, ranging upwards to about 300,000 (Hinckley et al. 2014, Nelson et al. 2015). Two other I. scapularis-borne Borrelia species are emerging as causes of human disease: the Lyme borreliosis group spirochaete Borrelia mayonii (Pritt et al. 2016) and the relapsing fever group spirochaete Borrelia miyamotoi (Molloy et al. 2015). Other human infections caused by I. scapularis-borne agents, including anaplasmosis and babesiosis, also are on the rise (Mead et al. 2015). I. scapularis-borne pathogens, most notably B. burgdorferi, present a major and still growing threat to human health in the United States, particularly in mid-atlantic, north-eastern and north-central Figure 1. The blacklegged tick (image from the Centers for Disease Control and Prevention). 430 Ecology and prevention of Lyme borreliosis

432 29. Lyme borreliosis prevention strategies Figure 2. Generalised distributions in the United States of the blacklegged tick and the western blacklegged tick (image from the Centers for Disease Control and Prevention). Figure 3. Reported cases of Lyme borreliosis (Lyme disease) in the United States in 2001 (left) versus 2014 (right) (images from the Centers for Disease Control and Prevention). states. The increase in Lyme borreliosis cases and geographic expansion of endemic areas from the early 1990s to present is juxtaposed against notable progress during the same time period for methods to manage the I. scapularis vector or disrupt enzootic B. burgdorferi transmission (Eisen et al. 2012, Ginsberg and Stafford 2005, Piesman and Eisen 2008, Stafford and Kitron 2002). Europe The main vector of Lyme borreliosis spirochaetes in Europe, Ixodes ricinus (L.), is the most studied of all tick species, initially because of its impact on livestock, but since the 1950s because of its role as a vector of tick-borne encephalitis virus to humans and since the 1990s in relation to Lyme borreliosis. This tick species is replaced in parts of Eastern Europe by the closely related Eurasian species, Ixodes persulcatus Schulze, but in some regions of Estonia and Latvia, the two species Ecology and prevention of Lyme borreliosis 431

433 Lars Eisen and Jeremy S. Gray Cases 45,000 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 0 Confirmed cases Probable cases* Figure 4. Reported cases of Lyme borreliosis (Lyme disease) by year in the United States from (image from the Centers for Disease Control and Prevention). *National Surveillance case definition revised in 2008 to include probable cases; details are available at are conspecific. There have been recent indications that populations of both tick species have increased (Bugmyrin et al. 2013, Sormunen et al. 2016, Sprong et al. 2012), which may be behind the reported increase in tick-borne disease incidence in Europe, though socio-economic reasons, especially following the disintegration of the Soviet Union, are also of importance (Sumilo et al. 2007). I. ricinus was implicated as a European vector of Lyme borreliosis spirochaetes in 1983 (Burgdorfer et al. 1983), but clinical manifestations of the disease were reported in Germany a hundred years earlier by Buchwald (1883), as a chronic skin disorder, which was named acrodermatitis chronica atrophicans a few years later (Herxheimer and Hartmann 1902). Other manifestations, such as erythema migrans, were subsequently described in Sweden (Afzelius 1910), borrelia lymphocytoma in Germany (Burckhardt 1911) and meningo-polyradicultis in France (Garin and Bujadoux 1922). But although the clinical manifestations were associated with ticks by Afzelius (1910), as well as by Garin and Bujadoux (1922), another 60 years were to elapse before the connection with I. ricinus was established. The spirochaete was therefore evidently causing disease in Europe for some decades before it emerged as a definitive pathogen in the United States and elsewhere. Determination of the precise incidence of the disease throughout Europe is impossible, but recent estimates for 17 countries in western Europe indicate a range from 0.001/100,000 in Italy to 464/100,000 in local regions of Sweden (Sykes and Makiello in press), with an overall estimate of annual case numbers as high as 232,125. Disease incidence has risen in several countries over the last 20 years, for example in Germany (Fülöp and Poggensee 2008), the Netherlands (Hofhuis et al. 2006) and the United Kingdom (Smith et al. 2000), and while higher awareness of Lyme borreliosis has undoubtedly contributed to an increase in reporting, there has evidently been a genuine rise in transmission rates in some regions (Kampen et al. 2004). Lyme borreliosis in Europe should therefore be regarded as a continuing emergent disease, though in some regions there is now evidence of stabilisation in the numbers of early cases (Hofhuis et al. 2016). 432 Ecology and prevention of Lyme borreliosis

434 29. Lyme borreliosis prevention strategies The uneven distribution of the disease in Europe is due mainly to differences in availability of suitable habitat for both vector and pathogen. Two surveys (Gray et al. 1998b, Estrada-Peña et al. 2011) reported that tick infection rates tend to be lower in western Europe than in the east, with high rates also in southern Scandinavia, and that the highest risk, as defined by the presence of large numbers of infected nymphal I. ricinus, occurs in heterogeneous woodland with a diverse fauna, including deer. This trend is reflected to a large extent by Lyme borreliosis incidence (Hubálek 2009) and suggests that increases in forestation and/or deer abundance may be driving the observed increased disease rates. Evolution of tick/pathogen control strategies United States The evolution of I. scapularis/b. burgdorferi control strategies has progressed along multiple tracks in the United States, including a human Lyme borreliosis vaccine (no longer available), different types of personal protective measures to avoid tick bites or reduce the risk of infection following bites by infected ticks, and various environmentally-based tick/pathogen control methods aimed at reducing the density of infected host-seeking ticks (Figure 5). This has been accompanied by research evaluations of specific control strategies, for example within the context of the TickNET Personal protective measures Residential landscape/ vegetation management Killing of hostseeking ticks Rodent-targeted methods Deer-targeted methods Avoid tick habitat Physically protective clothing Regular tick checks & prompt tick removal Synthetic repellent Natural product repellent Permethrin-treated clothing Human Lyme borreliosis vaccine* Human anti-tick vaccine (disrupting tick feeding)* Xeriscaping/ Hardscaping Keep grass short, remove weeds Remove leaf litter and brush Remove rodent harborage Do not use plants that attract deer Move play structures to low risk areas in the yard Ecotone barrier to tick movement Synthetic chemical acaricide Natural productbased chemical acaricide Biological fungal acaricide Acaricide enhanced by tick arrestment pheromones* Topical acaricide Oral tick growth regulator/ acaricide* Oral antibiotic* Oral Lyme borreliosis vaccine* Deer fencing Deer reduction Topical acaricide Oral tick growth regulator/acaricide* Deer anti-tick vaccine (disrupting tick feeding or reproduction)* * Potential personal protective measure or control method that is not yet available for public use Figure 5. Overview of currently available (black text) and potentially forthcoming (red text) personal protective measures and environmentally based control methods in the United States. Ecology and prevention of Lyme borreliosis 433

435 Lars Eisen and Jeremy S. Gray national public health network s Emerging Infections Program (Mead et al. 2015), as well as educational messages from state and federal public health agencies to promote the use of safe and presumably effective means to reduce tick bites or prevent infection following a bite by an infected tick (CDC 2016, Hayes and Piesman 2003, Mead 2015). However, as noted by Mead et al. (2015), case reports for common tick-borne diseases continue to increase despite decades of education about these prevention measures. There is no question that the re-emergence of a human vaccine against B. burgdorferi would be the most effective way to reduce Lyme borreliosis cases. The rise and fall of the LYMErix vaccine, which briefly was available from , and the prospects for and problems associated with first developing and then gaining and sustaining public acceptance for a new Lyme borreliosis vaccine have been addressed elsewhere (Embers and Narasimhan 2013, Shen et al. 2011, Steere and Livey 2012). Bites by infected I. scapularis nymphs account for the majority of Lyme borreliosis cases (Piesman et al. 1987, Spielman et al. 1985). Recommended personal protective means of preventing tick bites include avoiding tick habitat, showering or bathing within two hours after coming inside, using tick repellents on skin or clothing and wearing permethrin-treated clothing (CDC 2016). Despite access to a wide range of synthetic and botanical repellents (Dolan and Panella 2011, Eisen and Dolan 2016), frequent use of tick repellents range from only 15-40% for people living in Lyme borreliosis endemic areas (Butler et al. 2016, Connally et al. 2009, Finch et al. 2014, Gould et al. 2008, Hook et al. 2015, Vázquez et al. 2008). The realisation that transmission from a single B. burgdorferi-infected nymph is unlikely to occur within the first h of attachment (Piesman 1993, Piesman and Dolan 2002) led to the recommendation to check for and remove attached ticks daily to prevent transmission by attached infected nymphs (CDC 2016, Hayes and Piesman 2003). The main roadblock for this seemingly simple infection prevention strategy is unwillingness to conduct or difficulty in effectively conducting daily whole body searches for the small and hard-to-spot I. scapularis nymphs. One recent study conducted at the national scale (Hook et al. 2015) found that less than 50% of respondents do tick checks in Lyme borreliosis endemic regions of the United States. There is evidence that antibiotic prophylaxis can prevent disease manifestations following exposure to a B. burgdorferi-infected tick, if the treatment is given promptly (Nadelman et al. 2001, Warshafsky et al. 2010). However, most Lyme borreliosis patients are unaware of having been bitten by a (nymphal) tick prior to becoming ill (Benach and Coleman 1987, Gerber et al. 1996, Nadelman et al. 1996, Steere et al. 1983, Wormser et al. 2005) and therefore cannot be treated prophylactically before symptoms occur. Testing of individual ticks removed from a person for presence of B. burgdorferi is not recommended for several reasons, including that having been bitten by an infected tick does not necessarily result in infection and that negative testing results can lead to false assurance (CDC 2016). Early studies in the Northeast pointed to residential properties as the primary environment in which people are bitten by B. burgdorferi-infected ticks (Falco and Fish 1988, Maupin et al. 1991). Reduction of infected nymphs on residential properties therefore became a strong focus for the development of environmentally-based tick/pathogen control methods. Individual homeowners shoulder the responsibility for prevention of Lyme borreliosis on their own properties. Control efforts on public land generally are restricted to messaging to encourage the use of personal protective measures. 434 Ecology and prevention of Lyme borreliosis

436 29. Lyme borreliosis prevention strategies Environmentally-based tick control methods available to homeowners to protect individual properties include: (1) landscape and vegetation management to reduce tick habitat and decrease the risk of tick bites on the property; (2) suppression of host-seeking ticks with synthetic or natural product-based chemical acaricides or fungal biological agents; (3) rodent reservoirtargeted topical acaricides; and (4) deer fencing (CDC 2016, Eisen et al. 2012, Eisen and Dolan 2016, Stafford 2007). In the cases of acaricides to kill host-seeking ticks and rodent reservoir-targeted topical acaricides, homeowners have the options of using over-the-counter products or to use the services of licensed pest control operators (Schulze et al. 1997, Stafford 1997). At larger spatial scales, e.g. neighbourhoods, small communities or on islands, there has been interest in targeting adult ticks on deer, via deer reduction or use of topical acaricides, to suppress I. scapularis across the landscape rather than on individual properties (Pound et al. 2009, Stafford 2007). Other important issues include the efficacy of environmentally-based control measures to suppress host-seeking B. burgdorferi-infected I. scapularis ticks, especially nymphs, acceptability of specific control measures, and willingness of homeowners to pay for tick control. Although the rationale for landscape and vegetation management to suppress infected host-seeking ticks on residential properties is biologically sound, the efficacy of this control strategy remains to be determined. Numerous studies have shown that synthetic or natural product-based acaricides or fungal biological agents can effectively suppress host-seeking I. scapularis ticks in woodlands and residential settings. Single application of a synthetic acaricide is a robust method to suppress hostseeking I. scapularis ticks within a treated area, regardless of formulation and application method. Highly controlled experimental studies typically have achieved >85% control of host-seeking I. scapularis nymphs for up to 6-8 weeks following application of pyrethroids (Curran et al. 1993, Eisen and Dolan 2016, Schulze et al. 2005, Solberg et al. 1992), and a large-scale, effectiveness study, of pyrethroid applied by commercial companies resulted in 69% control on treated properties in one of two evaluation years (Hinckley et al. 2016). Because many homeowners are reluctant to use synthetic acaricides on their properties, substantial efforts were made to develop alternative means of killing host-seeking ticks. Resulting natural product-based acaricides or fungal biological agents can achieve similar levels of control, but require more frequent applications and are more sensitive to application method and environmental conditions as compared with synthetic acaricides (Allan and Patrican 1995, Bharadwaj and Stafford 2010, Bharadwaj et al. 2012, Dolan et al. 2009, Eisen and Dolan 2016, Jordan et al. 2011, Rand et al. 2010, Stafford and Allan 2010). Following the realisation that the white-footed mouse is a key reservoir for B. burgdorferi in the Northeast (Mather et al. 1989), there has been interest in approaches that aim to either reduce rodent-tick contact or to prevent or cure infection in the rodent in order to suppress or interrupt enzootic spirochaete transmission. Two commercial products for topical application of acaricide to rodents have emerged (Damminix Tick Tubes, Ecohealth, Inc., Brookline, MA, USA; Select TCS Tick Control System, Tick Box Technology Corporation, Norwalk, CT, USA), and oral baits containing a rodent-targeted vaccine against B. burgdorferi or an antibiotic have been developed and evaluated in the field. Results for application of Damminix Tick Tubes have ranged from no impact on the density of host-seeking infected I. scapularis nymphs to >95% control across field evaluations (Daniels et al. 1991, Deblinger and Rimmer 1991, Mather et al. 1988, Stafford 1991). The Select TCS Tick Control System and use of a rodent-targeted vaccine or antibiotic show variable degrees of promise to suppress infected nymphs but each of these methods have yet to be evaluated in more than a single published field study (Dolan et al. 2004, 2011, Meirelles Richer et al. 2014). Benefits of a rodent-targeted approach include that an acaricide, antibiotic, or vaccine can be contained within a treatment device, such as a bait box, rather than being widely broadcast in the environment, and that the impact of the intervention will extend across rodent home ranges Ecology and prevention of Lyme borreliosis 435

437 Lars Eisen and Jeremy S. Gray and, thus, likely will protect all portions of an individual residential property. Problems with a rodent-targeted approach arise if the intervention fails to achieve a high coverage rate in the locally most important B. burgdorferi reservoirs or if non-rodent B. burgdorferi reservoirs, such as shrews and birds, contribute substantially to local enzootic spirochaete transmission and thus diminish the effect of the rodent-targeted intervention. Deer-targeted approaches, such as deer reduction and topical application of acaricide to kill I. scapularis adults on deer, have strong potential to suppress the tick across the entire landscape but most likely will require reduction or treatment levels beyond what is acceptable to the public or logistically feasible in order to succeed. Field observations (Brei et al. 2009, Jordan et al. 2007, Kilpatrick et al. 2014, Rand et al. 2004, Stafford 2007, Wilson et al. 1988) and results from simulation modelling (Mount et al. 1997) collectively suggest that >70% of deer must be treated with topical acaricide, or deer reduced to <3 animals per km 2, to produce a large reduction in the abundance of infected nymphs within 5-10 years. Massive reduction of deer numbers may only be acceptable and feasible in very specific settings, such as on islands. Topical application of acaricide to deer with the United States Department of Agriculture s 4-Poster device was evaluated in five states in a suite of linked studies (Pound et al. 2009). The overall reduction in abundance of hostseeking I. scapularis nymphs within areas with a high density of 4-poster devices (1 per ha) approached 50% by the third year after the intervention began and reached 60% and 70% in the fourth and sixth years, respectively (Brei et al. 2009). However, there was no significant impact on the prevalence of infection with B. burgdorferi in host-seeking I. scapularis nymphs within the treatment areas (Gatewood Hoen et al. 2009), indicating that the interventions failed to reduce the burden of I. scapularis immatures on rodent reservoirs to the point where it negatively impacted enzootic B. burgdorferi transmission. Problems with use of the 4-poster device include variable homeowner acceptance leading to patchy deployment, regulatory issues preventing placement in optimal locations and during optimal time periods (which also coincide with the deer hunting season), interference with devices by non-target mammals such as tree squirrels and raccoons, acorn mast providing a competing food source in some years, and the contribution to tick feeding by non-treated deer (Carroll et al. 2008, 2009, Miller et al. 2009, Stafford et al. 2009). Integrated tick/pathogen management combines two or more control methods and aims to reduce distribution of chemicals in the environment (Eisen and Dolan 2016, Ginsberg 2001, Ostfeld et al. 2006, Stafford 2007). The published literature on the use of such strategies to suppress I. scapularis is very limited. Schulze et al. (2007, 2008) evaluated the integrated use of barrier spraying of the woods/lawn edge using a synthetic acaricide (year 1 only) with topical acaricide applications targeted to rodents (years 1-2 only) and deer (years 1-3) to suppress I. scapularis in a residential landscape. This intervention attacked both immature and adult tick stages as well as host-seeking ticks and ticks on hosts; and the successive withdrawal of methods served to minimise the amount of acaricide used. The abundance of host-seeking nymphs was reduced by 86% in the year after the intervention was put in place and by 86-94% in the two following years. Gould et al. (2008) reported on public attitudes toward and willingness to pay for tick control in Lyme borreliosis endemic settings in Connecticut. Among the most important findings were that respondents: (1) in most cases (63%) were unwilling to spend more than $ 100 per year on tick control; (2) most often (>80%) were in favor of landscape management to reduce tick habitat; (3) were less willing to spray a chemical acaricide to kill host-seeking ticks (47%) or consider deer-targeted solutions such as erecting deer fences (52%); and (4) more often than not approved of community-wide interventions including deer reduction (63%) and topical application of acaricide to deer (70%). No more than 6-12% of respondents in Lyme borreliosis 436 Ecology and prevention of Lyme borreliosis

438 29. Lyme borreliosis prevention strategies endemic settings reported current use of yard-based acaricides (Connally et al. 2009, Hook et al. 2015). Despite their demonstrated efficacy in suppressing host-seeking B. burgdorferi-infected I. scapularis ticks or preventing tick bites, many available methods are underutilised because people either are unwilling to use them or consider them too expensive. As noted by Hook et al. (2015), future research should determine the specific reasons why people choose not to implement certain measures so that educational messages can be crafted to overcome barriers to the use of prevention and control methods that are both acceptable and proven to be effective. Europe The first coordinated attempt to address our understanding of Lyme borreliosis in Europe occurred with the funding in 1993 by the European Union of a network of more than 30 scientists and physicians in 14 countries. The networking project Risk assessment in Lyme borreliosis (EUCALB) enabled the participants to review the current state of knowledge and to determine research priorities. The outcome of the three year project was a series of short papers published by Zentralblatt für Bakteriologie in 1998 (Anonymous 1998a, 1998b, 1998c, Cimmino et al. 1998, Estrada-Peña et al. 1998, Gern et al. 1998, Gray et al. 1998a, 1998b, Guy et al. 1998, Kahl et al. 1998a, O Connell et al. 1998, Saint Girons et al. 1998, Smith et al. 1998), and a website, that is now the information resource for ESGBOR, a study group for Lyme borreliosis, hosted by the European Society of Clinical Microbiology and Infectious Diseases. Other notable Pan-European initiatives have included the EDEN project, the VBORNET network, the One Health programme and ANTIDotE, but none of these have focused specifically on Lyme borreliosis. Although more is now known about Lyme borreliosis in Europe, little progress in the development and implementation of measures that could reduce its incidence has occurred. As in the United States, hopes were initially high that a vaccine could be developed for use in Europe. The main pathogenic Lyme borreliosis spirochaete occurring in North America, B. burgdorferi, also occurs in Europe. However, two additional pathogenic spirochaetes of the B. burgdorferi sensu lato complex, B. afzelii and B. garinii, are widespread in Europe (Saint-Girons et al. 1998), and others, B. spielmanii and B. bavariensis, have been identified more recently (Margos et al. 2013, Richter et al. 2006). This diversity of pathogenic European Lyme borreliosis group spirochaetes could make it more difficult to develop an effective and marketable vaccine than in the United States, although promising protection against B. afzelii, B. burgdorferi and B. garinii resulted when mice were vaccinated with Osp A, the same antigen target as in the withdrawn vaccine in the United States (Gern et al. 1997). Despite considerable research on this and other vaccination targets, such as OspC, there is no immediate prospect of a Lyme borreliosis vaccine for use in humans in Europe (Schuijt et al. 2011, Steere and Livey 2012). The simplest preventive approach is prompt removal of attached ticks, and although there is evidence that I. ricinus in Europe transmits at least some B. burgdorferi sensu lato spirochaetes more rapidly than I. scapularis in the United States (Kahl et al. 1998b), daily inspection for ticks after exposure is generally recommended in most European countries. However, this sensible measure on its own will not suffice because many Lyme borreliosis patients are not aware of having been bitten by a tick. The testing of detached ticks for Lyme borreliosis spirochaetes, followed by antimicrobial treatment of that person if positive, was suggested by Maiwald et al. (1998), following American studies, and was based on infection rates determined in ticks detached from people in a local study in Germany. However, in a review by Stanek and Kahl (1999) it was concluded that this approach should not be recommended, because of the uncertainty in predicting transmission or subsequent development of disease. This conclusion is still valid despite the improvement of Ecology and prevention of Lyme borreliosis 437

439 Lars Eisen and Jeremy S. Gray testing since that time, and recently a position paper discouraging tick testing was published online by ESGBOR (2013). This viewpoint is supported by studies in Sweden (Fryland et al. 2011), Switzerland (Huegli et al. 2011) and the Netherlands (Tijsse-Klasen et al. 2011), which determined the risk of disease developing as a result of a tick-bite to be very low. Furthermore, Fryland et al. (2011) reported that in a case of neuroborreliosis, the tick submitted for analysis proved to be uninfected, illustrating the point that this approach is prone to error. Ticks are now routinely included on the list of target arthropods for marketed repellents, of which DEET-based products are still regarded as the bench-mark. Most repellents are designed for skin application, but some, such as those based on permethrin, are applied to clothing. Research and development on repellents continues in Europe (Garboui et al. 2006, Faulde and Uedelhoven 2006, Schwantes et al. 2008), but this preventive approach is limited by the need for multiple applications, general inconvenience and also public suspicion because of perceived undesirable side-effects. Its increased use in the future relies heavily on both improved products and improved communication to the public. There has been little interest in Europe in environmentally-based preventive measures. Only two limited studies on the effects of vegetation management on tick populations have been conducted (Hubálek et al. 2006, Tack et al. 2013), the general view being that the marginal benefits are far outweighed by the expense, though this approach remains to be adequately explored for tickinfested parks to which the public have access. An interesting indirect approach was suggested by Gassner et al. (2008) who observed that the introduction of cattle reduced the amount of tickpermissive habitat in the Netherlands sufficiently to result in a fall in the tick population. However, this approach is probably habitat-specific, because cattle are very efficient hosts of I. ricinus, and where the rainfall is high enough can boost tick populations enormously (Gray et al. 1995). Biological control is often perceived as environmentally-friendly, and both the parasitoid wasp Ixodiphagus hookeri (Howard) and the fungus Metarhizium anisopilae (Metschnikoff) Sorokin have been suggested as possible control agents. However, neither is currently considered to be a viable option in Europe, although some preliminary studies have been undertaken (Ramos et al. 2015, Wasserman et al. 2016). Area-wide spraying with pesticides to control ticks was carried out on a large scale and longterm basis in Russia during the 1970s and 1980s in an attempt to control I. persulcatus, the main vector of the tick-borne encephalitis virus (Uspensky 1999). This approach was never adopted in Europe because, firstly tick-borne encephalitis is less prevalent there, and secondly by the time Lyme borreliosis had emerged as a significant disease in the late 1980s environmental politics made such an approach impractical. In contrast to some regions in the United States, pest control companies in Europe do not even offer pesticide-based control in small-scale domestic situations, primarily because of environmental concerns, but also possibly because of a perception that the disease threat does not justify the trouble and expense. Considerable amounts of pesticides are sold in Europe to control ticks on domestic livestock and pets, but these animals contribute very little to the maintenance of I. ricinus populations and the use of such pesticides therefore has no meaningful role in the control of this tick. By far the most important hosts for the maintenance of I. ricinus populations in European Lyme borreliosis habitats are wild deer (Gray et al. 1992, Jaenson et al. 2012, Ruiz-Fons et al. 2012, Tagliapietra et al. 2011), but no attempt has been made to emulate the 4-poster device for application of pesticides to deer, an approach that has been researched in depth in the United States (Pound et 438 Ecology and prevention of Lyme borreliosis

440 29. Lyme borreliosis prevention strategies al. 2009). The deer species mainly involved in tick population maintenance in Europe is the roe deer, Capreolus capreolus L., which is more solitary than the American white-tailed deer so that bait stations are unlikely to have the same impact as in the United States. Until recently, the main risk of infection in most European countries has been perceived to be in parks and woodland, rather than in domestic and peridomestic situations as in the northeastern United States. Effective use of this particular control measure in Europe would therefore be much more expensive, except perhaps in high-risk parks and nature reserves. However, Mulder et al. (2013) reported that in the Netherlands, an unexpectedly high proportion of tick bites (31%) were found to occur in private gardens and in some European countries the risk factors for transmission may be similar to the northeastern United States. In Europe the potential for deer exclusion as a control measure was first demonstrated by fencing, which reduced deer faecal pellet scores by 79%, I. ricinus larvae on mice by 89% and flagged nymphs by 79% (Gray et al. 1992). More recently deer exclusion of both roe and red deer (Cervus elaphus L.) in Scotland resulted in an % reduction in questing nymphs, and deer culling in a % reduction (Gilbert et al. 2012). Despite these encouraging results, no details of large-scale control schemes involving deer exclusion or removal have yet been published in Europe. In many parts of Europe rodents are significant reservoir hosts of B. burgdorferi sensu lato spirochaetes so that treating these animals with pesticides might be a logical approach if the practical difficulties could be overcome. However, attempts to emulate an American product (Damminix Tick Tubes) that consisted of providing the main rodent reservoir, the white-footed mouse, with pesticide-treated cotton-wool bedding, came to nothing because the equivalent rodents in Europe, Apodemus spp. mice, were insufficiently interested in the treated bedding (Mejlon et al. 1995). A variant of this approach, the provision of bait boxes containing the pesticide fipronil for direct topical application to the back of rodents as they navigate toward a food bait (Select TCS Tick Control System), has not been tested in Europe. The targeting of birds, which are now recognised as a major source of some B. burgdorferi sensu lato genospecies in Europe, is recognised as being totally impractical. Public attitudes to control and prevention measures in a high-risk region in Switzerland (Neuchâtel) were recently investigated by Aenishaenslin et al. (2016) and compared with those in a low-risk region in Canada. The options included biological control, deer reduction, deer exclusion, deer treatment, rodent treatment, rodent vaccination, area-acaricide application and landscaping. The latter two measures were the least acceptable for both regions (at about 10% for Neuchâtel) and biological control and deer treatment scored the highest in Neuchâtel, at 75 and 50%, respectively. A high knowledge of Lyme borreliosis was negatively associated with area-wide acaricide application and this was explained by concerns about cost, the environment, and effectiveness in relation to the disease threat. The high rating of biological control and deer treatment did not take into account the practicalities of effective implementation. Although various tools are theoretically available, disease prevention measures in Europe have consisted so far of providing information and education for the public in order to promote personal preventive action. Such actions include avoiding high-risk behaviour and habitats, wearing of protective clothing, application of repellents, checking the skin for ticks, and prompt removal of attached ticks by using fine forceps without the application of chemicals or heat. Account should be taken of the practicality of some of these protective measures. For example, avoidance of highrisk habitats when these are often not clearly defined, and the wearing of protective clothing, which some may view as sufficiently restricting to negate the point of visiting the countryside for recreation. A study on public perceptions in the Netherlands established, logically enough, Ecology and prevention of Lyme borreliosis 439

441 Lars Eisen and Jeremy S. Gray that preventive measures were more likely to be adopted with increased knowledge of Lyme borreliosis, but also that the use of protective clothing and repellents were least acceptable (Beaujean et al. 2013). In the absence of evidence for the effectiveness of personal protective measures, even expert opinion is divided on some issues, and the only measure that receives unanimous support is prompt removal of attached ticks. An alerting system for tick activity and thus for the risk of tick-borne disease, has been available in Germany since This on-line service, tick-radar ( Zeckenwetter in German), monitors tick activity in especially designed tick-plots (Dautel et al. 2008). It avoids most of the artefacts associated with conventional tick-flagging, such as the effects of weather and the time of day, and accurately reflects the situation in nature. Another system, established in 2012, curiously also called tick-radar ( tekenradar in Dutch), though with no connection to the former service, offers a forecasting system in the Netherlands. This is based on a model that was constructed from tick-flagging data obtained over one summer. The intention of both systems is to improve preventive behaviour, but in neither case is it known whether this has been achieved. Most European countries now have official websites underwritten by national departments of health or institutes and there are also a few Pan-European sites, such as the European Concerted Action on Lyme Borreliosis, the European Centre for Disease Control and Prevention, and the European Federation of Neurological Societies. Evidence base for tick/pathogen control to reduce Lyme borreliosis United States It has been noted that currently available Lyme borreliosis prevention approaches in the form of personal protective measures or environmentally-based suppression of I. scapularis and B. burgdorferi suffer from the limitations of poor rates of compliance and their effectiveness having been difficult to demonstrate in terms of reducing disease cases (Shen et al. 2011). Indeed, in the absence of a human vaccine, no single approach has proven to consistently reduce Lyme borreliosis cases, and no integrated tick/pathogen management strategy has yet been evaluated with Lyme borreliosis cases as an outcome. This unfortunate knowledge gap also limits the potential for educational messages to aid in reduction of Lyme borreliosis cases. Use of personal protective measures, i.e. regular tick checks and removal of attached ticks, physically protective clothing, or use of repellent, was associated with decreased risk of Lyme borreliosis in individual case-control or cross-sectional studies in the eastern United States but no specific measure was consistently protective across all studies in which it was examined (Armstrong et al. 2001, Connally et al. 2009, Finch et al. 2014, Klein et al. 1996, Orloski et al. 1998, Phillips et al. 2001, Schwartz and Goldstein 1990, Smith et al. 1988, 2001, Vázquez et al. 2008). Use of repellents was significantly associated with reduced risk of Lyme borreliosis in only three of these 10 studies, and tick checks in only two of the studies. Moreover, even in the few studies that showed significantly reduced risk the protective effect was at best moderate (odds ratios of ). Prospective studies are still lacking to clarify whether or not use of repellents or permethrintreated clothing can reduce Lyme borreliosis cases in the general public, and to elucidate how specific use patterns may impact the level of protection. Use of acaricides on the residential property to kill host-seeking ticks was included in five of the above-mentioned case-control or cross-sectional studies, with no evidence of protection against Lyme borreliosis. A prospective study comparing occurrence of Lyme borreliosis for individuals 440 Ecology and prevention of Lyme borreliosis

442 29. Lyme borreliosis prevention strategies living on residential properties treated with barrier spraying with a synthetic pyrethroid, as compared with untreated control properties, similarly showed no reduction in Lyme borreliosis cases (Hinckley et al. 2016). No study has examined whether a rodent-targeted approach can reduce Lyme borreliosis cases. Outcomes of deer reduction studies are mixed, likely resulting in part from variable end points for deer density. Jordan et al. (2007) found no impact on Lyme borreliosis incidence in a New Jersey community after deer density was gradually reduced to ~24/km 2, whereas Kilpatrick et al. (2014) reported an 80% reduction in resident-reported cases of Lyme borreliosis in a Connecticut community after deer density was reduced to ~5/km 2. The interpretation of the results from the latter study is complicated, however, by the lack of data from a comparison area where deer were not reduced. Such data from comparison areas were included in a previous study on the same Connecticut community where the epidemiological outcome was physician diagnosed erythema migrans (EM) rather than resident-reported Lyme borreliosis (Garnett et al. 2011). In that study, there was no evidence for EM rash incidence decreasing to a greater extent in the community where deer were reduced relative to the comparison areas. Garnett et al. (2011) also examined the impact of use of deer-targeted topical acaricide on EM rash incidence in another set of Connecticut communities, again compared with non-treated comparison areas, and found some evidence suggestive of a reduced EM rash incidence in the treatment area. Europe The only preventive measure currently in use in Europe is the dissemination, in a somewhat haphazard way, of information on the nature of Lyme borreliosis, how it may be contracted, and personal preventive measures that may be undertaken. While a plethora of information leaflets and websites (of variable quality) exist, there are no published studies showing that such information is effective in modifying behaviour, let alone reducing disease rates. However, the first step is awareness and a limited study by Gray et al. (1998a) showed that, not surprisingly, the greatest awareness of Lyme borreliosis occurred in countries with a high incidence of the disease, and that most information was obtained from the media. It was also shown that with a carefully designed leaflet accompanied by before and after questionnaires, it is possible to markedly improve knowledge that is relevant to prevention and control. The next step should be to determine whether specific preventive behaviours, promoted by educational campaigns, can have a real impact on disease levels. The future of Lyme borreliosis prevention United States The continuing spread of I. scapularis and the lack of a human vaccine against B. burgdorferi together present a formidable obstacle to stem the rising tide of Lyme borreliosis. Based on current use, no single existing personal protective measure or environmentally-based control method has consistently reduced Lyme borreliosis cases. In addition to exploring novel approaches, such as an anti-tick vaccine (Merino et al. 2013, Sprong et al. 2014), there is an urgent need for strengthening the evidence base for the capacity of existing personal protective measures and environmentally-based control methods to reduce Lyme borreliosis cases when used singly or in combination. Until this is accomplished we do not have enough information to discern whether or not the re-emergence of a human vaccine may ultimately be the only feasible solution to the Lyme borreliosis problem in the United States. Ecology and prevention of Lyme borreliosis 441

443 Lars Eisen and Jeremy S. Gray Europe It is perhaps remarkable that more than 30 years after the detection of the causative agent of Lyme borreliosis and its mode of transmission in Europe, very little research has been conducted on the means to reduce the incidence of the disease, and no organised system or policy to prevent the disease is currently in place in Europe. This is certainly a reflection on the difficulty and expense of implementing effective preventive measures, but may also be due to the main thrust of research on the topic in Europe, which is still very much on the collection of basic data on the presence of pathogens in ticks, and to some extent on the dynamics of transmission. Such data are necessary for the implementation of effective control and prevention, but it could be argued that we now know enough about the ecology of the disease to commence researching effective intervention more intensively. There is a need to test the efficacy of information systems in reducing exposure to infection, as well as to research practical intervention to prevent disease. Studies on vaccination in Europe have largely ceased since the withdrawal of industrial support for this topic a few years ago, although the approach is still in focus (Šmit and Postma, 2015) and technical advances will probably eventually result in a marketable vaccine if the economic case can be made. An innovative approach to vaccination has recently been adopted by a consortium of seven European institutions, whose EU funded programme, AntiDotE, has the aim of developing vaccines against the tick vector itself, I. ricinus (Sprong et al. 2014). An effective vaccine of this nature would not only reduce the health burden of Lyme borreliosis and tick-borne encephalitis in humans, but could also be deployed to prevent other tick-borne diseases. Public health relevance Lessons learned from years of Lyme borreliosis prevention research: The continuing spread of vector ticks together with the lack of a human vaccine against Lyme borreliosis spirochaetes present a formidable obstacle to stem the rising tide of Lyme borreliosis in the United States and Europe. Numerous approaches to prevent tick bites or suppress tick populations have emerged, including spray-on tick repellents and permethrin-treated clothing to prevent human-tick contact; synthetic chemicals, natural products, and biological agents to suppress host-seeking ticks; deer reduction to suppress tick populations; topical application of pesticides to reduce tick burdens on rodents and deer; and antibiotic treatment or vaccination of rodent reservoirs against Lyme borreliosis spirochaetes. The difficulties encountered in conventional vaccine development has led to interest in anti-tick vaccine development against Ixodes spp. vectors, which is currently under investigation. The evidence base for existing personal protective measures and environmentally-based control methods to reduce, or fail to reduce, Lyme borreliosis cases needs to be strengthened to clarify the value of employing such measures, singly or in combination. 442 Ecology and prevention of Lyme borreliosis

444 29. Lyme borreliosis prevention strategies Disclaimer The findings and conclusions are by the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Acknowledgements We thank Marc Dolan, Rebecca Eisen, Ken Gage, Alison Hinckley, and Anna Perea of the Centers for Disease Control and Prevention for helpful discussions and assistance with illustrations. References Aenishaenslin C, Michel P, Ravel A, Gern L, Waaub JP, Milord F and Bélanger D (2016) Acceptability of tick control interventions to prevent Lyme disease in Switzerland and Canada: a mixed-method study. BMC Public Health 16: 12. Afzelius A (1910) Verhandlungen der dermatologischen Gesellschaft zu Stockholm, Sitzung vom 28. Oktober Arch Dermatol Syph 101: Allan SA and Patrican LA (1995) Reduction of immature Ixodes scapularis (Acari: Ixodidae) in woodlots by application of desiccant and insecticidal soap formulations. J Med Entomol 32: Armstrong PM, Brunet LR, Spielman A and Telford SR III (2001) Risk of Lyme disease: perceptions of residents of a lone star tick-infested community. Bull WHO 79: Anonymous (1998a) Contributors. Zentralbl Bakteriol 287: Anonymous (1998b) Editorial. Zentralbl Bakteriol 287: Anonymous (1998c) EUCALB participation. Zentralbl Bakteriol 287: Bacon RM, Kugeler KJ and Mead PS (2008) Surveillance for Lyme disease United States, Morb Mort Wkly Rep 57: 1-9. Beaujean DJMA, Bults M, Van Steenbergen JE and Voeten HACM (2013) Study on public perceptions and protective behaviors regarding Lyme disease among the general public in the Netherlands: implications for prevention programs. BMC Public Health 13: 225. Benach JL and Coleman JL (1987) Clinical and geographic characteristics of Lyme disease in New York. Zentralbl Bakteriol Mikrobiol Hyg A 263: Bharadwaj A and Stafford KC III (2010) Evaluation of Metarhizium anisopliae strain F52 (Hypocreales: Clavicipitaceae) for control of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 47: Bharadwaj A, Stafford KC III and Behle RW (2012) Efficacy and environmental persistence of nootkatone for the control of the blacklegged tick (Acari: Ixodidae) in residential landscapes. J Med Entomol 49: Brei B, Brownstein JS, George JE, Pound JM, Miller JA, Daniels TJ, Falco RC, Stafford KC III, Schulze TL, Mather TN, Carroll JF and Fish D (2009) Evaluation of the United States department of agriculture northeast area-wide tick control project by meta-analysis. Vector-Borne Zoonotic Dis 9: Buchwald A (1883) Ein fall von diffuser idiopathischer Haut-Atrophie. Arch Dermatol Syph 10: Bugmyrin SV, Bespyatova LA, Korotkov YS, Burenkova LA, Belova OA, Romanova L, Kozlovskaya LI, Karganova GG and Ieshko EP (2013) Distribution of Ixodes ricinus and I. persulcatus ticks in southern Karelia (Russia). Ticks Tick Borne Dis 4: Burckhardt JL (1911) Zur Frage der Follikel- und Keimzentrenbildung in der Haut. Frankf Z Pathol. 6: Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E and Davis JP (1982) Lyme disease a tick-borne spirochetosis? Science 216: Burgdorfer W, Barbour AG, Hayes SF, Peter O, Aeschlimann A (1983) Erythema chronicum migrans a tick-borne spirochaetosis. Acta Trop 40: Butler AD, Sedghi T, Petrini JR and Ahmadi R (2016) Tick-borne disease preventive practices and perceptions in an endemic area. Ticks Tick Borne Dis 7: Ecology and prevention of Lyme borreliosis 443

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447 Lars Eisen and Jeremy S. Gray Hinckley AF, Connally NP, Meek JI, Johnson BJ, Kemperman MM, Feldman KA, White JL and Mead PS (2014) Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis 59: Hinckley AF, Meek JI, Ray JAE, Niesobecki SA, Connally NP, Feldman KA, Jones EH, Backenson PB, White JL, Lukacik G, Kay AB, Miranda WP and Mead PS (2016) Effectiveness of residential acaricides to prevent Lyme and other tick-borne diseases in humans. J Infect Dis 214: Hofhuis A, van der Giessen JWB, Borgsteede FHM, Wielinga PR, Notermans DW, and van Pelt W (2006) Lyme borreliosis in the Netherlands: strong increase in GP consultations and hospital admissions in past 10 years. Euro Surveill 11: E Hofhuis A, Bennema S, Harms M, Van Vliet AJH, Takken W, Van den Wijngaard CC and Van Pelt W (2016) Decrease in tick bite consultations and stabilization of early Lyme borreliosis in the Netherlands in 2014 after 15 years of continuous increase. BMC Publ Health 16: 425. Hook SA, Nelson CA and Mead PS (2015) US public s experience with ticks and tick-borne diseases: results from national HealthStyles surveys. Ticks Tick-borne Dis 6: Hubálek Z (2009) Epidemiology of Lyme borreliosis. Curr Probl Dermatol 37: Hubálek Z, Halouzka J, Juricová Z, Sikutová S and Rudolf I (2006) Effect of forest clearing on the abundance of Ixodes ricinus ticks and the prevalence of Borrelia burgdorferi s.l. Med Vet Entomol 20: Huegli D, Moret J, Rais O, Moosmann Y, Erard P, Malinverni R and Gern L (2011) Prospective study on the incidence of infection by Borrelia burgdorferi sensu lato after tick bite in a high endemic area of Switzerland. Ticks Tick Borne Dis 2: Jaenson TGT, Jaenson DGE, Eisen L, Petersson E, Lindgren E (2012) Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden. Parasit Vectors 5: 8. Jordan RA, Dolan MC, Piesman J and Schulze TL (2011) Suppression of host-seeking Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs after dual applications of plant-derived acaricides in New Jersey. J Econ Entomol 104: Jordan RA, Schulze TL and Jahn MB (2007) Effects of reduced deer density on the abundance of Ixodes scapularis (Acari: Ixodidae) and Lyme disease incidence in a northern New Jersey endemic area. J Med Entomol 44: Kahl O, Gern L, Gray JS, Guy EC, Jongejan F, Kirstein F, Kurtenbach K, Rijpkema SGT and Stanek G (1998a) Detection of Borrelia burgdorferi sensu lato in ticks: immunofluorescence assay versus polymerase chain reaction. Zentralbl Bakteriol 287: Kahl O, Janetzki-Mittman C, Gray JS, Jonas R, Stein J and de Boer R (1998b) Risk of infection with Borrelia burgdorferi sensu lato for a host in relation to the duration of nymphal Ixodes ricinus feeding and the method of tick removal. Zentralbl Bakteriol 287: Kampen H, Rötzel DC, Kurtenbach K, Maier WA, and Seitz HM (2004) Substantial rise in the prevalence of Lyme borreliosis spirochetes in a region of Western Germany over a 10-year period. Appl Environ Microbiol 70: Kilpatrick HJ, Labonte AM and Stafford KC III (2014) The relationship between deer density, tick abundance, and human cases of Lyme disease in a residential community. J Med Entomol 51: Klein JD, Eppes SC and Hunt P (1996) Environmental and life-style risk factors for Lyme disease in children. Clin Pediatr 35: Kugeler KJ, Farley GM, Forrester JD and Mead PS (2015) Geographic distribution and expansion of human Lyme disease, United States. Emerg Infect Dis 21: LoGiudice K, Ostfeld RS, Schmidt KA and Keesing F (2003) The ecology of infectious disease: effects of host diversity and community composition on Lyme disease risk. Proc Natl Acad Sci USA 100: Maiwald M, Oehme R, March O, Petney TN, Kimmig P, Naser K, Zappe HA, Hassler D and Von Knebel Doeberitz M (1998) Transmission risk of Borrelia burgdorferi sensu lato from Ixodes ricinus ticks to humans in southwest Germany. Epidemiol Infect 121: Margos G, Wilske B, Sing A, Hizo-Teufel C, Cao WC, Chu C, Scholz H, Straubinger RK and Fingerle V (2013) Borrelia bavariensis sp. nov. is widely distributed in Europe and Asia. Int J Syst Evol Microbiol 63: Mather TN, Ribeiro JMC, Moore S and Spielman A (1988) Reducing transmission of Lyme disease spirochetes in a suburban setting. Ann NY Acad Sci 539: Ecology and prevention of Lyme borreliosis

448 29. Lyme borreliosis prevention strategies Mather TN, Wilson ML, Moore SI, Ribeiro JMC and Spielman A (1989) Comparing the relative potential of rodents as reservoirs of the Lyme disease spirochete (Borrelia burgdorferi). Am J Epidemiol 130: Maupin GO, Fish D, Zultowsky J, Campos EG and Piesman J (1991) Landscape ecology of Lyme disease in a residential area of Westchester County, New York. Am J Epidemiol 133: Mead PS (2015) Epidemiology of Lyme disease. Infect Dis Clin N Am 29: Mead PS, Hinckley AF, Hook S and Beard CB (2015) TickNET a collaborative public health approach to tickborne disease surveillance and research. Emerg Infect Dis 21: Meirelles Richer L, Brisson D, Melo R, Ostfeld RS, Zeidner N and Gomes-Solecki M (2014) Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission. J Infect Dis 209: Mejlon HA, Jaenson TGT and Mather TN (1995) Evaluation of host-targeted applications of permethrin for control of Borrelia-infected Ixodes ricinus (Acari: Ixodidae). Med Vet Entomol 9: Merino O, Alberdi P, de la Lastra JMP and de la Fuente J (2013) Tick vaccines and the control of tick-borne pathogens. Front Cell Infect Microbiol 3: 30. Miller NJ, Thomas WA and Mather TN (2009) Evaluating a deer-targeted acaricide applicator for area-wide suppression of blacklegged ticks, Ixodes scapularis (Acari: Ixodidae), in Rhode Island. Vector-Borne Zoonotic Dis 9: Molloy PJ, Telford SR III, Chowdri HR, Lepore TJ, Gugliotta JL, Weeks KE, Hewins ME, Goethert HK and Berardi VP (2015) Borrelia miyamotoi disease in the northeastern United States. A case series. Ann Intern Med 163: Mount GA, Haile DG and Daniels E (1997) Simulation of management strategies for the blacklegged tick (Acari: Ixodidae) and the Lyme disease spirochete, Borrelia burgdorferi. J Med Entomol 34: Mulder S, Van Vliet AJ, Bron WA, Gassner F and Takken W (2013) High risk of tick bites in Dutch gardens. Vector Borne Zoonotic Dis 13: Nadelman RB, Nowakowski J, Fish D, Falco RC, Freeman K, McKenna D, Welch P, Marcus R, Aguero-Rosenfeld ME, Dennis DT and Wormser GP (2001) Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med 345: Nadelman RB, Nowakowski J, Forseter G, Goldberg NS, Bittker S, Cooper D, Aguero-Rosenfeld M and Wormser GP (1996) The clinical spectrum of early Lyme borreliosis in patients with culture-confirmed erythema migrans. Am J Med 100: Nelson CA, Saha S, Kugeler KJ, Delorey MJ, Shankar MB, Hinckley AF and Mead PS (2015) Incidence of cliniciandiagnosed Lyme disease, United States, Emerg Infect Dis 21: O Connell S, Granström M, Gray JS and Stanek G (1998) Epidemiology of European Lyme Borreliosis. Zentralbl Bakteriol 287: Orloski KA, Campbell GL, Genese CA, Beckley JW, Schriefer ME, Spitalny KC and Dennis DT (1998) Emergence of Lyme disease in Hunterdon County, New Jersey, 1993: a case-control study of risk factors and evaluation of reporting patterns. Am J Epidemiol 147: Ostfeld RS, Price A, Hornbostel VL, Benjamin MA and Keesing F (2006) Controlling ticks and tick-borne zoonoses with biological and chemical agents. Bioscience 56: Phillips CB, Liang MH, Sangha O, Wright EA, Fossel AH, Lew RA, Fossel KK and Shadick NA (2001) Lyme disease and preventive behaviors in residents of Nantucket Island, Massachusetts. Am J Prev Med 20: Piesman J (1993) Dynamics of Borrelia burgdorferi transmission by nymphal Ixodes dammini ticks. J Infect Dis 167: Piesman J and Dolan MC (2002) Protection against Lyme disease spirochete transmission provided by prompt removal of nymphal Ixodes scapularis (Acari: Ixodidae). J Med Entomol 39: Piesman J and Eisen L (2008) Prevention of tick-borne diseases. Annu Rev Entomol 53: Piesman J and Gern L (2004) Lyme borreliosis in Europe and North America. Parasitology 129: S191-S220. Piesman J, Mather TN, Dammin GJ, Telford SR III, Lastavica CC and Spielman A (1987) Seasonal variation of transmission risk of Lyme disease and human babesiosis. Am J Epidemiol 126: Pound JM, Miller JA, George JE, Fish D, Carroll JF, Schulze TL, Daniels TJ, Falco RC, Stafford KC III and Mather TN (2009) The United States Department of Agriculture s northeast area-wide tick control project: summary and conclusions. Vector-Borne Zoonotic Dis 9: Ecology and prevention of Lyme borreliosis 447

449 Lars Eisen and Jeremy S. Gray Pritt BS, Mead PS, Hoang Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL and Petersen JM (2016) Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infect Dis 15: Ramos RAN, Campbell BE, Whittle A, Lia RP, Montarsi F, Parisi A, Dantas-Torres F, Wall R and Otranto D (2015) Occurrence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus (Acari: Ixodidae) in southern Italy. Ticks Tick Borne Dis 6: Rand PW, Lacombe EH, Elias SP, Lubelczyk CB, St Amand T and Smith RP (2010) Trial of a minimal-risk botanical compound to control the vector tick of Lyme disease. J Med Entomol 47: Rand PW, Lubelczyk C, Holman MS, Lacombe EH and Smith RP (2004) Abundance of Ixodes scapularis (Acari: Ixodidae) after the complete removal of deer from an isolated offshore island, endemic for Lyme disease. J Med Entomol 41: Richter D, Postic D, Sertour N, Livey I, Matuschka FR and Baranton G (2006) Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov. Int J Syst Evol Microbiol 56: Ruiz-Fons F, Fernández-de-Mera IG, Acevedo P, Gortázar C and de la Fuente J (2012) Factors driving the abundance of Ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci. Appl Environ Microbiol 78: Saint-Girons I, Gern L, Gray JS, Guy EC, Korenberg E, Nuttall PA, Rijpkema SG, Schönberg A, Stanek G and Postic D (1998) Identification of Borrelia burgdorferi sensu lato species in Europe. Zentralbl Bakteriol 287: Schuijt TJ, Hovius JW, Van der Poll T, Van Dam AP and Fikrig E (2011) Lyme borreliosis vaccination: the facts, the challenge, the future. Trends Parasitol 27: Schulze TL, Jordan RA and Hung RW (1997) Availability and nature of commercial tick control services in established and emerging Lyme disease areas of New Jersey. J Spirochetal Tick-Borne Dis 4: Schulze TL, Jordan RA and Krivenko AJ (2005) Effects of barrier application of granular deltamethrin on subadult Ixodes scapularis (Acari: Ixodidae) and non-target forest floor arthropods. J Econ Entomol 98: Schulze TL, Jordan RA, Dolan MC, Dietrich G, Healy SP and Piesman J (2008) Ability of 4-poster passive topical treatment devices for deer to sustain low population levels of Ixodes scapularis (Acari: Ixodidae) after integrated tick management in a residential landscape. J Med Entomol 45: Schulze TL, Jordan RA, Schulze CJ, Healy SP, Jahn MB and Piesman J (2007) Integrated use of 4-poster passive topical treatment devices for deer, targeted acaricide applications, and Maxforce TMS bait boxes to rapidly suppress populations of Ixodes scapularis (Acari: Ixodidae) in a residential landscape. J Med Entomol 44: Schwantes U, Dautel H and Jung G (2008) Prevention of infectious tick-borne diseases in humans: comparative studies of the repellency of different dodecanoic acid-formulations against Ixodes ricinus ticks (Acari: Ixodidae). Parasit Vectors 1: 8. Schwartz BS and Goldstein MD (1990) Lyme disease in outdoor workers: risk factors, preventive measures, and tick removal methods. Am J Epidemiol 131: Shen AK, Mead PS and Beard CB (2011) The Lyme disease vaccine a public health perspective. Clin Infect Dis 52: S247-S252. Šmit R and Postma MJ (2015) Lyme borreliosis: reviewing potential vaccines, clinical aspects and health economics. Expert Rev Vaccines 14: 12. Smith G, Wileyto EP, Hopkins RB, Cherry BR and Maher JP (2001) Risk factors for Lyme disease in Chester County, Pennsylvania. Publ Health Rep 116: Smith M, Gettinby G, Granström M, Gray JS, Guy EC, Revie C, Robertson JN and Stanek G (1998) The European Union Concerted Action World Wide Web Site for Lyme Borreliosis. Zentralbl Bakteriol 287: Smith PF, Benach JL, White DJ, Stroup DF and Morse DL (1988) Occupational risk of Lyme disease in endemic areas of New York State. Ann NY Acad Sci 539: Smith R, O Connell S and Palmer S (2000) Lyme disease surveillance in England and Wales, Emerg Infect Dis 6: Ecology and prevention of Lyme borreliosis

450 29. Lyme borreliosis prevention strategies Solberg VB, Neidhardt K, Sardelis MR, Hoffmann FJ, Stevenson R, Boobar LR and Harlan HJ (1992) Field evaluation of two formulations of cyfluthrin for control of Ixodes dammini and Amblyomma americanum (Acari: Ixodidae). J Med Entomol 29: Sormunen JJ, Penttinen R, Klemola T, Hänninen J, Vuorinen I, Laaksonen M, Sääksjärvi IE, Ruohomäki K and Vesterinen EJ (2016) Tick-borne bacterial pathogens in southwestern Finland. Parasit Vectors 9: 168. Spielman A (1994) The emergence of Lyme disease and human babesiosis in a changing environment. Ann NY Acad Sci 740: Spielman A, Wilson ML, Levine JF and Piesman J (1985) Ecology of Ixodes dammini borne human babesiosis and Lyme disease. Annu Rev Entomol 30: Sprong H, Hofhuis A, Gassner F, Takken W, Jacobs F, Van Vliet AJH, van Ballegooijen M, Van der Giessen J and Takumi K (2012) Circumstantial evidence for an increase in the total number and activity of Borrelia-infected Ixodes ricinus in the Netherlands. Parasit Vectors 5: 294. Sprong H, Trentelman J, Seemann I, Grubhoffer L, Rego ROM, Hajdušek O, Kopáček P, Šíma R, Nijhof AM, Anguita J, Winter P, Rotter B, Havlíková S, Klempa B, Schetters TP and Hovius JWR (2014) ANTIDotE: anti-tick vaccines to prevent tick-borne diseases in Europe. Parasit Vectors 7: 77. Stafford KC III (1991) Effectiveness of host-targeted permethrin in the control of Ixodes dammini (Acari: Ixodidae). J Med Entomol 28: Stafford KC III (1997) Pesticide use by licensed applicators for the control of Ixodes scapularis (Acari: Ixodidae) in Connecticut. J Med Entomol 34: Stafford KC III (2007) Tick management handbook. An integrated guide for homeowners, pest control operators, and public health officials for the prevention of tick-associated disease. The Connecticut Agricultural Experiment Station, New Haven, CT, USA. Stafford KC III and Allan SA (2010) Field applications of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae F52 (Hypocreales: Clavicipitaceae) for the control of Ixodes scapularis (Acari: Ixodidae). J Med Entomol 47: Stafford KC III and Kitron U (2002) Environmental management for Lyme borreliosis control, pp In: Gray J, Kahl O, Lane RS and Stanek G (eds.) Lyme borreliosis biology, epidemiology and control. CABI Publishing, New York, NY, USA. Stafford KC III, Denicola AJ, Pound JM, Miller JA and George JE (2009) Topical treatment of white-tailed deer with an acaricide for the control of Ixodes scapularis (Acari: Ixodidae) in a Connecticut Lyme borreliosis hyperendemic community. Vector-Borne Zoonotic Dis 9: Stanek G and Kahl O (1999) Chemoprophylaxis for Lyme borreliosis? Zentralbl Bakteriol 289: Steere AC and Livey I (2012) Lyme disease vaccines. In: Plotkin SA, Orenstein WA and Offit PA (eds.) Vaccines. Elsevier, Philadelphia, PA, USA, pp Steere AC, Bartenhagen NH, Craft JE, Hutchinson GJ, Newman JH, Rahn DW, Sigal LH, Spieler PN, Stenn KS and Malawista SE (1983) The early clinical manifestations of Lyme disease. Ann Inter Med 99: Sumilo D, Asokliene L, Bormane A, Vasilenko V, Golovljova I and Randolph SE (2007) Climate change cannot explain the upsurge of tick-borne encephalitis in the Baltics. PLoS ONE 2: e500. Sykes RA and Makiello P (in press) An estimate of Lyme borreliosis incidence in Western Europe. J Public Health DOI: Tack W, Madder M, Baeten L, Vanhellemont M and Verheyen K (2013) Shrub clearing adversely affects the abundance of Ixodes ricinus ticks. Exp Appl Acarol 60: Tagliapietra V, Rosà R, Arnoldi D, Cagnacci F, Capelli G, Montarsi F, Hauffe HC and Rizzoli A (2011) Saturation deficit and deer density affect questing activity and local abundance of Ixodes ricinus (Acari: Ixodidae) in Italy. Vet Parasitol 183: Tijsse-Klasen E, Jacobs JJ, Swart A, Fonville M, Reimerink JH, Brandenburg AH, Van der Giessen JW, Hofhuis A and Sprong H (2011) Small risk of developing symptomatic tick-borne diseases following a tick bite in the Netherlands. Parasit Vectors 4: 17. Ecology and prevention of Lyme borreliosis 449

451 Lars Eisen and Jeremy S. Gray Uspensky I (1999) Ticks as the main target of human tick-borne disease control: Russian practical experience and its lessons. J Vector Ecol 24: Vázquez M, Muehlenbein C, Cartter M, Hayes EB, Ertel S and Shapiro ED (2008) Effectiveness of personal protective measures to prevent Lyme disease. Emerg Infect Dis 14: Warshafsky S, Lee DH, Francois LK, Nowakowski J, Nadelman RB and Wormser GP (2010) Efficacy of antibiotic prophylaxis for the prevention of Lyme disease: an updated systematic review and meta-analysis. J Antimicrob Chemother 65: Wassermann M, Selzer P, Steidle JL and Mackenstedt U (2016) Biological control of Ixodes ricinus larvae and nymphs with Metarhizium anisopliae blastospores. Ticks Tick Borne Dis 7: Wilson ML, Telford SR III, Piesman J and Spielman A (1988) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following elimination of deer. J Med Entomol 25: Wormser GP, Masters E, Nowakowski J, McKenna D, Holmgren D, Ma K, Ihde L, Cavaliere LF and Nadelman RB (2005) Prospective clinical evaluation of patients from Missouri and New York with erythema migrans-like skin lesions. Clin Infect Dis 41: Ecology and prevention of Lyme borreliosis

452 30. Concluding remarks Hein Sprong National Institute for Public Health and the Environment, Centre for Infectious Disease Control, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; Ixodes ricinus can feed on many wild and domestic animals, but its distribution and abundance appears to be strongly associated with just a few widespread and abundant European wildlife vertebrates: deer, thrushes and small rodents. Because of the expansion and protection of their natural habitats, the distribution and abundance of tick hosts and Lyme spirochaetes has increased. Other environmental factors, such as (micro)climate and vegetation structure, also affect the survival of ticks, and therefore the probability for ticks to find a feeding or reproduction host. How all these factors interact and affect the spatiotemporal variation of infected ticks requires further investigation. Ticks and wildlife harbour many more pathogens of human and veterinary concern. Therefore, future research should aim to link ecological parameters, such as wildlife abundance and diversity, to the abundance and variety of the most important wildlife and vector-borne pathogens. Infected ticks only become a health risk when their biotopes are frequently visited by humans, either for professional or for recreational activities. The One Health approach described in this book is predominantly preached and practiced by health professionals, aiming at the prevention of Lyme borreliosis. Clearly, personal protection, including the compliance with hygiene procedures, can still be improved, both by persons working in the green sector as well as by the general public. In the (near) future, a Lyme-vaccine might become available, which would be advantageous for the control of Lyme borreliosis, particularly in high-risk groups. A major advantage of environmental management control options over vaccines is the fact that the knowledge and tools have been developed and that they can already be applied in various practical situations. Furthermore, controlling tick abundance or tick exposure reduces the risk of acquiring any tick-borne disease for both humans and domestic animals, and not only (human) Lyme borreliosis. Still, there is room for the development of innovative environmental control options, which might be more (cost-)effective than the existing ones. Two promising new developments are anti-tick vaccines and sheep-mopping. The successful implementation of environmental management measures requires full collaboration of all stakeholders involved in nature management. Of key importance is that the environmental control options for Lyme borreliosis (and other wildlife and vector-borne diseases) are put into the context of their aims and ambitions. A One Health approach focuses predominantly on the control and mitigation of diseases, and not directly on nature conservation and biodiversity. Sectors involved in nature management and environmental planning are more familiar with a so-called Health in All Policies -approach. The Health in All Policies -approach integrates and articulates many health considerations, far broader than infectious diseases alone, into policymaking across sectors. The next challenge for public health is to integrate the risk of wildlife and vectorborne diseases into the Health in All Policies and several organisational levels. Acknowledgements All or part of the research described in Chapter 1, 4-10, 12, 16-20, 22-23, 25-26, and 30 was financially supported by the Dutch Ministry of Health, Welfare and Sport (VWS), and by the Strategic Program RIVM (SPR). Parts of the work described in this book were performed under the umbrella of a COST (TD1303) (Chapter 3, 7-8, 15-16) or FP7-ANTIDotE (Chapter 21) project. Marieta A.H. Braks, Sipke E. van Wieren, Willem Takken and Hein Sprong (eds.) Ecology and prevention of Lyme borreliosis Ecology and and control prevention of vector-borne of Lyme diseases borreliosis Volume DOI / _30, Wageningen Academic Publishers 2016

453

454 About the editors Marieta Braks (1969) is a medical entomologist with the Dutch National Institute for Public Health and the Environment (RIVM), the Netherlands. After studying biology at the University of Utrecht, she obtained her PhD in Entomology from Wageningen University in Subsequently, she worked seven years in the USA first as a postdoc at the Florida Medical Entomology Laboratory and at the Entomology Laboratory of the University of California at Riverside and later as an employee of the Department of Vector-borne diseases of the California State Department of Public Health in Sacramento. With extensive experience in the surveillance and control of vectors and diseases, she returned to the Netherlands to work at the Centre for Zoonoses and Environmental Microbiology of the RIVM in In addition to national activities, she takes part in international networks and projects related to vector-borne diseases. Sip van Wieren (1951) is Associate Professor in Wildlife Ecology and Management at Wageningen University & Research, the Netherlands. He studied in Groningen and obtained his PhD degree in 1996 based on research on the digestive physiology of ruminants and non-ruminants. Early in his career he worked on grazing ecology and grazing management, especially in rewilding conservation projects. When he started in Wageningen in 1991, his focus shifted more towards studying the community ecology of herbivore assemblages in both savannas and the dry grasslands of the Trans Himalaya. In recent years he started working on tick ecology with an emphasis on the role of mammals in the tick and the Borrelia cycle, but also on finding ways to reduce ticks in the field through the use of large herbivores. He serves on several advisory boards. Ecology and prevention of Lyme borreliosis 453

455 About the editors Willem Takken (1951) is Professor in Medical and Veterinary Entomology at Wageningen University & Research, the Netherlands. He studied in Wageningen and obtained his PhD degree in 1980 based on research on the biology and feeding behaviour of tsetse flies. He worked in several African countries on the control of animal trypanosomiasis. Upon his return to Europe, he was appointed as lecturer at the Laboratory of Entomology in Wageningen, where he introduced medical and veterinary entomology to the Wageningen academic society. His work involved mosquitohost interactions, in particular the host-seeking behaviour of malaria mosquitoes. He later expanded this work to include field research in Tanzania, Kenya, and other tropical countries. Today, his work involves the ecology of mosquitoes, biological control of mosquitoes and the impact of environmental change on malaria vectors. In Europe, Willem studies the ecology of Lyme disease vectors and vectors of other, emerging infectious diseases. He emphasises collaboration with other institutions, and has an extensive network of national and international collaborators. He serves on several editorial boards and advisory committees. Hein Sprong (1971) is a medical biologist with the Dutch National Institute for Public Health and the Environment (RIVM), the Netherlands. After studying Biomedical Sciences at the Utrecht University, he obtained his PhD in Medicine from the University of Amsterdam in Subsequently, he worked as a postdoc at the Max-Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, until He was an Assistant Professor at the Faculty of Chemistry, Utrecht University, the Netherlands, until Since then, he worked at the Centre for Zoonoses and Environmental Microbiology of the RIVM as a researcher and research coordinator on wildlife and vector-borne diseases. 454 Ecology and prevention of Lyme borreliosis