Wild animals as hosts for anthropophilic tick species in Serbia

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Wild animals as hosts for anthropophilic tick species in Serbia Snežana Tomanović,, PhD Laboratory for Medical Entomology, Center of excellence for food and vector borne zoonoses Institute for Medical Research, University of Belgrade Laboratory for Medical Entomology, Institute for Medical Research, University of Belgrade Founded almost 70 years ago with the foundation of the Institute for Medical Research (Laboratory for Medical Arachnoentomology) Since 2009 Center of excellence for food and vector borne zoonoses Research of ticks and tick-borne pathogens started on the Institute on 1981 (Dr. Marija Milutinović)

Area of research Arthropod vectors and vector borne pathogens Biology, ecology, physiology and genetics of vectors and vector borne pathogens Detection of vector borne diseases in animal and human populations Design and development of strategies for control and prevention of vector borne diseases Team Dr. sci. bio. Snežana Tomanović, associate research professor Sanja Ćakić, PhD student Darko Mihaljica, PhD student Ratko Sukara, PhD student Cooperation Faculty of Biology, University of Belgrade Faculty of Pharmacy, University of Belgrade Military medical academy, Belgrade Texas A&M University, College of Veterinary Medicine, Department of Veterinary Pathobiology, USA Institute for Microbiology and Immunology, Faculty of medicine, Ljubljana, Slovenia Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, WHO Collaborating Center for Research and Training in Mediterranean Zoonoses, Heraklion, Crete, Greece University of Veterinary Medicine, Vienna, Austria

Current research Focused on ticks-different aspects, mostly impact on human health 1. Tick fauna, tick-borne pathogens (TBP), parasite-host-pathogen relationships, enzoonotic cycles of tick borne pathogens 2. Markers of tick bite, anti-tick vaccines - antigenic determinants of tick saliva 3. TBP Local strains - Isolation, cultivation and characterization of tick-borne pathogens (Borrelia burgdorferi sensu lato) 4. Antimicrobial susceptibility of local strains Grants: National grant International grants (mobility - COST, bilateral projects ) COOPERATION people! Human and animal pathology Vectors significant Do not couse diseases it self, but enable spreading of the pathogens-causative agents of diseases 10% of described species transmit pathogenic microorganism Early XX century it was determined that ticks may act as vectors of human diseases 80s recognised as vectors of Lyme diseases Hard ticks more significant vectors of human diseases than soft ticks Lyme borreliosis CCHF Tularemia Rickettsiosis

Human and animal pathology Ticks were recognised as parasites of humans in Ancient greece today over 30 species are parasites of humans Direct influence (blood feeding) limited - Mechanical injuries (especially species with long mouth parts-ixodes, Hyalomma, Amblyomma) - Local irritation, anemia, alergic reactions, - Paralysis (North America-Dermacentor andersoni, D. variabilis; South Africa- Ixodes rubicundus, Rhipicephalus eversti eversti; Australia-I.holocyclus) Local irritation (Ixodes sp.) Mechanical injury (Amblyomma sp.) Paralysis (Ixodes holocyclus) Allergic reaction (Ixodes holocyclus) Studies on ticks in Serbia Studies on ticks in Serbia commenced at the beginning of the XX century (mainly hard, exophilous tick species). Hard ticks 5 genera 27 species Soft ticks 1 genus 3 species Osvald Petrović Milutinović Anthropophilic ticks

Studies of tick borne pathogens in Serbia Expansion of tick-borne pathogens in Europe Serbia is an endemic area for a number of tickborn diseases (Lyme disease,cchf, tularaemia, Q fever, human granulocytic anaplasmosis, TBE) Autochthonous cases of these diseases were continuously or periodically registered, but it is considered that the real epidemiological situation is underestimated. Tick-pathogen relationships in Serbia Relationship between viral and bacterial pathogen species and tick species in Serbia (Abbreviations: CCHF- Crimean-Congo Hemorrhagic Fever virus, TBE-Tick borne encephalitis virus, A.p.-Anaplasma phogocytophilum, B.v.-Babesia venatorum, B.m.-B. microti, A.o.-Anaplasma ovis, B.b.s.l.-Borrelia burgdorferi sensu lato, C.b.-Coxiella burnetii, F.t.-Francisella tularensis, R.h.-Rickettsia helvetica, R.m.-R. monacensis, R.r.- R. raoultii, R.ma.-R. masiliae)

Enzootic cycles and interrelationships Coevolution = equilibrium Pathogen Tick Human Domestic animals Host Pathogen Conservation medicine-one health The concepts of One health and Conservation medicine Target species: 1. Wild canids (golden jackal and red fox) sinantrophic species 2. European ground squirrel nests/burrows on open habitats 3. Bats caves

Wild canids - red fox (Vulpes vulpes) Widely distributed species, inhabiting the whole territory of the state, adopted to live in different habitats The most abundant predatory species in our country Coming to the close proximity of human settlements-close contact with domestic or stay dogs In competition with golden jackals Hunting season throughout year Foxes play an important role in epidemiology of tick-borne diseases and in the maintenance of tick populations Wild canids - red fox (Vulpes vulpes) Study PhD thesis Salem Juwaid During the period from 2010 to 2013 (2 winter seasons 2010/2011 and 2012/2013) 22 localities Hunted and roadkilled animals Data for each collected individual fox: sex, date of death and the most precise locality. Collected ticks were preserved in 70% ethanol until final identification Spleen samples Detection of pathogens in ticks and spleens

Wild canids - red fox (Vulpes vulpes) 67 red foxes - 41 26 I.h. I.r., I.h. I.r., I.h. Tick from 27 animals 10 localities, 80 ticks (49 18 13n) 5 five ixodid species D.r., I.r., I.c. I.c. H.c. I.r., I.h. I.r., I.c. I.c. I.r.,I.c., I.h. Ixodes ricinus - 38 13 Ixodes canisuga - 2 2 9n Ixodes hexagonus - 5 2 Heamaphysalis concinna 4n Dermacentor reticulatus - 4 1 Wild canids golden jackal(canis aureus) High spreading potential and sinantropic preferences. Dinamic population size and range Europe declination (beginning XX cent.) recolonization rapidly increasing populations, widening ranges toward Central and Eastern Europe In Serbia - neared extinction World War II (poisoning). Only two relic populations in Srem and eastern Serbia near Bulgarian borders. Beginning of the 1980s, started to spread quickly along the rivers (Timok, Morava, Danube, and Sava) and to increase in number -fusion of the two relic populations and widening of the range. Local populations - the highest density at the Balkan Peninsula. An opportunistic species, capable of using a wide range of food sources, come close to human settlements In competition with red fox The increase in densities and the widening of populations have not been accompanied by research on ectoparasites and pathogens of interest both to animals and to humans.

Wild canids golden jackal(canis aureus) Study PhD thesis Ratko Sukara During the period from 2010 to 2016 33 localities Hunted and roadkilled animals Data for each collected individual fox: sex, date of death and the most precise locality. Collected ticks were preserved in 70% ethanol until final identification Spleen samples Detection of pathogens in ticks and spleens Bats With approximately 1200 species - around quarter of all mammalian species on Earth. Bats are hosts to a large number of ectoparasites, including ticks, which can act as vectors of zoonotic agents. Three hard tick species have adapted to live exclusively on bats (Ixodes simplex mainly on Miniopterus schreibersii, I. kopsteini on mastiff bats (Tadarida sp.), I. vespertilionis parasitizes different bat species. in Europe I. simplex and I. vespertilionis are recorded, together with the recently described species I. ariadnae, collected from caves and bats in Hungary. Increasingly recognized as reservoirs of emerging, mostly zoonotic, viral and bacterial pathogens. In comparison to other mammalian hosts, ticks parasitizing bats have been largely understudied, especially in the central part of the Balkan Peninsula, where the last data from the field research date from almost 25 years ago.

Bats Study PhD thesis Jelena Burazerović Balkans (Serbia, Montenegro, Bosnia and Herzegovina, and Former Yugoslav Republic of Macedonia -FYROM). April October 2013 2015. 21 locality, from 149 bats belonging to 7 species Ticks were identified to the species level using morphological keys (Pomerancev 1950, Arthur 1956). Molecular analysis of the cytochrome oxidase subunit I (COI) gene as a complementary. 265 ticks 3 species Ixodes simplex -217 (24, 105 nymphs,78 larvae) Ixodes vespertilionis - 47(5, 24 nymphs and 18 larvae) Argas vespertilionis (1 larva) M.sc 117 212 217 Miniopterus schreibersii 6 Myotis myotis 2 3 6 Rhinolophus hiposideros 5 9 12 Ixodes simplex 8 Rhinolophus euryale 12 8 47 10 1 Ixodes vespertilionis Rhinolophus ferrumequinum 1 Myotis mystacinus 1 1 Argas vespertilionis 1 Pipistrellus pipistrellus European ground squirrel (Spermophilus citellus) Medium size rodent species Inhabits open habitats with short-grass (steppe, dry meadows, pastures) Lives in colonies, in long complex burrow systems, with each individual having its own burrow system Behavioral characteristics make S. cittelus exquisitely suitable host for ticks in the mentioned ecosystems. Spermophilus citellus is distributed in Southeastern and Central Europe, and its range is divided in two main geographic areas by the Carpathian Mountains. IUCN Red List of Threatened Species as vulnerable since 2008. By IUCN Red List of Threatened Species

European ground squirrel (Spermophilus citellus) summer months (July-early September) 2007-2013, as a part of continuous population monitoring in Vojvodina (northern part of Serbia). 12 localities 1009 ticks were collected from 151 animals Morphological identification of ticks was performed using standard taxonomic key Molecular analysis of the cytochrome oxidase subunit I (COI) gene as a complementary. 2 species Ixodes laguri 796 (102, 2, 666n, 26l) Haemaphysalis concinna 213 (213n) Fig. Phylogenetic analysis based on COI (a) and ITS2 (b) sequences of Ixodes sp. ticks obtained in this study (accession numbers KM096850- KM096863) and those previously deposited in the GenBank Ongoing research tick borne pathogens in ticks and reservoirs Molecular detection (ticks, different tissues) Babesia spp., Hepatozoon spp. Borrelia spp. Rickettsia spp., Anaplasma /Ehrlichia spp. Coxiella burnetii, Francisella spp., Bartonella spp. Leishmania Bartonella Golden jackals Red fox, golden jackal ticks, tissue bats, ground squirrel - ticks

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