increase in host-finding efficacy

Transcription

1 Appl Acarol (2008) : Exp /s DOI infestation risk Borrelia burgdorferi s.l. Tick increase in host-finding efficacy infection-induced 20 July 2007/Accepted: 22 January 2008/Published online: 1 February 2008 Received: Science+Business Media V (C) An investigation of the risk of human tick infestation, together with the Abstract of Borrelia burgdorferi s.l. infection, was conducted in a sylvatic habitat in prevalence Germany to provide data needed for future risk-benefit evaluations of acaricides western for clothing impregnation. Additionally, data were collected on behavioural changes used Borrelia burgdorferi s.1.-infected adult female L ricinus ticks the possible impact of in changes on host-finding efficacy. The risk of L ricinus-infestation was determined by such from the protective clothing of volunteers by dragging in known tickinfested collecting sites in the Kiihkopf Mountain area, Koblenz, Germany, from June through The overall tick infestation rate per person per hour was , with the October sex- stage-specific differences: males , females , following Although the difference in infection rates was statistically significant volunteers. < 0.023) only in adult female ticks, our data indicate that burgdorferi s.l. infection (P increase host-finding efficacy in adult L ricinus. The overall exposure risk was 1.0 may burgdorferi s.l.-infected ticks per person per hour of exposure, or 0.25 ticks per 100 m walking distance in the study area. Ixodes ricinus Host-seeking activity Borrelia burgdorferi s.1. Keywords exposure risk Human K. Faulde ([) M. of Medical Entomology/Zoology, Central Institute of the Federal Armed Forces Department Service, P.O. Box 730, Koblenz, Germany Medical G. Robbins R. Pest Management Information Analysis Center, Armed Forces Pest Management Board, Defense of female Ixodes ricinus under natural conditions Michael K. Faulde Richard G. Robbins , larvae Concurrent dragging revealed an average nymphs times higher infestation rate as well as a markedly lower infection rate with 19. borreliae in adult I. ricinus.,ticks when compared to ticks collected from exposed human Walter Reed Army Medical Center, Washington, DC , USA

2 Report Documentation Page Form Approved OMB No Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering maintaining the data needed, completing reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations Reports, 1215 Jefferson Davis Highway, Suite 120, Arlington VA Respondents should be aware that notwithsting any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE JUL REPORT TYPE 3. DATES COVERED to TITLE AND SUBTITLE Tick infestation risk Borrelia burgdorferi s.l. infection-induced increase in host-finding efficacy of female Ixodes ricinus under natural conditions 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Defense Pest Management Information Analysis Center (DPMIAC),Armed Forces Pest Management Board (AFPMB),Walter Reed Army Medical Center,Washington,DC, PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES 1. ABSTRACT see report 15. SUBJECT TERMS 11. SPONSOR/MONITOR S REPORT NUMBER(S) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT unclassified b. ABSTRACT unclassified c. THIS PAGE unclassified Same as Report (SAR) 18. NUMBER OF PAGES 9 19a. NAME OF RESPONSIBLE PERSON Stard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

3 the increasing number of vector-borne diseases that are currently emerging or Among worldwide, only a few are vaccine-preventable (e.g., tick-borne encephalitis, resurging fever, Japanese encephalitis, plague). For this reason, personal protective yellow against hematophagous vectors constitute the first line of defense against measures diseases in endemic areas. In Europe, tick-borne diseases pose the arthropod-transmitted risk, with over 10,000 reported clinical cases of tick-borne encephalitis, greatest of hundreds of thouss of clinical cases of Lyme disease occurring annually estimates Faulde, in press). A major advance in protecting at-risk personnel, such as (Ginsberg hikers soldiers, against ticks tick-borne diseases has been the development foresters, of topical repellents residual acaricides that can be impregnated into clothing et al. 1982; Rey 1998; Faulde Uedelhoven 2006). (Schreck skin or fabric repellents are likely to be used by thouss of inexperienced Since exposed to vector ticks, a safety factor should be calculated by analyzing the riskbenefit people ratio of the biocides employed (Young Evans 1998; WHO 2001a, b). In order evaluate a substance's toxicology the efficacy of its formulation for human use, it is to to quantify (1) the tick infestation rate on humans during field exposure in necessary tick habitats, (2) tick population densities, (3) local prevalence of associated known agents. Although toxicological evaluations of many skin repellents are available disease Yap 1997; WHO 2001a, b), studies of permethrin-based fabric repellent (Chavasse are well under way (Snodgrass 1992; Rossbach et al. 2005), no quantitative formulations of the risk of human tick infestation, associated disease agents, currently exists study Central Europe. for Europe, the castor bean or sheep tick, Ixodes ricinus, is the most common tick species In the principal vector of burgdorferi s.s., afzelii, garinii, spielmani, valaisiana, lusitaniae, tick-borne encephalitis virus (Central European encephalitis), virus, Erve virus, Eyach virus, Anaplasma phagocytophilum, Rickettsia Uukuniemi (Gem Humair 2002; Siiss Schrader 200; Siiss et al. 200). Ixodes helvetica is exophilic, passively questing from vegetation for its hosts, which include a wide ricinus diseases in Central Europe. borne L ricinus has been studied for more than 50 years, knowledge of this species' Although remains incomplete, partly because of geographical genetic heterogeneity, but biology because of the diversity of habitats in which it may be found (Gem Humair 2002). also L ricinus is active only when the humidity exceeds 80%. By contrast, Borreliainfected Generally, adults of L persulcatus, the principal vector of burgdorferi s.1. in Eastern Asia, appear to be significantly more tolerant of lower relative humidity Europe air soil temperatures than non-borrelia-infected adults (Alekseev Dubinina higher Additionally, exoskeleton anomalies in Borrelia-infected female L persulcatus 2000). in a 1.3-fold increase in locomotor activity (Alekseev et al. 2000). Whether resulted burgdorferi s.1.-induced changes in the behaviour of I. persulcatus I. ricinus result greater questing host seeking activity, leading to increased infestation efficacy, in unknown. remains study was designed to quantitatively analyze human exposure to L ricinus ticks, This 138 Exp Appl Acarol (2008): Introduction of reptiles, birds mammals. Due to the medical importance, great abundance, range distribution of both vector disease agent, L ricinus-associated Borrelia burg- broad s.1. infection constitutes an excellent model for investigating the impact of tickdorferi tick density by dragging determining the prevalence of Borrelia burgdorferi measuring in ticks removed from humans drags under field conditions in Germany. s.1.

4 Materials methods Tick field exposure sampling

5 every m. The "relative Tick Infestation Ratio" (rtir)mthe number of ticks counted 100 the blanket per m versus the number collected from an exposed volunteer to attached per 100 minis defined as follows: Number of ticks attaching to drag per 100 m Borrelia genospecies: burgdorferi s.s., garinii, afzelii. Borrelia burgdorferi three s.l.-specific fluorogenic primer probe sequences, targeting the Osp A gene, as as positive controls of burgdorferi s.s., were purchased from TibMolBiol Co. well Germany); DNA from garinii, afzelii was kindly provided by the (Berlin, Baden-Wiirttemberg (Stuttgart, Germany). Positive controls a Lesgesundheitsamt (Borrelia-DNA-free))-DNA control (LightCycler Fast Start DNA Master negative Roche Diagnostics Co.), simultaneously acting as a run-specific internal HybProbe, control, were employed during each run. amplification burgdorferi s.1. positive, borderline, unclear results were independently All by a second LC-PCR run using the commercially available RealArt Borrelia LU confirmed Kit (Artus GmbH, Hamburg, Germany) for combined detection of burgdorferi s.s., PCR garinii, afzelii. were reported as mean - SD. Differences in mean burgdorferi s.1. infection Values by tick sex stage were analyzed using Pearson's Z 2 test, including error degrees of rates (df), two-sided asymptotic significances against the residual error at the 5% freedom (statistical significance) (SPSS 8.0 program, SPSS Software GmbH, Munich, level 18 h of human exposure in known tick-infested habitats, 132 ticks were collected; During these, 6 (.5%) were males, 16 (12.1%) females, 6 (8.5%) nymphs, 6 (3.9%) of Figure 2 shows the tick infestation rates obtained by human field exposure. The larvae. tick infestation rate per person per hour was (range 0-23), with the overall sex- stage-specific differences: males (range 0-2), females following (range 0-), nymphs (range 0-17), larvae (range 0-12) Exp Appl Acarol (2008) : rtir Number of ticks infesting an exposed human per 100 m ticks collected from humans (except one adult female, which escaped during All or by dragging were further investigated for Borrelia burgdorferi s.1. infection. collection) Borrelia burgdorferi s.l. detection in ticks ticks were mechanically homogenized using a MagNA Lyser device (Roche Single Co., Mannheim, Germany). DNA was isolated using a High Pure PCR Diagnostics Preparation Kit (Roche Diagnostics Co., Mannheim, Germany) subsequently Template for burgdorferi s.l tested burgdorferi s.1. PCR was carried out using a LightCycler 2.0 (Roche Specific Co., Mannheim, Germany), as described by Rauter et al. (2002), targeting the Diagnostics Data analysis Germany). Results

6 Exp Appl Acarol (2008) : Mean sex- stage- 1 Fig. tick attachment rates per specific per hour of exposure, person the crude risk of tick. 10 constituting measured in the.2 infestation, recreational area, Kiihkopf Fig. 3 Mean sex- stage- tick infestation rates per specific per 100 m walking person versus dragging results, distance in the Kiihkopf measured area, Koblenz, recreational during 2006 Germany, 16 males B []females 1 [] larvae Total I'-! males females nymphs m walking distance, of which were males, were females, were nymphs, were larvae (Fig. 3). During 2006, L ricinus 0.91 per 2 m the following distribution by tick sex stage: males , ticks , nymphs , larvae (Fig. 3). Dragging resulted in a 1.0 females times (n 10, range ) higher tick infestation rate when compared to 19. numbers collected from humans. The mean rtir was 19., with the following sex- the differences: males 15.2, females 8.3, nymphs 18.9, larvae stage-specific the ticks collected from exposed volunteers (Table 1), burgdorferi s.l. Among was detected in 33.3% (2/6) of males, 56.25% (9/16) of females, 9.% (6/6) of infection 0% (0/6) of larvae, with a mean positivity rate of 12.9%. Out of the 17 nymphs, tick samples, 7 (1.2%) were burgdorferi s.s., 5 (29.%) were Borrelia-positive garinii, were afzelii (23.5%), including one (5.9%) burgdorferi s.s.ib, afzelii infection (Table 1). These results amount to an overall exposure risk of 1.0 double burgdorferi s.l.-infected tick per person per hour of exposure, or 0.25 per 100 m walking in the tick-infested sites studied. Ticks collected by dragging showed the distance burgdorferi s.l. positivity rates: males 17.8% (5/28), females 25% (11/), following 8.9% (10/112), larvae 2.9% (3/102), with a mean infection rate of 10.2%. nymphs genospecies differentiation showed the following pattern: 15 (51.7%) burg- Borrelia s.s., 8 (27.6%) garinii, 6 (20.7%) afzelii. Differences in burgdorferi s.l. dorferi in ticks sampled from humans versus dragging were statistically significant in infection (Z2= 5.2, df 1, P < 0.023), but not in males (g2= 0.7, df 1, P < 0.395), females (Z2= 0.1, df 1, P < 0.71), larvae (Z2= 1., df 1, P < 0.2) (Fig. ). nymphs 11.r 12 larvae 8 o Koblenz, Germany, during =llnymphs o = 10 flagging human exposure These results correspond to a mean tick infestation rate of ticks per person per activity was bimodal, peaking in June September. questing conducted concurrently with human exposure, yielded a mean of Dragging,

7 1 Overview on tick pattern collected by exposed volunteers, associated burgdorferi-positive Table ricinus ticks, climatic conditions!xodes Total tick No. of ticks Corresponding Temperature (C)/relative Volunteer exposure collected during Borrelia-positive humidity (%) during no. (h): exposure (M/F/N/L): ticks (M/F/N/L): exposure 12 (0/0/2/10) 0/0/0/0 23/51 (0/0/2/2) 0/0/0/0 23/5 (0/0/0/) 0/0/0/0 25/9 (0/0/0/6) 0/0/0/0 23/ (0/2/0/12) 0/1 Ba/0/0 23/51 2 (2/0/0/2) Bgss/0/0/0 23/5 (2/0/2/0) Bgss/0/0/0 25/9 (0/0/0/0) 0/0/0/0 23/ (0/1/0/0) 0/0/0/0 25/0 3 (0/0/6/0) 0/0/1 Ba/0 2/5 6 (2//2/0) 0/1 Bgss + 2 Bg/l Bg/0 23/ (0/0/6/0) 0/0/0/0 20/50 (0/2/5/0) 0/1 Bgss + Ba/l Bgss 3/32 7 1Ba/0 + (0/0/8/) 0/0/1 Bg/0 3/ (0/2//2) 0/1 Bgss + Bg/l Bgss/0 28/30 5 (0/1/2/0) 0/0/0/0 28/36 3 (0/2/8/0) 0/0/0/0 23/ (0/2/17/) 0/1 Bgss-Bg double 23/50 6 infection/0/0 males; F females; N nymphs; L larvae; Bbss Borrelia burgdorferi sensu stricto; M Borrelia afzelii; Bg Borrelia garinii Ba in burgdorferi s.l. differences in Ixodes ricinus ticks infection from humans collected to field exposure subsequent collection by dragging, versus in the Kiihkopf measured area, Koblenz, recreational during 2006 Germany, ,7 8,9 human I"l,flagging occupational health safety reasons, human field exposure to L ricinus ticks was For by walking very slowly in known tick-infested areas using full-body protection conducted leather military boots plus "buddy checks" designed to prevent tick bite possible of endemic tick-borne diseases. It is therefore impossible to ascertain whether transmission 12 Exp Appl Acarol (2008) : Fig. Sex- stage-specific 56, ,9 larvae males females nymphs Discussion

8 Exp Appl Acarol (2008) : humans have a differing stimulatory effect on tick host seeking infestation unprotected to more rapid volitalization of attractants. Additionally, different human exposure due footwear may result in varying tick infestation ratios, as reported for both behaviours of the western black-legged tick, Ixodes pacificus, the lone star tick, nymphs americanum (Carroll Kramer 2001; Lane et al. 200). Because of the Amblyomma high mobility of L ricinus ticks on our volunteers' legs, we did not attempt to relatively this species' "attachment area" or "attachment height". Given the density of the determine abundance during the 2006 activity season resulted in the second highest infestation great on humans, exceeded only by nymphs. It should be borne in mind that, because of their rate size cryptic colouration, attached larvae can easily be overlooked on the human small while remaining potential vectors of human pathogens acquired via transovarial body transmission. larvae collected from humans tested negative for burgdorferi s.1. infection, All three of eight larvae collected by dragging tested positive for burgdorferi s.s. whereas all three larvae were collected at a single location on the same day time Interestingly, were infected with the same Borrelia genotype, indicating that they were almost the progeny of the same female. Our field data underscore the focal nature of certainly transmission support recent findings that the efficacy of transovarial transovarial is usually low but, when it occurs, the prevalence of infection may be high, transmission burgdorferi s.s. prevailed in 7.8% of cases, garinii in 28.3%, afzelii in 21.7%; was also one (2.2%) burgdorferi s.s.lb, afzelii double infection. Kipp et al. (2006), there in six different habitats in Thuringia, Germany, determined that the prevalence working genetic diversity of burgdorferi s.1. are highly variable habitat-specific. burgdorferi s.s. is unusually high within the mesotrophic sylvatic Luzulo-Fagionbiotopes along the Rhine Valley in Rhinel-Palatinate, while garinii afzelii due to the harsh winter spring of 2005/2006 (the mean temperature between been March 2006 was 0.1 C, whereas the 10-year average for this area is 2.5C), January may have delayed tick activity until late April/early May. Simultaneous dragging which that the mean rtir was 19. times (range ) higher on low vegetation revealed litter, indicating that tick infestation risk increases dramatically in persons who come leaf contact with that microhabitat (Carroll Kramer 2001; Lane et al. 200), especially into prolonged periods (e.g., soldiers, hunters, foresters). Under such conditions, tick for measures should include a combination of skin fabric repellents (Faulde avoidance al. In Central Europe, the highest tick densities that have been reported to date are et 2006). 11 per m at foci in Alsace, France (Ferquel et al. 2006); by comparison, the 105 L ricinus population, as measured by dragging, the risk of tick infestation, tick bite, local tick-borne disease transmission may exceed our estimates. Moreover, in light of micro- differences, tick dragging is not directly comparable with human exposure. ecological the mean rtir values for male L ricinus indicate that male host-seeking Nonetheless, is considerably lower than it is in females. Larvae showed the highest rtir value, efficacy corresponds to the lowest host-seeking efficacy with respect to humans, but their which from 3 to 100% (Bellet-Edimo 1997; Gem Humair 2002). ranging respect to the overall Borrelia genospecies pattern detected in L ricinus, With on area habitat investigated, burgdorferi s.s. prevalence varied from 0 to Depending garinii from 0 to 39%, afzelii from 7 to 52%. Clearly, the prevalence of 20%, rates fit the expected range. prevalence are the stage of L ricinus most often found on humans, followed by larvae, Nymphs males. The unexpectedly high peak in larval questing during 2006 may have females, tick density in the Kiihkopf area, t- 9.2 ticks per 100 m 2, is moderate. In highly

9 areas, a tick infestation rate exceeding 100 per person per exposure hour can be infested But if we consider the corresponding burgdorferi s.1. positivity rates, which expected. between 9.6% (nymphs) 36.% (adults), with a reported mean of 19.%, then the vary of attached burgdorferi s.1.-infected ticks may exceed 20 per person per hour of number within the Alsatian foci, almost 20 times the risk measured in the Ktihkopf exposure area. recreational adults of L ricinus collected from humans showed a higher burgdorferi Intriguingly, infection rate than ticks collected by the dragging method, although this difference was s.l. significant only in females. Confirmation of this observation is needed because statistically the relatively small numbers of adult ticks in our study. Apparently, infection with of burgdorferi s.l. may induce behavioural changes in adult ticks, especially adult females, leading to an increase in host-finding efficacy as well as an underestimation of possibly yet it is unclear how far the suppression of locomotor activity, which occurs in adult As immature Borrelia burgdorferi s.1.-infected L ricinus as opposed to uninfected spec- (Alekseev et al. 2000), may positively or negatively influence host seeking efficacy. imens burgdorferi s.1. infection can cause behavioural changes in adult L ricinus, leading to If been reported for the sister species L persulcatus (Alekseev Dubinina 2000), then has acquisition may be facilitated, with it, transmission of disease agents. Whether host other than burgdorferi s.l. also influence tick behaviour, especially as coinfections, pathogens remains unknown. at infested sites in western Germany underscore the considerable risk of acquiring humans borreliosis in Central Europe. Since no licensed vaccine exists for Lyme borreliosis Lyme personal protective measures remain the first line of defense against tick bite worldwide, tick-borne diseases (Young Evans 1998; WHO 2001a, b; Mencke 2006). It is their assistance with fieldwork. TibMolBiol, Berlin, kindly offered technical support, DNA from Borrelia for burgdorferi s.s., helpful guidance. We are also grateful to the Lesgesundheitsamt Baden- AN, Dubinina HV (2000) Abiotic parameters diel seasonal activity of Borrelia-infected Alekseev uninfected Ixodes persulcatus (Acarina: lxodidae). J Med Entomol 37:9-15 AN, Jensen PM, Dubinina HV, Smirnova LA, Makrouchina NA, Zharkov SD (2000) Peculiarities Alekseev behaviour of taiga (lxodes persulcatus) sheep (lxodes ricinus) ticks (Acarina: Ixodidae) of by different methods. Folia Parasitol 7: determined OR (1997) Importance de la transmission transstadiale et de la transmission transovarienne du Bellet-Edimo Switzerl Neuchtel, JF, Kramer M (2001) Different activities footwear influence exposure to host-seeking nymphs of Carroll scapularis Amblyomma americanum (Acari: Ixodidae). J Med Entomol 38: Ixodes DC, Yap HH (1997) Chemical methods for the control of vectors pests of public health Chavasse importance. WHO/CTDAVHOPES/97.2. World Health Organization Distribution Sales, Geneva 1 Exp Appl Acarol (2008) : their impact in burgdorferi s.1. transmission. preference for higher air, soil subsoil temperatures lower relative humidity, as a data on tick infestation burgdorferi s.l. prevalence rates in ticks attaching to Our that the results reported here will prove useful in future risk-benefit analyses cf hoped formulated for use on fabrics. acaricides Acknowledgements We thank Marco Isack, Sabine Barz, Thorsten Lange, Bernd Bocklet Dirk Hiller Wtirttemberg, Stuttgart, Germany, for providing DNA samples from Borrelia afzelli garinii. References Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) chez la tique Ixodes ricinus (Acari: spirochete dans l'6pidemiologie de la borrliose de Lyme. PhD thesis, University of Neuchtel, Ixodidae)

10 Exp Appl Acarol (2008) : M, Uedelhoven W (2006) A new clothing impregnation method for personal protection against ticks Faulde biting insect. Int J Med Microbiol 296(Suppl 1): MK, Uedelhoven WM, Malerius M, Robbins RG (2006) Factory-based permethrin impregnation of Faulde residual activity against Aedes aegypti Ixodes ricinus in battle dress uniforms worn uniforms: field conditions, cross contamination during the laundering storage process. Mil Med under 171:72-77 E, Gamier M, Marie J, Bernede-Bauduin C, Baranton G, Perez-Eid C, Postic D (2006) Prevalence of Ferquel burgdorferi sensu lato Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a Borrelia of lyme borreliosis endemicity in France. Appl Environ Microbiol 72: focus L, Humair P-F (2002) Ecology of Borrelia burgdorferi sensu lato in Europe. In: Gray JS, Kahl O, Lane Gem Stanek G (eds) Lyme borreliosiswbiology, epidemiology control. CABI International, RS, pp HS, Faulde MK Ticks. In: Urban pests health. WHO, European Centre for Environment Ginsberg Bonn Office, Bonn, Germany (in press) Health, S, Dorn W, Wilske B, Fingerle V (2006) Heterogeneity in prevalence genetic diversity of Borrelia Kipp sensu lato in Ixodes ricinus ticks collected in different localities in Thuringia, Germany. Int burgdorferi Microbiol 296(Suppl 1): Med RS, Steinlein DB, Mun J (200) Human behaviors elevating exposure to Ixodes pacificus (Acari: Lane 1: T (2007) Anwendung der Biostoffverordnung bei Borrelienexposition: Auswirkungen auf den Lieboldt der Bundeswehr. Wehrmed Wehrpharm 31:66-69 Dienstbetrieb N (2006) Acaricidal repellent properties of permethrin, its role in reducing transmission of Mencke pathogens. Parassitologia 8: vector-borne C, Oehme R, Diterich I, Engele M, Hartung T (2002) Distribution of clinically relevant Borrelia Rauter in ticks assessed by a novel, single-run, Real-Time PCR. Clin Microbiol 0:36-3 genospecies JL (1998) Moyen actuel de protection contre les maladies transmises par les tiques. Med Mal Infect Rey 28: B, Scharnbacher J, Heinrich K, Mross KG, Letzel S, Egerer E (2005) Influence of permethrin Rossbach uniforms to the internal pyrethroid exposure of soldiers during deployment. Arbeitsmed impregnated Praeventivmed 3:127 Sozialmed W (198) Exkursionsflora, vol 2. Volk und Wissen Volkseigener Verlag, Berlin, pp -5 Rothmaler CE, Mount GA, Carlson DA (1982) Pressurized sprays of permethrin on clothing for personal Schreck against the lone star tick (Acari: Ixodidae). Econ Entomol 75: protection HL (1992) Permethrin transfer from treated cloth to the skin surface: potential for exposure to Snodgrass Toxicol Environ Health 35: humans. J, Schrader C (200) Durch Zecken iibertragene humanpathogene und bisher als apathogen geltende Siiss in Europa. Teil I: Zecken und Viren. Bundesgesundheitsbl Gesundheitsforsch Mikroorganismen 7:392-0 Gesundheitsschutz J, Fingerle V, Hunfeld K-P, Schrader C, Wilske B (200) Durch Zecken tibertragene humanpathogene Siiss bisher als apathogen geltende Mikroorganismen in Europa. Teil II: Bakterien, Parasiten und und Bundesgesundheitsbl Gesundheitsforsch Gesundheitsschutz 7:70-86 Mischinfektionen. (2001a) Vectors of diseases: hazards risks for travellersmpart I. WER 25: WHO (200 b) Vectors of diseases: hazards risks for travellersmpart II. WER 26: WHO D, Evans S (1998) Safety efficacy of DEET permethrin in the prevention of arthropod attack. Young lxodidae) nymphs their associated bacterial zoonotic agents in a hardwood forest. Med Entomol Mil Med 163:1-7