The Seroepidemiology of Lyme Borreliosis in Zoo Animals in Germany
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Epidemiol. Infect. (2003), 131, 975–983. f 2003 Cambridge University Press DOI: 10.1017/S0950268803008896 Printed in the United Kingdom The seroepidemiology of Lyme borreliosis in zoo animals in Germany K. STOEBEL1*, A. SCHOENBERG2 AND W. J. STREICH1 1 Institute for Zoo and Wildlife Research Berlin (IZW), Berlin, Germany 2 Federal Institute for Risk Assessment, Berlin, Germany (Accepted 27 March 2003) SUMMARY We conducted the first seroepidemiological study to evaluate the exposure of zoo animals to Borrelia burgdorferi s.l. in German zoos and wildlife parks. A total of 1487 individuals representing 148 ungulate and carnivore species belonging to 19 families were examined using a non-species dependent ELISA. Specific antibodies were detected in 154 (10.4%) animals; 168 (11.3%) sera produced borderline results. The percentage of seropositive individuals was related to species and origin (zoo), and increased with age of the animals. Sex and season did not influence seroprevalence. Examination of 600 ticks (Ixodes ricinus; caught from vegetation in the zoos) by darkfield microscopy and indirect immunofluorescence technique revealed infection rates within the range typical for Central Europe. The results substantiate that there is an infection risk for zoo animals. A differential diagnosis of Lyme borreliosis should be taken into account in case of suspicious clinical symptoms and possible contact to ticks. INTRODUCTION Spirochaetaceae causes a chronic multisystem disease with a great variety of clinical manifestations [6, 7]. Emerging infectious diseases do not only involve Lameness as a result of either arthralgia or arthritis many wildlife species as reservoirs of pathogens that is the main symptom in all domestic animal species threaten domestic animal and human health, but pose examined so far [8–13]. In contrast to its increasing also a substantial threat to the conservation of global importance in veterinary medicine, very few reports biodiversity [1]. One of these ‘new’ diseases possibly exist on clinical investigations on Lyme borreliosis in affecting endangered species is Lyme borreliosis: a free-ranging animals [14–18]. Although clinical symp- zoonosis with world-wide distribution which is now toms have been reported in a few wild species only regarded as the most important tick-borne disease in [15, 17, 19], this disease might nevertheless directly humans [2–4]. Unlike the situation in the USA, no affect free-ranging as well as captive wild animals and region of endemic occurrence of Borrelia-infected should therefore be considered as a possible cause of ticks has so far been discerned in Germany and inexplicable population declines in endemic areas [20]. neighbouring European countries. The main vector in Unfortunately, there exists hardly any information on Europe – the hard tick Ixodes ricinus – is indigenous Lyme borreliosis in the literature on both zoo and to the entire continent (except Iceland) between sea wildlife medicine. level and altitudes up to 2000 m [5, 6]. The pathogen A hitherto unique serologic survey in zoo animals Borrelia burgdorferi sensu lato (s.l.) of the family was carried out at the St. Louis Zoo [21]. In extension to that investigation the epidemiological study pres- * Author for correspondence: Institute for Zoo and Wildlife Research Berlin, Alfred-Kowalke-Strasse 17, D-10315 Berlin, ented here is the first one which evaluates the ex- Germany. posure of a broad range of zoo animals in different Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 00:19:14, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268803008896 976 K. Stoebel, A. Schoenberg and W. J. Streich age classes to B. burgdorferi s.l. and assesses the dis- tribution of Lyme borreliosis in a variety of places and settings during different seasons throughout the year. 6 Hamburg METHODS Sampling 7 1, 2 Hodenhagen A total of 1667 blood samples (1241 ungulates and Berlin 426 carnivores) were examined originating from 1487 individuals and representing 148 ungulate and carni- 3, 4 vore species belonging to 19 mammalian families. In 8 Leipzig 5 case of two or more sera tested from one animal the Cologne Dresden mean of all test-values was calculated. The majority of sera came from 11 zoos and wildlife parks all over 9 Frankfurt Germany (Fig. 1), the remaining samples were col- lected at 13 other German zoos. Sera were taken within a period of almost 30 years (1969–1998), with the majority being collected in the 1990s. Blood spe- 10 Stuttgart cimens were obtained from healthy as well as from diseased animals during various veterinary interven- 11 tions. Data concerning the sex of the animals was Munich available for 96% and regarding the age for 64% of the samples. Fig. 1. Main study sites of Germany: 1. Zoological Garden A total of 600 ticks were caught by sweeping low- Berlin, 2. Tierpark Berlin-Friedrichsfelde, 3. Zoological lying vegetation of all accessible areas at the zoos with Garden Leipzig, 4. Game Park Leipzig, 5. Zoo Dresden, 6. a cotton flag (flagging method). At the Serengeti Tierpark Hagenbeck, Hamburg, 7. Serengeti Safaripark Safaripark Hodenhagen, tick collection was only Hodenhagen, 8. Zoological Garden Cologne, 9. Zoological possible along the fence outside of the park. All ticks Garden Frankfurt, 10. Wilhelma Stuttgart, 11. Tierpark Hellabrunn Munich. were collected between June 1997 and October 1998, identified as unfed stages of Ixodes ricinus. Until examination for the presence of B. burgdorferi s.l., the indirect immunofluorescence technique (IFT): they were kept cool and dark (refrigerator) in small nymphs and adult ticks were tested individually and larvae in pools of three. The infection rates of indi- screw-top tubes equipped with a piece of moist tissue pffiffiffiffiffiffiffiffiffiffiffi for high humidity. vidual larvae ( p) were calculated with: p=1x 4 1xf , where f is the proportion of infected larvae pools [23]. Serological test for exposure of zoo animals to The unfed ticks were homogenized in 50 ml PBS/ B. burgdorferi s.l. MgCl2 (5 mM), and one drop of this suspension was examined under a darkfield microscope at a magnifi- Blood samples were tested for B. burgdorferi s.l. cation of 250. For examination by IFT marked antibodies by means of a modified non-species depen- microscope slides with 12 patches (medco, Germany) dent enzyme-linked immunosorbent assay (ELISA). were prepared by coating them with 10 ml tick sus- Here, specific B. burgdorferi s.l. IgG antibodies were pension as well as with positive (culture of B. burg- detected by peroxidase (PO) conjugated protein A or dorferi s.l., strain 1B29=B. garinii) and negative protein G as an (almost) universal label instead of (PBS) control fluid. The preparations were allowed to species-specific secondary antibodies. The test was air dry and subsequently fixed in acetone for 5 min at performed exactly as described elsewhere [22]. x20 xC. A polyclonal rabbit anti-Borrelia burgdorferi s.l. (strain 1B29)-serum (generated in the laboratory) Detection of B. burgdorferi s.l. in ticks was added at a dilution of 1:200 in PBS. Sub- The ticks were examined for the presence of spiro- sequently, the slides were incubated in a wet chamber chetes by darkfield microscopy (DFM) and by for 30 min at 33 xC, rinsed three times in PBS, and Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 25 Sep 2021 at 00:19:14, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268803008896 Lyme borreliosis in zoo animals 977 dried carefully between sheets of filter paper. Finally, RESULTS fluorescein (DTAF)-conjugated AffiniPure goat anti- Blood samples rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, USA) was added at a dilution of 1:200 A total of 1487 ungulate and carnivore blood samples in PBS/Evans-blue. Final dilution of Evans-blue was examined by means of a modified non-species (Sigma-Aldrich, Germany) in PBS was 1:100 000. The dependent enzyme-linked immunosorbent assay samples were incubated, washed and dried as de- (ELISA). Specific antibodies were detected in 154 scribed above. The slides were coverslipped using (10.4%) of the animals tested; 11.3% of the ungulates glycerol buffer (90% glycerol in PBS) as enhancing and 7.5% of the carnivores were seropositive. In ad- fluid and examined under a fluorescence microsope at dition, 168 (11.3%) sera produced borderline results. a magnification of 400. Seropositive samples came from 10 of the 11 main study sites (with the exception of Hodenhagen) and from 2 additional zoos. Seroprevalence varied from Statistics 3.4% (Hamburg) to 22.2% (Cologne). The results of Blood samples of zoo animals cannot be collected ELISA testing for B. burgdorferi s.l. antibodies in sera following a pre-set statistical design. Since the animals of captive ungulates and carnivores are listed in Table providing the sera were not chosen randomly from a 1a for all speices from which at least 10 samples population, the whole statistical analysis of sera must were available. Positive sera were found in all families be regarded as exploratory data analysis. Statistical with more than 30 specimens tested, namely Equidae, methods were used to look for patterns in data, but a Camelidae, Cervidae, Bovidae, Ursidae, and Felidae. ‘significant’ result is to be regarded as a tendency in Positive individuals were found in 57 of the 148 the sample rather than as a statistical proof. species included in this study (Table 1). In species Stepwise logistic regression [24] was performed to of which at least 10 samples were accessible, the fre- evaluate the contribution of the independent variables quency of antibody response was highest in musk origin/zoo (categories 1–11 are explained in Fig.