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European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4th scientific meeting,

joint with the

annual meeting of the European Wildlife Disease Association (EWDA)

May 8-12, 2002, Heidelberg, Germany.

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European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4th scientific meeting, joint with the annual meeting of the European Wildlife Disease Association (EWDA) May 8-12, 2002, Heidelberg, Germany. DISEASE RISK ASSESSMENT: PIROPLASMOSIS AT THE REINTRODUCTION SITE OF

THE PRZEWALSKI HORSE (Equus przewalskii) IN THE DSUNGARIAN GOBI, MONGOLIA

S. RÜEGG 1, C.WALZER 2,3, N. ROBERT 1, M. DOHERR 4 and K.T. FRIEDHOFF 5

Affiliation:

1. Institute for Animal Pathology, University of Bern (Director: Prof. Dr. M. SUTER) 2. Salzburg Zoo Hellbrunn (Dr. R. REVERS) 3. International Takhi Group 4. Institute of Animal Neurology, University of Bern (Prof. Dr. M. VANDEVELDE) 5. Institute of Parasitology, Tierärztliche Hochschule Hannover Abstract Piroplasmosis has been identified as a possible cause of mortality in reintroduced Przewalski horses (Equus caballus przewalskii) in the Dsungarian Gobi. The disease risk has been assessed with a descriptive model of Babesia caballi and Theileria equi in the vector (Dermacentor nuttalli) and the equid host, and by determining the age-stratified seroprevalence in the domestic horses sharing pasture and water points with the Przewalski horses. In order to predict the infection risk on long term however, more research is requir ed regarding the population dynamics of the ticks and the small mammals, as well as the immune response of the equine host against ticks and piroplasms. Zusammenfassung Piroplasmose wurde als mögliche Ursache für eine erhöhte Mortalität bei ausgewilderten Przewalskipferden (Equus caballus przewalskii) identifiziert. Das Infektionsrisiko für neu ausgewilderte Individuen wurde eruiert. Dazu wurde die Entwicklung von Babesia caballi und Theileria equi im Vektor (Dermacentor nuttalli) und im Wirtequiden in einem deskriptiven Model zusammengestellt und eine altersstratifizierte Seroprävalenz bei domestizierten Pferden, die sich auf den gleichen Weiden und an den gleichen Wasserlöchern aufhalten wie die Przewalskipferde, wurde erhoben. Weitere Untersuchungen be züglich der Zeckenentwicklung, der Kleinsäugerpopulation und der Immunreaktionen des equinen Wirtes gegen Zecken und Piroplasmen sind notwendig, um langfristig das Infektionsrisiko abzuschätzen. Résumé Babesia caballi et Theileria equi ont été indentifiées comme cause importante de mortalité chez les chevaux de Przewalski (Equus caballus przewalskii) réintroduits dans le désert de Gobi. Afin d'évaluer le risque d'infection pour les chevaux réintroduits, un modèle décrivant le développement des parasites en question dans le vecteur (Dermacentor nuttalli) et le cheval a été établi. La séroprévalence chez les chevaux domestiques partageant les mêmes pâturages et les mêmes points d'eau a été déterminée. Afin d'estimer le risque d'infection à long terme, des études supplémentaires sont nécessaires, parmi lesquelles l'étude du développement des tiques, l'étude des populations de petits mammifères, que l'étude des réaction immunitaires des chevaux contre les tiques et les piroplasmes.

Key words: przewalski horse, takhi, Equus przewalskii , Babesia caballi, Theileria equi, equine piroplasmosis, Dermacentor nuttalli, disease risk assessment, modelling, Mongolia



Introduction The Przewalski horse became extinct in the wild in the 1960‘s. The last sightings were recorded in the Dsungarian Gobi in SW Mongolia. At present 6 different reintroduction projects exist of which one is located in Takhin Tal (N45°32’20”, E093°39’06”) in the Dsungarian Gobi (20). A retrospective study and the standard necropsy protocol and sampling procedures revealed, that there is an increase of mortality during the months of April and May. The pathological findings suggest that equine piroplasmosis is likely to be responsible for these deaths. In addition the serological analysis of 3 serum samples from Przewalski horses (takhi), and the fact that the reintroduced takhis are naïve to piroplasmosis, indicate that the newly introduced individuals are probably infected with piroplasms during their first summer in Mongolia (20). Domestic hors es share pasture and water points with the takhis and are an important reservoir of diseases. Therefore samples were taken primarily from domestic herds, but also individual takhis were sampled at different occasions. Equine Piroplasmosis Equine piroplasmosis is caused by two intra-erythrocytic protozoan pathogens: Babesia caballi and Theileria equi (formerly Babesia equi (15)). They are both transmitted by ticks and cause primarily haemolytic anaemia and associated lesions in their mammalian host (8). In combination with exertion and stress they were shown to be an important mortality factor in horses.(8,16). Because of the disease risk for newly introduced takhi it is of primary interest to gain an insight into the parasitic cycle and epidemiological status at the reintroduction site at Takhin Tal and to determine the probability of a newly introduced takhi to become infected with either B.caballi or T.equi. A simple descriptive model of the disease will help determining the relevant data, which will need to be collected. Horses infected with T.equi remain inapparent carriers for life. B.caballi carriers harbour the parasite for 12-42 months (10). Mortality of the disease depends on the general immune status of the affected host and on the virulence of the piroplasm strain (8,16,18). Old horses are more seriously affected than young ones (17). Acquired immunity protects against clinical disease. There is no cross immunity between B.caballi and T.equi. In enzootic areas, constant super infections induce stable immunity (18). If no super infection occurs, antibody titres reach a peak after infection and decline thereafter (12). The foals of inapparently infected mares are already infected during the first postnatal year. Under protection of maternal antibodies, they usually go through an inapparent infection (18). Abortions are common and foetal erythrocytes are parasitised. Intrauterine transmission of T.equi appears to be relatively common. The foetus can be infected with both, B.caballi and T.equi during the various stages of pregnancy without the mare showing clinical symptoms (18). Immunosuppression: Babesiae suppress the immune reactions from the host against ticks. This leads to a higher egg production in ticks engorging on infected horses than in ticks from non-infected horses (6). Infectivity for ticks: Even extremely low parasitaemias in the horse lead to infection of 100% of the engorging ticks . Infectivity for ticks is highest at the beginning of the parasitaemia, but if more than 1% of the erythrocytes are infected with piroplasms the mortality in the ticks is high (6). T.equi completes a transstadial cycle in the vector, whereas B.caballi is transmitted transovarially. In replete tick females an infection with either pathogen causes a lower egg production and a higher mortality (6). Friedhoff and Smith have shown that without reinfection from the mammalian host the vector population would be cleared from Babesia sp. after 3-4 generations (6). The transmitting tick stages depend on the vector and the piroplasm species. Here we try to answer several questions related to the epidemiology of the disease:

1. How many horses are affected? 2. Which tick species is the vector? 3. When is the disease transmitted in Takhin Tal?



Material and Methods Preliminary evaluation The first question is best addressed by determining the seroprevalence. In addition we made blood smears from each sampled individual to determine the parasite rate. Because of the high number of horses kept in vicinity of the release site, the sample size had to be restricted. This was accomplished by choosing only those horses that share pasture and water points with the takhis at any given season during the year. This population was estimated to include 450 horses based on animal counts of the previous year (Ganbaatar and Walzer, personal communication). With Win Episcope 2.0 (freeware, http://www.clive.ed.ac.uk/winepiscope/) the sampling proportion for a level of confidence of 95% with an accepted error of 5% was calculated to be 30.90% and 45.92%, for expected prevalence of 84.5% (2) and 46.4% (11), respectively. Sampling logistics Mongolian herders live as nomads. Their herds are kept unfenced and especially the horse and camel herds are only checked sporadically. Their transhumance pattern does not vary much, adapting slightly every year according to the pasture quality and other- traditional considerations. A Mongolian co-worker travelled to the various water points within a perimeter of 60km of the releasing site, asking about herd size, migratory route, and arranged sampling dates at which the herders would bring the horses to their homes. For compensation the horses were given Ivermectin s.c. (0.02 mg/kg). This allowed us to plan the sampling and identify the relevant herds. Serial samples from 15 foals and 15 yearlings were taken to evaluate the incidence of piroplasmosis. A contract was signed with the Mongolian Army, which allowed us to take blood four times from horses in a herd of 250 animals that was moving around close to our research area. Sampling The horses in the serial study were marked with a microchip for identification (AEG ID 162 FDX-B, Fa. Richter Pharma, Wels, Austria). Each animal was caught with a lasso by the military staff or the private owner. Then blood (EDTA) and serum samples were taken. After sampling, blood smears were made. The samples were left for sedimentation over night and then the plasma, serum and buffy coat were isolated and stored at –11°C in a solar powered freezer. Ticks were collected from all horses. Their abundance and position on the host was recorded. 50% were first stored at 4°C in order to trigger the babesian cycle and then incubated for 8 days at 28°C for development of the infectious protozoan stages and egg production of the tick. Thereafter the ticks were dissected and the relevant organs extirpated and stored in 70% alcohol. B.caballi completing a transovarial cycle, is specifically detected in the ovaries and eggs, whereas T.equi being transmitted only transstadially is best identified in the salivary glands of ticks having fed for more than 3 days (4,13,19). To determine the topographical site of infection, the tick prevalence in 5 different habitats was investigated. Transects were laid along the river in the research area, 100m distant of it, in a habitat dominated by Achnatherum splendens grassland and one by Halophilus sp. scrubs, and in a mountainous region. For each sample a flag was drawn over the vegetation for 500m and the attached ticks collected. Laboratory On site a microscope allowed rough evaluation of the Giemsa-stained blood smears. All other samples were returned to Europe and were analysed there. Previous studies on piroplasmosis have shown the Immunofluorescence Antibody Test (IFAT) to be the most economic (3) and efficient test for serology with a specificity of up to 100%, a sensitivity of 100% and predictive values from 99 to 100% (9). The recovered ticks were all identified as Dermacentor nuttalli using the key from Arthur (1). The salivary glands were exposed to a Feulgen reaction, and eggs and ovaries subjected to Giemsa staining (19). Developmental stages of T.equi or B.caballi were identified microscopically. The disease can best be described with a flow chart for both B.caballi and T.equi.



Descriptive model (figure 1 and 2). Every arrow describes a proportion or a transition factor from one population to the next. For simplicity, only the most relevant transitions are mentioned.

Figure 1 Discussion For equine piroplasmosis in general many factors are known, but there are practically no data available for the specific case of B.caballi and T.equi in Dermacentor nuttalli in Mongolia. However in Dermacentor sp. intrastadial infection has been seen and males are known to change host for up to 6 times. Co-feeding of infected and uninfected ticks on a small mammal can cause infection in the naïve ticks without acquisition of the infection by the mammalian host. (Friedhoff, personal communication) This explains transmission by this tick species, although it is a three-host tick (feed on a different host species in every stage) (7) and T.equi was shown not to be transmissible to rodents (5). A simulation model of piroplasmosis in Takhin Tal would give a relatively good estimate of the disease risk and allow predictions on the dynamics of the disease in the coming years. Such a model would require a thorough examination of additional factors involved in the development of the disease:

• Population dynamics of D.nuttalli • Population dynamics of rodents and small mammals • Immune reactions of the horses to piroplasms and ticks affecting the survival rate

of ticks and the antibody titres of the equine hosts.

rodents and small mammals

Susceptible horses S

Latent horses L

Babesia caballi

Non - infected eggs

Infected eggs Surviving infected females

Infected Larvae

Infected Nymphs

Infected Larvae

Infected Nymphs

Larvae

Nymphs

Larvae

Nymphs

Proportion of infective bites

Survival rate of superinfections in females

Non - infected females

Susceptible ticks ST

adult females adult males

Susceptible ticks ST

adult females adult males

Infectious Ticks IT adult males

adult females Infectious Ticks IT adult males

adult females

Superinfected females

Superinfection Superinfection

Transovarially infec ted females

Death

Death

Mortalities due to Piroplasmosis

Cc Infectious healthy horses Ih

Infectious clinical horses Ic

Horse mortalities C

100% infectivity Proportion infected females

Births



Figure 2 A further, more instantaneous approach is the analysis of the serologic data. The samples are grouped according to the age of the horses. Assuming the humoral immunity remains for life after an infection, the seropositive individuals cumulate over time. The age-stratified seroprevalence can be formulated as a function of age: ( ) htetI −−= 1 I= infection rate h= inoculation rate t= mean age of group in days corrected for the time

post infection to develop antibodies This function describes a sigmoid curve, whose slope is dependent on the inoculation rate h.. This value represents the rate of new successful infections with piroplasms and it includes many undetermined factors such as infectivity of horses for ticks, survival rate of the ticks after infection, egg production (only for B.caballi), prevalence in the ticks, transmission efficiency of the ticks, prevalence in the horses. It does not take into account that there are annual fluctuations in the tick population as well as the populations of larvae and nymph hosts, meteorological influences on parasite development, genetic variation for host susceptibility etc. In addition the formula assumes that the inoculation rate is constant over time and between different age groups (homogeneous distribution). This simplifies the actual epidemiological mechanisms dramatically and should therefore be considered as a momentary image only. Previously this model has been applied to bovine babesiosis in Australia and used as a tool to decide whether to imply a parasite control program or not (14). In the case of piroplasmosis in Takhin Tal, the graph helps evaluating the risk of an outbreak and therefore allows decisions on treatment of newly introduced takhis.

rodents and small mammals

Theileria equi

Infectious ticksIT

adult females and males

DeathLarvae

Nymphs

Larvae

Nymphs

Proportion of infective bites

Proportion of successfull infections and biting twice

(!)

Susceptible ticksST

adult males adult females

Susceptible ticksST

adult males adult females

Eggs

Susceptible horses

S

Latent horses

L

Mortalities due to Piroplasmosis

CcInfectious healthy horses

Ih

Infectious clinical horses

Ic

Horse mortalities C

Births



In cattle the risk of infection with babesia was shown to be cyclic over several years (Doherr, personal communication) and therefore introduction of susceptible individuals into an endemic region could be planed at times when the risk of infection is minimal. Since clinical disease in horses is associated with stress, it would be preferable to introduce naïve takhis to Takhin Tal at times of low infection risk. This will rise the proportion of individuals that are infected during their second year in Mongolia (when they are already partially acclimatised and less susceptible to clinical piroplasmosis). To predict the variations of infection risk on long term it would be necessary to develop a simulation model of the disease. This, however, would require further research regarding the presently unknown factors mentioned above. Acknowledgements This project is financed in a large part by the Austrian National Bank (Jubiläumsfond der Österreichischen Nationalbank, Nr. 8977). I would like to thank the Salzburg Zoo, the International Takhi Group and the Department of Wildlife Pathology at the University of Bern for their support. My particular thanks go to Mr. Sukhbaatar, Mr. Tumur, Mr. Badsuur, Mr. Toolbaatar, Mr. Ganbaatar and Mr. Enksaikhaan, without their help this project would have been impossible. References 1. Arthur DR. Part V on the genera Dermacentor, Anocentor, Cosmiomma, Boophilus & Margaropus. Ticks, a

monograph of the Ixodidea. Great Britain: The Syndics of the Cambridge University Press, 1960. 2. Avarzed A, De Waal DT,and Igarashi I . Prevalence of equine piroplasmosis in Central Mongolia. Onderstepoort J

Vet Res 1997; 64(2):141-5. 3. Böse R, Jorgensen WK, Dalgliesh RJ, Friedhoff KT, and de Vos AJ. Current state and future trends in the diagnosis

of babesiosis. Vet Parasitol 1995; 57(1-3):61-74. 4. Büs cher G, and Tangus J. Quantitative studies on Theileria parva in the salivary glands of Rhipicephalus

appendiculatus adults: search for conditions for high infections. Int J Parasitol 1986; 16:121-9. 5. Frerichs WM and Holbrook AA. Feeding mechanisms of Babesia equi. J Protozool 1974; 21(5):707-9. 6. Friedhoff KT and Smith RD. Transmission of Babesia by Ticks. Ristic M KJ Ed. Babesiosis. Academic Press Inc.,

1981: 267 -321. 7. Anastos G Editor. Translation of "Fauna of USSR Arachnida, Vol IV, No.2, Ixodid Ticks" by B.I. Pomerantzev.

Washington 6, DC: American Institute of Biological Sciences, 1959. (Pavlovski Editor in Chief; ). 8. Hailat NQ, Lafi SQ, al-Darraji AM and al-Ani FK. Equine babesiosis associated with strenuous exercise: clinical and

pathological studies in Jordan. Vet Parasitol 1997; 69(1-2):1-8. 9. Heuchert CM, de Giulli V Jr, de Athaide DF, Böse R and Friedhoff KT. Seroepidemiologic studies on Babesia equi

and Babesia caballi infections in Brazil. Vet Parasitol 1999; 85(1):1-11. 10. Holbrook AA, Anthony DWand Johnson AJ. Observations on the development of Babesia caballi (Nuttall) in the

tropical horse tick Dermacentor nitens Neumann. J Protozool 1968; 15(2):391-6. 11. Ikadai H, Osorio CR and Xuan X . Detection of Babesia caballi infection by enzyme-linked immunosorben t assay

using recombinant 48-kDa merozoite rhoptry protein. Int J Parasitol 2000; 30(5):633-5. 12. James MA. Immunology of Babesiosis. Ristic M Editor. Babesiosis of Domestic Animals and Man. CRC Press, Inc.,

1988: 119 -30. 13. Klinckmann G. Sporozoitenstabilate vo n Babesia equi aus Hyalomma anatolicum anatolicum und Rhipicephalus

turanicus Hannover: Tierärztliche Hochschule Hannover, 1981. 14. Mahoney DF and Ross DR. Epizootiological factors in the control of bovine babesiosis. Aust Vet J 1972; 48(5):292-

8. 15. Mehlhorn H and Schein E. Redescription of Babesia equi Laveran, 1901 as Theileria equi . Parasitol Res 1998;

84(6):467 -75. 16. Oladosu LA and Olufemi BE. Haematology of experimental babesiosis and ehrlichiosis in steroid

immunosuppressed horses. Zentralbl Veterinärmed [B] 1992; 39(5):345-52. 17. Purnell RE. Babesiosis in Various Hosts. Ristic M, Kreier JP Editors. Babesiosis. London: Academic Press, Inc.Ltd.,

1981: 25-64. 18. Schein E. Equine Babesiosis. Ristic M Editor. Babesiosis of Domestic Animals and Man. CRC Press, Inc., 1988:

197-208. 19. Sigrist B. Übertragung von Babesia equi durch Hyalomma anatolicum anatolicum und Rhipicephalus turanicus

Hannover: Tierärztliche Hochschule Hannover, 1983. 20. Walzer C, Baumgartner R, Robert N, Suchebaatar Z, Bajanlamgaa N. Medical considerations in the reintroduction of

the Przewalski horse (Equus przewalskii) to the Dsungarian Gobi, Mongolia. Zwart P, Proceedings Editor. Proceedings of the annual conference. EAZWV, 2000: 147-50.



European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4th scientific meeting, joint with the annual meeting of the European Wildlife Disease Association (EWDA) May 8-12, 2002, Heidelberg, Germany

VETERINARY SUPERVISION OF LYNX TRANSLOCATION WITHIN THE SWISS ALPS

M.-P. RYSER-DEGIORGIS 1,2, H. LUTZ3, K. BAUER3, H. SAGER4, A. RYSER2, F. ZIMMERMANN2, CH. BREITENMOSER-WÜRSTEN2 and U. BREITENMOSER2

Affiliation: 1. Centre for Fish- and Wildlife Health, Institute of Animal Pathology, University of Bern 2. KORA (co-ordinated projects for the conservation and management of carnivores in Switzerland) 3. Clinical Laboratory, Dept. of Internal Veterinary Medicine, University of Zurich 4. Institute of Parasitology, University of Bern

Abstract In 2001, six free-ranging Eurasian lynx were caught in the north-western Swiss Alps and translocated to north-eastern Switzerland. Veterinary supervision of lynx translocation was important not only to improve the chances of success of this reintroduction project, but also for scientific documentation of the work and for reasons of animal welfare. All lynx caught in the frame of the project were clinically healthy. To prevent problems associated with the stress of the translocation procedure, all animals were systematically treated with anti-parasitic and antibiotic drugs before and after quarantine. Quarantine did not only allow the observation of the animals, but also blood and faeces analysis, assessment of laboratory results, and the detailed organisation of the release procedure. The project has been successful so far, and translocation of more animals is planned for the winter 2002/03. Zusammenfassung In 2001 wurden sechs freilebenden Eurasische Luchse in den Schweizer Nordwestalpen gefangen und in die Nordostschweiz umgesiedelt. Die veterinärmedizinische Betreuung der umgesiedelten Luchse war nicht nur wichtig, um die Chancen eines Erfolges zu vergrössern, sondern auch aus wissenschaftlichen und tierschützerischen Gründen. Alle Luchse, die im Rahmen des Projektes gefangen wurden, waren klinisch gesund. Trotzdem wurden die Tiere systematisch mit Antiparasitica und einem Antibiotikum behandelt, dies vor und nach der Quarantäne, um den Tieren optimale Startbedingungen zu geben. Die Quarantäne erlaubte nicht nur die Beobachtung der Tiere, sondern auch die Durchführung der Blut- und Kotuntersuchungen, die Beurteilung der Laborresultate, sowie eine sorgfältige Organisation der Freilassung. Das Projekt war bisher erfolgreich; weitere Umsiedlungen sind für den Winter 2002/03 geplant. Résumé Au cours de l’année 2001, six lynx eurasien en liberté ont été capturés dans les Alpes suisses du nord-ouest et déplacés dans le nord-est de la Suisse. La supervision vétérinaire des translocations était importante non seulement afin d’augmenter les chances de réussite de ce projet de réintroduction, mais également pour des raisons scientifiques et pour le bien des lynx concernés. Tous les lynx capturés dans le cadre du projet étaient en bonne santé. Les animaux ont quand même systématiquement reçu un traitement antiparasitaire et antibiotique, ceci avant et après la quarantaine, afin d’éviter tout problème lié à une infection. La quarantaine a permis non seulement d’observer les animaux, mais surtout d’effectuer les analyses de sang et de crottes, d’apprécier les résultats de laboratoire, et d’organiser en détails la procédure des lâchers. Jusqu’à présent, le projet s’est déroulé avec succès et d’autres translocations sont prévues pour l’hiver 2002/03. Key words: Eurasian lynx, Lynx lynx, parasites, quarantine, reintroduction, Switzerland, translocation, viral

diseases



Introduction Lynx vanished from Switzerland during the 19th century as a result of persecution, loss of habitat and natural prey base. Since 1962 the species has been protected by law, and in the 1970s, some 25 lynx were reintroduced. Two populations arose from these releases: one in the Jura Mountains and one in the north-western Swiss Alps (1,10). However, the population in the Alps expands very slowly. Although in the north-western Swiss Alps, lynx abundance was increased from 1996–2000 (2), barriers such as high mountain ridges or densely settled valleys with highways, rivers and lakes fragment the Alps and hinder the emigration of lynx. In order to help the lynx population to spread into new areas and to reduce at the same time local high densities leading to conflicts with sheep breeders and hunters, the Swiss Agency of Environment, Forest and Landscape implemented the Swiss Lynx Concept (3), a management plan for the lynx in Switzerland. An important consequence of the concept was the agreement between the federal authorities and several cantons to translocate 8–12 lynx (six in the first year) from the north-western Alps (cantons of Bern, Fribourg and Vaud) to eastern Switzerland (cantons of St. Gallen and Zurich). The foundation of a new population nucleus in the eastern Swiss Alps will help to join the isolated lynx populations in the north-western Alps and in the triangle of Austria, Slovenia and Italy, as it is recommended in the pan-Alpine conservation strategy for the lynx (11). The LUNO Project (Luchsumsiedlung Nordostschweiz, Engl: lynx translocation north-eastern Switzerland) was thoroughly planned, for scientific and political reasons, following e.g. the IUCN recommendations for reintroduction (9). Several cases of mange in lynx in the north-eastern Swiss Alps in 1999 (13) caused uncertainty regarding the health status of the source population, and therefore the veterinary supervision of the translocations became of particular importance. Planning of the veterinary supervision The success of wildlife translocations, i.e. the transport of wild animals from an area to another one, depends on many factors. Sanitary aspects (including veterinary public health) play an important role, as translocations represent a potential danger for disease transmission. This danger can be summarised in two main scenarios: (i) Introduction of a disease into the new area through the translocated animals (transmission to wildlife and/or to domestic animals), and (ii) transmission of a local disease to the translocated animals in the new area (from wildlife and/or from domestic animals). In addition, translocated animals risk injury before or during the transport to the new area; an infected wound could cause septicaemia and finally lead to death. Stress as a result of the capture, transport and change of territory is a predisposing factor for the outbreak of a disease (appearance of clinical symptoms) in infected animals. Although this last scenario does not necessarily represent a danger for other animals, it is a considerable risk for the individuals translocated and hence for the success of the whole project. In the frame of a translocation project, three points must be clarified during the planning part: a. The susceptibility to disease and the eventual role as a reservoir of the species to be

translocated; b. The presence of diseases and other health problems in the source population; c. The situation of disease in the new area. In the LUNO Project, the three points were evaluated as follows: a. Causes of mortality and susceptibility of lynx (Lynx sp.) to disease: First, the present knowledge on disease susceptibility and causes of death in lynx was reviewed (12). b. Health status of the lynx population in the north-western Swiss Alps : The lynx population in the north-western Swiss Alps was considered to be healthy. The only disease observed in lynx in this area during the past 13 years was mange: five cases were observed for the first time in 1999 (13). A retrospective pathological study (16) revealed that only 11 out of 53 (20%) animals necropsied from 1987-1999 died of an infection. Six lynx were infected with parasites – five of them had



mange, and one suffered of a strong infestation with Toxocara worms – two animals died of a bacterial infection, and in two further cases the aetiology of the disease remained unknown. The bacterial infections were not caused by specific agents, but were in some cases a consequence of infected wounds. There was no indication of any viral disease. Switzerland is officially free of rabies since April 1999 (18). c. Presence of infectious agents in north-eastern Switzerland which might infect the released lynx: The only disease for which the lynx is susceptible and which is found in north-eastern Switzerland but not in the north-western Alps is Borna disease. But there is very little chance that a lynx could be infected by the Borna virus: clinical cases in domestic cats are rare in Switzerland (8), and in Sweden, were the disease is common in endemic areas, only one single case has been described in a lynx (4). Other infectious agents which could possibly infect lynx are widespread (e.g. cat viruses) but they do not represent a higher risk of infection in north-eastern Switzerland than in the north-western Alps. We concluded that (i) the risk that the lynx would introduce new infectious agents from the north-western Alps to the north-eastern area of Switzerland was very low and (ii) the risk that the released lynx would get an infectious disease which would be new for them was minimal. On the base of this knowledge, a veterinary protocol was set up. Our goals were to release absolutely healthy animals and to gather as many data as possible for scientific documentation. Realisation of the project I. Capture The plan was to capture 8-12 lynx over two years, and at least half of them had to be females. For biological reasons, the capture took place between February and April: Young lynx are born in May-June and stay with their mother until the next mating season (February-March). To avoid separation of dams from their kittens, captures could take place only during the mating season. Lynx were caught by means of three capture systems. Foot snares or an remote controlled teleinjection system (14) were set at a kill over night and monitored by means of a transmitter by the team of Swiss lynx project. Or cage traps were set on paths in lynx territories and monitored by means of a transmitter by local game wardens. During the season 2001, three males and three females were caught and translocated. Two more males caught in traps were released at the site of capture. II. Veterinary supervision Anaesthesia The animals were immobilised with medetomidin-ketamin, and atipamezol was used for reversal. Anaesthesia was monitored by assessment of respiratory and heart rate, colour of the mucous membranes, capillary refill time, eye reflexes and body temperature. All animals (except those, which were released) were examined and marked in a protected place (garage, alpine hut, stable) to reduce the risk of hypothermia. Physical exam All animals caught in a box trap had fresh wounds: usually quite large superficial abrasions on the head with contusions in the ocular region and reddened conjunctivae, damages of claws (splintering, bleedings, ruptures) especially of the forepaws. Two animals had wounds on the balls and three had broken teeth. Ectoparasites were found on several lynx: two had ear mites and four had ticks. In fections with Otodectes cynotis and ticks have already been described in Eurasian lynx (5,16). However, the presence of ear mites in 2 out of 8 lynx indicates that free-ranging lynx harbour these parasites more frequently than previously assumed.



Further findings were: 1x unilateral severe cataract, 1x monorchidia (not translocated), 1x old scar consisting in a deep splitting of the upper lip, 3x small old scars on the ears, some canini of two older males were quite used, 1x ulceration on the palate (not translocated). Treatments and marking procedure Each animal was marked with a radio-collar and with a microchip. All lynx received a preventive treatment consisting in a long acting antibiotic (amoxycillin) and anti-parasitic drugs (Doramectin and Praziquantel). Treatment doses were chosen according to the conventional recommendations for felines. Wounds were disinfected, in case of ear mites infestation, ears were cleaned and treated locally with an antibiotic ointment. If ocular inflammation was present, an antibiotic ointment was put in the eyes. Quarantine Animals to be translocated were put in a so-called quarantine enclosure for three purposes: (I) To obtain sufficient time to catch a lynx pair and to organise its release (since lynx were translocated during the mating season, it was considered as important to release animals in couples). (ii) « Break » in order to prevent a return to the original home range. (iii) To be able to observe the animals in order to make sure that all animals were in a good health The animals were transported in flat transport boxes covered with a grid to allow anaesthesia monitoring. The car used for the transport to the quarantine station had to be heated as much as possible to keep a stable body temperature. Anaesthesia was prolonged until arrival and antagonised in the quarantine enclosure. Enclosures, animal care and hygiene Animals were housed alone in four 18.5 m2 enclosures close to each other in a wildlife station not accessible for the public. To reduce the risk of injuries and to prevent lynx to be stressed by the surroundings (especially humans), all lateral grids were covered with wooden panels. Between the enclosures, small sliding doors that could be opened from outside gave the possibility to push an animal to the next enclosure or to join two enclosures. In each enclosure there was a platform with branches between the platform and the ground, giving the animals the possibility to climb and hide. In an edge of the enclosure, there was also a large transport box containing some hay, that might have been used as a “den”. Water was continuously running from a tap and also offered in a separated basket. The ground was covered with sand. Discrete observation of lynx was possible through small holes in the main door. Two animal keepers were present 24h a day to take care of the captive animals. The enclosures were cleaned twice a week (the lynx was first pushed into the next enclosure, then rests of food and faeces were removed, and the basket was filled with fresh water). After release, the whole enclosure was disinfected. Feeding Exclusively roe deer meat (exception: one hare) was fed to the animals. Keepers put a piece of roe deer (mainly road kills) once a day on a wood panel placed on the ground close to the main door. All lynx ate for the first time in the night from the 3 rd to the 4th day in captivity (one exception: 2nd to 3rd day) and ate then about 2 kg meat per day. A lynx consumed about one roe deer per week, leaving just bones and pelt, and three animals used the hay of the box to cover their “prey”. It is known from studies about free-ranging lynx that an adult consumes about one ungulate per week and hides its prey with leaves and grass until it is completely consumed (7). Duration of quarantine and behaviour The lynx spent an average of 19 days in captivity (13-26 days). Females (younger than males) were shier and used to hide several days before climbing on the platform. They sprang sometimes against the walls, trying to escape. Males seemed to be calmer but after release we noticed damages in the enclosure: platform and wood panels had been scratched and bitten, indicating that males had tried to break out as well.



Blood sampling and analysis Blood samples were taken with vacutainers from the Vena cephalica for haematology blood chemistry, serology, genetic and research (cell culture). The samples were sent by train or brought directly to the laboratory (Clinical laboratory, University of Zurich) within the 12h following sampling Blood analysis Blood results (haematology, blood chemistry) were compared with reference data for domestic cats. Erythrocytes: Results of all eight lynx were within the reference area but PCV, haemoglobin and RBC were rather at the upper reference limit. Leukocytes and leukocyte differential count: There was big variation. In most lynx there was indication of stress, more or less evident depending on the capture technique. Blood chemistry: most medians were within the reference ranges, some values being rather at the upper respectively lower reference limit. None of the animals had values indicating any kind of serious disorder or infection. Serology All eight animals were tested for selected infectious diseases using established procedures (6). Antigens: feline leukaemia virus (FeLV). Antibodies: feline coronavirus (FCoV), Feline immunodeficiency virus (FIV), feline parvovirus (FPV), feline calicivirus (FCV), feline herpesvirus (FHV), Bartonella henselae, Ehrlichia sp. Positive results were found only for FPV (4 lynx) and FCV (1 lynx). Parvovirus infections have been described in free-ranging lynx from the Jura Mountains (16,17) but not from the north-western Swiss Alps. However, these results indicate that the virus is present in this population as well. Analysis of a larger number of blood samples collected by the Swiss Lynx Project during capture and marking procedures are on-going to determine the seroprevalence in both Swiss lynx populations. Parasitology For each lynx, the first faeces excreted in the enclosure was collected and analysed at the Institute of Parasitology (University of Bern). Toxocara sp. – which is known to be the most common parasite in free -ranging lynx (12) – was found in the faeces of all six lynx, five animals were also infected with Capillaria sp., and in one case Cystoisospora sp. was found as well. To check if the anti-parasitic treatment had been effective, a second analysis was performed for four lynx just before release. The results indicated that the treatment had been only partially effective. III. Release Second physical exam During captivity, all animals regularly ate and excreted normal faeces. All wounds caused in the box trap healed without problem. For release, all animals were again immobilised with medetomidine-ketamine. Since the lynx were very stressed by the presence of humans, the best procedure was to blind them with a light before sunrise and shoot them with a blow pipe. The animals were then brought inside, weighed and examined again. It was important to plan enough time for all the procedure. From our arrival at the quarantine station to the start of the drive to the release site, we needed 2-5 hours for 1 -2 animals. At the time of release, all animals had the same weight (+/- 1kg) as when they were caught. Four lynx had erosions of the lips and paws, possibly due to the sand but also because they bit wood objects and tried to escape. For the last animal brought to the quarantine station, the ground of the enclosure was covered with peat instead of sand and the lesions were smaller but still present. One male, which had caused more damages in the enclosure, had broken one of its teeth.



Treatments Doramectin was injected again into all animals: first because some intestinal worm eggs were still present in the faeces, second because it was particularly important to be sure that lynx were not infected with mange mites. The treatment with amoxycillin was repeated as well, in case the lynx would get injuries during the transport. Transport to the release site The transport boxes were the ones standing in the enclosure as “den” boxes. Their size makes it possible for an adult lynx to sit inside. A sliding door allows anaesthesia monitoring and release. As soon as awake, the animals seemed to consider the breathing holes of the box as possible exits and scratched them, trying to escape. It was therefore very important to have boxes with many small holes instead of a few big ones. Boxes were transported in a minibus. As long as the animals were asleep, it was heated as much as possible to avoid hypothermia, and as soon as they were awake, the heating was turned off. Anaesthetics were not antagonised (one exception) so that the animals slowly awaked during transport. The anaesthesia lasted 2h +/- 15 min. First, the animals just turned themselves in the boxes. Then there was some excitation phase during which they were quite nervous, scratched the walls and moved in the box a lot (few minutes – 1h). Finally, the animals became quiet again but more and more attentive and reacted (growling) when humans came close to the box. Once they were awake, it proved to be particularly important not to speak since human voices made them very nervous. IV. Conclusion and outlook The first four lynx were released in couples in March 2001, and the two last animals were released separately in surrounding areas in April 2001. All animals bounced out of the boxes as soon as the door was open. At that time they had completely recovered from the anaesthesia, and they ran away to disappear in the forest. Since then, the six lynx have been closely followed by radio tracking. So far, all animals showed expected behaviour concerning habitat use and prey choice (15). Within the first few months, they established a typical land tenure system. The LUNO project has been successful so far. In the winter 2002/03, another three lynx will be translocated to north-eastern Switzerland. Acknowledgements We acknowledge the Swiss Office of Environment, Forest and Landscape and the cantonal hunting inspectors who initiated the project. Many thanks go also to all people and institutions who helped in any way to the realisation of the project, in particularly: the canton of Bern for transforming enclosures of the cantonal wildlife station into quarantine enclosures; Dr. M. Janovsky (University of Bern) for his help in the fields; Prof. Dr. B. Gottstein (University of Bern) for his logistic, and Mrs. Ursula Mäusli for her technical support; Prof. Dr. B. Spiess (University of Zurich) for assessment of eye disorders; Dr. G. Neiger-Aeschbacher (Royal Veterinary College, University of London, UK) for uncomplicated support and advises for the establishment of the anaesthesia protocol; and Dr. D. Grobler (Kruger Park, South Africa) for sharing his experience with transport and mange treatment in large wild cats. References 1. Breitenmoser U, Breitenmoser-Würsten Ch, and Capt S. Re-introduction and present status of the lynx in

Switzerland. Hystrix 1998; 10: 17-30. 2. Breitenmoser-Würsten Ch, Zimmermann F, Ryser A, Capt S, Laass J, Siegenthaler A, and Breitenmoser U.

Untersuchungen zur Luchspopulation in den Nordwestalpen der Schweiz 1997–2000. KORA Bericht 2001; 9: 88 pp. 3. BUWAL. Konzept Luchs Schweiz. Berne, 28 August 2000. 4. Degiorgis M-P, Berg A-L, Hård af Segerstad C, Mörner T, Johansson M, and Berg M. Borna disease in a free-

ranging lynx (Lynx lynx). J Clin Microbiol 2000; 38: 3087-91. 5. Degiorgis M-P, Hård af Segerstad C, Christensson B, and Mörner T. Otodectic Otoacariasis in free-ranging

Eurasian lynx in Sweden. J Wildl Dis 2001; 37: 626-9.



6. Hofmann-Lehmann R, Fehr D, Grob M, Elgizoli M, Packer C, Martenson JS, O'Brien S, and Lutz H. Prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in East Africa. Clin Diagn Lab Immunol 1996; 3: 554-562.

7. Jobin A, Molinari P, and Breitenmoser U. Prey spectrum, prey preference and consumption rates of Eurasian lynx in the Swiss Jura Mountains. Acta Theriol 2000; 45: 243-252.

8. Melzer K. Untersuchungen zur Aetiologie von ZNS-Erkrankungen bei der Katze in der Schweiz mit besonderer Berücksichtigung der Borna Disease Virus Infektion. Diss Univ Zürich 1999; 70 pp.

9. IUCN. Guidelines for re-introductions. Gland and Cambridge, IUCN 1998; 10 pp. 10. Molinari-Jobin A, Zimmermann F, Breitenmoser-Würsten Ch, Capt S, and Breitenmoser U. Present status and

distribution of the lynx in the Swiss Alps. Hystrix 2001; 12: 21-31. 11. Molinari-Jobin A, Molinari P, Breitenmoser-Würsten Ch, Wölfl M, Stanisa C, Fasel M, Stahl P, Vandel JM, Rotelli L,

Kaczensky P, Huber T, and Breitenmoser U. Pan-Alpine conservation strategy for the lynx. Council of Europe, Strasbourg 2002; 19pp. (in press)

12. Ryser-Degiorgis M-P. Todesursachen und Krankheiten beim Luchs – eine Übersicht. KORA Bericht 2001; Nr. 8, 18 pp.

13. Ryser-Degiorgis M-P, Ryser A, Bacciarini L, Angst C, Gottstein B, Janovsky M, and Breitenmoser U. Notoedric and sarcoptic mange in free-ranging lynx from Switzerland. J Wildl Dis 2002; 38: in press.

14. Ryser A, Scholl M, et al. A remote controlled teleinjection system for the stress-free capture of larger mammals. In prep.

15. Ryser A, von Wattenwyl K, et al. Land tenure system and feeding behaviour of translocated lynx in the Swiss Alps. In prep.

16. Schmidt-Posthaus H, Breitenmoser-Würsten C, Posthaus H, Bacciarini L, and Breitenmoser U. Causes of mortality in reintroduced Eurasian lynx in Switzerland. J Wildl Dis 2002; 38: 84-92.

17. Stahl P, and Vandel J-M. Mortalité et captures de lynx (Lynx lynx) en France (1974-1998). Mammalia 1999; 63 : 49-59.

18. Zanoni R, Kappeler A, Müller UM, Müller Ch, Wandeler AI, and Breitenmoser U. Tollwutfreiheit der Schweiz nach 30 Jahren Fuchstollwut. Schweiz Arch Tierheilk 2000; 142: 423-429.



European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4 th scientific meeting, joint with the annual meeting of the European Wildlife Disease Association (EWDA) May 8-12, 2002, Heidelberg, Germany.

THE ROLE OF ZOO VETERINARIANS IN CONSERVATION PROJECTS - 6 YEARS IN TAPIR CONSERVATION.

S. M. HERNANDEZ-DIVERS Affiliation: Small Animal Department, College of Veterinary Medicine, University of Georgia, Athens, GA 30605. [email protected] Abstract This presentation will outline the need for veterinarians to become more involved in conservation projects. It will aim to explain the history of veterinary involvement in field projects, their suitability and their limitations for this task. The skills that zoo veterinarians have acquired will be discussed as to how they relate to conservation projects. The author’s involvement in the Baird’s Tapir Project will be discussed as an example of such involvement. Zusammenfassung In dieser Presentation wird der Nutzen von Veterinären bei Konservierungsprojecten besprochen. Es wird versucht die Geschichte der veterinären Beteiligung an Feldprojecten, ihr Nutzen und die Einschränkungen für diese Aufgabe dar zu stellen. Die Fähigkeiten, die Zooveterinäre erlernt haben werden besprochen, sowie ihr Bezug zu den Konservierungsprojecten. Die Beteiligung des Autors an Bairds Tapir Project wird als Beispiel angeführt. Résumé Cet exposé cherche à souligner l'importance de l'implication des vétérinaires dans les projets de conservation. Nous expliquerons l'histoire de l'engagement vétérinaire dans les projets de terrain, ainsi que leurs compétences et leurs limites dans ce domaine. Le savoir faire que les vétérinaires de zoo ont acquis par rapport aux projets de conservation sera commenté. L'implication de l'auteur dans le projet du Baird's tapir sera citée en exemple. Key words: Tapirus bairdii, conservation, in situ, zoo, veterinarians, free-ranging Introduction The history of veterinary involvement in in situ projects Historically there has been friction between the two professional groups that should be considered essential for conservation of wildlife: wildlife managers (biologists, ecologists etc) and veterinarians. It could be that either group felt threatened by the other’s level of education, or that in the past veterinarians knew little about the natural history of non-domestic species or that biologists have got along for decades without the apparent need for a veterinarian to help with their projects… I can only speculate on the reasons for why this friction developed; however, it is time to overcome these issues and understand the value in both groups coming together to work as one. Although the general public views veterinarians as “dog/cat doctors”, our profession has grown to be extremely diverse. In the USA a veterinarian can work with livestock in production medicine, pet animals, work for the state on free-ranging populations, conduct molecular research or work for private



industry. Obviously, it is the type of training we receive that allows us to become qualified for such diverse careers. Who else, if not veterinarians, are experts in animal anatomy, physiology and disease? The tools we receive during our veterinary training give us the foundation needed to tackle any animal disease issue. Teams made of ecologists, wildlife managers and veterinarians can collaborate to study animal populations and research the best ways to conserve them. The need for veterinary involvement The unfortunate truth about free-ranging populations is that they are diminishing at alarming rates. Human population growth, leading to a decrease in available habitat for wildlife, coupled with increases in pollution, overuse of natural resources etc are causing the disappearance of flora and fauna on a global scale. There are many steps to the conservation of a population of animals. The first step and the most basic is understanding what makes that population flourish. Unfortunately, for a large number of species, we cannot answer those simple questions. Knowing the basic habitat and nutritional requirements, the reproductive behaviour and life span of an animal can often take long years of extensive, involved research. Secondly, we must appreciate what threatens the stability or growth of a population. Sometimes disease can be an important factor. Endemic disease can be a normal component of population dynamics. However, under unusual pressures (overcrowding, decrease in available resources, weather changes, natural disasters etc) disease can have a significant negative affect on populations. New diseases can also be introduced, through human or livestock vectors or other displaced free-ranging populations. The introduction of a new disease into a naïve population can prove disastrous. Understanding the “normal” cycles of disease can help wildlife managers work through risk assessment scenarios to develop prevention plans. More importantly they can develop management plans in the event of a disaster. As available habitat decreases, free-ranging populations are forced into smaller areas. Often, populations are restricted to “protected” areas. These populations are isolated from other groups, which can result in a lack of genetic diversity. Essentially, the future of animal populations will be restricted to islands of semi-free-ranging populations surrounded by livestock and human activity. Wildlife managers will be faced with the challenges that those conditions will create. What can a veterinarian do? Veterinarians can play a significant role in the conservation of animal species in a variety of ways: 1) Little information is known about some species. Veterinarians can aid in the collection of basic

biological and ecological data by: a) Anaesthetising/immobilising animals for data collection, translocation, reintroduction and/or

radiocollar attachment while ensuring the safety the animal and involved personnel. b) Perform procedures such as collection of blood, biopsies, coprological examinations, c) Perform more complicated procedures such as radiotransmitter implantation.

2) Veterinarians are trained to understand both the virtues and the limitations of diagnostic tests (cytology, serology, PCR etc).

3) Epidemiology is an important part of the veterinary curriculum. Veterinarians are trained to investigate the prevalence and significance of disease in populations of animals.

4) Through the above, veterinarians can then begin to asses the role of livestock and human populations on the disease status of a given wildlife population.

5) In addition, veterinarians can provide valuable information on reproduction, reproductive behaviour and reproductive physiology.

6) A veterinarian is trained to understand gastrointestinal physiology and nutritional requirements. They can be an integral part of a nutrition study.

7) Finally, veterinarians can be a significant part of a team that determines the minimum requirements a species will need to maintain a steady population.



Limitations of veterinarians-what we are not Veterinarians by nature are not ecologists. The veterinary curriculum offers little in the way of training about animal populations, animal predator-prey relationships, an animal’s interaction with its environment, natural history of non-domestic animals etc etc…It is the responsibility of a veterinarian interested in working with wildlife to learn these aspects on their own. As part of a traditional veterinary curriculum, a veterinarian is essentially a mammalian biologist. In other words, most of the anatomy and physiology learned during veterinary school is based on the mammalian model. Individuals hoping to pursue careers in free-ranging animals will need to learn basic reptilian and avian anatomy and physiology on their own. In order for the veterinarian to pursue a career in conservation, he or she will need some degree of further training. Although it is not essential, there are now several programs, which will formally supplement the traditional veterinary medicine curriculum through internships and residencies in the fields of zoo, wildlife and exotic pet medicine. Alternatively, there are many resources (textbooks, workshops, courses etc) that can help any veterinarian supplement their basic knowledge to deal with wildlife issues. Why zoo veterinarians? Zoo veterinarians, by definition, have typically gone above and beyond the traditional curriculum to teach themselves the natural history, anatomy and physiology of non-domestic species. They are capable of adapting methods utilised in domestic animals and applying them to a variety of animals. They are well versed in chemical restraint and immobilisation. They have been faced with understanding the limitations of diagnostic test designed for domestic species. As part of their job, they are faced with issues such as reproduction control on a daily basis. Most zoo veterinarians play an active role in the nutrition (and nutritional problems) for a variety of species. The zoo veterinarian is often faced with looking beyond the individual animal and treating groups of animals. Why would a zoo veterinarian want to become involved in an in situ project? Zoos have long claimed to be important hubs in the wheels of conservation. The public demands a strong justification to keep exotic animals in captive environments. As zoo advocates, we cite that zoos educate. Although it is true that zoos are an important educational tool, I propose we go further. Zoos can, and should be, major centers of research. Ideally, every zoo would be more involved in “in situ” projects. Information sharing between personnel that work with an animal in captivity and those that work on free-ranging populations is invaluable. Not only can we best learn how to adequately manage animals in captivity by learning more about their free-ranging counterparts, but there are often pieces of the natural history of an animal that cannot be studied adequately in the wild, but can be researched in captive settings. In conclusion, veterinarians should become involved in conservation projects as often as possible. Wildlife managers can utilise our skills to attain their goals. We have much to offer and much to learn. How to become involved in field projects There are many ways for zoo veterinarians to become involved in field projects. Species, which belong to SSP programs, are typically linked to field projects directly. The AZA has information on field projects. The local university is likely to have a group involved in animal investigation such as the ecology department. Large, non-governmental institutions typically support field projects. Although most veterinary work in field projects is voluntary, costs should be covered by grants. State or federal agencies typically employ veterinarians to aid in the management of their local wildlife populations. National parks or reserves may employ an individual to co-ordinate projects, which can provide information on existing research. Zoological parks offer the infrastructure to initiate and develop research. Zoos should be encouraged to devote their financial and personnel resources to support field research.



The Baird’s Tapir Project The Baird’s tapir is currently listed as Vulnerable as established by the 1996 IUCN Red List of Threatened Animals. Despite its general category of vulnerable, this species of tapir is likely to be considered endangered with extinction in most countries (4). Historically, this species ranged from southeastern Mexico, from the northern portion of Colombia to the Gulf of Guayaquil in Ecuador (3). Today this species occurs west of the Andes in Colombia and Ecuador (2). Its confirmed current range in Central America is limited to areas sparsely populated by humans and isolated populations in protected areas of Mexico, Belize, Honduras, Costa Rica, Panama and possibly Nicaragua. It is considered extinct in El Salvador (4). As with most large, solitary mammals, tapir populations are normally found in low densities. The primary pressures on their numbers are habitat loss and excessive hunting. Due to the tapir’s low reproductive potential and slow growth, even minimal hunting has proven to significantly decrease their populations (1). Consequentially, these smaller populations become even more susceptible to extinction from natural disasters and disease epidemics. In Costa Rica, the primary threat to the Baird’s tapir is deforestation due to logging and farming practices. It is estimated that 80% of Costa Rica has been deforested (4). In Corcovado National Park, gold mining also poses an important threat to their habitat (4,5). Without reliable information on the basic ecological requirements of tapirs (habitat use, home range, demographics, etc), it will be impossible to develop an effective strategy for sustaining free-ranging populations. Until recently, researchers have relied on indirect methods such as track counts, faecal examination and transect counts to estimate population densities, habitat use and diet. These methods, though useful, are limited in the scope of data they can provide. Until 1995, only two previous studies had utilised direct methods to gather such data in tapirs (6-9). During the last few years, more tapir researchers have elected to employ radio telemetry to monitor the movements of their study animals. As a result, there has arisen a need for an effective protocol for the immobilisation of free-ranging tapirs that is safe for the animals as well as the researchers. Since 1994, the authors have studied the ecology and health of a small population of Baird’s tapirs in Corcovado National Park, Costa Rica. The project aimed to define basic ecological details such as home range, habitat use, activity patterns, diet, disease susceptibility and reproduction. The veterinary aspect of this project included immobilisation for application of radiocollars and collection and interpretation of biological data for a health assessment study. From 1995-2000, 19 individuals were captured at least once to radiocollar them. A total of 32 immobilisation took place. During that time morphometric measurements, complete cell counts, serum biochemistry panels, mineral analysis, ectoparasite collection, skin biopsies and ultrasound examination of some females were performed. Almost as important as the scientific data obtained during this project, a large network of other tapir researchers and other veterinarians working with the Tapirus genus was established. In 1999, a small group of researchers focusing on tapirs met at the Wildlife Management for Amazonia Conference. There the plans for a tapir symposium began. This symposium was realised Nov. 3-7, 2001 in San Jose, Costa Rica. A significant component of this conference was the level of zoo participation. Ten years ago there was little or no collaboration between zoos and tapir field researchers. Today, modern zoos are focusing more on their primary mission of conservation rather than just exhibition. It is the responsibility of these zoos to use their animals as ambassadors for their wild counterparts. A good example of the modern zoos' new commitment to conservation was the support they gave to the tapir symposium. The budget for the symposium was just over $50,000, most of which went to funding people to attend the conference. Over 80% of this budget was covered by donations from four major zoos (Houston, Los Angeles, Disney and San Diego). The Directors of two of these contributory zoos, Rick Barongi from the Houston Zoo and Manuel Mollinedo from the Los Angeles Zoo, attended the symposium. There were 120 participants, representing 22 countries. Participants included field researchers, husbandry and captive management specialists, non-governmental organisation representatives, university representatives and other key players in the development and implementation of tapir conservation programs.



Specific topics of the paper sessions were Ecological Studies, Population Management, Husbandry/Education, Veterinary Issues/Diseases and Tapir Bio-Politics. In all, 48 papers and 9 posters were presented. All of these presentations and posters provided the audience with a very complete overview of current tapir research (in-situ and ex-situ), conservation, education, veterinary, husbandry and management issues. The keynote speakers were Richard Bodmer (Kent University, England), Daniel Janzen (University of Pennsylvania, United States) and, William Konstant (Conservation International, United States). Special evening presentations were conducted by Wally Van Sickle (Idea Wild, United States) and Matthew Colbert (Department of Geological Sciences, University of Texas, United States. Four workshops were held to discuss more specific topics related to tapir research and conservation. Wally Van Sickle conducted an amazing workshop on fundraising and exchanged ideas with participants on how to acquire funds to sustain their projects for the long term. Patty Peters (Columbus Zoo, United States) and Diane Ledder (Disney, United States) conducted a Marketing and Media Affairs workshop and shared their experience on how to get the conservation message out to the general public. Pati Medici from Brazil conducted two other workshops. The first one, the IUCN/SSC Tapir Specialist Group workshop, outlined the next steps needed to take in order to make the IUCN/Tapir Specialist Group more active in terms of tapir conservation. The species co-ordinators, Emilio Constantino from Colombia (Mountain Tapir Co-ordinator), Denis Alexander Torres from Venezuela (Lowland Tapir Co-ordinator), Eduardo Naranjo from Mexico (Baird's Tapir Co-ordinator) and, Nico van Strien from The Netherlands (Malay Tapir Co-ordinator) were introduced to the audience and made brief comments about their views on the future of the group. Alfredo Cuaron from Mexico helped chair the second workshop, which focused on the Tapir Action Plan (TAP). During that session the need to review the 1997 version of the Tapir Action Plan was discussed. The last session of the symposium was a plenary session called "Plans for Action". Susie Ellis (Conservation International, United States) who helped the symposium group to establish priorities conducted it and got participants committed to the tasks and challenge ahead of us. The symposium group was divided into smaller groups, which outlined their tasks for the upcoming year. Twelve veterinarians attended this meeting. The tasks outlined by this group of veterinarians included forming a more meaningful network of communication. Also formulating a list of benefits that the involvement of a veterinarian can offer a field biologist, make publications by other colleagues more available and contribute to the Tapir Gallery Web Site link specific for veterinarians with veterinary information and a bibliography. The most important benefit is that this group of veterinarians was brought into contact and is now sharing information. Several projects are scheduled to take place as a result of this meeting. Zoo veterinarians are in a unique situation in that they possess the skills, contacts and typically, the interest to become actively involved in in situ conservation projects. Due to the pressure from the public, zoological parks are becoming much more involved in the true definition of conservation, which means supporting field projects. Veterinarians, as an integral part of the zoo staff should take the lead in this involvement. Acknowledgements The Baird’s tapir project could not have been completed without the financial support of the Wildlife Conservation Society, The Zoological Society of San Diego, Zoo Conservation Outreach Group, Idea Wild and Wildlife Preservation Trust International. The authors would like to thank Drs. Don Janssen, Roberto Aguilar, James Bailey and Danilo Leandro for their significant contributions to the Baird’s tapir project. References 1. Bodmer RE and Brooks DM. Status and Action Plan of the Lowland Tapir (Tapirus terrestris). In: Brooks DM, Bodmer

RE, et al, eds. Tapirs -Status Survey and Conservation Action Plan. IUCN/SSC Tapir Specialist Group. IUCN Gland, Switzerland and Cambridge, 1997; 46-56.

2. Eisenberg JF. Mammals of the Neotropics, Vol. 1: The Northern Neotropics. Univ. Chicago Press, Chicago.1989.



3. Hershkovitz P. Mammals of Northern Colombia, preliminary report No. 7: Tapirs (Genus Tapirus), with a systematic review of American species. In: Proc US Nat Mus 1954; 103: 465-96.

4. Matola S, Cuaron, AD et al. Status and Action Plan of Baird’s Tapir (Tapirus bairdii). In: Brooks DM, Bodmer RE, et al, eds . Tapirs -Status Survey and Conservation Action Plan. IUCN/SSC Tapir Specialist Group. IUCN Gland, Switzerland and Cambridge, 1997; 29-45.

5. Naranjo EJ. Abundancia y uso de habitat del tapir (Tapirus bairdii) en un bosque tropical humedo de Costa Rica. Vida Silv Neotrop. 1995; 4: 20-31.

6. Williams, KD. Trapping and immobilization of the Malayan tapir in West Malaysia. Malay Nat J 1979; 33: 117-22. 7. Williams, KD. Radio-tracking tapirs in the rainforest of west Malaysia. Malay Nat J 1979; 32: 253-8. 8. Williams KD. and Petrides, GA. Browse use, feeding behavior, and management of the Malayan tapir. J Wldlfe Mgmt

1980; 44: 489-94. 9. Williams KD. The Central American tapir in Northwestern Costa Rica. PhD Thesis, Michigan State University, East

Lansing, Michigan.1984; 84 pp.



European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4th scientific meeting, joint with the annual meeting of the European Wildlife Disease Association (EWDA) May 8-12, 2002, Heidelberg, Germany.

MYCOPLASMA AND AVIAN POLYOMA VIRUS INFECTION IN CAPTIVE SPANISH IMPERIAL EAGLES (Aquila adalberti)

U. HÖFLE1,2, J.M. BLANCO1, J. SPERGSER3, R.M. JOHNE4 and E.F. KALETA2

Affiliation: 1. Centro de Estudios de Rapaces Ibéricas, 45671 Sevilleja de la Jara, Spain, e-mail: [email protected] . 2. Institut für Geflügelkrankheiten, JLU-Giessen, Germany. 3. Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, Austria.

Institute of Virology, 4. Faculty of Veterinary Medicine, University of Leipzig, Germany. Abstract

Both avian polyomavirus (APV) and mycoplasma spp. can cause respiratory disease in birds. In birds of prey the detection of either of the two pathogens in relation to disease has been scarce until now. A Spanish Imperial Eagle (Aquila adalberti ) that died despite treatment from severe anaemia was found positive for mycoplasma spp. in all organs. Although cultures were negative for cytopathogenic viruses, avian polyomavirus could be demonstrated by PCR (Polymerase Chain Reaction) in spleen and kidney. Mycoplasma spp. of yet unidentified species was cultured from 12 (67%) out of 18 captive Spanish Imperial Eagles examined subsequently, and APV could be isolated from two (11%) of the 18 healthy eagles. Mycoplasma or APV or both may have played a role in respiratory diseases such as Aspergillosis observed frequently in captive and free-living Spanish Imperial eagles in which analysis for these pathogens had not been performed. There is a need to better understand the role of these pathogens in captive and free living populations of eagles, especially the Spanish Imperial eagle.

Zusammenfassung

Aviäre Mykoplasmen und aviäre Polyomaviren (APV) können schwere, häufig mit respiratorischen Symptomen verbundene Erkrankungen bei verschiedenen Vögeln hervorrufen. Bei Greifvögeln werden derartige Infektionen bisher nur selten diagnostiziert. Ein in Gefangenschaft gehaltener Spanischer Kaiseradler (Aquila adalberti) verstarb trotz Behandlung an einer schweren Anämie. Postmortal konnte aus allen Organen des Adlers hochgradig Mycoplasma spp. isoliert werden. Obwohl in der Zellkultur keine zytopathogenen Viren isoliert werden konnten, wurde mittels PCR (Polymerase-Ketten Reaktion) in der Niere und der Milz des Adlers aviäres Polyomavirus nachgewiesen. Von 18 weiteren, in menschlicher Obhut befindlichen, klinisch gesunden Kaiseradlern wurden Rachen-, Tracheal- und Bindehautsacktupfer untersucht. Dabei konnten bei 12 (67%) der Adler Mykoplasmen und bei zwei Adlern (11%) APV nachgewiesen werden. Die Spez ies der isolierten Mykoplasmen konnte bisher nicht bestimmt werden. Sowohl Mykoplasmen als auch APV könnten eine Rolle bei den, bei freilebenden und in Gefangenschaft befindlichen Kaiseradlern häufig auftretenden Atemwegserkrankungen, meist Aspergillosen, stehen, bei denen bisher keine Untersuchung auf diese Erreger durchgeführt wurde. Weitere Untersuchungen zur Bedeutung von Mykoplasmen und APV für frei lebende und in menschlicher Obhut befindliche Kaiseradler wären von Interesse.

Résumé

La mycoplasmose et l’infection avec le polyomavirus aviaire sont des maladies graves souvent respiratoires dans les oiseaux. Au contraire ce ne sont pas de diagnoses fréquents chez les rapaces. Un aigle imperial espagnol (Aquila adalberti ) qui présentait une anémie grave s’était mort malgré le traitement d’émergence. Les cultures de mycoplasmes étaient positives des toutes les organes de l’aigle. Utilisant des fibroblastes des embrions de poule il n’était pas possible d’isoler des virus cytopatogènes, mais avec le PCR (Polymease chain reaction) nous avons trouvé du APV dans le rein y la rate de l’aigle. Des echantillons de 18 aigles imperiaux ont éte examinés en culture par mycoplasmes et dans des cultures des fibroblastes des embrions de poule. Dans 12 (67%) des oiseaux nous avons isolé du mycoplasme des espèces sans identifier, et dans les cultures du pharynx de deux aigles (11%) il y’ avait APV. Dans les aigles imperiaux sauvages et on captiveté il y a regulièrement des maladies respiratoires, fréquentement des aspergilloses. Les deux patogènes isolés dans cette étude pouvraient avoir un rôle dans ces episodes, mais jusqu’ á maintenant il n’y avait pas d’analise de



mycoplasme ou de APV. Il faut des études supplémentaires pour mieux entendre l’importance de ces deux patogènes par l’aigle imperial sauvage et on captivité. Key words: mycoplasma, polyomavirus, imperial eagle Materials and Methods Avian polyomavirus virus (APV) infections have only recently been demonstrated to affect birds of prey (2), such as the common buzzard (Buteo buteo) and the common kestrel (Falco tinnunculus ). The virus was described originally as budgerigar fledgling disease virus and is known to cause severe fatal disease in psittacine and non-psittacine birds worldwide (3, 6). Mycoplasmosis is an important disease in poultry that is also known in birds of prey (1). Recent investigations have led to the detection of a number of species -specific new mycoplasma species from birds of prey with respiratory disease (5). Demonstration of mycoplasma infection on cytology or by other direct methods is difficult, as well as the isolation and identification of Mycoplasma spp. (4). As a result mycoplasmosis seems to be an underdiagnosed disease especially in free-living birds of prey admitted to rehabilitation centres (5). This extended abstract describes a case of mycoplasmosis and APV infection in a captive Spanish Imperial eagle and summarises briefly the results on the prevalence of mycoplasma and APV in 18 captive Spanish imperial eagles. An apparently healthy adult female imperial eagle was found to be severely anaemic during a routine health survey. The bird was in very good body condition and showed very moderate respiratory distress. Despite the application of emergency treatment including homologous blood transfusions the eagle died within 24 hours. Macroscopic lesions observed during necropsy included the presence of numerous small (0.5 to 1 cm) granulomatous lesions in the fascia adjacent to the left shoulder joint, as well as in all air sacs (including the clavicular air sacs) and a very pale and atrophic spleen. Microscopically severe haemosiderosis was observed in the liver, in combination with a moderate diffuse infiltrate of mono- and polymorph-nuclear cells. In the lung few large pale eosinophilic intranuclear inclusions could be observed in the present macrophages and some epithelial cells. Samples were processed for microbiology, isolation and PCR of Mycoplasma spp., Virus isolation and for PCR for the detection of avian Polyomavirus DNA. Mycoplasma spp. Was cultured in large amounts from all organs, as well as from swabs from the mentioned shoulder joint. The isolate could not yet be identified, but using antisera against M. Synoviae and M. Gallisepticum it proved to be neither. Cultures in chicken embryo fibroblast (CEF) cultures were negative for cytopathogenic viruses. Using PCR, APV could be detected in the spleen and kidney of the bird. No pathogenic bacteria or fungi were found in the microbiological cultures. Subsequently, tracheal, choanal and conjunctival swabs of 18 captive Spanish Imperial eagles were examined for the presence of Mycoplasma spp. By culture and PCR. In 12 (67%) of the analysed, clinically healthy birds Mycoplasma spp. Were detected. In five birds Mycoplasma spp. Could be grown from choanal as well as from tracheal swabs, while in five eagles only the choanal swabs were positive. Only on one occasion Mycoplasma spp. Could be cultured from a conjunctival swab. Pharyngeal and cloacal swabs of the same 18 eagles were examined for the presence of cytopathogenic viruses in CEF-cultures. APV was isolated from pharyngeal swabs of two eagles (11%), while no cytopathogenic virus was found in the other samples. The two APV positive eagles had both presented respiratory symptoms upon admission at the rehabilitation and captive breeding centre a few month ago, but were clinically healthy at the time of sampling. Both, Mycoplasma spp. And APV are known to cause respiratory disease in their hosts (1, 2, 3, 4). However, Mycoplasma spp. Have also been isolated from wild injured or debilitated raptors that did not show signs of respiratory disease or affection of joints (4). A common kestrel from which APV could be isolated showed anaemia, ascites and fatty degeneration of the liver (2). In previous years, cases of Aspergillosis with severe pulmonary fibrosis have been observed in young, free-living Imperial eagles found debilitated or injured, in which viral cultures were negative but no PCR for APV or cultures for Mycoplasma spp. Had been performed. Either one or both of the aforementioned pathogens may have played a role in these cases. Additional analysis are under



way in order to better understand the importance, pathogenesis and epidemiology of APV and Mycoplasma spp. Infections in captive and free-living populations of the Spanish Imperial eagle. References

1. M. Greifvögel: Krankheiten, Haltung, Zucht. Blackwell Wissenschafts -Verlag, Berlin 1996; 123. 2. Johne R and Muller H. Avian Polyomavirus in wild birds: genome analysis of Is olates from Falconiformes and

Psittaciformes. Arch Virol 1998 143: 1501-12. 3. Khan MS, Johne R, Beck I, Pawlita M, Kaleta EF and Muller H. Development Of a blocking enzyme-linked

immunosorbent assay for the detection of avian Polyomavirus-specific antibodies. J Virol Methods 2000; 89: 39-48. 4. Lierz M, Schmidt R, Goebel T, Ehrlein J and Runge M. Detection of Mycoplasma spp. In raptorial birds in Germany

In: Lumeij, JT., Remple, JD. 5. Redig PT, Lierz M and Cooper JE. Raptor Biomedicine III. Zoological Education Network, Inc. Lake Worth (FL)

2000; 25-33. 6. Poveda JB, Giebel J, Flossdorf J, Meier J and Kirchhoff H. Mycoplasma Buteonis sp. Nov., Mycoplasma falconis sp.

Nov. And Mycoplasma gypis sp. Nov., Three species from birds of prey. Intern J Sys Bacteriol 1994; 44: 94-8. 7. Sandmeier P, Gerlach H, Johne R and Muller H. Polyomavirus infections in Exotic birds in Switzerland. Schweiz

Arch Tierheilkd 1999; 141: 223-9.



European Association of Zoo- and Wildlife Veterinarians (EAZWV) 4 th scientific meeting, joint with the annual meeting of the European Wildlife Disease Association (EWDA) May 8-12, 2002, Heidelberg, Germany.

Mycobacterium tuberculosis INFECTION IN ASIAN ELEPHANTS (Elephas maximus) IN SWEDEN

D. GAVIER-WIDEN1, C. HÅRD AF SEGERSTAD1, B. RÖKEN2, TORSTEN MØLLER2, G. BÖLSKE 1and S. STENBERG1

Affiliation: 1. National Veterinary Institute, 751 89 Uppsala, Sweden 2. Kolmården Zoo, 618 92 Kolmården, Sweden Extended abstract Key words: tuberculosis, mycobacteria, M. tuberculosis, elephant, zoonosis Tuberculosis caused by Mycobacterium tuberculosis is an important zoonotic disease of captive Asian elephants. Elephant tuberculosis has acquired renewed attention following the outbreaks in USA in the past years. It is estimated that 3% of the captive elephant population in North America is infected (1). Although tuberculosis was recognised in captive elephants in Europe as early as in the 18th century, only limited information, mostly isolated reports, is available about the tuberculous status in captive elephants in Europe. Recently two infected Asian elephants were detected in a Swedish zoo during a routine control based on mycobacterial culture of trunk lavage. The elephants were a 30-years-old female born in Burma, and a 26-years-old female. Both had arrived to the zoo in the late 1980-s, after having spent 12 and 10 years respectively at European circuses. The two elephants had shown occasional weak bovine reaction to the comparative tuberculin skin test, the 30-years old in 1993 and the 26-years-old in 1997. Thereafter their skin test was negative or inconclusive four times and twice respectively. Both animals seroconverted between 1995 and 1997 and developed a weak positive antibody titre at the ELISA (ELIB and MB70- antigen, at ID-Lelystad, The Netherlands). Culture of trunk lavage has been made in all the elephants of the zoo since 1998. In late summer 2001 the 30-years-old female became tired, developed polydipsia but retained normal appetite and body weight. A significant increase in beta- and gamma globulins and a drop in albumin/globulin-quota were noted, starting in early 2001. The trunk lavaged cultured positive for M. tuberculosis and she was euthanised. The 26-year-old elephant showed positive culture of all three trunk washes in December 2001, while the cultures from lavage obtained in October and November had been negative. This animal is to the date (end of January 2002) in excellent physical condition but will be euthanised in February 2002. The post mortem examination of the 30-years-old elephant revealed lesions consistent with pulmonary tuberculosis. Solid granulomatous pneumonia involved approximately one fourth of the left lung. Two large (20 x10 x10 cm diameter) granulomas were present in the right lung. The rest of both lungs’ parenchyma had widespread numerous small foci. Lesions were also found in bronchomediastinal lymph nodes and in an axillary lymph node. Multiple granulomas of up to 10 mm were observed along the mucosa of the trachea and the epiglottis.



Histopathology showed diffuse granulomatous consolidation, formed by whirls of epithelioid cells surrounded by macrophages, eosinophils and lymphocytes. There were multiple small foci of central caseous necrosis and several microabscesses within the consolidation. Large organised epithelioid cell granulomas were present in the lungs, lymph nodes and spleen. These lesions had irregular encapsulation, cellular outer layers and large necrotic centres. Calcification was mild. Some of the lesions were extending into the lumen of the smaller airways in the lungs. Large necrotic granulomas with abundant acid fast bacilli were present in the mucosa of the trachea, opening through the epithelium into the lumen. A few acid-fast bacilli were found in the lung lesions. Multinucleated giant cells were not observed. Mycobacterium tuberculosis was isolated from the lesions on Löwenstein-Jensen medium. Tuberculosis in elephants is most frequently of pulmonary forms (2). The lesions observed in this case were indicative of shedding via aerosol and respiratory secretions. It is generally recognised that the diagnosis of pre-clinical tuberculosis in elephants is difficult. The present diagnostic tests can detect exposure to mycobacteria and immunological response, but do not provide information on the progression of the infection. The definitive diagnosis can be made by culture of clinical samples first when shedding is in place, which appears to occur at later stages, when well established and productive lesions have developed in natural passages, such as respiratory tract. Tuberculosis acquired by humans from elephants is considered to result most often from close and repeated contact, such as that with elephant handlers. There is also a potential risk for the transmission of M. tuberculosis from elephants to other exotic species. It is important that monitoring and control programs for the detection of tuberculous elephants in captivity is implemented in order to prevent infection in humans and animals. References 1. Mikota SK., Larsen RS and Montali R. (2000). Tuberculosis in elephants in North America. Zoobiology 19 (5), 393-403. 2. Montali R.., Mikota SK and Cheng LI. (2001) Mycobacterium tuberculosis in zoo and wildlife species. Rev. sci. tech. Off.

Int. Epiz, 20 (1), 291-303


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