Posters Session. Scroll down to view documents. European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5th Scientific Meeting

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1 Posters Session Scroll down to view documents European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5th Scientific Meeting May 19-23, 2004, Ebeltoft, Denmark This manuscript is reproduced in the IVIS website with the permission of EAZWV

2 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. AURAL ABSCESSATION IN FREE-LIVING EASTERN BOX TURTLES (Terrapene carolina carolina): A PATHOLOGIC AND EPIDEMIOLOGIC DESCRIPTION J. D. BROWN 1, J. ROBERTSON 1, S. HOLLADAY 1, F. ELVINGER 1 and J. M. SLEEMAN 1,2 Affiliation: 1. Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U.S.A. 2. Wildlife Center of Virginia, P.O. Box 1557, Waynesboro, Virginia 22980, U.S.A. Abstract A recent study found aural abscessation to be a significant cause of morbidity and mortality in free-living eastern box turtles (Terrapene carolina carolina) in Virginia (1). Though its etiology remains unknown, hypovitaminosis A has been suggested based on a similar lesion occurring in captive chelonia fed diets deficient in vitamin A (4). This hypothesis was supported by significantly greater body burdens of organochlorine compounds, a reported disruptor of vitamin A metabolism (5), and a non-significant trend toward lower serum and vitamin A levels found in free-living box turtles with this lesion (3). The tympanic epithelium was evaluated in twentyseven turtles (ten with aural abscessation and seventeen without) in order to describe the pathology of this lesion. Pathological changes to the tympanic epithelium of turtles with aural abscessation included: hyperplasia, squamous metaplasia, hyperemia, cellular sloughing, desquamation, granulomatous inflammation, and bacterial infection. These changes were more severe in box turtles with aural abscesses as opposed to those without, and were greater in tympanic cavities containing an abscess as opposed to one without (when the lesion was unilateral). The epidemiology of 46 cases of aural abscessation in free-living eastern box turtles admitted to the Wildlife Center of Virginia (WCV, Virginia, USA) from 1991 to 2000 was also evaluated using univariate analysis followed by multivariable logistic regression. County human population density, year and season of admission, weight, and gender were not associated with an increased risk for box turtles developing aural abscesses. Spatial location of cases (based on county) was analyzed for clustering by Grimson s method (2). Counties with cases of aural abscessation were not randomly distributed, but rather were clustered into two multi-county regions. The histopathological changes in box turtles with aural abscessation and the epidemiological profile are consistent with a syndrome that may involve hypovitaminosis A and/or environmental organochlorine compound exposure, and is being further investigated. Acknowledgements This study was funded by the Geraldine R. Dodge Foundation Frontiers For Veterinary Medicine initiative, and the Environmental Quality Technology Program of the U.S. Army, with continued support from the Morris Animal Foundation. This study was approved by the Virginia Tech Animal Care Committee, and the samples were collected with permission from the Virginia Department of Game and Inland Fisheries under Scientific Collection Permit number

3 References 1. Brown JD, and Sleeman JM. Morbidity and mortality of reptiles admitted to the Wildlife Center of Virginia, J Wildl Dis 2002; 38: Grimson RC. A versatile test for clustering and a proximity analysis of neurons. Methods of Information in Medicine 1991; 30: Holladay SD, Wolf JC, Smith SA, Jones DE and Robertson JL. Aural abscesses in wild-caught box turtles (Terrapene carolina): Possible role of organochlorine-induced hypovitaminosis A. Ecotoxicology and Environmental Safety 2001; 48: Murray MJ. Aural abscesses. In: Mader DR (ed). Reptile Medicine and Surgery. WB Saunders Company Philadelphia etc 1996; Poon R, Lecavalier P, Chan P and Viau C. Subchronic toxicity of medium-chained chlorinated paraffin in the rat. Journal of Applied Toxicology 1995; 15:

4 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. NORMAL BLOOD CONCENTRATIONS OF COPPER, MAGNESIUM AND ZINC IN STONE CURLEW AND FIVE SPECIES OF BUSTARDS IN THE UNITED ARAB EMIRATES T.A. BAILEY 1, C.D. SILVANOSE 1, O. COMBREAU 2 AND J.C.HOWLETT 2 Affiliation: 1. Dubai Falcon Hospital, PO Box 23919, Dubai, UAE 2. National Avian Research Center, P.O. Box 45553, Abu Dhabi, United Arab Emirates. Abstract This study defined normal ranges for blood concentrations of copper, magnesium and zinc in the sera of five species of healthy bustards and stone curlew (Burhinus oedicnemus). Inter-species differences were evident. Adult houbara bustards had significantly higher copper and magnesiu m levels than adult buff-crested and kori bustards. White-bellied and buff-crested bustards had significantly higher zinc levels than houbara and kori bustards. Key words: trace element, copper, magnesium, zinc, stone curlew, bustard, United Arab Emirates Introduction Minerals are nutrients that are essential for good health in animals. Mineral deficiency or excess can cause disease, so animal health evaluation often requires the determination of mineral status. Mineral status can be determined by analysis of serum, bone, tissues (e.g. liver) and feed (Scheideler et al, 1994). The normal mineral concentration ranges in the blood or tissues of healthy animals must be known to determine mineral status. Trace minerals, including copper, manganese, selenium and zinc function as accessory factors to enzymes and are required in small amounts in the diet (National Research Council, 1994). Trace minerals have been extensively studied in the blood and tissues of domestically farmed animals such as poultry and health examination of flocks frequently involves an assessment of mineral status. However, in comparison there is little data on levels in wildlife and extrapolation from domestic species may not be reliable (Blakley et al, 2000). To the knowledge of the authors, no data exists in the accessible literature on normal baseline values of trace minerals in bustards. Similarly, although mineral toxicity in avian species is well reported, to the knowledge of the authors, only one report on lead toxicity has been reported in bustards (Bailey et al, 1995). The purpose of this study was to define the normal range for blood concentrations of copper, magnesium and zinc in the sera of healthy bustards and stone curlew (Burhinus oedicnemus). Such information will assist in the ability to diagnose metal toxicities or deficiencies in these species. Materials and Methods Blood samples were obtained from 56 (36 female, 20 male) houbara bustards (Chlamydotis undulata macqueeni), 45 kori bustards (Ardeotis kori) (35 fema le, 10 male), 33 (15 female, 18 male) white -bellied bustards (Eupodotis senegalensis), 31

5 (16 female, 15 male) buff-crested bustards (E. gindiana ), four female Heuglin s bustards (Neotis heuglinii), three male black bustards (E. afra) and nine (seven female, two male) stone curlews. Of the birds sampled, 15 koris, six white-bellied and five buff-crested bustards bred during the subsequent breeding season. Samples were collected during routine health checks in October 2000 to January The birds were 1.5 to 6 yr old and were maintained in outdoor aviaries with natural vegetation and galvanised wire mesh. These birds were maintained at the breeding facilities of NARC (24 N, 55 E, Al Ain, Abu Dhabi Emirate, United Arab Emirates). They were kept to outdoor pens with natural vegetation. Their diet consisted of a proprietary pellet supplemented with vegetables, fruit, mice and invertebrates (Sleigh and Samour, 1996). Food was provided in stainless steel food dishes and water was provided in plastic bowls. Clean tap water was provided on a daily basis. Table 1 summarises the micronutrient composition of the diet fed to bustards in the investigation (Anderson, 1995). Table 2 summarises differences in the management of each species. Table 1. Micronutrient composition of diet fed to bustards in the investigation (Source Anderson, 1995). Estimated levels on a fresh weight (73.46 percent dry matter) basis. Micronutrient Unit Diet Copper mg/kg Magnesium % 0.24 Zinc mg/kg Table 2. Management regime of each bustard species and age group at the time of blood sample collection (adapted from Anderson et al, 2002). Availability of Species Diet offered natural food a Pen size b Social grouping Houbara Maintenance Limited Small Mixed groups of varying sizes, up to 30 Kori Maintenance Available Large Large groups of up to 25, 1 trio Buff-crested Production Available Small Pairs, singles White - Production Available Large Pairs, singles bellied Black Maintenance Negligible Small Single Heuglin s Production Available Large Small group (5) Stone curlew Maintenance Available Large Small mixed group a Negligible: The black bustards only had access to invertebrates that may have entered their pens. Limited: Adult houbara bustards had access to invertebrates and small skinks that may have entered their pens; some pens also had cultivated alfalfa beds. The juveniles had access to invertebrates and small skinks that may have entered their pens; their pens contained shrubs and cultivated alfalfa beds. Available: Pens contained shrubs, varying quantities of grasses and herbaceous vegetation, cultivated alfalfa beds (except for the kori bustard pens), invertebrates, and small vertebrates (such as skinks). b Small: 6 x 3 m; large: 50 x 100 m. All birds had been vaccinated against Newcastle disease 1 yr previously (Newcavac Nobilis, Intervet, Cambridge, UK) and had received prophylactic treatment against gastro-intestinal helminth (Droncit, Bayer plc, Bury St Edmunds, UK; Ivomec, MSD

6 AGVET, Hoddesdon, UK) and protozoan parasites (Emtryl, Rhône Mérieux Ltd, Harlow, UK) at least 3 mo prior to sampling. The houbara, white -bellied, buffcrested, black and Heuglin s bustards, and the stone curlews were captured by hand between 06:30 to 09:00 AM, placed in a transport box and taken to a nearby (<1 km) veterinary clinic at the same NARC site. They were manually restrained for the sampling procedure, during which approximately 2 ml of blood was obtained within 1 to 2 hr of capture. The kori bustards were captured by hand and were manually restrained for the sampling procedure, which took place in the aviary within 15 min of capture. Prior to sampling, all birds were examined by a veterinarian and declared clinically normal. After collection from the brachial (basilic) vein using a 3 ml disposable syringes without a rubber plunger (Fortuna, W. Graf GmbH & Co, 6980 Wertheim, Germany) and a 25 g butterfly cannulae, all samples were immediately put into a commercially available serum gel storage tubes (Sarstedt, Numbrecht, Germany). After 2 hours the samples were centrifuged at 10,000 g for 10 min using a bench -top centrifuge (Hawksley, Lancing, UK). Serum was removed and stored in microcentrifuge tubes at - 20 C until analyses were carried out within 3 months). A wet chemistry ACE Schiappareli Biosystems Analyser (Schiappareli Biosystems, AL Woerdan, The Netherlands) at the Abu Dhabi Falcon Hospital Laboratory (Abu Dhabi, UAE) was used to analyse the serum. Magnesium was determined by the xylidyl-blue reaction (ACETM Magnesium Reagent, Schiappareli Biosystems, AL Woerdan, The Netherlands), copper was determined by the 3, 5-Dibromo-2- pyridylazo -N-Ethyl-N-aniline method (Randox Laboratories, Antrim, UK) and zinc was determined by the dimethylglyoxime method (Audit Diagnostics, Cork, Ireland). Quality control was carried out using the following reference sera; magnesium (Gemcal, Schiappareli Biosystems, AL Woerdan, The Netherlands), copper (Control Multi-Sera, Randox Laboratories, Antrim, UK) and zinc (Multi-Sera, Randox Laboratories, Antrim, UK). Results are expressed as mean ± standard error (SE). Statistical analyses were performed using the computer program MEDCALC (Medcalc software, Mariakerke, Belgium). Means were compared between the species using one-way-analysis of variance (ANOVA). Values of P 0.05 were considered statistically significant. The black bustards, Heuglin s bustards and stone curlew were excluded from analyses due to small sample size. Results Serum concentrations of copper, magnesium and zinc for the six of bustard species and stone curlew maintained by NARC are presented in Table 3. Inter-species differences were evident. Adult houbara bustards had significantly higher copper (P<0.01) and magnesium (P<0.01) levels than adult buff-crested and kori bustards. White-bellied and buff-crested bustards had significantly higher zinc levels than houbara and kori bustards (P<0.01). Discussion Trace element levels in the blood are useful aids to diagnose deficiencies of magnesium and zinc (National Research Council, 1994). The means and ranges observed of magnesium found in sera from adult birds in the current study was comparable to values reported in ratites (Stewart, 1989) and previous studies in houbara bustards and stone curlew at NARC (Samour, 1998; Bailey et al, 1999). The most widely used method for assessing zinc status is the measurement of plasma levels (Keen and Graham, 1989). Typical plasma or serum levels of zinc in most species range from µg/dl (Keen and Graham, 1989), while the normal range for zinc in Hispaniolan amazons (Amazona ventralis) is µg/dl (Osofsky

7 et al, 2000). These levels are comparable to the levels detected in bustards and stone curlew in the current study. Serum copper levels are also useful in suspected cases of deficiency, as low levels are considered to be diagnostic (Keen and Graham, 1989). The normal range of copper in the blood of most healthy animals is between 50 and 150 µg/dl, although birds, fish and marsupials are characterised by copper levels that are half of these values (Keen and Graham, 1989). Levels of copper in the sera of bustards and stone curlew in the current study (range µg/dl for kori bustards) are higher than sera levels reported in ratites (15-28 µg/dl) by Angel (1996) and in Hispaniolan amazons (Amazona ventralis) ( µg/dl) by Osofsky et al (2000). The birds sampled in this study were kept in naturalistic aviaries where it is known that approximately 25% of the diet consists of natural food (annual plants and terrestrial invertebrates) from their surroundings, in addition to the same standard diet (Warren, 1996). Seasonal changes in the serum concentration of micronutrients have been reported in spe cies such as llamas (Bechert and Smith, 1996) and are considered to be related to levels in forage. The reasons for the high copper levels, relative to other avian species in bustards is not known and further studies ware warranted to determine copper levels in the tissues of bustards maintained on diets, unsupplemented by natural foods would be helpful. Elevated serum copper levels can also reflect a number of stress syndromes in animals (Keen and Graham, 1989), so a finding of high blood copper should not be the sole criterion for diagnosis of copper toxicity. Liver levels are considered to be a reliable indicator of copper status in domestic species (Keen and Graham, 1989). While the collection of blood samples is a practical and minimally invasive technique to screen flocks of birds, further studies are warranted to correlate tissue (liver) and blood levels. Reported copper levels in the livers of wild birds varies widely, from 2,000 to 100,000 µg/dl wet weight in sea ducks and swans respectively (Sigurdson and Franson, 2000) and from 1,500 to 3,000 µg/dl in poultry (National Research Council, 1980). Copper deficiency has been diagnosed in an ostrich chick on the basis of low liver copper, clinical signs (star-gazing and weakness) and histopathological lesions of encephalomalacia in the CNS (Speer, 1995). These data must be considered preliminary. Further investigations to determine tissue (e.g. liver, bone) levels of these and other trace elements in bustards will be necessary to correlate serum levels with feed and tissue levels. Table 3. Serum copper, magnesium and zinc levels in six species of captive bustards and stone curlew in the United Arab Emirates. Species n a Copper (mg/dl) Magnesium (mmol/l) Zinc (mg/dl) Houbara b ± 0.71 c ( ) d 1.2 ± 0.04 ( ) ± 2.53 ( ) Kori ± 0.88 ( ) 1.08 ± 0.05 ( ) ± 5.53 ( ) White-bellied ± 0.59 ( ) 1.12 ± 0.04 ( ) ± 4.33 ( ) Buff-crested ± 0.82 ( ) 1.01 ± 0.03 ( ) 174 ± 3,37 ( ) Stone curlew ± 1.28 ( ) 0.88 ± 0.03 ( ) ± 7.89 ( ) Black ± 0.81 ( ) 1.03 ± 0.09 ( ) ± 8.84 ( ) Heuglin s ± 4.14 ( ) a number of samples (mean b ± SE c, range d ) 1.12 ± 0.05 ( ) ± 4.92 ( )

8 Acknowledgements This study was conducted while Tom Bailey and Christudas Silvanose were working for the National Avian Research Center. We thank H. H. Sheikh Khalifa bin Zayed Al Nahyan, President of the ERWDA Board of Directors and H. H. Sheikh Hamdan bin Zayed Al Nahyan, Vice President of the ERWDA and Mr. M. Al Bowardi, ERWDA Managing Director, for their support of the veterinary science programme of NARC. We thank H.H. Sh. Hamdan bin Rashid al Maktoum and Mr Humaid Obaid al Muhairi, Dubai Falcon Hospital Director, for their support of the senior author. References 1. Anderson, S. (1995) Bustard micronutrient review. NARC external Report No Anderson, S.J., Dawodu, A., Patel, M., Bailey, T.A. & Silvanose, C. (2002) Plasma concentrations of vitamin E in six species of bustards (Gruiformes: Otididae). Journal of Wildlife Diseas es. 38: Angel, C.R. (1996) Serum chemistries and vitamin D metabolites in ostriches, emus, rheas and cassowaries. International Conference on Improving our understanding of ratites in a farming environment, Manchester, UK. Pp Bailey, T.A., Samour, J.H., Naldo, J. and Howlett, J.C. (1995) Lead toxicosis in captive houbara bustards (Chlamydotis undulata maqueenii). Veterinary Record. 137: Bailey, T.A., Wernery, U., Howlett, J., Naldo J., and Samour J.H. (1999) Age-related plasma chemistry changes in houbara (Chlamydotis undulata) and kori bustards (Ardeotis kori). Journal of Wildlife Diseases. 35: Blakley, B.R., Kutz, S.J., Tedesco, S.C. and Flood, P.F. (2000) Trace mineral and vitamin concentrations in the liver and serum of wild muskoxen from Victoria Island. Journal of Wildlife Diseases. 36: Bechert, U.S. and Smith, B.B. (1996) Serum Macro and Micro Element Concentrations in the Llama. Veterinary Clinical Nutrition. 3: Sigurdson, C.J. and Franson, J.C. (2000) Avian toxicologic diagnosis. In: Laboratory medicine: avian and exotic pets (ed, Fudge, A.M.) W.B. Saunders Co., Philadelphia. Pp Keen, C.L. and Graham, T.W. (1989) Trace elements. In: Clinical biochemistry of domestic animals (ed Kaneko, J.J.). Academic Press, Harcourt Brace Jovanovich, San Diego. Pp National Research Council (1980) Mineral tolerance of domestic animals. Washington DC: National Academy Press. 11. National Research Council (1994) Nutrient requir ements of poultry, 9 th revised edition. National Academy press, Washington. 12. Osofsky, A., Jowett, P.L., Hosgood, G., and Tully, T. (2000) Normal blood concentrations for lead, zinc, iron and copper in Hispaniolan amazons (Amazona ventralis). Proceedings of the Association of Avian Veterinarians. Pp Samour, J., Howlett, J., Silvanose, C.-D., Bailey, T.A. and Wernery, U. (1998) Normal haematology and blood chemistry of stone curlews. Comparative Haematology International. 8: Scheideler, S.E., Wallner -Pendleton, E.A., Schneider, N., and Carlson, M. (1994) Determination of baseline values for skeletal (leg bone) growth, calcification and soft tissue mineral accretion. Proceedings of the Association of Avian Veterinarians. Pp Speer, B.L. (1995) Copper nutritional deficiency in the ostrich (Struthio camelus). Proceedings of the Association of Avian Veterinarians Pp Stewart, J.S. (1989) Husbandry, medical and surgical management of ratites, part two. Proceedings of the Association of Zoo and Wildlife Veterinarians. Pp Warren, S. (1996) An evaluation of the availability and importance of natural food in the diet of captive houbara bustard. National Avian Research Center Internal Research Report No. 43, Abu Dhabi, United Arab Emirates, pp 1-36.

9 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. IS BARK PEELING BY RED DEER (Cervus elaphus) A RESULT OF HUMAN DISTURBANCE? J. DRACK and S. HUBER Affiliation: Research Institute of Wildlife Ecology, University of Veterinary Medicine Vienna, Savoyenstr. 1, 1160 Vienna, Austria. Key words: Bark peeling, Cervus elaphus, human disturbance, red deer, stress Extended abstract Forest damage due to bark peeling by red deer (Cervus elaphus) is a major problem in wildlife management and causes high economic losses. It is assumed that bark peeling may be intensified as a consequence of stress resulting from human disturbance. The study was therefore aimed at investigating whether human disturbances lead to increased bark peeling by red deer. Faecal samples were collected once a week for one year in two meadow habitats of comparable structure but with different levels of human disturbance: habitat 1 Untere Lobau with a high level of human disturbance because of its location near the central city of Vienna; habitat 2 Eckartsau with a low level of disturbance due to its location in a low populated area. The collected faecal samples (n=620) were analyzed for remnants of bark. One dropping of each sample was dried, pulverized and centrifuged. As a result crude and undigested material concentrated on the upper surface. Three subsamples were taken from this coarse material and then the bark remains were quantified under a binocular microscope. As soon as 5% or more of this crude material were identified as bark, a faecal sample was classified as bark positive. The relevance of this method was validated with a feeding experiment in a group of captive red deer. The animals were fed with either standard red deer diet or standard red deer diet supplemented with bark formed into pellets. Analysing the faecal samples (n=57), we were able to classify more than 95% of bark-containing faecal samples correctly. Applying this method to faeces collected in the field, approximately 15% of the samples contained significant amounts of bark remnants. Bark peeling varied with season in both habitats. Bark positive samples were identified from March through to November whereas almost no faecal bark remains were found between December and February. However, the extent of bark peeling also differed between the two habitats. In March and April, significantly more bark positive faecal samples were detected in habitat 1 Untere Lobau than in habitat 2 Eckartsau, whereas the opposite happened in November. These results indicate that the extent of bark peeling depended on the season, apparently coinciding with the beginning of the vegetation period. In addition, disturbances also appeared to play a role as maximum amounts of bark peeling were detected in habitat 1 Untere Lobau in March and April, at a time when human disturbances were particularly high and foliage cover for red deer was still poor. Acknowledgements Supported by the Austrian Science Fund via a Hertha Firnberg program for women (T3-BIO) commissioned by the BMBWK.

10 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. INDONESIAN ZOOS OR CAGED CRUELTY? J. DRACK Affiliation: Research Institute of Wildlife Ecology, Veterinary University of Vienna, Savoyenstr. 1, 1160 Vienna, Austria Key words: caged cruelty, illegal market and trade, Indonesian zoos Extended abstract A zoo is supposed to be a place of animal welfare and conservation of endangered species. Therefore the five freedoms should be the basis for each place in the world were wild animals are kept in captivity: to be free of 1. hunger and thirst, 2. physical discomfort, 3. pain and injury, 4. fear and distress and 5. to be able to express normal behaviour. Animals in Indonesian zoos (Ragunan Zoo Jakarta, Surabaya Zoo, Gembiraloka Zoo Yoygakarta, Taman Safari near Bogor) are suffering under horrifying conditions: Their enclosures are entirely inadequate and lack of sufficient space. There is no access to clean water and no shelter from the elements in a lot of cases. Cages are often just single fenced or open enclosures which makes it easy for people to throw food, rubbish at the animals and to get in direct contact with them. When they do get their food it s almost rotten and they start begging visitors. Lots of wild animals don t even get their appropriate diet and in general the diets are very poor and without any variety. As something like an enrichment plan doesn t exsist and no proper training is done the animals a re often sluggish, seemingly depressed or traumatized. Unable to cope with all the distress they start to mutilate themselves. Concerning veterinary tasks there is very little done to improve the situation as there are no proper feeding and enrichment planes, almost no faecal samples are taken for parasitic controlls and periodical deworming is not happening. Very often open wounds are left untreated and just a few post mortems are carried out. As there is no breeding programme zoos end up having too many individuals of some species without any reintroduction programme and neither suitable nor additional enclosures. In an attempt to provide amusement for visitors zoos also use their animals in circus-like exhibits. They often beat and starve endangered species such as sunbears (Helarctos malayanus), tigers (Panthera tigris spp.), orang-utans (Pongo pygmaeus, Pongo abelii) to make them perform. In addition they offer people rides on elephants (Elephas maximus), camels (Camelus spp.) and pictures together with lion cubs (Panthera leo ) or orang -utans. Another layer of corruption is that the zoo falsely reports the death of an animal and then sell it to another facility or collector. Even zoostaff is often involved in illegal trading and sometimes zoo animals just disappear. The biggest bird market called Pramuka is situated in Jakarta were you are not just able to buy endangered birds like: Sulphur-crested cockatoo (Cocatua galerita ), Palm cockatoo (Probosciger aterrimus), Eclectus parrot (Eclectus roratus), they also sell protected species such as: Slow loris (Nycticebus coucang), Siamang (Hylobates syndactylus), Gibbons (Hylobates spp.), Orang -utans, Cats (Felis spp.), Prevost s squirrel (Callosciurus prevosti), ; Conditions in zoos in Indonesia has revealed an industry which shows little care for the most basic requirements of animal welfare and conservation seems to be a forgein word almost nobody has heard about.

11 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. EMERGENCE OF CONGENITAL MALFORMATIONS IN FREE-RANGING LYNX (Lynx lynx) FROM SWITZERLAND: FIRST EVIDENCE OF INBREEDING DEPRESSION? M.-P. RYSER-DEGIORGIS 1, A. RYSER 2, G. OBEXER-RUF 3, U. BREITENMOSER 4, J. LANG 5 and CH. BREITENMOSER WÜRSTEN 2 Affiliation: 1. Centre for Fish and Wildlife Health, Institute of Animal Pathology, University of Berne, Postfach, CH-3001 Berne, Switzerland 2. KORA, Berne, Switzerland 3. Institute of Animal Genetics, Nutrition and Housing, University of Berne, Switzerland 4. Institute of Veterinary Virology, University of Berne, Switzerland 5. Small Animal Clinic, University of Berne, Switzerland Abstract The Eurasian lynx (Lynx lynx) vanished from Switzerland during the 19 th century and was reintroduced in the 1970ies. Re -introductions are known to create artificially a bottleneck situation, which can lead to a reduction in genetic variability of the newly developing population. In recent years, several congenital malformations were observed in free -ranging lynx from Switzerland. Furthermore, genetic analyses revealed a remarkably reduced genetic variability. In this paper we report these observations and discuss the possibility of a correlation between the emergence of inborn defects and loss of genetic diversity. On the basis of the actual knowledge, the link between congenital defects and inbreeding can neither be confirmed nor refuted, and our goal is not to be ala rming but vigilant. Key words: congenital, Eurasian lynx, genetics, inbreeding, Lynx lynx, malformation, Switzerland Introduction One of the goals of conservation is to predict and try to advert the genetic deterioration of species, to preserve species potential for adaptation to both short- and long -term environmental variation, and thereby reduce their chances of extinction (4). Heterozygosity is important for the conservation of species because all populations carry recessive alleles and a proportion of these alleles are lethal when homozygous and a larger proportion are deleterious. The increasing homozygosity from breeding between relatives usually results in reduced fertility, survivorship, disease resistance and growth rates, and is termed inbreeding depression. For example, in the endangered Florida panther (Felis concolor coryi), a remarkable reduction in genetic diversity was reported in correlation with skeletal, reproductive and congenital abnormalities (17). The Eurasian lynx (Lynx lynx) vanished from Switzerland during the 19 th century and was reintroduced in the 1970ies. The animals used for reintroduction were free-ranging lynx from the Carpathian mountains. The number of animals released was very low: in the Swiss Alps and Jura Mountains, only eight lynx were officially released. Further reintroductions raised the number to in the Alps and 8-10 in the Jura Mountains, and two distinct populations arose from these releases (2). Nowadays, the lynx populations are estimated to resident animals in the Jura and in the northwestern Alps (23). As they are still isolated and have developed out of a few individuals, small genetic diversity can be expected in both populations. Re-introductions are indeed known to create artificially a bottleneck situation. This can lead to a reduction in genetic variability of the newly developing population. In this This manuscript is repro duced in the IVIS website with the permission of EAZWV

12 context, a genetic study on the re-introduced lynx populations in Switzerland was designed (3). In recent years, several congenital malformations were observed in free-ranging lynx from Switzerland. We report here these observations and discuss the possibility of a correlation between the emergence of congenital defects and loss of genetic diversity as a consequence of inbreeding. Material and Methods Animals Both Swiss lynx populations have been studied in the frame of several research projects: in the northwestern Alps, in the Jura Mountains, and again in the northwestern Alps. In addition, a translocation project was started in 2001 and since then nine lynx were moved from the northwestern Alps and the Jura Mountains to eastern Switzerland (18, 20). All together, about 100 lynx were captured and radio -collared, and 11 of them were submitted to clinical examination by a veterinarian at time of capture. Furthermore, each autumn 1-2 orphans were caught in villages while looking for easy prey like chickens. Most of them were brought to the wildlife rescue centre of Landshut, close to the city of Berne, where they were usually submitted to a veterinary checkup. If the animals were healthy, they were forwarded to interested zoological gardens. Lynx is a protected species in Switzerland and it is compulsory to report any lynx mortality. Since 1987, lynx carcasses found by means of telemetry or reported to the state game wardens have been sent to the Centre for Fish and Wildlife Health (FIWI), University of Berne, for post-mortem examination. Complete necropsies were performed on 98 lynx that died from 1987 to Genetic analysis We used microsatellite markers isolated from domestic cats (14, 15, 16) and Canada lynx (6) to measure genetic variability. To assess the impact of a bottleneck we compared the results of the re-introduced populations with the autochthonous source population from the Carpathian Mountains of Slovakia. Currently, results of 17 markers are available to present preliminary conclusions. Results Case reports 1) In November 1992, a juvenile male was killed by a hunting dog, which was very unusual as lynx are normally able to escape such attacks. Besides the fatal injuries, necropsy revealed multiple skeletal deformities: scoliosis, kyphosis and fusion of two vertebrae in the thoracal area; deformity of the ribs (with indications of old factures); and pronounced symmetrical deformity of the anterior extremities at the level of the elbow that prevented a normal extension of the limbs. 2) In 1995, a juvenile male was found alive in a private garden in the village of Gstaad in the canton of Berne. It was brought to the wildlife rescue centre of Landshut. According to the animal keepers, the lynx ate and behaved normally for five days. He then suddenly stopped eating and was found dead one day later. At necropsy, the animal appeared very emaciated and dehydrated, and a congenital diaphragmatic hernia with following displacement of a liver lobe into the pericardial sac was diagnosed (peritoneo -pericardial hernia). 3) In 1998, an adult male was caught in the canton of Berne and equipped with a radio -collar. At capture, the biologists noticed a prognathism of the lower jaw. The animal died two years later for reasons independent of this malformation. 4) In the frame of the translocation project, an adult male from the canton of Vaud was caught in a box trap in early spring 2001 but not translocated. At clinical examination, it appeared that this animal was monorchid. 5) Another adult male ( Vino ) from the canton of Fribourg was caught the same year and translocated. At clinical examination, this animal presented a discrete heart murmur. Since this lynx seemed rather old but was in a good general condition, and no congenital abnormalities had ever been reported, neither from lynx from Switzerland nor from any other This manuscript is repro duced in the IVIS website with the permission of EAZWV

13 Eurasian lynx population, he was considered as healthy and was translocated. During the two following years, intensive radio-tracking revealed no abnormality, neither of feeding nor of social behaviour. However, at re-capture in April 2003, the biologists noticed a very enlarged abdomen and an abnormally slow response to anaesthetics. About one month later, the lynx almost stopped to move and caught small prey only; he was found dead two weeks later. At necropsy, the main findings were an accumulation of fluid in all body cavities as well as in the subcutaneous tissues, and a highly fibrotic liver consequently to a circulatory failure. A subaortic stenosis, arteriosclerosis and severe fibrosis were found in the heart. 6) During the fall 2002, a juvenile female was caught on a farm close to the village of Spiez in the canton of Berne and was brought to the wildlife rescue centre of Landshut. At clinical examination, the animal was emaciated but otherwise healthy. During the following days, it ate and behaved normally. Five days after its arrival to the station, the animal keeper found it laying on the ground in agony. Vomit was found everywhere in the enclosure. The animal was hypothermic and dehydrated, and showed an elevated respiratory rate. Attempts to treat it failed. Necropsy revealed a very large esophageal hiatus with no sign of any old or recent trauma, thus indicating a congenital malformation. According to the clinical symptoms, it was assumed that a part of the stomach had been temporarily displaced into the mediastinum (hiatal hernia). 7) Furthermore, a young adult female from the Bernese Alps translocated in 2001 has not given birth to any kitten so far, although intensive radio-tracking showed a normal behaviour and evidence of meetings with the adult male Vino during the breeding season for the two past years. According to the knowledge gathered up to now, this lack of reproduction appears as very unusual and we suspect either this female or the male to be infertile. Genetic analysis The heterozygosity value was higher in the autochthonous source population than in the reintroduced populations in the Jura Mountains and in the Alps (0.63 vs 0.54 and 0.42). The same was true for the mean number of alleles per locus (4.65 vs 3.47 and 2.87). In the Jura Mountains, 27% and in the Alps 39% of the alleles are missing today compared to the source population. The proportion of rare alleles (having a share of less than 20%) dropped from 56% to 38%. Discussion Intensive monitoring of the Swiss lynx populations during the past 20 years by means of captures, radio-tracking and post-mortem examinations hadn t revealed any congenital abnormalities until Since then, one lynx from the Jura mountains and five from the northwestern Swiss Alps were found to present inborn defects: one juvenile suffered of skeletal deformities, two died of the consequences of a diaphragmatic hernia, one adult male is cryptorchid, one presented a deformity of the lower jaw, and another one a heart defect. Furthermore, there are indications that an adult female (or a male) from the Alps might be infertile. Cryptorchidism can have different etiologies, including genetic factors. Low genetic diversity has indeed been shown to be responsible for a high prevalence of cryptorchidism within the Florida panther population (13, 17). Th e incidence of cryptochidism in this puma subspecies has increased dramatically from 0% of pre birth to 80% of the males born after 1989 and is associated with documented consanguineous matings. However, other factors can result in cryptorchidism, including prolonged breech labor, navel infections during testicular descent, exposure to the fetus to an increased maternal estrogen concentration, or antiandrogenic chemicals, or maternal vitamin A deficiency during fetal development (review in 7). The prevalence of cryptorchidism in non-inbred domestic animals varies by species and breed from 1-2% (review in 13). In apparently non -inbred mountain lions (F. concolor), Barone et al. (1) found a prevalence of cryptorchidism of 3.7%. Subaortic stenosis is one of the most common congenital heart disease in dogs but the condition is uncommon in the cat. Subaortic stenosis was shown to be hereditary in Newfoundland dogs and is likely to be hereditary in other breeds (10). However, the etiology of this condition is not fully understood. This manuscript is repro duced in the IVIS website with the permission of EAZWV

14 Diaphragmatic hernias in veterinary medicine are mostly traumatic in origin, but developmental and heredity etiologies have been identified in several species (11, 22, 24). Hiatal hernias occur in a variety of dog and cat breeds; however, males and Chinese sharpei dogs appear to be predisposed to this condition. These congenital hernias are usually the consequence of a lax or stretched phrenicoesophagal ligament (8). Congenital pericardioperitoneal diaphragmatic hernias are not uncommon in domestic cats, and longhaired breeds seemed to be predisposed to this condition (25). This malformation is believed to be the result of a teratogen, genetic defect, or prenatal injury (9). Skeletal abnormalities can have diverse origins, e.g. nutritional imbalance, metabolism or endocrine dysfunction, and genetic defect. In humans many constitutional disorders of the bones occur as sporadic cases and represent new mutations or the expression of rare recessive traits; the same is true in animal populations. Abnormal metaphyseal modelling, scoliosis, fusion of vertebrae and prognathism are all malformations described in cats and/or dogs that might be inherited (5, 12). Small populations are susceptible to genetic deterioration through genetic drift, which may lead to the loss of genetic variability and the expression of deleterious, recessive alleles. Our genetic analyses revealed a remarkably reduced genetic variability, especially in the Alpine population. Interestingly, except the first case, all animals observed with congenital malformations so far belonged to the Alpine population. The congenital defects recently observed in lynx from Switzerland were so far all isolated cases and might be just accidental findings without any hereditary origin. The reported prevalence is very low and might be considered as normal in comparison with observation in domestic animal populations. However, the emergence of such defects in the past years is worrying. Indeed, a retrospective study performed in Sweden on more than 500 lynx necropsies revealed only one case of skeletal disorder (a scoliosis); congenital malformations of the diaphragma, heart, genitals or any other organ were not observed (Ryser-Degiorgis et al., in prep.). To our knowledge, data from other Eurasian lynx populations are not available, making further comparisons impossible. Furthermore, prevalence of congenital malformations in Swiss lynx might indeed be fully underestimated: All three adult lynx were radio -collared, and two of three juveniles were found in villages as orphans looking for food; these five animals might not have been found in other circumstances. In field studies about wildlife diseases, animals that die due to human activities (e.g. traffic accidents, hunting) are more likely to be found than diseased animals that might hide in retired areas of their habitat. In a retrospective study performed on Eurasian lynx from Switzerland by Schmidt-Posthaus et al. (21), it was shown that the distributions of causes of mortality clearly differed between the radio -tagged animals and the animals found by chance and it was assumed that radio -tagged animals represent better the actual situation in the wild because these animals are found independent of the cause of death. Overall, 20% of the lynx died of infectious diseases; however, based upon the data from radio-tagged animals, 40% died due to infections. This figure might be true for congenital malformations as well. Telemetry studies of lynx populations are over in both the Jura Mountains and the northwestern Swiss Alps and detailed data won t be available in the future any more. If the prevalence of congenital malformation increases, it might stay undiscovered. On the basis of the reported observations and of the actual knowledge, the link between congenital defects and inbreeding can neither be confirmed nor refuted. Our goal is not to be alarming but vigilant. Therefore, we suggest giving particular attention to the genital organs, the heart, the diaphragm and the skeleton during future clinical and post-mortem examinations of Eurasian lynx from Switzerland as well as from other countries. Furthermore, we advise to be cautious regarding genetic diversity in the frame of future reintroduction projects. Acknowledgements Many thanks go to all people who helped in any way to collect these data, in particular (in alphabetical order): Philipp Burri, Martin Janovsky, Cristina Köppel, Daniel Mayer, Nadia Robert, Michel Schmidt, Kuno von Wattenwil, Christian Willisch, and Fridolin Zimmermann. This manuscript is repro duced in the IVIS website with the permission of EAZWV

15 References 1. Barone MA, Roelke ME, Howard JG, Brown JL, Anderson AE and Wildt DE. Reproductive characteristics of male Florida panthers: comparative studies from Florida, Texas, Colorado, Latin America, and North American Zoos. J Mammal 1994; 75(1) Breitenmoser U, Breitenmoser -Würsten Ch and Capt S. Re-introduction and present status of the Lynx (Lynx lynx ) in Switzerland. Hystrix 1998; 10: Breitenmoser-Würsten, Ch and G. Obexer-Ruff. Population and conservation genetics of two re-introduced lynx (Lynx lynx) populations in Switzerland a molecular evaluation 30 years after translocation. Proc 2nd conf on the status and conservation of the Alpine lynx population, Amden, Switzerland 2003; Burgman MA, Ferson S and Akçakaya HR. Risk assessment in conservation biology. 1 st ed London. Chapman & Hall 1993; Byrne MJ and Byrne GM. Inheritance of 'overshot' malocclusion in German shorthaired pointers. Vet Rec 1992; 130(17): Carmicheal LE., Clark W and Strobeck C. Development and characterization of microsatellite loci from lynx (Lynx canadiensis), and their use in other felids. Mol Ecol 2000; 9: Dunbar MR, Cunningham MW, Wooding JB and Roth RP. Cryptorchidism and delayed testicular descent in Florida black bears. J Wildl Dis 1996; 32(4): Fossum TW, Hedlund CS, Hulse DA, Johnson, AL, Seim HB III, Willard MD and Carroll GL. Small animal surgery St. Louis, Missouri. Mosby -Year Book, Inc 1997; Fossum TW, Hedlund CS, Hulse DA, Johnson, AL, Seim HB III, Willard MD and Car roll GL. Small animal surgery St. Louis, Missouri. Mosby -Year Book, Inc 1997; Fossum TW, Hedlund CS, Hulse DA, Johnson, AL, Seim HB III, Willard MD and Carroll GL. Small animal surgery St. Louis, Missouri. Mosby -Year Book, Inc 1997; Fox RR and Crary DD. Hereditary diaphragmatic hernia in the rabbit. Genetics and Pathology. J Hered 1973; 64: Jezyk FP. Constitutional disorders of the skeleton in dogs and cats. In: Newton DC and Nunamaker DM. Textbook of small animal orthopedics , Chapter 57. HYPERLINK Mansfield KG and Land ED. Cryptorchidism in Florida panthers: prevalence, features, and influence of genetic restoration. J Wildl Dis 2002; 38(4): Menotti-Raymond M and O'Brien SJ. Evolutionary conservation of ten microsatellite loci in four species of Felidae. J Hered 1995; 86: Menotti-Raymond M, David VA, Stephens JC, Lyons LA and O'Brien SJ. Genetic individualisation of domestic cats using feline STR loci for forensic applications. J Forensic Sci 1997; 42: Menotti-Raymond M, David VA, Lyons LA, Schaffer J., Hutton MK and O'Brien SJ. A genetic linkage map of microsatellites in the domestic cat (Felis catus ). Genomics 1999; 57: Roelke ME, Martenson JS and O Brien SJ. The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Curr Biol 1993; 3: Ryser-Degiorgis M-P, Lutz H, Bauer K, Sage H, Ryser A, Zimmermann F, Breitenmoser -Würsten Ch, and Breitenmoser U. Veterinary supervision of lynx translocation within the Swiss Alps. Proc EAZWV and EWDA scientific meeting, Heidelberg, Germany 2002; Ryser-Degiorgis M-P, Bröjer C, Hård af Segerstad C, Bignert A, Bornstein S, Gavier-Widén D, Jansson D and Mörner T. Assessment of the health status of the free-ranging lynx population in Sweden. In prep. 20. Ryser A, von Wattenwyl K., Ryser-Degiorgis M-P, Willisch Ch, Zimmermann F and Breitenmoser U. Luchsumsiedlung Nordostschweiz , Schlussbericht Modul Luchs des Projektes LUNO. KORA - Bericht 2004; 22: in press. 21. Schmidt-Posthaus H, Breitenmoser-Würsten Ch, Posthaus H, Bacciarini L and Breitenmoser U. Causes of mortality in reintroduced Eurasian lynx in Switzerland. J Wildl Dis 2002; 38(1): Valentine BA, Cooper BJ, Dietze AE and Noden DM. Canine congenital diaphragmatic hernia. J Vet Intern Med 1988; 2(3): von Arx M, Breitenmoser -Würsten Ch and Breitenmoser U. Status and distribution of the Eurasian lynx (Lynx lynx ) in Europe in KORA -Bericht 2004; 19: in press. 24. Wilson G and Muir W. Diaphragmatic hernias. In: Bojrab MJ (ed). Current techniques in small animal surgery. 2 nd ed Philadelphia, Pennsylvania. Lea and Feiber 1983; Wotton PR and Gruffydd-Jones TJ. The cardio-vascular system. In: Chandler EA, Gaskell CJ and Gaskell RM (eds). Feline medicine and therapeutics. 2nd ed London. Blackwell Sciences Ltd 1994; 398. This manuscript is repro duced in the IVIS website with the permission of EAZWV

16 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. FEEDING TRIALS WITH A GROUP OF CAPTIVE ANOA (Bubalus depressicornis) AND A DIET SURVEY K. FLORES-MIYAMOTO 1,M. CLAUSS 2, S. ORTMANN 3 and A. W. SAINSBURY 1 Affiliation: 1. The Institute of Zoology, Zoological Society of London, London, UK 2. Institute of Animal Physiology, Physiological Chemistry and Animal Nutrition, Schönleutner Str. 8, Oberschleissheim, Germany 3. Institute of Zoo and Wildlife Research (IZW) Berlin, Germany Poster abstract On the one hand, members of the Bovini genus are classified as grazers. On the other hand, smaller species of ruminants are not expected to be able to digest particularly fibrous diets and are more often classified as intermediate feeders or browsers. Anoas (Bubalus spp.) are interesting in this respect as they are the smallest representatives of the Bovini, being 5-10 times smaller than other species of the same tribe. A feeding trial was carried out with four lowland anoas (Bubalus depressicornis) at London Zoo, investigating diet digestibility by total faecal collection and passage rates by the simultaneous administration of a fluid (Co -EDTA) and a particle (Cr-mordanted fibre <2mm) marker. The diet consisted of legume hay, dairy cow pellets, browse, and fruits and vegetables. The achieved digestibility coefficients averaged 70 ± 4 % for dry matter and 57 ± 7 % for cell walls (NDF). Mean retention times for the total gastrointestinal tract were 25 ± 4.1 h for fluid and 39 ± 6.7 h for particles, respectively. The ratio of forestomach particle:fluid retention was 2.14 ± Additional information regarding anoa diets in captivity was collected through a survey targeting all institutions that currently have anoa in their collection. Suitability of the provided diet was evaluated using the ratio of unstructured:structured feeds (unstructured feeds being pellets, grains and produce; structured feeds being roughage and browse) on a dry matter basis and an assumed complete consumption of offered unstructured diet items, with only the remaining intake capacity being met by structured items. The use of this ratio reliably predicted one facility that reported chronic diet-related problems. As other ruminants, anoas should receive a diet with restricted amounts of concentrates and fruits. Both the comparatively high fibre digestibility and the high selective particle retention in the forestomach suggest a classification of an intermediate/grazing ru minant. The lowland anoa might represent a typical example of insular dwarfism; its original grazer-type digestive strategy allowed it to exploit a feeding niche typical for intermediate feeders/browsers.

17 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. ENERGY AND MINERAL NUTRITION IN CAPTIVE HIPPOPOTAMIDAE A. SCHWARM 1, M. CLAUSS 2, S. ORTMANN 1, J. CASTELL 2, E. J. FLACH 3, B. R. KÜHNE 4, C. TACK 3 and H. HOFER 1 Affiliation: 1. Institute of Zoo and Wildlife Research (IZW), Alfred-Kowalke-Str. 17, Berlin, Germany 2. Institute of Animal Physiology, Physiological Chemistry and Animal Nutrition, Munich, Germany 3. The Zoological Society of London, Whipsnade Wild Animal Park, Bedfordshire, UK 4. Zoological Garden Berlin, Germany Poster abstract The captive management of the common (Hippopotamus amphibius) and the pygmy hippopotamus (Hexaprotodon liberiensis) is characterised by few veterinary or dietary problems. With increasing attention to possible overfeeding and obesity of animals kept in zoos, the adequacy of the usual dietary regime with a high proportion of energy-dense feeds such as bread or concentrates (2) should be re-evaluated. Hippos are a particularly challenging taxon for this purpose, since (a) conventional assessments that use body mass are difficult to apply to male common hippos because males grow indefinitely (3), and (b) owing to their amphibious lifestyle, digestion trials are difficult to perform. This study was designed to assess the digestibility of diets that include or exclude concentrates and supplements. The experiments were carried out with four common and four pygmy hippos at Whipsnade and Berlin Zoological Garden. All animals were considered to be in good body condition and did not have a history of any dietary problems. Animals received either a regular diet of roughage and concentrates (RC diet) followed by a roughage -only diet (R diet), or a diet of fresh grass in restricted amounts (restr. diet) followed by a diet of fresh grass in unrestricted amounts (ad lib. diet). In a pilot study, the feasibility of such trials using acid detergent lignin as an internal marker had been established (4). Pygmy hippos were weighed on a scale; body weights (BW) of common hippos had to be estimated. All feed items offered to and rejected by study animals were weighed and faeces regularly collected. Feeds and faeces were analysed for calcium (Ca), phosphorus (P), sodium (Na) and gross energy content (GE). Dry matter intake was low compared to other herbivores and ranged from % BW in common and % BW in pygmy hippos. Mass-specific digestible energy (DE) intake ranged from MJ/kg 0.75 in common and MJ/kg 0.75 in pygmy hippos. Highest values were obtained for RC diets. DE intake for the ad lib. diet was about 0.3 MJ/kg 0.75 in common and 0.2 MJ/kg 0.75 in pygmy hippos. Ca, P and Na contents of the rations consumed were adequate by ruminant standards except for the R diet where Na supply was marginal. The results indicate that known mass-specific DE intake values from domestic animals (e.g. horse DE maintenance requirement of 0.6 MJ/kg 0.75 ) are not suitable to predict the lower energetic needs of hippos, supporting the idea that hippos may have lower metabolic rates than the mammalian average (1). The good health and body condition of animals receiving the fresh grass diets indicate that a higher energy provision by the addition of concentrate feeds may be unnecessary; ideally, this should be corroborated by regular weighing of the animals. Data on the Na content of the diets used suggest that mineral supplementation may be necessary; this could be achieved by providing a mineral supplement rather than a complete pelleted feed.

18 References 1. Eltringham SK. The Hippos, Natural History and Conservation. Academic Press, UK, 1999; 2. Frost J. A survey of the Hippopotamuses in captivity in the British Isles. North of England Zoological Society 3. (ed.), Zoological Gardens Chester Marshall PJ and Sayer JA. Population ecology and response to cropping of a hippopotamus population in eastern 5. Zambia. J appl Ecol 1976; 13: Schwarm A, Clauss M, Flach EJ and Tack C. Passage rate and digestibility coefficients in captive hippopotamidae a pilot study. Verh ber Erkrg Zootiere 2003; 41:

19 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. EVALUATION OF LEMUR NECROPSY REPORTS WITH SPECIAL REFERENCE TO HAEMOSIDEROSIS C. SCHWITZER 1, N. PETERS 1,2, W. KAUMANNS 1, O. BEHLERT 1 and M. CLAUSS 3 Affiliation: 1. Zoologischer Garten Köln, Riehler Str. 173, Köln, Germany 2. Institute of Comparative Tropical Medicine and Parasitology, Munich, Germany 3. Institute of Animal Physiology, Physiological Chemistry and Animal Nutrition, Munich, Germany Poster abstract Individual cases of excessive iron deposits in lemurs have been reported as early as the 1960 s (2,5). This was later recognized to be a common problem in captive individuals (1,4,7). After screening material from a number of American and European facilities, Benirschke et al. (1) concluded that in all adult lemurs iron deposits were found. Additional observations of haemosiderosis in captive lemurs have recently been reported (3,6). In order to evaluate the scope of the problem, we evaluated necropsy reports for captive lemurs at Cologne Zoo, Germany. Sixty-nine necropsy reports were available, covering a time period from 1978 to 2003 and 13 species of lemurs altogether. The reports included 25 newborns/juveniles, 35 adult individuals and 9 individuals of unknown age. Among the newborns/juveniles, more than 50% of cases were due to a bacterial infection; the same was true for adult individuals as well. Neoplasia was the diagnosed cause of death in 23% of adult lemurs. In only 6 cases (17%) was haemosiderosis reported in the post mortem record. An absence of haemosiderosis was never noted. Haemosiderosis occurred with liver tumors, as reported earlier (2), and with liver rupture. The youngest animal in which haemosiderosis was reported was 11 years old. These results allow to different interpretations: either haemosiderosis was detected in all cases in which it was looked for, but was only looked for in a limited number of cases; or haemosiderosis was detected only in a limited number of cases, but the according (negative) investigations were not documented on the necropsy reports. Given the fact that other negative observations were, however, noted on the reports, the interpretation that haemosiderosis was found whenever it was looked for seems more likely. For the evaluation of species-specific diseases, such as haemosiderosis/haemochromatosis in lemurs, it is prerogative that necropsy reports address these diseases specifically, noting not only the observation, but also the absence of any indication for the disease in question. References 1. Benirschke K, Miller C, Ippen R and Heldstab A. The pathology of prosimians, especially lemurs. Adv Vet Sci Comp Med 1985; 30: Brygoo ER, Levaditi J, Destombes P and Guillon JC. Adéno-cancer avec «cirrhose bronzée» observé à Madagascar chez un Lemur m. macaco. Bull Soc Pathol Exot 1964; 57: Clauss M, Lechner-Doll M, Hänichen T and Hatt JM. Excessive iron storage in captive mammalian herbivores a hypothesis for its evolutionary etiopathology. Proc Sci Meeting EAZWV 2002; 4: Gonzales J, Benirschke K, Saltman P, Roberts J and Robinson PT. Hemosiderosis in lemurs. Zoo Biol 1984; 3: Griner LA Pathology of zoo animals. San Diego: The Zoological Society of San Diego. 6. Schwitzer C, Dunkel V and Lork A Nahrungsaufnahme und verwertung bei Lemuren. Z Kölner Zoo 45: Spelman LH, Osborn KG and Anderson MP Pathogenesis of hemosiderosis in lemurs: role of dietary iron, tannin, and ascorbic acid. Zoo Biol 8:

20 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. PULMONARY ADENOMATOSIS IN TWO SUGAR GLIDERS (Petaurus breviceps) M.F. BERTELSEN 1,2,3, G.J. CRAWSHAW 1 AND I.K. BARKER 2 Affiliation: 1. Toronto Zoo, 361A Old Finch Avenue, Scarborough, Ontario M1B 5K7, Canada 2. Ontario Veterinary College, University of Guelph, Ontario, Canada 3. Present address: Copenhagen Zoo, Søndre Fasanvej 79, 2000 Frederiksberg, Denmark Poster Abstract Pulmonary adenomatosis was diagnosed in two mature male sugar gliders (Petaurus breviceps) at the Toronto Zoo, Ontario, Canada. The animals were euthanised due to other disease processes. Both animals were born at the Zoo and had been housed in neighbouring enclosures. Case 1 (PM 18128) involved a 7 -year old male sugar glider euthanised December 10 th 2002 due to severe cloacitis. Grossly both lungs were found to contain a single raised tan nodule of 2-3mm. Histologically the pulmonary masses consisted of glandular-looking tissue forming cystic spaces lined by tall epithelial cells and more solid areas of adenomatous tissue. Within the spaces formed by the mass was a mixture of liquid, proteinaceous debris and basophilic granular material forming cholesterol clefts. In the more solid areas the roughly cuboidal cells formed tubules containing large foamy macrophages, granular material with cholesterol clefts and heterophils. There was a gradual transformation from normal ciliated bronchial lining to large irregular cuboidal cells, with three -fold anisokaryosis. No mitotic figures were observed. Case 2 (PM 18201) was a 9-year old male sugar glider euthanised march 14 th 2003 due to severe osteolysis of the mandible and cerebral cryptococcosis. Grossly the lungs were considered normal. On histological evaluation a single, fairly well-demarcated area of adenomatous tissue of approximately 0.4 x 0.5 mm was identified. The roughly cuboidal cells formed tubules and papillomatous protuberances intermixed with low numbers of foamy macrophages. Long cords of epithelial cells extend from the mass into the surrounding lung tissue, lining the adjacent airways. There was mild generalised pulmonary oedema, and in many areas the interstitium contained accumulations of foamy, slightly granular material and low numbers of heterophils. No mitotic figures were observed. The histological lesions in both cases were similar, and closely resemble those of jaagsiekte in sheep (1), and of pulmonary adenomatosis in Virginia opossums (Didelphis virginiana) (3, 4). In sheep the disease is induced by a retrovirus, while the etiology of the opossum disease is unresolved. An attempt to amplify DNA extracted from Case 1 with degenerate primers targeting a region from the viral protease to the reverse transcriptase gene according to (2) yielded two bands of ~830 and ~650 bp. Both were cloned and sequenced. However analysing these sequences failed to identify similarities with known retrovirus genomes. Key words: Sugar glider, Petaurus breviceps, pulmonary adenomatosis, tumour, neoplasia, lung Acknowledgements We thank D Bienzle and X Wen for assistance with PCR analysis.

21 References 1. Dungworth DL. The respiratory system. In: Jubb KVF, Kennedy PC and Palmer N (eds.) Pathology of Domestic Animals. Academic Press, Toronto, 1993; Herniou E, Martin J, Miller K et al. Retroviral diversity and distribution in vertebrates. J Virol. 1998; 72, Kim DY, Mitchell MA, De Las Heras M et al. Spontaneous squamous cell carcinoma of t he tongue and multiple bronchioalveolar carcinomas in a Virginia opossum (Didelphis virginiana). J Comp Path 2002; 126, Sherwood BF, Rowlands DT and Hackel DB. Pulmonary adenomatosis in opossums (Didelphis virginiana). J Am Vet Med Assoc 1969; 155,

22 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. CASEOUS LYMPHADENITS IN A CAPTIVE GROUP OF ALPINE IBEX (Capra ibex) - MANAGEMENT AND IMPLICATIONS S. SILINSKI and C. WALZER Affiliation: Zoo Salzburg, Natur- und Artenschutzzentrum, Anifer Landesstr. 1, 5081 Anif, Austria Extended Abstract In a group of 23 ibex (Capra ibex), held in a private park situated in an alpine environment, several individuals with recurrent swelling of the retropharyngeal and prescapular lymph nodes were observed. Necropsy of one affected animal revealed severe abscessation of the lungs. Caseous lymphadenitis (CLA) was suspected. The diagnosis was confirmed by bacterial culture of C. pseudotuberculosis in caseous aspirates of abscessed lymph nodes. CLA, a contagious bacterial disease, is caused by Corynebacterium pseudotuberculosis - a well-recognised pathogen of farm animals, particularly sheep and goats. Clinical signs include swelling and abscessation of superficial lymph nodes, but dissemination of the bacillus to viscera, most commonly the mediastinal lymph nodes or lungs, often results in secondary respiratory disease. CLA can spread rapidly through a herd and is difficult to eradicate due to its poor response to treatment and the ability of C. pseudotuberculosis to survive for extended periods in the environment. Therefore, identification of infected animals and their removal from the group is considered the most effective method of control (1, 5). When considering CLA control in the ibex population of this private park, economic constraints had to be taken in account. In order to determine the rate of infection in the group blood samples were drawn from 20 ibex (3 animals could not be sampled) and serologically screened for antibodies against C. pseudotuberculosis by ELISA. Anaesthesia also permitted a thorough clinical examination. Similar to a previous report in a group of infected dwarf goats at the Zoological Garden Basel, clinical symptoms and serology were poorly correlated (2), Animals with a positive or a doubtful result were separated from the group and will be culled (e.g. trophy hunting) as soon as possible. As vaccination against CLA may reduce the prevalence and number of abscesses in an endemic group by more than 70%, all animals were vaccinated with an auto-vaccine (3). However, keeping in mind the zoonotic potential (4) and risk for wild ruminant populations (Capra ibex and Rupicapra rupicapra) living in the surroundings of the park any form of therapy and management is more than questionable. Legally, it was not possible to prevent the therapy of CLA in this group. Key words: Caseous lymphadenitis, CLA, Corynebacterium pseudotuberculosis, Capra ibex Acknowledgements We thank Markus Kutscher for his assistance and entertainment during the sampling procedure. References 1. Anderson DE, Rings DM and Pugh DG. Caseous lymphadenitis.in: Pugh DG (ed). Sheep and Goat Medicine. WB Saunders Company Philadelphia 2001; Bacciarini LN, Völlm J, Janovsky M, et al. Corynebacterium pseudotuberculosis in the Zoological Garden Basel, Switzerland: Detection and eradication strategies. Proc EAZWV 4 th scientific meeting, Heidelberg 2002; Paton MW, Sutherland SS, Rose IR, et al. The spread of Corynebacterium pseudotuberculosis infection to unvaccinated and vaccinated sheep. Aust Vet J 1995; 72(7): Peel MM, Palmer GG, Stacpoole AM, et al. Human lymphadenitis due to Corynebacterium pseudotuberculosis: report of ten cases from Australia and review. Clin Infect Dis 1997; 24(2): Williamson LH. Caesous lymphadenitis in small ruminants. Vet Clin North Am Food Anim Pract 2001; 17(2): Bush M, Montali RJ, Brownstein D, et al. Vaccine-induced canine distemper in a lesser panda. J Am Vet Med Assoc 1976; 169(9):

23 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. SPIRURID INFECTION IN CAPTIVE GOELDI`S M ONKEYS A. FLOTO 1, K. MÄTZ-RENSING 1, C. EPE 2, F.-J. KAUP 1 and E. ZIMMERMANN 3 Affiliation: 1. Department of Infectious Pathology, German Primate Center, Kellnerweg 4, Göttingen, Germany. 2. School of Veterinary Medicine Hannover, Germany. 3. Zoo Dresden, Germany. Abstract Necropsy of a Callimico Goeldii at the German Primate Center confirmed the suspicion of Spiruridiasis in a group of Goeldi`s monkeys from Dresden Zoo. The parasites were mainly located in the epithelium of the lips, but also in hyperkeratotic lesions around the nose. Specific and consequent treatment of still infected monkeys primarily reduced the clinical signs and finally cured the animals from parasite infection. Key words: Goeldi`s monkey, Callimico goeldii, Spiruridiasis. Introduction A Goeldi`s monkey (Callimico goeldii) from the Dresden Zoo, was send to the Ge rman Primate Center for necropsy after dying by accident. Oral, perioral and nasal alterations, including swelling, edema and emphysema were reported during lifetime. The macroscopic examination revealed lots of nematodes (up to 1 cm in length) in and on the mucous membranes of the oral and pharyngeal cavity. Histologic and parasitologic examination confirmed the suspicion of Spirurid infection: the nematodes were predominantly found in the lips and the tongue, causing only mild inflammation (1, 2). Nematode species that belong to the group of Spirurida have an indirect life cycle. They have a wide variety in final hosts (mammal species), intermediate hosts (arthropods) and sites of infection (mucous membranes of alimentary tract) (3, 5). In order to their route of infection they may course severe oral inflammation especially after secondary bacterial colonisation (3). In our case the Dresden Zoo cured the monkeys with consequent ivermectin treatment and by eradicating the intermediate host (cockroach population) at the same time. Materials and Methods In spring 2003 a Goeldi`s monkey (female, 1 year old) from the Dresden Zoo was necropsied at the German Primate Center. Prior to death the monkey had shown swollen lips and nasal lesions covered with crusts. As reported from the owner clinical signs were first observed in August 2002, leading to take a swap from the alterated tissue. The probe was positive for Staphylococcus sp. and a local treatment with antibiotics was performed. To ease the breathing, which became more and more difficult because of the hyperkeratotic epithelium around the nose, the overlying crusts were removed frequently. In the following weeks the alterations almost disappeared but there was no complete healing achieved. The swollen and hyperaemic lips were still remarkable when the animal finally came to the Primate Center. Results Clinical history Retrospectively, similar clinical signs were seen in the parents of the monkey. They were observed in May 2002 shortly after the birth of the junior. Both animals developed

24 hyperkeratotic and hyperaemic alterations accompanied by depigmentation of oral and nasal epithelium. Swaps were taken and probes were positive for Pseudomonas sp. The monkeys were treated with several antibiotics (Baytril/Citrofloxacin), but the recovering was only slowly proceeding. In November 2002 the male animal was secondly affected. His lips were hyperaemic and swollen up to 3-4 mm, which was probably enforced by continuously rubbing and scratching. Multiple lesion s with corrugated surfaces, mainly in the angle of the lips, were maybe the reasons why the monkey denied fruits, because the fruit acids were supposed to burn during food intake. In order to that the monkey lost lots of weight having also flatulence and constipation. Applied parasitologic examination of faecal samples were negative in all three affected monkeys. Post mortem The sudden death of the young monkey facilitates the decision to perform necropsy at the German Primate Center with subsequent histological, bacteriological and parasitological examinations. Already macroscopically the infection with Spirurida sp. was assumed, because of the typical hyperaemic and swollen oral tissues as well as the overgrowing crusts especially on the mukocutaneous membranes of the lips and nasal region. Representative tissue samples were fixed in 10% neutral buffered formalin, embedded in paraplast, sectioned and finally stained with H&E. The histologic examination confirmed the suspicion: multiple sections of adult worms and parasite eggs were found in the epithelium of lips and tongue. Reactive inflammatory cell infiltration in the surrounding tissues was only minimal or not seen. Spirurida identification finally was performed in the Department of Parasitology in the School of Veterinary Medicine in Hannover. The parasites were mainly differentiated by the presence of the anterior cuticular bosses, but also in order to the specific male morphology. Discussion Spiruridiasis is a rare, but widespread infection not on ly in nonhuman primates. There are reports about Spirurid spp. in ruminants, pigs, horses and even in humans (2, 5). In most cases the parasites are localised in the mucous membranes of oral epithelium, pharynx and larynx, but dependent on the species there are also manifestations in oesophagus, lung and stomach known (1, 2). The parasites belong to the order Spirurida and have an indirect life cycle with a regular development in specific final hosts and specific intermediate hosts. The known and proved intermediate hosts are dung beetles and cockroaches, which acquire the parasites for the development of their third stage larvae (3). In our case the Dresden Zoo was able to control and eliminate the invasive cockroach population which was assumed to be the main vector for Spirurid infection in the Goeldi`s monkeys. Additionally the animals were regularly treated every 14 days with ivermectin (0,2 mg/kg of body weight). Clinical signs were removed after a 8 week application period. Acknowledgements The autho rs thank N. Knöchelmann and H. Zuri for their perfect laboratory assistance. References 1. Brack M. Gongylonematiasis in the Common Marmoset (Callithrix jacchus). Lab Anim Sci 1996; 46 (3): Craig LE, Kinsella JM, Lodwick LJ, Cranfield MR and Strandberg JD. Gongylonema macrogubernaculum in captive African Squirrels (Funisciurus substriatus and Xerus erythropus ) and Lion-tailed Macaque (Macaca silenus ). J Zoo Wildl Med 1998; 29 (3): Duncan M, Tell L, Gardiner CH and Montali R. Lingual gongylonemiasis and pasteurellosis in Goeldi`s Monkeys (Callimico Goeldii). J Zoo Wildl Med 1995; 26: Montali RJ, Gardiner CH, Evans RE, Bush M. Pterygodermatites nycticebi (Nematoda: Spirurida) in Golden Lion Tamarins. Lab Anim Sci 1983; 33(2): Yue MY, Jensen JM, Jordan HE. Spirurid infections (Rictularia sp.) in Golden Marmosets (Leontopithecus rosalia). J Zoo An Med 1980; 11:

25 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. GASTRIC AMEBIASIS DUE TO ENTAMOEBA HISTOLYTICA INFECTION IN A MANDRILL (Mandrillus sphinx) K. MÄTZ-RENSING 1, A. FLOTO 1, F.-J. KAUP 1 and K. SCHALLER Affiliation: 1. Department of Infection Pathology, German Primate Center, Kellnerweg 4, D Göttingen, Germany. 2. Westfälischer Zoologischer Garten Münster GmbH, Germany. Abstract A six year old female captive mandrill (Mandrillus sphinx) died unsuspected. Main pathologic alterations were found in the gastric mucosa. Histopathologic examination revealed numerous Entamoeba trophozoites within the gastric mucosa and less frequent within the gastric submucosa and submucosal vessels. Prospect microplate assay testing confirmed the identity of the trophozoites as Entamoeba (E.) histolytica. E. histolytica most commonly causes necrotizing colitis in humans and captive nonhuman primates. Gastritis is described in primates with sacculated stomachs. In vivo diagnosis and identification of asymptomatic carriers shedding infective cysts are difficult. E. histolytica can therefore cause a self perpetuating problem in housing facilities. The zoonotic potential of nonhuman primate born amebiasis must also be kept in mind for public health reasons. Diagnosis, therapy and zoonotic potential of this rare disease is discussed. Key words: Amebiasis, Entamoeba histolytica, Gastritis, Mandrillus sphinx Introduction Several parasitic intestinal amoeba species are reported in a number of nonhuman primates (4). Most are considered non-pathogenic with the exception of Dientamoeba species, E. histolytica and Balamuthia mandrillus. E. histolytica is the cause of amebiasis in humans. The parasite has a world wide distribution. Clinical signs of infection may be absent in E. histolytica infection especially in Old World monkeys. The life cycle of this important intestinal parasite is direct. The trophozoites of E. histolytica are µm and those of small strains are µm in diameter. Cysts of both are µm in diameter. Only the large strains are considered to be pathogenic (2). The excystment of the infective cysts appears to take place in the small intestine and the infection is established in the lumen of the large intestine. Ingestion of cysts causes infection and development of trophozoites. The resistant cyst stage is excreted in the feces. Trophozoites reside in the lumen of the intestine and may invade the intestine wall resulting in diarrhea and dysentery. Infections are often transient and are self-limiting in most cases. E. histolytica may invade blood vessels in the large intestine and be transported to other extraintestinal organs, especially the liver, the lung and the central nervous system. A consecutive amebic hepatitis may result. Hematogenous invasion of other organs is rare and nearly always associated with liver abscesses (1). Establishment of disease is multifactorial and depends on host factors and infectious dose.

26 Case report A six year old mandrill (Mandrillus sphinx) named Kaduna was sent to the German Primate Center (DPZ) by the Allwetter Zoo Münster for post mortem examination. The monkey weighted kg and had been ill for two days. She suffered with unspecific symptoms like anorexia, depression and apathia and died unsuspected over night. Gross pathologic findings were a multifocal severe ulcerative gastritis and enlarged kidneys with irregular granular surface and mottled to streaky cut surface as indicator for a chronic interstitial glomerulonephritis. Parasitological examination of gastric and small bowel ingesta showed Entamoeba sp. and Giardia sp.. In the colon only Giardia sp. could be detected. The parasitological results were confirmed by PRoSpect Entamoeba histolytica microplate assay ELISA for the detection of Entamoeba histolytica specific antigen. ProspecT Entamoeba histolytica Microplate Assay ELISA This highly specific and highly sensitive enzyme-immunoassay is established for the detection of E. histolytica specific (EHSA) antigen within feces even in the absence of cysts and trophozoites. The detection limit of EHSA is 40ng/ml. It is a simple and easy to use system based on the test principle of an antigen capture ELISA. EHSA is shed during the reproduction oftrophozoitesan cysts. Histological results Predominant finding in routine histology were multiple confluent ulcera and necrotic foci within the gastric mucosa. In multiple sections of the stomach numerous E. histolytica trophozoites had invaded the mucosa and displaced gastric glands (Fig. 1,2). The invading trophozoites were approximately µm in length and round, oval or amoeboid. The organisms were best demonstrated with the periodic acid-schiff reaction (PAS) or with giemsa stain. They had multifocally invaded the muscularis mucosa, submucosa and were rarely present within submucosal blood vessels. The gastric mucosa overlying the invading trophozoites was eroded. There was severe chronic active inflammation in the surrounding gastric tissue to the organisms. In parallel, the monkey exhibited a severe chronic-active glomerulonephritis. Discussion Amebiasis due to E. histolytica is most frequent in captive nonhuman primates and probably associated with extensive contact to human carriers. In the present case no hints were found for the route of infection. No entamoeba infection of man neither of other animal species within the zoo was reported. Till now it is unclear how the infection was established in this animal. Infection may be asymptomatic or produce mild to severe clinical signs. There is a great variability in virulence among different strains of organisms. Pathogenicity is affected by the host species infected, the nutritional status of the host and environmental factors. Most cases of gastric amebiasis were described in colobus and silver-leaved monkeys, species with a sacculated stomach and a neutral ph in presaccular portions presenting good environment for excystation of amoeba cysts. In the present case it is assumed that the chronic kidney disease may have induced uramic gastric alterations which were invaded secondary by amoeba cysts. Diagnosis and prevention can be difficult due to unspecific clinical signs and absence of cysts or trophozoites in the feces, especially in extraintestinal amebiasis. Symptoms may be anorexia, vomiting, diarrhea and hepatomegalia in cases of hepatic amoebic abscesses. In the case discussed unspecific symptoms like weight loss, apathia and separation from the group two days prior to death were the only clinical findings. Clinical laboratory was not done. In other cases clinical laboratory may reveal leucocytosis, liver enzyme elevations like AP, SGOT, LDH and low serum protein, albumin and cholesterol levels. The treatment of choice for both intestinal and extraintestinal amoebias is metronidazole in a dose of mg/kg/day given orally in three divided doses for 5-10 days. However, metronidazole is bitter in taste and efficient oral administration might fail unless a pediatric sirup is given or the intravenous formula and route is used. This recommended therapy

27 seems to be unpracticable for the treatment of a whole group. In our case a therapy over 3 days with a single dosage was efficient Three negative stools obtained on three successive days are indicative of cure! Reexamination of fecal samples at 1, 3 and 6 months after treatment and screening for asymptomatic carrier shedding cysts is recommended (3). In any case, amebiasis due to E. histolytica should be considered as potentially zoonotic cause of gastrointestinal disease in captive nonhuman primates. Acknowledgements We thank W. Henkel, E. Lischka and H. Zuri for their skilful technical assistance. References 1. Biagi FF and Beltran F. The challenge of amoebiasis: Understanding pathogenic mechanisms. Int Rev Trop Med 1969; 3: Levine ND. Protozoan parasites of non human primates as zoonotic agents. Lab Anim Care 1970; 20: Renquist DM and Whitney RA. Zoonoses acquired from pet primates. Vet Clin North Am Small Anim Pract 1987; 71: Toft JD and Eberhard ML. Parasitic diseases. In: Benett BT, Abee CR and Hendricks R (eds.). Nonhuman Primates in Biomedical Research. Diseases. Academic Press, San Diego, CA, 1998: Figure legends Fig. 1: The alterations were characterized by dense inflammation of a mixed inflammatory infiltrate and several crypt abscess formations (arrow) within the gastric mucosa; Hemalaun & Eosin stain, 376x; Fig. 2: In the center of the alterations multiple entamoeba trophozoites were easily demonstrable within gastric pits and mucosa (arrow), (Fig. 8, 9); PAS-reaction, 376x and 590x.

28 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. SELENIUM AND VITAMIN E DEFICIENCY IN DIFFERENT SPECIES OVER A PERIOD OF 8 YEARS A. LIESEGANG 1 and K. BAUMGARTNER 2 Affiliation: 1. Institute of Animal Nutrition, VETSUISSE. Faculty of the University of Zurich, Winterthurerstr. 260, CH-8057 Zurich, Switzerland 2. Zoo Nürnberg, Am Tiergarten 30, Nürnberg, Germany Abstract Several cases of white muscle disease were observed at the Nürnberg Zoo over a period of 8 years. Due to the loss of animals many analyses were made and a new feeding strategy was developed. This paper presents the practical problems and preliminary results. Key words: Selenium deficiency, vitamin E deficiency, different species, supplementation Introduction Selenium (Se) is always of interest because, in many areas of the world, plants contain concentrations that are either deficient in or toxic to the animal metabolism. One of the major functions of selenium is as a component of the enzyme glutathione peroxidase, which prevents the oxidation of unsaturated fats in cell membranes. When dietary selenium is insufficient to prevent this oxidation, muscle membranes rupture and leak cellular enzymes into the extracellular circulation. These damaged muscles become non-functional and turn white, hence the name white muscle disease. Animals differ in their requirements for selenium and their susceptibility to white muscle disease. Some of these species variations may be due to differences in the amount of nonselenium-dependent glutathione peroxidase activity. Dietary requirements for particular species vary, because high levels of dietary sulphur reduce selenium availability and chlorinated hydrocarbons interfere with selenium metabolism. The primary function of vitamin E (Vit. E) is to maintain the functional integrity of cellular and subcellular membranes, by preventing lipid peroxidation of unsaturated fatty acids (8). Nutritional muscular dystrophy, liver necrosis in swine and rats, foetal resorption in rats, and encephalomalacia in poultry are possible signs of deficiency. Vit. E deficiency has been observed in virtually all taxa of captive wildlife (3), especially in herbivores (4). Although low dietary Se may have been a contributing factor, Vit. E deficiency can develop in herbivores when they are fed hay with a low Vit. E content, due to late cutting, leaching or excessive storage, leading to oxidation of the vitamin. Reductions of 80 to 90% during the maturation of grasses are common. Another problem may be the degradation of Vit. E by gastrointestinal microflora when animals consume high grain diets, often fed in captivity (10). With a rising grain content the destruction of Vit. E increases from 8 to 42%. The Nürnberg area, where most of the forage used at the Nürnberg Zoo is grown, is known to be low in selenium. Although vitamin and mineral supplements were given to the animals at the zoo, there were many animal losses. In addition to clinical and pathological investigations, serum

29 levels were evaluated as often as possible. In this study several cases of white muscle disease in different species and age-groups are discussed. A new strategy was developed: While parenteral treatment of newborns and oral Vit. E/Se supplementation of pregnant animals was continued, higher amounts of Vit. E/Se were added to the zoo pellets that are fed to the herbivores. Material and Methods Summary of cases Over a period of 8 years ( ) 38 animals died of white muscle disease, with a peak of 16 animals in All post-mortems were performed at the Institute of Pathology (LGL) in Nürnberg. Many different species were affected, e.g. antelopes, donkeys, goats, sheep and deer. With the exception of an adult Cameroon sheep, all cases were either newborns, stillbirths or dystocias. Table 1 gives an overview of the different species and the age of the animals at death. Table 1 Animals dying between 1996 and 2003 Species Number Age Ammotragus lervia months Equus asinus asinus 1 1 week Bos taurus 3 1 day Lama glama pacos months Gazella subgutturosa 1 1 day Antelope cervicapra days Rangifer tarandus 2 3 days Lama glama guanicoe 1 1 day Capra hircus 8 1 day to 3 months Ovis aries 7 1 day (1* 5 years) Equus przewalskii 1 stillbirth Elaphurus davidianus 1 1 day Cervus elaphus canadensis 1 1 day Cervus elaphus canadensis 2 dystocia Bison bison 1 4 days Cervus nippon dybowskii Boselaphus tragocamelus days foetus 38 Most of the animals died within 1 or 2 days after birth. Pathological investigation always showed damage of the skeletal and/or myocardial muscles. In some cases stillbirths and dystocias were observed. Especially the adult Cameroon sheep* (see table 1) showed severe myocardial lesions and had to be euthanised after 2 days of unsuccessful treatment with 2 ml Novalgin, 3 ml Catosal, 2.5 ml Tardomyocel compositum, 0.5 ml Acutol, 3 ml Selenit E, 400 ml Ringer, 60 ml Amynin and 2 ml Buscopan compositum. 3 days before these acute symptoms occurred, two females were brought into his enclosure. The mating stress probably triggered the white muscle disease. Two animals died in 1996 and 1997 of white muscle disease, with a peak of 16 deaths in In 1999 and 2000 there were 3 and 4 losses, respectively. Five newborns died in 2001 and 2 in Of the 4 animals lost in 2003, one was an adult Cameroon sheep, the others being a stillborn nilgai antelope and 2 dystocias in a sheep.

30 The animals were fed a species-dependent diet, consisting of grass (summer), hay and silage (winter), pellets and mineral supplements. Tables 2 and 3 list the contents of vitamin E and selenium in the zoo pellets and mineral supplements. Table 2: Pellets and their content of Vitamin E and Selenium Pellets Vitamin E Selenium mg/kg mg/kg Kofu (DEWA, 1998, Emskirchen, Germany) Zoo animal pellets Kofu (DEWA, 2001, Emskirchen, Germany) Zoo animal pellets 520 1,2 Altromin (DEWA, 2000, Emskirchen, Germany) Savanna pellets 90 - Reindeer pellets (Mazuri, Special Diet Service Witham, England) 50 - Goat pellets (DEWA, Emskirchen, Germany) Combi-Pellets for horses (Eggersmann, Rinteln, Germany) 300 0,40 Table 3: Mineral Pellets and their content of Vitamin E and Selenium Mineral Pellets Vitamin E mg/kg Selenium mg/kg Sheep mineral (Salvana, Elmshorn, Germany) Cattle mineral (Salvana, Elmshorn, Germany) Horse mineral (Salvana, Elmshorn, Germany) Pig mineral (RKW Süd, Würzburg, Germany) Salvana PS Bisquits (Salvana, Elmshorn, Germany) 400/bisq. 1.8/bisq. Vitakalk (Marienfelde GmbH, Hamburg, Germany) EL-E-VITE (Mazuri, England) 2000 I.U. - Equistro Super E (IDT, Münster, Germany) Standard treatment and analyses Within the first 2 days all newborn animals are treated with Vit. E/Se parenterally (Selenit-E, WDT: Vit. E 100 mg/ml, Se 450 µg/ml). Beginning in 1997 adult animals with low serum Vit. E concentrations were individually supplemented with El-E-VITE pellets (Mazuri: Vit. E 2000 I.U./kg) or, if serum Vit. E and Se concentrations were low, with Equistro Super E (IDT, Vit. E: mg/kg; Se 10 mg/kg). To control the Vit. E/Se status in the animals on the basis of serum levels, as many blood samples as possible were taken. In the case of one bongo with dystocia, for example, serum levels revealed a low selenium status (Se 42?µg/l). Compared to references for domestic animals this value seemed far too low and the animal was supplemented with Equistro Super E for 3 weeks. This increased the serum concentration slightly (Se 53 µg/l) and the supplementation was continued. All analyses were conducted at the Vet Med Laboratory in Ludwigsburg, Germany (selenium references for cattle: µg/l sufficient, µg/l marginal, <50 µg/l low; for horses: µg/l sufficient, <50 low. Vit. E references for cattle > 3 mg/l, for horses > 1 mg/l, for pigs mg/l). The hay and grass were analysed (AGROLAB Labor für landwirtschaftliche Untersuchungen, Oberhummel, Germany) and contained mg Vit. E /kg DM (in 1985). The grass silage had a Vit. E concentration of 60 mg/kg DM (in 2002). Selenium analyses in adjacent areas gave the following values: Grass silage 1 st cut: GR < , 2 nd cut: GR < mg/kg DM

31 table 4. serum levels in different species at thennürnberg Zoo: Species Age Sex Reason for blood Feed/ Supplement Vit. E Se Year taking mg/l mg/l Taurotragus adult female dystocia Kofu / Vitakalk euryceros Gazella 2 years female lameness Altromin / Salvana bisq subgutturosa Addax naso- 1 year male transport Kofu / Vitakalk maculatus Equus burchelli adult female lameness Kofu / Salvana Bisq bohmi Equus burchelli adult female pregnancy Kofu / Salvana Bisq bohmi testing Equus asinus adult male hoof trimming Kofu / Vitakalk + Salvana somalicus Bisq. Equus asinus 1 week male poor condition Kofu / Vitakalk + Salvana somal icus Bisq. Equus 1 year male transport Kofu / Salvana Bisq przewalski Equus caballus adult female general blood test Combi-Pellets + Goatpellets / Salvana Bisquits Equus caballus adult female general blood test Combi-Pellets + Goatpellets / Salvana Bisquits Cervus nippon adult female dystocia Kofu / Vitakalk dybow Rangifer 1 month female juvenile control mother milk / Selenit E s.c tarandus Cervus elaph. ad female dystocia Kofu / no suppl canadensis Cervus elaph. adult female dystocia Kofu / no suppl canadensis Cervus elaph. adult female dystocia Kofu / no suppl canadensis Cervus elaph. adult male euthanasia Kofu / no suppl canadensis Bison bison adult female hoof trimming Kofu / Cattle-mineral Capra ibex ibex adult female x-ray Kofu / Vitakalk Ovis aries adult male poor condition SelenitE i.m (camerun) Ovis aries adult female control No pellets / no suppl (camerun) Ovis aries adult female control No pellets / no suppl (camerun) Ammotragus 3 weeks female general weakness Kofu / Cattle-mineral lervia Ammotragus adult female hind leg problems Kofu / Cattle-mineral lervia Ammotragus adult male hoof trimmimg Kofu / Cattle-mineral lervia Capra hircus adult female digestive problems Goat-pellets Lama glama adult male penis prolaps Kofu / Salvana bisq pacos Lama glama 1 year male new arrival Kofu / Salvana Bisq pacos Lama glama 1 week male juvenile control Kofu / Salvana Bisq guanicoe Rhinoceros adult male hoof trimming Kofu / Salvana bisq. + EL unicornis E-VITE Tapirus terrestris adult male abscess, lower jaw Kofu / Vitakalk New feeding strategy As many different species were affected by white muscle disease, a new feeding strategy was introduced in the summer of The pellets for zoo animals (Kofu), which are given on a daily

32 basis, were supplemented with Vit. E and Se. 2.5 kg of VITAMIN E-50 (ANIMEDICA: 1 kg powder contains 500 g Vit. E acetate) and 3.6 g of selenium were added to 6 tons of Kofu pellets. Sheep, goats, goitered gazelles and shire horses do not receive these enriched Kofu pellets and thus do not benefit from the supplement. Discussion With the new, enriched Kofu pellets, containing 520 mg vitamin E and 1.2 mg selenium per kg (before 2001 the content was 175 mg Vit. E and 0.45 mg Se per kg) the situation improved considerably. There were no more deaths in the animal species that had previously given the most problems, such as bison, wapitis and equines. But there were still a few cases of white muscle disease in 2002 and The most recent deaths occurred in the nilgai antelope (Boselaphus tragocamelus) and black buck (Antilope cervicapra), two species kept in large groups in a mixed enclosure. The amounts of Vit. E and Se may not have been sufficient for the individual animals, since individual administration of pellets is not possible for animals living in a group. Depending on its status in the herd and general health (e.g. pregnancy), an individual animal may receive less than the requirement. There are still some deficiency problems in goats and sheep. This is due to the fact that these animals live in an area of the zoo where they are fed by the public. They are offered low-protein goat pellets instead of the regular pellets for zoo animals (Kofu, which contain 18 % protein). The goat pellets have not yet been supplemented with Vit. E and Se, but this is planned for The adult Cameroon sheep, mentioned above, was one of these cases. Stress and physical exercise can increase the requirement and may have been the reason for its sudden death during mating season. The regime of treating all newborns parenterally with Selenit E and seasonally supplementing pregnant giraffes, bongos and rhinos with EL -E-VITE pellets and Equistro Super E was continued. This oral supplement is especially important in autumn and winter, when forage is low in vitamin E, depending on cutting time and the storage. However, it is important to feed the enriched Kofu pellets year-round. In spring and summer, when the grass is still young, the Vit. E content is high. But if the grass is cut too late or if excessive leaching occurs during haying, the Vit. E content decreases. Hay storage also plays an important role in Vit. E. content. If stored too long, the vitamin is oxidised. The high content of unsaturated fatty acids in young grass leads to a higher requirement of Vit. E for the animal, since this vitamin is needed to oxidise unsaturated fatty acids to saturated fatty acids. Thus, although the relative amount of Vit. E in young grass is high, the absolute content is not high enough to compensate for the large amounts of unsaturated fatty acids. In pigs, supplementation of the diet caused a three-fold increase in serum Se within the first week, with a slight tendency to further increases in subsequent weeks. The serum vitamin E of unsupplemented pigs declined by fifty percent during the experiment, whereas supplemental vitamin E resulted in increased serum vitamin E (12). In beef cattle, a Vit. E supplement to the diet also led to increased serum levels (1). Interestingly, various breeds of dairy cattle exhibited breed-related differences in Se metabolism (9). This may be the basis for the assumption that the health improvement obtained by vitamin E and/or Se supplements differs according to the animal species. Since the metabolism of Se and Vit. E probably varies in different species, not all species react in the same way. The black rhinoceros and elephant appear to have higher Vit. E requirements (6). In the wild, the plants that these two species browse on have much higher Vit. E concentrations than grasses and pelleted diets. One study (7) suggested that lower plasma vitamin E levels in captive versus wild rhinos reflect a lack of sufficient bile to emulsify and absorb Vit. E efficiently in animals fed intermittently. It is also known that the fat-soluble form of Vit. E is absorbed poorly in elephants whereas the water-soluble form is absorbed rapidly (11). Therefore EL -E-VITE pellets are used for some very delicate species at the Nürnberg Zoo. The form of Vit. E supplemented in the present study is thought to be stable (5). Recent

33 recommendations for captive wildlife are IU/kg DM, unless the higher levels produce excessively high serum concentrations (2). Significant increases in plasma concentrations through dietary supplements usually require weeks to months. Regular blood tests over longer periods of time, as done at the Nürnberg Zoo, are important to control the status of these animals. Low serum levels can show the necessity of higher supplementation, whereas high serum concentrations have to be corrected since excessive Se supplementation can cause selenium intoxication. Compared to other grass silage, with Vit. E contents ranging from mg/kg DM, the 60 mg/kg DM contained in the silage of Nürnberg Zoo is about average, but the selenium content of grass silage in the area ( mg/kg DM) is compared to other regions (e.g. switzerland mg/kg DM) lower. Last but not least, it is also important to mention the economical and practical factors. Vitamin E and selenium products are very expensive. The supplementation of the Kofu pellets at the Nürnberg Zoo is very economical and, as almost all herbivores are fed with these pellets, easy to handle. In the future, sensitive species will be observed closely, taking as many blood samples as possible to determine whether the supplementation is sufficient or if another approach is needed. References 1. Bass RT 2 nd, Swecker WS jr, and Stallings CC. Effects of supplemental administration of vitamin E and selenium to jerseys and holsteins during the nonlactating period. Am J Vet Res 2000, 61: Calle PP, Dierenfeld ES, and Robert ME. Serum α-tocopherol in raptors fed vitamin E-supplemented diets. J Zoo Wildl Med 1989, 20: Dierenfeld ES. Vitamine E deficiencies in zoo reptiles, birds and ungulates. J Zoo Wildl Med 1989, 20: Dierenfeld ES, du Toit R, and Miller RE. Vitamin E in captive and wild black rhinoceros (Diceros bicornis). J Wildl Dis 1988, 24: Dierenfeld ES, and Citino SB. Circulating plasma α-tocopherol following a single injection in a black rhinoceros (Diceros bicornis). J Wildl Dis 1989, 25: Dierenfeld ES. Vitamin E in exotics: Effects, evaluation and ecology. J Nutr 1994, 124: 2579S-2581S. 7. Ghebremeskel K, Williams G, Brett RA, Burek R, and Harbige LS. Nutrient composition of most plants most favoured by black rhinoceros (Diceros bicornis) in the wild. Comp Biochem Physiol 1991, 98A: Machlin LJ. Vitamin E: A comprehensive treatise. Dekker New York O Grady MN, Monahan FJ, Fallon RJ, and Allen P. Effects of dietary supplementation with Vitamin E and organic selenium on the oxidative stability of beef. J Anim Sci 2001, 79: Oksanen HE. Apspects of vitamin E deficiency in ruminants. Acta Agric Scand Suppl 1973, 19: Papas AM, Cambre RC, Citino SB, and Sokol RJ. Efficacy of absorption of various vitamin E forms by captive elephants and black rhinoceroses. J Zoo Wildl Med 1991, 22: Young LG, Lumsden JH, Lun A Claxton J, and Edmeades DE. Influence of dietary levels of Vitamin E and selenium on tissue and blood parameters in pigs. Can J Comp Med 1976, 40: Corresponding author: Dr. A. Liesegang Institute of Animal Nutrition VETSUISSE Faculty of the University of Zurich Winterthurerstr. 260 CH Zurich

34 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. RESULTS OF A FEEDING QUESTIONNAIRE AND DIGESTIBILITIES OF DIFFERENT FEEDSTUFFS IN LLAMAS (Lama glama) A. LIESEGANG 1, I. HENGRAVE 2 and M. MEYLAN 2 Affiliation: 1. Institute of Animal Nutrition, Vet Suisse Faculty of the University of Zurich. 2. Clinic for Ruminants, Vet Suisse Faculty of the University of Berne, Berne, Switzerland Abstract Over a period of a year several new world camelid farms were visited in Switzerland. A questionnaire was submitted to the owners and feedstuff digestibilities were tested in 4 herds. Key words: Llama, digestibility, Switzerland Introduction The importance of new world camelids is constantly growing in Switzerland. In order to allow for natural behavior of the animals, the keeping, feeding and breeding must be optimized. Most of the European Llamas are held for breeding or as companion animals, and a smaller part are used as packing animals on trekking tours or as therapy animals for interacting with emotionally or physically disabled children and adults. The llamas are social animals which must be held in herds. Some experience in managing the feeding of lamoids in zoos and private ownership has been reported (4). In the mountains of South America, the original habitat of these animals, camelids typically eat grass containing a high proportion of crude fiber, which is poorly digestible. In Europe, South American camelids often receive fodder (roughage and concentrates) which is highly digestible, often in excessive quantities. The animals tend to become obese, which leads to decreased reproduction performances and reduced life expectancy. Interestingly, llamas and alpacas defecate on special spots, i.e. the entire herd uses the same spots to urinate or defecate. Therefore, the animals not only have enough dry surfaces in order to lie down, but also the rest of the pastures do not become contaminated with parasite eggs. This leads to reduced parasitic burdens for the animals as they do not graze around the dung piles. In the present study, the feeding behavior, pasture systems and the different stable facilities used for new world camelids in Switzerland were evaluated and the digestibilities of the feeds were calculated in selected herds. Material and Methods 40 herds were visited all over Switzerland. At that time, a questionnaire was submitted to the animal owners. The questionnaire included questions about the animals in the herd (number, age, gender, origin), management methods, pastures, watering and feeding facilities for the animals, hay supplementation, composition of the ration and fodder additives used. Additionally, feces from 15 adult animals (12 females, 3 males) were collected in 4 herds and the exact diet was determined, either of the group or of individual animals. A proximate analysis was performed for the feedstuffs and the feces, to determine dry matter, ash, crude protein, crude fiber and fat contents. Lignin (ADL, Acid Detergent Lignin) was used as an endogenous indicator in order to calculate the apparent digestibilities. The apparent digestibility was calculated according to the following formula:

35 I (F) = concentration of the indicator in the fodder I (K) = concentratio n of the indicator in the feces N (F) = concentration of the nutrient in the fodder N (K) = concentration of the nutrient in the feces I (F) * N (E) Apparent digestibility = 1 - * 100 I (E) * N (F) (in %) Results The 40 farms examined kept an average of 10 animals, from which 9 were female and 1 male, between less than 1 year and over 15 years of age. The group of the 2-4-year old was the largest. In 30 of the 40 herds, a total of 430 animals were kept as companion animals. The pastures had a size between 1000 and m 2 depending on the number of pastures available. On 13 farms straw was used as bedding. On average 1.5 waterers were available per group. Generally the animals were fed with hay and special pellets for camelids. Ninety-five animals on 19 farms additionally received mineral salt. On 4 farms where fe cal samples were collected for analysis, a total of 19 animals were kept as companion animals (12 adult females, 2 adult intact males, one adult castrated male, one 1-year old male, and 3 juvenile animals). None of them had been imported. Llamas were the only animals kept in one of the herds, whereas they were kept together with chickens, horses, goats, cattle, and/or sheep on the other 3 farms. In these 4 herds, pasture size was between 1000 and 2000 m 2, and the farms had 1 to 6 pastures available. Two farms had a rotation pasture system with an average service life of 3 weeks. On three farms, the animals were held outside in all seasons with a shelter bedded with straw. In the shelter, the animals had access to water from an automated waterer. In summer, the llamas received mainly grass and additionally some hay and corn cubes, in winter the diet consisted mainly of hay and corn cubes. All 4 farms additionally fed special pellets for camelids and minerals. Analysis of the feed revealed an average apparent digestibility in the original substance of 71 ± 15 % for the organic matter, 71 ± 5 %,for crude protein, 90 ± 2 % for crude fat, 62 ± 2 % for calcium, 60± 20 % for phosphorus, 66 ± 18 % for magnesium, and 81 ± 7 % for crude fiber, respectively. Discussion These results only give exact information about the number of animals examined and for the keeping systems in use on the selected farms. The feeding management was investigated as precisely as possible, yet it turned out that the exact ration of individual animals was very difficult to evaluate, since these animals are often kept in groups and not fed individually. The examination of feces and fodder samples on the 4 selected farms showed digestibilities of the different compounds comparable to those described by Lopez et al. (2000) (5). Interestingly, Carmean et al. (1992) (1) described similar digestibilities of dry matter, but the digestibility of crude fiber and the other components was not determined in his study. In other herbivores, the apparent dige stibility of crude protein was comparable to that in the present study with 71 %, but a lower digestibility of crude fiber (48 %) was observed (6). Llamas, which are considered to be functional ruminants although they only possess a modified three -chambere d stomach, can digest cellulose easily. In a study by Schneider et al. (1974) (7), it was shown that sheep and llamas digest the organic matter and NfE (nitrogen-free extracts) in hay equally efficiently with digestibilities between 63% and 64% of the organic matter and 69% and 67% of the nitrogen-free extracts, respectively. The digestibilities of crude fiber with values of 81 ± 7 % were high in comparison with horses, which are fed similar rations and have an apparent digestibility around 40 to 61% (2).If the digestibilities of crude fiber of the llamas in the present study are compared to those of other ruminant species (goat, sheep), the crude fiber digestibility is still higher than in other species. Llamas are known to have a more efficient digestive system than other ruminants (4). This becomes especially evident if poorly digestible hay with high crude fiber content is fed. The protein supply of llamas in the wild seems to be much lower (protein content: 5 to 6%) compared to llamas kept as companion animals (protein content > 8%) (3). As far as the total

36 protein content is not higher than 12%, the animals seem to cope well with a higher protein intake. On the 4 farms included in this study, the protein contents were approx. 8% in hay and approx. 10% in the total ration. The results of the present study indicate that the provided feedstuffs had high apparent digestibilities. The nutritional requirements of the animals also depends on the production and physical work of the animals. If the animals are kept only on pasture and no intensive physical effort (trekking-tours) is performed, the provided fodder quantities and the supplementation with minerals were on average sufficient for the examined animals. Individual feeding of each animal in the herd is practically impossible. In larger herds, groups of animals with the same requirements can be formed, i.e. the mating season can be planned so that the females give birth over a time period as short as possible. Such a scheme would be highly beneficial since the feeding strategy would be easy to manage and, in addition, the social structure of the herd would benefit of such a schedule. However, the herds in Switzerland are mostly too small for a change of management in this direction. The llama owners must therefore try to give animals with a low social status the chance to compete with the other animals for sufficient fodder and water. Due to the number of feeding crips and waterers on the investigated farms, this seems not to be a problem in Swiss herds. In order to collect exact data about the apparent digestibilities of different feedstuffs for llamas in Switzerland, further studies will be necessary to examine the individual ration and weight of each animal during the investigation period. References 1. Carmean BR, Johnson KA., Johnson DE, et al. Maintenance energy requirement of llamas. Am J Vet Res 1992, Fehrle S. Untersuchungen zur Verdaulichkeit von Mischfutter beim Pferd in Anhängigkeit von der Rauhfutteraufnahme. Doctoral thesis, 1999, München. 3. Fowler ME. Feeding and Nutrition in Camelids. In: Fowler ME (ed). Zoo & Wild Animal Medicine. 2 nd edition. WB Saunders Company Philadelphia etc 1986; Fowler ME. Feeding and Nutrition. In: Fowler ME (ed). Medicine and Surgery of south american camelids. 2 nd edition. Iowa State University Press Ames 1998, Lopez V, Morales S, Cabrera C, et. al. Ingestion y digestibilidad aparente de forrajes por la lama (Lama glama) : I. Heno de alfalfa y paja de trigo en diferentes proporciones. Arch. Med. Vet. 2000, Moeini MM, Souri M, Hozabri, et al. Nutrient digestibility of alfalfa at different growth stages on sheep and goat. Proc Brit Society Anim Sci 2000, Schneider W, Hauffe R and Engelhardt W. Energie- und Stickstoffumsatz beim Lama. In: Energy metabolism of farm animals, European Association of Animal Production, Hohenheim; Dok. 14, Author s address: Dr. A. Liesegang Institute of Animal Nutrition VETSUISSE Faculty of the University of Zurich Winterthurerstr. 260 CH Zurich

37 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. DIETARY AND VETERINARY MANAGEMENT OF A LINGUAL ABSCESS IN A GERIATRIC CAPTIVE BLACK RHINO (Diceros bicornis) WITH IRON STORAGE DISEASE J-M. HATT 1, C. WENKER 2, J. CASTELL 3 and M. CLAUSS 3 Affiliation: 1. Division of Zoo Animals and Exotic Pets, Veterinary Faculty of the University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland 2. Basle Zoo, Switzerland 3. Ins titute of Animal Physiology, Physiological Chemistry and Animal Nutrition, Ludwig- Maximilians-University of Munich, Munich, Germany Key words: Digestion, dental plaques, quidding Poster Abstract The present case reports extensive investigations regarding the gastrointestinal tract in a captive female black rhinoceros (Diceros bicornis) showing symptoms of chronic quidding, described as reluctance to eat fibrous foods, hypersalivation and ejection of lumps of fibrous food material on which the animal had been chewing. At the age of approximately 15 years the animal started showing symptoms of intermittent quidding and from the age of 30 years on the quidding was constantly noticed. Several one-week periods of complete intake and faecal output documentation and recording allowed to estimate intake and digestibility coefficients in the following two years. The amount of saliva lost due to the ejection of lumps was quantified in three different weeks and varied between 2.2 and 5.7 kg of saliva. It was observed that the animal lost weight initially, which was seen by increasing prominence of bone structures such as the pelvis or spinous processes on the back. At this stage, on its regular diet (diet A) of Lucerne hay, grass hay, produce and concentrates, the animal had a dry matter intake (DMI) of 1.2 % of its estimated body weight (BW), with an according digestible energy (DE) intake of 0.76 MJ/kg Dry matter (DM) digestibility was 69 %, but crude fibre (CF) digestibility was only 38 %. In an attempt to increase fibre utilization and reduce the vain attempts to ingest whole roughage, green meal pellets were additionally introduced to the animal (diet B). In two according feeding periods, DMI averaged 1.2 % BW, DE intake was 0.73 MJ/kg DM and CF digestibility were 67 and 48 %, respectively. Therefore, it seemed that, with energy supply not being compromised, fibre utilization was increased. At the same time, the particle fraction in the faeces retained on a 4 mm sieve decreased from 57 to 36 %, whereas fine particles retained on a mm sieve increased from 11 to 20 %. Increasing bad breath odor lead to a first intervention under general anaesthesia to examine the oral cavity in February Anaesthesia was induced with 10 mg of detomidine, 6 mg butorphanol and 1.2 mg etorphine by blow dart followed by 100 mg ketamine i.v.. For anaesthesia antagonisation 60 mg naltrexone und 200 mg atipamezole i.v. were used. Large amounts of dental plaque and calculus were noted and removed. In the upper mandible between PM 1 and 2, an abscess was diagnosed, which was treated locally by flushing. Bacteriology revealed no specific bacteria. The treatment resolved in an improvement which lasted approximately six months.

38 From October 2000 until December 2002 a total of 17 blood samples were taken from the unsedated animal. Compared to reference data for female black rhinoceros aged >5 years (International Species Inventory System. ISIS Physiologic Data Reference Values. International Species Inventory System. 1999) no consistent abnormalities were noted. However, serum iron was high compared to values of free-ranging animals, mean transferrin saturation was 90 % (Reference value 28% (1)), and mean serum ferritin was 6046 ng/ml (Reference value 133 ng/ml (1)), indicating excessive iron absorption and excessive iron stores. When iron contents of diet A and diet B were compared, it was realized that the addition of green meal pellets had increased the iron concentration of the whole ration from app. 270 to app. 590 mg/kg DM. Additionally, the faecal consistency had deteriorated on diet B. Therefore, it was decided to replace the green meal pellets with a combination of grass and Lucerne chaff (diet C), which was well consumed; in contrast to whole hays, quidding and ejection of lumps of half-chewed hay was not observed. DMI was 1.2 % BW on this diet, DE intake was 0.67 MJ /kg DM digestibility was 66 %, but CF digestibility dropped again to %. As foetor ex ore was again noted, a second examination of the oral cavity was performed in February 2002 with a flexible endoscope. The anaesthetic regimen was identical to the first procedure and no new findings could be disclosed. Ten months later, in January 2003 the animal died suddenly, at the age of 32 years. Hours prior to death the animal showed minor bleeding from the mouth. Post-mortem examination revealed an abscess which opened laterally on the tongue and extended approximately 15 cm towards the base of the tongue. The main finding however was a severe hemochromatosis with consequent severe hepatocellular degeneration and fibrosis. Hemochromatosis was further diagnosed in lungs, spleen and small intestines. Anatomical preparation of the skull revealed minor osteolysis were during the first anaesthesia an abscess was found. It is reasonable to assume that the black rhinoceros in this case suffered from chronic iron storage disease (ISD). The intensive diagnostic measures and monitoring which were initiated in this case did not provide any clinical evidence for a disease process, confirming the fact that the clinical consequences of ISD are unclear in live animals (1). The difficulty of intra vitam diagnosis of ISD is well known. In human medicine the most efficient approach to the diagnosis of ISD is to measure the serum transferrin saturation and ferritin levels (2). Increased transferrin saturation is an important hallmark of ISD but phlebotomy will not give any information on the location of iron deposits, quantification of iron deposits and presence of cirrhosis. The latter being an important factor for the estimation of prognosis. Liver biopsies would allow such information to be gained (see Hatt et al. in this proceedings). However, liver biopsy sampling is not current practice in rhinoceros. Furthermore no explanation could be found for the chronic quidding in the present case. A possible explanation may be the absces s on the base of the tongue. Hanson et al. (1993) reported quidding in a horse suffering of a rhabdomyosarcoma of the dorsal aspect of the tongue (3). Following surgical removal of the neoplasia quidding resolved. Although two thorough oral examination were performed in the rhinoceros, the abscess in the tongue was not diagnosed. The oral anatomy in black rhinoceros make oral examination very challenging. Nevertheless in future case, quidding in rhinoceros should be linked to possible pathologies in the tongue and further diagnostic investigations, such as radiography attempted. Acknowledgments The authors would like to thank H. Lutz, P Ossent, M. Rütten and M. Wehrli for their support in the diagnostic work-up of this case. M. Studer and his team are acknow l-

39 edged for the outstanding management of the animal which made the procedures possible. References 1. Paglia D and Dennis P. Role of chronic iron overload in multiple disorders of captive black rhinoceroses. In: Proc Am Assoc Zoo Vet; 1999; Columbus; p Beutler E, Hoffbrand AV and Cook JD. Iron deficiency and overload. Hematology 2003; Hanson PD, Frisbie DD, Dubielzig RR, et al. Rhabdomyosarcoma of the tongue in a horse. J Am Vet Med Assoc 1993; 202:

40 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. ANALYSIS OF MAJOR COMPONENTS IN ASIAN ELEPHANT (Elephas maximus) MILK DURING DIFFERENT STAGES OF LACTATION S. KÖLBL 1, M. FLÜGGER 2, C. KUNZ 3, J. PETER-KATALINIC 1 and G.POHLENTZ 1 Affiliation: 1. Department of Biomedical Analysis, Institute for Medical Physics and Biophysics, University of Münster,Robert-Koch-Str. 31, Münster, Germany 2. Tierpark Hagenbeck, Hamburg, Germany 3. Institute for Nutrition Science, University of Giessen, Germany Key words: Asian Elephant, Elephas maximus milk major nutrients infant formula hand-rearing Introduction Experiences made in different zoos showed considerable difficulties with the artificial rearing of elephant calves born in captivity. Besides loose stools or diarrhea, e.g. skin dryness and umbilical infection seem to be more likely in hand -raised calves [1,4,6]. Therefore - and to prevent other nutrition-dependent stressors it is necessary to provide a milk replacer which closely resembles the nutrient makeup of the dam s natural breast milk. Milk is the sole food for infants during the first six month [7]. It is well known that calves are milkdependent for the first two years and will often suckle to the age of four or five. After the age of two the volume of the consumed milk begins to decrease gradually [1,7,8]. The makeup of natural milk is extremely complex and in most species, the milk composition changes during lactation. In order to achieve a successful hand -rearing it is necessary to optimise the formula over the whole period of lactation. Therefore as much information as possible is required about the milk composition during different stages of lactation. Material and Methods Since up to date only a few data on the milk composition during the whole lactation time of elephants are available we started our project in cooperation with Tierpark Hagenbeck, Hamburg, Germany. Up to now we analysed the mature milk of one Asian elephant taken during early ( days post partum; every four weeks, samples taken on three consecutive days, n=18) and late (4,5 years post partum, n=3) lactation for major components. The content of protein was determined according to the method of Lowry [2], total lipids gravimetrically, and lactose by use of a ß-galactosidase/glucoseoxidase test. Results Our results show that the contents of proteins (2.97% ± 0.37), fat (8.86% ± 1.61) and lactose (2.64% ± 0.24) have not changed significantly during the first seven months post partum. Protein composition and phospholipid pattern remained unaltered within this early observation period. However evident changes of the nutrient composition were observed in the milk of the late lactation. Protein levels in samples taken after 4.5 years were enhanced (4.98% ± 0.18) and the ratio of caseins and whey proteins was shifted to the latter. In contrast the lactose concentration was significantly lower (0.24% ± 0.09). The values obtained for the fat content were inconsistent (3.9% (231 weeks post partum); 10.7% (241 weeks post partum)). This might be due to difficulties in milk sampling [3,5].

41 Discussion and Outlook Since our results suggest obvious variations in the contents of at least proteins and lactose in the course of lactation we are very interested to find out when these changes take place and whether this process is creeping or spontaneous and is accompanied by a physiological development/event. In addition we intend to get a closer look at the milk of the first two month post partum, including colostrum. Thus, our further analysis may be helpful for adjusting a formula to the requirements at different stages of lactation. To get more sophisticated knowledge on milk constituents we plan to extent our investigations on a larger amount of samples. Therefore we would like to increase the number of collaborating zoos. Another objective is the analysis of the fatty acid pattern and its composition during lactation. A long-term aim will be the examination of minor milk components e.g. vitamins and minerals. Acknowledgements We thank the Stiftung Hagenbeck, Hamburg, Germany, for the financial support of the project. References 1. Emanuelson KA, Knizley CE. Elephants. Gage LJ (eds.). In: Hand-Rearing Wild and Domestic Mammals. Iowa State Press, Ames, Iowa 2002; Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: Mainka SA, Copper RM, Black SB, Dierenfeld ES. Asian Elephant (Elephas maximus ) milk composition during the first 280 days of lactation. Zoo Biol 1994; 13: Ochs A. Erste Erfahrungen bei der Handaufzucht eines Asiatischen Elefanten ( Elephas maximus) im Zoologischen Garten Berlin. Verh ber Arbeitstg Zootierärzte im deutschsprach Raum 2000; 20: Parrott JJ. Analysis of African elephant mature Milk in early lactation and formulation of an elephant calf milk replacer. Proceedings American Association of Zoo Verterinarians 1996; Rietkerk FE, Hiddingh H, Van Dijk S. Hand-rear ing an Asian elephant (Elephas maximus ) at the Noorder Zoo, Emmen. Int Zoo Yb 1993; 32: Sheldrick D. Raising baby orphaned elephants: part II. SWARA 1990; 13: Welsch U, Feuerhake F, von Aarde R, Buchheim W, Patton S. Histo- and cytophysiology of the lacting mammary gland of the African elephant (Loxodonta africana). Cell Tissue Res 1998; 294:

42 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. A CONTRIBUTION TO THE MORPHOLOGY OF THE BLOOD CELLS IN ASIAN ELEPHANT (Elephas maximus) K. STIEF 1, K. EULENBERGER 2, J. SEEGER 3, J. KACZA 3 and CHRISTA BACHMANN 2 Affiliation: 1. Animal hospital Dr. Karsten Stief, Uferstr. 1, D Heidenheim, Germany, 2. Zoological garden Leipzig, Pfaffendorferstr. 29, D Leipzig, Germany. 3. Institute of Veterinary Anatomy, Department of Histology and Embryologie, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43, D Leipzig, Germany Abstract In the literature concerning elephant haematology one can find various quotations of the different kinds of white blood cells. Especially in the number of lymphocytes and monocytes there are large differences, giving a range from 3 to 60 % of the leucocytes. The problem behind is that there are blood cells with two or three segmented nuclei which you can differentiate as lymphocytes or monocytes as well. Sometimes there are two segmented lymphocytes also in other species. We have seen them for example in kangaroos, black rhinos and giraffes as well. We assume that these cells belong to the lymphocytes. Also neutrophilic und eosinophilic granulocytes are in contrast to other species in elephants very similar to each other. Key words: Asian elephant, Blood cell morphology Introduction For the laboratory diagnostics it is very important to know the morphology of the blood cells. In the literature concerning elephant haematology one can find various quotations of the different kinds of white blood cells. This is a result of different interpretation of the morphology of these cells. For example there are mononuclear cells with two or three segmented nuclei which you can estimate as monocytes or as lymphocytes. Material and Methods The blood smears come from the archive of the Leipzig Zoological Garden. Coloration of blood smear according to Pappenheim. As microscope we used a light optical microscope with an oil immersion lens with hundredfold magnification as well as a high -resolution digital video camera. Results We assume that the mononuclear monocyte -like cells belong to the lymphocytes. Because first the zytoplasma is more similar to the lymphocytes than to monocytes, second sometimes there are two segmented lymphocytes seen also in other species and last the number of monocytes is similar known for other species with about 5% of the total leucocytes. Furthermore there are difficulties to differentiate neutrophilic granulocytes - in elephants also named heterophiles and eosinophilic granulocytes because of the large granules of the neutrophilic ones. The last ones are often hypersegmented. Remarkable are also the rather light, large and very commonly seen vacuoles in the cytoplasm of the eosinophilic granulocytes. Large lymphocytes in their morphology very similar to the lymphocytes in other mammalian species - are named lymphogenic effector cells by the authors. The nuclei of the basophilic granulocytes are completely overlapped by the basophilic granules. They are arranged towards the nuclei. Very often, a thin cytoplasm band is visible, which is free of basophilic granules. In the thrombocytes granules are well seen. Discussion In this paper, morphology of blood cells in elephant will be described in detail where especially the problem of the two or three segmented mononuclear monocyte-like cells is taken up. These cells

43 fall out of the typical morphology pattern what causes problems in the laboratory diagnostics. These problems could be solved by using special markers or colorations only that are, however, not available in practice. Furthermore there may be problems in differentiation of neutrophilic granulocytes and eosinophilic granulocytes by automatically counting in non adapted programs. References 1. Allen, J., E. Jacobson, J. Harveye u. W. Boyce. Hematological and serum biochemical reference range in healthy African Elephants. J. Zoo Anim. Med. 1985; 16, Cheryk, L. A., P. A. Gentry, T. Bast u. S. Yamashiro. Alterations in blood platelets morphology during aggregate formation in the Asian Elephant. J. Zoo Wildl. Med. 1998; 29(2), Jainudeen, M. R., u. J. B. Jayasinghe: Hemogram of the domesticated Asiatic elephant (Elephas Maximus). J. Zoo Anim. Med. 1971; 2, Kendal, H., R. Isaza u. J. Harvey. Clinicopathologic findings in mycobacterium tuberculosis culture-positive elephants in comparison to clinically normal elephants. In: Proceedings AAZV, AAWV, ARAV, NAZWV Joint Conference Nirmalan, G., S. G. Nair u. K. J. Simon: Hematology of the Indian elephant (Elephas Maximus).Can. J. Physiol. Pharmacol. 1967; 45, Ratnasooriya, W. D., S. B. U. Fernando, A. M. V. R. Manatunga, H. S. Caldera; G. K. Liyanage u. G. A. S. Premakumara. Haematological values for adult Asian elephants (Elephas maximus maximus) at the Pinnawala Elephant Orphanage, Sri Lanka. Med. Sci. Res. 1990; 18, Stief, K. Morphologie der Blutzellen ausgewählter pflanzenfressender Zootiere. Vet. med. Dissertation, university of Leipzig 2002; html 8. Silva, I., u. V. Kuruwita. Hematology, plasma and serum biochemistry values in free-ranging elephants in Sri Lanka.J. Zoo Wildl. Med. 1993, 24(4), Tell, L., M. B. Kabur, W. L. Smith, L. Gage u. J. S. Cullor. Oxygen radical production by Asian elephant heterophils and Holstein cattle neutrophiles. J. Zoo Wildl. Med. 1999, 30(3), Hawkey, C. M., u. T. B. Dennett: Farbatlas der Hämatologie Säugetiere, Vögel und Reptilien. Schlütersche Verlagsanstalt, Hannover 1990 pict.1: monocyte (l.), lymphocyte (r.) pict.2.: neutrophilic granulocyte (l.), eosinophilic granulocyte (r.) pict 3: neutrophilic granulocyte pict.4: basophilic granulocyte

44 pict.5: lymphogenic effector cell pict.6: 3 lymphocytes pict.7: thrombocytes

45 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. DETERMINATION OF PREGNANCY AGE IN RABBIT BY ULTRASONOGRAPHY SARANG SOROORI Affiliation: Department of Clinical Scien ces, Faculty of veterinary Medicine, University of Tehran, Tehran -Iran. Key words: Ultrasound, rabbit, pregnancy Abstract: To assess gestational age by Ultrasonography, 10 New Zealand White does and 10 bucks of the same breed were placed together in cages. The does were separated from bucks 24 hours after mating, and Ultrasonography program was performed. In order to assess the earliest time of pregnancy, Ultrasonography was performed daily from the day 4 to 10 of mating, and one day interval from day 10 until parturition.ultrasonographgy diagnosis was performed using transcutaneous technique. The best criterion pregnancy diagnosis in primary days of pregnancy was observation of embryonic vesicle. The earliest assessment of pregnancy was day 8 in which pregnancy could be diagnosed in eight does. By increasing of gestational age some criteria such as BPD, CRL and TD (thoracic Depth) were measured. Regarding to the results of the present study some morphometric values were gained by which the gestational age could be assessed in this breed. References: 1. Curley S.Ultrasound imaging and pregnancy monitoring of rabbits.j Animal technology 2001; 25(2): Rinck I. Sehic M.Butkovic V. Stanin D. Kadunc I, Ultrasonography diagnosis of pregnancy in rabbit.j Veterinerski-Archiv 1993; 36(2): Ypsilantis P. Saratris P. Early pregnancy diagnosis in the rabbit by real time Ultrasonography Word rabbit science 1999; 7(2):95-98.

46 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. EXPERIENCES IN SETTLING DOWN SNAKES, DIRECTLY IMPORTED FROM INDONESIA A. BERNHARD 1, R. SCHMÄSCHKE 2,W.-E. ENGELMANN 1, K. EULENBERGER 1, CHRISTA BACHMANN 1, H. BEHNCKE 1 and REGINA SCHELLER 3 Affiliation: 1. Zoological Garden Leipzig, Leipzig, Germany. 2. Department of Parasitology, Veterinary faculty Univ. Leipzig, Leipzig, Germany. 3. Landesuntersuchungsanstalt f. Gesundheits- und Veterinärwesen Sachsen, Standort Leipzig, Leipzig, Germany. Abstract On the 31 st of July 2002, 37 snakes (10 different species) arrived at the Zoological Garden of Leipzig, imported from Indonesia. By importing the snakes directly to the zoo, it could be avoided to involve a zoo dealer as an intermediate station with the aim, in order to minimize stress for the animals and the risk of infection between the reptiles during the transport. Nevertheless 26 snakes died within 6 month caused by different reasons. Key words: snake, import, Indonesia, quarantine, Rhabdias sp., Kalicephalus sp. Monocercomonas sp. Introduction Imports of snakes from Indonesia are to be considered as problematically. In order to minimize the stress for the animals during an import via an animal trader who seldom provides optimum transport and keeping conditions, animals were imported directly. Material and Methods 37 snakes of 10 different species have been kept individually under quarantine conditions. According to clinical symptoms (ano rexia, insufficient turgor, stomatitis, dyspnoea, diarrhoea), a symptomatic treatment was applied at first, later a therapy according to the diagnosis was given. Weekly parasitological and microbiological investigations of the faeces have been carried out. Fresh faecal samples have been controlled for flagellates. All animals deceased have been necropsied. Results Despite these measurements, 26 snakes died within the first six months. In the first year, only five snakes survived. Bacteriological examinations of post-mortem and faeces samples revealed the presence of pathogens, typically found in reptiles. In two snakes, inclusion bodies were found in the liver. This allowed the diagnosis of a virus infection. As the main problem, different parasites were identified: nematodes of the genus Rhabdias (seven times) and of the genus Kalicephalus (six times). Trematodes were diagnosed three times and cestodes twice. Protozoa Monocermonas sp. were found three times, serositis and tubulonephrosis each twice. These findings were mainly related to the parasitic infestations, complicated through secondary bacterial infections. Discussion Despite the fact that everything was done to optimise the requirements for the settling down of the snakes directly imported from In donesia, the survival rate was absolutely unsatisfactory. In future imports, nematodes of the intestines as well as of the lung should preventively be treated before diagnostic investigations have proven positive. Treatment with Fenbendazol ( mg/kg ) is possible. The role of the Monocercomonas sp. is also looked upon differently in literature. Next to the difficulties to come up with a diagnosis, the occurrence of a pathogenic or a nonpathogenic

47 Protozoa caused difficulties in the evaluation of the infection. Three days long, the animals were treated with 100 mg/kg BM Metronidazol. This led to a clear reduction of the flagellates in the faeces. References 1. Barnads, S.M. a. S.J. Upton. A veterinary guide to the parasites of reptiles. Volume I: Protoz oa. Krieger Publishing Company Malabar, Florida Frank, W. Amphibien und Reptilien. Ed. Isenbügel, E. a. W. Frank. Heimtierkrankheiten. Verlag Eugen Ulmer, Ortlepp, R.J. Observattions on the nematod genera Kalicephalus, Diaphanocephalus and Occipitodontus g.n., and on the larval development of Kalicephalus phylodryatus sp.n.. J. Helminthology 1923; 1,

48 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. PYOMETRA IN A FERRET V. JEKL Affilitation: Avian and Exotic Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmacetical Sciences Brno, Palackého 1 3, Brno, Czech Republic Key words: ferret, pyometra, ovarial tumor Extended abstract A six-year-old castrated female ferret weighing 0,91 kg suffered from bilateral alopetia of the caudal part of the body. The ferret was castrated (ovariectomy) 5 years ago and since then the animal has been suffering from the seasonal alopetic changes. This problem have been solved by three aplication of human gonadotropine. This year s aplication of the hcg failed to produce any effect, that s what made the owner visit our clinic. Further clinical examination revealed generalized hypotrichosis, abdomen distension, vulva without any enlargement. A piece of tough polycystic mass (size 2,5*4 cm) was found behind the right kidney and the whole abdomen was filled up with fluid containing tissue mass. Radiography and ultrasonography confirmed the diagnosis of pyometra. Biochemistry and hematology showed light azotemia and regenerative anemia. The level of progesterone four days prior to the surgery was 0,7 ng/l and the level of estradiol reached 1377 pmol/l. Two days after the operation the level of progesterone and estradiol dropped to 0,2 ng/l and 153 mmol/l. Urine examination did not show any pathological changes. Laparotomy helped discover neoplastic right ovary, massive enlargement of uterus filled with brown fluid and subcapsular and stromal cyst on the left kidney. Histology confirmed hormonal active leiomyoma of the right ovary and hyperplasia of the uterine wall. Bacteriology of the uterine swab gave negative results. Ten days after the surgery hair began to grow again. Acknowledgements I thank to prof. Gerry Dorrestein, DVM, PhD for histology findings and to technical staff of the Avian and Exotic Animal Clinic, University of Veterinary and Pharmacetical Sciences Brno for their skillful assistance

49 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. BLOOD ANALYSIS OF WILDLIVING ALDABRA-TORTOISES (Gigantea gigantea) B. FLEDELIUS AND G. JORGENSEN. Abstract: During a re search expedition to Aldabra 2003 blood samples were collected from wildliving Aldabra-tortoises (G. gigantea) to determine their normal bloodplasma chemistry values. The analysis included determination of ALT, ALB, ALKP, AMYL, Ca 2+, PHOS, CREA, GLU, K +, TBIL, BUN, GLOB and was performed on the MScan, profile 13 N from Melet-Schloesing.

50 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. Helicobacter pylori INFECTION OF RHESUS MACAQUES EXPERIENCE WITH A CLOSE TO HUMAN ANIMAL MODEL IN PRIMATES K. MÄTZ-RENSING 1, E. KUNZ 2, C. KRAFT 3, S. SUERBAUM 3 and F.-J. KAUP 1 Affiliation: 1. Department of Infection Pathology, German Primate Center, Kellnerweg 4, D Göttingen, Germany. 2. Altana Pharma AG, Konstanz, Germany 3. Institute of Hygiene and Microbiology, University of Würzburg, Germany Key words: chronic gastritis, Helicobacter pylori, Rhesus monkey Abstract Spiral-shaped bacteria of the genus Helicobacter have been associated with subclinical and clinical gastritis in humans and animals. Two main groups of gastric Helicobacter spezies can be distinguished: the first group includes species similar to Helicobacter pylori (H.p.). The second group includes larger Helicobacter species, such as H. heilmannii like organisms.the bacteria are involved in the development of gastritis in man and monkeys. H.p. appears to be enzootic in some breeds of rhesus monkeys (1,2). The associated diseases are quite similar in humans and monkeys. H.p. strains from macaques were indistinguishable from human isolates in biochemical and phenogenotypical analysis (3). In both species infection is associated with chronic gastritis. Persistent infection is considered to be a risk factor in the development of gastric lymphoma and gastric adenocarcinoma. Due to the close evolutionary relationship between humans and primates a primate model of this human relevant disease bears promise to mimick human H.p. infection more closely than any other animal model can do. The experimetal design of this study based on an intragastral application of different human pathogenic H.p. strains in a mixture containing 10 8 bacteria in three male rhesus monkeys prior tested negative for naturally H.p.-infection. The animals were investigated by endoscopic, histologic, bacteriologic and microbiologic methods. To cure a preexsisting infection with large gastrointestinal spirals all monkeys were treated with oral application of a monkey specific quadrupel therapy. Three weeks after the end of antibiotic therapy experimental infection started. Bacterial strains selected for the experimental infection presented the pathogenic factors vaca, caga and flab. Three days after infection the first endoscopic investigation was performed. This endoscopic examination was repeated at two weeks, three weeks, five weeks, 3, 5, 10, 12, 18 and 24 month after infection. After three years a superinfection was setted with a new combinations of H.p. strains. At each investigation point 5-6 pinch biopsies of gastric mucosa of the corpus, fundus and antrum were obtained endos copically. They were brought either into urea medium, or in brucella broth for bacteriological investigations, fixed in formalin or glutaraldehyd for morphologic investigations or stored natively at 80 C for PCR-techniques.

51 The results indicated that all three animals were successfully colonized with H.p.. It was possible to reisolate the inoculated H.p. from biopsies first taken two weeks after inoculation. All animals remained infected during the five years of the study. All animals developed the same clinical course with mild modificatons in severity. In order to characterize the reisolated bacteria, multiple single colony isolates of H.p. recovered from biopsies were analysed by RAPD fingerprinting and 16S rdna sequence analyses. The inoculum strains differed in three nucleotide positions in the V6 region of the 16S rdna genes, which permitted unequivocal discrimination between these strains.it was possible to demonstrate that one strain had outcompeted other bacterial strains and posess successful attributes to colonize the rhesus monkey gastric mucosa. In the present study we confirm, that rhesus macaques can be successfully colonized by human pathogenic isolates of H.p.. Our results are consistent with previous reports indicating that the rhesus monkey is of special value concerning the establishment of these infections as a model for human disease (3,4). In addition, the similarities to the human disease render the rhesus monkey useful not only for infection studies but even more for follow-up studies of longer duration, given the long lifespan of Macaca mulatta. The great similarities between primates and humans make this animal model attractive for investigating the onset and progression of H.p. associated gastointestinal diseases and to evaluate potential vaccine candidates in preclinical studies. Acknowledgements We thank A. Schrod, T. Eggers, W. Henkel, E. Lischka, H. Zuri, K. Kaiser-Jarry and N. Knöchelmann for their skilful technical assistance. References 1. Drazek ES, Dubois A and Holmes RK. Characterization and presumptive identification of Helicobacter pylori isolates from rhesus monkeys. J Clin Microbiol 1994; 32: Dubois A, Tarnawski A, Newell DG, Fiala N, Dabros W, Stachura J, Krivan H and Heman Ackah LM. Gastric injury and invasion of parietal cells by spiral bacteria in rhesus monkeys. Are gastritis and hyperchlorhydria infectious diseases? Gastroenterol 1991; 100: Dubois A, Berg DE, Incecik ET, Fiala N, Heman Ackah LM, Perez-Perez GI and Blaser MJ. Transient and persistent experimental infection of nonhuman primates with Helicobacter pylori: implications for human disease. Infect Immun 1996; 64: Dubois A, Berg DE, Incecik ET, Fiala N, Heman Ackah LM, Del Valle J, Yang M, Wirth HP, Perez- Perez GI and Blaser MJ. Host specificity of Helicobacter pylori strains and host responses in experimentally challenged nonhuman primates. Gastroenterol 1999; 116:

52 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. IDENTIFICATION OF CHLAMYDOPHILA ABORTUS FROM ABORTED SPRINGBOK: STRAIN ISOLATION, MOLECULAR CHARACTERIZATION AND EFFICACY EVALUATIONOF AN OVINE LIVE VACCINE M. BERRI 1, F. BERNARD 1, A. LECU 2, F. OLLIVET-COURTOIS 2 and A. RODOLAKIS 1 Affiliation: 1: INRA, Pathologie Infectieuse Immunologie, Nouzilly, France. 2: Museum National d'histoire Naturelle, Parc Zoologique de Paris, 53 Av. de St Maurice, Paris, France. Key words: Springbok, abortion, chlamydiosis, Chlamydophila abortus, DNA, RFLP-PCR, vaccine. Extended Abstract: The family of Chlamydiaceae has recently been reclassified based on 16S and 23S rrna gene sequences into two genera Chlamydia and Chlamydophila and nine species (2). Chlamydophila abortus and Chlamydophila pecorum can infect a wide variety of mammals including domestic and wild animals, birds and occasionally human's (5). C. abortus is zoonotic, and although most human infections are mild and often not noticed, pregnant women can develop severe life-threatening illness and abort (1). C. abortus has a tropism for ruminant's placenta and causes the disease commonly referred to as ovine enzootic abortion (OAE). It is one of the most important causes of ovine and caprine abortion worldwide. Additionally there are some reported cases of its causing abortion in wild animals such llamas' (3) and black buck antelope (4) but the infectious strain has not been isolated. From 1999 to 2003, abortion cases were reported among wild ruminants of the Zoo Park of Paris (France) including springbok, Beisa Oryx and Sable antelope. The seroprevalence of several abortion diseases as chlamydiosis, brucellosis, coxiellosis, toxoplasmosis were therefore investigated showing that chlamydial infection was widespread among wild ruminants. In February of 2003, an abortion occurred in springbok (antidorcas marsupialis) herd, then vaginal swab, placenta samples as well as liver, spleen, lung of delivered foetus were taken. DNA was purified and PCR assay using primers targeting the polymorph membrane protein gene (pmp) family of C. abortus was performed. PCR assay using pmp primers revealed the presence of Chlamydophila strain in vaginal swab and placenta samples. However, no PCR product was obtained with DNA purified from the organs taken from the delivered the foetus. In order to isolate C. abortus strain, the extract of infected sample was firstly inoculated to McCoy mo nolayer cell cultures then the bacteria were grown in specific pathogen -free chicken eggs. The inoculation of infected placenta extract led to successful isolation of C. abortus strain referenced ASb1. Genomic DNA of the harvested bacteria was purified and subjected to PCR reaction. The omp1 gene coding the major outer membrane protein (momp) and the 16S-23S rrna spacer region of the springbok isolate were compared to those of various strains by restriction fragment length polymorphism (RFLP). The RFLP analysis based on the omp1 gene and the 16S-23S sequence confirmed that this isolate belonged to C. abortus species and was highly related to known ruminant's abortion strains. A live vaccine designed 1B, based on temperature -sensitive mutant of the ovine abortion reference strain AB7 is commercially available. The efficacy evaluation of this vaccine against the springbok C. abortus strain was assessed using protection -challenge experiments in a mouse model. Protection -challenge experiments show that ASb1 strain was highly abortive in mice and vaccine 1B provided them effective protection. References 1. Buxton D. Potential danger to pregnant women of Chlamydia psittaci from sheep. Vet Rec 1986; 118: Everett K, Bush R, Andersen A et al. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam.nov.and Simkaniaceae fam. nov. each containing one monotypic genus, revised taxonomy of the family Chlamydiacea, including a new species, and standards for the identification of organisms. Int J syst Bacteriol 1999; 49: Jonhson LW. Llama reproduction. Vet Clin North Am Food Anim Pract 1989; 5: Mansell JL, Tang KN, et al. Disseminated chlamydial infection in antelope. J Vet Diagn invest 1995; 7: Rodolakis A, Salinas J, Papp J et al. Advances on ovine chlamydial abortions. Vet Res 1998; 29:

53 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. ENDOPARASITE MANAGEMENT IN CAPTIVE BIRDS OF PARADISE AT AL WABRA WILDLIFE PRESERVATION J. SCHULZ, S.B. JENSEN and S. HAMMER Affiliation: Al Wabra Wildlife Preservation, P.O. Box 44069, Doha, State of Qatar; alwabra@qatar.net.qa Extended abstract Even though Birds of Paradise (BoP) are well known as spectacular animals, they are only kept in a few facilities worldwide. Therefore, information on diseases - including endoparasites in captive BoP - is scarce. Our facility is currently housing more than 70 BoP of six different species; the animals are kept individually or in pairs in large aviaries in six different houses with soil ground and tropical vegetation, with a small attached concretefloored feeding cage; successful breeding has been achieved (2). Since 2001, faecal samples of individuals/pairs were examined for parasites with increasing frequency till 2003 a monthly routine screening was established, using a standard flotation technique and microscopic examination. Findings were documented in a semi-quantitative way (0, +, ++, +++, ++++). All examinations were performed by the same investigator. The percentage of positive samples decreased during the last three years (2001: 59.3%, n=59; 2002: 36.6%, n=290; 2003: 25.7%, n=962). The main findings were capillaria, coccidian, railletina cestodes, and various other nematodes. Amo ng the positive samples, the percentage of coccidian was relatively constant throughout the three years (45.7, 37.7 and 45.7%), whereas that of capillaria was declining (62.9, 50.9, 31.2%) and that of the cestodes was increasing (8.6, 19.8, 26.7%). There were notable differences between the individual bird houses. Less than 20 % of our BoP have never tested positive for parasites so far. Birds with a positive result are treated with fenbendazole or mebendazol, ivermectine, clazuril or praziquantel. In retrospect, it is not possible to decide whether the results indicate real developments in the incidences of parasite infestation in our facility, or whether this shift is due to the fact that in the beginning, only clinically suspicious individuals were tested. This would mean that capillaria infestations result in clinically ill birds and decrease in incidence when clinically healthy specimens included in the survey as well, whereas the opposite would hold true for cestode infestations. Up to now, one major problem in the adjustment of the anti-parasite treatment is the uncertainty whether a positive faecal sample after a period of medication denotes a persistent infestation or a new infestation from the enclosure environment. Several animal- and diet-related factors make an efficient parasite protection difficult. Like other frugivorous birds (5), BoP have very short gastrointestinal tracts and probably fast short ingesta passage rates. This could make oral medication less effective and necessitate higher dosages than in other bird species. At the same time, overdosing with potentially negative side-effects such as a fenbendazole toxicity (3) should be avoided. An enclosure design using natural vegetation and soil ground is regarded necessary for the well-being and particularly the breeding success in BoP (2, 4). While direct contact to faeces might be less probable, such an enclosure design allows the development of parasitic stages in intermediate hosts (earthworms, ants, beetles) which then might be ingested by the birds.

54 The increasing percentage of tapeworm infestations among positive cases for tapeworms, an intermediate host is obligatory could underline this problem. Interestingly, the BoP species most frequently diagnosed with tapeworms the twelve wire bird (Seleucidis melanoleuca ) is reported to have the highest proportion of insect food of all BoPs in its natural environment (1). The impression that the percentage of capillaria infestations has decreased could be an indication that the predominant capillaria species at Al Wabra does not rely on an intermediate host, but this suspicion needs to be corroborated by a species identification of the parasite. For the near future, such species identifications are planned, as well as controlled treatment trials with different dosages. As a new management procedure, all birds are now kept only in the feeding cages during deworming medication in order to reduce infestation of enclosure soil with parasite eggs, and to allow a reliable control of treatmen t success. References 1. Beehler BM, Pruett-Jones SG. Display dispersion and diet of birds of paradise, a comparison of nine species. Behavioral Ecology and Sociobiology, 1983; 13: Bruslund Jensen S and Hammer S. Breeding birds of paradise at Al Wabra Wildlife Preservation. International Zoo News, 2003; 5: Papendick R, Stalis I, Harvey C, Rideout B, Zuba J, Allen J and Sutherland-Smith M. Suspected fenbendazole toxicity in birds. Proc AAZV AAWV Joint Conference, 1998; Searle KC. Breeding Count Raggi s bird of paradise ( Paradisaea raggiana salvadorii) at Hong Kong. International Zoo Year Book, 1980; 20: Stevens CE and Hume ID. Comparative physiology of the vertebrate digestive system. Cambridge: Cambridge Univ Press, 1995.

55 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. MELENGESTROL ACETATE (MGA) AND ITS USE IN NON-HUMAN PRIMATES REPRODUCTION A. FILIPPI 1, K.C. GAMBLE 2 and S. ROMAGNOLI 3 1. Veterinary praxis, Padua, Italy, November 2003 graduate. Preceptorship student (Aug-Oct 2003) at the Lincoln Park Zoo, Chicago IL, USA. 2. DVM, MS, Dipl. ACZM, Director of Veterinary Services of Lincoln Park Zoo Chicago, IL, USA. 3. DVM, MS, PhD, Dipl ECAR, Associate Dean Professor, Clinical Veterinary Reproduction President, European Society of Feline Medicine Dept of Veterinary Clinical Sciences Faculty of Veterinary Medicine, University of Padua. Abstract Contraception is a tool to manage populations (Asa, 1996). It is necessary to control the number of animals in an ethically acceptable manner, in order to have space availability. This is necessary to achieve conservation. The goal of this work was to review melengestrol acetate (MGA) as a contraceptive method in non-human primate species housed in zoos. Ten cases of MGA usage in non-human primates (Lincoln Park Zoo, Chicago, IL, USA) are described with a total of 28 implants used. These cases are drawn from the historical database (using MedARKS) of a single collection for Working with these data, the efficacy was shown to be very high with no failures, the most frequent side effect was implant loss, and reversibility was never required. Two further case reports are presented to analyze the methodology of using the implant, from acquisition through placement. In conclusion, some considerations about the future of MGA are discussed. Key words : contraception, melengestrol acetate, MGA, non-human primate, implant. Introduction Improved conditions in zoological gardens have led to an increased life span, as well as to enhanced breeding success, and decreased neonatal mortality in many species. Even in endangered species, uncontrolled breeding inevitably creates a surplus of animals, for example, the threatened golden lion tamarin (Leontopithecus rosalia ) has a captive population that is voluntary kept at 500 individuals. In order to achieve this point, only 40 pairs are bred yearly and the others are contracepte d (Wood, 2001). A time has been reached where the life span of many captive animals is much longer than that of its wild counterpart. This fact, together with a higher reproduction rate, can lead to the potential overpopulation of different species in the zoo. In the past, the surplus of animals was solved by more space availability, and by the existence of a less prohibitive legislation. As mortality rate of captive animals was much higher than today, prevention of reproduction was seen not as a management tool but as an antithesis of a breeding program. All of these elements made the problem of overpopulation a minor one. Today, a real problem that zoos as a collective ark are facing is space (Asa, 1996). They need space to preserve the population and, parallel to this, a strategy of birth control in order to maintain a balance between the space available and the number of genetically valuable animals. In the United States, the breeding of many particular species is managed by Species Survival Plans (SSP) of the American Association of Zoos and Aquariums (AZA). However, not all species are in planned breeding programs. As euthanasia options are legally and ethically controversial, so an increasing demand for an effective way to control reproduction exists. Morality demands respect for animal life. This respect, in turn, demands fertility control rather than culling, if numbers of animals absolutely must be limited (Cohn, 2001).

56 Briefly, the best solution to the surplus animal is to prevent it in a way that possibly does not harm the animal s fertility. The importance of reversible contraception as a method to aid in the establishment of a genetically viable captive population within the constraints of limited captive habitat was recognized early ( Sea l et al., 1976) and is today widely recognized by the working experience of zoos. The manager s goal through the SSP and EEP (European Endangered Species Survival Plan) is thus to make every birth a planned birth. The ideal contraceptive method should be safe, have minimal or no effect on social organization or behaviour, prevent all pregnancies, and be reversible and inexpensive. Many contraceptive methods are available nowadays for zoo animals. But unfortunately, none of the existing methods fully satisfies these criteria. As a result, contraception in zoo is made on a case-by-case method. In response to the need of contraception, in 1989, the Contraception Advisory Group (hereafter referred to as CAG) was established by the AZA. The contraceptive meth ods available for exotic animal are: separation of sexes, gonadectomy, synthetic hormones, vaccines, antispermatogenenics, and mechanical devices (Asa 1999). The MGA (Fig. 1) is a progestinic drug. It is 100 times more potent than progesterone. It is one of the most frequently used contraceptive methods because it is: - very effective; - long acting (2 years), due to the silastic implant that allows slow drug delivery; - acceptably reversible; - it can be used in a wide variety of animals; - it is inexpensive and easy to produce. Figure 1: Melengestrol acetate (MGA) chemical structure (left) compared with progesterone (right). However, if the animal has a great genetic value, MGA is used with caution. As this relates to length of treatment, the use of one or two implants followed by a pregnancy is preferred. This is due mainly to the progestinic nature of MGA that increases the level of physiological progesterone thus overstimulating the genital tract. The disadvantages of MGA are: -infection of the surgery site and loss of the implant; -effects on the uterine mucosa (hyperplasia, infection); -weight gain and decrease in spontaneous activity; As MGA is still an experimental treatment and not everything is known about its mechanism of action, other disadvantages typical of progestins such as development of benign and malignant mammary tumours, adrenocortical suppression, acromegaly, diabetes mellitus, or congenital malformation of female foetuses when treating during pregnancy may be present. Melengestrol acetate works as a contraceptive by : 1 thickening the cervical mucus which impedes the movement of mucus 2 desynchronize sperm and ovum transportation in uterus and oviducts 3 blocking the lutenizing hormone (LH) surge necessary for ovulation (Asa, 99).

57 Studies in New World monkeys and felids demonstrate that MGA does not suppress folliculo genesis and luteal activity may occur during treatment (felids: Kazensky et al.,1998; common marmoset (Callithrix jacchus):mohle et al. 1999). The anti-estrogenic effect has some species- and dose-dependent effects, both on the genital tract and behavior. As with all the contraceptive methods used in captive or free ranging wildlife, the MGA is considered experimental. Despite the decades of use, the Food and Drug Administration (FDA) still designates it as an Investigational New Animal Drug (INAD) when used in zoo animals. This is due to the fact that sufficient data on both the efficacy and safety has not been generated for any of the contraceptive methods used in non-domestic species. However, the CAG maintains a database to monitor all contraceptives used in captive wildlife. Melengestrol acetate has been successfully used as a contraceptive in ruminants, chickens, rodents, swine, horses, domestic dogs, domestic and wild catsat various dosages (Wood, 2001), and, since 1981, in primates (Asa, 1990). It is supplied by a single source (E.D. Plotka Marshfield, WI) in silastic depots to provide continuous, long -term contraception. It is also active orally. One of the CAG scientific advisors ( Karen Dematteo) provided the data from the species that were evaluated (tab. n. 1). This demonstrates that implant loss is the most frequent side effect and that the implant is typically very effective. The failure rate is under 1% for all of the non-human primates, except the howler monkey (Alouatta caraya), where the failure rate is estimated at 3.3%. It is evident from the data that the biggest problem is the implant loss with an average around 10%. Reversibility is not yet completely achieved but an interesting point from this chart is that there are not as many reversal trials, suggesting that this method is used even if reversibility is not required. MGA IMPLANT 2003 SURVEY. Family Genus / Species: # total used MGA #stopped for medical reason # stopped for reproduction # reversal # incomplete reversal attempts # implant lost # unplanned birth # planned birth # stopped for other # birth control failure # pregnant at start Cebidae Alouatta caraya Callicebus donacephalus Callitrichidae Saguinus oedipus Cercopithecidae Allenopithecus nigrovirdis Mandrillus sphinx Cercopithecidae Colobus guereza Table. 1 Summary of the 2003 contraception survey on MGA implants. Data from the annual (Contraception Advisory Group) survey. The named species are the once evaluated in this study.

58 Case description.of the nearly 20 non-human primate species housed at the Lincoln Park Zoo, Chicago, IL, twelve are actually contracepted with MGA implants to prevent reproduction and were considered as subjects for this work. NUMBER OF ANIMAL COMMON NAME SCIENTIFIC NAME 5 Howler monkey Alouatta caraya 1 Mandrill Mandrilus (Papio) sphinx 1 Cotton topped tamarin Saguinus oedipus 1 Bolivian gray titi monkey Callicebus donacephalus 1 Allen s swamp monkey Allenopithecus nigrovirdis 3 Eastern Black and white colobus Colobus guereza Material. Lincoln Park Zoo s clinical medical history (from MedARKS ) was reviewed to obtain data about side effects, efficacy and reversibility of MGA implant. A total of 28 implants were evaluated from 1994 to For the first ten animals, a data sheet was created to easily collect information (fig n.1) from the records. It has been divided into 3 sections: -1-Animal Information: species; scientific and common name; age; sex; weight; reason for using contraception; reproductive history. -2-MGA implant information: dosage; method of sterilization; placement (site and tissue were it was placed). It is very important to assess these factors as one of the side effects is the formation of an abscess around the implant causing either loss of the implant and non-function as well as affecting animal health -3-Observation: if the animal tried to remove the implant, copulation; sexual swelling; behavioral changes; uterine size during examinations; presence of lactation; alopecia at the implant site. Two Eastern black and white colobus monkey (Colobus guereza) were implanted and evaluated as to the surgical procedure of implantation. The gas sterilized implant was put into the right quadriceps muscle belly. Results. Over 28 implants were placed and only five were lost due to infection of the surgical site. This infection is either due to grooming or improper sterile procedure during placement. None of the 7 implants placed into the muscle belly were lost. Unfortunately the type of sterilization method used to sterilize the implant was not reported in the MedARKS data. Mating behaviour was maintained in some New World primate species. It was not reported for any Old World primate nor for the Bolivian Gray titi monkey (Callicebus donacephalus), probably because they were housed as a family group where this disrupts the cycling of younger females. No failures were reported when defined as parturition and the implant is found in place. However, one unplanned birth occurred from an implant loss. A cotton-topped tamarin was implanted while pregnant and gave birth to two healthy offspring and one stillborn. This demonstrates the acceptable safety of the implant during pregnancy and parturition.

59 Concluding discussion. Discordant data in literature proved that, despite the years of trials, contraception for exotic animals, is still experimental and there is a need of more scientific trials that have to be coordinated by the SSP and EEP to study this subject in more depth. MGA is widely used in primates. It appears very safe and effective, but not enough data were available to assess reversibility. It is suggested that more effort should be put in controlling reproduction, and reversible contraception should not be seen as an alternative methods for controlling surplus instead of sterilization. On the contrary, permanent methods should be re - evaluated, since they are the safest and most recommended ones, especially when long-term reproduction control is required. However, managers feel more secure if contraception method is reversible if anything were to happen to the breeding pair. The implant loss is the most common problem, although it seems to diminish if the implant is placed within the muscular mass it is suggested that increased blood supply and tissue protection helps to diminish infection and implant loss. Acknowledgements We thank Lincoln Park Zoo and its staff for their support as well as Karen Dematteo, Dr. Ed Plotka, and Dr. Tom Meehan for their pertinent information. We also thanks Dr. Christian Walzer, Dr. Paolo Cavicchio, Dr. Gidona Goodman, Dr. Thierry Petit, Dr. Klaus Friederich for the fruitfull discussions. References <<Journals>> 1. Abbott DH, Barnett DK, Colman RJ, Yamamoto EM, Schultz -Darken NJ. Aspect of Common Marmoset Basic Biology and Life History Important for Biomedical Research. Comparative Medicine Vol 53 n 4 August 200 3: Asa CS, Porton I. Primate contraception methods in use and development. Proceedings American Association of Zoo Veterinarians annual conference 1990: Cohn, PN, International Symposium on Reproduction in Wildlife Population proceeding 2001: Kazensky C A, Munson L, Seal US the effect of melengestrol acetate on the ovaries of captive wild felids. Journal of Zoo and Wildlife Medicine 29(1) 1998: Mohle U., Heistermann m, Einspainier A, Hodges JK. Efficacy and effects of short- and medium- term contraception in the common marmoset (Callitrix jacchus) using melengestrol acetate implants. J Med Primatol feb; 28(1): Murnane R D, Zdziarski J M, Walsh T F, Kinsel M J, Meehan T P, Kovarik P, Briggs M Reverty S A, Phillips G Jr: Melengestrol Acetate induced exuberant endometrial decidualization in Goeldi s Marmoset (Callimico goeldii) and Squirrel Monkey (Saimiri sciureus). Journal of Zoo and Wildlife Medicine 27(3), 1996: Plotka E D, and Seal U S. Fertility control in deer. Journal of Wildlife Disease 25,1989: Wood C, Ballou J D, Houle C S: Restoration of reproductive potential following expiration or removal of melengestrol acetate contraceptive implants in golden lion tamarins (Leontopithecus rosalia). Journal of Zoo and Wildlife Medicine 32(4) 2001: <<Books>> 1. Asa C. Mammalian Contraception. In: Fowler ME, Miller RE (eds). Zoo & Wild Animal Medicine. Current Therapy 4. WB Saunders Company Philadelphia etc 1999: Asa CS, Porton I, Baker AM, Plotka ED. Contraception as a Management tool for Controlling Surplus Animals. In: Wild Mammals in Captivity. Kleiman D G, Allen M E, Thomson K V and Lumpki S (Eds. The university of Chicago Press, Chicago) 1996: <<Internet references >> 1. Dematteo K, MS. Saint Louis Zoo. Member and Scientific Advisor of the Contraception Advisory Group (CAG). dematteo@stlzoo.org 2. Saint Louis Zoo internet site.

60 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. ENTEROTOXEMIA ASSOCIATED WITH BETA2 TOXIN-PRODUCING CLOSTRIDIUM PERFRINGENS TYPE A IN 2 ASIATIC BLACK BEARS (Selenarctos thibetanus) GRAZIA GRECO 1 *, ANNA MADIO 1, VITO MARTELLA 1, MARCO CAMPOLO 1, MARIALAURA CORRENTE 1, MARTA TOTARO 1 and DOMENICO BUONAVOGLIA 1 Affiliation: From the Department of Health and Animal Well-being 1 of the Faculty of Veterinary Medicine of Bari Valenzano, Italy Poster Abstract C. perfringens is a Gram positive spore-forming, anaerobic rod responsible for necrotic and haemorrhagic enteritis and enterotoxemia in humans and in several animal species. This bacterial species can produce: i) four major lethal toxins namely alpha, beta, epsilon and iota toxin (α, β, ε, ι) on which the toxin typing of the species is based; ii) nine minor toxins (delta, theta, kappa, lambda, mu, nu, gamma, eta, and neuraminidase); iii) the enterotoxin, which is responsible for foodborne illnesses caused by C. perfringens. Another toxin of C. perfringens of recent identification is the β2. The β2 toxin is produced by C. perfringens isolated from piglets with haemorrhagic enteritis and from other animal species (horses, calves, lambs and elephants) sometimes showing haemorrhagic enteritis or diarrhoea. We report the characterization of β2-toxigenic C. perfringens type A strains isolated from 2 asiatic black bears (Selenarctos thibetanus) (GG17/02, GG18/02), died suddenly of enterotoxemia. The animals, of approximately 7 years of age, had been raised in captivity in the Fasano Safari Zoo (Bari-Southern Italy) and belonged to a population of 35 asiatic black bears. The 2 bears developed the disease after receiving a meal with spoiled herrings, that had been thawed three days before and kept at room temperature until administration. The bears developed depression, anorexia, pale mucosae and bloody diarrhea. One animal died on the third day after the index meal and the other bear a week after showing the same clinical signs. Necropsy, per formed on both animals within 6 h after death, revealed necrotic and haemorrhagic enteritis in the small and large intestine, petechia and ecchymotic haemorrhages on myocardium, pale friable liver and congested edematous lungs. Specimens from the liver, lungs, heart and small and large intestine of both the animals were collected and processed within 2 h. Each specimen was plated out on 5% sheep blood agar and cultured aerobically, at 37 C for 2 days, to exclude the presence of aerobic pathogenic organisms. Anaerobic culturing was done onto eggs yolk agar with D-cycloserine 400 µg ml -1 (TSC) and 5% sheep blood agar, at 37 C for 18 h. For the anaerobic growth, colonies with zone of double hemolysis and lecithinase activity on TSC agar were considered to be C. perfringens and inoculated in fluid thioglycollate medium (FTG). The overnight cultures were used for DNA extraction. To type the C. perfringens isolates by their toxin genes, multiplex-pcr was employed. Samples were tested for presence of cpa, cpb, cpb2, etx, iap and cpe genes, which encode the α, β, ε, ι, β2 and enterotoxins, respectively. Large numbers of C. perfringens in pure culture were isolated from livers, lungs, hearts and the small and large intestine content of both animals. By multiplex PCR analysis it was possible to classify all the isolates, obtained from the animals, as C. perfringens type A β2-toxinpositive. The role of C. perfringens type A isolates in enteric diseases, and particularly the role of the α-toxin as a virulence factor of type A and of the other toxin types, is still uncertain. However the α- toxin alone may not account for the development of severe necrotic lesions at the intestinal level. It was possible to isolate C. perfringens type A β2-toxin-positive from the intestine and tissues of the bears and the contemporaneous presence of the α and β2 toxins would be more consistent with the severity of the lesions observed. The administration of partially spoiled meat in the bears' diet may have decreased the intestinal motility, with reduction of the normal bacterial flora and overgrowth of C. perfringens in the small and large intestine, with the concomitant production of extracellular toxins responsible for the enterotoxemia. It is unclear whether the bears were carriers at the intestinal level of the microrganisms isolated or they had ingested clostridia with food-stuff. However the spoiled meat could have been the source and the predisposing factor of the infection. A similar toxin-type, indeed has been identified in the intestinal content of healthy Atlantic codfish and as the animals usually

61 receive a meal based on frozen herrings from North Sea, it may be hypothesized a food-born origin of the unusual infection of the bears. Our investigations showed that β2 toxin-positive C. perfringens type A may be associated with enterotoxemia and severe necrotic enteritis in asiatic black bears. This is, to our knowledge, the first report of enterotoxemia associated with β2-toxigenic C. perfringens type A in asiatic black bears in Italy. Address of authors : * Dott.ssa Grazia GRECO Dipartimento di Sanità e Benessere Animale, Facoltà di Medicina Veterinaria di Bari, S.p. per Casamassima Km 3, Valenzano (Bari), Italia Tel.: , Fax: g.greco@veterinaria.uniba.it

62 European Association of Zoo- and Wildlife Veterinarians (EAZWV) 5 th scientific meeting, May , Ebeltoft. Denmark. COMPARATIVE LIGHT AND SCANNING ELECTRON MICROSCOPIC STUDY OF THE EPITHELIAL LAYER OF THE TERM PLACENTA OF THE ASIAN ELEPHANT (Elephas maximus) A. HOFFMANN 1, S. LODERSTEDT 1, K. EULENBERGER 2, M. FLÜGGER 3, J. JUNHOLD 2 and J. SEEGER 1 Affiliation: 1. Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 43, Leipzig, Germany 2. Zoo Leipzig, Pfaffendorfer Str. 29, Leipzig, Germany 3. Tierpark Hagenbeck, Lokstedter Grenzstr. 2, Hamburg-Stellingen, Germany Keywords: Asian elephant - term placenta - epithelium - trophoblast - allantochorion - light microscopy - scanning electron microscopy Poster Abstract The combined light and scanning electron microscopic (SEM) analyses are the first findings to investigate the morphological features of the fetale membrane of the placenta in the Asian elephant (Elephas maximus). The detailed characterisation of the epithelial layers on different locations documented with ultrastructural procedures (SEM) are poorly understood. We used 2 term placentas with gestation period of 634 days (Zoological Garden Leipzig) and 657 days (Tierpark Hagenbeck, Hamburg) and selected unequal sites as specimen, particularly - free membrane of the allantois, - lateral edge of the placentar band, the "haemophagous zone", - allantochorion near the placental band and, - allantochorion on the end of the chorioallantoic sac. Macroscopically we confirmed the completely zonary placenta with marginal haemophagous zone. In our cases the whole weight of the placentas measured kg and the placental band indicated cm in circumferential diameter, cm in width and 4-5 cm in thickness. On the outer surface of the end of allantochorionic sac numerous white "pustules" have been seen. The allantoic membrane is distinguished in 4 compartments and determined the formation of the umbilical cord, which possessed one vein, two arteries and one urachus. Microscopically the free membrane of the allantois shows a simple, cuboidal epithelium with apical domains of microplicae and microvilli. In the SEM analyses we documented a cobblestone -like architecture of the epithelial cells with various cell sizes, small, middle and giant. Furthermore, we found pear-shaped cells with long pedicle, which are attached to the basement membrane and cell openings, presumable due to cell desquamation. The haemophagous zone was characterised by a lamellate system between the long fingerlike chorionic villi and the maternal blood lacunae. The simple, squamous and columnar trophoblast cells are here bathed in the maternal blood. In some chorionic villi deposits brown-yellow material in the fetale connective tissue. A new finding was the appearance of strip-like microplicae in the middle of chorionic villi. The allantochorion near the placental band indicated simple and ramified chorionic villi with smooth, gyrus-like trophoblast cells and foamy cytoplasma, sometimes. The attached allantois possessed a simple, columnar epithelium with microvilli. Furthermore, we analysed villi-like projections, which rising up to allantoic cavity. The white "pustules" on the outer surface of the end of the chorioallantoic sac consist of stratified squamous epithelia and in the basal domain of the pustules numerous mitosis have been seen. Surprisingly, the findings of intraepithelial capillaries intend the trophoblast cells

63 presume a nutrient exchange also outside of the placental band with her gross functional metabolism. This study revealed some unknown and interesting features of the epithelial layer of different locations in the placenta of an Asian elephant. Perhaps, with a detailed knowledge of the morphology of the epithelium we convey a better understanding of functional cohesions of elephant placentation. figure 1: The free membranes of the allantois of the term placenta of the Asian elephant (Elephas maximus) shows a cobblestone-like architecture of the simple cuboidal epithelial cells with various sizes. The surface morphology are determined through microplicae and microvilli. figure 2: Among the cuboidal epithelial cells exist numerous pear -shaped cells with long pedicle exist, which attached on the basement membrane. The cells possessed modifications of their surface, particularly microplicae. Some pear -shaped cells shows only the pedicle, perhaps that is a loss through preparation or its presumable due to cell desquamation. Between these interesting cells one erythrocyte lies. Acknowledgement We are extremely grateful Mrs. Bachmann of the Zoological Garden Leipzig for their practical help in gathering samples.