Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring.

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng µL-1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80-120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

Retrospective Study of Intertarsal Joint Inflammation in Avian Species From a Zoological Institution.

A retrospective cross-sectional study was performed to measure the prevalence of avian intertarsal inflammation over a 5-year period, identify risk factors, and discuss treatment options. The authors hypothesized that: 1) long-legged birds would be more affected, 2) participation in a bird show would be a significant risk factor, and 3) young animals would be more frequently affected. Thirty-five clinical cases from 9 avian orders were included in the study. Statistical analysis indicated that the orders Ciconiiformes (9/150; 6%, 95% confidence interval [CI]: 3.2-11), Gruiformes (4/132; 3%, 95% CI: 1.2-7.5), and Pelecaniformes (7/152; 2.8%, 95% CI: 1.4-5.6) were significantly more affected than other orders (P < 0.01). Similarly, long-legged birds (21/35) had 9.8 times greater chance (P < 0.001, 95% CI: 4.7-21) of developing the condition compared with other birds. Participation in a free-flight show (22/35) was a significant risk factor (P < 0.001; odds ratio: 7.0, 95% CI: 3.3-15). Mean age at onset of clinical signs was 5.7 years, and being < 2 years-of-age during the study period was not a significant predictor of disease (P = 0.054). The tibial cartilage, a fragile fibrocartilaginous structure, was frequently affected (34%, 12/35). Treatment protocols included anti-inflammatory drugs, analgesic drugs, or both (94%, 33/35), low-level laser therapy (54%, 19/35), joint immobilization (34%, 12/35), intra-articular corticoid injections (20%, 7/35), surgical stabilization (17%, 6/35), physiotherapy (9%, 3/35), intra-articular hyaluronic acid (6%, 2/35) or platelet-rich plasma (3%, 1/35) injections, and chiropractic care (3%, 1/35). Overall recovery rate was 49% (17/35), and the condition was associated with a poor prognosis in chronic cases.

Influence of Freezing Temperature, Freezing Duration, and Repeated Freeze/Thaw Cycles on Electrophoretic Profiles in the White Stork (Ciconia ciconia).

Plasma electrophoresis is an ancillary diagnostic tool in avian medicine, with agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) being the most common techniques. Frozen samples can be used for quantitative studies or comparative diagnostic purposes, but stability of avian plasma proteins under freezing is poorly described. To evaluate the influence of plasma freezing on electrophoretograms in white storks (Ciconia ciconia), heparin blood was sampled from 30 individuals during annual health examinations. Plasma samples were obtained after centrifugation of fresh samples and divided into aliquots. Both AGE and CZE were performed on fresh aliquots. The remaining aliquots were frozen at -20°C (-4°F) or -180°C (-292°F) and thawed following different protocols: 1 freeze/thaw cycle after 6 months at -20°C; 1, 2, 4, and 7 cycles over 12 months at -20°C; and 1 cycle after 18 months at -180°C. For both techniques, electrophoretic profiles obtained from these thawed aliquots were compared to fresh electrophoretograms. Quantitatively, significant differences (P < 0.05) in most fractions were seen from 6 months postfreezing at -20°C for both techniques. Fewer statistically significant differences were observed after 18 months under cryogenic preservation (-180°C). Qualitatively, AGE provided more repeatable and stable results than CZE over time on samples stored at -20°C, and electrophoretograms were stable after 18 months of cryogenic storage. An electromigration distortion associated with freezing was seen with CZE only. Plasma samples stored in a conventional freezer (-20°C) should not be compared to fresh plasma. For quantitative studies, cryogenic storage should be privileged.

Conserved core microbiota in managed and free-ranging Loxodonta africana elephants.

The gut microbiota plays a crucial role in animal health and homeostasis, particularly in endangered species conservation. This study investigated the fecal microbiota composition of European captive-bred African savanna elephants (Loxodonta africana) housed in French zoos, and compared it with wild African savanna elephants. Fecal samples were collected and processed for DNA extraction and amplicon sequencing of the 16S rRNA gene. The analysis of α and ß diversity revealed significant effects of factors such as diet, daily activity, and institution on microbiota composition. Specifically, provision of branches as part of the diet positively impacted microbiota diversity. Comparative analyses demonstrated distinct differences between captive and wild elephant microbiomes, characterized by lower bacterial diversity and altered co-occurrence patterns in the captive population. Notably, specific taxa were differentially abundant in captive and wild elephants, suggesting the influence of the environment on microbiota composition. Furthermore, the study identified a core association network shared by both captive and wild elephants, emphasizing the importance of certain taxa in maintaining microbial interactions. These findings underscore the impact of environment and husbandry factors on elephant gut microbiota, highlighting the benefits of dietary enrichment strategies in zoos to promote microbiome diversity and health. The study contributes to the broader understanding of host-microbiota interactions and provides insights applicable to conservation medicine and captive animal management.

Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) under managed care: a clinical and environmental investigation in a French zoological park.

Aspergillosis is pervasive in bird populations, especially those under human care. Its management can be critically impacted by exposure to high levels of conidia and by resistance to azole drugs. The fungal contamination in the environment of a Humboldt penguin (Spheniscus humboldti) group, housed in a French zoological park next to numerous large crop fields, was assessed through three serial sessions of surface sampling in nests, in 2018-20: all isolates were counted and characterized by sequencing. When identified as Aspergillus fumigatus, they were systematically screened for resistance mutations in the cyp51A gene and tested for minimal inhibitory concentrations (MICs) determination. At the same time, the clinical incidence of aspergillosis was evaluated in the penguin population by the means of systematic necropsy and mycological investigations. A microsatellite-based analysis tracked the circulation of A. fumigatus strains. Environmental investigations highlighted the substantial increase of the fungal load during the summer season (>12-fold vs. the other timepoints) and a large overrepresentation of species belonging to the Aspergillus section Fumigati, ranging from 22.7 to 94.6% relative prevalence. Only one cryptic species was detected (A. nishimurae), and one isolate exhibited G138S resistance mutation with elevated MICs. The overall incidence of aspergillosis was measured at ∼3.4% case-years, and mostly in juveniles. The analysis of microsatellite polymorphism revealed a high level of genetic diversity among A. fumigatus clinical isolates. In contrast, one environmental strain appeared largely overrepresented during the summer sampling session. In all, the rural location of the zoo did not influence the emergence of resistant strains.

Assessment of the Safety and Efficacy of an Oral Probiotic-Based Vaccine Against Aspergillus Infection in Captive-Bred Humboldt Penguins (Spheniscus humboldti).

Aspergillosis is a fungal infection caused mainly by Aspergillus fumigatus that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental A. fumigatus infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (Spheniscus humboldti). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Escherichia coli Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti-A. fumigatus Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in E. coli Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti-E. coli IgY and anti-α-Gal IgY in the E. coli Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that E. coli Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.

EQUID ALPHAHERPESVIRUS 9 OUTBREAK ASSOCIATED WITH MORTALITY IN A GROUP OF GREVY'S ZEBRA (EQUUS GREVYI) HOUSED IN A MIXED-SPECIES EXHIBIT.

A herd of seven captive-born Grevy's zebras (Equus grevyi) experienced an outbreak of nasal discharge and sneezing. Clinical signs, including lethargy and anorexia, were severe and acute in three animals, including a 16-mo-old male that died within 48 h. Treatment of two severely affected zebras included valacyclovir (40 mg/kg PO), meloxicam (0.6 mg/kg IM/PO), and cefquinome (2.5 mg/kg IM q48h). An adult female improved rapidly, and clinical signs resolved within 48 h of treatment. Administration of valacyclovir pellets was very complicated in a 2-mo-old female, and death occurred within 48 h. Histologic examination of the two individuals that died revealed severe fibrinonecrotic interstitial pneumonia with prominent hyaline membranes and type II pneumocyte hyperplasia. Additionally, the 16-mo-old male presented systemic endothelial activation with vascular thrombosis and necrosis and mild nonsuppurative meningoencephalitis. Herpesviral DNA was detected in the lungs of both individuals by nested polymerase chain reaction. The nucleic acid sequence of the amplicons showed 100% similarity with previously published equid alphaherpesvirus 9 sequences. Three additional animals developed mild nasal discharge only and recovered spontaneously. The zebras shared housing facilities with other species, including white rhinoceros (Ceratotherium simum), reticulated giraffe (Giraffa camelopardalis reticulata), and several antelope species. None of these animals showed clinical signs. Additionally, nasal swabs and whole blood samples were collected from cohoused white rhinoceroses (n = 3) and springboks (Antidorcas marsupialis, n = 3) as well as nasal swabs from cohoused reticulated giraffes (n = 4). Nucleic acid sequence from equid herpesviruses was not detected in any of these samples. The source of the infection in the zebras remains unclear.

ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE ADULT SPRINGBOK (ANTIDORCAS MARSUPIALIS), IMPALA (AEPYCEROS MELAMPUS), ALPACA (VICUGNA PACOS), AND RED-NECKED WALLABY (MACROPUS RUFOGRISEUS) OVER A YEAR.

Enterotoxemia is an important issue in various zoological taxa. In this study, serologic responses over a 1-yr period after vaccination with a multivalent clostridial vaccine were evaluated in 10 adult springboks (Antidorcas marsupialis), 12 impalas (Aepyceros melampus), seven alpacas (Vicugna pacos), and five red-necked wallabies (Macropus rufogriseus). Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay and determined as the percentage of inhibition (% inhib). Initial % inhib was (0.01-18.9)%. All animals received initial vaccination with a booster vaccine 4 weeks apart. Serum samples were collected at T0 (nonvaccinated), 15, 30, 60, 180, and 360 days postvaccination (dpv) for analysis. The vaccine induced a high antibody response that peaked at 15, 30, and 60 dpv in springboks, 30 and 60 dpv in impalas (P < 0.01), and 60 dpv in alpacas and wallabies (P < 0.01). The booster vaccine was followed by a high antibody response, which slowly decreased with time. The antibody response was significantly higher at 360 dpv than at T0 in wallabies and alpacas (P < 0.01). In impalas and springboks, it appeared that a booster every 6 mo might be required to maintain an antibody response above baseline (P < 0.01). Because no challenge studies were performed, it is unknown whether the measured humoral immune responses would have been protective. Further research is warranted to investigate protective effects of antibodies to inoculation challenge in nondomestic species.

Malignant Melanoma in a Long-legged Buzzard (Buteo rufinus): Characterization of Clinical and Histologic Lesions.

A 17-year-old, male long-legged buzzard (Buteo rufinus) presented for acute lethargy and black gelatinous tissue in both nostrils. Despite intensive care, the bird died within 2 days. A postmortem computed tomographic scan of the head revealed a complete obstruction of the rostral infraorbital sinus, later confirmed by necropsy. Postmortem examination also highlighted multifocal, black, infiltrating nodules of various sizes (2 mm to 2 cm in diameter) in almost all internal organs: lungs, heart (pericardium, myocardium, endocardium), kidneys, pancreas, adrenals, muscles, and bones (periosteum and medulla). Histologically, the nodules were composed of anaplastic cells containing abundant melanin pigment, consistent with a disseminated melanoma. Malignant melanomas are aggressive neoplasms frequently reported in mammals but rarely found in avian species. In Accipitridae and Falconidae, only 2 cases have been reported.

First case of Plasmodium relictum lineage pGRW11 infection in a captive-bred common eider (Somateria Mollissima) in Europe.

A 4-year-old, female common Eider (Somateria mollissima) was presented for mild lethargy with no previous medical history. Numerous intraerythrocytic, round-shaped inclusions were visualized on blood smears, later morphologically identified as Plasmodium relictum parasites. Despite oral doxycycline treatment, clinical condition declined 48 h later. Supportive care was initiated, but the bird died rapidly. Necropsy revealed acute, internal hemorrhages (lungs, air sacs) and subcutaneous, diffuse cervical hematoma, associated with resuscitation attempts. Marked, multicentric amyloidosis (kidney, liver, spleen) was the main histological finding. Molecular analysis identified lineage pGRW11 of P. relictum. This is the first reported case of P. relictum lineage pGRW11 infection in a common Eider. This report describes the clinical features, diagnosis, treatment and associated pathological findings of infection by P. relictum lineage pGRW11 in a common Eider.

New insights in Cercopithecinae spermatozoa.

Sperm morphometric and morphologic data have been shown to represent useful tools for monitoring fertility, improving assisted reproduction techniques and conservation of genetic material as well as detecting inbreeding of endangered primates. We provide here for the first time sperm morphologic and morphometric data from Cercopithecus neglectus, Cercopithecus cephus, Papio papio and critically endangered Cercopithecus roloway, as well as comparative data from other Cercopithecinae species, i.e. Allochrocebus lhoesti, Mandrillus sphinx and Papio anubis. Following collection from the epididymis, spermatozoa were measured for each species for the following parameters: head length, head width, head perimeter, head area, midpiece length and total flagellum length, and the head volume, ellipticity, elongation, roughness and regularity were then calculated. Our data are consistent with both the general morphology and the morphometric proportions of Cercopithecinae sperm. Some specificities were observed, with C. cephus displaying a narrow head (width = 2.76 ± 0.26 µM) and C. roloway displaying a short midpiece (6.65 ± 0.61 µM). This data set represents an important contribution, especially for Cercopithecus roloway, one of the most endangered monkeys in the world, and further data on additional specimens coupled to data on mating systems and reproductive ecology should allow a better understanding of the mechanisms underlying these morphological differences across primate species.

Laparoscopic ovariectomy with a single-port multiple-access device in seven African lionesses (Panthera leo).

CASE DESCRIPTION 7 privately owned female African lions (Panthera leo) that had been bred for public exhibition and were housed in outdoor pens were evaluated prior to undergoing elective ovariectomy. CLINICAL FINDINGS All animals were healthy. Median age was 15 months (range, 9 to 34 months), and median body weight was 71 kg (156 lb; range, 48 to 145 kg [106 to 319 lb]). TREATMENT AND OUTCOME Surgical sterilization by means of single-incision laparoscopic ovariectomy was elected. A 2- to 3-cm-long skin incision was made just caudal to the umbilicus, and a single-port multiple-access device was bluntly inserted through the incision. Traction was maintained with stay sutures to provide counterpressure, and three 5-mm-diameter cannulae were introduced through the device's access channels with a blunt trocar. The abdomen was insufflated to a pressure of 12 mm Hg with CO2. Each ovary was grasped and suspended with a standard 36-cm-long laparoscopic grasper, and ovariectomy was performed with a 5-mm vessel sealer and divider device. Because of the depth of subcutaneous fat, extensive subcutaneous dissection was necessary to insert the single-port device. In contrast, fat content of the mesovarium was minimal and did not vary markedly among animals. Subjectively, single-incision laparoscopic ovariectomy was easily performed, but all surgeons had experience in laparoscopic surgery. Median duration of the surgical procedure was 29 minutes (range, 21 to 49 minutes). No perioperative complications were encountered. CLINICAL RELEVANCE Findings suggested that the single-incision laparoscopic technique may be an acceptable, minimally invasive option for ovariectomy of large felids.

Clinical and histopathological aspects of an alopecia syndrome in captive Andean bears (Tremarctos ornatus).

BACKGROUND: Captive Andean bears (Tremarctos ornatus) develop a distinct alopecic syndrome of unknown aetiology. HYPOTHESIS/OBJECTIVES: To describe the histological features of healthy Andean bear skin, to define the clinical and histopathological features of Andean bears with signs of alopecia, and to propose an aetiopathogenesis. ANIMALS: Eighteen healthy Andean bears housed in 12 European zoos and 13 Andean bears with mild to severe alopecia housed in nine European zoos. METHODS: Two surveys describing signalment and clinical features of affected bears; follicular density was measured in a single healthy bear using a dermatoscope; cytological samples were collected by tape stripping from two healthy and three alopecic bears; skin biopsies were collected for histological evaluation from healthy and alopecic bears; immunohistochemistry (CD3, AE1/AE3 cytokeratins) was performed when lymphocytic inflammation was observed. RESULTS: The syndrome is an acquired, slowly progressive alopecia. Bears are otherwise healthy. Histological features include a dermal inflammatory infiltrate composed of T lymphocytes and eosinophils; atrophy of hair follicles at the level of or below the isthmus, and lymphocytic infiltration of hair follicles and the epidermis. Multinucleated giant cells were present in the outer root sheaths of hair follicles in five bears. CONCLUSION AND CLINICAL IMPORTANCE: Andean bear alopecia syndrome is an acquired, progressive alopecia with histological features consistent with a lymphocytic immune-mediated reaction directed against follicular sheaths and the epidermis. Trigger factors have not been identified. Further studies are indicated to define the features of this multifactorial syndrome.

TREATMENT SUCCESS IN THREE ANDEAN BEARS (TREMARCTOS ORNATUS) WITH ALOPECIA SYNDROME USING OCLACITINIB MALEATE (APOQUEL®).

Andean bear (Tremarctos ornatus) alopecia syndrome (ABAS) commonly affects captive bears, particularly sexually mature females. ABAS is characterized by bilaterally symmetrical predominantly flank alopecia with or without profound pruritus and secondary bacterial and Malassezia infections. There is no effective treatment and severely affected bears have been euthanized. This paper describes the successful management of ABAS in three female Andean bears. Skin biopsies and cytology revealed a mixed dermal inflammatory infiltrate, alopecia, hyperkeratosis, and Malassezia dermatitis. Allergen specific serology was positive for environmental allergens in one case. Hematology, serum biochemistry, and thyroid and adrenal function were normal in all cases. There was no consistent response to novel diet trials, antifungals, antihistamines, allergen specific immunotherapy, or topical antimicrobials. There was a partial response to ciclosporin (Atopica® cat, Novartis Animal Health; 5 mg/kg po, sid) in one case and oral glucocorticoids in all cases (dexamethasone sodium phosphate, [Colvasone 0.2%, Norbrook], 0.15 mg/kg po, sid or prednisolone [Deltacortene, Bruno Farmaceutici, and Megasolone 20, Coophavet], 0.3-1.2 mg/kg po, sid), but treatment was withdrawn following adverse effects. Treatment with oclacitinib maleate (Apoquel®, Zoetis; 0.46-0.5 mg/kg po, bid) resulted in rapid and complete resolution of the pruritus with subsequent improvement in demeanor and fur regrowth. After 5 mo, the bears were almost fully furred and off all other medication. Treatment was tapered to the lowest dose that prevented relapse of the pruritus (0.23-0.4 mg/kg po, sid). No adverse effects have been noted. ABAS is usually an intractable condition, and, to our knowledge, oclacitinib is the first treatment shown to result in sustained clinical improvement. Further studies on the etiology of ABAS, and on efficacy and long-term safety of oclacitinib are needed.

SQUAMOUS CELL CARCINOMA IN TWO SNOW LEOPARDS (UNCIA UNCIA) WITH UNUSUAL AURICULAR PRESENTATION.

Squamous cell carcinoma (SCC) is well documented in snow leopards ( Uncia uncia ) and most common locations are oral, facial, or pedal. These two cases illustrate an unusual auricular presentation, which is more often reported in white domestic cats. The animals were aged and presented clinical signs of otitis such as head shaking and ear scratching. Clinical examinations showed auricular canal masses with chronic purulent otitis. In both cases, clinical deterioration led to euthanasia and histology of the ear canal was consistent with SCC and showed numerous vascular emboli. These cases illustrate an unreported aggressive localization for SCC in snow leopards, which should be included in the differential diagnosis of otitis in this species. Auricular SCC may be underdiagnosed as the ear canal is infrequently sampled for histopathology. This auricular localization should be considered when metastases are found upon necropsy without internal primary tumor.

STRESS INFLUENCE ON PLASMA PROTEIN ELECTROPHORESIS IN THE RED-BREASTED GOOSE (BRANTA RUFICOLLIS) AND HAWAIIAN GOOSE (BRANTA SANDVICENSIS).

The objective of this study was to evaluate the effects of stress (as measured by total heterophil + eosinophil counts [THECs] and plasma corticosterone [PC] levels) on plasma protein electrophoresis (PPE) in two Anseriform species. Ten red-breasted geese ( Branta ruficollis ) and eight Hawaiian geese (Branta sandvicensis) were received into quarantine at Beauval Zoo and housed together with other Anseriformes. Two days later, all were examined as part of routine quarantine procedures, and blood was collected. THECs were performed using a Malassez hemocytometer after a 1 : 200 dilution with an eosinophil dilution liquid containing phloxine B. PPE, as well as total protein and PC assays, was performed on lithium heparin plasma. Twenty days after arrival, all birds were similarly restrained, examined, and sampled. Pododermatitis lesions were identified on four geese that were excluded from statistical analysis. For each goose, THECs and PC values were sorted by value (lower or higher) independently from the day of sampling. A Wilcoxon signed rank test showed no significant differences between lower and higher values of THECs for any of the PPE fractions. Higher values of corticosterone were associated with higher values of prealbumin, but none of the other fractions were significantly different. A Spearman rank correlation coefficient showed that THECs and PC were not correlated; this suggested differences in kinetics between these stress markers. Results did not show significant alterations of electrophoresis patterns associated with stress as measured with THECs and PC. The results also highlight the complexity of precisely assessing acute and chronic stress in avian species.

GREATER KUDU (TRAGELAPHUS STREPSICEROS) MORTALITY IN EUROPEAN ZOOLOGICAL INSTITUTIONS: A RETROSPECTIVE STUDY.

A questionnaire was sent to 39 European institutions holding greater kudus (Tragelaphus strepsiceros), in order to determine the causes of captive greater kudu mortality. All reported macroscopic lesions and histopathologic observations, as well as other information regarding individuals that died, were analyzed to determine the most affected body systems and causes of death. Overall response rate was 31%, and 131 individuals were included in the study. The most frequently affected body systems were the digestive system (47%), respiratory system (38%), musculoskeletal system (37%), and cardiovascular system (32%). Most frequent causes of death were infectious diseases (27%) and trauma/accidents (18%); the cause was undetermined in 28% of cases. Nutrition-related disorders were difficult to assess, but results highlight possible nutritional imbalances. This retrospective study represents the first overview of greater kudu mortality in a captive population.

CLINICAL MANAGEMENT OF BILATERAL CUTANEOUS SQUAMOUS CELL CARCINOMA OF THE HIND FEET PADS IN A SOUTHERN WHITE RHINOCEROS (CERATOTHERIUM SIMUM SIMUM).

The current report describes the temporary regression, due to intensive symptomatic treatment, of ulcerative skin lesions caused by squamous cell carcinoma in a white rhinoceros. A captive, 40-yr-old southern white rhinoceros (Ceratotherium simum simum) developed profound, ulcerative skin lesions on the pads of both hind feet. At the peak of the disease, at least one quarter of the pads was affected. A diagnosis of squamous cell carcinoma was made via biopsy. Treatment included anti-inflammatory drugs, antibiotics, and local care. The lesions regressed on both feet until they seemed clinically healed. It was presumed that long-term, anti-inflammatory treatment and local bandaging had induced the temporary regression of the lesions. Two years later, however, a small ulcerative lesion reappeared on one pad and post mortem examination confirmed that the carcinoma was also histologically present in the clinically intact tissue. No metastasis was found and computed tomography showed normal digital bones.

Ovarian down Regulation by GnRF Vaccination Decreases Reproductive Tract Tumour Size in Female White and Greater One-Horned Rhinoceroses.

Reproductive tract tumours, specifically leiomyoma, are commonly found in female rhinoceroses. Similar to humans, tumour growth in rhinoceroses is thought to be sex hormone dependent. Tumours can form and expand from the onset of ovarian activity at puberty until the cessation of sex-steroid influences at senescence. Extensive tumour growth results in infertility. The aim of this study was to down regulate reproductive function of tumour-diseased and infertile females to stop further tumour growth using a Gonadotropin releasing factor (GnRF) vaccine. Four infertile southern white (Ceratotherium simum simum) and three Greater one-horned rhinoceroses (rhinoceros unicornis) with active ovaries and 2.7 ± 0.9 and 14.0 ± 1.5 reproductive tract tumours respectively were vaccinated against GnRF (Improvac®, Zoetis, Germany) at 0, 4 and 16 weeks and re-boostered every 6-8 months thereafter. After GnRF vaccination ovarian and luteal activity was suppressed in all treated females. Three months after vaccination the size of the ovaries, the number of follicles and the size of the largest follicle were significantly reduced (P<0.03). Reproductive tract tumours decreased significantly in diameter (Greater-one horned rhino: P<0.0001; white rhino: P<0.01), presumably as a result of reduced sex-steroid influence. The calculated tumour volumes were reduced by 50.8 ± 10.9% in Greater one-horned and 48.6 ± 12.9% in white rhinoceroses. In conclusion, GnRF vaccine effectively down regulated reproductive function and decreased the size of reproductive tract tumours in female rhinoceros. Our work is the first to use down regulation of reproductive function as a symptomatic treatment against benign reproductive tumour disease in a wildlife species. Nonetheless, full reversibility and rhinoceros fertility following GnRF vaccination warrants further evaluation.

Morphologic and molecular study of hemoparasites in wild corvids and evidence of sequence identity with Plasmodium DNA detected in captive black-footed penguins (Spheniscus demersus).

A morphologic and molecular epidemiologic investigation was conducted on a captive African black-footed penguin (Spheniscus demersus) colony with a history of Plasmodium infections at La Palmyre Zoo (France). Each penguin received 12.5 mg of pyrimethamine twice a week as a prophylaxis every year from April to November. Although Plasmodium parasites were not detected in blood smears and tissues collected from the penguins, various blood parasites were recorded in blood smears from wild Eurasian magpies (Pica pica) and carrion crows (Corvus corone) sampled at the same time in the study area. These parasites consisted of several Plasmodium spp. (P. lenoblei, P. dorsti, P bioccai, P. relictum, P. dherteae, P. beaucournui, P. maior, P. tranieri, and P. snounoui), Parahaemoproteus spp., Trypanosoma spp., and Leucocytozoon spp. On the other hand, nested polymerase chain reaction enabled detection of Plasmodium DNA in 28/44 (64%) penguins, 15/25 (60%) magpies, and 4/9 (44%) crows. Sequencing and phylogenetic analyses indicated that the parasite DNA amplified from the penguins, magpies, and crows were similar. Magpies and crows could therefore act as a reservoir for penguin Plasmodium infections, which may be more prevalent than previously thought. Morphologic characterization of the Plasmodium spp. detected in the penguins, as well as further biological and epidemiologic studies, are needed to fully understand the transmission of Plasmodium parasites to captive penguins.