Approaches and methods to study wildlife cancer.

The last few years have seen a surge of interest from field ecologists and evolutionary biologists to study neoplasia and cancer in wildlife. This contributes to the One Health Approach, which investigates health issues at the intersection of people, wild and domestic animals, together with their changing environments. Nonetheless, the emerging field of wildlife cancer is currently constrained by methodological limitations in detecting cancer using non-invasive sampling. In addition, the suspected differential susceptibility and resistance of species to cancer often make the choice of a unique model species difficult for field biologists. Here, we provide an overview of the importance of pursuing the study of cancer in non-model organisms and we review the currently available methods to detect, measure and quantify cancer in the wild, as well as the methodological limitations to be overcome to develop novel approaches inspired by diagnostic techniques used in human medicine. The methodology we propose here will help understand and hopefully fight this major disease by generating general knowledge about cancer, variation in its rates, tumour-suppressor mechanisms across species as well as its link to life history and physiological characters. Moreover, this is expected to provide key information about cancer in wildlife, which is a top priority due to the accelerated anthropogenic change in the past decades that might favour cancer progression in wild populations.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species. IMPORTANCE: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Cutaneous apocrine gland neoplasia in 16 captive African wild dogs (Lycaon pictus).

This retrospective study documents the occurrence of single and multiple cutaneous apocrine gland tumours (CATs) on the dorsal midline of 16 captive African wild dogs (AWDs, Lycaon pictus) derived from 161 submissions to diagnostic laboratories in South Africa, France and Germany between 1997 and 2022. Animals included in the study came from zoological institutions in South Africa (n = 2), France (n = 5) and Germany (n = 1) and ranged from 5 to 14 years of age. Fifteen affected animals were female (94%) and one was male. CATs presented as raised, hairless, multilobular, grey firm masses, consistently located along the dorsal midline. Apart from a single cutaneous apocrine adenoma and a cystadenoma occurring concurrently with two non-cystic adenocarcinomas, neoplasms were consistent with malignant cutaneous apocrine adenocarcinomas with lymphatic spread and visceral metastases. Advanced age and female sex were identified as risk factors. A genetic component or association with the increasing use of GnRH agonist contraceptives was suspected but could not be established. This study highlights the need for close clinical monitoring of AWDs over the age of 5 years for the development of CATs along the dorsal midline and supports early surgical intervention. More research is needed to determine the role of inbreeding, endocrine changes and husbandry factors that may play a role in the development of CATs on the dorsal midline of AWDs.

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.

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.

Impact of Annual Bacterial Epizootics on Albatross Population on a Remote Island.

The reduced species richness typical of oceanic islands provides an interesting environmental setup to examine in natura the epidemiological dynamics of infectious agents with potential implications for public health and/or conservation. On Amsterdam Island (Indian Ocean), recurrent die-offs of Indian yellow-nosed albatross (Thalassarche carteri) nestlings have been attributed to avian cholera, caused by the bacterium Pasteurella multocida. In order to help implementing efficient measures for the control of this disease, it is critical to better understand the local epidemiology of P. multocida and to examine its inter- and intra-annual infection dynamics. We evaluated the infection status of 264 yellow-nosed albatrosses over four successive breeding seasons using a real-time PCR targeting P. multocida DNA from cloacal swabs. Infection prevalence patterns revealed an intense circulation of P. multocida throughout the survey, with a steady but variable increase in infection prevalence within each breeding season. These epizootics were associated with massive nestling dies-offs, inducing very low fledging successes (≤ 20%). These results suggest important variations in the transmission dynamics of this pathogen. These findings and the developed PCR protocol have direct applications to guide future research and refine conservation plans aiming at controlling the disease.

AMYLOIDOSIS IN CARACALS (CARACAL CARACAL).

Nine cases of amyloidosis in caracals (Caracal caracal) from three different institutions in Europe were reviewed and evaluated histopathologically. The six males and three females died between 2008 and 2018 at an age of 6 yr ± 2.5 mo (median ± interquartile range). In two out of nine (2/9) animals, amyloidosis was an incidental postmortem finding; the animals died of bronchopneumonia and gastric ulceration due to Helicobacter spp., respectively. Seven (7/9) animals suffered from acute renal failure due to amyloidosis, one of them additionally of cardiac decompensation. The predominant clinical signs were weight loss, lethargy, dys- or anorexia, dehydration, increased BUN and creatinine, and azotemia. The main gross lesion was a pale renal cortex on cut surface; in two animals, the kidneys appeared enlarged. Histologically, glomerular amyloid was present in every animal (9/9), and was the predominant renal manifestation of amyloidosis. Additional findings included splenic amyloid (8/8), amyloid in the lamina propria of the intestine (5/5), and amyloid in the lingual submucosa (4/4). Gastric mineralization was present in four animals suffering from renal failure. In the animal dying from bronchopneumonia, severe pancreatic amyloid deposits mainly affecting the exocrine pancreas (1/5) were identified. Immunohistochemistry was employed to identify amyloid AA in eight cases; only in the caracal dying from bronchopneumonia AA was amyloid confirmed. In several organs, especially in those where only small amyloid deposits were detected, a Congo red stain was often necessary to confirm the deposition. The etiology of the amyloidosis remains unknown. Three caracals were related within two generations, another three within four generations, so one might hypothesize a familial trait. In conclusion, amyloidosis should be considered as a significant disease in the caracal. Particularly in cases with renal disease, it should be included as a major differential diagnosis.

Severe parasitism by Versteria mustelae (Gmelin, 1790) in the critically endangered European mink Mustela lutreola (Linnaeus, 1761) in Spain.

The riparian European mink (Mustela lutreola), currently surviving in only three unconnected sites in Europe, is now listed as a critically endangered species in the IUCN Red List of Threatened Species. Habitat loss and degradation, anthropogenic mortality, interaction with the feral American mink (Neovison vison), and infectious diseases are among the main causes of its decline. In the Spanish Foral Community of Navarra, where the highest density of M. lutreola in its western population has been detected, different studies and conservation measures are ongoing, including health studies on European mink, and invasive American mink control. We report here a case of severe parasitism with progressive physiological exhaustion in an aged free-ranging European mink female, which was accidentally captured and subsequently died in a live-trap targeting American mink. Checking of the small intestine revealed the presence of 17 entangled Versteria mustelae worms. To our knowledge, this is the first description of hyperinfestation by tapeworms in this species.

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.

Evidence for the possible occurrence of Grave's disease in a blue-eyed black lemur (Eulemur flavifrons).

The blue-eyed black lemur (Eulemur flavifrons) is classified by the International Union for Conservation of Nature (IUCN) as critically endangered. A 23-year-old male housed at Mulhouse Zoo presented with lethargy, polyphagia, alopecia, and chronic weight loss. Clinical examination suggested an endocrine pathology such as hyperthyroidism. Secondary examinations included cervical ultrasound, thyroid biopsy, and scintigraphy. The latter revealed elevated thyroid activity. Blood analysis was performed to measure the level of anti-receptor thyroid-stimulating hormone antibodies, which allowed us to test the autoimmune hypothesis. The high level of antibodies together with levels of thyroid-stimulating hormone and the scintigraphy images led to the diagnosis of Grave's disease. Carbimazole treatment followed by thyroidectomy resulted in a quick weight gain and general improvement in health status. The following breeding season, the treated individual sired an offspring. To the authors' knowledge, this is the first report of likely Grave's disease in a non-human primate.

Pathology and Epidemiology of Oxalate Nephrosis in Cheetahs.

To investigate cases of acute oxalate nephrosis without evidence of ethylene glycol exposure, archived data and tissues from cheetahs ( Acinonyx jubatus) from North America ( n = 297), southern Africa ( n = 257), and France ( n = 40) were evaluated. Renal and gastrointestinal tract lesions were characterized in a subset of animals with ( n = 100) and without ( n = 165) oxalate crystals at death. Crystals were confirmed as calcium oxalate by Raman spectroscopy in 45 of 47 cheetahs tested. Crystals were present in cheetahs from 3.7 months to 15.9 years old. Cheetahs younger than 1.5 years were less likely to have oxalates than older cheetahs ( P = .034), but young cheetahs with oxalates had more oxalate crystals than older cheetahs ( P < .001). Cheetahs with oxalate crystals were more likely to have renal amyloidosis, interstitial nephritis, or colitis and less likely to have glomerular loop thickening or gastritis than those without oxalates. Crystal number was positively associated with renal tubular necrosis ( P ≤ .001), regeneration ( P = .015), and casts ( P ≤ .001) but inversely associated with glomerulosclerosis, renal amyloidosis, and interstitial nephritis. Crystal number was unrelated to the presence or absence of colitis and was lower in southern African than American and European animals ( P = .01). This study found no evidence that coexisting chronic renal disease (amyloidosis, interstitial nephritis, or glomerulosclerosis), veno-occlusive disease, gastritis, or enterocolitis contributed significantly to oxalate nephrosis. Oxalate-related renal disease should be considered as a potential cause of acute renal failure, especially in young captive cheetahs. The role of location, diet, stress, and genetic predisposition in the pathogenesis of oxalate nephrosis in cheetahs warrants further study.

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.

DESCRIPTION OF GASTRIC ULCERS AND OF THEIR SUSPECTED, ASSOCIATED RISK FACTORS IN DECEASED WILD EQUIDS AT THE RÉSERVE AFRICAINE DE SIGEAN, FRANCE (2010-2016).

Gastric ulcers are common in domestic horses and foals, affecting at least 90% of unmedicated racehorses in active training. Despite these high prevalences in domestic horses, literature about this condition in wild equids is almost nonexistent. The presence of gastric ulcers was evaluated at necropsy in six species of wild equids that died at the Réserve Africane de Sigean, a safari park in the south of France from 2010 to 2016. Among the 55 individuals that died during that period, a description of the gastric mucosa was available in 82% (45/55) of cases. Considering the cases for which a description of the gastric mucosa was available, the prevalence of gastric ulcers was 64% (29/45). The highest prevalences were noted in Grant's zebra (Equus quagga boehmi) and Hartmann's mountain zebra (Equus zebra hartmannae) at 83% and 100%, respectively. In contrast to what is reported in domestic foals, gastric ulcerations were only diagnosed in one foal (out of 11 foals necropsied). The higher prevalence was noted in young individuals (3-36 mo old) at 93% (14/15); the lesions observed consisted mainly of single to multiple, superficial lesions, of which, only the mucosa was missing; these superficial lesions are often considered not clinically significant. The prevalence was lower for adults (74%; 14/19), but lesions were deeper or with a hyperemic or inflammatory appearance. All the lesions observed were located in the gastric, nonglandular, stratified squamous mucosa, along the margo plicatus. No statistical correlation could be found between the development of gastric ulcers and an ongoing, chronic pathologic process or a digestive tract pathology. The detection of gastric ulcers was, therefore, significantly greater in wild equids isolated in smaller enclosures. Nevertheless, additional larger-scale research is needed to point out predisposing factors in equids under human care.

COLONIC ADENOCARCINOMAS IN A FAMILIAL GROUP OF CAPTIVE AMUR RAT SNAKES (ELAPHE SCHRENCKI).

This article reports five cases of colonic adenocarcinomas in a family group of captive Amur rat snake (Elaphe schrenckii) from the Réserve Africaine de Sigean, France. This tumor was detected in three females and two males, all adults, and accounted for 16% of causes of death of adults of this species at this institution from 1986 to 2013. Grossly, mild to marked thickening of the intestinal wall cranially to the cloaca was found in four cases; tan to yellow firm masses were noted in the distal intestinal wall in the other case. Microscopically, neoplasms were characterized by infiltrating, poorly circumscribed, and unencapsulated nests of epithelial cells. Marked anisokaryosis and anisocytosis were seen in all neoplasms. The etiology of the neoplasms was not determined, but the familial clustering suggests a common etiologic factor.

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.

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.

FATAL ENCEPHALOMYOCARDITIS VIRUS INFECTION IN AN AFRICAN SAVANNA ELEPHANT (LOXODONTA AFRICANA) IN A FRENCH ZOO.

A fatal case of encephalomyocarditis virus (EMCV) involving an African elephant ( Loxodonta africana ) occurred in November 2013 at the Réserve Africaine de Sigean, France. An adult female was found dead without any preliminary symptoms. Gross pathologic changes consisted of petechiae and hemorrhages on mucosae and internal organs, abundant transudate in the abdominal and pericardial cavities, and myocarditis. Histopathologic examination showed extensive degeneration and necrosis of ventricular cardiomyocytes with concurrent lymphoplasmocytic and eosinophilic infiltrate. An EMCV was isolated from several organs and considered the causative agent of the myocarditis. The same strain of virus was also isolated in rodents captured on zoo premises and considered to be the reservoir of the virus. To the authors' knowledge, this is the first EMCV case in a captive African elephant in Europe.

Bilateral Granulomatous and Fibrinoheterophilic Otitis Interna due to Pseudomonas aeruginosa in a Captive Little Bustard ( Tetrax tetrax ).

A captive juvenile little bustard ( Tetrax tetrax ) was presented for acute onset of right head tilt and right circling. The bird failed to respond to supportive care and systemic antibiotic therapy. A bilateral granulomatous and fibrinoheterophilic otitis interna due to Pseudomonas aeruginosa was diagnosed postmortem by histopathologic examination and bacterial culture. In bustards, Pseudomonas species have been documented in the normal bacterial flora of the oropharynx and are frequently reported in upper respiratory tract infections. This is the first report of a peripheral vestibular syndrome due to P aeruginosa otitis interna in a bustard species. Pseudomonas aeruginosa should be included as a possible cause of otitis and peripheral vestibular syndrome in bustards.