SERUM VITAMIN D AND SELECTED BIOMARKERS OF CALCIUM HOMEOSTASIS IN ASIAN ELEPHANTS (ELEPHAS MAXIMUS) MANAGED AT A LOW LATITUDE.

An understanding of species-specific vitamin D metabolism and its role in calcium homeostasis is essential for correct diet formulation and development of husbandry protocols for managed nondomestic species. This study documented serum vitamin D metabolites and other analytes involved in calcium homeostasis in Asian elephants (Elephas maximus) managed at a latitude similar to their wild natural habitat. Serum values for 33 elephants managed at a low latitude were measured in the peak of summer, revealing low vitamin D2 (25(OH)D2 2.3 ± 0.6 ng/ ml and 24,25(OH)2D2 2.17 ± 0.52 ng/ml) and nondetectable vitamin D3. Serum minerals (calcium, phosphorus, magnesium), ionized calcium, and parathyroid hormone were within normal reported ranges. In comparison with previously reported values in elephants managed at a high latitude, 25(OH)D2 (P < 0.001), 24,25(OH)2D2 (P = 0.001), and magnesium (P = 0.013) were significantly lower, and parathyroid hormone was significantly higher (P < 0.001). The lack of D3 production during ample sun exposure at a low latitude suggests that Asian elephants are incapable of cutaneous photobiosynthesis of vitamin D, and that low serum D2 is normal for this species.

Widespread, symmetric, noninflammatory alopecia associated with follicular dysplasia in the American red squirrel (Tamiasciurus hudsonicus).

This report describes a unique pattern of alopecia in 8 American red squirrels (Tamiasciurus hudsonicus) from 2013 to 2021. All animals were juveniles; 6 were female and 2 were male. Seven presented between September and November, and one presented in April. All squirrels had widespread, bilaterally symmetric, noninflammatory, well-demarcated alopecia involving the entire trunk and legs and normal hair on their muzzle and dorsal surfaces of their paws. Six months later, a normal hair coat had grown on 2 of the animals, which were littermates. Hair fully grew 2 months later in another animal. Histopathology of the alopecic skin was performed in 7 of 8 animals. The following changes were noted: bent and coiled hairs, perforating folliculitis, melanin clumping, and distortion of hair shafts. Based on features of follicular dysplasia and apparent seasonality, this condition has some similarities to canine seasonal flank alopecia. A genetic etiology is suspected.

Histopathologic and molecular characterization of Erethizon dorsatum papillomavirus 1 and Erethizon dorsatum papillomavirus 2 infection in North American porcupines (Erethizon dorsatum).

Erethizon dorsatum papillomavirus 1 (EdPV1) and Erethizon dorsatum papillomavirus 2 (EdPV2) are associated with cutaneous papillomas in North American porcupines (Erethizon dorsatum). This study defined gross, histopathologic, and molecular characteristics of viral papillomas in 10 North American porcupines submitted to the New York State Animal Health Diagnostic Center. Investigation for the presence of EdPV1 and EdPV2 DNA via polymerase chain reaction (PCR) was performed in 9 of the 10 (90.0%) porcupines, and all porcupines were investigated for the detection and localization of EdPV1 and EdPV2 E6 and E7 nucleic acid via chromogenic in situ hybridization (CISH). Next-generation sequencing (NGS) was performed in 2 porcupines. Papillomas were diagnosed on the muzzle (n = 4), caudal dorsum (n = 1), upper lip (n = 1), chin (n = 1), gingiva (n = 2), and nasal planum (n = 1). Histologically, the lesions consisted of hyperplastic epidermis or epithelium with orthokeratotic keratin, prominent keratohyalin granules, and intranuclear inclusion bodies. PCR identified EdPV1 in 6 of 9 samples and EdPV2 in the remaining 3 samples. NGS resulted in 100% genome coverage of EdPV1 and 76.20% genome coverage of EdPV2 compared with GenBank reference sequences, with 99.8% sequence identity to the complete EdPV2 L1 gene of a novel subtype recently identified in France. Hybridization patterns in 9 of the 10 (90.0%) porcupines were characterized by strong nuclear signals in the superficial epidermis, with strong nuclear and punctate cytoplasmic signals in the stratum spinosum and basale. In one animal, CISH suggested dual EdPV1 and EdPV2 infection.

EVALUATION OF ß-HYDROXYBUTYRATE, AMINO ACIDS, AND SELECTED CHEMISTRY VALUES IN THE PLASMA OF FREE-RANGING RED-TAILED HAWKS (BUTEO JAMAICENSIS).

The red-tailed hawk (Buteo jamaicensis) is a raptor species native to North America that is commonly presented to wildlife rehabilitators and veterinarians, but little is known about biomarkers of metabolic status in this species. This study evaluates plasma ß-hydroxybutyrate (BHB) and free amino acids in 24 free-ranging red-tailed hawks in good body condition in order to establish reference intervals for these values. Standard biochemical analytes were also measured. The mean plasma BHB level measured was 13.9 mg/dl. Plasma amino acids were not similar to the few available reports in avian species. Standard biochemical analytes were similar to those previously reported for red-tailed hawks. These data serve as a foundation for further investigation into the use of these biomarkers to assess metabolic status of this species in health and disease.

EXPERIMENTAL CHOLECALCIFEROL SUPPLEMENTATION IN A HERD OF MANAGED ASIAN ELEPHANTS (ELEPHAS MAXIMUS).

Vitamin D supplementation may pose a significant health risk in species where levels of deficiency, sufficiency, and toxicity have not been clearly established, and species-specific research on vitamin D supplementation should be performed. This study documented the effect of vitamin D supplementation on serum vitamin D metabolites and other analytes of Ca homeostasis in Asian elephants (Elephas maximus). Six adult Asian elephants received PO supplementation with cholecalciferol at 300 IU/kg of body weight (BW) once a week for 24 wk. Serum was analyzed every 4 wk for 25-hydroxyvitamin D2/D3 [25(OH)D]; 24,25-dihydroxyvitamin D2/D3 [24,25(OH)2D]; 1,25-dihydroxyvitamin D [1,25(OH)2D]; parathyroid hormone (PTH); total Ca; ionized Ca (iCa); P; and Mg. After the supplement was discontinued, serum 25(OH)D2/D3 was measured every 4 wk until levels returned to baseline. At the start of the study, the average serum 25(OH)D3 was nondetectable (<1.5 ng/ml). With cholecalciferol supplementation, 25(OH)D3 increased at an average rate of 2.26 ng/ml per month and reached an average concentration of 12.9 ± 3.46 ng/ml at 24 wk. Both 24,25(OH)2D3 and 1,25(OH)2D increased over time with supplementation from an average of <1.5 to 12.9 ng/ml and from 9.67 to 36.4 pg/ml, respectively. PTH, iCa, Ca, P, and Mg remained within reported normal ranges throughout supplementation. After the supplement was discontinued, serum 25(OH)D3 demonstrated a slow decline to baseline, taking an average of 48 wk. Elephants demonstrated significant individual variation in response to supplementation and subsequent return to baseline. Supplementation of Asian elephants with a weekly dose of 300 IU/kg BW cholecalciferol for 24 wk appears to be effective and safe. Additional clinical studies would be necessary to investigate the safety of other routes of administration, dosages, and duration of vitamin D supplementation, as well as associated health benefits.

IN VIVO CONFOCAL MICROSCOPY FOR CHARACTERIZATION OF MYCOTIC KERATITIS IN OWLS (BUBO SCANDIACUS, STRIX VARIA) AND A WOODCOCK (SCOLOPAX MINOR): THREE CASES.

This case series describes the use of in vivo confocal microscopy in the diagnosis and treatment of mycotic keratitis in two owls (one Bubo scandiacus, one Strix varia) and one woodcock (Scolopax minor). Each bird was at increased risk of fungal infection due to recent injury or stress. Ophthalmic findings in all birds included blepharospasm, ocular discharge, ulcerative keratitis, white or yellow corneal plaques, and anterior uveitis. Fungal hyphae were identified in corneal samples from all three eyes examined cytologically and in all three eyes by using in vivo confocal microscopy. Aspergillus fumigatus was isolated from a corneal culture in one bird. Despite medical treatment, progressive ocular disease prompted enucleation in two birds. Fungal hyphae were detected by histopathology in one of the two enucleated eyes. In vivo confocal microscopy aided the diagnosis of fungal keratitis in all birds and was the only diagnostic method that allowed immediate, real-time quantification of the extent (area and depth) and severity of mycotic keratitis.

Cutaneous concentrations of 7-dehydrocholesterol in Hoffmann's two-toed sloths (Choloepus hoffmanni) in managed care.

Hoffmann's two-toed sloths (Choloepus hoffmanni) are unique animals popular in zoological settings. The role of vitamin D in the maintenance of calcium homeostasis in this species is unexplored, although disorders of calcium homeostasis have been reported in managed sloths, including urolithiasis and soft tissue mineralization. This study assessed cutaneous concentrations of 7-dehydrocholesterol (7-DHC) in nine indoor-housed Hoffmann's two-toed sloths, as a means of evaluating the potential for cutaneous photobiosynthesis of vitamin D3 . Samples were obtained from both abdominal and lumbar regions of the skin to assess for regional variation in 7-DHC concentration. The average concentration of 7-DHC in the sloth skin was low (0.197 ± 0.02 nmol/cm2 ). Location had a significant effect on 7-DHC level only when concentrations were expressed based on the wet weight of the sample (ng/g), but levels were similar when values were normalized to biopsy surface area (nmol/cm2 ).

VITAMIN D STATUS OF INDOOR-HOUSED HOFFMANN'S TWO-TOED SLOTHS (CHOLOEPUS HOFFMANNI): A PILOT STUDY.

Disorders of calcium homeostasis have been reported with some frequency in two-toed sloths, yet little investigation has been performed on vitamin D and mineral metabolism in these species. This study evaluates biomarkers involved in vitamin D and calcium metabolism in a group of nine managed Hoffmann's two-toed sloths (Choloepus hoffmanni), correlated with diet analysis. Serum was analyzed for both vitamin D2 and D3 metabolites, minerals (calcium [Ca], phosphorus [P], and magnesium [Mg]), parathyroid hormone (PTH), and ionized calcium (iCa). The diet was analyzed for proximate nutrients, minerals and vitamins D2 and D3, and feed intake was estimated. Average values reported for D3 metabolites, including 25(OH)D3, 1,25(OH)2D3, and 24,25(OH)2D3, were 25.1 ng/ml, 52.23 pg/ml, and 7.65 ng/ml, respectively. Average PTH was 0.22 pmol/L while average iCa was 1.46 mmol/L. Values reported for Ca, P, Mg were within expected ranges. Dietary concentration of D3 was 6.7 ng/g and the average daily intake per sloth was 113.7 IU/kg body weight. No detectable levels of vitamin D2 metabolites 25(OH)D2, 1,25(OH)2D2, or 24,25(OH)2D2 were found in the diet or serum. These data will serve as a starting point for future investigations into the vitamin D metabolism and calcium homeostasis of two-toed sloths to improve the health in managed settings.

THE VITAMIN D STATUS OF ASIAN ELEPHANTS (ELEPHAS MAXIMUS) MANAGED IN A NORTHERN TEMPERATE CLIMATE.

Knowledge about the normal metabolism and involvement of vitamin D in elephant calcium homeostasis is essential to understanding the possible role of vitamin D in Asian elephant (Elephas maximus) health, as well as to informing accurate diet formulation. This study provides an evaluation of analytes involved in vitamin D metabolism, in conjunction with dietary intake and ultraviolet light (UV) exposure, in Asian elephants managed in a northern temperate climate. Once monthly, for a total of 12 mo, serum from six adult Asian elephants was analyzed for 25-hydroxyvitamin D [25(OH)D], 24,25-dihydroxyvitamin D [24,25(OH)2D], 1,25-dihydroxyvitamin D [1,25(OH)2D], parathyroid hormone (PTH), total calcium (Ca), ionized calcium (iCa), phosphorus (P), and magnesium (Mg). The diet was analyzed monthly for vitamin D, Ca, and P. Monthly average vitamin D-weighted UV daily sums were determined to gauge average UV light exposure within the vitamin D action spectrum. No serum or diet parameters were affected by time or season. Average serum 25(OH)D2 was 7.02 ± 0.85 ng/ml. 25(OH)D3 levels were nondetectable in all samples despite supplementation of the diet with recommended levels of vitamin D3, and UV exposure was at sufficient levels for cutaneous vitamin D synthesis for 6 mo of the year. Levels of 24,25(OH)2D averaged 31.7% higher than 25(OH)D, and average 1,25(OH)2D2 was 11.24 ± 1.04 pg/ml. Values for PTH, Ca, iCa, P, and Mg were within expected ranges for Asian elephants. The information gained from this research expands the knowledge base for these analytes, evaluates 24,25-dihydroxyvitamin D for the first time, and provides new information regarding vitamin D metabolism and test interpretation in the Asian elephant.

EVALUATION OF 25-HYDROXYVITAMIN D IN HOFFMANN'S TWO-TOED SLOTHS (CHOLOEPUS HOFFMANNI) USING DRIED BLOOD SPOTS ANALYZED BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.

Although biochemical analytes have typically been measured using serum or whole blood samples, an increasing number of assays are validated for measurement of analytes from dried blood spots (DBS) on filter paper. DBS techniques are minimally invasive, require only a small sample volume, and simplify processing, storage, and shipment of samples. These qualities make DBS-based assays ideal for sampling of wildlife species in both captive and field settings. In this study, a liquid chromatography-tandem mass spectrometry assay was evaluated for measurement of 25-hydroxyvitamin D in sloths. Paired serum and DBS samples were collected from nine healthy captive Hoffmann's two-toed sloths (Choloepus hoffmanni). Statistical analysis using Passing-Bablok regression analysis, Bland-Altman plots, and the Wilcoxon signed-rank tests found good agreement between 25-hydroxyvitamin D3 measurements in serum and DBS samples. Constant and proportional bias were absent. Results from this study support the use of DBS samples for the evaluation of vitamin D status in Hoffmann's two-toed sloths and provide a foundation for further studies to validate this technique.

SURGICAL ANATOMY OF CELIOTOMY APPROACHES TO THE STOMACH IN THE COMMON SNAPPING TURTLE (CHELYDRA SERPENTINA).

Entanglement in or ingestion of fishing gear is a common cause of morbidity and mortality in chelonians. Commercial and recreational fishing activities exert bycatch pressures sufficient to cause population declines in the common snapping turtle (Chelydra serpentina) and the alligator snapping turtle (Macrochelys spp.). Gastrotomy for the removal of fish hook foreign bodies from the stomach of freshwater turtles typically requires a plastron osteotomy but in sea turtles has been successfully accomplished via soft tissue approaches. This study compared the anatomy and feasibility of soft tissue surgical approaches to the stomach in the common snapping turtle in both the prefemoral and the axillary regions. Anatomical dissections were performed on cadavers of five adult common snapping turtles, and the surgical anatomy of the left axillary and left prefemoral regions was characterized. The left axillary approach required extensive transection of musculature and provided poor exposure of the coelomic cavity and stomach. In contrast, the left prefemoral approach was simple to perform and provided sufficient access to the stomach for gastrotomy. The prefemoral celiotomy has significant advantages over the axillary celiotomy in the common snapping turtle and should be considered the surgical approach of choice for gastrotomy in this species.

HEPATIC OSTEODYSTROPHY IN A GOLDEN LION TAMARIN (LEONTOPITHECUS ROSALIA).

An 8-yr-old, captive, female golden lion tamarin ( Leontopithecus rosalia ) with a 6-yr history of hyperbilirubinemia was examined for inappetence and weight loss. Physical examination and blood pressure monitoring under anesthesia revealed hypothermia and hypotension, and blood work revealed hypoglycemia, markedly elevated liver enzymes, including serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase, and confirmed the hyperbilirubinemia. A complete blood count suggested chronic lymphoid leukemia. The animal's condition deteriorated during recovery, and the animal died despite aggressive treatment. Grossly, there was micronodular cirrhosis of the liver, severe icterus, and diffuse osteopenia of all examined bones. Microscopic examination of the liver confirmed the micronodular cirrhosis and bone lesions were compatible with diffuse osteopenia and osteomalacia. This brief communication presents a case of chronic liver disease and lesions indicative of metabolic bone disease, also known as hepatic osteodystrophy. To the authors' knowledge, this is the first documented case of hepatic osteodystrophy in the veterinary literature.

RADIATION THERAPY OF A PRESUMPTIVE URETHRAL TRANSITIONAL CELL CARCINOMA IN AN EASTERN GRAY SQUIRREL (SCIURUS CAROLINENSIS).

An adult female Eastern gray squirrel (Sciurus carolinensis), with a previous history of primary renal transitional cell carcinoma treated by nephrectomy, was diagnosed with a metastatic urethral transitional cell carcinoma (TCC) utilizing the veterinary bladder tumor antigen test in combination with other noninvasive diagnostic tests. The squirrel was treated with piroxicam and external beam radiation therapy given in 18 treatments over 30 days to achieve a total of 54 gray. Mild to moderate side effects from the pelvic irradiation were self-limiting and easily managed. Resolution of clinical signs was achieved for approximately 6 mo until recurrence of metastasis. This report represents the first published account of both TCC and external beam radiation therapy in an Eastern gray squirrel.

Ciprofloxacin-resistant ST198 Salmonella Kentucky in a hospitalized American black bear (Ursus americanus), with evidence of subsequent nosocomial transmission.

Global emergence of ciprofloxacin-resistant ST198 Salmonella Kentucky poses an important public health threat. While conducting Salmonella surveillance among wildlife patients admitted to our veterinary medical teaching hospital in central New York, we isolated multidrug-resistant (MDR) ST198 Salmonella Kentucky from an American black bear (Ursus americanus) in September 2020. The isolate was phenotypically resistant to numerous antimicrobial agents, including ceftriaxone and ciprofloxacin, and several antimicrobial resistance genes and mutational resistance determinants were detected. Between April and July 2021, the same strain of MDR ST198 Salmonella Kentucky was also isolated from seven other wildlife patients and multiple hospital environmental locations, suggesting nosocomial transmission. Ciprofloxacin resistance is conferred by triple point mutations in the quinolone resistance-determining regions (QRDRs), a genotypic profile indicative of Clade ST198.2. To our knowledge, this is the first report of this ciprofloxacin-resistant clade being identified in animals or animal products in the United States. Timely resolution of the outbreak was achieved following efforts to further enhance environmental disinfection protocols and biosecurity measures at the hospital, with no known cases or positive environmental samples after July 2021.

Characterization of Listeria monocytogenes isolated from wildlife in central New York.

BACKGROUND: Listeria monocytogenes (Lm) present in farming soil and food-processing facilities threatens food safety, but little is known about the carriage of Lm by wildlife. OBJECTIVES: We estimated the prevalence of faecal Lm shedding among wildlife admitted to a veterinary medical teaching hospital in central New York and characterized a subset of the Lm isolates. METHODS: Wildlife samples were collected between May 2018 and December 2019. We characterized the Lm isolates by assessing the growth at three temperatures approximating the body temperatures of reptiles (25°C), mammals (37°C), and birds (42°C) and identifying genotypic characteristics related to transmission and virulence. RESULTS: The apparent prevalence of faecal Lm shedding was 5.6% [18/324; 95% confidence interval (CI), 3.3%-8.6%]. Among 13 isolates that represented two lineages and 11 clonal complexes, three and five isolates were grouped into the same SNP clusters with human clinical isolates and environmental isolates, respectively. However, specific SNP difference data showed that Lm from wildlife was generally not closely related (>22 SNP differences) to Lm from human clinical sources and the food-processing environment. While the stress response locus SSI-2 was absent, SSI-1 was found in four isolates. Virulence genes prfA, plcA, hly, mpl, actA, plcB, inlA, inlB, inlC, inlE, inlH, inlJ, and inlK were present, without any premature stop codons, in all isolates. Virulence loci Listeria pathogenicity island 3 (LIPI-3) and LIPI-4, which have been linked to hypervirulence, and inlG were found in four, three, and seven isolates, respectively. CONCLUSIONS: Wildlife represents a potential reservoir for genetically diverse and putatively hypervirulent Lm strains. No statistically significant association between growth parameters and hosts was observed. However, compared to lineage I isolates, lineage II isolates showed significantly (p < 0.05) faster growth at 25°C and significantly slower growth at 42°C, suggesting that wildlife Lm isolates that belong to lineages I and II differ in their ability to grow at 25°C and 42°C.

SALMONELLA ISOLATED FROM CENTRAL NEW YORK WILDLIFE ADMITTED TO A VETERINARY MEDICAL TEACHING HOSPITAL.

The role of wildlife as a source of zoonotic Salmonella transmission is poorly understood, as are the clinical implications of this pathogen among wildlife species. Wildlife hospitals represent an important location to conduct Salmonella surveillance, given the wide variety of species admitted for medical and surgical care. Our objectives were to estimate the prevalence of fecal Salmonella shedding among wildlife admitted to a veterinary medical teaching hospital, to identify risk factors for infection, and to fully characterize the isolates. Voided fecal samples (birds and mammals) and cloacal swab samples (reptiles and amphibians) were collected between May 2018 and March 2020. Standard bacteriologic culture methods were used to detect Salmonella, and isolates were characterized via serotyping, antimicrobial susceptibility testing, and whole-genome sequencing. Samples were collected from 348 wildlife patients representing 74 wildlife species, and the apparent prevalence of fecal Salmonella shedding was 1.4% (5/348; 95% confidence interval, 0.5-3.3%). Four serotypes were identified, and isolates were phenotypically susceptible to all antimicrobial agents tested. Two isolates were closely related to human clinical isolates, demonstrating the overlap between wildlife and human pathogens. Fecal Salmonella shedding among hospitalized wildlife appears to be uncommon, and the risk of either nosocomial or zoonotic Salmonella transmission is presumably low. Nevertheless, the occurrence of Salmonella in wildlife, particularly among common species found in a wide array of habitats, poses a potential threat to public health and may result in transmission to more-vulnerable wildlife populations.

Yersinia pseudotuberculosis in a closed colony of Egyptian fruit bats (Rousettus aegyptiacus).

An outbreak of Yersinia pseudotuberculosis (Yptb) occurred in a closed colony of Egyptian fruit bats (Rousettus aegyptiacus) and resulted in the death of seven bats over a 6-week period. An initial survey of the remaining bats revealed visceral abscessation characteristic of pseudotuberculosis in five of the 12 bats examined (41.7%), inciting depopulation of the colony. At necropsy, 70% of the 115 bats in the colony exhibited gross evidence suggestive of Yptb infection, including mesenteric lymphadenopathy (ML), hepatic abscessation (HA), and/or splenomegaly (SPM). Thirty of these bats (13 females and 17 males of various ages) were chosen at random and their tissues submitted for bacterial culture and histopathologic examination. Twenty-three of these 30 bats had one or more gross lesions considered consistent with Yptb, including ML, HA, and SPM. On histopathology, four of the 30 bats had necrotizing lesions containing Gram-negative bacteria in multiple organs, while 18 others exhibited mild mesenteric lymphadenitis and hepatitis. Four of the 30 bats had positive cultures for Yptb. Bats with gross evidence of mesenteric lymphadenopathy, splenomegaly, or histopathologic presence of demodicosis or bacteria in tissues were more likely (P < 0.05) to have a positive Yersinia culture. Examination of the correlation between population density and mortality rates of the colony revealed that the mortality rate of subadult bats increased dramatically at the time of the outbreak, when the population density was at its highest. It is suspected that stress, primarily from severe overcrowding, predisposed the bat colony to morbidity and mortality from this organism, which likely originated from a rodent reservoir.

Sarcomatoid renal cell carcinoma in a binturong (Arctictis binturong).

An adult, female binturong (Arctictis binturong) was examined due to lethargy, inappetence, and an abdominal mass. Diagnostic investigations, including radiographs, abdominal ultrasound, clinical laboratory findings, and a fine-needle aspirate of the mass, were suggestive of a sarcoma with metastasis. Necropsy and histopathologic findings confirmed a widely disseminated sarcomatoid variant of a renal cell carcinoma, likely originating in the left kidney, with metastasis to the right kidney, spleen, pancreas, liver, mesenteric lymph nodes, and lungs. This is the first report of this neoplasm in a binturong and only the second report in the veterinary literature. Sarcomatoid renal cell carcinoma is a rare histologic variant of renal cell carcinoma that is aggressive, commonly metastatic, and associated with a very poor prognosis in humans. Accurate antemortem diagnosis of this tumor may be complicated by its biphasic morphology, which may resemble carcinoma or sarcoma (or both), often necessitating the use of immunohistochemical techniques.