Amdoparvovirus-associated disease in red pandas (Ailurus fulgens).

The roster of amdoparvoviruses (APVs) in small carnivores is growing rapidly, but in most cases, the consequences of infection are poorly understood. Red panda amdoparvovirus (RPAV) is highly prevalent in zoo-housed red pandas and has been detected in both healthy and sick animals. Clarifying the clinical impact of RPAV in this endangered species is critical, and zoological collections offer a unique opportunity to examine viral disease association in carefully managed populations. We evaluated the potential impact of RPAV in captive red pandas with a combination of prospective and retrospective analyses. First, we collected feces from 2 healthy animals from one collection over a 6-year period and detected virus in 72/75 total samples, suggesting that RPAV can be a long-term subclinical infection. We next investigated the infections using a retrospective study of infection status and tissue distribution in a cohort of necropsied animals. We performed polymerase chain reaction and in situ hybridization on 43 necropsy cases from 4 zoo collections (3 from the United States, 1 from Europe, 1997-2022). RPAV was present in these populations for at least 2 decades before its discovery and is detectable in common and significant lesions of zoo-housed red pandas, including myocarditis (3/3 cases), nephritis (9/10), and interstitial pneumonia (2/4). RPAV is also detectable in sporadic lesions, including multisystemic pyogranulomatous inflammation, oral/pharyngeal mucosal inflammation, and dermatitis. The colocalization of virus with lesions supports a role in causation, suggesting that despite the apparently persistent and subclinical carriage of most infections, RPAV may have a significant impact in zoo collections.

Multifocal Hepatocellular Carcinoma in a Malayan Wreathed Hornbill (Rhyticeros undulatus).

A 30-year-old female intact Malayan wreathed hornbill (Rhyticeros undulatus) was presented for presumed nesting behavior, progressive anorexia, dropping food, and coelomic distension. A complete blood count and plasma biochemistry analysis revealed marked inflammation, severe electrolyte abnormalities, elevated liver enzyme activities and bile acids, and normal plasma iron concentrations. Radiographic images of the patient were consistent with hepatomegaly and loss of serosal detail in the coelomic cavity. A computed tomography study revealed multiple poorly contrast-enhancing hepatic nodules, hepatoperitoneal and intestinal peritoneal fluid and gas, and a contrast-enhancing mass in the ventral coelom. Cytologic samples of the liver were consistent with necrosis, and the coelomic effusion was characterized as an aseptic suppurative exudate. An exploratory coeliotomy was performed and biopsy samples of the liver and a mesenteric mass were histologically interpreted as a tubular carcinoma with metastasis to the liver and secondary portal hepatitis. Euthanasia was elected and multiple liver masses and a peripancreatic mass were identified on necropsy. Histopathological samples collected during the postmortem gross examination showed multiple well-demarcated hepatic masses consisting of neoplastic hepatocytes encapsulated by fibrous tissue and proliferation of dysplastic biliary ductules, as well as a peripancreatic heterophilic granuloma with adjacent pancreatic atrophy and ductular proliferation. Ultimately, the patient was diagnosed with multifocal hepatocellular carcinoma and chronic granulomatous and heterophilic pancreatitis, steatitis, and coelomitis with intralesional bacteria. Malignant hepatobiliary neoplasia has been poorly documented in hornbills despite high anecdotal incidence in this and other avian species predisposed to iron storage disease. This report illustrates clinical and pathological information, including advanced imaging, which could aid in the diagnosis of this condition in hornbills and other avian species.

Amdoparvovirus-associated disease in striped skunks (Mephitis mephitis).

Disease caused by the archetypical amdoparvovirus (APV), Aleutian mink disease virus (AMDV), has been well studied, but APV infections in other carnivores are poorly understood. Skunk amdoparvovirus (SKAV), one of a handful of newly discovered APVs, is apparently species-specific in striped skunks (Mephitis mephitis) and has a high prevalence across North America. We have evaluated the infection status and viral tissue distribution in a cohort of 26 free-ranging California skunks from a single rehabilitation facility who were euthanized due to poor prognosis for recovery from neurologic disease. SKAV was detected in the majority of this cohort, and virus was associated with a spectrum of lesions including tubulointerstitial nephritis, meningoencephalitis, myocarditis, and arteritis. Affected tissue and patterns of inflammation were partially overlapping with those of AMDV infection but were notably distinct in the kidney.

AMDOPARVOVIRUS INFECTIONS ARE PREVALENT, PERSISTENT, AND GENETICALLY DIVERSE IN ZOO-HOUSED RED PANDAS (AILURUS FULGENS).

Red pandas (Ailurus fulgens) are a globally endangered small carnivoran species and subjects of a robust ex situ conservation effort that includes animals housed in zoos. In 2018, red panda amdoparvovirus (RPAV) was discovered by metagenomics analyses of tissues from two geriatric red pandas, and in one case it was associated with significant lesions. Because RPAV was discovered in a single zoo cohort, it was unclear whether these infections represented a widely distributed, enzootic virus of red pandas or a localized 'spillover' from a different host species into this collection. The first goal of this study was to estimate the prevalence of RPAV in US zoos. The authors amplified RPAV from feces of 104 individual red pandas from 37 US zoos, and the virus was detected in 52/104 samples (50.0%). Next, to establish persistence of infection in individual animals, the authors tested serial samples in a single cohort over a 4.5-yr period, and virus was consistently shed by infected animals throughout the sampling period. Finally, full viral coding sequences were amplified and sequenced from three cases, and partial sequences of both the nonstructural and capsid genes were obtained for an additional 19 cases. RPAV is a genetically diverse but monophyletic viral species, and multiple viral lineages are present in US zoo-housed red pandas. The authors do not know how red pandas were originally infected, but RPAV is very common in red pandas in the United States, and infections are persistent-presumably for the lifetime of the animal.

Amdoparvovirus Infection in Red Pandas ( Ailurus fulgens).

Aleutian mink disease virus is the type species in the genus Amdoparvovirus, and in mink and other Mustelidae can cause either subclinical disease or fatal chronic immune stimulation and immune complex disease. The authors describe a novel amdoparvovirus in the endangered red panda ( Ailurus fulgens), discovered using viral metagenomics. The authors analyzed the prevalence, tissue distribution, and disease association by PCR, in situ hybridization, electron microscopy, and histology in a group of 6 red pandas from a single zoological collection. The study incorporates a fecal shedding survey and analysis of tissues from 4 necropsied animals over a 12-year span. The tentatively named red panda amdoparvovirus (RpAPV) was detected in the feces and/or tissues of all animals tested. At necropsy of 1 geriatric animal, infection was associated with pyogranulomatous peritonitis, pancreatitis, and myocarditis. Other animals had detectable low-level viral nucleic acid in lymph nodes and both oral and intestinal epithelium at the time of necropsy. Full-length genome sequences of RpAPV strains from 2 animals had 12% sequence divergence, demonstrating genetic diversity even among in-contact animals. RpAPV is a persistent infection in this cohort of red pandas, and has variable clinical expression.

A practical approach to the sampling, fixation, softening, and sectioning of whole honey bees for histologic evaluation.

The Western honey bee (Apis mellifera) is economically important as the primary managed pollinator of many agricultural crops and for the production of various hive-related commodities. Honey bees are not classically or thoroughly covered in veterinary pathology training programs. Given their unique anatomic and biological differences from the other species more traditionally evaluated by veterinary pathologists, establishing routine and consistent methods for processing samples for histology ensures accurate diagnostic and research conclusions. We developed and tested several field protocols for the sampling of honey bees. We compared the tissue-quality outcomes for worker bees fixed, collected, and/or softened under the following protocols: 1) routine formalin fixation; 2) softening chitin via exposure to Nair for 2 d or 3) 5 d; 4) shortened times between formalin submersion and trimming of body segments to enhance penetration of formalin into internal tissues; 5) ethanol submersion of specimen prior to formalin fixation; 6) indirect dry ice exposure; and 7) prolonged -80°C storage. Routine formalin fixation, exposure to Nair for 2 d, indirect dry ice exposure, and trimming body segments within 2 h of formalin submersion resulted in the highest quality histologic tissue sections. The poorest quality sections resulted from softening of chitin by exposure to Nair for 5 d, submersion in ethanol for 3 d before formalin fixation, and prolonged storage at -80°C. Our results indicate that routine formalin fixation is adequate, and that immobilizing bees with indirect dry ice exposure aids in sample collection without negatively impacting the quality of histologic sections.

Fatal cerebrovascular accident in a captive red panda (Ailurus fulgens fulgens) with concurrent amdoparvovirus infection.

We report the pathological and molecular findings in an adult male Himalayan red panda (Ailurus fulgens fulgens) whose death was attributed to parenchymal brain haemorrhage (PBH) of the thalamus. Post-mortem examination revealed severe, acute PBH and intraventricular haemorrhage with major involvement of the thalamus, as well as scattered chronic microinfarctions. Vascular disease in the brain and other organs was suggestive of systemic hypertension. Histological lesions included arteriolar hyalinosis and varying degrees of arteriosclerosis, arterial tunica media hypertrophy and hyperplasia and infiltration of arterial walls by lipid-laden macrophages. Other relevant findings included marked myocardial fibrosis, lymphoplasmacytic tubulointerstitial nephritis, lymphoplasmacytic meningoencephalitis and chronic mitral valve degeneration. The changes in the cerebral vasculature were consistent with hypertensive encephalopathy and a cerebrovascular accident, specifically PBH, which has not been previously reported in this species. Additionally, polymerase chain reaction analysis for red panda amdoparvovirus (RPAV) was positive in the brain and kidneys. Preceded by hypertensive vascular changes and brain microinfarctions, sudden death in this animal likely resulted from fatal PBH with intraventricular haemorrhage. The clinicopathological role of RPAV infection is unknown in this case, although its contribution to the chronic renal disease is considered possible in the context of our current understanding of RPAV-associated pathology.

Natural disease and evolution of an Amdoparvovirus endemic in striped skunks (Mephitis mephitis).

Striped skunks (Mephitis mephitis) densely populate the human-animal interface of suburbia throughout North America. Skunks share that habitat with numerous related mesocarnivores, where increased contact, competition for shared food and water sources and other stressors contribute to increased exposure and susceptibility to viral infection. The recently identified skunk amdoparvovirus (SKAV) has been detected at high prevalence in skunks and occasionally in mink, but its distribution in North America is unknown. To understand the impact of SKAV in striped skunks and the risk posed to related species, we investigated the geographic distribution of SKAV, analysed its genetic diversity and evolutionary dynamics and evaluated viral distribution in tissues of infected animals to identify possible mechanisms of transmission. SKAV was detected in 72.5% (37/51) skunks and was present at high rates at all locations tested across North America. Analysis of the complete genomic sequence of 29 strains showed a clear geographic segregation, frequent recombination and marked differences in the evolutionary dynamics of the major structural (VP2) and non-structural (NS1) proteins. NS1 was characterized by a higher variability and a higher percentage of positively selected codons. This could indicate that antibody-mediated enhancement of infection occurs in SKAV, an infection strategy that may be conserved across amdoparvoviruses. Finally, in situ hybridization revealed virus in epithelium of the gastrointestinal tract, urinary tract and skin, indicating that viral transmission could occur via oronasal, faecal and/or urinary secretions, as well as from skin and hair. The endemicity of SKAV over large geographic distances and its high genetic diversity suggest a long-term virus-host association. Persistent shedding and high environmental stability likely contribute to efficient viral spread, simultaneously offering opportunities for cross-species transmission with consequent risk to sympatric species, including domestic animals and wildlife.

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus).

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the genera Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus (Ursus americanus circovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanus parvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases by in situ hybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears.

Case Series: Virulent hemosporidiosis infections in juvenile great horned owls (Bubo virginianus) from Louisiana and California, USA.

A total of eight juvenile great horned owls (Bubo virginianus) were found lethargic and on the ground in spring 2015, 2016, and 2017, including one fledgling from Louisiana, USA and seven nestlings from California, USA. One bird survived to release after rehabilitation; seven birds died or were euthanized due to poor prognosis and were necropsied. Necropsy findings were similar and included general pallor of all tissues, particularly the subcutis and lungs, and enlarged liver and spleen. Histopathology revealed multi-organ necrosis, abundant meronts containing merozoites, and intracytoplasmic pigmented haemosporidian parasites in blood cells in one bird. Leucocytozoon lineages lSTOCC16 and BUVIR06 were identified by polymerase chain reaction and genetic sequencing. The systemic Leucocytozoon infections were likely associated with morbidity and mortality in these owls. A second parasite, Haemoproteus lineage hSTVAR01, was also identified in an owl from Louisiana. This is the first identification of Leucocytozoon lineages that have been associated with mortality in young great horned owls.