Vesicular stomatitis virus in two species of rhinoceros at a California zoological park.

OBJECTIVE: To describe an outbreak of vesicular stomatitis virus (VSV) in southern white rhinoceros (SWR; Ceratotherium simum simum) and greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) at a safari park in San Diego, CA, from May to September 2023. ANIMALS: 21 SWR and 5 GOHR in professionally managed care. METHODS: Rhinoceros of both species presented with a range of clinical signs and severities. Lesion locations were categorized as cutaneous (coronary bands, heels and soles, limbs, ventrum, neck folds, and ears) and mucocutaneous (lips, nostrils, mucous membranes of the oral cavity, and vulva). Clinical signs included lethargy, lameness, difficulty with prehension, hyporexia to anorexia, and hypersalivation. Severely affected rhinoceros had clinical pathology findings consistent with systemic inflammation. RESULTS: Vesicular stomatitis New Jersey virus was confirmed via PCR from swabs of lesions in 10/26 (38%) rhinoceros. Of these 10 confirmed cases, 9 (90%) were SWR and 1 (10%) was a GOHR. A further 6/26 (24%) were considered probable cases, and 10/26 (38%) were considered suspect cases based on clinical signs, but the inability to appropriately sample due to the housing environment precluded confirmation. Histopathology samples from 3 rhinoceros were consistent with VSV, and viral RNA was localized in histologic lesions via RNA in situ hybridization for 1 case. All rhinoceros survived infection despite severe systemic illness in 2 animals. CLINICAL RELEVANCE: This case series describes the clinical appearance and progression of VSV in 2 rhinoceros species. To the authors' knowledge, this is the first report of VSV in a rhinoceros.

Systemic avian poxvirus infections associated with the B1 subclade of canarypox virus.

Avian poxvirus infections typically manifest as 2 forms: cutaneous ("dry") pox, characterized by proliferative nodules on the skin, and diphtheritic ("wet") pox, characterized by plaques of caseous exudate in the oropharynx and upper respiratory and gastrointestinal tracts. Systemic spread of virus to visceral organs beyond the skin and mucous membranes is rarely reported. Out of 151 cases diagnosed with avian poxvirus over a 20-year period at a zoological institution, 22 were characterized as having systemic involvement based on histopathology and molecular findings. Gross lesions in systemic cases included soft white nodules scattered throughout the liver, spleen, and kidneys. Two histopathologic patterns emerged: (1) widespread histiocytic inflammation in visceral organs with intrahistiocytic viral inclusions and (2) severe, localized dry or wet pox lesions with poxvirus-like inclusions within dermal and subepithelial histiocytes. In situ hybridization targeting the core P4b protein gene confirmed the presence of poxvirus DNA within histiocytes in both patterns. Polymerase chain reaction was performed targeting the reticuloendothelial virus long terminal repeat (REV LTR) flanking region and the core P4b protein gene. Sequences of the REV LTR flanking region from all systemic pox cases were identical to a previously described condorpox virus isolated from an Andean condor with systemic pox. Sequences of the core P4b protein gene from all systemic pox cases grouped into cluster 2 of the B1 subclade of canarypox viruses. Systemic involvement of avian poxvirus likely occurs as a result of infection with certain strain variations in combination with various possible host and environmental factors.

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.

CAUSES OF MORTALITY AND COMORBIDITIES IN CAIMAN LIZARDS (DRACAENA GUIANENSIS) AT SIX ZOOLOGICAL INSTITUTIONS.

An understanding of the main causes of mortality in caiman lizards (Dracaena guianensis) under managed care is imperative to promote optimal husbandry, health, and welfare. A retrospective review of morbidity and mortality in caiman lizards from North American zoological institutions between 2005 and 2020 was conducted. Postmortem data, including gross necropsy and histopathology findings, were available for 32 caiman lizards (n = 12 subadults, n = 20 adults) from six zoological institutions. Necropsy reports were evaluated to collect general demographic data, categorize cause of death (accident/trauma, congenital/genetic, degenerative/geriatric, infectious, deposition disease, neoplastic, unknown, and multifactorial), and assess common comorbidities. Infectious disease was the most common cause of mortality in adult lizards (8/20; 40%) with amoebiasis and bacterial etiologies being overrepresented. Demise due to traumatic/accidental injury was the second most common cause of death in adult lizards (3/20;15%) and included blunt force trauma or suspected drowning. Infectious disease (4/12; 33.3%) and trauma/accidental injury (4/12; 33.3%) were also the most common causes of death in subadults. The most common comorbidities or other incidental findings identified during necropsy included trematode parasitism (15/32; 46.9%), arteriosclerosis (11/32; 34.4%), and adrenocortical hyperplasia (6/32; 18.8%). This retrospective review suggests that management practices to prevent and control infectious diseases and mitigate traumatic injury play a pivotal role in the long-term care and survival of caiman lizards in managed care.

Mitochondrial Gene Diversity and Host Specificity of Isospora in Passerine Birds.

Isospora infections are common in both wild and captive passerine species. Many bird species have been shown to have co-evolved with a particular species of Isospora. Disease can range from subclinical to severe and fatal, making infection and transmission of this parasite a concern for birds under managed care, particularly in institutions housing endangered species for breeding and reintroduction purposes. Whether birds in mixed-species enclosures represent a risk factor for severe isosporiasis due to infection with non-host-adapted strains is of concern for institutions managing these populations. To begin answering this question, we sought to characterize the host-specificity of Isospora spp. in a large number of passerine birds via retrospective sequencing of mitochondrial gene cytochrome c oxidase subunit I (COI). Despite outliers, Isospora sequences largely grouped by host species and/or host family. Additional research is warranted into the degree of interspecies transmission and host-switching of Isospora parasites, and risk factors for the development of severe disease in passerine birds.

Unique Isospora-associated histologic lesions in white-rumped shama (Copsychus malabaricus).

Twenty-one white-rumped shamas (19 necropsied, 2 biopsied) (Copsychus malabaricus) housed at the San Diego Zoo between 1992 and 2020 were diagnosed with Isospora infection based on evaluation of histologic sections. Review of these cases revealed a consistent histologic lesion characterized by nodular aggregates of atypical epithelioid macrophages containing few intracytoplasmic protozoa, with or without lymphocytic infiltrates. Of the 19 necropsied cases, 16 (84%) had systemic lesions variably affecting the liver, spleen, gastrointestinal tract, lung, pancreas, connective tissues, or bone marrow, while all 21 diagnosed cases had skin involvement. The findings suggest that white-rumped shamas have a unique inflammatory response to isosporosis with a predilection for the skin. Skin may be a diagnostically sensitive sampling site for histologic diagnosis of Isospora in this species.

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.

Herpesvirus surveillance and discovery in zoo-housed ruminants.

Gammaherpesvirus infections are ubiquitous in captive and free-ranging ruminants and are associated with a variety of clinical diseases ranging from subclinical or mild inflammatory syndromes to fatal diseases such as malignant catarrhal fever. Gammaherpesvirus infections have been fully characterized in only a few ruminant species, and the overall diversity, host range, and biologic effects of most are not known. This study investigated the presence and host distribution of gammaherpesviruses in ruminant species at two facilities, the San Diego Zoo and San Diego Zoo Safari Park. We tested antemortem (blood, nasal or oropharyngeal swabs) or postmortem (internal organs) samples from 715 healthy or diseased ruminants representing 96 species and subspecies, using a consensus-based herpesvirus PCR for a segment of the DNA polymerase (DPOL) gene. Among the 715 animals tested, 161 (22.5%) were PCR and sequencing positive for herpesvirus, while only 11 (6.83%) of the PCR positive animals showed clinical signs of malignant catarrhal fever. Forty-four DPOL genotypes were identified of which only 10 have been reported in GenBank. The data describe viral diversity within species and individuals, identify host ranges of potential new viruses, and address the proclivity and consequences of interspecies transmission during management practices in zoological parks. The discovery of new viruses with wide host ranges and presence of co-infection within individual animals also suggest that the evolutionary processes influencing Gammaherpesvirus diversity are more complex than previously recognized.

Phaeohyphomycosis due to Veronaea botryosa in cultured white sturgeon (Acipenser transmontanus Richardson) from California USA during 2006 to 2015.

Infection with Veronaea botryosa can result in rare cutaneous or disseminated, granulomatous to pyogranulomatous phaeohyphomycosis in humans, although disease due to the fungus has also been reported in non-mammalian vertebrates. This report documents disease due to V. botryosa in captive, juvenile to subadult or young adult white sturgeon (Acipenser transmontanus Richardson) from California USA and complements a previous report of the disease in captive Siberian sturgeon (Acipenser baerii) from Florida USA. Pathological examinations revealed granulomatous to pyogranulomatous inflammation of multiple organs. Isolates of the fungal agent were phenotypically consistent with V. botryosa, and molecular analyses of the D1/D2 region of the fungal 28S rRNA gene and the internal transcribed spacer (ITS) region located between the fungal 18S and 28S rRNA genes confirmed the aetiologic agent as V. botryosa. The disease in captive sturgeon results in a considerable economic encumbrance to the producer due to the loss of the cumulative financial resources invested in the production of older subadult to young adult sturgeon.

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

The first reptilian circovirus identified infects gut and liver tissues of black-headed pythons.

Viral metagenomic analysis of the liver of a black headed python (Aspidites melanocephalus) euthanized for a proliferative spinal lesion of unknown etiology yielded the first characterized genome of a reptile-infecting circovirus (black-headed python circovirus or BhPyCV). BhPyCV-specific in situ hybridization (ISH) showed that viral nucleic acids were strongly expressed in the intestinal lining and mucosa and multifocally in the liver. To investigate the presence of this virus in other snakes and its possible pathogenicity, 17 snakes in the python family with spinal disease were screened with ISH yielding a second BhP positive in intestinal tissue, and a Boelen's python (Morelia boeleni) positive in the liver. BhPyCV specific PCR was used to screen available frozen tissues from 13 of these pythons, four additional deceased pythons with and without spinal disease, and fecal samples from 37 live snakes of multiple species with unknown disease status. PCR detected multiple positive tissues in both of the ISH positive BhP and in the feces of another two live BhP and two live annulated tree boas (Corallus annulatus). Preliminary analysis indicates this circovirus can infect BhPs where it was found in 4/5 BhPs tested (2/2 with spinal disease, 2/3 live with unknown status), Boelen's python (1/2 with spinal disease), and annulated tree boa (2/6 live with unknown status) but was not detected in other python species with the same spinal lesions. This circovirus' causal or contributory role in spinal disease remains speculative and not well supported by these initial data.

A Highly Divergent Picornavirus Infecting the Gut Epithelia of Zebrafish (Danio rerio) in Research Institutions Worldwide.

Zebrafish have been extensively used as a model system for research in vertebrate development and pathogen-host interactions. We describe the complete genome of a novel picornavirus identified during a viral metagenomics analysis of zebrafish gut tissue. The closest relatives of this virus showed identity of <20% in their P1 capsids and <36% in their RdRp qualifying zebrafish picornavirus-1 (ZfPV-1) as member of a novel genus with a proposed name of Cyprivirus. Reverse transcription (RT)-PCR testing of zebrafish from North America, Europe, and Asia showed ZfPV-1 to be globally distributed, being detected in 23 of 41 (56%) institutions tested. In situ hybridization of whole zebrafish showed viral RNA was restricted to a subset of enterocytes and cells in the subjacent lamina propria of the intestine and the intestinal mucosa. This naturally occurring and apparently asymptomatic infection (in wild-type zebrafish lineage AB) provides a natural infection system to study picornavirus-host interactions in an advanced vertebrate model organism. Whether ZfPV-1 infection affects any immunological, developmental, or other biological processes in wild-type or mutant zebrafish lineages remains to be determined.

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.

Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses.

Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV. IMPORTANCE: Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.

Circovirus in tissues of dogs with vasculitis and hemorrhage.

We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Susceptibility of white-tailed deer (Odocoileus virginianus) to experimental infection with epizootic hemorrhagic disease virus serotype 7.

During the fall of 2006, in Israel, epizootic hemorrhagic disease virus (EHDV) serotype 7 caused an intense and widespread epizootic in domestic cattle that resulted in significant economic losses for the dairy industry. The susceptibility of potential North American vector and ruminant hosts to infection with EHDV-7 is not known but is essential to understanding the potential for establishment of this exotic orbivirus in North America if it were introduced. Our primary objective was to determine whether white-tailed deer (WTD; Odocoileus virginianus) are susceptible to infection with EHDV-7. Six, 8-mo-old WTD were experimentally infected with EHDV-7, and all became infected and exhibited varying degrees of clinical disease. Clinical signs, clinicopathologic abnormalities, and postmortem findings were consistent with previous reports of orbiviral hemorrhagic disease (HD) in this species. Four of six animals died or were euthanized because of the severity of disease, one on postinoculation day (PID) 5 and the remaining WTD on PID 7. All deer had detectable viremia on PID 3, which peaked on PID 5 or 6 and persisted for as long as PID 46 in one animal. Deer surviving the acute phase of the disease seroconverted by PID 10. Based on the 67% mortality rate we observed, this strain of EHDV-7 is virulent in WTD, reaffirming their role as a sentinel species for the detection of endemic and nonendemic EHDV. Further, the observed disease was indistinguishable from previous reports of disease caused by North American EHDV and bluetongue virus serotypes, highlighting the importance of serotype-specific diagnostics during suspected HD outbreaks.

An outbreak of equine botulism type A associated with feeding grass clippings.

In September 2010, an outbreak of type A botulism involved 4 horses in northern California that were fed grass clippings obtained from a nearby park. All 4 animals developed a progressive flaccid paralysis syndrome clinically consistent with exposure to preformed Clostridium botulinum neurotoxin (BoNT). Within 48 hr of consuming the grass clippings, all 4 horses showed marked cervical weakness (inability to raise their heads to a normal position) and died or were euthanized within 96 hr. One horse was submitted for diagnostic examination and subsequent necropsy. At necropsy, extensive edema was observed in areas of the nuchal ligament and inguinal fascia. A sample of the grass clippings tested positive for preformed BoNT type A by the mouse bioassay test. Emphasis should be placed on early case recognition, rapid initiation of treatment with the trivalent antitoxin product, and preventing exposure to BoNT in spoiled forages.