An outbreak of SARS-CoV-2 with high mortality in mink (Neovison vison) on multiple Utah farms.

The breadth of animal hosts that are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may serve as reservoirs for continued viral transmission are not known entirely. In August 2020, an outbreak of SARS-CoV-2 occurred on five mink farms in Utah and was associated with high mink mortality (35-55% of adult mink) and rapid viral transmission between animals. The premise and clinical disease information, pathology, molecular characterization, and tissue distribution of virus within infected mink during the early phase of the outbreak are provided. Infection spread rapidly between independently housed animals and farms, and caused severe respiratory disease and death. Disease indicators were most notably sudden death, anorexia, and increased respiratory effort. Gross pathology examination revealed severe pulmonary congestion and edema. Microscopically there was pulmonary edema with moderate vasculitis, perivasculitis, and fibrinous interstitial pneumonia. Reverse transcriptase polymerase chain reaction (RT-PCR) of tissues collected at necropsy demonstrated the presence of SARS-CoV-2 viral RNA in multiple organs including nasal turbinates, lung, tracheobronchial lymph node, epithelial surfaces, and others. Localization of viral RNA by in situ hybridization revealed a more localized infection, particularly of the upper respiratory tract. Whole genome sequencing from multiple mink was consistent with published SARS-CoV-2 genomes with few polymorphisms. The Utah mink SARS-CoV-2 strains fell into Clade GH, which is unique among mink and other animal strains sequenced to date. While sharing the N501T mutation which is common in mink, the Utah strains did not share other spike RBD mutations Y453F and F486L found in nearly all mink from the United States. Mink in the outbreak reported herein had high levels of SARS-CoV-2 in the upper respiratory tract associated with symptomatic respiratory disease and death.

FATAL SYSTEMIC FUNGAL INFECTION IN EASTERN BONGO ANTELOPE (TRAGELAPHUS EURYCERUS ISAACI): SIX CASES.

Over a span of 6 yr, six adult eastern bongo antelope (Tragelaphus eurycerus isaaci) from a single institution died due to systemic mycotic infections. All animals were of the same genetic lineage and in good body condition at the time of death. Gross findings in all cases included multifocal white-to-tan nodules up to 10 cm in diameter that were most numerous in the heart, lung, and kidney. Histologic examination identified these nodules as foci of granulomatous inflammation containing branching, septate, broad, undulating fungal elements. Identification of the fungal species was pursued using PCR with sequencing, immunohistochemistry, and culture. Multiple fungal species were identified using the various modalities, and commonality of species identification was limited to Cladosporium sp. in four of the cases. The clinical and postmortem findings in these cases were identical and were considered to be the same infectious disease. The Cladosporium sp. was considered a candidate as an emerging fatal infectious agent in this population of bongo antelopes. In all of these cases, death was attributed to conduction abnormalities associated with the cardiac lesions or euthanasia.

Avian coxiellosis in nine psittacine birds, one black-browed barbet, and one paradise tanager.

Infection with a novel species of the genus Coxiella was first described in three Swainson's blue mountain rainbow lorikeets from a zoological collection, and days later in a group of seven other psittacine birds and a toucan. We provide an update on coxiellosis in nine additional psittacines, and two non-psittacines. Psittacines originated in New England, the mid Atlantic, the Midwest, the South, and the Northwest. Psittacines most commonly had lesions in the brain, spleen, liver, and lungs, consisting of meningoencephalitis, hepatosplenomegaly, and interstitial pneumonia. Lesions contained histiocytic infiltrate, with intracytoplasmic, Gimenez-positive coccobacilli. Transmission electron microscopy revealed bacteria with trilaminar cell walls, electron dense cores, and spore-like forms. PCR revealed homology to the organism in index cases. In addition, one black-browed barbet and one paradise tanager were found with systemic coxiellosis; sequencing identified the same pathogen. These are the second piciforme and the first passerine affected by this pathogen, indicating expanded infectivity and pathogenicity.RESEARCH HIGHLIGHTS Report of coxiellosis in nine psittacines; lesions often in brain, spleen, liver, lung.Second piciforme with coxiellosis, a black-browed barbet.First case of avian coxiellosis described in a passerine, a paradise tanager.

TUBERCULOSIS CAUSED BY MYCOBACTERIUM ORYGIS IN A GREATER ONE-HORNED RHINOCEROS (RHINOCEROS UNICORNIS): FIRST REPORT IN THE WESTERN HEMISPHERE.

Mycobacterium orygis, a newly identified member of the Mycobacterium tuberculosis complex, has been isolated predominantly from hoofstock in eastern Africa and the Arabian Peninsula, and sporadically in cattle (Bos taurus indicus), rhesus monkeys (Macaca mulatta), humans, and a greater one-horned rhinoceros (Rhinoceros unicornis) in South Asia. In rhinoceros, tuberculosis typically presents as a chronic progressive respiratory disease. The report describes the postmortem diagnosis of tuberculosis caused by Mycobacterium orygis in a greater one-horned rhinoceros with hind limb paresis due to neural granulomatosis. Serologic assays for detection of antibodies to M. tuberculosis complex proteins before culture results allowed for appropriate herd management protocols to be initiated. Mycobacterium genus-specific polymerase chain reaction assays with direct sequencing allowed timely confirmation of the serologic results. This is the first isolation of M. orygis in the western hemisphere, showing the need for mycobacterial testing of rhinoceros before international shipments and the urgency for validated antemortem M. tuberculosis complex screening assays in rhinoceros species.

DERMATITIS AND RHINOSINUITIS CAUSED BY CURVULARIA SPECIES IN A CHINESE GORAL (NAEMORHEDUS GRISEUS).

Curvularia spp. are globally distributed saprophytic fungi, classified in the literature as dematiaceous, or darkly pigmented fungi. These fungi have been increasingly recognized as causing cutaneous, ocular, respiratory, and central nervous system infections in humans, but have been infrequently documented as pathogens in the veterinary literature. A 5-yr-old male Chinese goral (Naemorhedus griseus) presented with bilateral fungal dermatitis of the pinnae, and subsequent pyogranulomatous rhinosinusitis. Clinical signs included epistaxis, mucosanguineous nasal discharge, and dyspnea. Sequential histologic examinations of cutaneous and nasal lesions revealed pyogranulomatous inflammation with extracellular and phagocytized nonpigmented yeasts. Fungal culture and polymerase chain reaction identified Curvularia sp. The absence of pigmentation in tissue in this case suggests that pigmentation may not be a consistent histologic finding for this fungus, emphasizing the importance of molecular identification to prevent misidentification. Despite intensive interventions in this goral, the disease progressed, and was ultimately fatal.

Sarcocystis calchasi Outbreak in Feral Rock Pigeons ( Columba livia) in California.

Twenty-two feral rock pigeons ( Columba livia) from 10 counties in California with ataxia, torticollis, and difficulty standing and flying were admitted to rehabilitation centers in late winter and spring of 2017 and died or were euthanized. Common necropsy findings included thin body condition, generalized deep red discoloration of organs, and hemorrhagic subcutaneous neck tissues. Meningoencephalitis was observed microscopically in 16 pigeons, and 3 also had protozoal schizonts in the brain. The most consistently affected regions of the brain were cerebellum and brainstem. Skeletal muscles, and less frequently the heart, contained large intrasarcoplasmic bradyzoites typically without inflammation. Fifteen of the 22 birds tested positive using pan- Sarcocystis polymerase chain reaction. The sequence of the amplicon was most closely related to S. calchasi, and the 8 subtyped sequences had 100% homology with S. calchasi. This investigation demonstrated the transcontinental and North American spread of S. calchasi causing a disease outbreak in free-ranging rock pigeons, thus warranting increased surveillance in susceptible native columbids.

MYCOBACTERIUM GENAVENSE IN AN AFRICAN PENGUIN (SPHENISCUS DEMERSUS).

A 19-yr-old female African penguin (Spheniscus demersus) presented with labored breathing and anorexia. Radiographs revealed soft-tissue density lesions in the left lung fields and fluid in the right. The penguin died during the night. Postmortem examination demonstrated multiple granulomas in the lungs and air sacs. The right coelom was filled with opaque fluid. Histopathology of the lung, liver, kidney, and spleen identified Mycobacterium as a primary disease etiology. Large numbers of acid fast-positive, rod-shaped bacteria were recognized on tissue staining. Mycobacterium genavense was detected by polymerase chain reaction (PCR) using primers specific for the species. Further confirmation of M. genavense was accomplished using PCR with universal Mycobacterium spp. primers followed by sequencing of the amplicon obtained. To our knowledge, this is the first reported case of mycobacteriosis-and specifically M. genavense -in an African penguin. This case also demonstrates the similarities of presentation between the more commonly suspected and encountered aspergillosis and mycobacteriosis.

T-CELL LYMPHOCYTIC LEUKEMIA IN A FALLOW DEER (DAMA DAMA).

A 17-yr-old female fallow deer presented with ataxia, inappetence, decreased fecal output, and decreased mentation. A complete blood count demonstrated leukocytosis (24.1×10(3)/µl, n=1.16-7.38×10(3)/µl), characterized by lymphocytosis (22.89×10(3)/µl, n=0.18-3.65×10(3)/µl), anemia (packed cell volume 20%, n=29.0-55.8%), decreased red blood cell count (4.1×10(3)/µl, n=6.86-14.72×10(3)/µl), and decreased hemoglobin (7.5 g/dl, n=9.4-19.2 g/dl). Numerous mature, well-differentiated lymphocytes were noted on the blood film. Despite treatment and clinical improvement, the decision was made to euthanize the deer. Histopathology identified a monomorphic population of CD3 positive, CD79a negative small lymphocytes replacing most of the hematopoietic tissue in the bone marrow without evidence of tissue invasion. Results of viral screening were negative.

Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

Intestinal coccidioidomycosis in a red coachwhip snake (Masticophis flagellum piceus).

An adult female, wild-caught red coachwhip snake (Masticophis flagellum piceus) was euthanized at the Phoenix Zoo due to severe neurologic signs. Necropsy and histopathology revealed an invasive liposarcoma of the vertebral column, which likely caused the neurologic signs. Histology of the small intestine revealed a granuloma with intralesional yeasts morphologically compatible with the genus Coccidioides. The diagnosis of coccidioidomycosis was confirmed with immunohistochemistry staining. Coccidioides posadasii is endemic to Arizona and is an important cause of disseminated fungal infections in mammals in this region. This is the first known report of intestinal coccidioidomycosis in a veterinary species and the second report of coccidioidomycosis in a reptile.

Investigation of laboratory methods for characterization of aquatic viruses in fish infected experimentally with infectious salmon anemia virus.

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

The effects of exposure of susceptible alpacas to alpacas persistently infected with bovine viral diarrhea virus.

Reports of bovine viral diarrhea virus (BVDV) infections in alpacas have been increasing in recent years but much is still unknown about the mechanisms of disease in this species. This report characterizes the transmission of BVDV from persistently infected (PI) alpacas to BVDV naïve alpacas, documents shedding patterns, and characterizes the disease effects in both PI and transiently infected alpacas. Two PI alpacas shed BVDV Type 1b virus in most body fluids, and commonly available diagnostic tests verified their status. Bovine viral diarrhea virus Type 1b transient infections produced only mild signs of disease in BVDV naïve alpacas. Viremia was detected in whole blood, but viral shedding during the acute phase was not detected and antibody appeared to be protective upon re-exposure to the virus.

Disseminated, histologically confirmed Cryptococcus spp infection in a domestic ferret.

CASE DESCRIPTION: A 4-year-old castrated male domestic ferret from central Massachusetts was evaluated for weight loss over a 1.5-month period and for 2 days of retching, diarrhea, and signs of lethargy. It had been housed indoors, with 2 other ferrets, 2 cats, and humans that lacked signs or symptoms of disease. CLINICAL FINDINGS: Physical examination revealed a thin body condition, tachypnea, an increase in respiratory effort, and retching. Splenomegaly was detected during abdominal palpation. Clinicopathologic analysis revealed lymphopenia, lactic acidosis, hypoglycemia, hypocalcemia, hypoalbuminemia, and hyperglobulinemia. A pulmonary bronchointerstitial pattern was evident on radiographs, and abdominal ultrasonography revealed a suspected pancreatic mass and mesenteric lymphadenopathy. TREATMENT AND OUTCOME: After 2 weeks of medical treatment and once clinical signs resolved, an exploratory laparotomy was performed and a lymph node biopsy specimen was collected. Histologic evaluation of the specimen revealed Cryptococcus-like organisms. Antifungal treatment was initiated with itraconazole (PO) and amphotericin B (IV). The ferret died after 2 days of treatment. A full necropsy was performed, revealing multicentric cryptococcosis affecting the lungs, brain, spleen, and multiple lymph nodes. Paraffin-embedded, formalin-fixed lung tissue was submitted for DNA extraction, and the organism was identified as Cryptococcus neoformans var grubii. CLINICAL RELEVANCE: To the authors' knowledge, this is the first report of disseminated cryptococcosis in a North American ferret. This case is unique in that the ferret lived indoors, in a geographic region in which reports of cryptococcosis are rare. The genotyping technique used to identify the Cryptococcus strain can aid in better understanding the epidemiology of cryptococcosis.

Disseminated Bovine viral diarrhea virus in a persistently infected alpaca (Vicugna pacos) cria.

Bovine viral diarrhea virus (BVDV) is an emerging infectious pathogen of concern to the alpaca industry. A 4-month-old, intact, male alpaca cria was diagnosed as persistently infected with BVDV on the basis of repeated positive antemortem polymerase chain reaction (PCR) and virus isolation (VI) assays and negative serologic titers to BVDV. Immunohistochemistry, real-time reverse transcription PCR, and VI performed on tissues collected at necropsy demonstrated disseminated BVDV-1b infection. Virus was detected in multiple tissues, including parotid salivary gland, testes, prostate, kidneys, skin, and gastrointestinal tract. Demonstration of BVDV in previously unreported tissues suggests additional potential routes of BVDV transmission in alpacas.

Phaeohyphomycosis of the carapace in an Aldabra tortoise (Geochelone gigantea).

An adult male Aldabra tortoise (Geochelone gigantea) presented with a deep flaking area of the carapace, and histologic examination of biopsies from this area revealed phaeohyphomycosis of the superficial keratinized layers. The disease progressed rapidly and spread to numerous sites on the carapace. After several weeks of regular debridement, deep bone involvement was evident and was confirmed through histologic examination. Fungal culture was attempted but was unsuccessful at isolating the infectious agent. Polymerase chain reaction analysis of extracted DNA from the fixed tissue block identified the fungus as Exophiala oligosperma. Initial treatment included weekly debridement and oral and topical antifungal agents. A nuclear scintigraphy bone scan was performed to determine the extent and status of the infection. Multiple foci of uptake of the radiopharmaceutical marker were present within the carapace, indicating active lesions. The tortoise was maintained on oral antifungal treatment, and lesions resolved over several months. A repeat bone scan performed 1 yr after initial presentation showed reduction in marker uptake, indicating a response to treatment in the deeper lesions. Phaeohyphomycosis should be considered as a differential diagnosis for cases of shell lesions in chelonians.

Proventricular nematodiasis in wrinkled hornbills (Aceros corrugatus).

Three immature Sunda wrinkled hornbills (Aceros corrugatus) were diagnosed postmortem with proventricular spirurid nematodiasis. Concurrent severe disseminated larval granulomatosis in other visceral organs was considered contributory to mortality in each case. Clinical signs of nematodiasis were vague but generally consisted of weight loss, anorexia, and lethargy. Frequent antemortem fecal examinations were negative for spirurid eggs. In these present cases, based on routine histopathology, both prophylactic and empirically based therapeutic anthelmintic treatments had no evident benefit in the elimination of the proventricular nematodes. Spirurid nematodiasis may be an important cause of mortality in young hornbills.

Rhodococcus equi-specific hyperimmune plasma administration decreases faecal shedding of pathogenic R. equi in foals.

Rhodococcus equi is the most common cause of pneumonia in young foals. Pneumonic foals are an important source of environmental contamination as they shed higher amounts of R. equi in their faeces than unaffected foals. As R. equi-specific hyperimmune plasma (HIP) lessens clinical pneumonia, we hypothesise that its use would result in decreased faecal shedding of R. equi by foals. Neonatal foals were either given HIP (n=12) or nothing (n=9, control) shortly after birth and were then experimentally infected with R. equi Faeces were collected before and on weeks 2, 3, 5 and 7 after infection. Presence of virulent R. equi was tested using qPCR. There was strong evidence of an association between HIP administration and a decrease in faecal shedding of virulent R. equi (P=0.031 by Pearson chi-squared test). Foals in the control shed significantly more R. equi (colony-forming units/ml) than foals that received HIP (P=0.008 by Mann-Whitney rank-sum test). While our study is the first to report this additional benefit of HIP administration, future studies are needed to evaluate the implications of its use under field conditions.

Evidence for persistent Bovine viral diarrhea virus infection in a captive mountain goat (Oreamnos americanus).

Bovine viral diarrhea (BVD) viruses are pestiviruses that have been isolated from domestic and wild ruminants. There is serologic evidence of pestiviral infection in more than 40 species of free-range and captive mammals. Vertical transmission can produce persistently infected animals that are immunotolerant to the infecting strain of Bovine viral diarrhea virus (BVDV) and shed virus throughout their lives. Seven species (white-tailed deer, mouse deer, eland, domestic cattle, alpaca, sheep, and pigs) have been definitively identified as persistently infected with BVDV. This study provides serological, molecular, immunohistochemical, and histological evidence for BVDV infection in 2 captive mountain goats from a zoological park in Idaho. The study was triggered by isolation of BVDV from tissues and immunohistochemical identification of viral antigen within lesions of a 7-month-old male mountain goat (goat 1). Blood was collected from other mountain goats and white-tailed and mule deer on the premises for BVDV serum neutralization, viral isolation, and reverse transcription polymerase chain reaction. One 3-month-old mountain goat (goat 2) was antibody negative and BVDV positive in serum samples collected 3 months apart. This goat subsequently died, and though still antibody negative, BVDV was isolated from tissues and identified by immunohistochemistry within lesions. Sequencing and phylogenetic analysis identified the isolates as BVDV-2. These findings provide evidence of persistent infection in a mountain goat, underscoring the need for pestivirus control strategies for wild ruminants in zoological collections.

Acute disseminated mycobacteriosis in captive Atlantic guitarfish ( Rhinobatos lentiginosus).

An adult female captive-born Atlantic guitarfish ( Rhinobatos lentiginosus) was found acutely moribund on exhibit and died soon after presentation. Abnormalities on autopsy were focal cutaneous erythema on the tail, a small liver, many variably sized friable ovarian follicles, and coelomic effusion. Histologic examination revealed systemic bacterial embolization, and yolk coelomitis with minimal associated inflammation and some mineralization. Bacterial culture of blood and coelomic effusion grew a rapidly growing Mycobacterium species that was further identified as Mycobacterium chelonae by PCR amplification and sequencing of the RNA polymerase subunit beta ( rpoB) gene of isolated genomic DNA. Concurrent reproductive disease may have caused immunosuppression, thus predisposing to the mycobacterial infection. At another institution, an adult male wild-caught Atlantic guitarfish was found dead on exhibit with no premonitory signs. Abnormalities on autopsy were a thin body condition, small liver, and coelomic effusion. Histologic examination revealed acute mycobacterial septicemia. M. chelonae was also identified in this fish by PCR amplification and sequencing. Mycobacteriosis has rarely been reported in captive elasmobranchs. Guitarfish may have greater susceptibility to mycobacteriosis than other elasmobranchs, and acute and chronic manifestations of the disease may exist in this species.

Systemic Mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat.

Dogs may be infected by Mycobacterium (M.) tuberculosis, M. bovis, and M. avium complex, and the clinical signs associated with each of these infections may be indistinguishable. Rapid speciation of the infecting organism is desirable because of the public health concerns associated with M. bovis and M. tuberculosis infections. A mycobacterial infection was suspected in the dog of this report based on acid-fast staining of organisms in macrophages obtained from liver aspirates and buffy-coat preparations. Polymerase chain reaction (PCR) analysis of a buffy-coat preparation identified M. avium.