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.

Household Transmission of SARS-CoV-2 from Humans to Pets, Washington and Idaho, USA.

SARS-CoV-2 likely emerged from an animal reservoir. However, the frequency of and risk factors for interspecies transmission remain unclear. We conducted a community-based study in Idaho, USA, of pets in households that had >1 confirmed SARS-CoV-2 infections in humans. Among 119 dogs and 57 cats, clinical signs consistent with SARS-CoV-2 were reported for 20 dogs (21%) and 19 cats (39%). Of 81 dogs and 32 cats sampled, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive. This discordance might be caused by delays in sampling. Respondents commonly reported close human‒animal contact and willingness to take measures to prevent transmission to their pets. Reported preventive measures showed a slightly protective but nonsignificant trend for both illness and seropositivity in pets. Sharing of beds and bowls had slight harmful effects, reaching statistical significance for sharing bowls and seropositivity.

Autochthonous Transmission of Coccidioides in Animals, Washington, USA.

We report 5 cases of coccidioidomycosis in animals that were acquired within Washington, USA, and provide further evidence for the environmental endemicity of Coccidioides immitis within the state. Veterinarians should consider coccidioidomycosis in animals with compatible clinical signs that reside in, or have traveled to, south central Washington.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Novel Eurasian highly pathogenic avian influenza A H5 viruses in wild birds, Washington, USA, 2014.

Novel Eurasian lineage avian influenza A(H5N8) virus has spread rapidly and globally since January 2014. In December 2014, H5N8 and reassortant H5N2 viruses were detected in wild birds in Washington, USA, and subsequently in backyard birds. When they infect commercial poultry, these highly pathogenic viruses pose substantial trade issues.

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.

Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale.

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale.

An outbreak of Chlamydophila psittaci in an outdoor colony of Magellanic penguins (Spheniscus magellanicus).

An outbreak of Chlamydophila psittaci occurred in an outdoor colony of 63 Magellanic penguins (Spheniscus magellanicus) at the San Francisco Zoo. Affected penguins presented with inappetence, lethargy, and light green urates. Hematologic and serum biochemical findings were consistent with chronic inflammation. Penguins did not respond to initial supportive and antimicrobial therapy, and 3 died. Necropsy results of the 3 birds revealed hepatomegaly and splenomegaly, and histologic lesions included necrotizing hepatitis, splenitis, and vasculitis. Chlamydophila psittaci infection was confirmed by results of Gimenez staining, immunohistochemistry, and tissue polymerase chain reaction assay. As additional birds continued to present with similar clinical signs, the entire colony of penguins was prophylactically treated with a 30-day minimum course of doxycycline, administered orally or intramuscularly or as a combination of both. Despite treatment, 9 additional penguins died during a 3-month period. Pathologic results from these birds revealed renal and visceral gout (n = 4), cardiac insufficiency (n = 2), sepsis from a suspected esophageal perforation (n = 2), and no gross lesions (n = 1). During the outbreak, 4 birds presented with seizures, 5 developed dermatitis, and nearly 90% of birds in the colony showed severe keratoconjunctivitis, believed to be related to drug therapy with doxycycline. We report the clinical and pathologic features of Chlamydophila psittaci infection in an outdoor colony of penguins and the associated challenges of treatment.

Household transmission of SARS-CoV-2 from humans to pets in Washington and Idaho: burden and risk factors.

SARS-CoV-2 is believed to have emerged from an animal reservoir; however, the frequency of and risk factors for inter-species transmission remain unclear. We carried out a community-based study of pets in households with one or more confirmed SARS-CoV-2 infection in humans. Among 119 dogs and 57 cats with completed surveys, clinical signs consistent with SARS-CoV-2 were reported in 20 dogs (21%) and 19 cats (39%). Out of 81 dogs and 32 cats sampled for testing, 40% of dogs and 43% of cats were seropositive, and 5% of dogs and 8% of cats were PCR positive; this discordance may be due to delays in sampling. Respondents commonly reported close human-animal contact and willingness to take measures to prevent transmission to their pets. Reported preventative measures showed a slightly protective trend for both illness and seropositivity in pets, while sharing of beds and bowls had slight harmful effects.

Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

Identification of Anaplasma marginale proteins specifically upregulated during colonization of the tick vector.

The transition between infection of the mammalian host and colonization of an arthropod vector is required for the ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either chemotherapy or immunization. We used an unbiased proteomic screen to identify Anaplasma marginale proteins specifically upregulated in the tick compared to the mammalian host. Comparative mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis of uninfected and infected ISE6 cells and infected mammalian cells identified 15 proteins exclusively expressed or upregulated in tick cells. All 15 had originally been annotated as hypothetical proteins. We confirmed quantitative upregulation and expression in situ within the midgut epithelial and salivary gland acinar cells of vector ticks during successful transmission. The results support the hypothesis that A. marginale gene expression is regulated by the specific host environment and, in a broader context, that the core genome evolved in the arthropod vector with differential regulation, allowing adaptation to mammalian hosts. Furthermore, the confirmation of the in situ expression of candidates identified in ISE6 cell lines indicates that this approach may be widely applicable to bacteria in the genera Anaplasma and Ehrlichia, removing a major technical impediment to the identification of new targets for vaccine and chemotherapeutic blocking of transmission.

Coxiella burnetii infection of a Steller sea lion (Eumetopias jubatus) found in Washington State.

A pregnant sea lion stranded in the State of Washington was found to have placentitis caused by a unique strain of Coxiella burnetii. This is the first description of coxiellosis in a sea lion and suggests that exposure to sea lions may be a risk factor for contracting Q fever.

Outbreak of malignant catarrhal fever among cattle associated with a state livestock exhibition.

CASE DESCRIPTION: Severe disease and death were identified in cattle exhibited at a state fair that were naturally infected with ovine herpesvirus type 2 (OvHV-2). CLINICAL FINDINGS: Most affected cattle had anorexia, signs of depression, diarrhea, fever, and respiratory distress ultimately leading to death. Mean duration of clinical signs prior to death was 6 days (range, 1 to 26 days). Mean number of days between apparent exposure and death was 71 days (range, 46 to 139 days). TREATMENT AND OUTCOMES: 19 of 132 cattle cohoused in 1 barn died of malignant catarrhal fever (MCF). The diagnosis of sheep-associated MCF was confirmed on the basis of results of an OvHV-2-specific PCR assay performed on tissue samples obtained from affected cattle. The disease was associated but not significantly with distance from the center of the barn and was not associated with distance from the center of the sheep pens. CLINICAL RELEVANCE: Outbreaks of MCF in cattle are unusual, particularly in association with livestock exhibitions. Because the clinical signs may be similar to those of some transboundary diseases, cases of MCF should be reported and investigated. Findings for this outbreak provided evidence to suggest that fair boards and veterinarians should reexamine biosecurity recommendations for livestock exhibitions.

Evaluation of an arthroscopic approach for transection of the equine collateral sesamoidean ligament.

OBJECTIVE: To evaluate: (1) an arthroscopic technique for transection of the collateral sesamoidean ligament (CSL); and (2) the healing response using magnetic resonance (MR) and microscopic examination. STUDY DESIGN: Experimental study. ANIMALS: Adult horses (n=6). METHODS: Six sound horses with normal front foot radiographic and MR examinations were used. Lameness examination was performed before surgery and monthly for 12 months. Front foot radiography was performed at 180 and 360 days after surgery. Front foot MR was performed before, and at 7, 90, 180, and 360 days after surgery. Arthroscopic CSL desmotomy was performed on 1 forelimb. Gross and microscopic examination was performed on the CSL from both forelimbs at 360 days after surgery. Lameness scores were compared over time using the nonparametric Friedman's test for paired groups. CSL measurements were compared using paired t-tests with a 2-tailed significance level of P<.05. RESULTS: Radiographs remained normal throughout study period. Surgery resulted in lameness on the operated limb for up to 2 months, after which all horses returned to soundness. CSL transection was confirmed during arthroscopy and with MR examination 7 days after surgery. Gross and microscopic evaluation confirmed ligament healing. CONCLUSIONS: CSL desmotomy resulted in short-term lameness after surgery followed by healing of the CSL confirmed by gross and microscopic analysis.

Interlaboratory comparison of heavy metal testing in animal diagnostic specimens and feed using inductively coupled plasma-mass spectrometry.

We compared inductively coupled plasma-mass spectrometry (ICP-MS) test results for the analysis of heavy metals (As, Ba, Cd, Hg, Pb, and Se) in pet foods and routine veterinary diagnostic specimens using intralaboratory and interlaboratory comparisons. Four laboratories, 1 principal laboratory and 3 collaborating laboratories, conducted instrument comparison (limit of detection [LOD], limit of quantification [LOQ], and linear dynamic range [LDR] on 24 data sets), in-house method comparison (accuracy and precision on 120 data sets), and interlaboratory comparison (reproducibility on 528 data sets using Horwitz equation analysis). Matrices tested included 2 types of pet food jerky treats (chicken and sweet potato), bovine blood, and bovine liver and kidney. The instrument comparison study confirmed that ICP-MS provided the sensitivity necessary for the analysis of all heavy metals tested at concentrations below the level of concern for routine diagnostic testing. The "in-house" method comparison samples, spiked at low (0.04 µg/g), medium (0.4 µg/g), and high (8.0 µg/g; note: the high validation level spike for mercury was 2 µg/g) concentration levels, indicated that ICP-MS can meet U.S. FDA acceptance criteria for both accuracy (90-105% recovery) and precision (< 6% coefficient of variation). The interlaboratory comparison studies showed that ICP-MS is a reproducible method for the analysis of heavy metals (HorRat value of 0.5-2.0) except for mercury in one laboratory, which used a different sample preparation method (open block rather than microwave digestion). Overall, our study showed that ICP-MS is a reproducible method for the analysis of heavy metals in spite of minor differences in methodology.

Quantitative differences in salivary pathogen load during tick transmission underlie strain-specific variation in transmission efficiency of Anaplasma marginale.

The relative fitness of arthropod-borne pathogens within the vector can be a major determinant of pathogen prevalence within the mammalian host population. Strains of the tick-borne rickettsia Anaplasma marginale differ markedly in transmission efficiency, with a consequent impact on pathogen strain structure. We have identified two A. marginale strains with significant differences in the transmission phenotype that is effected following infection of the salivary gland. We have proposed competing hypotheses to explain the phenotypes: (i) both strains are secreted equally, but there is an intrinsic difference in infectivity for the mammalian host, or (ii) one strain is secreted at a significantly higher level and thus represents delivery of a greater pathogen dose. Quantitative analysis of pathogen replication and secretion revealed that the high-efficiency St. Maries strain replicated to a 10-fold-higher titer and that a significantly greater percentage of infected ticks secreted A. marginale into the saliva and did so at a significantly higher level than for the low-efficiency Israel vaccine strain. Furthermore, the transmission phenotype of the vaccine strain could be restored to that of the St. Maries strain simply by increasing the delivered pathogen dose, either by direct inoculation of salivary gland organisms or by increasing the number of ticks during transmission feeding. We identified morphological differences in the colonization of each strain within the salivary glands and propose that these reflect strain-specific differences in replication and secretion pathways linked to the vector-pathogen interaction.

Limited transcriptional response of ovine microglia to prion accumulation.

The conversion of normal cellular prion protein to disease-associated prion protein (PrP(Sc)) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion conversion and the impact of PrP(Sc) accumulation on cellular biology are not fully understood. To further define the molecular changes associated with PrP(Sc) accumulation in cultured cells, the transcriptional profile of PrP(Sc)-accumulating primary ovine microglia was compared to the profile of PrP(Sc)-lacking microglia using the Affymetrix Bovine Genome Array. The experimental design included three biological replicates, each with three technical replicates, and samples that were collected at the point of near maximal PrP(Sc) accumulation levels as measured by ELISA. The array analysis revealed only 19 upregulated genes and 30 downregulated genes in PrP(Sc)-accumulating microglia. The results support the hypothesis that chronic PrP(Sc) accumulation in cultured microglia results in a limited transcriptional response.

Small-ruminant lentivirus enhances PrPSc accumulation in cultured sheep microglial cells.

Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C) [C superscript stands for cellular]) to disease-associated prion protein (PrP(Sc) [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrP(Sc), sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrP(Sc) accumulation over time. The accumulated PrP(Sc) demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrP(Sc) demonstrated an approximately twofold increase in PrP(Sc) accumulation compared to that of primary microglia infected with PrP(Sc) alone. The results demonstrate the in vitro utility of PrP(Sc)-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

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.

Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories.

This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.