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.

Genomic characterization of antimicrobial resistance in 61 aquatic bacterial isolates.

Antimicrobial resistance (AMR) in pathogens important to aquatic animal health is of increasing concern but vastly understudied. Antimicrobial therapy is used to both treat and prevent bacterial disease in fish and is critical for a viable aquaculture industry and for maintenance of wild fish populations. Unfortunately, phenotypic antimicrobial susceptibility testing is technically difficult for bacteria recovered from aquatic animal hosts resulting in challenges in resistance monitoring using traditional methods. Whole-genome sequencing provides an appealing methodology for investigation of putative resistance. As part of the ongoing efforts of the FDA CVM Vet-LIRN to monitor AMR, source laboratories cultured and preliminarily identified pathogenic bacteria isolated from various fish species collected in 2019 from across the United States. Sixty-one bacterial isolates were evaluated using whole-genome sequencing. We present here the assembled draft genomes, AMR genes, predicted resistance phenotypes, and virulence factors of the 61 isolates and discuss concurrence of the identifications made by source laboratories using matrix-assisted laser desorption/time-of-flight mass spectrometry.

Geographic Distribution and Neuropathology of Elaeophora schneideri in Moose (Alces alces shirasi) in Idaho, USA.

Elaeophorosis, infection by the filarial worm Elaeophora schneideri, is a parasitic disease of wild ungulates in North America; however, our understanding of the relevance of E. schneideri to moose (Alces alces) morbidity and mortality is incomplete. Between March 2020 and July 2022, necropsy and histopathology were performed on 61 Shiras moose (Alces alces shirasi) in Idaho, US. Among the 41 adults (greater than 1 yr old), 21 moose were from northern Idaho, and 20 were from southeastern Idaho. Elaeophorosis was diagnosed in 24% (10 of 41). All 10 infected moose were from southeastern Idaho; none of the 21 moose from northern Idaho were infected. No juvenile moose (nine from northern and 11 from southeastern Idaho) were infected. Microfilariae were detected histologically in 9 of 10 infected moose, most consistently in brain tissue associated with lesions indicative of ischemic injury to the neuroparenchyma attributed to occlusion of arterioles and capillaries by microfilariae or fibrin thrombi, including edema, necrosis, and glial nodules. Microfilariae found in other tissues of the head, including the eye, tongue, and pinnae of some animals, as well as in lung, heart, liver, and kidney, typically were associated with inflammation. Three of the 10 infected moose had cropped ears attributed to elaeophorosis, and four exhibited abnormal behavior, which may have been due to neuropathology associated with E. schneideri microfilariae in the brain.

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.

Utility of postmortem bacterial culture of abdominal organs at autopsy of young calves.

Postmortem bacterial culture is controversial in human medicine, and veterinary-specific research in this area is lacking. To address this knowledge gap, we cultured liver, kidney, and spleen individually from on-farm calf mortalities to determine the number of bacterial species present, concordance between organ cultures, and agreement with gross and histologic findings. We hypothesized that the spleen, a filtering organ, would be the most useful organ with the least amount of postmortem contamination given that it does not have a direct conduit to a bacterial population. Fresh liver, kidney, and spleen were collected for culture from 30 calves 5-28-d-old with various causes of mortality. Bacterial growth of ≥2 species was observed in ~48% of cultures, with Escherichia coli and Streptococcus spp. being most frequent. One bacterial species was present in 20% of cultures, with E. coli predominating. No growth was observed in ~32% of cultures. In 43% of cases, there was agreement in the culture results for all 3 organs; however, the majority were mixed bacterial growth. The best agreement was observed when there were no gross and/or histologic septic lesions in target organs and no bacterial growth on culture. The spleen was not helpful in determining bacterial significance in comparison to kidney or liver.

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.

3D Printing in alloy design to improve biocompatibility in metallic implants.

3D Printing (3DP) or additive manufacturing (AM) enables parts with complex shapes, design flexibility, and customization opportunities for defect specific patient-matched implants. 3DP or AM also offers a design platform that can be used to innovate novel alloys for application-specific compositional modifications. In medical applications, the biological response from a host tissue depends on a biomaterial's structural and compositional properties in the physiological environment. Application of 3DP can pave the way towards manufacturing innovative metallic implants, combining structural variations at different length scales and tailored compositions designed for specific biological responses. This study shows how 3DP can be used to design metallic alloys for orthopedic and dental applications with improved biocompatibility using in vitro and in vivo studies. Titanium (Ti) and its alloys are used extensively in biomedical devices due to excellent fatigue and corrosion resistance and good strength to weight ratio. However, Ti alloys' in vivo biological response is poor due to its bioinert surface. Different coatings and surface modification techniques are currently being used to improve the biocompatibility of Ti implants. We focused our efforts on improving Ti's biocompatibility via a combination of tantalum (Ta) chemistry in Ti, the addition of designed micro-porosity, and nanoscale surface modification to enhance both in vitro cytocompatibility and early stage in vivo osseointegration, which was studied in rat and rabbit distal femur models.

Algal Lymphadenitis in a Dog Caused by Scenedesmus Species.

A 6-year-old, spayed female Labrador/Weimaraner cross-breed dog that had previously lived in Arizona presented in Montana for an annual examination with an incidentally enlarged popliteal lymph node, which was subsequently biopsied. Histologically, the lymph node was expanded by eosinophil-rich granulomas with both extracellular and intrahistiocytic green algae. These algae had intracytoplasmic, birefringent, and refractile granules; readily formed 2 to 3 mm green colonies on Columbia blood agar medium; and ultrastructurally had a multilayered cell wall and intracytoplasmic chloroplasts. Amplified product from the internal transcribed spacer and D1/D2 regions of the 28S ribosomal RNA gene had high sequence identity to Scenedesmus sp. Despite similar infection in the retropharyngeal lymph node 1 year later, the animal remained otherwise healthy with no clinical signs. To the authors' knowledge, this is the first case of Scenedesmus species infection in a dog and is a differential diagnosis for Coccidioides immitis.

A Novel Neorickettsial Infection in 3 Dogs in the Pacific Northwest.

The genus Neorickettsia includes obligate, intracellular bacteria responsible for diseases including Potomac horse fever caused by Neorickettsia risticii and salmon poisoning disease (SPD) caused by Neorickettsia helminthoeca. The Stellanchasmus falcatus (SF) agent is a member of this genus previously associated only with mild clinical signs in dogs. Between 2013 and 2016, 3 dogs in Washington State (USA) presented with disease suggestive of SPD, but N. helminthoeca was not detected by molecular techniques. Clinical signs included depression, anorexia, and diarrhea. Cytologic examination of aspirates supported a diagnosis of granulomatous lymphadenitis with organisms suggestive of Neorickettsia. Dogs either died or were humanely euthanized due to poor response to therapy. Necropsy findings included lymphadenomegaly and hepatomegaly. Histopathology identified granulomatous and lymphoplasmacytic splenitis, lymphadenitis, enteritis, and hepatitis with extensive necrosis. Neorickettsia DNA was detected using genus-specific primers and direct sequencing showed 100% sequence identity to the SF agent in all 3 dogs. This is the first clinicopathologic description of severe disease in dogs attributed to the SF agent. These findings may suggest the emergence of a novel neorickettsial disease in the Pacific Northwest.

Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis.

Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned cats with various forms of feline infectious peritonitis (FIP). Methods Twenty cats from 3.3-82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study. Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However, disease signs recurred 1-7 weeks after primary treatment and relapses and new cases were ultimately treated for a minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1-7 weeks of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged 3.3-4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment and at the time of writing for 5-14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12 weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat with only mesenteric lymph node involvement that went into remission after three relapses that required progressively longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption of permanent teeth in cats treated before 16-18 weeks of age. Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has opened the door to targeted antiviral drug therapy.

Central and peripheral reservoirs of feline immunodeficiency virus in cats: a review.

Infection with feline immunodeficiency virus (FIV), a lentivirus similar to human immunodeficiency virus (HIV), results in lifelong viral persistence and progressive immunopathology in the cat. FIV has the ability to infect and produce infectious virus in a number of different cell types. FIV provirus can also be maintained in a replication-competent but transcriptionally quiescent state, facilitating viral persistence over time. Immediately after the initial infection, FIV infection quickly disseminates to many anatomical compartments within the host including lymphoid organs, gastrointestinal tract and brain. Collectively, the anatomic and cellular compartments that harbour FIV provirus constitute the viral reservoir and contain foci of both ongoing viral replication and transcriptionally restricted virus that may persist over time. The relative importance of the different phenotypes observed for infected cells, anatomic compartment, replication status and size of the reservoir represent crucial areas of investigation for developing effective viral suppression and eradication therapies. In this review, we discuss what is currently known about FIV reservoirs, and emphasize the utility of the FIV-infected cat as a model for the HIV-infected human.

Levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection.

Twenty specific pathogen free cats were experimentally infected with a virulent cat-passaged type I field strain of FIPV. Eighteen cats succumbed within 2-4 weeks to effusive abdominal FIP, one survived for 6 weeks, and one seroconverted without outward signs of disease. A profound drop in the absolute count of blood lymphocytes occurred around 2 weeks post-infection (p.i.) in cats with rapid disease, while the decrease was delayed in the one cat that survived for 6 weeks. The absolute lymphocyte count of the surviving cat remained within normal range. Serum antibodies as measured by indirect immunofluorescence appeared after 2 weeks p.i. and correlated with the onset of disease signs. Viral genomic RNA was either not detectable by reverse transcription quantitative real-time PCR (RT-qPCR) or detectable only at very low levels in terminal tissues not involved directly in the infection, including hepatic and renal parenchyma, cardiac muscle, lung or popliteal lymph node. High tissue virus loads were measured in severely affected tissues such as the omentum, mesenteric lymph nodes and spleen. High levels of viral genomic RNA were also detected in whole ascitic fluid, with the cellular fraction containing 10-1000 times more viral RNA than the supernatant. Replicating virus was strongly associated with macrophages by immunohistochemistry. Virus was usually detected at relatively low levels in feces and there was no evidence of enterocyte infection. Viral genomic RNA was not detected at the level of test sensitivity in whole blood, plasma, or the white cell fraction in terminal samples from the 19 cats that succumbed or in the single survivor. These studies reconfirmed the effect of lymphopenia on disease outcome. FIPV genomic RNA was also found to be highly macrophage associated within diseased tissues and effusions as determined by RT-qPCR and immunohistochemistry but was not present in blood.