Characterization of a Novel Reassortant Epizootic Hemorrhagic Disease Virus Serotype 6 Strain Isolated from Diseased White-Tailed Deer (Odocoileus virginianus) on a Florida Farm.

We report an outbreak of a novel reassortant epizootic hemorrhagic disease virus serotype 6 (EHDV-6) in white-tailed deer (WTD) on a Florida farm in 2019. At necropsy, most animals exhibited hemorrhagic lesions in the lung and heart, and congestion in the lung, liver, and spleen. Histopathology revealed multi-organ hemorrhage and congestion, and renal tubular necrosis. Tissues were screened by RT-qPCR and all animals tested positive for EHDV. Tissues were processed for virus isolation and next-generation sequencing was performed on cDNA libraries generated from the RNA extracts of cultures displaying cytopathic effects. Six isolates yielded nearly identical complete genome sequences of a novel U.S. EHDV-6 strain. Genetic and phylogenetic analyses revealed the novel strain to be most closely related to a reassortant EHDV-6 strain isolated from cattle in Trinidad and both strains received segment 4 from an Australian EHDV-2 strain. The novel U.S. EHDV-6 strain is unique in that it acquired segment 8 from an Australian EHDV-8 strain. An RNAscope® in situ hybridization assay was developed against the novel U.S. EHDV-6 strain and labeling was detected within lesions of the heart, kidney, liver, and lung. These data support the novel U.S. reassortant EHDV-6 strain as the cause of disease in the farmed WTD.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

BRCA2-DSS1 interaction is dispensable for RAD51 recruitment at replication-induced and meiotic DNA double strand breaks.

The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.

Gastrointestinal entamoebiasis in captive anurans in North America.

Infections with Entamoeba spp. are recognized as a cause of clinical disease in many species including humans and reptiles; however, cases in amphibians are under-reported. Investigation of a mortality event among a captive population of Cranwell's horned frogs Ceratophrys cranwelli at a production facility in Florida, USA, revealed that deaths were due to the newly described Entamoeba species CT1. Infection caused severe necroulcerative gastroenterocolitis with a predilection for the colon. To date, this Entamoeba species has only been described in invasive cane toads Rhinella marina in Australia. Retrospective screening of archived anuran cases from a zoological pathology service identified 8 cases from captive populations that had histological evidence of gastrointestinal entamoebiasis. Molecular characterization was positive in 3 cases. Two cases, 1 in a Puerto Rican crested toad Peltophryne lemur and 1 in an Amazon milk frog Trachycephalus resinifictrix, showed 100% homology to E. ranarum and 1 case in a White's tree frog Litoria caerulea showed 100% homology to Entamoeba sp. CT1. This is the first report of novel Entamoeba sp. CT1 being associated with clinical disease in anurans within North America and also the first report of this Entamoeba species causing disease within managed collections as far back as 2003.

Shell Lesions Associated With Emydomyces testavorans Infection in Freshwater Aquatic Turtles.

A newly described onygenalean fungus, Emydomyces testavorans, has been isolated from ulcerative shell and skin lesions of freshwater aquatic chelonians. To investigate the shell lesions associated with infection and determine if any lesional features were unique to E. testavorans, tissues from turtles housed in zoological institutions (n = 45) in the United States and free-living turtles (n = 5) submitted for diagnostic biopsy or necropsy were examined. Free-living turtles were from geographically distinct habitats in Florida (n = 1) and Washington (n = 4) at the time of sampling. Histologic shell sections were evaluated for the presence or absence of specific lesional features. Infection with E. testavorans was evaluated in all cases by screening GMS (Grocott-Gomori's methenamine silver)-stained histologic sections for the presence of morphologically consistent fungi and by quantitative PCR (polymerase chain reaction) on representative frozen tissue or formalin-fixed paraffin-embedded sections. Additionally, culture was performed for 15 cases with available fresh/frozen tissue. In total, there were 17 PCR-confirmed E. testavorans cases, 29 cases with morphologically consistent fungi on GMS-stained sections, and 21 cases of shell lesions without histologic or molecular evidence of E. testavorans infection. Epithelial inclusion cysts, defined as cystic structures within the dermis lined by keratinized stratified squamous epithelium and containing necrotic bone and keratin debris, were significantly (P < .01) associated with E. testavorans infection. Other significantly associated shell lesions included squamous metaplasia, hyperkeratosis, inflammation, and osteonecrosis (P < .05). This study identified characteristic shell lesions associated with E. testavorans infection. Further studies to prove causality are needed.

Pulmonary Sarcomatoid Carcinoma Associated with Arterial Thromboembolism in a Cat.

A 14-year-old, neutered male domestic shorthair cat presented for acute monoparesis with physical exam findings and biochemical data supportive of a distal arterial thromboembolism. Thoracic radiographs revealed an alveolar pattern in the right middle lung lobe and multifocal nodules in other lung lobes. A pulmonary mass was found on necropsy, which was composed of both carcinomatous and sarcomatous components, confirmed with cytokeratin and vimentin immunohistochemistry. Using the World Health Organization classification scheme for mixed pulmonary tumors, this tumor would be characterized as a pleomorphic squamous cell carcinoma under the umbrella term of pulmonary sarcomatoid carcinoma. The World Health Organization classification of mixed pulmonary tumors and its application to previously reported mixed pulmonary tumors in companion animals is discussed. This is the first reported case of this tumor type in a cat, as well as the first report of this tumor type associated with an arterial thromboembolism in any veterinary species.

Cytologic, histologic, microbiologic, and electron microscopic characterization of a canine Prototheca wickerhamii infection.

An adult dog was presented for chronic cough and a recent development of ulcerated, erythematous nares with nasal discharge. Cytology of enlarged peripheral lymph nodes revealed many intracellular and extracellular organisms. These round or rarely oval organisms measured approximately 5-9 µm in diameter and frequently contained several globular structures, ranging from deeply basophilic to magenta. A thin, clear halo was present. Smaller 1-2 µm, magenta forms were also observed. Fungal culture yielded small, wet, raised, irregularly shaped, white to pale tan colonies. Microbiologic staining of cultured material revealed features suggestive of algae. Histopathology of the lymph nodes revealed marked granulomatous inflammation with intralesional algal organisms suggestive of Prototheca. Electron microscopic findings were also consistent with protothecosis. Polymerase chain reaction, followed by direct DNA sequencing, identified the organism as Prototheca wickerhamii. A brief literature review discussing protothecosis in veterinary medicine is included.

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability.

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment.

The Discovery of 2-Aminobenzimidazoles That Sensitize Mycobacterium smegmatis and M.†tuberculosis to ß-Lactam Antibiotics in a Pattern Distinct from ß-Lactamase Inhibitors.

A library of 2-aminobenzimidazole derivatives was screened for the ability to suppress ß-lactam resistance in Mycobacterium smegmatis. Several non-bactericidal compounds were identified that reversed intrinsic resistance to ß-lactam antibiotics in a manner distinct from ß-lactamase inhibitors. Activity also translates to M. tuberculosis, with a lead compound from this study potently suppressing carbenicillin resistance in multiple M. tuberculosis strains (including multidrug-resistant strains). Preliminary mechanistic studies revealed that the lead compounds act through a mechanism distinct from that of traditional ß-lactamase inhibitors.