Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Madin-Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75-31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5-7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-ß, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Lymphoproliferative Disease Virus in Wild Turkeys (Meleagris gallopavo) from Manitoba and Quebec, Canada.

This study describes the first recognized clinical case of lymphoproliferative disease virus (LPDV) in Canada and extends the range of LPDV in Canada through its detection in Manitoba and Quebec. We assessed the prevalence of LPDV in eastern wild turkeys (Meleagris gallopavo silvestris) with the use of whole, clotted blood from live birds in Manitoba (n = 65) and tissue samples collected postmortem in Quebec (n = 4). We tested for LPDV proviral DNA through PCR amplification and sequencing of a portion of the gag (p31) gene. Samples were also tested for reticuloendotheliosis virus (REV) by PCR. Twenty-four birds (34.8%) were positive for LPDV, including all diagnostic cases. One bird (1.4%) from Quebec had gross and microscopic lesions consistent with LPDV. Two turkeys (2.9%) were REV positive, one (1.4%) of which was co-infected with LPDV. Phylogenetic analysis of LPDV strains from Quebec and Manitoba grouped with previously sequenced samples from Ontario and publicly available sequences from a North American lineage. This study contributes valuable information toward ongoing surveillance and monitoring of LPDV in North America.

Virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo) de Manitoba y Quebec, en Canadá. Este estudio describe el primer caso clínico reconocido del virus de la enfermedad linfoproliferativa (LPDV) en Canadá y extiende el rango de detección de este virus a través de su detección en Manitoba y Quebec. Se evaluó la prevalencia del virus de la enfermedad linfoproliferativa en pavos silvestres (Meleagris gallopavo silvestris) de la parte oriental, mediante el uso de sangre coagulada de aves vivas en Manitoba (n = 65) y de muestras de tejidos recolectadas postmortem en Quebec (n = 4). Se analizó el ADN proviral del virus de la enfermedad linfoproliferativa del pavo a través de la amplificación por PCR y la secuenciación de una parte del gene gag (p31). Las muestras también se analizaron para detectar el virus de la reticuloendoteliosis (REV) mediante PCR. Veinticuatro aves (34.8%) resultaron positivas para la presencia del virus de la enfermedad linfoproliferativa, incluyendo todos los casos diagnósticos. Un ave (1.4%) de Quebec tenía lesiones macroscópicas y microscópicas compatibles con este virus. Dos pavos (2.9%) fueron positivos a la presencia del virus de la reticuloendoteliosis, uno (1.4%) de los cuales se co-infectó con el virus de la enfermedad linfoproliferativa. El análisis filogenético de cepas del virus de la enfermedad linfoproliferativa de Quebec y Manitoba agrupó a estos virus con muestras previamente secuenciadas de Ontario y secuencias disponibles públicamente de un linaje de América del Norte. Este estudio aporta información valiosa para la vigilancia y el monitoreo continuos del virus de la enfermedad linfoproliferativa en América del Norte.

Challenging diagnosis--icterus associated with a single perforating duodenal ulcer after long-term nonsteroidal antiinflammatory drug administration in a dog.

A dog developed icterus, vomiting, and anorexia 2 wk after orthopedic surgery and treatment with meloxicam for approximately 1 y. Exploratory laparotomy revealed a single perforated duodenal ulcer. The most likely cause of the hyperbilirubinemia was intrahepatic cholestasis resulting from peritonitis associated with the perforation.