Bovine astrovirus and its role in lymphocytic encephalitis in cattle in Ontario, Canada, 1988-2019.

Astroviruses have been found in cattle and other species with encephalitis. Our objective was to determine the frequency of neurotropic bovine astrovirus (BoAstV) in cases of encephalitis in cattle ≥ 4-mo-old. Of 56 cases of idiopathic lymphocytic encephalitis examined retrospectively (1988-2019), fixed brain from 11 cases (19%) tested positive by semi-quantitative RT-PCR for BoAstV CH13/NeuroS1. None of the control cases tested positive, including 32 with other forms of encephalitis and 40 with no neurologic disease. Most astrovirus-positive cases were 1-2-y-old, with a range of 7 mo to 7 y, and affected both beef and dairy breeds with wide geographic distribution. BoAstV-positive cases had acute onset of neurologic signs of 12 h to 7 d before death or euthanasia. Affected cattle had lymphocytic inflammation throughout the brain including cerebrum, thalamus, midbrain, cerebellum, medulla oblongata, and spinal cord, and affecting gray and white matter. Further PCR testing identified a possible cause in 9 of the 45 (20%) remaining idiopathic cases of lymphocytic encephalitis, including eastern equine encephalitis virus, Listeria monocytogenes, bovine viral diarrhea virus, bovine alphaherpesvirus 1, and ovine gammaherpesvirus 2 (malignant catarrhal fever); we found no cases of infection by West Nile virus, rabies virus, or Chlamydia spp. No cause was identified in 36 of 56 (64%) cases of lymphocytic encephalitis. We frequently identified neurotropic BoAstV in cases of lymphocytic encephalitis that had no previously identified cause. Neurotropic BoAstV infections had gone undetected for decades, but the frequency of BoAstV infections has not increased among contemporary cases.

Canine distemper virus infection of vaccinal origin in a 14-week-old puppy.

The body of a 14-wk-old puppy (Canis familiaris) was submitted to the Animal Health Laboratory, University of Guelph, Ontario for postmortem examination following a history of intermittent anorexia and lethargy progressing to pyrexia, pruritic skin rash, mucoid nasal discharge, decreased mentation, dysphagia, muscle twitches, and focal seizures. Gross examination revealed rhinitis and pulmonary edema. Histologically, there was fibrinonecrotizing bronchopneumonia, tracheitis, and neutrophilic and lymphohistiocytic rhinitis; rarely within the cortical gray and white matter of the brain were small clusters of glial cells, with rare individual neutrophils in the choroid plexus. Although canine distemper was suspected, none of the usual supportive histologic lesions of distinct syncytial cells, viral inclusion bodies, or demyelinating leukoencephalitis were observed. Lung and brain tissues were PCR-positive for canine distemper virus (CDV), and CDV was detected immunohistochemically in the brain. The agent from the PCR-positive sample from the brain was genotyped and was a 99.9% match to the CDV Rockborn strain, indicating that the disease agent in our case was vaccinal in origin. Our unusual case highlights the possibility of reversion to virulence in a modified-live virus vaccine, and the occurrence of a disease in the absence of a full complement of the usual and compatible histologic lesions.

Development of a multivalent adjuvanted inactivated vaccine against variant arthrotropic avian reoviruses.

Variant avian reoviruses (ARVs) are economically important emerging pathogens of poultry, which mainly affect young broiler chickens and cause significant production losses. Currently, there are no effective commercial vaccines available for control and prevention of emerging variant ARVs. In this study, monovalent inactivated adjuvated (20% Emulsigen D) broiler breeder vaccines containing antigens from ARV genotype cluster (C) group -2, -4, -5, or -6, and a multivalent vaccine containing antigens from all the four indicated genotypic cluster groups were developed and evaluated for their efficacy in protecting broiler progenies against homologous or heterologous ARV challenge. The use of monovalent or multivalent inactivated vaccines in a prime-boost immunization strategy induced the production of ARV specific antibodies in broiler breeders. The maternal antibodies were effectively transferred to broiler progenies. Broiler progenies obtained from immunized breeders demonstrated milder clinical symptoms and reduced gross and histopathological lesions after homologous ARV challenge. More severe gross and histological lesions were observed in challenged progenies from unvaccinated broiler breeders. However, cross protection was not observed when either of the monovalent-vaccine groups were challenged with a heterologous virus. In addition, the progenies from the unvaccinated ARV challenged control or heterologous ARV challenged vaccinated groups had significantly reduced body weight gain (p < 0.01) than the unchallenged-control, challenged-multivalent, or homologous ARV-challenged monovalent vaccine groups. However, homologous ARV challenged progenies in the multivalent or monovalent vaccine groups had similar body weight gain as the control unchallenged group with significantly reduced viral load (p < 0.01) in the gastrocnemius tendon tissue. This study indicates that broad-spectrum protection of broiler progenies from variant ARV infections is feasible through the development of multivalent vaccines after proper characterization, selection and incorporation of multiple antigens based on circulating ARV genotypes in targeted regions.

Development of interactive dashboards for monitoring endemic animal pathogens in Ontario, Canada: Ontario interactive animal pathogen dashboards.

The advancement of web-based technologies makes it possible to build user interfaces or web pages that present and summarize complex data in easy-to-read graphical formats that emphasize key information. Taking advantage of this technologic progress, we addressed the need for real-time visualizations of trends for major pathogens in the largest livestock industries in Ontario: poultry, swine, and cattle. These visualizations were built using test data from the laboratory information management system of the Animal Health Laboratory at the University of Guelph, a large veterinary diagnostic laboratory in Ontario. The data were processed using R software and used to construct interactive and dynamic visualizations using Tableau Desktop v.2021.4 (Tableau Software). We designed 12 dashboards: in chickens-influenza A virus, fowl adenovirus, infectious bronchitis virus, and infectious laryngotracheitis virus; in turkeys-influenza A virus; in swine, influenza A virus, rotavirus, and porcine reproductive and respiratory syndrome virus; in cattle-bovine viral diarrhea virus, Mycoplasma bovis, Salmonella Dublin in individual samples, and Salmonella Dublin in bulk tank milk samples. Data for each pathogen are presented in 2 dashboards. One shows the data of the last 10 y (general view) and the other the data of the last 3 y, but in more detail (comprehensive view). Information on gaining access to all dashboards is available at https://iapd.lsd.uoguelph.ca/. The visualizations provide near-real-time access to aggregated assay results for selected pathogens for veterinarians, animal health regulatory agencies, researchers, and other users who are interested in livestock pathogen surveillance.

CANINE DISTEMPER VIRUS ECOLOGY: INSIGHTS FROM A LONGITUDINAL SEROLOGIC STUDY IN WILD RACCOONS (PROCYON LOTOR).

Increasing reports of canine distemper virus (CDV) in a variety of hosts, and changing CDV dynamics, have led to renewed interest in the ecology of CDV infections in wildlife. Longitudinal serologic studies provide insights into intrapopulation and intraindividual pathogen dynamics, but few studies in wildlife have been conducted. We used data from 235 raccoons (Procyon lotor) captured on more than one occasion between May 2011 and November 2013 to investigate CDV dynamics in Ontario, Canada. Using mixed multivariable logistic regression, we found that juvenile raccoons were more likely to be seronegative from August to November than from May to July. Using paired titers from CDV-exposed individual raccoons, we determined that the winter breeding season, when there is high intraspecific contact and an increase in susceptible juveniles, may be a period of high risk for CDV exposure. Interestingly, CDV seropositive adult raccoons had nondetectable titers ranging from 1 mo to 1 yr later. Based on our preliminary investigation using two different statistical approaches, CDV exposure was associated with a decrease in parvovirus titer. This result raises important questions about whether virus-induced immune amnesia occurs after CDV exposure, which has been described for measles virus, a closely related pathogen. Overall, our results provide significant insights into CDV dynamics. Further research is needed to investigate whether CDV-induced immune amnesia occurs in raccoons and to determine the potential impacts of a reduced population immunity that may occur secondary to CDV exposure, particularly as it relates to rabies control efforts.

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.

Bronchopneumonia with interstitial pneumonia in beef feedlot cattle: Characterization and laboratory investigation.

Bronchopneumonia with interstitial pneumonia (BIP) has been considered a variant of acute interstitial pneumonia (AIP) rather than a distinct disease. This study compared 18 BIP, 24 bronchopneumonia (BP), and 13 AIP cases in feedlot beef cattle. Grossly, BIP cases typically had cranioventral lung lesions of similar morphology and extent as BP cases, but the caudodorsal lung appeared overinflated, bulged on section, and had interlobular edema and emphysema. Gross diagnosis of BIP had 83% sensitivity and 73% specificity relative to histopathology. Histologic lesions of BIP in cranioventral areas were of chronic BP, while caudodorsal lesions included alveolar and bronchiolar damage and inflammation, interstitial hypercellularity, and multifocal hemorrhages. In BIP cases, cranioventral lung lesions were more chronic than caudodorsal lesions. Histologic scores and microbiology data were comparable in cranioventral lung of BIP versus BP cases and caudodorsal lung of BIP versus AIP cases, with differences reflecting a more chronic disease involving less virulent bacteria in BIP versus BP. Mycoplasma bovis infection was similarly frequent among groups, and a viral cause of BIP was not identified. Lesion morphology and similar blood cytokine concentrations among groups argued against sepsis as a cause of lung injury. Surfactant dysfunction was identified in BIP and BP, and was only partially the result of protein exudation. These and other findings establish BIP as a distinct condition in which chronic cranioventral BP precedes acute caudodorsal interstitial lung disease, supporting a role of chronic inflammation in heightened sensitivity to 3-methylindole or another lung toxicant.

Assessment of seasonality of rotavirus PCR detection in swine from Ontario and Quebec between 2016-2020 using submissions to a diagnostic laboratory.

The goal of this study was to determine if seasonality of rotavirus A, B, and C infection is present in Ontario and Quebec swine herds by investigating submissions to a diagnostic laboratory. Samples (N = 1557) within 755 case submissions from Canadian swine herds between 2016 and 2020 were tested for rotaviruses A, B, and C using a real-time polymerase-chain reaction assay and described. Data from Ontario and Quebec were additionally analyzed using boxplots, 6-week rolling averages, time-series decomposition, and negative binomial regression models. Percentage positivity of submissions for rotaviruses A, B, and C were discovered to be highest in nursery/weaner (n = 100, 94.0%, 60.0%, 80.0%) and grower/finisher (n = 13, 84.6%, 46.2%, 61.5%) pigs and lowest in gilt/sow (n = 45, 68.9%, 20.0%, 40.0%) and suckling pigs (n = 102, 67.6%, 10.8%, 38.2%), respectively. The most common combination of rotavirus at the sample level was AC (n = 252, 17%) and ABC (n = 175, 23.2%) at the submission level. Percent positivity for rotavirus A, B, and C across all Canadian provinces included in the study were 69.9%, 32.6%, and 53.1%, respectively. Descriptive analysis suggested little to no evidence of seasonal patterns, although a spike in November was seen in the monthly total submissions and monthly total positive submissions. Statistically, the overall month effect could not be identified as statistically significant (P > 0.05) for any of the evaluated submission counts. Overall, there was no evidence supporting seasonality of rotavirus within Ontario and Quebec swine herds between 2016 and 2020.

Le but de cette étude était de déterminer si la saisonnalité de l'infection à rotavirus A, B et C est présente dans les troupeaux de porcs de l'Ontario et du Québec en examinant les soumissions à un laboratoire de diagnostic. Des échantillons (N = 1557) de 755 cas soumis de troupeaux de porcs canadiens entre 2016 et 2020 ont été testés pour les rotavirus A, B et C à l'aide d'un test de réaction d'amplification en chaîne par polymérase en temps réel et décrits. Les données de l'Ontario et du Québec ont également été analysées à l'aide de diagrammes en boîte, de moyennes mobiles sur 6 semaines, d'une décomposition de séries chronologiques et de modèles de régression binomiale négative. On a découvert que le pourcentage de positivité des soumissions pour les rotavirus A, B et C étaient le plus élevé en pouponnière/sevrage (n = 100, 94,0 %, 60,0 %, 80,0 %) et en croissance/engraissement (n = 13, 84,6 %, 46,2 %, 61,5 %) des porcs et le plus bas chez les cochettes/truies (n = 45, 68,9 %, 20,0 %, 40,0 %) et les porcs à la mamelle (n = 102, 67,6 %, 10,8 %, 38,2 %), respectivement. La combinaison la plus courante de rotavirus au niveau de l'échantillon était AC (n = 252, 17 %) et ABC (n = 175, 23,2 %) au niveau de la soumission. Les pourcentages de positivité pour les rotavirus A, B et C dans toutes les provinces canadiennes incluses dans l'étude étaient de 69,9 %, 32,6 % et 53,1 %, respectivement. L'analyse descriptive a suggéré peu ou pas de preuves de tendances saisonnières, bien qu'un pic en novembre ait été observé dans les soumissions totales mensuelles et les soumissions positives totales mensuelles. Statistiquement, l'effet mensuel global n'a pu être identifié comme statistiquement significatif (P > 0,05) pour aucun des nombres de soumissions évalués. Dans l'ensemble, il n'y avait aucune preuve à l'appui de la saisonnalité du rotavirus dans les troupeaux de porcs de l'Ontario et du Québec entre 2016 et 2020.(Traduit par Docteur Serge Messier).

Genotyping and In Silico Analysis of Delmarva (DMV/1639) Infectious Bronchitis Virus (IBV) Spike 1 (S1) Glycoprotein.

Genetic diversity and evolution of infectious bronchitis virus (IBV) are mainly impacted by mutations in the spike 1 (S1) gene. This study focused on whole genome sequencing of an IBV isolate (IBV/Ck/Can/2558004), which represents strains highly prevalent in Canadian commercial poultry, especially concerning features related to its S1 gene and protein sequences. Based on the phylogeny of the S1 gene, IBV/Ck/Can/2558004 belongs to the GI-17 lineage. According to S1 gene and protein pairwise alignment, IBV/Ck/Can/2558004 had 99.44-99.63% and 98.88-99.25% nucleotide (nt) and deduced amino acid (aa) identities, respectively, with five Canadian Delmarva (DMV/1639) IBVs isolated in 2019, and it also shared 96.63-97.69% and 94.78-97.20% nt and aa similarities with US DMV/1639 IBVs isolated in 2011 and 2019, respectively. Further homology analysis of aa sequences showed the existence of some aa substitutions in the hypervariable regions (HVRs) of the S1 protein of IBV/Ck/Can/2558004 compared to US DMV/1639 isolates; most of these variant aa residues have been subjected to positive selection pressure. Predictive analysis of potential N-glycosylation and phosphorylation motifs showed either loss or acquisition in the S1 glycoprotein of IBV/Ck/Can/2558004 compared to S1 of US DMV/1639 IBV. Furthermore, bioinformatic analysis showed some of the aa changes within the S1 protein of IBV/Ck/Can/2558004 have been predicted to impact the function and structure of the S1 protein, potentially leading to a lower binding affinity of the S1 protein to its relevant ligand (sialic acid). In conclusion, these findings revealed that the DMV/1639 IBV isolates are under continuous evolution among Canadian poultry.

Evaluation of five circulating strains of variant infectious bursal disease virus (varIBDV) for their immunogenicity as broiler breeder vaccines and protective efficacy in neonatal broiler chicks.

The majority of infectious bursal disease virus (IBDV) strains circulating in the broiler chicken industry in Canada are variant strains (varIBDV). Despite high levels of maternally derived antibodies (MtAb), the circulating varIBDVs can establish infection and cause severe immunosuppression in broiler chicks. The objective of this study was to evaluate circulating varIBDVs as broiler breeder vaccine candidates and investigate their protective efficacy against varIBDV challenge in their progeny chicks. Six groups of breeders (20 females/group) were vaccinated with varIBDV strains, SK09, SK10, SK11, SK12, and SK13 or saline at the age of 13 weeks and antibody response was determined by ELISA at 3-7-, and 20- weeks post-vaccination. We also included commercial chicks for the comparison. Results showed that SK-09 is the most antigenic strain, followed by SK-10, SK-12, and SK-13. In contrast, SK-11 showed the lowest antibody response, and over time, antibody titers steadily decreased. Eggs from breeders were collected at 21-week post-vaccination and incubated to produce their respective progenies. The serum antibody titer in day-old chicks showed a successful MtAb transfer. Progeny chicks (n = 40/group) were orally challenged with varIBDV-SK-09 strain at 6 days of age and serum antibody titer (19 d and 35 d of age), bursa to body weight ratio (19 d and 35 d of age), bursal viral load (9 d and 19 d of age) was examined to assess the protection against IBDV. Following the challenge, we found a significant increase in the antibody titers in MtAb-free and commercial vaccine groups than in the varIBDV groups, both at 19 d and 35 d of age. The BBW ratio and viral load data indicated a significant homologous and heterologous protection against varIBDV-SK-09 challenge by SK-09 and SK-10 MtAbs, respectively. Overall, this study demonstrated the feasibility of developing breeder vaccines using circulating varIBDV as candidate vaccine antigens.

Laboratory investigation of cases of fatal bacterial pneumonia in dairy cows.

Objective: Bacterial bronchopneumonia occurs in mature dairy cows but much of the information is extrapolated from knowledge of the disease in calves. The study was prompted by perceptions of an increasing occurrence and a paucity of information on fatal Mannheimia haemolytica pneumonia in dairy cows in Ontario. The study objectives were to describe the seasonality, main pathogens involved, and suggested predisposing factors for cases of fatal bacterial bronchopneumonia in mature dairy cows submitted for postmortem examination to a diagnostic laboratory, and to evaluate if the frequency of such submissions has increased over time. Animals: Mature dairy cows. Procedure: Retrospective study of cases submitted for postmortem examination to a diagnostic laboratory from 2007-2020 that were diagnosed as bacterial bronchopneumonia. Results: Most of the postmortem cases of bacterial bronchopneumonia in dairy cows were submitted from November to February (54% of cases). Mannheimia haemolytica was isolated from lung of 61/101 cases. Viruses were only identified in 8/55 cases tested. A minority (29/92) of bacterial isolates had in vitro resistance to antimicrobials used to treat pneumonia. Frequently suggested predisposing factors included recent introductions or movement of animals, recent or imminent calving, inclement weather, concurrent diseases, and poor ventilation in barns. Conclusion and clinical relevance: This study describes seasonal and annual trends, major pathogens, antimicrobial resistance profiles, and suggested predisposing factors in Ontario dairy cows submitted to a diagnostic laboratory for postmortem investigation of pneumonia and provides insights for understanding why outbreaks occur.

Objectif: La bronchopneumonie bactérienne survient chez les vaches laitières matures, mais une grande partie de l'information est extrapolée à partir de la connaissance de la maladie chez les veaux. L'étude a été motivée par la perception d'une occurrence croissante et d'un manque d'information sur la pneumonie mortelle à Mannheimia haemolytica chez les vaches laitières en Ontario. Les objectifs de l'étude étaient de décrire la saisonnalité, les principaux agents pathogènes impliqués et les facteurs prédisposants suggérés pour les cas de bronchopneumonie bactérienne mortelle chez les vaches laitières matures soumises à un examen post-mortem à un laboratoire de diagnostic, et d'évaluer si la fréquence de telles soumissions a augmenté au fil du temps. Animaux: Vaches laitières matures. Procédure: Étude rétrospective des cas soumis pour examen post-mortem à un laboratoire de diagnostic, entre 2007 et 2020, qui ont été diagnostiqués comme une bronchopneumonie bactérienne. Résultats: La plupart des cas post-mortem de bronchopneumonie bactérienne chez les vaches laitières ont été soumis de novembre à février (54 % des cas). Mannheimia haemolytica a été isolée du poumon de 61/101 cas. Des virus n'ont été identifiés que dans 8/55 cas testés. Une minorité (29/92) d'isolats bactériens présentaient une résistance in vitro aux antimicrobiens utilisés pour traiter la pneumonie. Les facteurs prédisposants fréquemment suggérés comprenaient des introductions ou des déplacements récents d'animaux, un vêlage récent ou imminent, des conditions météorologiques défavorables, des maladies concomitantes et une mauvaise ventilation dans les étables. Conclusion et pertinence clinique: Cette étude décrit les tendances saisonnières et annuelles, les principaux agents pathogènes, les profils de résistance aux antimicrobiens et les facteurs prédisposants suggérés chez les vaches laitières de l'Ontario soumises à un laboratoire de diagnostic pour une enquête post-mortem sur la pneumonie et fournit des informations pour comprendre pourquoi les épidémies se produisent.(Traduit par Dr Serge Messier).

Comparative full genome sequence analysis of wild-type and chicken embryo origin vaccine-like infectious laryngotracheitis virus field isolates from Canada.

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Phylogenetic analysis of porcine circovirus 3 circulating in Canadian pigs.

INTRODUCTION: Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. METHODS: To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. RESULTS: Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. CONCLUSION: Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.

Virulence of Emerging Arthrotropic Avian Reoviruses Correlates With Their Ability to Activate and Traffic Interferon-γ Producing Cytotoxic CD8+ T Cells Into Gastrocnemius Tendon.

Newly emerging arthrotropic avian reoviruses (ARVs) are genetically divergent, antigenically heterogeneous, and economically costly. Nevertheless, the mechanism of emerging ARV-induced disease pathogenesis and potential differences in virulence between virus genotypes have not been adequately addressed. In this study, the life cycle of ARV, including the formation of cytoplasmic ARV neo-organelles, paracrystalline structures, and virus release mechanisms, were characterized in the infected host cell by transmission electron microscopy (TEM). In addition, progressive changes in the structure of infected cells were investigated by time-lapse and field emission scanning electron (FE-SE) microscopy. ARVs from the four genotypic cluster groups included in the study caused gross and microscopic lesions in the infected birds. Marked infiltration of γδT cells, CD4+ and CD8+ T lymphocytes were observed in ARV infected tendon tissues starting day 3 post-infection. The ARV variant from genotype cluster-2 triggered significantly high trafficking of IFN-γ producing CD8+ T lymphocytes in tendon tissues and concomitantly showed high morbidity and severe disease manifestations. In contrast, the ARV variant from genotype cluster-4 was less virulent, caused milder disease, and accompanied less infiltration of IFN-γ producing CD8+ T cells. Interestingly, when we blunted antiviral immune responses using clodronate liposomes (which depletes antigen-presenting cells) or cyclosporin (which inhibits cytokine production that regulates T-cell proliferation), significantly lower IFN-γ producing CD8+ T cells infiltrated into tendon tissues, resulting in reduced tendon tissues apoptosis and milder disease manifestations. In summary, these data suggest that the degree of ARV virulence and tenosynovitis/arthritis are potentially directly associated with the ability of the virus to traffic massive infiltration of cytotoxic CD8+ T cells into the infected tissues. Moreover, the ability to traffic cytotoxic CD8+ T cells into infected tendon tissues and the severity of tenosynovitis differ between variants from different ARV genotype cluster groups. However, more than one virus isolate per genotype group needs to be tested to further confirm the association of pathogenicity with genotype. These findings can be used to further examine the interaction of viral and cellular pathways which are essential for the pathogenesis of the disease at the molecular level and to develop effective disease control strategies.

IN SITU HYBRIDIZATION AND VIRUS CHARACTERIZATION OF SKUNK ADENOVIRUS IN NORTH AMERICAN WILDLIFE REVEALS MULTISYSTEMIC INFECTIONS IN A BROAD RANGE OF HOSTS.

Skunk adenovirus-1 (SkAdV-1) has been reported infecting several North American wildlife species; however, lesions associated with disease have not yet been completely characterized, particularly in porcupines. We describe and characterize the tissue distribution and lesions associated with SkAdV-1 infection in 24 wildlife diagnostic cases submitted between 2015 and 2020, including 16 North American porcupines (Erethizon dorsatum), three striped skunks (Mephitis mephitis), and five raccoons (Procyon lotor), which constitute a new host species. The most common lesion in all species was severe necrotizing bronchopneumonia with (n=12) or without (n=10) interstitial involvement. Intranuclear inclusion bodies were common in respiratory epithelium (n=21) and less often in renal tubular (n=6) and biliary epithelium (n=1). Several cases (n=4) had secondary bacterial infections, including Bordetella bronchiseptica, Pasteurella multocida, and Streptococcus zooepidemicus. In situ hybridization in porcupine (n=6), raccoon (n=1), and skunk (n=1) revealed SkAdV-1 DNA in multiple tissue types, including lung, trachea, turbinates, liver, kidney, lymph node, and brain, and multiple cell types including epithelial, endothelial, and mesothelial cells. These findings were consistent across species. Comparison of viral genomes from a porcupine and a raccoon with that originally isolated from a skunk demonstrated DNA point mutations affecting several viral genes, including the fiber protein gene. Our findings show the spectrum of disease associated with SkAdV-1 infection in a broad host range of wildlife species.

Genetic characterization of canine distemper virus from wild and domestic animal submissions to diagnostic facilities in Canada.

Traditionally considered an agent affecting domestic dogs, canine distemper virus (CDV) is now well known for an ability to infect a broad range of hosts. In Ontario, domestic dogs are routinely vaccinated and clinical disease attributed to CDV infection in this population is infrequent. CDV has been regularly documented in Ontario wildlife spanning at least 4 decades however, the molecular identity of circulating CDV strains is currently unknown. Our objective was to investigate the molecular identities of and genetic relationships between CDV detected in wild and domestic animals from Canada, across multiple host species and over time. Samples were opportunistically collected from submissions to the Ontario-Nunavut node of the Canadian Wildlife Health Cooperative and the Animal Health Laboratory in Guelph, Ontario. RT-PCR was used to confirm CDV diagnosis, and the hemagglutinin gene was sequenced. Phylogenetic relationships were inferred, and the geographic distribution of clades was visualized using a geographic information system. Phenetic relationships between sequences were investigated with a median joining network analysis and through mixed multivariable linear regression. CDV sequences from ten wild and domestic species were characterized into seven lineages, that overlapped geographically and temporally. The predominant lineage circulating in Ontario wildlife, denoted Canada-1, has not been previously described to the authors knowledge. Our analysis indicates that the Canada-1 lineage is most genetically similar to America-1 sequences, however according to current methodology represents a distinct lineage. Multiple co-circulating CDV lineages were also identified, and raccoons appear to play an important role in the maintenance and transmission of these heterogeneous lineages in Ontario. This study also confirmed the presence of CDV from a lineage not found to be circulating in Ontario wildlife, in a domestic dog imported into Ontario from South America. Therefore, travel and the trade of animals may be an important avenue for the introduction of novel CDV lineages. It remains unclear whether and to what extent the genetic heterogeneity identified poses a risk to the efficacy of current vaccines. Increasing viral activity and continued antigenic drift resulting in partial protection or vaccine failure remains a concern.

Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

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.

Molecular Characterization of 4/91 Infectious Bronchitis Virus Leading to Studies of Pathogenesis and Host Responses in Laying Hens.

Infectious bronchitis virus (IBV) initially establishes the infection in the respiratory tract and then spreads to other tissues depending on its virulence. During 2011-2018, the 4/91 IBV strain was isolated from poultry flocks affected by decreased egg production and quality in Eastern Canada. One of the Canadian 4/91 IBV isolates, IBV/Ck/Can/17-038913, was propagated in embryonated chicken eggs and molecularly characterized using whole genome sequencing. An in vivo study in laying hens was conducted to observe if IBV/Ck/Can/17-038913 isolate affects the egg production and quality. Hens were infected with IBV/Ck/Can/17-038913 isolate during the peak of egg lay, using a standard dose and routes maintaining uninfected controls. Oropharyngeal and cloacal swabs were collected at predetermined time points for the quantification of IBV genome loads. At 6 and 10 days post-infection, hens were euthanized to observe the lesions in various organs and collect blood and tissue samples for the quantification of antibody response and IBV genome loads, respectively. Egg production was not impacted during the first 10 days following infection. No gross lesions were observed in the tissues of the infected birds. The IBV genome was quantified in swabs, trachea, lung, proventriculus, cecal tonsils, kidney, and reproductive tissues. The serum antibody response against IBV was quantified in infected hens. In addition, histological changes, and recruitment of immune cells, such as macrophages and T cell subsets in kidney tissues, were measured. Overall, data show that IBV/Ck/Can/17-038913 isolate is not associated with egg production issues in laying hens infected at the peak of lay, while it demonstrates various tissue tropism, including kidney, where histopathological lesions and immune cell recruitments were evident.

Classification of porcine reproductive and respiratory syndrome virus in Ontario using Bayesian phylogenetics and assessment of temporal trends.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine viruses globally, including in Ontario, Canada. Understanding the evolution and relation of the various PRRSV genotypes in Ontario can provide insight into the epidemiology of the virus. The objectives of this study were to i) describe the variability of PRRSV genotypes in Ontario swine herds, and ii) evaluate possible groupings based on PRRSV genomic data. Virus open reading frame 5 (ORF-5) sequences collected from 2010 to 2018 were obtained from the Animal Health Laboratory, University of Guelph and Bayesian phylogenetic models were created from these. The PRRSV population of Ontario was then categorized into 10 distinct clades. Model comparisons indicated that the model with a constant population assumption fit the data best, which suggests that the net change in the PRRS virus variation of the entire population over the last decade was low. Nonetheless, viruses grouped into individual clades showed temporal clustering during distinct time intervals of the entire study period (P < 0.01).

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est l'un des virus porcins les plus importants au monde, y compris en Ontario, au Canada. Comprendre l'évolution et la relation des divers génotypes du VSRRP en Ontario peut donner un aperçu de l'épidémiologie du virus. Les objectifs de cette étude étaient de i) décrire la variabilité des génotypes du VSRRP dans les troupeaux de porcs de l'Ontario et ii) évaluer les regroupements possibles en fonction des données génomiques du VSRRP. Les séquences du cadre de lecture ouvert 5 du virus (ORF-5) recueillis de 2010 à 2018 ont été obtenues auprès du Laboratoire de santé animale de l'Université de Guelph et des modèles phylogénétiques bayésiens ont été créés à partir de ceux-ci. La population de VSRRP de l'Ontario a ensuite été classée en 10 clades distincts. Les comparaisons de modèles ont indiqué que le modèle avec une hypothèse de population constante correspondait le mieux aux données, ce qui suggère que le changement net de la variation du virus SRRP de l'ensemble de la population au cours de la dernière décennie était faible. Néanmoins, les virus regroupés en clades individuels ont montré un regroupement temporel à des intervalles de temps distincts de toute la période d'étude (P < 0,01).(Traduit par Docteur Serge Messier).