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The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus, with high mutation rates and genetic variability ; which is evident by the large number of discrete strains that co-circulate in swine populations. Veterinary practitioners frequently identify certain discrete PRRSV strains as having a higher clinical impact on production. However, with exception of a few strains, production impact is not well characterized for the majority of PRRSV variants. Predictive analytics, coupled with routine diagnostic sequencing of PRRSV, provide opportunities to study the clinical impact of discrete PRRSV strains on production. Thus, the primary objective of this research was to evaluate clinical impact of discrete PRRSV clades observed in Ontario sow farms. PRRS viruses were classified into discrete clades using Bayesian analysis of the nucleotide sequences of the ORF-5 region of the genome. Production data were gathered through veterinary clinics from herds participating in the ongoing PRRSV surveillance system. Data about pre-weaning mortality, sow mortality, and abortion rates were measured up to 8 weeks post initial PRRSV outbreak. Through conventional regression analysis, results support that clinical impact of the viruses varied among clades over time for abortion rate (p = 0.05) and pre-weaning mortality (p < 0.01). Using predictive modelling approaches based on grouped K-fold cross-validation, it was identified that PRRSV clade designations and other measured factors showed low predictive performance for abortion (R2 = 0.07), pre-weaning mortality (R2 = 0.09), and sow mortality (R2 = 0.04). Clade designation consistently showed moderate importance for abortion and pre-weaning mortality, with clade 2 viruses being identified, on average, as having higher impact. These results demonstrate that the prediction of clinical impact, through production parameters, based on phylogenetic classification of PRRS viruses is possible. However, very high impact outbreaks were difficult to predict across production parameters. More surveillance- derived data are required to continue to improve predictive performance of the models.

Rabies and canine distemper virus infections in wildlife share similar presenting signs. Canine distemper virus was detected using real-time PCR of conjunctival swabs in rabies positive raccoons (22/32) and skunks (7/34) during a concurrent rabies and canine distemper outbreak in Ontario, Canada in 2015–16. Coinfections with both viruses should be considered, particularly in distemper endemic areas that are at risk of rabies incursion.

Influenza A virus commonly circulating in swine (IAV-S) is characterized by large genetic and antigenic diversity and, thus, improvements in different aspects of IAV-S surveillance are needed to achieve desirable goals of surveillance such as to establish the capacity to forecast with the greatest accuracy the number of influenza cases likely to arise. Advancements in modeling approaches provide the opportunity to use different models for surveillance. However, in order to make improvements in surveillance, it is necessary to assess the predictive ability of such models. This study compares the sensitivity and predictive accuracy of the autoregressive integrated moving average (ARIMA) model, the generalized linear autoregressive moving average (GLARMA) model, and the random forest (RF) model with respect to the frequency of influenza A virus (IAV) in Ontario swine. Diagnostic data on IAV submissions in Ontario swine between 2007 and 2015 were obtained from the Animal Health Laboratory (University of Guelph, Guelph, ON, Canada). Each modeling approach was examined for predictive accuracy, evaluated by the root mean square error, the normalized root mean square error, and the model's ability to anticipate increases and decreases in disease frequency. Likewise, we verified the magnitude of improvement offered by the ARIMA, GLARMA and RF models over a seasonal-naïve method. Using the diagnostic submissions, the occurrence of seasonality and the long-term trend in IAV infections were also investigated. The RF model had the smallest root mean square error in the prospective analysis and tended to predict increases in the number of diagnostic submissions and positive virological submissions at weekly and monthly intervals with a higher degree of sensitivity than the ARIMA and GLARMA models. The number of weekly positive virological submissions is significantly higher in the fall calendar season compared to the summer calendar season. Positive counts at weekly and monthly intervals demonstrated a significant increasing trend. Overall, this study shows that the RF model offers enhanced prediction ability over the ARIMA and GLARMA time series models for predicting the frequency of IAV infections in diagnostic submissions.

Canine distemper virus (CDV) is a widespread morbillivirus that causes subclinical to fatal infections in domestic and wild carnivores. Raccoons ( Procyon lotor) are CDV reservoirs and suffer from associated disease. Aspects of pathogenesis may lead to difficulty in the interpretation of commonly used testing modalities, such as reverse-transcription real-time (RT-rt)PCR and immunohistochemistry (IHC). The reliance upon such tests is greater for wildlife, which are often submitted as carcasses with no clinical history. We compared CDV RT-rtPCR results to immunohistochemistry (the gold standard) in tissues from 74 raccoons. These tests had high kappa agreement (lymph node: 0.9335; lung: 0.8671) and a negative correlation between IHC score and threshold cycle (Ct) value for lymph node and lung (Spearman rank correlation coefficient [ rs] = −0.8555 and −0.8179, respectively; p < 0.00001). An RT-rtPCR Ct value of 30 in lung and lymph node with sensitivity and specificity of 92.3 and 92.6% and 86.8 and 96.4%, respectively, was suitable for determining CDV involvement. Conjunctival swabs provide an alternative for distemper diagnosis, as there was a strong correlation between Ct values of conjunctival swabs and tissues ( rs = −0.8498, p < 0.00001, n = 46). This information will aid in more efficient and accurate diagnoses in individuals, small-scale outbreaks, and epidemiologic investigations in wildlife.

A one-year-old male Himalayan monal (Lophophorus impejanus) was presented for veterinary attention with a history of chronic wasting, weakness and ataxia. The bird died, and post-mortem findings included mild non-suppurative encephalitis and degenerative encephalopathy, lymphoplasmacytic myenteric ganglioneuritis (particularly of the proventriculus), and Wallerian degeneration of the sciatic nerves. Avian bornavirus (ABV) was identified in the brain by immunohistochemistry and reverse-transcriptase polymerase chain reaction. Sequencing of the reverse-transcriptase polymerase chain reaction product indicated the presence of ABV genotype 4, which is generally associated with disease in psittacine birds. Subsequent to the death of the pheasant, ABV genotype 4 was identified at autopsy from a juvenile white-bellied caique (Pionites leucogaster) in the same collection. We hypothesize that the pheasant became infected through contact with psittacine birds with which it shared an aviary. We believe this to be the first reported case of natural ABV infection in a bird in the Order Galliformes.

Avian reovirus (ARV) is an economically significant pathogen of broiler chickens. Our objective was to determine the prevalence, geographical distribution, and seasonal variation of ARV infection among commercial broiler flocks in Ontario, Canada during grow-out. A cross-sectional study of 231 randomly selected flocks was conducted from July 2010 to January 2012. Fifteen blood samples, 15 whole intestines, and 15 cloacal swabs per flock were collected at slaughter; ELISA and PCR were used to determine a flock’s ARV exposure status. Avian reovirus prevalence was 91% (95% CI: 87 to 94). District alone did not significantly explain the overall variation in the prevalence of ARV (univariable logistic regression; P = 0.073), although geographical differences were identified. The odds of ARV presence were significantly lower in the summer/autumn compared to the winter/spring (univariable exact logistic regression; P < 0.001). There was no association between flock mortality and flock ELISA mean titer or PCR status.

Summary In January 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (PED) in the USA, the first case of PED was confirmed in a swine herd in south-western Ontario. A follow-up epidemiological investigation carried out on the initial and 10 subsequent Ontario PED cases pointed to feed as a common risk factor. As a result, several lots of feed and spray-dried porcine plasma (SDPP) used as a feed supplement were tested for the presence of PEDV genome by real-time RT-PCR assay. Several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of PEDV into Canada. These findings led us to conduct a bioassay experiment in which three PEDV-positive SDPP samples (from a single lot) and two PEDV-positive feed samples supplemented with this SDPP were used to orally inoculate 3-week-old piglets. Although the feed-inoculated piglets did not show any significant excretion of PEDV, the SDPP-inoculated piglets shed PEDV at a relatively high level for ≥9 days. Despite the fact that the tested PEDV genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role.

Samples from 231 randomly selected commercial broiler chicken flocks in Ontario were tested at slaughter for exposure to chicken anemia virus (CAV), fowl adenovirus (FAdV), and infectious bursal disease virus (IBDV). Fifteen blood samples per flock were collected and analyzed for the presence of antibodies against CAV, FAdV, and IBDV by ELISA or agar gel immunodiffusion test. Fifteen cecal tonsils and cloacal swabs per flock were analyzed for the presence of CAV, FAdV, and IBDV by PCR. The prevalence of exposure to avian adeno-associated virus (AAAV) was estimated by a PCR test on a subset of FAdV-PCR-positive samples from 178 flocks. Genotypes of FAdV and IBDV were identified on a subset of isolates (n = 353 and 45, respectively). The flock-level period prevalence of exposure to AAAV, CAV, FAdV, and IBDV during grow-out were 88.76% (95% CI: 84.08-93.45%), 77.06% (95% CI: 71.59-82.52%), 96.54% (95% CI: 94.16-98.91%), and 48.92% (95% CI: 42.42-55.41%), respectively. Results of a multivariable logistic regression model showed a significant association of exposure to FAdV with exposure to AAAV (OR = 18.57, 95% CI: 3.67-93.86, P = 0.004) but not with exposure to CAV (P = 0.7752) or exposure to IBDV (P = 0.2274). Pathogenic FAdV genotypes (FAdV-02, FAdV-08, and FAdV-11) constituted 39.38% of the isolates. The most-common IBDV genotypes identified were IBDV NC171 (60%) and IBDV 05SA8 (28.89%). This is the first large-scale study to estimate the baseline flock prevalence of exposure to AAAV, CAV, FAdV, and IBDV in commercial broiler flocks in Canada. Potentially pathogenic genotypes of FAdV and IBDV that can guide vaccine development and disease control efforts in Ontario were identified.

Highly pathogenic avian influenza virus (HPAI) and virulent forms of avian paramyxovirus-1 (APMV-1) cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE). The clinical presentation of avian influenza (AI) and APMV-1 infections are difficult to differentiate, emphasizing the importance of rapid and sensitive serologic assays that are able to distinguish them. Currently, a variety of serological assays are used for the serologic diagnosis of both diseases, but these assays are not used in multiplex formats. In this study, development of a duplex fluorescent microsphere immunoassay (FMIA) based on Luminex xMAP Technology is described. The assay employs MagPlex magnetic microspheres that are covalently coated with recombinant avian influenza virus nucleoprotein and APMV-1 nucleocapsid antigens produced in a baculovirus insect cell expression system. The assay is able to detect AIV antibodies against all existing hemagglutinin (H1–H16) subtypes and simultaneously detect antibodies against APMV-1. In the process of this assay development different bead coupling conditions were compared. The assay has the capability of detecting serum antibodies from chickens and turkeys and optimization was accomplished by using 2462 chicken and 446 turkey field and experimental sera and had a comparable detection capability with currently used assays in the laboratory. Assay threshold values were calculated with Receiver Operating Characteristic Analysis (ROC) in non-parametric analysis due to a highly skewed data distribution; this analysis resulted in AIV nucleoprotein relative diagnostic sensitivity and specificity of 99.7%, and 97.3% respectively. The APMV-1 nucleocapsid relative diagnostic sensitivity and specificity were 95.4%, and 98.5% respectively.

Avian bornavirus (ABV), the cause of proventricular dilation disease in psittacine birds, has been detected in multiple tissues of infected birds using immunohistochemical staining (IHC) and reverse transcription polymerase chain reaction (RT-PCR). In the current study, real-time RT-PCR, using primers targeting the ABV matrix gene, was used to detect ABV in 146 tissues from 7 ABV-infected psittacine birds. Eighty-six percent of the samples tested positive, with crossing point values ranging from 13.82 to 37.82 and a mean of 22.3. These results were compared to the findings of a previous study using gel-based RT-PCR and IHC on the same samples. The agreement between the 2 RT-PCR techniques was 91%; when tests disagreed it was because samples were negative using gel-based RT-PCR but positive on real-time RT-PCR. Agreement with IHC was 77%; 16 out of 74 samples were negative using IHC but positive on real-time RT-PCR. The results suggest that real-time RT-PCR is a more sensitive technique than gel-based RT-PCR and IHC to detect ABV in tissues. The tissues that were ranked most frequently as having a high amount of viral RNA were proventriculus, kidney, colon, cerebrum, and cerebellum. Skeletal muscle, on the other hand, was found to have a consistently low amount of viral RNA.

Nine hundred and fifty-five pathology cases collected in Ontario between 1992 and 2011 from wild free-ranging Canada geese, trumpeter swans and mute swans were retrospectively evaluated for the pathology associated with avian bornavirus (ABV) infection. Cases were selected based on the presence of upper gastrointestinal impaction, central nervous system histopathology or clinical history suggestive of ABV infection. The proportion of birds meeting at least one of these criteria was significantly higher at the Toronto Zoo (30/132) than elsewhere in Ontario (21/823). Central, peripheral and autonomic nervous tissues were examined for the presence of lymphocytes and plasma cells on histopathology. The presence of virus was assessed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) on frozen brains and on formalin-fixed paraffin-embedded tissues. Among selected cases, 86.3% (44/51) were considered positive on histopathology, 56.8% (29/51) were positive by immunohistochemistry, and RT-PCR was positive on 88.2% (15/17) of the frozen brains and 78.4% (40/51) of the formalin-fixed paraffin-embedded samples. Histopathological lesions included gliosis and lymphoplasmacytic perivascular cuffing in brain (97.7%), spinal cord (50%), peripheral nerves (55.5%) and myenteric ganglia or nerves (62.8%), resembling lesions described in parrots affected with proventricular dilatation disease. Partial amino acid sequences of the nucleocapsid gene from seven geese were 100% identical amongst themselves and 98.1 to 100% identical to the waterfowl sequences recently described in the USA. Although ABV has been identified in apparently healthy geese, our study confirmed that ABV can also be associated with significant disease in wild waterfowl species.

While the prevalence of infectious bursal disease virus (IBDV) on chicken farms in some provinces of Canada has been documented, the economic impact of variant IBDV infection on the broiler chicken industry in Saskatchewan has not. The objectives of this study were to identify the variant strains of IBDV circulating on Saskatchewan chicken farms and evaluate their economic impact on broiler production. Infection due to IBDV was detected in 43% of Saskatchewan chicken farms, with variant strains detected in infected birds closely related predominantly to NC171, 586, and Delaware-E. Infected flocks showed an IBDV antibody titer of 4236 geometric mean (GM), whereas an antibody titer of 157 GM was measured in uninfected flocks. Infected flocks had very low (0.06) bursa-to-body-weight (BBW) ratio (an indicator of immunity) compared to high BBW ratio (0.17) in uninfected flocks, which suggests a significant immunosuppression in the former. Flocks positive for IBDV had mean mortality of 8.6% and mean condemnation of 1.5%. In contrast, mean mortality in uninfected flocks was 6.1% and mean condemnation was 1.1%. The live market weight per grow area at 37 d of age was 29.3 kg/mBien que la prévalence du virus de la maladie infectieuse de la bourse (VMIB) sur les fermes de poulets dans quelques provinces canadiennes ait été documentée, l’impact économique d’infections par des variants du VMIB sur l’industrie du poulet à griller en Saskatchewan ne l’est pas. Les objectifs de la présente étude étaient d’identifier les souches variantes du VMIB circulant au sein des fermes de poulet de la Saskatchewan et d’évaluer leur impact économique sur la production de poulets à griller. L’infection due au VMIB a été détectée sur 43 % des fermes de poulet de la Saskatchewan, avec des souches variantes détectées chez des oiseaux infectés étant fortement apparentées en prédominance aux souches NC171, 586, et Delaware-E. Les troupeaux infectés présentaient une moyenne géométrique (MG) des titres d’anticorps contre le VMIB de 4236, alors que la MG des titres d’anticorps des oiseaux provenant de troupeaux non-infectés était de 157. Les troupeaux infectés avaient un très faible ratio (0,06) du poids bourse-poids corporel (BPC) (un indicateur de l’immunité) comparativement au ratio BPC élevé (0,17) dans les troupeaux non-infectés, ce qui suggère une immunosuppression significative dans les troupeaux infectés. Les troupeaux positifs au VMIB avaient un taux de mortalité moyen de 8,6 % et un taux de condamnation moyen de 1,5 %. À l’opposé, dans les troupeaux non-infectés le taux de mortalité moyen était de 6,1 % et le taux de condamnation moyen de 1,1 %. Le poids vif de marché à 37 j d’âge par surface de croissance était de 29,3 kg/m

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×103 EID50) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8+ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.

Summary Soon after the emergence of 2009 pandemic H1N1, the first outbreaks in breeder turkey operations were reported that implicated human-to-turkey transmission. In the spring of 2016, the reoccurrence of 2009 pandemic H1N1 lineage viruses infecting breeder turkey flocks in Ontario and Manitoba, Canada, also implicated human-to-turkey transmission. In addition to raising concerns over biosecurity and vaccine failures, these cases once again raise the issue of whether turkeys have the potential to act as a bridge species to generate novel influenza A virus reassortants with public health implications.

Antibiotic-free and safe animal products are most desirable among consumers. However, ensuring safe poultry products is a challenging task when the chicken immune system is compromised. Infectious bursal disease virus (IBDV) causes immunosuppression and predisposes chickens to secondary infections. Breeder vaccination against IBDV is routinely practiced for producing chicks with maternally-derived antibody (MAb) to prevent infection in newly hatched chicks. The majority of IBDV circulating in Canadian farms are variant strains (vIBDV). Whether circulating vIBDV strains are immunosuppressive in chicks or are amenable to current vaccine regimens has not previously been tested through challenge studies. In this study, one-day-old broiler chicks (n=240) carrying MAb were obtained from broiler breeders vaccinated with commercial IBDV vaccines. In the first set of experiments (n=40/group), at six days post-hatch, one group was challenged with a Canadian field isolate, vIBDV (strain-SK09) (3×10(3) EID50). The second and the third groups (controls) were inoculated with non-immunosuppressive IBDV D-78 (10×10(3) TCID50) and saline, respectively. Histopathological examination on days 14 and 30 post-challenge revealed that despite the high level of MAb, vIBDV (SK09) caused severe bursal damage in chicks. Another set of experiments with treatment groups as above, demonstrated that pre-exposure of chicks with vIBDV (SK09) caused immunosuppression resulting in significantly higher mortality and disease severity in chicks challenged with a virulent strain of Escherichia coli (E. coli). Our data provide evidence that IBDV strains circulating in Canada are immunosuppressive, not amenable to current anti-IBDV vaccination strategy, and a potential threat to antibiotic-free chicken farming.

Proventricular dilatation disease (PDD) is a neurologic disease of psittacine birds suspected to be caused by a recently identified Avian bornavirus (ABV). In the current report, data supporting the causal association of ABV with PDD are presented. Immunohistochemistry (IHC) with rabbit polyclonal antiserum raised against ABV nucleocapsid protein was used to identify cell and organ distribution of viral antigen. The ABV antigen was most consistently detected in brain, spinal cord, adrenal gland, pancreas, and kidney. Histopathologic evaluation was correlated with ABV-specific polymerase chain reaction (PCR) and immunohistochemical tests in multiple tissues from 16 psittacine birds with and without PDD. Using histopathologic diagnosis as the gold standard, the sensitivity and specificity of IHC for ABV antigens were found to be 100% and 100%, respectively. Many more tissues were positive for ABV RNA by reverse transcription PCR than were positive for pathologic changes or viral antigens by IHC, indicating the presence of subclinical or asymptomatic infection at many sites.

Influenza A viruses infect a range of host species, including various mammals and more than 100 species of birds. For avian influenza viruses (AIV), prevalence varies between different groups of birds, with waterfowl showing the highest prevalence. We have sequenced the complete genome of A/Thick-billed Murre/Newfoundland/031/2007(H11N2), an AIV identified in the pelagic seabird, Thick-billed Murre (Uria lomvia). This represents the first complete genome sequence of an AIV from this host species, and only the second complete genome sequence from a seabird in the alcid group. All of the virus segments fall within the American avian lineage. Several of the segments show a close relationship to AIV identified in other marine host species, and also a strong geographic association with other AIV sequences from the northeastern coast of North America from recent years. The identification of this virus, and the growing number of AIV identified in seabird species, indicates these marine birds could be underappreciated host species. This has potential consequences for global influenza dynamics because of the seasonal distributions and migratory patterns of this group of birds.

A multi-agency, Canada-wide survey of influenza A viruses circulating in wild birds, coordinated by the Canadian Cooperative Wildlife Health Centre, was begun in the summer of 2005. Cloacal swab specimens collected from young-of-year ducks were screened for the presence of influenza A nucleic acids by quantitative, real-time reverse transcription-polymerase chain reaction (RRT-PCR). Specimens that produced positive results underwent further testing for H5 and H7 gene sequences and virus isolation. In addition to live bird sampling, dead bird surveillance based on RRT-PCR was also carried out in 2006 and 2007. The prevalence of influenza A viruses varied depending on species, region of the country, and the year of sampling, but generally ranged from 20% to 50%. All HA subtypes, with the exception of H14 and H15, and all NA subtypes were identified. The three most common HA subtypes were H3, H4, and H5, while N2, N6, and N8 were the three most common NA subtypes. H4N6, H3N2, and H3N8 were the three most common HA-NA combinations. The prevalence of H5 and H7 subtype viruses appears to have a cyclical nature.

Avian influenza virus (AIV) was studied in ring-billed gulls (Larus delawarensis) in one breeding colony on Lake Erie in 2000, and two on Lake Ontario in both 2000 and 2004. Antibodies to H13 AIV were detected in 92% of adults in 2000 and 82% in 2004. Antibody prevalence in 3-wk-old chicks was 5%-30% (overall 15%) in 2000 and 21% and 76% (overall 48%) in 2004. In 5-wk-old chicks, antibody prevalence was 23%-75% (overall 53%) in 2000 and 53% and 79% (overall 66%) in 2004. Geometric mean antibody titers at 3 and 5 wk did not differ in 2000, but increased significantly at one colony in 2004. In 2000, overall prevalence of AIV isolation from cloaca in embryonated chicken eggs was 32% (3 wk old), 13% (5 wk old), and 0 (adults), but AIV was also isolated from kidney and lung in a high proportion of tissues cultured from 3-wk-old birds in one colony. Isolates from cloaca were characterized as subtype H13 by serology; all 15 tested for neuraminidase were H13N6. However, three AIV detections considered on the basis of nucleotide sequence to be subtype H16 were among the 28 detected retrospectively by PCR in archived cloacal swabs; the remainder were subtype H13. Outcome of virus isolation was not related to presence of antibody titers in chicks. The presence of antibody to AIV in chicks was associated significantly with inflammation in heart, kidney, pancreas, and liver. AIV was not isolated in 2004. AIV infected chicks annually within the first 3 wk of life, ultimately infecting the majority of birds in most colonies, but did not appear to cause clinical disease.

Increased mortality was reported in two flocks of Muscovy ducklings from two consecutive hatches originating from the same breeder flock. Coughing, dyspnea, and gasping were observed in some ducklings between 6 and 11 days of age. Opaque white plugs of exudate were seen in the tracheas with some ducklings having multiple tracheal plugs. Tracheal and bronchial epithelium was hyperplastic and superficial epithelial cells contained eosinophilic intranuclear viral inclusions. Virus particles compatible with adenovirus morphology were observed in tracheal epithelial cells by electron microscopy and in the supernatant from cell cultures inoculated with filtered tracheal homogenates. The isolated virus was genetically indistinguishable from duck adenovirus 1 (DAdV-1). Our report confirms for the first time the presence of DAdV-1 in Canada and also reports for the first time adenovirus-associated respiratory disease in ducklings and supports previous findings that some DAdV-1 can be pathogenic even in waterfowl.

The course of turkey coronavirus (TCoV) infection in young turkey poults was examined using a field isolate (TCoV-MG10) from a diarrhoeal disease outbreak on a commercial turkey farm in Ontario, Canada. Two-day-old and 28-day-old poults were inoculated orally with TCoV-MG10 to examine the effect of age on viral shedding and serum antibody responses to the virus. The presence of coronavirus particles measuring 105.8+/-21.8 nm in the cloacal contents was confirmed using transmission electron microscopy. The pattern of cloacal TCoV shedding was examined by reverse-transcription polymerase chain reaction amplification of the nucleocapsid gene fragment. TCoV serum antibody responses were assessed with two recently developed TCoV enzyme-linked immunosorbent assays that used TCoV nucleocapsid and S1 polypeptides as coating antigens. Poults were found equally susceptible to TCoV infection at 2 days of age and at 4 weeks of age, and turkeys of either age shed virus in their faeces starting as early as 1 day post-inoculation and up to 17 days post-inoculation. Poults infected at 2 days of age were immunologically protected against subsequent challenge at 20 days post-inoculation. The protection was associated with measurable serum antibody responses to both the nucleocapsid and S1 structural proteins of TCoV that were detectable as early as 1 week post-infection.

Genomes of two low pathogenic H5N1 avian influenza (LPAI) viruses, A/Turkey/ON/84/1983 and A/Mallard/ON/499/2005 from Ontario, Canada were cloned and genetically characterized. Phylogenetic analysis showed that the Canadian isolates cluster with other North American AIVs and are distinct from the Euro-Asian H5N1 isolates. Individual gene comparisons demonstrated that the Ontario isolates were most similar to the viruses isolated from around the same time period and geographical area. A long deletion of 22 amino acids was identified in the stalk region of NA of A/Turkey/ON/84/1983 isolate, a characteristic mutation related to its adaptation to domestic birds. To our knowledge A/Turkey/ON/84/1983 genomic sequence is the first and only available entire genomic sequence of a H5N1 AIV from domestic birds in Canada and USA.

Turkey coronavirus (TCoV) causes diarrhea in young turkey poults, but little is known about its prevalence in the field. To address this, the complete nucleocapsid gene was cloned and expressed inEscherichia coli. Expressed nucleocapsid gene produced two distinct proteins (52 and 43 kDa); their specificity was confirmed by Western blotting using two different monoclonal antibodies. Recombinant N protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay (ELISA) for the serological detection of TCoV that was then validated using experimentally derived turkey serum. The N-based ELISA showed (97%) sensitivity and (93%) specificity for TCoV, which was significantly higher than an infectious bronchitis coronavirus-based commercial test for TCoV. To assess the utility of this recombinant ELISA, 360 serum samples from turkey farms in Ontario, Canada, and 81 serum samples from farms in Arkansas were tested for TCoV-specific antibodies. A high seroprevalence of TCoV was found in turkeys from the Ontario farms with 73.9% of breeders and 60.0% of meat turkeys testing seropositive using the N-based ELISA. Similarly, a high field prevalence was found in the turkeys from Arkansas, for which 64.2% of the serum samples tested seropositive.

Turkey coronavirus (TCoV), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in Ontario, Canada, and the full-length genomic sequence was determined. The full-length genome was 27,632 nucleotides plus the 3′ poly(A) tail. Two open reading frames, ORFs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream ORFs were identified. A gene for hemagglutinin-esterase was absent in TCoV. The region between the membrane (M) and nucleocapsid (N) protein genes contained three potential small ORFs: ORF-X, a previously uncharacterized ORF with an associated putative TRS within the M gene (apparently shared among all group III coronaviruses), and previously described ORFs 5a and 5b. The TCoV genome is organized as follows: 5′ UTR – replicase (ORFs 1a, 1b) – spike (S) protein – ORF3 (ORFs 3a, 3b) – small envelop (E or 3c) protein – membrane (M) protein – ORF5 (ORFs X, 5a, 5b) – nucleocapsid (N) protein −3′ UTR – poly(A). TCoV genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (IBV). This is the first complete genome sequence for a TCoV and confirms that TCoV belongs to group III coronaviruses.

For this retrospective study, infectious bursal disease virus (IBDV) was detected in 134 bursal samples that originated from flocks with conditions such as airsacculitis, tracheitis, pneumonia, septicaemia, inclusion body hepatitis, coccidiosis, and/or a history of production problems without overt clinical symptoms. Samples were from seven Canadian provinces: Ontario, Quebec, Manitoba, British Columbia, Nova Scotia, Alberta, and Newfoundland and Labrador. Viral RNA was identified in bursae with moderate to severe and acute to chronic bursal damage. The ages of the flocks from which samples were collected ranged from 3 to 63 days. Following reverse transcriptase-polymerase chain reaction the nucleotide sequence of the VP2 hypervariable region was determined and compared with sequences available in GenBank. The most common Canadian IBDV field strains were North-American variant viruses. Forty-four viruses were highly related (97.5% to 100.0%) to the US IBDV strain NC171. Moreover, 16 field viruses whose VP2 sequences were 99.2% to 100% identical to the South African 05SA8 IBDV strain appeared closely related to the NC171 group. Delaware E-related field viruses, 98.3% to 100.0% identical to the prototype virus, were identified in 33 samples. Thirty-four Canadian IBDVs showed the highest identity, 94.2% to 98.3%, to US IBDV strain 586. Five samples contained vaccine-related viruses, while two field strains showed the best match to Del A (United States) and IBDV strains SP_04_02 (Spain). Very virulent IBDVs were not detected in Canada.

In January, 2014, increased mortality was reported in piglets with acute diarrhea on an Ontario farm. Villus atrophy in affected piglets was confined to the small intestine. Samples of colon content were PCR-positive for porcine epidemic diarrhea virus (PEDV). Other laboratory tests did not detect significant pathogens, confirming this was the first case of PED in Canada.

Since January 2005, H3N2 influenza viruses have been isolated from pigs and turkeys throughout Canada and from a swine farmer and pigs on the same farm in Ontario. These are human/classical swine/avian reassortants similar to viruses that emerged in US pigs in 1998 but with a distinct human-lineage neuraminidase gene.

Summary The main goal of this study was to investigate the occurrence of porcine reproductive and respiratory syndrome virus (PRRSV)-specific genotypes in swine sites in Ontario (Canada) using molecular, spatial and network data from a porcine reproductive and respiratory syndrome (PRRS) regional control project. For each site, location, animal movement service provider (truck companies), PRRSV status and sequencing data of the open reading frame 5 (ORF5) were obtained. Three-kilometre buffers were created to evaluate neighbourhood characteristics for each site. Social network analysis was conducted on swine sites and trucking companies to assemble the network and define network components. Three different PRRSV genotypes were used as outcomes for statistical analysis based on the region's phylogenetic tree of the ORF5. Multivariable exact logistic regression was conducted to investigate the association between being positive for a specific genotype and two main exposures of interest: (i) having at least one neighbour within three km also positive for the same genotype outside the production system and (ii) having at least one positive site for the same genotype in the same truck network component outside the production system. Results showed that the importance of area spread and truck network on PRRSV occurrence differed according to genotype. Additionally, the Ontario PRRS database appears suitable for conducting regional disease investigations. Finally, the use of relatively new tools available for network, spatial and molecular analysis could be useful in investigation, control and prevention of endemic infectious diseases in animal populations.

To investigate the possibility of in ovo infection with avian bornavirus (ABV) in wild Canada geese (Branta canadensis), 53 eggs were opportunistically collected at various stages of embryonic development from 16 free-ranging goose nests at a large urban zoo site where ABV infection is known to be present in this species. ABV RNA was detected in the yolk of one of three unembryonated eggs using real-time reverse transcription polymerase chain reaction. ABV RNA was not identified in the brains from 23 newly hatched goslings or 19 embryos, nor from three early whole embryos. Antibodies against ABV were not detected in the plasma of any of the hatched goslings using an enzyme-linked immunosorbent assay. Possible reasons for the failure to detect ABV RNA in hatchlings or embryos include low sample size, eggs deriving from parents not actively infected with ABV, the testing of only brain tissue, and failure of the virus to replicate in Canada goose embryos. In conclusion, this preliminary investigation demonstrating the presence of ABV RNA in the yolk of a Canada goose egg provides the first evidence for the potential for vertical transmission of ABV in waterfowl.

We surveyed free-ranging Canada Geese (Branta canadensis), Trumpeter Swans (Cygnus buccinator), Mute Swans (Cygnus olor), and Mallards (Anas platyrhynchos) to estimate the prevalence of antibodies to avian bornavirus (ABV) and of cloacal shedding of ABV RNA in southern Ontario, Canada. Blood samples and cloacal swabs were collected from 206 free-ranging Canada Geese, 135 Trumpeter Swans, 75 Mute Swans, and 208 Mallards at 10 main capture sites between October 2010 and May 2012. Sera were assessed for antibodies against ABV by enzyme-linked immunosorbent assay and swabs were evaluated for ABV RNA using real-time reverse-transcription PCR. Serum antibodies were detected in birds from all four species and at each sampling site. Thirteen percent of the geese caught on the Toronto Zoo site shed ABV RNA in feces compared with 0% in geese sampled at three other locations. The proportions of shedders among Mute Swans, Trumpeter Swans, and Mallards were 9%, 0%, and 0%, respectively. Birds that were shedding viral RNA were more likely to have antibodies against ABV and to have higher antibody levels than those that were not, although many birds with antibodies were not shedding. We confirmed that exposure to, or infection with, ABV is widespread in asymptomatic free-ranging waterfowl in Canada; however, the correlation between cloacal shedding, presence of antibodies, and presence of disease is not fully understood.

Infectious bronchitis virus (IBV) was detected in 185 samples originating from chicken flocks of various commodity groups in Canada. Flocks with clinical signs such as respiratory challenge, sudden death, egg production problems, or nephropathogenic conditions, and randomly selected flocks sampled at slaughter as part of an Ontario broiler surveillance project, were included. Most samples were from Ontario and Quebec; however, a small number from British Columbia, Nova Scotia, and Newfoundland and Labrador were also analysed. The nucleotide sequence of the spike (S) protein gene was compared with sequences available in GenBank. Based on their S gene sequence similarities, Canadian IBVs could be divided into nine genotypes belonging to four groups: Canadian variant virus, strain Qu_mv; the classic, vaccine-like viruses, Connecticut and Massachusetts; US variant-like virus strains, California 1734/04, California 99, CU_82792, Pennsylvania 1220/98 and Pennsylvania Wolg/98; and non-Canadian, non-US virus, strain 4/91. Based on the field situation, the effectiveness of current vaccination practices mostly based on Massachusetts and Connecticut-type vaccines appeared generally satisfactory for minimizing the damage due to infection with Canadian variant and US variant-like viruses. However, the recent outbreaks of severe respiratory disease and production problems in Ontario chicken flocks related to the incursion of IBV strain 4/91 were not prevented by standard vaccination protocols. It appears that IBV strain 4/91 has now become endemic in Ontario and the need for 4/91-type vaccines must be evaluated.

Gulls are important hosts of avian influenza A viruses (AIVs) and gull AIVs often contain gene segments of mixed geographic and host lineage origins. In this study, the prevalence of AIV in gulls of Newfoundland, Canada from 2008 to 2011 was analyzed. Overall prevalence was low (30/1645, 1.8%) but there was a distinct peak of infection in the fall. AIV seroprevalence was high in Newfoundland gulls, with 50% of sampled gulls showing evidence of previous infection. Sequences of 16 gull AIVs were determined and analyzed to shed light on the transmission, reassortment and persistence dynamics of gull AIVs in Atlantic North America. Intercontinental and waterfowl lineage reassortment was prevalent. Of particular note were a wholly Eurasian AIV and another with an intercontinental reassortant waterfowl lineage virus. These patterns of geographic and inter-host group transmission highlight the importance of characterization of gull AIVs as part of attempts to understand global AIV dynamics.

Influenza A viruses infect a wide range of hosts, including many species of birds. Avian influenza A virus (AIV) infection appears to be most common in Anseriformes (ducks, geese, and swans) and some Charadriiformes (shorebirds and gulls), but many other birds also serve as hosts of AIV. Here, we evaluated the role of seabirds as hosts for AIV. We tested 3,160 swab samples from 13 seabird species between May 2008 and December 2011 in Newfoundland and Labrador, Canada. We also tested 156 serum samples for evidence of previous infection of AIV in Common Murres (Uria aalge) and Atlantic Puffins (Fratercula arctica). Avian influenza A virus was detected in breeding Common Murres and nonbreeding Thick-billed Murres (Uria lomvia), and Common Murres also had high antibody prevalence (44%). From these findings, combined with other studies showing AIV infection in murres, we conclude that murres are important for the ecology of AIV. For other species (Razorbill, Alca torda; Leach's Storm-Petrel, Oceanodroma leucorhoa; Black-legged Kittiwake, Rissa tridactyla; Atlantic Puffin) with good coverage (>100 samples) we did not detect AIV. However, serology indicates infection does occur in Atlantic Puffins, with 22% antibody prevalence found. The possibility of virus spread through dense breeding colonies and the long distance movements of these hosts make a more thorough evaluation of the role for seabirds as hosts of AIV important.

The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.

As part of our efforts to develop bovine adenoviruses into a vector system, we sequenced the region predicted to encode the terminal protein precursor of either bovine adenoviruses type 2 and bovine adenoviruses type 3. We examined the regions containing the terminal protein precursors and showed that the bovine adenovirus early region 2B had an identical organization to all adenoviruses so far examined. The bovine adenovirus terminal protein precursors and those of other adenoviruses were also compared in a sequence alignment analysis and several highly conserved structural domains were identified. Finally, we showed how the various terminal protein precursors were related in a sequence pair similarity analysis and concluded that the terminal protein precursors of bovine adenoviruses types 2 and 3 are significantly different from each other at the protein sequence level.

Avian influenza A viruses (AIVs) often infect waterfowl, gulls and shorebirds, but other bird groups including pelagic seabirds also serve as hosts. In this study, we analyzed 21 AIVs found in two distant breeding colonies of Common Murre (Uria aalge) in Newfoundland and Labrador, Canada, during 2011. Phylogenetic analyses and genotype assignments were performed for the 21 Common Murre viruses together with all Common and Thick-billed Murre (Uria lomvia) AIV sequences available in public sequence databases. All fully characterized viruses from the Common Murres in 2011 were H1N2 subtype, but the genome sequences revealed greater diversity and the viruses belonged to four distinct genotypes. The four genotypes shared most segments in common, but reassortment was observed for PB2 and M segments. This provided direct genetic data of AIV diversification through segment reassortment during an outbreak of AIV infection in high-density breeding colonies. Analysis of the total collection of available murre viruses revealed a diverse collection of subtypes and gene lineages with high similarity to those found in viruses from waterfowl and gulls, and there was no indication of murre-specific AIV gene lineages. Overall, the virus gene pool in murres was predominantly made up of AIV lineages associated with waterfowl, but also featured considerable gull lineage genes and inter-continental reassortments. In particular, all but one of the 21 Common Murre viruses from 2011 in Newfoundland contained 1 or 2 Eurasian segments and 16 contained 1 gull lineage segment. This mosaic nature of characterized murre AIV genomes might reflect an under-recognized role of these pelagic seabirds in virus transmission across space and between bird host taxa.

Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.

A temporal study was carried out to determine Salmonella prevalence, trends, major serovars, and their clusters from environmental samples, in poultry breeder flocks in Ontario between January 1998 and December 2008. Surveillance data were obtained from the Ontario Hatchery and Supply Flock Policy. Logistic regression with a random effect for flock was used to identify factors [poultry type, year (trend) and season] associated with the prevalence of Salmonella. A cluster detection test was used to identify clusters of common serovars. The period prevalence of Salmonella was 47.4% in broiler-breeder, 25.7% in layer-breeder, and 19.6% in turkey-breeder flocks. The overall trend in the prevalence of Salmonella was decreasing for all breeder types, due primarily to decreasing trends of Salmonella Heidelberg. The seasonal effects varied by year with the highest probability of Salmonella occurring in different seasons. The 4 most common serovars identified were Salmonella Heidelberg, Kentucky, Hadar, and Typhimurium in broiler-breeders; Salmonella Heidelberg, Brandenburg, Thompson, and Typhimurium in layer-breeders; and Salmonella Heidelberg, Saintpaul, Brandenburg, and Muenster in turkey-breeders. Salmonella Enteritidis was infrequently isolated in all poultry breeder types. Temporal clusters of different serovars were identified in all poultry breeder types. Clusters of Salmonella Heidelberg, Typhimurium, and Hadar from environmental samples from breeder flocks were detected during a similar period to clusters from hatchery fluff samples from the same population. Therefore, interventions at the breeder flock-level might help to reduce transmission of Salmonella from breeder flocks to hatcheries and possibly, to lower levels of the poultry production chain.Une étude temporelle a été menée afin de déterminer la prévalence de

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

Raccoons (Procyon lotor) live at high densities, often in close association with people, in urban areas in Ontario and have been implicated as potential reservoirs of numerous zoonotic disease agents. We collected 137 blood samples from 61 apparently healthy raccoons in a small area of Toronto, Ontario, from June to October 2007 as part of a longitudinal study to determine the seasonal patterns of seroprevalence of Francisella tularensis, avian influenza, and Leptospira. In addition, we collected 35 urine samples by cystocentesis from 23 animals to look for evidence of urinary shedding of Leptospira. All samples were serologically negative for F. tularensis and avian influenza. Nineteen of 61 animals (31%) were positive for Leptospira antibodies in one or more trapping periods. The seroprevalence of Leptospira increased from 5% in June to 38% in October. Of the 19 positive animals, 14 were seropositive for serogroup Grippotyphosa, 4 for serogroup Pomona, and 1 for both serogroups Australis and Grippotyphosa. Raccoons were seronegative to serovars representative of serogroups Autumnalis, Canicola, Icterohaemorrhagiae, and Sejroe. Only one urine sample was culture positive for Leptospira (2.9%). Although we found evidence that raccoons in this study were exposed to leptospires belonging to serogroup Grippotyphosa, likely serovar Grippotyphosa, during the summer and able to shed leptospires in urine, further work is required to determine the importance of raccoons as reservoirs of Leptospira in Ontario.

The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.

Turkey coronavirus (TCoV) is an important viral pathogen causing diarrhoea of young turkey poults that is associated with sizeable economic losses for the turkey industry. Using a field isolate that was found to be free from turkey astrovirus and avian reovirus we were able to reproduce the clinical disease associated with TCoV. Clinical signs and weight gain of poults during experimental infections were compared with age-matched, uninfected controls. Poults infected at 2 days of age had 100% morbidity and 10% mortality, and birds infected at 28 days of age showed 75% morbidity and no mortality. Diarrhoea was consistently seen in infected poults at 2 to 3 days post infection (d.p.i.) with a duration of about 3 to 5 days. Mean body weights of birds infected at 2 or 28 days of age were significantly reduced compared with uninfected birds by 7 d.p.i. and remained significantly lower for the duration of the study. At 44 days of age, poults infected at 2 or 28 days of age weighed only 68.1% or 77.7%, respectively, compared with uninfected turkeys of the same age on the same diet, a mean difference in body weights of 683 or 477g, respectively. Infected birds had profound villus atrophy with some compensatory crypt hyperplasia at 5 to 7 d.p.i. Villus heights in the duodenum were significantly reduced at 7 d.p.i. We were able to reproduce enteric disease using only a pathogenic field isolate (MG10) of TCoV that negatively affected growth performance and intestinal morphology of young turkey poults.

Background Since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. Both SARS Coronavirus which quickly spread to 29 countries in February 2003 and H1N1 swine influenza that recently spread from Mexico to 30 countries in three weeks represent major pandemic threats for mankind. Diagnostic assays are required to detect novel influenza strains with pandemic potential. Objective In this report we evaluate the ability of a multiplex PCR test (xTAG™ RVP) to detect new, “non-seasonal” influenza viruses including the H1N1 swine influenza A/swine/California/04/2009. Study design Laboratory based study using retrospective and prospective specimens. Results This multiplex PCR test detected the present of non-seasonal (non-H1, non-H3) influenza in 20 of 20 patients infected with H1N1 swine flu virus. In addition to detecting the current swine flu the xTAG™ RVP test detected the H5N1 A/Vietnam/1203/2004 high pathogenicity avian influenza virus that circulated in South East Asia in 2003 as well as 17 out of 17 influenza A viruses representing 11 HA subtypes isolated from birds, swine and horses not yet seen in the human population. Conclusion Based on these results we believe that this molecular test can perform an important role as a sentinel test to detect novel non-seasonal influenza A viruses in patients presenting with influenza-like illness (ILI) and therefore act as an early warning system for the detection of future pandemic influenza threats.

Turkey coronavirus (TCoV) causes diarrhoea in young turkey poults but little is known about its prevalence in the field. To address this, a portion of the S1 region of the spike glycoprotein of TCoV carrying relevant B cell epitopes (amino acid positions 54-395) was cloned and expressed in Escherichia coli. This protein was purified and used to develop an indirect ELISA for detection of antibodies against TCoV. Using experimentally derived positive and negative turkey serum samples this ELISA showed high sensitivity (95%) and specificity (92%) for TCoV. To further evaluate the potential of the ELISA, 360 serum samples from commercial turkey farms in Ontario were tested for TCoV-specific antibodies using the recombinant TCoV ELISA. High seroprevalence of TCoV was found with 71.11% of breeders and 56.67% of meat turkeys testing seropositive. Although there was significant positive correlation with a TCoV-N protein-based ELISA, there was little to no correlation with the whole IBV antigen-based ELISA when field sera were tested for antibodies against TCoV.

The objective of this study was to estimate the apparent and true prevalence of exposure to Toxoplasma gondii in Ontario finisher pigs. During the study period (2001 to 2004), sera from 6048 pigs were tested with a commercial enzyme-linked immunosorbent assay (ELISA); 103 farms were included 1 to 3 times in the study. True prevalence was estimated using a Bayesian approach. Apparent prevalence at the pig level was 1.59% [95% confidence interval (CI): 0.45, 2.99] in 2001, 0.06% (95% CI: 0.00, 0.46) in 2003, and 0.26% (95% CI: 0.00, 0.82) in 2004. Apparent prevalence at the herd-level was 13.7% (95% CI: 7.5, 22.3) in 2001; 1.25% (95% CI: 0.03, 6.77) in 2003, and 3.75% (95% CI: 0.78, 10.6) in 2004. Similarly, posterior Bayesian estimates of true prevalence at the pig level were 1.7% [95% probability interval (PI): 1.2, 2.2] in 2001, 0.2% (95% PI: 0.04, 0.4) in 2003, and 0.3% (95% PI: 0.1, 0.7) in 2004. At the herd level, posterior estimates of prevalence were 11.6% (95% PI: 7.4, 16.8) in 2001, 0% (95% PI: 0.0, 2.5) in 2003, and 1.2% (95% PI: 0.0, 5.0) in 2004 when a herd cut-pointor = 1 was used. Exposure to T. gondii in finishing pig farms in Ontario appears to be infrequent.

Of 4,268 wild ducks sampled in Canada in 2005, real-time reverse transcriptase–PCR detected influenza A matrix protein (M1) gene sequence in 37% and H5 gene sequence in 5%. Mallards accounted for 61% of samples, 73% of M1-positive ducks, and 90% of H5-positive ducks. Ducks hatched in 2005 accounted for 80% of the sample.

Canine leptospirosis has been described as having re-emerged in North America around the mid-1990s, with a change in the epidemiology of the infecting serovars responsible for the disease emergence. A retrospective case-control study was conducted to examine the re-emergence of seroprevalent cases of canine leptospirosis in Ontario using serology submission records from 1406 dogs from January 1, 1998 to December 31, 2006. The data collected [results of the microscopic agglutination test (MAT), veterinary clinic postal code, age, sex, neutering status, and breed] were analyzed by multivariable logistic regression, generalized linear mixed modeling, and Cochran-Armitage test for trends in proportions. Dogs in urban areas appeared to be at significantly higher risk than dogs in rural areas for the entire study period [odds ratio (OR) = 1.6, confidence interval (CI) = 1.2–2.3], though this was not as marked as in other studies. Results indicated that canine leptospirosis in Ontario is a disease of all breeds and ages, regardless of gender. No geographic clustering was noted, but clustering of cases by clinic within geographic areas suggested differences in awareness or in diagnosis by veterinarians. A distinctive seasonal pattern of leptospirosis, with more cases occurring during the summer and fall, as found in previous studies, was also observed in this study. The temporal trend analysis was consistent with an increasing proportion or re-emergence of seroprevalent cases of canine leptospirosis since 1998, suggesting that the putative increase in canine leptospirosis has been genuine.

Five hundred and seventy-three clinical submissions with fowl adenovirus (FAdV) involvement were examined to investigate the association of different types of FAdV with clinical problems related to FAdV infection. Samples were received from 2000 to 2006 and originated from seven Canadian provinces. Four hundred and eighty-seven submissions were inclusion body hepatitis (IBH) related, while 86 were not IBH related. Viruses isolated from 287 samples were further analysed by hexon gene loop 1 sequencing. Twenty-seven genotyped FAdVs were from Alberta, 20 from British Columbia, 16 from Manitoba, one from Nova Scotia, 82 from Ontario, 64 from Quebec and 77 from Saskatchewan. Two hundred and forty-six analysed FAdVs were from IBH cases, confirmed by liver histopathology, by FAdV isolation from the liver, or both. Based on hexon gene loop 1 sequencing analysis, FAdVs associated with IBH outbreaks were genetically related to FAdV02 (nine isolates, 99.4%), FAdV08a (100 isolates, 99.4% to 100%) and FAdV11 (98 isolates, 99.4% to 100%). Thirty-nine viruses were 93.7% to 94.3% identical to FAdV07 strain x11a, but the genetic and immunogenic properties of this strain require further investigation. In IBH cases, the co-infection rates for infectious bursal disease virus, infectious bronchitis virus, reoviruses and Newcastle disease virus were 3.47%, 1.04%, 6.25% and 0.69%, respectively. Forty-one genotyped FAdVs were from "non-IBH" cases. Viruses isolated from non-IBH cases consisted of 22 FAdV01, 15 FAdV11, two FAdV08a and one each of FAdV02 and FAdV04 viruses. Co-infection rates in non-IBH submissions were 50.00% for infectious bursal disease virus, 40.70% for infectious bronchitis virus, 27.91% for reoviruses and 1.16% for Newcastle disease virus.

In recent years inclusion body hepatitis (IBH) has emerged as an economically important disease in Western Canada. Historically, infections with infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) have been known to suppress the immune system of broilers and make them more susceptible to a secondary disease such as IBH. Recently it has been reported that virulent adenoviruses are able to cause IBH as a primary disease in broilers without apparent involvement of IBDV and CAV. The objectives of this study were to examine the possible association of IBH with IBDV and CAV infections in Western Canada and to identify adenoviruses involved in outbreaks. Serum samples from 17 broiler-breeder flocks and their progeny were collected when broilers were hatched and then again from broilers at the time of slaughter, and these samples were tested for IBDV and CAV antibodies by enzyme-linked immunosorbent assay (ELISA). Based on the ELISA titers the antibody response to vaccination against IBDV and CAV was at an expected level in all broiler flocks. Therefore, IBH outbreaks in these flocks were not due to inadequate levels of antibodies against IBDV and CAV. Moreover, there was no correlation found between occurrences of IBH outbreaks in broilers and their IBDV or CAV titers at the time of processing. Viruses that were isolated from livers of birds suffering from IBH could be classified into four different genotypes. Their hexon gene loop 1 sequences showed high percentages of identity to FAdV-7, FAdV-8a, FAdV-8b, and FAdV-11. The results of this study could not demonstrate an association of IBH with IBDV and CAV infections, but they supported the hypothesis that IBH in broilers in Western Canada is a primary disease with no apparent immunosuppressive involvement.