Whole Genome Sequencing of a Canadian Bovine Gammaherpesvirus 4 Strain and the Possible Link between the Viral Infection and Respiratory and Reproductive Clinical Manifestations in Dairy Cattle.

Bovine gammaherpesvirus 4 (BoHV-4) is a herpesvirus widespread in cattle populations, and with no clear disease association. Its genome contains a long unique coding region (LUR) flanked by polyrepetitive DNA and 79 open reading frames (ORFs), with unique 17 ORFs, named Bo1 to Bo17. In 2009, a BoHV-4 strain was isolated (FMV09-1180503: BoHV-4-FMV) from cattle with respiratory disease from Quebec, Canada, and its LUR was sequenced. Despite the overall high similarity, BoHV-4-FMV had the most divergent LUR sequence compared to the two known BoHV-4 reference strain genomes; most of the divergences were in the Bo genes and in the repeat regions. Our phylogenetic analysis based on DNA polymerase and thymidine kinase genes revealed that virus isolate was BoHV-4 gammaherpesvirus and clustered it together with European BoHV-4 strains. Because BoHV-4-FMV was isolated from animals presenting respiratory signs, we have updated the BoHV-4 Canadian cattle seroprevalence data and tried to find out whether there is a link between clinical manifestation and BoHV-4 seropositivity. An indirect immunofluorescence assay (IFA) was performed with nearly 200 randomized sera of dairy cattle from two Canadian provinces, Quebec (n = 100) and Ontario (n = 91). An additional set of sera obtained from Quebec, from the healthy (n = 48) cows or from the animals experiencing respiratory or reproductive problems (n = 75), was also analyzed by IFA. BoHV-4 seroprevalence in Canadian dairy cattle was 7.9% (Quebec: 6% and Ontario: 9.9%). Among animals from the Quebec-based farms, diseased animals showed higher BoHV-4 seropositivity than healthy animals (P < 0.05), with a significant 2.494 odds ratio of being seropositive in sick compared to healthy animals. Although there is no established direct link between BoHV-4 and specific diseases, these seroprevalence data suggest the possible involvement of BoHV-4 in dairy cattle diseases.

Characterization of a novel adenovirus isolated from a skunk.

Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus.

Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012.

The objective of this study was to characterize H1N1 and H1N2 influenza A virus isolates detected during outbreaks of respiratory disease in pig herds in Ontario (Canada) in 2012. Six influenza viruses were included in analysis using full genome sequencing based on the 454 platform. In five H1N1 isolates, all eight segments were genetically related to 2009 pandemic virus (A(H1N1)pdm09). One H1N2 isolate had hemagglutinin (HA), polymerase A (PA) and non-structural (NS) genes closely related to A(H1N1)pdm09, and neuraminidase (NA), matrix (M), polymerase B1 (PB1), polymerase B2 (PB2), and nucleoprotein (NP) genes originating from a triple-reassortant H3N2 virus (tr H3N2). The HA gene of five Ontario H1 isolates exhibited high identity of 99% with the human A(H1N1)pdm09 [A/Mexico/InDRE4487/09] from Mexico, while one Ontario H1N1 isolate had only 96.9% identity with this Mexican virus. Each of the five Ontario H1N1 viruses had between one and four amino acid (aa) changes within five antigenic sites, while one Ontario H1N2 virus had two aa changes within two antigenic sites. Such aa changes in antigenic sites could have an effect on antibody recognition and ultimately have implications for immunization practices. According to aa sequence analysis of the M2 protein, Ontario H1N1 and H1N2 viruses can be expected to offer resistance to adamantane derivatives, but not to neuraminidase inhibitors.

Equid herpesvirus 9 (EHV-9) isolates from zebras in Ontario, Canada, 1989 to 2007.

The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.

L'objectif de la présente étude était d'identifier et de caractériser partiellement trois herpesvirus équins isolés de zèbres décédés en Ontario, Canada en 1989, 2002, et 2007. Ces trois isolats viraux furent caractérisés par morphologie des plages de lyse, par polymorphisme de taille des fragments de restriction (RFLP) de leur ADN génomique, par épreuve de réaction d'amplification en chaîne par la polymérase (PCR) en temps réel, et analyse de la séquence de la toute la longueur du gène de la glycoprotéine G (gG) (ORF70) et une portion du gène de la polymérase de l'ADN (ORF30). Les isolats furent également comparés à trois souches de référence d'herpesvirus équin de type 1 (EHV-1). L'examen de la culture des virus sur des cellules rénales de lapin a permis de constater que les plages de lyse causées par les isolats provenant des zèbres étaient beaucoup plus grandes que celles causées par les souches de référence d'EHV-1. Les patrons de RFLP des virus de zèbres différaient entre eux ainsi que des souches de référence d'EHV-1. Les analyses par PCR en temps réel et l'analyse de séquence d'une portion du gène de la polymérase de l'ADN ont permis de déterminer que les isolats d'herpesvirus provenant de zèbres avaient un G comme nucléotide à la position 2254 et un acide aminé N correspondant à la position 752, ce qui suggère qu'il pourrait s'agir de souches neuropathogènes d'EHV-1. Toutefois, des analyses phylogénétiques subséquentes du gène gG suggèrent qu'il s'agirait plutôt d'EHV-9 et non d'EHV-1.(Traduit par Docteur Serge Messier).

Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012.

BACKGROUND: Data about molecular diversity of commonly circulating type A influenza viruses in Ontario swine are scarce. Yet, this information is essential for surveillance of animal and public health, vaccine updates, and for understanding virus evolution and its large-scale spread. METHODS: The study population consisted of 21 swine herds with clinical problems due to respiratory disease. Nasal swabs from individual pigs were collected and tested by virus isolation in MDCK cells and by rtRT-PCR. All eight segments of 10 H3N2 viruses were sequenced using high-throughput sequencing and molecularly characterized. RESULTS: Within-herd prevalence ranged between 2 and 100%. Structurally, Ontario H3N2 viruses could be classified into three different groups. Group 1 was the most similar to the original trH3N2 virus from 2005. Group 2 was the most similar to the Ontario turkey H3N2 isolates with PB1 and NS genes originating from trH3N2 virus and M, PB2, PA and NP genes originating from the A(H1N1)pdm09 virus. All Group 3 internal genes were genetically related to A(H1N1)pdm09. Analysis of antigenic sites of HA1 showed that Group 1 had 8 aa changes within 4 antigenic sites, A(1), B(3), C(2) and E(2). The Group 2 viruses had 8 aa changes within 3 antigenic sites A(3), B(3) and C(2), while Group 3 viruses had 4 aa changes within 3 antigenic sites, B(1), D(1) and E(2), when compared to the cluster IV H3N2 virus [A/swine/Ontario/33853/2005/(H3N2)]. CONCLUSIONS: The characterization of the Ontario H3N2 viruses clearly indicates reassortment of gene segments between the North American swine trH3N2 from cluster IV and the A(H1N1)pdm09 virus.

Assessment of seasonality of influenza in swine using field submissions to a diagnostic laboratory in Ontario between 2007 and 2012.

BACKGROUND: Seasonality of any infectious disease is important for its control and monitoring. While influenza seasonality in people has been evaluated extensively, this question has not been studied well in swine populations. OBJECTIVE: The goal of this study was to investigate seasonality of influenza in swine, using diagnostic submissions to a diagnostic laboratory. METHODS: Two thousand seven hundred and eleven virological tests within 685 submissions and 5471 serological tests within 193 submissions in Ontario swine between 2007 and 2012 were included in the study and converted to total monthly number of virological and serological submissions, and the number of positive submissions. Data were analyzed by time-series decomposition, fixed-effect Poisson, random-effect Poisson regression with month as uncorrelated and correlated random effects. RESULTS: All approaches identified seasonality in virological submissions (P < 0.02) with peak in January and April, and a trough in July, but were not able to detect seasonality of influenza-positive virological submissions (P > 0.13). Seasonality of positive serological submissions was identified only if independence between months was assumed (P < 0.03). Almost 50% of serological submissions had evidence of exposure to H3N2 and H1N1. CONCLUSIONS: Thus, this study identified evidence of seasonality in influenza-like disease in swine herds, but not in circulation of influenza virus. Evidence of seasonality in exposure to influenza was dependent on assumptions of between-month correlation. High exposure to H3N2 and H1N1 subtypes warrants more detailed investigation of within-herd influenza virus circulation. The study provides initial insight into seasonality of influenza in swine and should be followed with herd-level studies.

Experimental infection of New Zealand white rabbits (Oryctolagus cuniculi) with Leporid herpesvirus 4.

Leporid herpesvirus 4 (LHV4) is a novel alphaherpesvirus recently identified in domestic rabbits (Oryctolagus cuniculi). Little is known about the pathogenesis or time course of disease induced by this virus. We therefore intranasally inoculated 22 female New Zealand white rabbits with 8.4 × 10(4) CCID50 of a clinical viral isolate. Rabbits were monitored for clinical signs, viral shedding in oculonasal secretions, and development and persistence of serum antibodies. Rabbits were euthanized at 3, 5, 7, 14, and 22 d postinfection (dpi) to evaluate gross and microscopic changes. Clinical signs were apparent between 3 to 8 dpi, and included oculonasal discharge, respiratory distress, and reduced appetite, and viral shedding occurred between 2 and 8 dpi. Seroconversion was seen at 11 dpi and persisted to the end of the study (day 22). Severe necrohemorrhagic bronchopneumonia and marked pulmonary edema were noted by 5 dpi and were most severe at 7 dpi. Pulmonary changes largely resolved by 22 dpi. In addition, multifocal splenic necrosis was present at 5 dpi and progressed to submassive necrosis by 7 dpi. Eosinophilic herpesviral intranuclear inclusion bodies were detected in the nasal mucosa, skin, spleen, and lung between 3 to 14 dpi. LHV4 is a pathogen that should be considered for rabbits that present with acute respiratory disease. LHV4 infection can be diagnosed based on characteristic microscopic changes in the lungs and spleen and by virus isolation. Serum antibody levels may be used to monitor viral prevalence in colonies.

The spread of type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in North America: a phylogeographic approach.

The emergence and spread of Type 2 Porcine Reproductive and Respiratory Syndrome virus (Type 2 PRRSV) in North America is heavily influenced by the multiple site production system used in the hog industry. However, it is unclear how anthropogenic factors such has this have shaped the current spatial distribution of PRRSV genotypes. We employed Bayesian phylogeographic analyses of 7040 ORF5 sequences to reveal the recent geographical spread of Type 2 PRRSV in North America. The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada into the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.

A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens.

From 2009 to 2011, 163 sheep and 96 goat abortion submissions were received at the Animal Health Laboratory, University of Guelph, Ontario, Canada, for gross and histologic examination, as well as real-time polymerase chain reaction (PCR) testing for Chlamydophila abortus and/or Coxiella burnetii. Additional testing included immunohistochemistry for Toxoplasma gondii and Chlamydophila spp., routine bacterial culture and selective culture for Campylobacter spp., examination of modified acid-fast-stained placenta smears, enzyme-linked immunosorbent assay testing for Chlamydophila spp., and virus isolation. The final diagnosis made for each case by individual pathologists, based on gross and histologic lesions, as well as ancillary testing, was used as a standard to determine the significance of C. abortus and C. burnetii infection. Coxiella burnetii was identified by real-time PCR in 113 of 163 (69.0%) and 72 of 96 (75%) sheep and goat abortion submissions, respectively, but was considered to be significant in causing abortion in only 11 of 113 (10%) sheep and 15 out of 72 (21%) goat submissions that tested positive. Chlamydophila abortus was identified by real-time PCR in 42 of 162 (26%) and 54 of 92 (59%) sheep and goat submissions, respectively, but was considered the cause of the abortion in 16 of 42 (38%) sheep and 34 of 54 (63%) goat submissions that tested positive. Optimal sensitivity and specificity cut points for the real-time PCR copy number for C. abortus and C. burnetii were determined using the final pathology diagnosis as the reference test.

Microbiological identification and analysis of swine tonsils collected from carcasses at slaughter.

The primary objective of this 7-month study was to determine the prevalence of porcine pathogens of the tonsil of the soft palate of swine at slaughter. Additional objectives were to determine if sampling the carcasses of normal or abnormal hogs provided different microbiological profiles and if the slaughter plant provides a feasible sampling frame and environment for detecting and monitoring important pathogens in tonsils that have health implications for both swine and humans. A total of 395 samples were collected from 264 farms. Of these, 180 tonsils were collected from normal carcasses and 215 tonsils were collected from carcasses that were diverted to the hold rail. Laboratory testing included bacteriological culture and identification as well as real time-polymerase chain reaction (PCR) testing for porcine reproductive and respiratory syndrome virus (PPRSV) and immunohistochemistry (IHC) for porcine circovirus-2 (PCV-2). The most commonly isolated bacteria included: Streptococcus suis (53.7%), Arcanobacterium pyogenes (29.9%), Pasteurella multocida (27.3%), and Streptococcus porcinus (19.5%). Virus screening revealed evidence of PRRSV and PCV-2 in 22.0% and 11.9% of the samples, respectively. Salmonella Typhimurium and Yersinia enterocolitica were isolated in 0.5% and 1.8% of the samples, respectively. Tonsils collected from the hold rail were more likely to be positive for Staphylococcus hyicus [odds ratio (OR) = 7.51, confidence interval (CI) = 2.89 to 19.54], Streptococcus porcinus (OR = 9.93, CI = 4.27 to 23.10), and Streptococcus suis (OR = 2.16, CI = 1.45 to 3.24). Tonsils collected from abnormal carcasses were less likely to be positive for Staphylococcus aureus (OR = 0.05, CI = 0.005 to 0.482).

Interlaboratory comparison of Porcine circovirus-2 indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay results on experimentally infected pigs.

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n  =  7), 2) PCV-2a (n  =  8), 3) PCV-2b (n  =  8), 4) PCV-1 (n  =  8), 5) PCV-2 vaccine A (n  =  8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B (n  =  8; Circumvent® PCV2), and 7) PCV-2 vaccine C (n  =  8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti-PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14-49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.

Porcine reproductive and respiratory syndrome virus in Ontario, Canada 1999 to 2010: genetic diversity and restriction fragment length polymorphisms.

Classification of Ontario porcine reproductive and respiratory syndrome virus (PRRSV) field isolates (n = 505) from 1999 to 2010, based on a global type 2 PRRSV ORF5 phylogenetic framework, revealed genetic diversity comparable to PRRSV in the USA, with sequences assigned to five of nine lineages (1, 2, 5, 8 and 9). Importantly, the tree topology indicated a Canadian ancestry for the highly virulent MN184-related strains that first emerged in 2001 in the USA. Mapping of the RFLP patterns onto the phylogenetic tree revealed numerous examples of different RFLP patterns located within the same phylogenetic cluster. Statistical analysis showed occurrences where similar RFLP patterns masked diverse genetic distances and instances of close genetic proximity with divergent RFLP patterns. Collectively, extensive genetic diversity prevails in type 2 PRRSV in one region of the North American swine industry, and it is not described adequately by RFLP typing, which might have value in differentiating strains at the local farm level.

Seroprevalence of bovine viral diarrhea virus neutralizing antibodies in finisher hogs in Ontario swine herds and targeted diagnostic testing of 2 suspect herds.

A pilot study was initiated to determine the seroprevalence of bovine viral diarrhea virus (BVDV) neutralizing antibodies in finisher hogs in Ontario swine herds, including 2 swine herds with clinical syndromes suspicious of BVDV. No herds were positive for BVDV antibodies by virus neutralization. The 2 swine herds with clinical disease suggestive of pestivirus infection were also negative for antibodies to BVDV in indirect fluorescent antibody assays. Prevalence of BVDV in Ontario swine farms is negligible.

Clinical signs and their association with herd demographics and porcine reproductive and respiratory syndrome (PRRS) control strategies in PRRS PCR-positive swine herds in Ontario.

The purposes of this study were to describe the clinical signs observed in PRRS positive herds during a porcine reproductive and respiratory syndrome (PRRS) outbreak in Ontario and to determine associations between these clinical signs and herd demographics and PRRS control strategies. All PRRS polymerase chain reaction-(PCR)-positive submissions to a diagnostic laboratory between September 1, 2004 and August 31, 2007 were identified (n = 1864). After meeting eligibility requirements and agreeing to voluntary study participation, producers from 455 of these submissions were surveyed for information on clinical signs observed in their herds, herd demographics, and PRRS control strategies used in their herds at the time that the PCR-positive samples were taken. Larger herd size was associated with an increased risk of reporting abortion, weakborn piglets, off-feed sows, and sow mortality in sow herds, and with an increased risk of reporting mortality in finishing herds. When disease control strategies were examined, use of a commercial PRRS vaccine in sows and gilts was associated with a decreased risk of reporting weakborn pigs and high pre-weaning mortality, while the use of serum inoculation in breeding animals was associated with an increased risk of reporting off-feed sows and sow mortality. Providing biofeedback of stillborn/mummified piglets, placenta or feces to gilts was associated with an increased risk of reporting respiratory disease and mortality in finishing pigs while all-in/all-out flow in farrowing rooms was associated with an increased risk of reporting sow mortality and weakborn piglets.

Distribution of genotypes of porcine reproductive and respiratory syndrome virus in Ontario during 2004-2007 and the association between genotype and clinical signs of disease.

Restriction fragment length polymorphism (RFLP) was first proposed to classify porcine reproductive and respiratory syndrome virus (PRRSV) in 1998. The primary objective of this study was to identify associations between different PRRSV RFLP types in swine herds in southern Ontario and clinical signs of disease in those herds. Herds included in the study submitted samples to the Animal Health Laboratory at the University of Guelph between September 2004 and August 2007. Each farm owner was surveyed to describe the clinical disease in the herd and the RFLP pattern of an isolate of PRRSV was obtained from a diagnostic sample. The most frequent isolates were RFLP types 1_4 (25.1%), 252 (14.7%), 134 (12%), and 1_2 (7.7%). The distribution of RFLP types in this study was found to be different from a previous investigation in Ontario. Those RFLP types most associated with clinical disease in the farrowing phase of production were 1_4, 1_2, and 134. The only virus type to be significantly associated with disease in the finisher phase was RFLP type 262. During the study period RFLP type 184 emerged in the population in November 2005.

Surveillance of equine respiratory viruses in Ontario.

The objective of this project was to develop and implement an active surveillance program for the early and rapid detection of equine influenza viruses in Ontario. For this purpose, from October 2003 to October 2005, nasopharyngeal swabs and acute and convalescent serum samples were collected from 115 client-owned horses in 23 outbreaks of respiratory disease in Ontario. Sera were paired and tested for antibody to equine influenza 1 (AE1-H7N7), equine influenza 2 (AE2-H3N8), equine herpesvirus 1 and 4 (EHV1 and EHV4), and equine rhinitis A and B (ERAV and ERBV). Overall, the cause-specific morbidity rate of equine influenza virus in the respiratory outbreaks was 56.5% as determined by the single radial hemolysis (SRH) test. The AE2-H3N8 was isolated from 15 horses in 5 outbreaks. A 4-fold increase in antibody levels or the presence of a high titer against ERAV or ERBV was observed in 10 out of 13 outbreaks in which AE2-H3N8 was diagnosed as the primary cause of disease. In conclusion, AE2-H3N8 was found to be an important contributor to equine respiratory viral disease. Equine rhinitis A and B (ERAV and ERBV) represented an important component in the equine respiratory disease of performing horses.

Acute hemorrhagic and necrotizing pneumonia, splenitis, and dermatitis in a pet rabbit caused by a novel herpesvirus (leporid herpesvirus-4).

A 1.5-year-old female rabbit (doe) was presented with a 3-day history of lethargy, anorexia, and mild facial swelling. The animal died shortly after examination and severe, acute hemorrhagic pneumonia was noted grossly. An alphaherpesvirus consistent with leporid herpesvirus-4 was isolated and characterized from this animal. This is the first confirmed report of the disease in Canada.

Serologic cross-reactivity with pandemic (H1N1) 2009 virus in pigs, Europe.

We tested serum samples from pigs infected or vaccinated with European swine influenza viruses (SIVs) in hemagglutination-inhibition assays against pandemic (H1N1) 2009 virus and related North American SIVs. We found more serologic cross-reaction than expected. Data suggest pigs in Europe may have partial immunity to pandemic (H1N1) 2009 virus.

Seroprevalence of antibodies to canine influenza virus in dogs in Ontario.

A cross-sectional study evaluating the seroprevalence of antibodies to canine influenza virus in dogs in Ontario was performed. The prevalence was 0.4% (1/225), and the only seropositive dog was a greyhound that originated in Florida.

The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease--2004-2006.

In the late fall of 2004 more severe lesions of porcine circovirus-2 associated disease (PCVAD) than usual occurred during an outbreak of porcine circovirus-2 (PCV-2) infection in Ontario nursery and grower/finisher pigs. The lesions were of unprecedented severity and included diffuse bronchointerstitial pneumonia, granulomatous enteritis, vasculitis, interstitial nephritis, and new lesions of splenic infarction. Some affected herds had up to 50% mortality. The outbreak correlated with the sudden emergence of a variant PCV-2, with PCR restriction fragment length polymorphism (RFLP) type 321. Phylogenetic comparison of ORF2 sequences and full genome sequences showed the new variant to be different from the previously dominant RFLP type 422 viruses, and similar to viruses that had occurred in France and other European and Asian countries. A subsequent retrospective study showed a statistically significant increase in the frequency of histological lesions in lymph node, spleen, lung, small intestine, colon and kidney, for pigs spontaneously infected with RFLP type 321, compared with the older RFLP type 422 strain. Viral burden, based on IHC staining in lymph node, also showed a statistically significant increase in pigs infected with the newer variant RFLP type 321, compared with the older RFLP type 422 strain. This enhanced virulence in pigs infected with PCV-2 RFLP type 321 strain may be related to the genetic differences in this new strain of PCV-2. This virus is now the dominant strain of PCV-2 virus found in Ontario and Quebec swine.