Impact of alignment algorithm on the estimation of pairwise genetic similarity of porcine reproductive and respiratory syndrome virus (PRRSV).

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a major threat to the swine industry. It is caused by the PRRS virus (PRRSV). Determination and comparison of the nucleotide sequences of PRRSV strains provides useful information in support of control initiatives or epidemiological studies on transmission patterns. The alignment of sequences is the first step in analyzing sequence data, with multiple algorithms being available, but little is known on the impact of this methodological choice. Here, a study was conducted to evaluate the impact of different alignment algorithms on the resulting aligned sequence dataset and on practical issues when applied to a large field database of PRRSV open reading frame (ORF) 5 sequences collected in Quebec, Canada, from 2010 to 2014. Five multiple sequence alignment programs were compared: Clustal W, Clustal Omega, Muscle, T-Coffee and MAFFT. RESULTS: The resulting alignments showed very similar results in terms of average pairwise genetic similarity, proportion of pairwise comparisons having ≥97.5% genetic similarity and sum of pairs (SP) score, except for T-Coffee where increased length of aligned datasets as well as limitation to handle large datasets were observed. CONCLUSIONS: Based on efficiency at minimizing the number of gaps in different dataset sizes with default open gap values as well as the capability to handle a large number of sequences in a timely manner, the use of Clustal Omega might be recommended for the management of PRRSV extensive database for both research and surveillance purposes.

A descriptive study on spatial and temporal distributions of genetic clusters of porcine reproductive and respiratory syndrome virus infecting pig sites in Quebec, Canada, between 2010 and 2019.

BACKGROUND: The wide diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains combined with incomplete heterologous cross-protection complicates the management of the disease at both the herd and the regional levels. The objectives of this study were to describe the spatial and temporal distribution of various PRRSV genetic clusters infecting pig sites in Quebec, Canada, and to compare PRRSV regional diversity of wild-type sequences over the years. MATERIALS AND METHODS: A retrospective surveillance-based study was conducted on all pig sites which had PRRSV ORF5 sequences from field submissions transferred into the Laboratoire d'épidémiologie et de médecine porcine database from January 1, 2010 to December 31, 2019. A maximum likelihood phylogenetic tree inferred from multiple sequence alignment was used to identify genetic clusters. For each wild-type cluster gathering ≥ 15 sequences, the number of pig sites in which the cluster was detected per administrative region and per year were displayed on bubble charts and the spatiotemporal distribution of pig sites was illustrated using pie chart maps. A molecular analysis of variance was performed to compare PRRSV wild-type sequence diversity according to the administrative region for each year. RESULTS: A total of 32 wild-type clusters gathering 1653 PRRSV2 sequences from 693 pig sites were described. Each cluster was detected on up to 132 pig sites and 7 administrative regions over the 10-year period. Annually, the mean (min-max) number of wild-type clusters detected in at least one pig site reached 24 (17-29). Some clusters remained localized on a few sites over time whereas others were widespread over the territory during a few or many years. For each year, regional differences were also observed in PRRSV diversity of wild-type sequences. CONCLUSIONS: The differences observed in both the spatiotemporal distributions of PRRSV clusters and in the regional diversity of wild-type sequences highlight the importance of ongoing provincial surveillance to improve collective PRRS management strategies.

Correlation among genetic, Euclidean, temporal, and herd ownership distances of porcine reproductive and respiratory syndrome virus strains in Quebec, Canada.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has a major economic impact for the swine industry. Its control is mostly directed towards preventing its spread which requires a better understanding of the mechanisms of transmission of the virus between herds. The objectives of this study were to describe the genetic diversity and to assess the correlation among genetic, Euclidean and temporal distances and ownership to better understand pathways of transmission. RESULTS: A cross-sectional study was conducted on sites located in a high density area of swine production in Quebec. Geographical coordinates (longitude/latitude), date of submission and ownership were obtained for each site. ORF5 sequencing was attempted on PRRSV positive sites. Proportion of pairwise combinations of strains having ≥98% genetic homology were analysed according to Euclidean distances and ownership. Correlations between genetic, Euclidean and temporal distances and ownership were assessed using Mantel tests on continuous and binary matrices. Sensitivity of the correlations between genetic and Euclidean as well as temporal distances was evaluated for different Euclidean and temporal distance thresholds. An ORF5 sequence was identified for 132 of the 176 (75%) PRRSV positive sites; 122 were wild-type strains. The mean (min-max) genetic, Euclidean and temporal pairwise distances were 11.6% (0-18.7), 15.0 km (0.04-45.7) and 218 days (0-852), respectively. Significant positive correlations were observed between genetic and ownership, genetic and Euclidean and between genetic and temporal binary distances. The relationship between genetic and ownership suggests either common sources of animals or semen, employees, technical services or vehicles, whereas that between genetic and Euclidean binary distances is compatible with area spread of the virus. The latter correlation was observed only up to 5 km. CONCLUSIONS: This study suggests that transmission of PRRSV is likely to occur between sites belonging to the same owner or through area spread within a 5 km distance. Both should be considered in the perspective of prevention.

Alopecia areata and humpy-back syndrome in suckling piglets.

This report describes an uncommon variant of humpy-back syndrome associated with multiple rib fractures and multisystemic vasculitis in several nursing piglets and, for the first time, a skin disease in swine consistent with alopecia areata. Both conditions were observed concurrently on the farm and occasionally in the same piglets.

RésuméAlopécie areata et syndrome du porc bossu chez des porcelets à la mamelle. Les auteurs rapportent chez plusieurs porcelets sous la mère une variante peu fréquente du syndrome de lordose et xyphose associée à des fractures costales multiples et à une vasculite multisystémique et, pour la première fois chez le porc, une maladie cutanée compatible avec l'alopécie areata. Les 2 conditions ont été observées simultanément sur la ferme et même chez certains porcelets.(Traduit par les auteurs).

Risk factors at slaughter associated with presence of Salmonella on hog carcasses in Canada.

Despite the application of hazard analysis and critical control point systems at slaughter and during processing, Salmonella contamination is still a significant biological hazard associated with pork products. A better understanding of risk factors in slaughterhouses and of contamination sources is therefore critical to improve control of this bacterium in the abattoirs. The objectives of this study were to identify the risk factors at slaughter that are associated with the presence of Salmonella on hog carcasses and to assess possible sources of contamination. A questionnaire on potential risk factors was developed. Over 7,400 hogs originating from 312 randomly selected production lots were tested. The lots were from 10 different abattoirs located in five different Canadian provinces. At slaughter, blood was collected for serological analysis, and mesenteric lymph nodes (MLN) and carcass swabs were collected for Salmonella analysis. Furthermore, pulsed-field gel electrophoresis was conducted to establish the genetic profiles of selected isolates from carcasses and MLN and to compare these profiles with those recovered from the slaughter environment. Multivariate regression analysis results indicated that the cleanliness of the hogs and the status of the scald water were factors significantly associated with the Salmonella status of the carcasses at the end of the slaughter process. Pulsed-field gel electrophoresis analysis showed that most isolates from carcasses were similar to those from animals (MLN) or the preevisceration environment.

Positioning Quebec ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) within Canada and worldwide diversity.

Sequencing of ORF5 gene is widely used and considered essential for diagnostics and control of porcine reproductive and respiratory syndrome (PRRS) in Canada. The objective of this study was to position Quebec ORF5 sequences of PRRS virus within Canada and worldwide diversity. Overall, 76.8% of the 5204 sequences gathered from Quebec (n = 5031), Ontario (n = 151) and Manitoba (n = 18) were classified into one of 34 genetic clusters defined as groupings including ≥15 sequences and having ≥70% rapid bootstrap support value from a maximum likelihood (ML)-phylogeny. Following the addition of PRRSV 2 international reference dataset from Shi et al. (2010), the most predominant lineages in our dataset were wild-type 1 and vaccine-like 5.1 (MLV) and 8.9 (ATP). No strains or only a very few (1 or 2) were assigned to lineages 1.3-1.5, 3, 4, 5.2, 6, 7 or 9. Most wild-type clusters (97%) detected in a dataset from Canada did not include any sequence from the international reference dataset. It might reflect recent subpopulations that were absent at the time of Shi's publication. As an example, cluster #25 first appeared in 2007, but since then had expanded considerably and is now the most prevalent wild-type cluster found in Quebec. A total of 117 RFLP patterns were identified and those were poorly correlated with genetic clusters based on phylogeny. Factors modulating PRRSV diversity such as pig movement that occurred within and between provinces should be further investigated in a perspective of disease control.

Porcine reproductive and respiratory syndrome virus: web-based interactive tools to support surveillance and control initiatives.

BACKGROUND: Control of porcine reproductive and respiratory syndrome (PRRS) represents a tremendous challenge. The trend is now toward managing the disease collectively. In Quebec, area and regional control and elimination (ARC&E) initiatives started in 2011; diagnostic testing, including ORF5 sequencing, and sharing of information among stakeholders are largely promoted. At the provincial level, a data-sharing agreement was signed by Quebec swine practitioners allowing PRRS virus (PRRSV) sequences to be transferred to a database maintained by the Laboratoire d'épidémiologie et de médecine porcine (LEMP-DB). Several interactive tools were developed and are available to veterinarians to allow comparison of PRRSV ORF5 sequences within ARC&E projects or provincially while managing confidentiality issues. RESULTS: Between January 1st 2010 and December 31st 2018, 4346 PRRSV ORF5 sequences were gathered into the LEMP-DB, involving 1254 sites and 43 practicing veterinarians. Approximately 34% of the submissions were from ARC&E projects. Using a novel web-based sequence comparison tool, each veterinarian has access to information on his/her client sequences and can compare each sequence with 1) commercial vaccine strains, 2) historical samples from the same site, and 3) all sequences submitted to the database over the last 4 years. Newly introduced PRRSV into breeding herds can be monitored using a new sequence comparison tool based on comparison of sequences at the provincial level. Each month, graphs providing the number of introductions per month and the yearly cumulative are updated. Between August 1st 2014 and December 31st 2018, 233 introductions were detected on 180 different breeding sites. Following a data-sharing agreement, veterinarians involved in ARC&E projects have access to an interactive mapping tool to locate pig sites, compare sequence similarity between participating sites and visualize the results on the map. CONCLUSIONS: The structure developed in Quebec to collect, analyse and share sequencing data was efficient to provide useful information to the swine industry at both provincial and regional levels while dealing with confidentiality issues.

Evaluating an automated clustering approach in a perspective of ongoing surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) field strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a major economic impact on the swine industry. The important genetic diversity needs to be considered for disease management. In this regard, information on the circulating endemic strains and their dispersal patterns through ongoing surveillance is beneficial. The objective of this project was to classify Quebec PRRSV ORF5 sequences in genetic clusters and evaluate stability of clustering results over a three-year period using an in-house automated clustering system. Phylogeny based on maximum likelihood (ML) was first inferred on 3661 sequences collected in 1998-2013 (Run 1). Then, sequences collected between January 2014 and September 2016 were sequentially added into 11 consecutive runs, each one covering a three-month period. For each run, detection of clusters, which were defined as groups of ≥15 sequences having a≥70% rapid bootstrap support (RBS) value, was automated in Python. Cluster stability was described for each cluster and run based on the number of sequences, RBS value, maximum pairwise distance and agreement in sequence assignment to a specific cluster. First and last run identified 29 and 33 clusters, respectively. In the last run, about 77% of the sequences were classified by the system. Most clusters were stable through time, with sequences attributed to one cluster in Run 1 staying in the same cluster for the 11 remaining runs. However, some initial groups were further subdivided into subgroups with time, which is important for monitoring since one specific wild-type cluster increased from 0% in 2007 to 45% of all sequences in 2016. This automated classification system will be integrated into ongoing surveillance activities, to facilitate communication and decision-making for stakeholders of the swine industry.

Aplasia cutis congenita (epitheliogenesis imperfecta) in swine: observations from a large breeding herd.

Epitheliogenesis imperfecta has been reported in several animal species, and its inheritance is suspected to be autosomal recessive. This term has been used to describe two different diseases, namely epidermolysis bullosa and aplasia cutis congenita, which are both grossly characterized by an absence of epidermis or mucosal epithelium and are most frequently reported on the distal limbs and oral cavity. Epitheliogenesis imperfecta has been described in swine, but the literature on the subject is scarce. To better characterize this condition, 70 piglets with congenital skin defects macroscopically compatible with epitheliogenesis imperfecta were examined. In all but 1 case, only 1 piglet per litter was affected. Of the affected piglets, 65 (93%) were male, suggesting a sex-related problem. More than half of the piglets had multiple skin lesions. All defects were located on the caudal half of the body, and none was found in the oral cavity. Most lesions were characterized by an absence of epidermis and part of the dermis and adnexae. Adnexal dysplasia was also observed at several sites, both with and without epitheliogenesis imperfecta, suggesting a developmental problem. Fluid-filled, congenital subcutaneous bullae were noted grossly on 7 piglets; their relationship, if any, with epitheliogenesis imperfecta remains unknown. As the term epitheliogenesis imperfecta has been used in cases of epidermolysis bullosa, the term aplasia cutis congenita seems to be more appropriate to describe these lesions in swine.

Pathology of ear hematomas in swine.

The objectives of our study were to describe the pathology of ear hematomas in swine and to add to the comprehension of the pathogenesis of this condition. The pathogenesis of aural hematomas has been studied mainly in dogs; however, disagreements exist about the precise anatomic location of the hemorrhage. Sixteen pigs with ear hematoma at various stages of development were included in this study. The pigs were submitted for routine autopsy for various and unrelated reasons over a period of several years. Based on gross examination, the 16 cases of aural hematomas were subjectively classified as acute (n = 6), subacute (n = 3), and chronic (n = 7). The age of the animals at the time of autopsy ranged from 2 weeks to adulthood, with all acute cases being <7 weeks of age. Morphologic examination of all acute cases revealed that the hematoma developed predominantly in a subperichondral location on both sides of the cartilaginous plate simultaneously. Within these same cases, there were also some areas in which blood-filled clefts had formed within the cartilage itself. Besides fibroplasia, neoformation of cartilage was found to represent a significant part of the repair process. All chronic cases were characterized on cross-section of the ear by the presence of at least 2 distinct, wavy, focally folded, and roughly parallel plates of cartilage separated from each other by fibrous tissue.

Efficacy of Fostera PRRS modified live virus vaccine against a Canadian heterologous virulent field strain of porcine reproductive and respiratory syndrome virus.

Vaccination is a useful option to control infection with porcine reproductive and respiratory syndrome virus (PRRSV), and several modified live-PRRSV vaccines have been developed. These vaccines have shown some efficacy in reducing the incidence and severity of clinical disease as well as the duration of viremia and virus shedding but have failed to provide sterilizing immunity. The efficacy of modified live-virus (MLV) vaccines is greater against a homologous strain compared with heterologous PRRSV strains. The objective of this study was to evaluate the efficacy of Fostera PRRS MLV vaccine in protecting against challenge with a heterologous field strain widely circulating in the swine herds of eastern Canada. Forty-six piglets were divided into 4 groups: nonvaccinated-nonchallenged; nonvaccinated-challenged; vaccinated-challenged; and vaccinated-nonchallenged. The animals were vaccinated at 23 d of age with Fostera PRRS and challenged 23 d later with a heterologous field strain of PRRSV (FMV12-1425619). Overall, the vaccine showed some beneficial effects in the challenged animals by reducing the severity of clinical signs and the viral load. A significant difference between nonvaccinated and vaccinated animals was detected for some parameters starting 11 to 13 d after challenge, which suggested that the cell-mediated immune response or other delayed responses could be more important than pre-existing PRRSV antibodies in vaccinated animals within the context of protection against heterologous strains.

La vaccination est une option utile pour limiter l'infection par le virus du syndrome reproducteur et respiratoire porcin (VSRRP), et plusieurs vaccins VSRRP vivants modifiés ont été développés. Ces vaccins ont démontré une certaine efficacité à réduire l'incidence et la sévérité de la maladie clinique ainsi que la durée de la virémie et de l'excrétion virale mais ont failli à produire une immunité stérilisante. L'efficacité des vaccins vivants modifiés (VVM) est supérieure contre une souche homologue comparativement à des souches hétérologues de VSRRP. L'objectif de la présente étude était d'évaluer l'efficacité du vaccin Fostera, un VVM contre le VSSRP, à protéger contre une infection défi avec une souche de terrain hétérologue circulant librement dans les troupeaux porcins de l'est du Canada. Quarante-six porcelet ont été répartis en quatre groupes : non vaccinés-non infectés; non vaccinés-infectés; vaccinés-infectés; et vaccinés-non infectés. Les animaux ont été vaccinés à 23 jours d'âge avec Fostera SRRP et infectés 23 jours plus tard avec une souche de terrain hétérologue du VSRRP (FMV12-1425619). De manière générale, le vaccin a démontré quelques effets bénéfiques chez les animaux infectés en réduisant la sévérité des signes cliniques et la charge virale. Une différence significative entre les animaux non vaccinés et ceux vaccinés a été détectée pour quelques paramètres et débutant 11 à 13 j suite à l'infection, ce qui suggère que la réponse de l'immunité à médiation cellulaire ou d'autres réponses retardées pourraient être plus importantes que la présence d'anticorps anti-SRRP existants chez des animaux vaccinés dans le contexte d'une protection contre des souches hétérologues.(Traduit par Docteur Serge Messier).

Molecular epidemiology of porcine reproductive and respiratory syndrome virus (PRRSV) in Québec.

Porcine reproductive and respiratory syndrome virus (PRRSV) strains identified in samples from 226 field cases originating from Québec herds and submitted over a 4-year period (March 1998-July 2002) were studied. Sequencing of PRRSV strains was performed on the ORF5 gene amplified product and restriction fragment length polymorphism (RFLP) patterns for enzymes MluI, HincII and SacII were determined on these sequences. Twenty-four other PRRSV isolates including three vaccine strains were also included for comparison purposes and the total of 250 PRRSV strains were used in a phylogenetic analysis. Clinical and epidemiological data were collected through a questionnaire for each of the submitted field cases. About 75% of the cases were submitted during autumn and winter. Over 60% of the cases were submitted for reproductive problems, 33% for respiratory problems and 6% for increased PRRSV serological titers in the herd in absence of clinical signs. In 69% of the cases there was a PRRS vaccination program for the herd. However, only 26% of the animals from which samples were obtained had been vaccinated themselves. The genomic analysis of this large number of strains revealed a great variability of PRRSV ORF5 with 59% of amino acid positions being polymorphic. A total of 29 RFLP patterns were obtained. The main RFLP patterns obtained were 1-8-4 (28%), 1-4-4 (16%), 1-2-4 (9%) and 1-11-4 (9%). The global findings derived from the molecular analysis of 226 PRRSV strains suggest that PRRSV circulating in Québec represent a different sub-population of strains. Vaccine-like strains were identified in 10% of the cases. A phylogenetic tree enabled the identification of 44 groupings comprising two to 23 strains each. Of the 250 sequences analyzed, 183 (73%) could be included in one of these groupings. The data collected from the questionnaires were used to establish epidemiological links between strains within groupings. The main relationships between strains within a grouping were the introduction of infected animals (19%) and area spread (33%). In 40% of the cases from which an area spread was suspected, herds were located within 3 km from another. Aerosol transmission was suspected in several cases, more than half of which belonged to different owners. In 41 herds, more than one strain (2-8) were identified over a period varying from 3 months to 4 years. Data indicated that a PRRSV strain can persist in a herd up to 3.5 years displaying as little as 2% variation in ORF5 during this time. In 78% of the herds with multiple submissions genetically different strains were identified; often within 1 year of the original identification. These genetically distinct strains were often associated with a recrudescence of moderate to severe clinical signs. Coexistence on the same farm of different PRRSV strains was also observed.

Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions.

Porcine circovirus type 2 (PCV2) has been associated with a newly identified and described disease in swine called the postweaning multisystemic wasting syndrome (PMWS). An association of PCV2 with various other clinical conditions in pigs are also increasingly being reported. To date molecular studies to determine the extent of genetic variations of this virus have been limited. To more fully understand the extent of genetic diversity we report the sequencing of 34 PCV2 strains from Eastern Canada originating from a variety of clinical conditions spanning years 1990-2001 along with the phylogenetic analysis of these strains and that of 36 PCV2 sequences published in GenBank. Sequence analysis of the complete genome indicated that the Canadian PCV2 strains analyzed in the present study are closely related to each other but also to other PCV2 strains originating from Western Canada, US, Europe and Asia. Sequence analysis of ORF1 and ORF2 genes of the 34 strains revealed that the extent of nucleotide variation was greater for the ORF2 than for the ORF1. The amino acid sequences alignment of the PCV2 capsid protein identified three major regions of amino acid heterogeneity, two of which correspond with dominant immunoreactive areas. No repeatable amino acid motifs for these two regions could be associated with PCV2 strains identified from cases of PMWS or other clinical conditions. Phylogenetic analysis of all 70 strains revealed one large cluster composed of strains from Europe, Taiwan, China and Canada. This large cluster could be divided in several sub clusters, in two of which Canadian strains were found closely related to strains from Germany. The remaining strains from Canada and the US were spread in small groupings along the phylogenetic tree and an association with geographic origin could not be established. The genomic characterizations performed in this study indicate that PCV2 strains associated with PMWS are scattered throughout the phylogenetic tree often in groupings including PCV2 strains identified from cases other that PMWS such as, porcine reproductive and respiratory syndrome (PRRS), generalized tremors, porcine dermatitis and nephropathy syndrome (PDNS), arthritis, nervous signs, erysipelas and even healthy pigs.

Comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome.

A comparative serologic and virologic study was performed in pigs from 5 herds with postweaning multisystemic wasting syndrome (PMWS) and 2 herds without PMWS in Quebec. In each herd, 60 blood samples were collected at 4-wk intervals from pigs from 3 to 23 wk of age. The serum was evaluated for the presence of antibodies to porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as for the presence of nucleic acid of PCV2, PRRSV, an porcine parvovirus (PPV), by means of the polymerase chain reaction (PCR). Serologic profiles for PCV2 were very similar in 6 of the 7 herds, including the 2 without PMWS, and were characterized by a gradual decrease in antibody titres from 3 until 11 wk of age, followed by seroconversion at 15 wk, and high PCV2 antibody titres thereafter in all pigs. Only starting at 11 to 15 wk of age could PCV2 viremia be detected, except in 1 herd, in which clinical signs were observed at 6 to 7 wk of age. A PCV2 viremia could be detected within the same pigs for a minimum of 8 wk, and the virus could still be detected in 41% of the serum samples obtained at 23 wk of age. The antibody level did not appear to influence the occurrence of disease, since titres were similar in pigs in the herds with or without PMWS. Infection with PRRSV, as demonstrated by PCR and seroconversion, preceded that of PCV2 by at least 1 mo in both types of herd. Both PRRSV and PCV2 were detected in some pigs in 5 of the 7 herds, including 1 herd without PMWS. Porcine parvovirus could be detected in serum by PCR in 2 herds with PMWS after the onset of clinical signs and also in 1 herd without PMWS. Genomic analysis of PCV2 strains identified in the herds without PMWS indicated complete or very high homology (99.4% to 100%) with the PCV2 strains identified in 4 herds with PMWS. In our field study, the triggering of PMWS in the herds could not be linked to coinfection with either PRRSV or PPV or to the use of a specific immunostimulant, such as vaccines, or to particular genomic differences between the PCV2 strains identified.

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.

Epidemiological investigations in regard to porcine reproductive and respiratory syndrome (PRRS) in Quebec, Canada. Part 2: prevalence and risk factors in breeding sites.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat for swine industry and understanding factors involved in its epidemiology is undoubtedly essential for disease control. As a part of a larger project, a cross-sectional study was performed on breeding sites in a moderate density area of swine production in Quebec to estimate the prevalence of PRRSV infected sites and to evaluate if characteristics of sites and biosecurity practices, either as specific measures or as a global score, were associated with PRRSV status. A questionnaire and diagnostic procedures were performed on 54 breeding sites between September 2006 and August 2008. A biosecurity score that had been previously computed using two-step clustering procedure was used, classifying breeding sites into two biosecurity patterns (high vs. low) according to 21 specific biosecurity measures. The apparent prevalence of PRRSV infected sites was 74.0% (95% CI, 60.3-85.0). Univariable and multivariable logistic regression models with robust standard errors adjusting for potential clustering of sites due to same ownership were computed. In a first multivariable model evaluating characteristics of sites and specific biosecurity variables, four main effects were significantly associated (P<0.05) with PRRSV positive status: large pig inventory (OR: 10.7), proximity to closest pig site (OR: 7.3), absence of shower (OR: 8.7) and free access to the main entrance of the site by the rendering truck (OR: 7.0). In a second multivariable model including a global biosecurity score as a surrogate for a specific pattern of biosecurity measures, this score was not retained in the final model. The adjusted population attributable fractions were 16% for the proximity to closest pig site variable, 27% for the absence of shower variable, and 10% for the free access to main entrance of the site by the rendering truck. These two latter biosecurity measures, manageable directly on the site, should be prioritized and be part of any intervention strategy designed for PRRSV control.

Epidemiological investigations in regard to porcine reproductive and respiratory syndrome (PRRS) in Quebec, Canada. Part 1: biosecurity practices and their geographical distribution in two areas of different swine density.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a considerable threat to the swine industry and implementing biosecurity measures is essential for the control of its transmission. The aims of this study were: (1) to describe biosecurity practices in production sites located in a moderate density (MD) and a high density (HD) pig area according to production type; (2) to group sites in different patterns according to their biosecurity practices; and (3) to determine the geographical distribution of sites according to biosecurity patterns. Biosecurity practices were selected based on PRRS epidemiology. A questionnaire was completed on 125 breeding sites (MD=54; HD=71) and 120 growing (HD) sites, between 2005 and 2008. Depending on area and production type, the frequency of biosecurity practices used ranged from 0 to 2% for barrier at site entrance, 0 to 19% for use of shower, 25 to 35% for washing truck between loads of pigs, 51 to 57% for absence of rendering or rendering without access to the site, and 26 to 51% for absence of gilt purchase or purchase with quarantine. Better practices pertaining to entrance protocol (i.e. "no-entry" sign, shower, ≥24 h downtime) were reported more frequently on breeding sites in the MD than the HD area (P<0.05). In the HD area, growing sites had in general a lower level of biosecurity than breeding sites. Using a two-step clustering procedure performed separately for breeding and growing sites, two different patterns were obtained for each production type, which corresponded to a high and low level of biosecurity. For breeding sites, a higher biosecurity level was observed at sites located away from other pig sites, set at more than 300 m from the public road, having higher sow inventory, or being part of an integrated production (P<0.05). Spatial clusters of sites for each biosecurity pattern were detected. This study identified some shortcomings regarding biosecurity that should be addressed before implementing any PRRSV regional control. Vicinity of sites with different biosecurity levels also suggests difficulties in planning priorities of intervention based on geographical distribution of sites.

Porcine reproductive and respiratory syndrome virus diversity of Eastern Canada swine herds in a large sequence dataset reveals two hypervariable regions under positive selection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be genetically highly variable, but knowledge of sequence diversity from Eastern Canada and its degree of genetic plasticity in or near the principal neutralizing epitope (PNE) in association with evolutionary selective pressure is limited. The purposes of our study were to investigate the extent of strain diversity, the existing glycotypes and the amino acid sites under selective evolutionary pressure in its encoded protein, GP5, for a dataset of 1301 sequences (1998-2009). This was addressed by partitioning and clustering into subgenotypes a large number of open reading frame 5 sequences from the province of Quebec and analyzing the content of these subgenotypes. The overall pairwise diversity was 12% and was comparable to what has been reported around the world. The mean diversity for sequences within subgenotypes was around 7%. No marked variations in subgenotype emergence could be observed through time. Thirty-eight GP5 glycotype patterns were observed which included a newly identified site at position N57 which was already present in 1998. These patterns possessed one to six N-glycosylation sites in total and could be located in eight different positions. No obvious grouping of glycotypes could be established in relation to subgenotypes. Positions N44 and N51 were confirmed to be fixed N-glycosylation positions, whereas other positions where found to be shifting and located in or near hypervariable regions (HVRs) 1 and 2. Both HVRs were under selective evolutionary pressure in half of all subgenotypes including vaccine-like groups. Conversely, the PNE flanked by both HVRs was well conserved among most subgenotypes demonstrating potential molecular constraint in a probable viral binding region. The analysis of this dataset increased knowledge of evolutionary change inferred from genetic data, more specifically regarding the implications of both HVRs in PRRSV diversity.