Sylvie D'Allaire

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.

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.

Infectious agents identified in pigs with multifocal interstitial nephritis at slaughter

One kidney was taken from each of 100 pigs at slaughter; 50 had gross lesions of multifocal interstitial nephritis and 50 had no gross lesions. Forty-nine of the affected kidneys had lesions that were characterised by the presence of either a few randomly distributed or numerous widely disseminated pale foci, 1 to 3 mm in diameter, on the cortical surface (white-dotted kidneys). Microscopically, these focal inflammatory lesions often had a distinct lymphofollicular pattern (follicular nephritis). Lesions of chronic vasculitis were observed in 21 of the affected kidneys. Histologically, the control kidneys had only small and sparse inflammatory foci. Standard bacterial cultures of kidneys of both groups were not significant, and cultures for the isolation of leptospires were all negative. Virological examination of the kidney homogenates by PCR did not reveal any porcine reproductive and respiratory syndrome virus and only a few cases were positive for the porcine circovirus type 1. However, porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) were detected in many kidneys of both groups but in a significantly higher proportion of the kidneys with interstitial nephritis. There was a significant association between the lesions and the presence of PPV and PCV-2 with odds ratios of 7.5 (P<0.0001) and 3.4 (P=0.0074), respectively, and the odds ratio increased to 22.7 (P<0.0001) when both viruses were identified in the same kidney. However, a subsample of kidneys taken from both groups were negative by immunohistochemistry for the presence of PPV and PCV.2 antigens.

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.

Influence of management, housing and personality of the stockperson on preweaning performances on independent and integrated swine farms in Québec

Abstract A cross-sectional study was implemented to determine which factors related to management, housing and stockperson in the farrowing quarters were associated with preweaning mortality and piglets weaned per litter in swine breeding herds. The associations were sought separately for the two most common types of swine production in Quebec. Forty-eight randomly-selected independent farms and 38 others belonging to integrated organizations specialized in swine production conveniently chosen among the largest organizations in Quebec participated in the study. Preweaning performances were computed from the sow records. Information on housing features was gathered during the visit to the farrowing quarters. Management practices were obtained through a questionnaire from the stockperson working in the farrowing quarters and his or her personality traits were evaluated through a personality test. Backward elimination procedure was used to build multiple linear regression models for each measure of performance for each type of farm. The procedures started from the management, housing and stockperson groups of variables taken separately and altogether, leading to the building of different regression models. Piglets weaned per litter averaged 8.9 and 8.6 on the independent and integrated farms, respectively and preweaning mortality, 14.4 and 15.4%, respectively. On the independent farms, high preweaning performances (high number of piglets weaned per litter and low preweaning mortality) were associated with routinely washing the farrowing crates, vaccination of the sows against neonatal diarrhea, farrowing crates equipped with high bottom bars, partially-slotted floors in the farrowing pens and high self-discipline from the stockperson. Poor performances were associated with the use of oxytocin around farrowing, mixed nursery-farrowing quarters and a stockperson being exaggeratedly self-assured and sensitive. On the integrated farms, the use of oxytocin at farrowing was also associated with poor performances, as well as several rooms or all-in/all-out farrowing quarters, farrowing crates equipped with high or medium bottom bars, totally-slotted floors and a stockperson being rather bold, suspecting and tense. High performances were positively associated with warmth, emotional stability and self-discipline from the stockperson. These findings provided evidences of the influence of the stockpersons personality on performances in particular, on the integrated farms on which the management in the farrowing quarters was more proactive (implying a stronger relationship between the stockperson and the pigs). In contrast, the importance to preweaning performances of general hygiene and health control was emphasized on the independent farms.

The Evaluation of Postmortem Ocular Fluid Analysis as a Diagnostic Aid in Sows

This study was undertaken to evaluate the diagnostic usefulness of postmortem ocular fluid analysis in estimating the antemortem status of various serochemical constituents. Chemical values of serum and aqueous and vitreous humors were compared following different procedures. A blood sample and the 2 eyes were collected from each of 100 sows at a nearby abattoir. The results obtained from immediate centrifugation of ocular fluids after sampling were compared with those samples in which centrifugation was delayed by 2 hours. Two different postmortem intervals were used for sampling ocular fluids, 2 and 24 hours. Concentrations of urea, calcium, phosphorus, potassium, sodium, and chloride were determined from serum and humors. Delayed centrifugation did not affect chemical values of ocular fluids nor the relationships between serum and humors. Phosphorus and potassium values increased significantly with the postmortem interval in both aqueous and vitreous humors. The relationships between chemical values of ocular fluids and serum were determined using simple linear regression. There was a poor correlation between ocular fluid and serum values for all electrolytes; a significant correlation was found only for urea concentrations in both humors.

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.

Determination of Halothane Gene Mutation Associated with Malignant Hyperthermia in Sows Dead of Cardiac Failure

Significant losses due to sow mortality may be encountered in some swine breeding herds. Many conditions are responsible for death in sows; some, however, are more common Several investigations showed that cardiac failure is among the major causes of death in breeding females, accounting for up to 31% of the mortalities. 1,4,17,19,20 Parturition, heat stress, mating, fighting, and transport were identified as predisposing factors for cardiac failure in sows housed in total confinement. 1-6 Most of these events, which are demanding for the cardiovascular system, are also considered triggering factors for the development of malignant hyperthermia (MH) in pigs, a genetically transmitted disease. Differentiating a simple case of cardiac failure from MH might be difficult because these 2 conditions share many clinical and pathologic similarities. Recently, MH in swine, also called porcine stress syndrome (PSS), has been associated with a recessive mutation in the gene coding for porcine calcium release channel, also called the ryanodine receptor gene (ryr-1 locus) or halothane gene (Hal), which is located on chromosome 6. With molecular biology techniques, it is now possible to identify pigs that are MH susceptible, MH carrier, and norma1. The aim of this retrospective study was to determine if cases of cardiac failure in sows could be attributed to the defective gene responsible for MH. Formalin-fixed, paraffin-embedded tissues from 84 sows previously collected in a study on sow mortality in Canada were used in the present investigation. From these selected sows, 42 were identified to have died of cardiac failure and 42 died of various other causes (control group), such as torsions of abdominal organs, cystitis-pyelonephritis, gastric ulcers, and uterine prolapse. Paraffin-embedded tissue blocks from heart, skeletal muscle, liver, spleen, and kidney were used to determine the Hal genotype by a polymerase chain reaction (PCR) technique. 11,22 For each sow, a 10-μm-wide section was prepared from tissue blocks, and excess paraffin was trimmed. Sections were placed in 1.5-ml sterile microfuge tubes. The microtome blade, tweezers, and other equipment that could come into contact with the samples were carefully sterilized before processing each tissue block. Tissue sections were deparaffinized with toluene and washed twice with ethanol to remove the solvent. Ethanol was allowed to evaporate under vacuum for 10 minutes. To isolate genomic DNA, 100 μl of digestion buffer (50 mM Tris [pH