Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey

• Published in: Journal of veterinary diagnostic investigation 2008-09, Vol.20 (5), p.545-558
• Main Authors: Gagnon, Carl A, Del Castillo, Jérome R.E, Music, Nedzad, Fontaine, Guy, Harel, Josée, Tremblay, Donald
• Format: Article
• Language: English
• Subjects: Actinobacillus pleuropneumoniae; animal pathogenic bacteria; Animals; Circoviridae Infections - veterinary; Circovirus - classification; Circovirus - genetics; diagnostic techniques; disease surveillance; DNA Primers; epidemiology; Genotype; mixed infection; multiplex real-time quantitative polymerase chain reaction; pathogen identification; Plasmids; polymerase chain reaction; Polymerase Chain Reaction - methods; Porcine circovirus; Porcine circovirus-associated disease; Porcine reproductive and respiratory syndrome virus; Quebec; Sensitivity and Specificity; serotypes; strain differences; strains; Streptococcus suis; Swine; swine diseases; Swine Diseases - epidemiology; Swine Diseases - virology; Wasting Syndrome - epidemiology; Wasting Syndrome - veterinary; Wasting Syndrome - virology
• ISSN: 1040-6387; 1943-4936
• DOI: 10.1177/104063870802000503

Correspondence: 1 Corresponding Author: Carl A. Gagnon, Faculté de médecine vétérinaire, Université de Montréal, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada, J2S 2M2. carl.a.gagnon{at}umontreal.ca By the end of 2004, the Canadian swine population had experienced a severe increase in the incidence of Porcine circovirus –associated disease (PCVAD), a problem that was associated with the emergence of a new Porcine circovirus-2 genotype (PCV-2b), previously unrecovered in North America. Thus, it became important to develop a diagnostic tool that could differentiate between the old and new circulating genotypes (PCV-2a and PCV-2b, respectively). Consequently, a multiplex real-time quantitative polymerase chain reaction (mrtqPCR) assay that could sensitively and specifically identify and differentiate PCV-2 genotypes was developed. A retrospective epidemiologic survey that used the mrtqPCR assay was performed to determine if cofactors could affect the risk of PCVAD. From 121 PCV-2–positive cases gathered for this study, 4.13%, 92.56%, and 3.31% were positive for PCV-2a, PCV-2b, and both genotypes, respectively. In a data analysis using univariate logistic regressions, the PCVAD-compatible (PCVAD/c) score was significantly associated with the presence of Porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV viral load, PCV-2 viral load, and PCV-2 immunohistochemistry (IHC) results. Polytomous logistic regression analysis revealed that PCVAD/c score was affected by PCV-2 viral load ( P = 0.0161) and IHC ( P = 0.0128), but not by the PRRSV variables ( P > 0.9), which suggests that mrtqPCR in tissue is a reliable alternative to IHC. Logistic regression analyses revealed that PCV-2 increased the odds ratio of isolating 2 major swine pathogens of the respiratory tract, Actinobacillus pleuropneumoniae and Streptococcus suis serotypes 1/2, 1, 2, 3, 4, and 7, which are serotypes commonly associated with clinical diseases. Key Words: Epidemiologic survey • genotyping • molecular diagnostic test • Porcine circovirus-2