A. BROES , Centre de développement du porc du Québec inc. - Canadian Research Network on Bacterial Pathogens of Swine J. HAREL, GREMIP - FMV - Université de Montréal - Canadian Research Network on Bacterial Pathogens of Swine M. KOBISCH, AFSSA Ploufragan - Unité Mycoplasmologie et Bactériologie M. GOTTSCHALK, GREMIP -FMV- Université de Montréal - Canadian Research Network on Bacterial Pathogens of Swine
Title
Comparison and field validation of PCR tests for the detection of Actinobacillus pleuropneumoniae in carrier pigs.
Abstract Text
Actinobacillus pleuropneumoniae (App) is the causative agent of swine pleuropneumonia. Detection of subclinically infected animals is of main importance to control the disease. The aim of this study was to compare seven PCR tests for the detection of App in swine tonsils. Firstly, PCR tests were compared regarding their specificity using App and related bacterial species, and their sensitivity using in vitro experimentally infected tonsils. PCR were carried out directly on tonsils homogenates (“direct PCR”), or after a culture step (“after culture PCR”). Most tests demonstrated a good specificity. However, some tests gave false positive results with non-App species. A high variation in sensitivity among tests was observed for “direct PCR”. The sensitivity of “after culture PCR” was higher and very similar among tests. In a second phase, the effect of time and storage conditions of the sample was evaluated using tonsils from experimentally infected animals. Storage at –20ºC allowed the detection of App for at least 4 months. A frozen-thawing cycle of the tonsils did not significantly affect PCR detection of App. Finally, 2 PCR tests were validated using field samples. Their effectiveness was compared to that of standard culture and immunomagnetic-separation-based (IMS) bacterial isolation. A comparison between tonsil biopsies (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. Samples originated from: 1) a herd free of App; 2) a herd infected with App serotype 5, (high seroprevalence of infection); 3) a herd infected with App serotype 5, (medium seroprevalence); and 4) a herd infected with App serotype 7, (low seroprevalence). App was neither isolated nor detected by PCR in herd 1. In the 3 other herds, using whole tonsils, PCR was more sensitive than standard isolation and, in general, similar to IMS isolation. “After culture PCR” offered the highest sensitivity. PCR detection rate was higher from whole tonsils than from biopsies. A good agreement was found between demonstration of App in tonsils and serological status. PCR tests may thus be used as a complement of regular serological monitoring.
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