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Comparison of commercial DNA extraction and QPCR systems for a better sensitivity in detecting the causative paratuberculosis pathogen in dairy cow fecal samples.

Fock-Chow-Tho, D. E. Topp, E. Ibeagha-Awemu, and N. Bissonnette. Comparison of commercial DNA extraction and QPCR systems for a better sensitivity in detecting the causative paratuberculosis pathogen in dairy cow fecal samples. International Congress on Paratuberculosis, Nantes, France, June 19-24th.

Abstract

Mycobacterium avium ssp. paratuberculosis (MAP) is the pathogen inducing ruminant paratuberculosis (Johne’s disease) worldwide. While formerly of sporadic incidence, the prevalence of infected animals resulting from modern farming has raised paratuberculosis bovine disease to a global concern. Oral-fecal contamination is the most important mode of transmission of paratuberculosis. Hence, eradicating shedders could prevent MAP propagation. Whereas considered the standard method for MAP diagnosis, fecal culture requires specialised costly media and a long incubation time which sometimes resolve into disappointing bacterial contamination. To facilitate the efforts of control programs we evaluated the performance of direct fecal QPCR assays in terms of sensitivity. Several commercial kits use different strategies for DNA extraction and QPCR systems for capturing the presence of MAP in fecal samples. In this study, our aim is to compare the sensibility of detection of three commercially available DNA extraction kits broadly used in Canada, named A, B, and C, combined with two methods of QPCR detection (T and V). Forty-nine dairy cows from 5 different herds were sampled and diagnosed by fecal culture and ELISA assays to 6 months interval during 2 years. At the first sampling, their fecal samples were then tested 8 times with the respective DNA extraction method. While all of the three commercial DNA extraction kits were described as very efficient for the paratuberculosis diagnosis, method B allows a more sensitive detection than the two others. Indeed, 100% of cows declared positive for paratuberculosis by both fecal culture and ELISA assays were identified with method B while only 23% and 43% cows were confirmed with methods A and C, respectively. Interestingly, by using method B, low MAP shedders were detected. Moreover the QPCR system plays a critical role, with detection system T yielding QPCR reactions with the highest sensitivity. The results presented herein suggest that DNA extraction kit C in combination with QPCR system T allow successful amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. The current study demonstrates the importance of testing different kits for DNA extraction from fecal samples and the impact of a QPCR system to identify MAP shedding animals.

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