Abstract
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease in ruminants and a probable pathogen of Crohn’s disease in humans. Accurate, cost-effective, and time-relevant diagnostics are the basis for efficient control programs. This study was conducted as an attempt to re-evaluate MAP detection improvement by coupling solid media enrichment to a more specific IS900 conventional PCR and a very specific F57/IC real-time PCR. In a spiking experiment, we investigated the improvement of molecular-based MAP detection in feces after a culture-based enrichment step into Herrold’s egg yolk media with mycobactin J (HEYM-MJ) for different time intervals, when compared to traditional culture. Detection limit of culture was 0.33 × 104 bacteria × g−1 (33 CFU g−1), while that of IS900 PCR when coupled with an enrichment step for 2, 4, and 6 weeks was 0.33 × 105 (0.33 × 103 CFU g−1), 0.33 × 104 (33 CFU g−1), and 33 (>3.3 CFU g−1) bacteria × g−1, respectively. Whereas the detection limits of F57/IC real-time PCR after the enrichment step for the same time intervals were 0.33 × 105 (0.33 × 103 CFU g−1), 0.33 × 103 (3.3 CFU g−1), and 33 (>3.3 CFU g−1) bacteria × g−1, respectively. Altogether, enrichment of bovine fecal samples into solid media increased the sensitivity of specific molecular detection of MAP using IS900 conventional PCR and duplex F57/IC real-time PCR and offers an expedited and accurate alternative for MAP detection in bovine feces. Validation of these results is further recommended using field bovine fecal samples.
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Acknowledgments
This work was financed by a grant from Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection (HMUKLV). It was also carried out in cooperation with the German Academic Exchange Service (DAAD).
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Fawzy, A., Eisenberg, T., El-Sayed, A. et al. Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment. Trop Anim Health Prod 47, 721–726 (2015). https://doi.org/10.1007/s11250-015-0784-9
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DOI: https://doi.org/10.1007/s11250-015-0784-9