Petr Kralik1, Iva Slana1, Alena Kralova1, Vladimir Babak1, Robert H. Whitlock2, Ivo Pavlik1
1Veterinary Research Institute, Czech Republic; 2University of Pennsylvania, School of Veterinary Medicine, PA, USA
Mycobacterium avium subsp. paratuberculosis (MAP) causes losses in ruminants especially in cattle. The conventional control programmes are based on the traditional culture that suffers from the long time required for the incubation. In this study we have aimed on two main issues. Firstly, we have developed and optimised a reliable and cost-efficient DNA isolation procedure from faeces that could be coupled with previously developed IS900 and F57 quantitative real time PCR (qPCR) for the MAP detection. To determine the quantity of MAP as precise as possible, the recovery of MAP DNA from the spiked faecal samples was established. It ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 104 for F57 qPCR and 6.87 × 102MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.
This work was supported by the EC (ParaTBTools), the Ministry of Education, Youth and Sports of the Czech Republic “AdmireVet” (CZ 1.05/2.1.00/01.0006 and No. ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grants Nos. MZe0002716202 and QH81065).
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