Mycobacterium Paratuberculosis Detection in Bovine Feces is Improved by Coupling Agar Culture Enrichment to an IS900-Specific Polymerase Chain Reaction Assay

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Abstract
The feasibility of coupling an agar culture enrichment step with gene amplification (ACE-PCR) as a means to improve turnaround time and detect Mycobacterium paratuberculosis (Mpt) in the presence of contaminants was investigated. Fecal samples from 463 Pennsylvania dairy cows were cultured in duplicate sets. One replicate from each set was processed and interpreted according to standard culture (SC) protocol, whereas cultures from the second replicate were harvested at 6 weeks postinoculation; DNA extracts from the harvested material were evaluated by a polymerase chain reaction (PCR) test for the Mpt-specific IS900 gene. One hundred seventy-six of 463 culture sets were positive by either method. One hundred sixty-five of these (94%) were ACE-PCR positive, and 151 (86%) were positive by SC. Eleven SC-positive samples were ACE-PCR negative, and 9 ACE-PCR-positive samples were negative by SC; these discrepancies could be a consequence of a low organism burden (≤5 organisms/g) or slow growth rate of Mpt in cultures of these samples. One hundred thirty-nine of 463 culture sets (30%) were reported as inconclusive because of culture contamination according to SC protocol; 16 of these (11.5%) were ACE-PCR positive. Seventy-four ACE-PCR-positive sets (42% of all positives) were negative or inconclusive by SC at 6 weeks postinoculation. Agar culture enrichment prior to IS900 PCR testing significantly improves Mpt culture turnaround time and sensitivity.