Isolation of Mycobacterium avium subspecies paratuberculosis from muscle and peripheral lymph nodes using acid-pepsin digest prior to BACTEC culture

Meat has received little attention regarding human exposure to Mycobacterium avium subsp. paratuberculosis, a possible infectious trigger of Crohn's disease. Meat has less contamination with other organisms than gut tissues, facilitating modifications to existing decontamination protocols prior to BACTEC culture that could increase analytical sensitivity. Using spiked meat samples we trialled enzymatic and chemical digestion techniques to concentrate larger starting samples, and modifications to existing clinical mycobacteriological decontamination protocols. An acid-pepsin digestion method using a 20 g sample was considerably more sensitive (detection limit 0.88 log10 viable organisms per gram) than previous techniques. However, it was cumbersome for routine use, and subject to frequent contamination. Modifications to an existing centrifugation protocol yielded a simple, robust technique with slightly improved sensitivity (detection limit 1.77 log10 per gram). Use of these sensitive tests in parallel identified M. a. paratuberculosis in the muscle of 59% and peripheral lymph nodes (PLN) of 85% of clinically infected sheep. The numbers of M. a. paratuberculosis in these infected tissues were low (1.67 ± 0.92 log10 per gram in muscle and 2.06 ± 0.69 log10 per gram in PLN), such that many would not have been detected by routine methods. Fewer subclinically infected animals with gross lesions harboured M. a. paratuberculosis in meat (4.5%) or PLN (32%), and the numbers of organisms in such infected animals were lower. Because most animals raised specifically for meat production are young and unlikely to be heavily infected, and because meat is usually consumed cooked, the risk of human exposure to viable M. a. paratuberculosis via meat may be small. Measures to prevent heavily infected animals, especially those with clinical signs, from entering the human food chain would further reduce this risk.