Considerable attention has been given to milk and dairy products as potential vehicles of Mycobacterium avium subsp. paratuberculosis(Map) transmission to the food chain. Surveillance of such products for the presence of Map has been hindered by difficulties in culture methodology. Molecular detection methods for Map, including endpoint PCR (duplex, multiplex and nested) and real time quantitative PCR (qPCR) have been widely reported. However, these methods cannot differentiate between DNA recovered from dead and viable cells.

The method assessed in this study involved addition of test sample to enrichment broth, removal of initial sub-sample for Map-DNA extraction/qPCR followed by enrichment and a subsequent DNA extraction/qPCR assay. A significant reduction in the qPCR Ct value following enrichment is indicative of Map growth/recovery.

Samples containing < 2 cfu/ml Map were enriched into Middlebrook 7H9 broth, Dubos broth and a customised enrichment broth. Following Map-DNA extraction using the Adiapure® kit and real time PCR (IS900 and F57), Map was undetected at time zero but was detected in 7H9 enrichment medium after 7 d (Ct: 36); 14 d (Ct: 25); 21 d (Ct: 20). Enrichment in Dubos medium and the customised broth resulted in slower rates of growth and qPCR detection. Raw milk cheeses (n = 14) were subject to PANTA-supplemented 7H9 enrichment/qPCR. No viable Map were detected (enrichment (>100 d)/qPCR) despite a number of the samples revealing Map presence prior to enrichment. All samples were negative by culture (7H10 and HEYM medium) after 20 wk incubation suggesting no viable Map present in cheese samples. Map- spiked pasteurised and raw milk samples were heat treated and subsequently enriched. Decreases in Ct values (qPCR) in the enriched pasteurised samples after 5 wk was indicative of recovery and growth of heat-treated Map cells whereas enriched raw milk samples presented problems for the PCR assay.

This enrichment-qPCR strategy may represent a methodological option to screen samples for the presence of viable Map rather than liquid culture which can rely on continual monitoring, subculture and confirmation.