A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk

A possible mode of transmission for the ruminant pathogen Mycobacterium avium subsp. paratuberculosis (MAP) from cattle to humans is via milk and dairy products. Although controversially, MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. A method to detect MAP in milk with real-time PCR was developed for screening of bulk tank milk. Pellet and cream fractions of milk were pooled and subjected to enzymatic digestion and mechanical disruption and the DNA was extracted by automated magnetic bead separation. The analytical sensitivity was assessed to 100 organisms per ml milk (corresponding to 1-10 CFU per ml) for samples of 10 ml. The method was applied in a study of 56 dairy herds to compare PCR of farm bulk tank milk to culture of environmental faecal samples for detection of MAP in the herds. In this study, 68% of the herds were positive by environmental culture, while 30% were positive by milk PCR. Results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will probably occur in low numbers in the bulk tank milk due to dilution as well as general milking hygiene measures. The concentration of MAP can therefore be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be more useful for control of MAP presence in milk, in order to avoid transfer to humans, than for herd prevalence testing. It could also be of value in assessing human exposure to MAP via milk consumption. Quantification results also suggest that the level of MAP in the bulk tank milk of the studied Danish dairy herds was low, despite environmental isolation of MAP from the herds.