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. Isolation of MAP has been reported from milk of infected cows, bulk tank milk and from pasteurized milk. For screening of farm bulk tank milk, a method to detect MAP in milk with real-time PCR was developed.

With this method, both pellet and cream fraction of the milk were harvested for analysis. The bacteria were lysed enzymatically and by mechanical disruption and the DNA was extracted by robotized magnetic bead separation. The analytical sensitivity was determined to 100 organisms per ml milk (corresponding to less than 10 CFU per ml, as CFU measurement usually underestimates the actual numbers) for samples of 10 ml, although as few as 10 organisms per ml milk was detected in three of four replicates in spiked milk samples.

The method was applied in a study of 55 dairy herds to compare PCR of farm bulk tank milk to culture of environmental samples for detection of MAP in the herds. In this study, 17 herds (31%) were negative with both methods, 21 herds (38%) were positive with environmental culture but negative with milk PCR, one herd (2%) was positive with milk PCR but negative with environmental culture and 16 herds (29%) were positive with both methods. Hence the sensitivity for detection of MAP in a herd was considerably higher for the environmental culture method than PCR testing of farm bulk tank milk.

From the 37 herds that were proven positive by culture of environmental samples, 89 tank milk samples were tested. Eighteen of these milk samples (20%) tested PCR positive and altogether 16 of these 37 herds (43%) had at least one positive tank milk sample. By comparison with spiked milk samples, it was concluded that the positive milk samples contained low numbers of MAP, usually less than 100 organisms per ml and never more than a few hundred organisms per ml.

The results indicate that although MAP may be shed into milk or transferred to milk by faecal contamination, it will only occur in low numbers in the farm bulk tank milk due to the dilution and it can be assumed to often fall below the detection limit. Thus, PCR detection of MAP in milk would be less suitable for herd prevalence testing, but useful for control of MAP presence in milk, in order to avoid transfer to humans. The results also suggest that the level of MAP in the bulk tank milk of Danish dairy herds with paratuberculosis is low.