Survival kinetics of Mycobacterium bovis during manufacture and ripening of raw milk Cheddar and Caerphilly cheese produced on a laboratory-scale.

AIMS: Persistence of Mycobacterium bovis was investigated in UK raw milk cheeses. METHODS AND RESULTS: Replicating traditional cheese production methods under stringent CL3 containment conditions, Cheddar and Caerphilly cheeses were produced with Myco. bovis inoculated raw milk. High-inoculum investigations used three Myco. bovis genotypes; later low-inoculum investigations used only Myco. bovis AF2122/97. High-inoculum Cheddar (n = 9) and Caerphilly (n = 9) were matured for a minimum of 12 and 4 months respectively; maturation of low-inoculum Cheddar (n = 3) and Caerphilly (n = 3) was up to 11 weeks. Survival of Myco. bovis was monitored by enumeration at different points throughout cheese manufacture and ripening. D values were calculated as follows: 57 and 59 days in high-inoculum Cheddar and Caerphilly, respectively, and 41 and 24 days in low-inoculum Cheddar and Caerphilly respectively. CONCLUSIONS: Mycobacterium bovis is concentrated in cheese curd and a proportion lost with the whey. Reduction in viability during manufacturing is limited, while significant Myco. bovis inactivation occurs during maturation. Inactivation was improved, during Caerphilly ripening, when acid development was enhanced by increasing the proportion of starter culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Mycobacterium bovis inactivation data obtained could be used to inform assessment of the risk posed to consumers by raw milk dairy products.

Optimization of modified Middlebrook 7H11 agar for isolation of Mycobacterium bovis from raw milk cheese.

Reports have highlighted the absence of contemporary peer reviewed publications pertaining to Mycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese-making methodology and equipment were devised to produce Cheddar (n = 6) and Caerphilly (n = 3) artificially contaminated with M. bovis (three genotypes) under stringent laboratory-containment guidelines for handling hazardous microbiological material. Middlebrook 7H11, modified for M. bovis isolation, was assessed for capacity to enumerate M. bovis despite changing cheese microflora and prolonged M. bovis exposure to the cheese matrix using maturing cheese test portions (n = 63; up to 16 weeks). Malachite green (MG) containing media isolated M. bovis at significantly (P < 0·05) lower levels than unmodified Middlebrook 7H11 agar despite MG being a common adjunct of Middlebrook 7H11 agar modified for M. bovis growth. Subsequently, a selective MG-free Middlebrook 7H11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing (P < 0·05) medium for recovery of M. bovis from typical UK cheese types, Cheddar and Caerphilly. Significance and impact of the study: Following increased M. bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M. bovis in raw milk products are limited. Cheese-making protocols and M. bovis culture media reported here provide tools for further investigation of M. bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M. bovis contamination of unpasteurized dairy products.