Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: Effect of age of milk, centrifugation, and decontamination

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease in cattle and it has been suggested that this organism may be associated with Crohn’s disease in humans. Cows at the advanced stage of the disease shed this organism into both their milk and feces. The objective of this study was to develop a more efficient procedure for isolating MAP from bulk tank raw milk. Bulk tank raw milk (50 mL) samples 3 to 13 d old after collection without spiking were investigated to evaluate the effects of milk age on the efficacy of decontamination. Milk samples, 2 to 3 d old, were seeded with MAP at levels of 50 to 200 colony forming units/mL in experiments involving factorial design to evaluate 1) the effects of different decontaminating reagents and decontamination procedures on recovery of MAP, and 2) partition MAP in milk fractions after centrifugation in raw milk. Decontamination in 20 mL of 0.75% hexadecylpyridinium chloride (HPC) at room temperature (22°C) for 2 to 5 h, with shaking, at intervals was found to be the most effective procedure for decontaminating milk 2 to 3 d old. Prolonged exposure to decontaminants, additional incubations in antibiotics, or at higher temperature (37°C) significantly reduced recovery of live MAP. Enhanced growth of microbial contaminants was noticed in samples decontaminated overnight at room temperature compared to those decontaminated for 2 to 5 h. Decontamination of 6 d old milk samples required extra incubation in antibiotic brew. Decontamination of milk samples that are 8 d and older was not effective in removing microbial contaminants. The MAP cells preferentially partitioned into the cream fraction after centrifugation, and combining the milk cream and pellet fractions enhanced recovery of MAP. A recovery rate of 16.6% was estimated with the use of our improved protocol