Effectiveness of copper ions against Mycobacterium avium subsp. paratuberculosis and bacterial communities in naturally contaminated raw cow's milk.

AIM: The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow's milk. METHODS AND RESULTS: A copper ions treatment was evaluated in naturally contaminated cow's milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical-chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively). CONCLUSION: With the exception of some MAP-tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow's milk. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow's milk, information that could be useful as a tool for MAP control.

Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the peptide-mediated magnetic separation-phage assay.

AIM: To validate an optimized peptide-mediated magnetic separation (PMS)-phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in milk. METHODS AND RESULTS: Inclusivity, specificity and limit of detection 50% (LOD50 ) of the optimized PMS-phage assay were assessed. Plaques were obtained for all 43 MAP strains tested. Of 12 other Mycobacterium sp. tested, only Mycobacterium bovis BCG produced small numbers of plaques. LOD50 of the PMS-phage assay was 0·93 MAP cells per 50 ml milk, which was better than both PMS-qPCR and PMS-culture. When individual milks (n = 146) and bulk tank milk (BTM, n = 22) obtained from Johne's affected herds were tested by the PMS-phage assay, viable MAP were detected in 31 (21·2%) of 146 individual milks and 13 (59·1%) of 22 BTM, with MAP numbers detected ranging from 6-948 plaque-forming-units per 50 ml milk. PMS-qPCR and PMS-MGIT culture proved to be less sensitive tests than the PMS-phage assay. CONCLUSIONS: The optimized PMS-phage assay is the most sensitive and specific method available for the detection of viable MAP in milk. Further work is needed to streamline the PMS-phage assay, because the assay's multistep format currently makes it unsuitable for adoption by the dairy industry as a screening test. SIGNIFICANCE AND IMPACT OF THE STUDY: The inclusivity (ability to detect all MAP strains), specificity (ability to detect only MAP) and detection sensitivity (ability to detect low numbers of MAP) of the optimized PMS-phage assay have been comprehensively demonstrated for the first time.