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To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk.Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 106 CFU ml−1) were heated at 63°C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0·5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4°C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0·75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4°C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37°C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0·05) and after refrigerated storage for 48 h (P < 0·01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates.CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample.Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.