THE EVALUATION OF THERMAL INACTIVATION OF COXIELLA BURNETII NINE MILE PHASE II IN BOVINE AND NON-BOVINE MILKS BY MOST PROBABLE NUMBER-POLYMERASE CHAIN REACTION (MPN-PCR) ASSAY

As non-bovine milks become popular for human consumption, ensuring that standard bovine milk pasteurization conditions provide enough treatment for non-bovine milks is significant for food safety. Coxiella burnetii, an obligate intracellular bacterium, has been used as the reference microorganism for defining milk pasteurization conditions. To evaluate C. burnetii thermal inactivation in bovine and non-bovine milks at commercial pasteurization temperature, an MPN-PCR assay was developed to quantitate viable C. burnetii in milk. Using this assay, the thermal inactivation of C. burnetii and a potential nonpathogenic surrogate, Micrococcus luteus, was tested in bovine, buffalo, camel and goat milks. Milk in sealed glass vials was pre-heated in a water bath at 72°C and inoculated via a syringe with C. burnetii and M. luteus at a final concentration of ~6.5 log10 ge/mL (CFU/mL) each. The inoculated milk was heat-treated at 72°C for up to 16 sec, cooled in a crushed ice bath and serially diluted. Viable M. luteus was quantitated by plating on BHIA plates. For C. burnetii detection, 1 mL of each dilution was inoculated into 9 mL Acidified Citrate Cysteine Medium-2 (ACCM-2) in triplicate T-25 flasks to produce a 3- flask Most Probable Number (MPN) assay. Viability of C. burnetii was considered positive if an increase of ≥0.5 log10 ge/mL was detected by qPCR after 14 d growth in ACCM-2 media. The numbers of positive flasks at each dilution were used to calculate the remaining viable C. burnetii by MPN method. The average D-values for 72°C inactivation were 1.99 ± 0.21 sec, 0.79 ± 0.28 sec, 1.43 ± 0.30 sec, and 2.06 ± 0.71 sec for C. burnetii, and 5.47 ± 0.94 sec, 3.65 ± 0.45 sec, 3.48 ± 0.83 sec and 5.34 ± 1.54 sec for M. luteus in bovine, buffalo, camel and goat milks, respectively. For C. burnetii, D-values in camel and goat milks were not significantly different (p>0.05) from bovine milk, but the D-value in buffalo milk was significantly lower (p