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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
Title
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
Creator
Zhang, Cheng
Date
2016, 2016-05
Description
As non-bovine milks become popular for human consumption, ensuring that standard bovine milk pasteurization conditions provide enough...
Show moreAs 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<0.05). These results indicate that non-bovine milks may not be a safety concern under standard milk pasteurization conditions, and M. luteus could be a good surrogate for C. burnetii thermal inactivation in milk.
M.S. in Food Safety and Technology, May 2016
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ENRICHMENT OF COXIELLA BURNETII FROM BOVINE AND NON-BOVINE MILKS USING AN AXENIC LIQUID CULTURE MEDIUM
Title
ENRICHMENT OF COXIELLA BURNETII FROM BOVINE AND NON-BOVINE MILKS USING AN AXENIC LIQUID CULTURE MEDIUM
Creator
Shi, Manman
Date
2016, 2016-07
Description
Non-bovine milks are becoming more popular as beverages and for making cheeses, however little are understood regarding how pathogens are...
Show moreNon-bovine milks are becoming more popular as beverages and for making cheeses, however little are understood regarding how pathogens are inactivated in these milks during pasteurization. Recently, a tissue culture-PCR method has been developed to quantify inactivation of Coxiella burnetii, the obligate intracellular bacterium which is the reference pathogen for milk pasteurization. Unfortunately, this method is time-consuming and quite laborious. A potential improvement over the ICC-PCR method may be the use of a new specialized liquid medium which has been shown to allow growth of pure cultures of C. burnetii outside of host cells. In this study, ACCM-2 was evaluated for its’ ability to enrich C. burnetii from bovine and non-bovine milks as a potential alternative to tissue culture enrichment for Most Probably Number (MPN) quantification of inactivation of C. burnetii in milks. C. burnetii in bovine whole milk and cream (as a surrogate for a high fat product), camel, water buffalo, and goat milk were grown in ACCM-2 media (1:10) in 10 mL volumes in T-25 flasks at 37 °C under 5% CO2 and 2.5% O2 with sampling over 14 days. C. burnetii levels were determined by quantitative PCR (qPCR) against a standard curve for the Coxiella-specific IS1111a gene. C. burnetii in all milks grew at least 3 logs over 10 days with the exception of the water buffalo milk which did not allow growth. Bovine milk allowed the least growth and additional studies determined that the detection limit for growth of C. burnetii from this milk is about 6 ge/mL. Additional studies showed that adjustment of the pH from ~5.0 to 4.75 results in much improved growth of C. burnetii from bovine milk. Adjustment of pH did not allow growth from water buffalo milk, however additional dilution of the milk did allow improved C. burnetii growth but the added dilution also decreases the detection limit of the proposed new MPN-PCR assay. Further studies were completed to determine how thermal injury affects recover and growth of Coxiella burnetii in ACCM-2 media after thermal processing at 64 °C for up to 8 minutes. Although injury did increase the lag phase period and reduce the log phase growth rate from these samples, recovery and growth of at least 0.5 log was possible from all samples indicating that this media should be useful as an alternative enrichment method for use in an MPN format for quantification of thermal inactivation of C. burnetii in both bovine and non-bovine milks.
M.S. in Food Safety and Technology, July 2016
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