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THE EVALUATION OF THERMAL INACTIVATION OF COXIELLA BURNETII NINE MILE PHASE II IN SKIM MILK BY INTEGRATED CELL CULTURE-POLYMERASE CHAIN REACTION (ICC-PCR) ASSAY
Title
THE EVALUATION OF THERMAL INACTIVATION OF COXIELLA BURNETII NINE MILE PHASE II IN SKIM MILK BY INTEGRATED CELL CULTURE-POLYMERASE CHAIN REACTION (ICC-PCR) ASSAY
Creator
Zheng, Jiaojie
Date
2014, 2014-07
Description
Coxiella burnetii (C. burnetii) is an obligate intracellular bacterium and replicates exclusively in an acidified, lysosome-like vacuole which...
Show moreCoxiella burnetii (C. burnetii) is an obligate intracellular bacterium and replicates exclusively in an acidified, lysosome-like vacuole which means the analysis of C. burnetii is difficult than other bacterial which can growth on regular liquid medium. An Integrated Cell Culture-Polymerase Chain Reaction (ICC-PCR) assay has been developed as a potential alternative to animal bioassays for evaluating C. burnetii inactivation in milk. This thesis research is to demonstrate the usefulness of this assay for evaluating C. burnetii inactivation in skim milk and comparing the results found for whole milk which was completed by another researcher. Before the thermal studies, the thermal kinetics of heating skim milk in glass vials and the polymerase chain reaction (PCR) detection limit were determined. For thermal treatments, Ultra High Temperature (U.H.T.) skim milk containing C. burnetii at ~7.2 log10 genome equivalents/ml (ge/ml) was treated in submerged vials at 60 °C, 62 °C and 64 °C for various times. After serial dilution of milk to 10-6, triplicate Vero cell monolayers were infected at each level for 48 hours followed by 9 days incubation after inoculum removal and addition of fresh RPMI + 1% FBS media. Infected cells were freeze-thawed followed by deoxyribonucleic acid (DNA) extraction and real- time PCR (RT-PCR) for the C. burnetii IS1111a gene. C. burnetii in samples was considered as viability if the Day 9 post infection (p.i.) level increased by ≥0.5 log10 C. burnetii ge/ml over the most concentrated Day 0 p.i. sample. The numbers of positive wells from each dilution were used to calculate the remaining viable C. burnetii/ml by MPN method. The thermal kinetics profile for heating the skim milk showed that the come up and cool down time would not adversely affect the thermal x treatment at 60 °C and 62°C. The qPCR could detect the propagation of C. burnetii in skim milk containing as low as 120 C. burnetii ge/ml. The ICC-PCR assay demonstrated that the thermal inactivation of C. burnetii in skim milk was faster than in whole milk at 62 °C and 64 °C. For the 62 °C treatment, the infectious C. burnetii in skim milk was reduced by 1.3 log10 ge/ml at 10 minutes and was no longer infectious after 20 minutes, whereas C. burnetii in whole milk had no obvious reduction after 10 minutes, 3.7 log10 ge/ml after 20 minutes, and was no longer infectious after 26 minutes. After 6 minutes treatment at 64 °C, infectious C. burnetii was reduced by 6.2 log10 ge/ml for skim milk vs. 3.8 log10 ge/ml for whole milk with complete inactivation after 9 minutes for both milk types. This ICC-PCR assay is a specific and sensitive method to detect the inactivation of C. burnetii in skim milk and allows differentiation of the thermal inactivation kinetics of different types of milk, and may be useful for the evaluation of thermal and novel non-thermal processes for C. burnetii inactivation in milk.
M.S. in Food Safety and Technology, July 2014
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