Purpose: To demonstrate the usefulness of ICC-PCR assay for comparing Cbinactivation in skim and whole milk.
Methods: Cb resuspended in skim or whole milk at ~7.2 log genome equivalents/milliliter (ge/ml) was treated in sealed vials submerged in a circulating water bath at 62°C or 64°C for various times. After serial dilution of milk to 10-6, triplicate Vero cell monolayers were infected at each level for 48 h followed by 9 day incubation after inoculum removal and addition of fresh RPMI + 1% FBS media. Infected cells were freeze-thawed followed by DNA extraction and qPCR for the Cb IS111a gene. Cb viability was considered positive if the Day 9 post-infection (PI) level increased by ≥ 0.5 log Cb ge/ml from the most concentrated Day 0 PI sample. The numbers of positive wells from each dilution were used to calculate the remaining viable Cb/ml by MPN method.
Results: The ICC-PCR assay demonstrated that the thermal inactivation of Cb in skim milk was faster than in whole milk regardless of treatment temperature. For the 62°C treatment, the infectious Cb in skim milk was reduced by 1 log at 10 min. and was no longer infectious after 20 min., whereas Cb in whole milk by decreased 0.3 log after 10 min., 3.1 log after 20 min., and was no longer infectious after 26 min. After 8 min. treatment at 64°C, infectious Cb was reduced by 5.4 log for skim milk vs. 3.1 log for whole milk with complete inactivation after 10 min. for both milk types.
Significance: This ICC-PCR assay is a specific and sensitive method to detect differences in the inactivation of Cb in skim and whole milk, and may be useful for the evaluation of thermal and novel non-thermal processes for Cb inactivation in milk.