Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2003
Publication Date: 12/1/2003
Citation: Perdue, M.L., Karns, J.S., Higgins, J.A., Van Kessel, J.S. 2003. Detection and fate of Bacillus anthracis (Sterne) vegetative cells and spores added to bulk tank milk. Journal of Food Protection. 66:2349-2354. Interpretive Summary: The recent attacks using anthrax spores sent through the mail raises questions regarding the potential for other attacks including through the food system. Very little is known about the fate, stability or viability of anthrax spores in food and how we might approach detecting them if they were purposefully added. The milk supply system may be particularly vulnerable since contamination of a large bulk milk tank could conceivably be distributed to thousands of people. In this study we have determined that anthrax spores (derived from a vaccine strain of Bacillus anthracis that is not virulent for humans) are not killed by standard pasteurization temperatures and are stable for at least two weeks following pasteurization. It was further shown that the spores selectively concentrate into the cream fraction of raw milk. Finally we were able to use a rapid detection procedure in a portable real-time PCR device to detect down to 2500 spores per milliliter of milk in about 30 minutes. Although we have no data that tells us how many ingested spores are needed to cause disease in humans, the technology evaluated here gives us the potential to rapidly determine if significant contamination levels are present in raw or pasteurized milk.
Technical Abstract: A preparation of spores of Bacillus anthracis (Sterne strain) was utilized to evaluate commercially available reagents and portable equipment for detecting anthrax contamination using real time PCR and to assess the fate of spores added directly to bulk tank milk. The Ruggedized Automated Pathogen Identification Device (R.A.P.I.D.) was employed to detect spores in milk down to a concentration of 2500 spores per milliliter in raw milk. Commercially available primers and probes developed to detect either the protective antigen (PA) gene or the lethal factor (LF) gene both provided easily read positive signals in the RAPID following extraction from milk with the commercially available extraction kit. Nucleotide sequence analysis of the vrrA gene using DNA extracted from spiked milk provided molecular data that readily identified the spores as B. anthracis with a 100% BLAST match to the Sterne and Ames strains. Physical fate and thermal stability studies demonstrated that spores and vegetative cells have a strong affinity for the cream fraction in whole milk and a single treatment at standard pasteurization temperatures had no effect on the viability of cells 14 days after treatment. A second treatment at 72 degrees/15 seconds, 24 hours following the first, reduced viability of the population by approximately 99 percent, but still did not kill all the cells. From these studies we conclude that standard pasteurization techniques for milk would have little effect on viability of Bacillus anthracis spores but that raw or pasteurized milk pose no obstacles to rapid detection of the spores by molecular techniques.