Fate and detection of Bacillus anthracis spores in pasteurized milk, juice and eggs

Bacillus anthracis is a highly virulent, rapidly growing gram-positive bacterial pathogen that may cause inhalational, gastrointestinal or the cutaneous forms of anthrax in humans. The inhalational form of anthrax is the most lethal and feared form of the disease particularly if spores from a virulent strain are deliberately used as a source of human exposure. However, recently, gastrointestinal anthrax resulting from the consumption of meat from animals that die of the disease has been reported in humans from several parts of the world. Deliberate contamination of common foods such as milk, powdered milk, other milk products, fruit juices, liquid eggs and other foods with spores of this organism could potentially result in a massive epidemic and panic in unsuspecting populations. This could be a real threat to homeland biosecurity. In this study, survival of B. anthracis was evaluated by using freshly prepared spore suspensions of Bacillus anthracis Sterne (BAS) and Pasteur (BAP) of known concentration to spike different food matrices, followed by subjection of the matrixes to four different pasteurization temperatures and time protocols common to the dairy industry in the United States. These were: (i) the traditional---low temperature/longer time: 63° C for 30 minutes, (ii) high temperature/short time: 72°C for 16 seconds, (iii) higher temperature/short time: 78°C for 16 seconds and (iv) the most recent method of subjecting the milk to ultra high temperature/very short time: 100°C for 3 seconds. The detection and/or enumeration of spores was conducted using microscopic spore count, viable colony count, a real time PCR test (based on a widely used proprietary protocol run on Roche's LightCycler RTM platform) and an electrochemiluminescence assay (BioVerify Anthrax Test manufactured by BioVeris, run on BioVeris M1M platform). Viability of spores following pasteurization depended mostly upon the type of pasteurization, type of food matrix, spore concentration in the suspension and the B. anthracis spore strain used. All pasteurization processes were completely lethal to all vegetative cells. B. anthracis Pasteur strain spores were more susceptible to heat inactivation compared to the Sterne strain. Pasteurization had little or no effect on Sterne strain spores. Among the four common pasteurization methods selected, the ultra high temperature/short time pasteurization (100°C. for 3 sec.) was more lethal to spores, especially the Pasteur strain, resulting in a one to two log reduction in viability. These studies suggest that current pasteurization techniques, as practiced by the dairy industry for various products, would have little to no effect on viability of spores. Using 100°C for 3 seconds may be helpful in reducing the spore load, and at light contamination levels may be preferred in management of crisis related to milk contamination with B. anthracis spores. Technology for rapid detection of spores in raw or processed dairy products is not yet perfected and bacteriological culture remains the "gold standard" (standard method of diagnosis).