Concentration and extraction of Bacillus anthracis spores and ricin.

Abstract

Food is an essential part of life for every human and animal. In order to feed the world, food production has become a global industry. This globalization brings efficiency of production, transportation, and year round availability of many ingredients. However, mass production of food also means that any mistake made during production is magnified in scale and distribution. Recent incidents of food contamination have involved not only traditional food pathogens, such as Salmonella and Escherichia coli O157, but also have included chemical contaminants such as melamine. These incidents serve to highlight the inherent vulnerability of food to contamination. Although the majority of foodborne illnesses are caused by a small group of pathogens, this does not preclude other bacteria or agents from being transmitted through food sources. Many potential bioterrorist agents have also the potential to be transmissible through food and water sources. Some of these agents, such as Bacillus anthracis spores and ricin toxin, are also resistant to the effects of existing food processing technologies such as pasteurization. Given the inherent vulnerability of the food production system, it seems a likely target for potential bioterrorism attack. Many rapid and sensitive tests have been developed to detect biological agents in a variety of settings. However, the complex nature of food matrices often limits the application of these tests to food sources. In addition, the distribution of a select agent in a food source may not be homogeneous, and testing of small samples may not represent the whole batch. The goal of this project was to design and test pre-analytical extraction techniques for two potential bioterrorism agents, B. anthracis spores and ricin toxin, from liquid foods. The outcome of this project was the development of a rapid concentration and extraction protocol for milk and fruit juice potentially contaminated with B. anthracis spores. The resulting sample was compatible with detection via real-time PCR for both milk and fruit juice samples and juice samples were compatible with detection with a commercially available lateral flow immunoassay. This concentration and extraction procedure enhanced the limit of detection by 2 log CFU/ml spores, such that real-time PCR can consistently detect B. anthracis at a level of 10 spores/mL in the initial sample. This project also examined the application of immunomagnetic separation for extraction of ricin toxin from liquids. Results from this portion of the project suggested that immunomagnetic beads can specifically bind ricin in traditional immunomagnetic separation. However, recirculating immunomagnetic separation using the Pathatrix® system was not demonstrated to specifically bind ricin.