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Concentration and Extraction of Bacillus Anthracis Spores and Ricin

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Concentration and Extraction of Bacillus Anthracis Spores and Ricin Concentration and Extraction of Bacillus anthracis Spores and Ricin Toxin from Liquid Foods A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Oriana Nicole Leishman IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisors: Dr. Francisco Diez-Gonzalez and Dr. Theodore P. Labuza June 2009 © Oriana Nicole Leishman 2009 Acknowledgements I’d like to thank my family, Eric, Vanessa, Mom, and Papa, for their inspiration, love and support through this process. I would also like to thank my wonderful advisors, Dr. Francisco Diez-Gonzalez and Dr. Theodore Labuza for all their advice, support, and patience. Also thank you to Dr. Joellen Feirtag for all her great feedback and advice. I’d like to thank Dr. Michael O’Connor for helping me get excited about the world of academic research. Sincere thanks to Dr. Gary Krejcarek for his advice and help with this project, as well as for suggesting food science. I’d like to thank the Science Museum of Minnesota, Nora Geving, and Joanne Weber for building the foundations of my love for biological science. Thank you to everyone in the Diez lab group! Special thanks to Dr. Sa Xu - for teaching me about spores and getting me started on this journey, Dr. Amit Pal - for teaching me how to get a PhD while keeping at least 50% of your sanity, Stelios Viazis- for his friendship and humor through the grad school process, Miranda Johnson- for her good humor, all of her help with experiments, lighting quick media preparation and for cleaning so many bottles of milk, Matt Dodor – for all his excellent media making skills! Thanks to my computer and flash drives for keeping this document safe. Lastly, and most importantly, thank you to my brain for remaining partly functional throughout this process and for never letting me give up. i Dedication This dissertation is dedicated to the professors, teacher, and mentors who inspired me to take this journey. ii 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 iii 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. iv Table of Contents Acknowledgements ............................................................................................................ i Dedication .......................................................................................................................... ii Abstract.............................................................................................................................iii List of Tables ................................................................................................................... vii List of Figures................................................................................................................... ix Chapter 1 ........................................................................................................................... 1 Introduction and statement of the problem.................................................................... 1 1.1 Introduction............................................................................................................... 1 1.2 Statement of the problem.......................................................................................... 4 1.3 Research goals, hypotheses (Ho), and sub-objectives............................................... 5 Chapter 2 ........................................................................................................................... 8 Review of Bacillus anthracis, ricin toxin, the history of bioterrorism, milk processing, and orange juice processing ......................................................................... 8 2.1 Bacillus anthracis ................................................................................................... 10 2.1.1 Physiological properties of Bacillus anthracis .................................................... 11 2.1.1a Physical structure ............................................................................................... 11 2.1.1b Defining characteristics ..................................................................................... 11 2.1.1c Morphology....................................................................................................... 12 2.1.1.1 The vegetative cell and germination............................................................. 13 2.1.1.2 The spore and sporulation............................................................................. 15 2.1.2 Virulence factors.................................................................................................. 17 2.1.3 Regulatory pathways relating to virulence .......................................................... 26 2.1.4 Diseases caused by Bacillus anthracis ................................................................ 28 2.1.4.1 Animal disease.............................................................................................. 29 2.1.4.2 Cutaneous anthrax......................................................................................... 31 2.1.4.3 Inhalation anthrax ......................................................................................... 32 2.1.4.3a Inhalation anthrax outbreak in Sverdlosk, USSR 1979 .............................. 34 2.1.4.4 Gastrointestinal anthrax ................................................................................ 35 2.2 Population structure of B. anthracis ....................................................................... 39 2.3 B. anthracis vaccines .............................................................................................. 41 2.4 Current detection methods for B. anthracis............................................................ 44 2.5 Ricin........................................................................................................................ 46 2.5.1 Ricin toxicity and treatment............................................................................. 49 2.5.2 Potential therapeutic uses of ricin.................................................................... 51 2.6 Current methods for ricin detection ........................................................................ 51 2.7 Pathatrix® use for detection of foodborne pathogens ............................................ 52 2.8 Brief history of biological and chemical warfare ................................................... 53 2.9 Cow’s Milk ............................................................................................................. 64 2.9.1 Milk composition and definition...................................................................... 64 2.9.2 Milk Processing ............................................................................................... 66 2.9.3 Vulnerability of the milk processing system ..................................................
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