2007 Annual Report 1a.Objectives (from AD-416) Determine enteric dose-response relationships for crude preparations of botulinum neurotoxin (BoNT) and ricin in rodent models and document the pathophysiological, immunological, and histological responses, as well as potential toxin synergies. Optimize sample preparation for recovery of toxins from food. Develop rapid immunological, biochemical, and/or molecular biological tests for botulinum toxin and ricin or appropriate surrogates. The results should provide both basic and applied knowledge useful in countering intentional biothreats, including an understanding of the effects of complex food matrixes on the toxicity of BoNT and ricin following exposure by ingestion and new analytical technology to detect these biothreat toxins in foods. 1b.Approach (from AD-416) Toxicity studies will be performed to better define the safety and security problems and help to define the analytical needs. The stability of pure and crude toxins in food matrices will be determined, and dose-response relationships will then be established for enteric exposure to pure and crude toxins in three food matrices (raw milk, liquid eggs, and ground beef). Oral administration of toxins will include feeding and gavage. The acute toxicity as well as the histopathology of intoxication will be studied. Milk, liquid eggs, and ground beef are the three foods of primary interest in this project, primarily because they comprise major commodities processed in large batches via a highly decentralized system. Immune responses will be characterized with regard to specificity for A chain, B chain, hemagglutinins, and other components of crude toxins. We will determine whether these antibodies are protective by challenging immunized mice and naïve controls with toxin. Sample preparation technology for toxins of interest that is compatible with real-time and multi-analyte testing of large numbers of samples will be developed. Extraction and capture procedures that extract toxin(s)or marker(s)from the matrices, concentrate the analytes, and remove most of the impurities that would otherwise interfere with the assay will be developed. These will include techniques that could be used in a field setting, e.g., a mobile laboratory or "black box" assay machine. These sample preparation methods must be simple and extremely robust; immunoaffinity capture is an example of a possible methodology. We will determine matrix effects for existing assays and use this information as a starting point for developing preparative techniques applicable to these assays as well as new techniques developed in this project. Assay protocols will be validated using food spiked with active toxin. The best methods identified for each toxin-food combination will be compared to the mouse bioassay. All sample preparation procedures will be characterized for throughput and robustness, as well as their impact on the accuracy, precision, sensitivity, and dynamic range of assays for which they are used. New, rapid tests for BoNT and ricin that can be used to test a variety of food samples will be developed utilizing multiple methodologies. Monoclonal antibody ligands, nanoparticle labels, and immunosensor techniques offer the possibility of ultrasensitive assays. An additional analytical approach will be to develop biochemical and/or cellular assays that could possibly provide even greater biological relevance. FY01 program increase $269,370. 1 SY added. Bridging project replacing 5325-42000-027-00D (Feb. 05) FY05 Prog. Inc. %400,000. Add 1 SY. Formerly 5325-42000-042-00D (11/05). 3.Progress Report In FY07 we filled a critical SY vacancy with new Research Chemist Reuven Rasooly. We also filled our vacant Administrator-funded Research Associate position. 4.Accomplishments Toxicity of crude toxins in food matrices. In order to do a risk assessment for potential toxins in food matrices, it is necessary to elucidate the toxicity of crude toxins, in comparison to the better studied purified toxins. At WRRC, Albany, CA, Foodborne Contaminants Research Unit scientists investigated the effects of heating crude ricin toxin spiked into food matrices on their bioavailability and oral LD50, comparing results for purified ricin toxin. Additionally, the relative toxicities of crude and purified botulinum toxin was also investigated. The results of the studies will help guide government and food industry scientists in developing procedures to maintain a safe and secure food supply. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest. Specifically this research addresses Problem Statements: 1.2.1 Detection and Validation, 1.2.7 Risk Assessment, and 1.2.9 Food Security. Immunoassays for biothreat toxins. There is a need for rapid, sensitive assays for botulinum neurotoxin that can be used to detect intentionally contaminated foods, without the use of animal bioassay. At WRRC our Foodborne Contaminants Research Unit scientists discovered new monoclonal antibodies for ricin and botulinum neurotoxin, and then used them to create immunoassays with sufficient sensitivity to detect subtoxic levels of these toxins in food matrices. The new antibodies for botulinum toxin resulted in high assay sensitivity, similar to that of the mouse bioassay, and some antibodies were able to protect mice from the effects of toxin. The availability of new test-tube assays with sufficient sensitivity to detect subtoxic levels of these toxins in food matrices should help maintain the security and safety of the US food supply. New protective antibodies could prove useful in treating victims exposed to these toxins. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest. Specifically this research addresses Problem Statements: 1.2.1 Detection and Validation, and 1.2.9 Food Security. Sample preparation for PCR (polymerase chain reaction) analysis of crude ricin. There is a need for rapid, sensitive assays for detection of castor bean’s toxic protein, ricin, in intentionally contaminated foods, without the use of animal bioassay. We previously developed a rapid quantitative PCR test useful for ground beef, so we investigated the applicability of this method to other foods. At WRRC our Foodborne Contaminants Research Unit scientists developed satisfactory sample preparation methods for analysis of liquid egg and milk, permitting use of the PCR method for castor DNA, a surrogate analyte for the biothreat toxin, ricin, to detect low levels of crude ricin preparations in these foods. These results should enable rapid and extremely sensitive detection of potential crude ricin contamination of two important food commodities, thus contributing to the security and safety of our food supply. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest. Specifically this research addresses Problem Statements: 1.2.1 Detection and Validation, and 1.2.9 Food Security. Assays for active biothreat toxins. Some assays for toxins in food detect active as well as inactive forms of toxins such as ricin and botulinum neurotoxin. However, ensuring food safety and security requires assays that detect biologically active forms of these toxins. At WRRC our Foodborne Contaminants Research Unit scientists developed an assay for ricin based on the ability of this toxin to inhibit protein synthesis. In addition, we developed an assay for botulinum toxin in fruit juices and milk, based on the ability of that toxin to activate a fluorescent peptide resembling the toxin’s target in nerve cells. With further development, these assays could contribute to food safety and security by providing sensitive and specific detection of active biothreat toxins. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest. Specifically this research addresses Problem Statements: 1.2.1 Detection and Validation, and 1.2.9 Food Security. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of new CRADAs and MTAs 7 Number of active CRADAs and MTAs 7 Number of invention disclosures submitted 1 Number of patent granted 1 Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 8 Review Publications He, X., Brandon, D.L., Chen, G.Q., McKeon, T.A., Carter, J.M. 2007. Detection of castor contamination by real-time polymerase chain reaction. Journal of Agricultural and Food Chemistry. 55(2):545-550.