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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT OF DETECTION TECHNOLOGIES FOR TOXINS AND THEIR VALIDATION IN FOOD MATRICES
2008 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
Toxicity studies have been performed and analytical techniques developed to better define and address the food safety and security concerns for two major biothreat toxins, ricin and botulinum neurotoxin (BoNT). Studies in this project have particularly addressed the properties of crude toxins, the forms most likely encountered in intentional food adulteration. Ricin is produced by the castor bean and BoNTs, by bacteria (especially Clostridium botulinum). BoNTs are secreted as large complexes of various molecular sizes (referred to in this report as toxin complex and crude toxin) that contain non-toxic neurotoxin-associated proteins (NAPs).

Analytical studies were conducted using newly produced mAbs for ricin in ELISA and immuno-polymerase chain reaction (PCR) formats. The effects of food matrices on the activity of ricin was studied a cell-free translation assay that uses the enzyme luciferase as a reporter for protein translation. These assays complemented earlier studies of this project using mouse bioassay. The results demonstrated that ricin is relatively heat-stable in food matrices such as milk and ground beef, suggesting it may be stabilized by food components such as fats or by thermally induced aggregation.

Protein components secreted into culture supernatants along with BoNT serotype A (BoNT/A) were identified by mass spectrometry. The contribution of NAPs to the toxicity of BoNTs was determined in mouse models of botulinum intoxication. Although toxicities of pure, crude, and complex toxin were similar (on the basis of pure toxin content) when administered by intraperitoneal injection, the crude and complex toxins were about 17-fold more toxic orally. The nonfat milk matrix mitigated the oral toxicity of crude toxin about 2-fold, but no effect was seen with toxin complex. Observed histopathological changes in tissues of the immune system suggest that changes in immune responses could possibly serve as biomarkers of BoNT exposure, permitting earlier, more successful treatment of intoxications.

High-affinity monoclonal antibodies (mAbs) that specifically bind BoNT/A were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blot studies, and peptide-binding analysis. Two of the mAbs were used to develop a highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A in liquid food matrices such as milk. This assay offers sufficient sensitivity to provide a realistic alternative to the mouse bioassay for detection of BoNT/A. Further studies are in progress using recombinant peptide fragments and phage-display libraries to characterize the epitopes of BoNT/A – the precise parts molecule that bind antibody. One the new antibodies was also used in the form of immunomagnetic beads to aid in sample preparation for a new assay of BoNT/A activity in liquid food matrices. In this assay, BoNT/A specifically cleaves a synthetic peptide, generating a fluorescence signal that provides enough sensitivity to detect active toxin well below levels lethal to humans orally (about 70 mcg).

This relates to National Program 108, Component 1.


4.Accomplishments
1. Bioavailability of botulinum neurotoxin in food matrices. Along with potent neurotoxins (BoNT), the crude food poisoning mixture produced by Clostridium botulinum bacteria includes other non-toxic proteins. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA compared the toxicity of crude versus purified BoNT preparations presented to mice in food, and showed these accessory proteins protect BoNT from destruction and inactivation in the digestive tract. These results help scientists understand how these potent biothreat toxins work and to develop strategies for maintaining a safe and secure food supply. Understanding the stability of these biothreat toxins will help government agencies and food processors develop strategies for maintaining a safe and secure food supply. This accomplishment addresses NP108 Food Safety Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest; Problem Statements 1.2.1 Detection and Validation, 1.2.7 Risk Assessment, and 1.2.9 Food Security.

2. Activity assay for botulinum neurotoxin in food matrices. Rapid and simple detection of active botulinum neurotoxin (BoNT) is important for ensuring food safety and security. BoNT causes fatal paralysis by cutting a specific protein involved in nerve transmission. In the new test developed by ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA, BoNT is first concentrated using antibodies and magnetic beads. Then the BoNT cuts a specially designed peptide molecule, producing highly fluorescent fragments. This fast and effective assay system can be used for large-scale screening to detect BoNT , replacing widely used animal bioassays, and helping government agencies and food processors assure a safe and secure food supply. This accomplishment addresses NP108 Food Safety Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest; Problem Statements 1.2.1 Detection and Validation, and 1.2.9 Food Security.

3. Activity assay for ricin toxin in food matrices. To avoid false positives from heat-killed toxin, detection of bioactive ricin (from castor beans) requires a sensitive test of the unusual enzymatic activity, in which the toxin inactivates the ribosome, the cell’s protein-making apparatus. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA assessed the utility of a recently developed cell-free ribosomal translation assay and optimized conditions for detecting biologically active ricin in food samples. The results suggested that some components in these food matrices may protect the activity of ricin against heat. This information will help guide government agencies and food processors to provide effective food defense. This accomplishment addresses NP108 Food Safety Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest; Problem Statements 1.2.1 Detection and Validation, 1.2.7 Risk Assessment, and 1.2.9 Food Security

4. New assay for botulinum neurotoxin. Because botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum, is the most toxic agent known, tests must be extremely sensitive to be practical. Most testing for BoNT is currently performed using live mice. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA have created four new monoclonal antibodies (mAbs) that bind specifically and unusually strongly to BoNT. The mAbs were used to develop a new test for BoNT, called a sandwich ELISA, which represents a realistic alternative to the mouse bioassay. This assay could be adapted for field use by farmers, dairies, and regulatory agencies to ensure food safety and security for consumers. This accomplishment addresses NP108 Food Safety Component 1.2 Pathogens, Toxins, and Chemical Contaminants Postharvest; 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 CRADAS1
Number of the New MTAs (providing only)2
Number of New Patent Applications Filed1
Number of Non-Peer Reviewed Presentations and Proceedings5
Number of Other Technology Transfer1

Review Publications
Cheng, L.W., Onisko, B.C., Johnson, E.A., Reader, J.R., Griffey, S.M., Larson, A.E., Tepp, W.H., Stanker, L.H., Brandon, D.L., Carter, J.M. 2008. Effects of purification on the bioavailability of botulinum neurotoxin type A. Toxicology. 249(2008):123-129.

Stanker, L.H., Merrill, P.A., Scotcher, M.C., Cheng, L.W. 2008. Development and Partial Characterization of High-affinity Monoclonal Antibodies for Botulinum Toxin Type A and their use in Analysis of Milk by Sandwich ELISA. Journal of Immunological Methods, 336:1-8.

He, X., Chen, G.Q., Kang, S.T., Mckeon, T.A. 2007. Ricinus communis contains and acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester. Lipids Journal. 42:931-938.

Rasooly, R., Stanker, L.H., Carter, J.M., Do, P.M., Cheng, L.W., He, X., Brandon, D.L. 2008. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food. International Journal of Food Microbiiology. 126 (2008):135-139

Rasooly, R. 2007. Phloxine B, aversatile bacterial stain. International Journal of Food Microbiology.157: 29-34.

Rasooly, R., Do, P.M. 2008. In vitro peptide cleavage assay for detection of Botulinum Neurotoxin-A activity in food. Applied and Environmental Microbiology. 74(14):4309-4313

Last Modified: 9/10/2014
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