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United States Department of Agriculture

Agricultural Research Service

2009 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). Combining 5325-32000-006-00D (1/09)

3.Progress Report
Toxicity studies have been performed and analytical techniques developed to better define and address the food safety and security concerns regarding the following toxins: ricin, Shiga toxins (Stx), botulinum neurotoxin (BoNT), and staphylococcal enterotoxin type A. This project has particularly addressed the properties of crude toxins, the forms most likely encountered in food contamination and intentional adulteration. Ricin is produced by the castor bean and the others, by bacteria. BoNTs are secreted as large complexes of various molecular sizes (toxin complex and crude toxin) that contain additional neurotoxin-associated proteins.

Analytical studies were conducted using newly produced monoclonal antibodies for ricin in enzyme-linked immunosorbent assay (ELISA) and immuno-polymerase chain reaction (PCR) formats, complementing our earlier studies using mouse bioassay. Previous results demonstrated that ricin is stabilized by food matrices such as milk and ground beef. The new studies used a variety of sample preparation techniques to extract the toxin from these matrices.

To assess the contribution of neurotoxin-associated proteins to the toxicity of BoNTs, studies were conducted in mouse models of botulinum intoxication. Our previous studies suggested that changes in immune responses could possibly serve as biomarkers of BoNT exposure, permitting earlier, more successful treatment of victims. Collaboration is underway with the Pacific Southwest Regional Center of Excellence, funded, in part, by a grant from the National Institutes of Health.

Complementing previously reported high-affinity monoclonal antibodies that specifically bind BoNT serotype A, new antibodies were produced that bind to serotype E and were characterized by ELISA and peptide-binding analysis. Recombinant peptide fragments and peptide libraries were used to characterize the precise molecular regions that bind antibody.

Staphylococcal enterotoxins involved in food poisoning act as "superantigens" that cause large numbers of cells in the immune system to start dividing. A spleen cell assay was developed to measure this activity of toxin serotype A. This assay uses a color change to detect newly made DNA as a measure of cell division. The assay is highly sensitive, accurately measuring as little as 20 nanograms per liter of toxin and can also distinguish active toxin from inactive forms of the toxin in foods, thereby providing essential information to ensure safety.

Shiga toxins, produced by E. coli O157:H7 and other bacteria, cause about 73,000 illnesses and 60 deaths per year in the U.S. A cell-free protein translation assay was developed, validated, and shown to be effective in distinguishing between E. coli strains that produce Shiga toxin 2 and nonproducing strains. A second assay, employing cells grown in tissue culture, was used to monitor the stability of Stx2 in milk. Although most milk consumed in the U.S. is pasteurized, eliminating almost all pathogens, these studies demonstrated that Shiga toxin is stable under conditions used for pasteurizing milk. This information will help guide efforts to assure food safety and security.

1. Test for detection of staphylococcal enterotoxin A in food. Staphylococcal enterotoxin A (SEA) is a common cause of food poisoning in the US, but common tests for the presence of SEA in food require the use of live animals (kittens or monkeys) or are not sensitive enough to detect small but dangerous amounts of toxin. ARS scientists in Albany, CA developed a new test based on the known property of SEA to stimulate growth of immune cells, using cells grown in culture instead of live animals. The test uses magnetic microbead technology to concentrate toxin from large volumes of sample and has a color readout, with increasing color indicating higher amounts of SEA in the food sample. Because it is more sensitive and less expensive than other tests, it is useful for application by scientists in both research and regulatory labs.

2. Rapid test for Shiga toxin. E coli O157:H7 causes thousands of cases of foodborne illness in the US each year by producing Shiga toxin. Shiga toxin kills cells by blocking their manufacture of protein. ARS scientists in Albany, CA developed a test for the presence of Shiga toxin in food based on this property. Our new test is rapid, specific, and sensitive. The test can be used by laboratory scientists to detect E coli O157:H7, and to distinguish between E coli O157:H7 and non-toxic strains of E coli.

3. Shiga toxin 2 stability in food. Shiga toxin, which is expressed by some strains of E coli bacteria, causes serious and sometimes fatal foodborne disease. ARS scientists in Albany, CA made a biosensor for Shiga toxin by inserting a gene for Green Fluorescent Protein into Vero cells. These special Vero cells, which we grow in the laboratory, are highly fluorescent until they encounter Shiga toxin from E coli, and their subsequently reduced fluorescence is easy to notice and measure. We used the new test to determine that pasteurization of milk, at 63 degrees C for 30 minutes, or at 72 degrees C for 15 seconds, is not effective at destroying the activity of Shiga toxin. The test can be used to detect Shiga and help to prevent outbreaks of the associated foodborne disease.

4. Characterization of antibodies that bind botulinum neurotoxins. Current treatment for the fatal foodborne disease of botulism uses horse serum, which causes a high rate of severe side effects. ARS scientists at Albany, CA previously created new antibodies for use in sensitive detection of botulism toxin in food. We now show that these antibodies neutralize the toxin through very specific molecular interactions with the toxin protein. These results will help in the development of improved detection methods for the toxin and in the design of improved treatments and vaccines.

5. Field test for botulism toxin. The toxin that causes botulism is the most poisonous substance known. The tiny amount capable of causing human illness or death is extremely difficult to detect using laboratory methods without live animals. ARS scientists in Albany, CA have developed antibodies with unusually strong binding to botulism toxin and used them to make a “dipstick” test that is very simple to operate and can detect minimal amounts of toxin in food. The device can be used by farmers, dairies, regulatory agencies, and even consumers to ensure food safety and security.

6. Differences in toxicity between different strains of botulism bacteria. Botulism can be caused by several different strains of the bacteria Clostridium botulinum, which produce different toxins. Using mice, ARS scientists in Albany, CA studied the serotypes most likely to cause human botulism – serotypes A, B, and E. They showed that toxicity varied depending on the food in which the toxin was present. This knowledge of the factors affecting toxicity is critical to food safety and security risk assessments.

6.Technology Transfer

Number of the New/Active MTAs (providing only)2
Number of Other Technology Transfer2

Review Publications
Brandon, D.L., Hernlem, B.J. 2009. Development of monoclonal antibodies specific for Ricinus agglutinins. Food and Agricultural Immunology. 20(1):11-22

He, X., Lu, S.S., Cheng, L.W., Rasooly, R., Carter, J.M. 2008. Effect of food matrices on the biological activity of ricin. Journal of Food Protection 71(10):2053-2058.

He, X., Quinones, B., Carter, J.M., Mandrell, R.E. 2009. Validation of a Cell-Free Translation Assay for Detecting Shiga Toxin 2 in Bacterial Culture. Journal of Agriculture and Food Chemistry. 57(11):5084-5088

Rasooly, R., Do, P.M. 2009. In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food. FEMS Immunology and Medical Microbiology 56(2009): 172-178.

Scotcher, M.C., Mcgarvey, J.A., Johnson, E.A., Stanker, L.H. 2009. Epitope characterization and variable region sequencing of F1-40, a high-affinity monoclonal antibody to botulinum neurotoxin Type A (Hall Strain). PLoS ONE 4(3). Available:

Last Modified: 4/19/2014
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