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United States Department of Agriculture

Agricultural Research Service

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention Research

2013 Annual Report

4. Accomplishments
1. Test for 7 Shiga toxin-producing E. coli (STEC) strains. Some types of E. coli bacteria produce disease in humans by releasing Shiga toxin. The seven most common STEC are known as O26, O45, O103, O111, O121, O145, and O157. Control of these bacteria is dependent on detecting them, and in the case of outbreaks it is very helpful to know which type is present. A test that determines which of the 7 most common types of E. coli are present developed by ARS scientists in Albany, California, was compared to a polymerase chain reaction-based assay developed by Food and Drug Administration. Both tests use a microbead format and were used to characterize a panel of 160 field isolates of STEC. This test facilitates and automates testing of foods possibly contaminated with STEC and extends our capability to improve food safety.

2. Rapid biochemical assays for 3 serotypes of botulinum neurotoxin. Although it is very sensitive, the mouse bioassay for botulinum neurotoxins (BoNTs) takes 4-7 days to complete, uses death as an end point, and is only is available in a few laboratories. A test is needed that specifically measures the activity of individual toxin serotypes in food and serum samples within a few hours. ARS scientists in Albany, California, cooperated with BioSentinel Pharmaceuticals Inc. (Madison, Wisconsin) to develop a set of rapid biochemical assays that measures activity of BoNT serotypes A, B, and F, 3 of the 4 serotypes associated with human disease. The test uses specific monoclonal antibodies and magnetic beads to purify the toxin complexes from food samples such as whole milk. These tests will further our ability to monitor for botulinum neurotoxins and improve the safety and biosecurity of the food supply.

3. Purification, characterization, and immunoassay of Shiga toxin 2f. Shiga toxin 2 (Stx2) subtype f is commonly found in avian species. Its role in human disease may be underestimated because it has been difficult to detect. ARS scientists in Albany, California, developed a purification scheme for this toxin and characterized its binding to cell surface glycolipids that serve as receptors. Stx2f-specific monoclonal antibodies (mAbs) were then generated that were specific for Stx2f. An antibody-based test was developed with a detection limit of 1 ng/mL Stx2f. This highly sensitive immunoassay will contribute to understanding the role of Stx2f in Shiga toxin-producing E. coli (STEC) disease in humans and animals.

4. New means of detecting staphylococcal enterotoxins. Staphylococcal enterotoxins (SEs) are produced by the bacterium S. aureus, and generate a superantigen immune response and gastrointestinal disease at low concentrations. ARS scientists in Albany, California, measured the responses of mouse immune cells exposed to S. Aureus and Staphylococcal Enterotoxins A (SEA) and S. Aureus and Staphylococcal Enterotoxins B (SEB) using a flow cytometer. This instrument detects the binding if specially labeled antibodies to specific cell surface molecules by means of a laser-excited fluorescent signal. Cell surface changes were observed within 6 hours of exposure to Staphylococcal Enterotoxins (SEs). This response provided the basis for a test for SEs that proved effective in spiked samples of milk and other food matrices. This rapid new method detects only active toxin and does so with exquisite sensitivity.

5. Reagents for biothreat detection. As part of an intense research effort to develop detection reagents, vaccines and therapeutics for biothreat agents, ARS scientists in Albany, California, developed high-affinity monoclonal antibodies (mAbs) for the sensitive detection of botulinum neurotoxins (BoNTs) and other toxins. New methods using electrochemiluminescence were developed. Some of the mAbs were also tested for their ability to provide therapeutic protection. The timing of antibody neutralization in animal models revealed windows of opportunity for antibody therapeutic treatment as well as information on the movement of BoNTs between blood and other tissues. Technology has been transferred to federal and state laboratories via cooperative work with the Department of Homeland Security, participation in the Pacific Southwest Regional Center of Excellence, and other agreements. These reagents better equip the nation for assuring food safety and biosecurity.

Review Publications
Singh, A.K., Stanker, L.H., Sharma, S.K. 2013. Botulinum neurotoxin: Where are we with detection technologies. Critical Reviews in Microbiology. 39(1):43-56. doi:10.3109/1040841X.2012.691457.

Dubois, J., Piccirilli, A., Magne, J., He, X. 2012. Detoxification of castor meal through reactive seed crushing. Industrial Crops and Products. 43(1):194-199.

Scotcher, M.C., Cheng, L.W., Ching, K.H., Mcgarvey, J.A., Hnasko, R.M., Stanker, L.H. 2013. Development and characterization of six monoclonal antibodies to hemagglutinin-70 (HA70) of Clostridium botulinum and their application in a sandwich ELISA. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 32(1):6-15. doi: 10.1089/mab.2012.0071.

He, X., Mcmahon, S.A., Skinner, C.B., Merrill, P.A., Scotcher, M., Stanker, L.H. 2013. Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk. Journal of Immunological Methods. 389(1-2):18-28. doi:10.1016/j.jim.2012.12.005.

Clotilde, L.M., Bernard IV, C., Salvador, A., Lin, A., Lauzon, C., Muldoon, M., Xu, Y., Lindpaintner, K., Carter, J.M. 2012. A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli. Journal of Microbiological Methods. 92(2):226-230. doi:10.1016/j.mimet.2012.11.023.

Cheng, L.W., Stanker, L.H. 2013. Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electrochemiluminescent immunoassay in food and serum. Journal of Agricultural and Food Chemistry. 61(3):755-760 doi:10.1021/jf3041963.

Brandon, D.L., Korn, A.M., Yang, L. 2013. Immunosorbent analysis of ricin contamination in milk using colorimetric,chemiluminescence, and electrochemiluminescence detection. Food and Agricultural Immunology. doi:10.1080/09540105.2012.753515.

Skinner, C.B., Patfield, S.A., Rasooly, R., Carter, J.M., He, X. 2013. Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2. PLoS One. 8(3):e59760.

McKeon, T.A., Shim, K., He, X. 2012. Reducing the toxicity of castor seed meal through processing treatments. Biocatalysis and Agricultural Biotechnology.

Rasooly, R., He, X., Friedman, M. 2012. Milk inhibits the biological activity of ricin. Journal of Biological Chemistry. 287(33):27924-27929. doi:10.1074/jbc.M112.362988.

Bagramyan, K., Kaplan, B.E., Cheng, L.W., Rummel, A., Kalkum, M. 2013. Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples. Analytical Chemistry. 85(11):5569-76. doi: 10.1021/ac4008418.

Brandon, D.L. 2012. Electrochemiluminescence immunosorbent assay of ricin in ground beef: Biotinylated capture antibodies and matrix effects. Food and Agricultural Immunology. 23(4):329-337.

Friedman, M., Rasooly, R. 2013. Review of the inhibition of biological activities of selected food-related toxins by natural compounds. Toxins. 5:743-775. doi: 10.3390/toxins50x100x.

Singh, A.K., Sachdeva, A., Degrasse, J.A., Croley, T.R., Stanker, L.H., Hodge, D., Sharma, S.K. 2013. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5. The Protein Journal. 32(4):288-296. DOI: 10.1007/S10930-013-9486-1.

Last Modified: 09/20/2017
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