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United States Department of Agriculture

Agricultural Research Service

2010 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 were performed and analytical techniques developed to address the food safety and biosecurity concerns regarding ricin, Shiga toxins (Stx), botulinum neurotoxin (BoNT), and staphylococcal enterotoxin type A (SEA). Ricin was detected using enzyme-linked immunosorbent assay (ELISA), ECL and immuno-polymerase chain reaction (PCR) formats, in important food matrices such as ground beef and dairy products as well as in samples such as blood. These methods, data, and reagents will help to bolster defense of the US food supply and may help with attribution of sources of ricin in contamination incidents.

There are 7 distinct BoNT serotypes and several kinds of toxin complexes for each of these that comprise toxin and non-toxic neurotoxin-associated proteins (NAPs). Collaborative studies of BoNTs defined biomarkers of BoNT exposure and addressed the role of NAPs in mouse models of botulinum intoxication. The oral and systemic mouse dose responses were determined in food matrices. Toxicity was affected by serotype, size of the complex, and the food matrix (which can increase or decrease toxicity).

New monoclonal antibodies (mAbs) were produced for serotypes B and E, supplementing our previously produced and now patented (US Patent No. 7,732,579) mAbs. The epitopes (antibody-binding sites on the toxin surface) were characterized and toxin neutralization studies were performed. MAbs directed against BoNT/B also neutralized BoNT/E, protecting mice from botulism. These results correlated well with studies of toxin neutralization in vitro. The results will help in developing new detection methods for toxin in foods and better diagnostic and therapeutic reagents for treating victims of botulism. SEA is one of over 20 enterotoxins produced by Staphylococcus aureus, but is responsible for the vast majority of staphylococcal foodborne disease. We further developed our cell-based assay for SEA for greater sensitivity by using a fluorescence signal for readout. Using this test, we found that both fresh and processed apple juices could reduce the biological activity of SEA. The results provide an extremely sensitive method for measuring active SEA in food samples and potential approaches to inactivate SEA and possibly other bacterial toxins.

Shiga toxins are produced by E. coli O157:H7 and other strains of this bacterium. A cellular activity assay for Stx2 was previously developed by us, using a cell line transduced with adenovirus to express the GFP gene. Stx2 inhibited the fluorescence, measured using a microplate reader. Further studies defined the heat stability of this toxin and the impact of food matrices on its activity. Although Stx2 activity was not destroyed by pasteurization, in milk or apple juice, it was inhibited by components of apple juice. Polyphenolic compounds may be responsible for the apparently irreversible toxin inactivation. These results could be exploited to improve food safety and biosecurity.

1. Ultra-sensitive test for ricin. The potential use of ricin as a bioweapon in food highlights the necessity for developing detection methods that work well for food samples. ARS scientists in Albany, CA, used a new method for the detection of ricin in three economically important food matrices. The method exploits the specificity of antibodies with the enormous amplification provided by the polymerase chain reaction (PCR) technique, to enable measurement of about 1 billionth of a gram of this toxin in a meatball-sized portion of ground beef. This technique could be used in regulatory laboratories charged with defending the US food supply and by investigators seeking the source of foodborne contaminants.

2. Ingested Shiga toxin 2 can cause pathology. Shiga toxins are produced by the bacterium Escherichia coli strain O157:H7 and by several other strains of this bacterium. ARS scientists in Albany, CA, working with collaborators at the University of California, Davis, investigated whether Shiga toxin that is consumed by eating contaminated food can survive passage through the digestive system and cause pathology like the toxin-producing bacteria themselves. Using a mouse model they found that the toxin can remain active and cause damage in the kidneys and other tissues in studies. These studies showed the importance of testing foods for toxins, not just bacteria, and the importance of preventing the contamination of food with the toxin-producing bacteria.

3. Assay for botulinum neurotoxin B in milk. Because the soil bacterium Clostridium botulinum occurs so widely, the deadly neurotoxin it produces commonly causes severe food poisoning. ARS scientists in Albany, CA developed a sensitive test for botulinum neurotoxin serotype B, the second most common form of this toxin. The test was used to detect toxin in artificially contaminated milk. The method uses new monoclonal antibodies developed in the Albany laboratory and could detect less than one billionth of a gram of toxin in a teaspoonful of milk. This assay is fifty times more sensitive than the standard mouse bioassay, and will contribute to our ability to assure the safety of the food supply.

4. Sensitive assay for Shiga toxin-producing bacteria. Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens with high economic consequences. ARS scientists in Albany, CA, in collaboration with scientists in the San Francisco district office of the US Food and Drug Administration, developed new methodology for detecting these bacteria and the bacterial proteins, known as virulence factors, that contribute to the ability of the bacteria to cause disease in humans. This method is based on a magnetic microbead assay that permits sampling a larger volume of food extract than is possible with many other analytical methods. This reliable, sensitive, and rapid assay will directly benefit regulatory agencies and food producers and processors by minimizing analysis time, without producing false-positive or false-negative results.

Review Publications
Rasooly, R., Do, P.M. 2009. Shiga toxin Stx2 is heat-stable and not inactivated by pasteurization. International Journal of Food Microbiology. 136(2010):290-294

Hernlem, B.J., Hua, S.T. 2010. Dual fluorochrome flow cytometric assessment of yeast viability. Current Microbiology. 61:57-63.

Cheng, L.W., Stanker, L.H., Henderson Ii, T.D., Lou, J., Marks, J.D. 2009. Antibody Protection Against Botulinum Neurotoxin Intoxication In Mice. Infection and Immunity. 77(10):4305-4313.

He, X., McMahon, S.A., McKeon, T.A., Brandon, D.L. 2010. Development of a Novel Immuno-PCR Assay for Detection of Ricin in Ground Beef, Liquid Chicken Egg and Milk. Journal of Food Protection. 73(4):695-700

Scotcher, M.C., Cheng, L.W., Stanker, L.H. 2010. Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and its Application to Toxin Detection in Milk. PLos One. 5(6):e11047.doi:10.1371/journal.pone.0011047.

Rasooly, R., Do, P.M., Friedman, M. 2010. Inhibition of Biological Activity of Staphylococcal Enterotoxin A (SEA) by Apple Juice and Apple Polyphenols. Journal of Agricultural and Food Chemistry. 58:5421-5426.

Scotcher, M.C., Johnson, E.A., Stanker, L.H. 2009. Characterization of the epitope region of F1-2 and F1-5, two monoclonal antibodies to Botulinum Neurotoxin Type A. Hybridoma. 28(5):315-325

Last Modified: 4/19/2014
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