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United States Department of Agriculture

Agricultural Research Service

2011 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. Bridging project replacing 5325-42000-027-00D (Feb. 05). Formerly 5325-42000-042-00D (11/05). Combining 5325-32000-006-00D (1/09)

3.Progress Report
This project terminated in December 2010 and was replaced by #5325-42000-048-00D, emphasizing bacterial toxins. New detection methods were developed for ricin, botulinum neurotoxin (BoNT), Shiga toxin (Stx) and staphyloccocal enterotoxin A (SEA), as follows. A quantitative polymerase chain reaction (PCR) assay was developed, using castor DNA as a marker for crude ricin toxin. The exquisitely sensitive test is suitable for detection of potential bioterror incidents of food adulteration. New monoclonal antibodies (mAbs) for ricin were used in enzyme-linked immunosorbent assay (ELISA) and immuno-PCR formats. The assays were applied to the analysis of spiked milk, ground beef, eggs, and baby foods. A variation of the assay, with electrochemiluminescence detection, provided excellent recovery and sensitive detection in ground beef, milk, and eggs. The immuno-PCR afforded sensitivity 10 – 100-fold greater than standard ELISA formats. An activity assay for ricin was developed and utilized to study the effect of food matrices on activity. Ricin proved relatively heat-stable in food matrices such as milk and ground beef.

High-affinity mAbs against BoNT/A and BoNT/B were prepared and sandwich ELISAs were developed, detecting as little as 2 pg/mL BoNT/A or BoNT/B in food matrices. Studies of the epitopes (Ab-binding sites) of BoNTs were conducted and should help with detection method and vaccine development. After attachment to magnetic beads, mAbs were used to prepare samples for a new BoNT activity assay. BoNT captured by the beads cuts a specially designed peptide molecule, producing highly fluorescent fragments. Each of the new BoNT assays could be used for large-scale screening to detect BoNT and replace bioassays. Studies of the effect of the mAbs on BoNT pharmacokinetics provide a basis for future development of Ab therapeutics against botulism.

Data on ricin bioavailability were obtained, contributing to a threat assessment for pure and crude ricin in foods. The molecular biodistribution time for ricin following oral ingestion was estimated in mice using an Ab neutralization assay. Ricin was detected in the blood about 6 to 7 h after intoxication. In studies of the bioavailability of BoNT, crude and complexed BoNT/A were about 17-fold more toxic orally compared to purified toxin. The size of the toxin complex, the presence of non-toxin components of complexes, and the type of food matrix all impacted the toxicity and bioavailability of toxin. Histopathological changes in immune system tissues suggest potential new biomarkers of BoNT exposure, permitting earlier, more successful treatment of botulism.

A cell-based assay of the superantigen activity of SEA was developed. This alternative to bioassays is specific for active toxin. Apple juice inhibited SEA activity. A cell-free translation (CFT) assay for Stx was validated. The thermal stability of Stx2 was studied using the CFT and cell-based assays. Conventional pasteurization of milk or apple juice did not eliminate activity. In studies of Stx toxicity in mice, histopathology revealed severe changes in kidneys, spleen, and thymus.

1. Detection of ricin contamination in foods. In order to detect potential intentional contamination of complex foods with the toxic protein, ricin, ARS scientists in Albany, CA, developed antibodies that bind tightly and specifically to ricin. Electrochemiluminescence (ECL) were used to detect this binding in ground beef that was experimentally spiked with tiny amounts of ricin. The ECL detection method uses a weak electric current to produce a chemical reaction that leads to generation of light. The amount of light measured was directly related to the amount of ricin in the sample. The ricin test was able to detect very small amounts of the toxin – less than the amount that would make a person sick if consumed in a serving of food. This means that the test could be used for screening foods to detect intentional contamination with this potential biothreat and prevent poisoning.

2. Distribution of ricin in mice. To better understand the toxicity of ricin, ARS scientists in Albany, CA, obtained data on the fate of ricin following oral intoxication in a mouse model system. Such data were previously unavailable because of the lack of sensitive and specific detection tools. An immuno-polymerase chain reaction assay (IPCR) for ricin was developed and used to quantify ricin in mouse sera and feces, providing information on the time course of ricin distribution in an exposed animal. Understanding the distribution and pathological effects of ricin following oral exposure provides a better understanding of the causes of toxicity and will help in the design of more effective diagnostic tests and treatment methods.

Review Publications
Clotilde, L.M., Hartman, G.L., Lau, D.K., Carter, J.M. 2011. Microbead-based immunoassay for simultaneous detection of Shiga toxins and isolation of Escherichia coli O157 in foods. Journal of Food Protection. 74(3):373-379.

Rasooly, R., Do, P.M. 2010. Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization. Journal of Agricultural and Food Chemistry. 58:12557-12561.

Land, K.M., Cheng, L.W. 2010. Botulinum neurotoxin: a deadly protease with applications to human medicine. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. Spain. Formatex Research Center. 965-971.

Brandon, D.L. 2011. Detection of ricin contamination in ground beef by electrochemiluminescence immunosorbent assay. Toxins. 3(4):398-408.

Cheng, L.W., Henderson II, T.D. 2011. Comparison of oral toxicological properties of botulinum neurotoxin. Toxicon. 58:62-67.

He, X., Qi, W., Quinones, B., Mcmahon, S.A., Cooley, M.B., Mandrell, R.E. 2011. Sensitive detection of Shiga toxin 2 and some of its Stx 2 variants in environmental samples by a novel immuno-PCR assay. Applied and Environmental Microbiology. 77(1):3558-3564. doi:10.1128/AEM.02205-10.

He, X., Mcmahon, S.A., Henderson II, T.D., Griffey, S.M., Cheng, L.W. 2010. Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR. PLoS One. 5(9):e12858. doi:10.1371/journal.pone.0012858.

Keegan, J., O'Kennedy, R., Crooks, S., Elliott, C.T., Brandon, D.L., Danaher, M. 2011. Detection of benzimidazole carbamates and amino metabolites in liver by surface plasmon resonance-biosensor. Analytica Chimica Acta. 700(1-2):41-48.

Pellett, S., Tepp, W.H., Stanker, L.H., Band, P.A., Johnson, E.A., Ichtchenko, K. 2011. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A. Biochemical and Biophysical Research Communications. 405:673-677.

Last Modified: 12/1/2015
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