The overall objective of this project is to advance the development of technologies for detecting toxins that impact food safety and food defense and to determine their stability and bioavailability. Specifically, the project will focus on the following four objectives: Objective 1: Advance the development of structure- and activity-based detection methods for protein toxins. Subobjective 1A: Develop new antibodies (Abs) to botulinum neurotoxin (BoNT) serotype F, with serotypes C, D, and G as secondary priorities. Subobjective 1B: Determine the impact of different types of accessory proteins on the detection of BoNTs. Subobjective 1C: Develop activity-based detection methods for staphylococcal enterotoxin (SE) serotype E. Subobjective 1D: Develop monoclonal antibodies (mAbs) to Shiga toxin (Stx) subtypes and variants, including those from non-E. coli. Objective 2: Advance the development of detection methods for non-bacterial toxins. Subobjective 2A: Develop new detection methods for plant-derived protein toxins such as abrin. Subobjective 2B: Develop new detection methods for mushroom toxins such as amatoxins. Objective 3: Assess foodborne risks through examination of toxin stability and bioavailability in relation to intrinsic and extrinsic stresses. Subobjective 3A: Use activity-based assays to assess impact of food processing, matrices and accessory proteins on toxin activity. Subobjective 3B: Determine the factors that affect the bioavailability of toxins using rodent bioassay. Objective 4: Advance the development of instrumental, portable, and field-deployable testing methods. Subobjective 4A: Develop platforms such as optical array technologies to detect toxins. Subobjective 4B: Utilize instrumental methods to detect toxins based on mass spectra and/or other physicochemical characteristics.
Objective 1 has 2 general approaches: (1) Exploit ELISA and related technologies because of their versatility, robustness, and sensitivity. The mAbs developed for immunoassay will also be useful for sample preparation and for establishing design criteria for protective antibodies with clinical utility. (2) Develop activity assays for Stx2 variants and SEs. Activity assays will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are expected in processed food samples. Both approaches address practical analytical problems. The following hypotheses will be tested:(a) High affinity mAbs and recombinant Abs for BoNT serotypes and NAPs as analytical targets will provide useful reagents for ELISA and sensor methodology. (b) Neurotoxin-associated proteins (NAPs) influence the physicochemical properties of BoNT complexes, and their ease of detection in food matrices. These effects depend on the types of accessory proteins present. (c) A cell line can be engineered to provide a cell-based activity assay for SEE to measure active toxin in food matrices to replace bioassay and improve upon structure-based immunoassay. (d) New mAbs will be able to distinguish new subtypes of Stx1 or Stx2 produced by non-E. coli bacteria such as Enterobacter cloacae. Objective 2 will exploit immunoassays, especially ELISA and related technologies (as in Objective 1) and also develop and utilize activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation. The following hypotheses will be tested: (a) High affinity variant-specific mAbs will provide useful reagents for ELISA and sensor methodology for detecting nonbacterial protein toxins like abrin. (b) High affinity toxin-specific and group-specific mAbs will provide useful reagents for ELISA and sensor methodology for mushroom toxins. Objective 3 will exploit activity assays and rodent bioassay to better define the vulnerabilities of our food supply and the analytical needs. The following hypotheses will be tested: (a) Food processing conditions, food matrices, and accessory proteins impact toxin activity. (b) The oral bioavailabilities of BoNT and abrin vary among subtypes of toxin and state of the toxin (pure, complexed, crude) and depend on the food matrix. Objective 4 will exploit instrumental and portable technologies for toxin detection. Some of these technologies will will utilize binding molecules and activity assays developed under Objectives 1 and 2. To advance the development of instrumental and field-deployable testing, the following hypotheses will be tested: (a) A robust cell-based activity assay for SEE in a food matrix can be developed using a small fluorescence-detecting charge-coupled device to read data. (b) Mass spectral data and other physicochemical properties are useful for detection of toxin proteins and peptides. Contingency plans are built into the Approach for each objective and sub-objective. For example, contingencies for Objective 1 include use of alternative immunogens, sample preparation strategies, and assay formats.
This is the final report for project 2030-42000-049-00D, “Advance the Development of Technologies for Detecting and Determining the Stability and Bioavailability of Toxins that Impact Food Safety and Food Defense,” which expired on December 27, 2020, and has been replaced by the new project 2030-42000-053-00D, “Technologies for the Detection of Bacterial and Plant Toxins and Allergens that Impact Food Safety and Food Defense.” For additional information, see the new project. The objectives of the project were to develop critical reagents against toxins, develop sensitive detection assays, and assess the contribution of non-toxic components of complexes for toxin stability in relevant food matrices in order to protect the food supply and public health. Significant progress was achieved for all four objectives. For the life of this entire project, novel reagents, methodologies, and technologies that improve the detection of both bacterial and non-bacterial toxins have been published (63 peer-reviewed), patented (5) and licensed (16). The overall goal of Objective 1 was to advance the development of structure and activity-based detection methods for protein toxins, such as botulinum neurotoxins (BoNT) and their accessory proteins, staphylococcal enterotoxins (SE), and Shiga toxins (Stx). In support of Sub-objective 1A, immunogens were made via molecular biology while a novel inactivation strategy created toxoids. Antibodies to BoNT/E, BoNT/FA, and humanized BoNT/FA were developed and characterized. A BoNT/E sandwich enzyme-linked immunosorbent assay (ELISA) identified toxin in both buffer and food matrices at levels comparable to known detection assays. The goal of Sub-objective 1B was to determine the impact of accessory proteins on the detection of BoNTs. Immunogens to all neurotoxin-associated proteins (NAPs), accessory proteins of botulinum neurotoxins, were generated. Hemoagglutinin-70 mAb characterization did not reveal an improvement to BoNT/A detection. Characterization of BoNT/FA and NAPs monoclonal antibodies (mAbs) will continue in the new project. Over the course of this project, multiple cell-based activity assays for SE serotypes A, E and D were developed under Sub-objective 1C. Multiple cell lines were engineered to couple toxin activity with a defined read-out including light emission and cell proliferation. These assays revealed orders of magnitude increase in sensitivity as compared to ELISAs or in vivo animal models. These assays have been used in the detection of these active SEs in food matrices, hence improving our surveillance capabilities of our stakeholders for contaminated food. Refinement of these assays will continue in the new project to include new toxin targets. During the life of this project for Sub-objective 1D, we successfully developed antibodies to all Stx subtypes and variants. Of importance were mAbs to Stx1d, Stx1e, Stx1e-B-chain, Stx2b, Stx2f, Stx2h, Stx2k, and a rabbit polyclonal antibody to Stx1. New highly sensitive ELISAs were developed that were compatible with food matrices, environmental samples, and human sera. These antibodies were used to develop ELISAs capable of detecting all Stx1 toxins, a “Universal” Stx assay for both Stx1 and Stx2 toxins, and detection of Stx2k, a subtype poorly recognized by current methods. A new sample preparation method for human sera was established and enhanced detection of Stx2 in human sera from Shiga toxin-producing E.coli (STEC)-infected patients, allowing for early detection and better treatment outcomes for STEC-infected patients. Additional mAbs and detection assays have been developed to identify mobile colistin-resistance in meat samples and its prevalence in the U.S. food supply. These critical reagents and assays will help our stakeholders protect the U.S. food supply. The new project will continue the development of Stx reagents and detection assays. The overall goal of Objective 2 was to develop detection methods for non-bacterial toxins, such as abrin and the mushroom toxin, amanitin. Under Sub-objective 2A, seven mAbs to both the A- and B-chain subunits of abrin toxin were developed. These mAbs were incorporated into a new ELISA capable of abrin detection in buffer and milk equivalent to commercially available kits. A collection of abrin A-chain hybridoma cell lines were obtained, expanded, and monoclonal antibodies were purified. A sandwich ELISA comprised of these mAbs detected abrin at levels similar to available kits. Additionally, another nine abrin B-chain mAbs were made and partial characterization revealed no cross-reactivity to the A-chain of abrin. Further characterization will occur in the new project. Under Sub-objective 2B, new methodologies to link and conjugate (periodate) amanitin, a known mushroom toxin, to carrier proteins were established. Polyclonal and monoclonal antibodies to three different forms of amanitin, alpha-, beta-, and gamma, were successfully produced using these new methods and detection assays were developed using these antibodies. A competitive ELISA detected amanitin in both buffer and mushrooms with high sensitivity. A simple, cost-effective, dipstick lateral flow immunoassay (LFIA) using these mAbs distinguished all three forms of amanitin in human and dog urine along with mushroom extracts. The assay detection limits were as low as 10 nanograms per milliliter (ng/mL), well below the amounts needed to cause human disease. The LFIA is a vast improvement over available assays and comparable to the gold standard, liquid chromatography mass-spectrometry. This LFIA will allow for more rapid diagnosis of amanitin poisoning by the general public, public health departments, and veterinary clinics. The overall goal of Objective 3 was to assess toxin stability and bioavailability in relation to both intrinsic and extrinsic stresses. Under Sub-objective 3A, researchers at Albany, California, developed two types of in vitro assays, cell-free (in vitro protein translation) and cell-based (cell cytotoxicity), to detect active abrin, Stx, and SE toxins in different food and environmental matrices. Toxins were exposed to a variety of stress conditions (heat, pH, chemical, biological, foods, food processing conditions) and toxin activity was measured using one or both of these assays. A cell-based assay validated Stx toxin activity in environmental samples at low concentrations of STEC bacteria regardless of the presence of microflora or chlorine. Abrin was extremely tolerant to both heat and pH in both buffer and food matrices. Food matrices protected SEE and abrin when exposed to high heat. Common food processing temperatures and times were insufficient to inactivate abrin in different food matrices. These in vitro cell-free and cell-based assays are less time consuming, cheaper, and may obtain results similar to those using animal experiments. Under Sub-objective 3B, researchers at Albany, California, found that many factors, including accessory proteins and inhibitors, affect the toxicity and bioavailability of toxins in vitro and in vivo. BoNT/A accessory proteins enhanced the rate of toxin uptake and internalization but were not required in the oral intoxication animal model. Crude BoNT/E extracts were found to be more toxic than the holotoxin or BoNT/E complex, implicating a role for these accessory proteins in toxin stability and bioavailability in foodborne botulism. Bithionol, a Food and Drug Administration-approved drug, protected mice from BoNT/A toxin mediated death. Yeast and Lactobacilli probiotic strains inhibited the cellular uptake and internalization of BoNT/A in vitro. Heat-treated abrin were assessed for active toxin activity using both an in vitro cell-free assay and the in vivo mouse bioassay. The cell-free assay indicated a temperature-dependent dose response, but these results were contradicted by the in vivo mouse assay. These results implicate a need to validate in vitro assay results that only measure toxin catalytic activity with in vivo animal experiments that model the entire intoxication process. Researchers at Albany, California, also showed that witch hazel extracts were anti-bacterial and inhibited SEA toxin production. Addition of the extract improved the efficacy of commercially available teat dips used to treat bovine inflammatory infections by Staphylococcus. Integration of these results and practices by stakeholders may improve both human and animal health. The overall goal of Objective 4 was to advance the development of instrumental, portable, and field-deployable testing methods. Under Sub-objective 4A, a simple, sensitive, low-cost charge-coupled device (CCD) camera system was developed to detect and quantify active toxins. This technology can be coupled to other cell-based assays (luminescent, fluorometric, flow-cytometric) to detect active aflatoxin, SEA, SEE, Bacillus cereus non-hemolytic enterotoxin (Nhe), and abrin in different matrices that are orders of magnitude greater than commonly used detection assays or in vivo animal models. This technology can be further developed to target other food contaminants for the rapid detection of contaminated food and food outbreaks. A rapid, easy-to-use, portable cell-based biosensor assay was validated for use to detect BoNT/A in 10 different matrices, including economically important foods with good sensitivities. These portable technologies will greatly improve food safety surveillance for stakeholders in the food industry, governmental and regulatory agencies. Under Sub-objective 4B, researchers at Albany, California, developed a liquid chromatography mass spectrometry method that detected very low amounts of Shiga toxin in human sera and refinement improved its sensitivity 10-fold. These results implicate a vital role for this technology to diagnose STEC-infected patients early for better treatment outcomes.
1. Detection of active Stx in environmental waters. Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the Environmental Protection Agency’s current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. ARS researchers at Albany, California, developed a cell-based assay for the detection of metabolically active Stxs in environmental water. This assay is simple, affordable, performance is not affected by background flora and chlorine, and it can detect active Stxs even when Stx-producing bacteria are less than 0.4 CFU/mL. The multi-well format of the assay makes it ideal for high-throughput screening of water samples by environmental public health surveillance programs to reduce human risk of infection with STEC.
2. Simple, low-cost CCD camera system for active Bacillus cereus non-hemolytic enterotoxin complex. Bacillus cereus is a common foodborne pathogen and non-hemolytic enterotoxin (Nhe) is thought to cause intestinal distress and diarrhea. Assays to detect and quantify Nhe ideally respond only to the active form of the toxin and this usually employs animal testing. ARS researchers at Albany, California, built a low-cost charge-coupled device (CCD)-luminometer and applied it to cell-based assays. In the presence of active toxin, emitted light is reduced and this can be quantified. This method was able to detect as little as 7 ng/ml of active toxin. Simple and inexpensive methods add to the arsenal of tools in the accurate detection of Nhe in adulterated foods and as an attractive alternative to animal experiments.
Hughes, A.C., Patfield, S., Rasooly, R., He, X. 2020. Validation of a cell-based assay for detection of active Shiga toxins produced by Escherichia coli in water. International Journal of Environmental Research and Public Health. 17(21):7901. https://doi.org/10.3390/ijerph17217901.
Carter, M.Q., Pham, A., Huynh, S., Parker, C., Miller, A., He, X., Hu, B., Chain, P. 2020. DNA Adenine Methylase, not the Pstl restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli. Food Microbiology. 96. Article 103722. https://doi.org/10.1016/j.fm.2020.103722.
Rasooly, R., Do, P.M., Hernlem, B.J. 2020. Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex. PLoS ONE. 15(9): Article e0238153. https://doi.org/10.1371/journal.pone.0238153.
Carter, M.Q., Pham, A.C., Du, W.N., He, X. 2020. Differential induction of Shiga toxin in environmental Escherichia coli O145:H28 strains carrying the same genotype as the outbreak strains. International Journal of Food Microbiology. 339. Article 109029. https://doi.org/10.1016/j.ijfoodmicro.2020.109029.