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.
Under Sub-objective 1A, researchers at Albany, California, developed new monoclonal antibodies (mAbs) for botulinum neurotoxin serotype FA (BoNT/FA), also known as BoNT/H. These antibodies are known to recognize BoNT/FA as well as serotypes BoNT/A and BoNT/F, two of the four BoNTs that are known to cause botulism in humans. These antibodies will be used in new detection assays, such as sandwich enzyme-linked immunosorbent assay (ELISA) and lateral flow/dipstick assays, as well for new biosensor platforms. Additionally, under Sub-objective 1A, ARS researchers at Albany, California, also developed new mAbs for the detection of BoNT/E. The mAbs were then used in sandwich ELISA type immunoassays that were able to detect as little as 0.2 nanograms per milliliter (ng/mL) and 10 ng/mL of BoNT/E in standard buffer and fish product matrices, respectively. Abrin is a di-chain Select Agent toxin. Previously, under Sub-objective 2A, researchers at Albany, California, developed new mAbs against abrin A-chain where the toxin activity site is located. New mAbs have now been developed against the B-chain of abrin. New abrin B-chain mAbs can be combined with A chain antibodies to create potentially much more sensitive detection assays. Since these antibodies target the receptor-binding domain of abrin, they could potentially be used therapeutically in the neutralization of abrin. Botulinum neurotoxins are secreted from the bacterium along with a range of associated proteins that function to protect the toxin from digestive degradation or aid its absorption into the intestinal cells. Under Sub-objective 2B, researchers at Albany, California, developed mAbs against a previously uncharacterized accessory protein component of BoNT. These antibodies could be used to develop new detection assays or could potentially be used as a novel therapeutic target. Under Sub-objective 2B, researchers at Albany, California, developed mAbs that sensitively and selectively bind amatoxins. These reagents have been successfully incorporated into ELISA and dipstick lateral flow immunoassays (LFA). Both the ELISA and LFA have a detection limit of 1 ng/mL. The LFA has been used to detect amatoxins in three different sample types: human urine, dog urine, and from mushroom extracts. These methods of detection and mushroom toxin extraction represent a marked improvement over currently used methods that require laboratory expertise and use of expensive instruments. To date, 14 different wild mushroom species have been tested using these immunoassays with 100 percent accuracy in amatoxin detection. Under Objective 3, researchers at Albany, California, determined the stability and bioavailability of the Select Agent abrin in economically important food matrices of whole milk, non-fat milk, liquid egg and ground beef using in vitro assays (Vero cell cytotoxicity and cell free translation) as well as the mouse bioassay. Common food processing and pasteurization parameters used by processors for whole milk, non-fat milk, and liquid egg were insufficient to fully inactivate abrin activity. These studies suggest that different food matrices can have significant effects on the biological activity of abrin. Shiga toxin 2 (Stx2) is the main virulence factor produced by pathogenic E. coli strains responsible for hemorrhagic colitis and the life-threatening sequela hemolytic uremic syndrome in children. Under Objective 3, researchers at Albany, California, in collaboration with a scientist from the University of Bologna, Italy, determined how Stx2 was transferred to the target sites in the kidney and brain after accessing the circulatory system. Under Sub-objective 4A, researchers at Albany, California, and a cooperative research and development agreement (CRADA) partner evaluated a new biosensor for botulinum neurotoxin serotype A. This new biosensor assay is rapid (40-minute assay time), sensitive, portable, requires minimal processing, and uses small volumes. The biosensor was effective in the detection of BoNT/A in buffer with limits of detections in the low nanogram per milliliter (ng/mL) to 200 ng/mL range depending on the food matrix tested, which is comparable to the detection limits of some LFAs. This new detection platform, however, works better than LFAs because it can be used for more complex food matrices such as milks, acidic juices, ground beef, green bean baby puree, smoked salmon, and liquid eggs. The recent discovery and rapid spread of the mobile colistin-resistant (MCR) gene, mcr-1, in bacteria is undermining our ability to treat bacterial infections. Under Objective 4, researchers at Albany, California, developed and characterized new mouse and rabbit antibodies against MCR-1 and MCR-2, the proteins responsible for colistin resistance in bacteria. Sensitive immunoassays were built and tested for the detection of MCR-positive bacteria in meat samples. A patent application has been submitted and a commercial license for these antibodies was issued to a detection kit manufacturer. Staphylococcal enterotoxin A (SEA) is regarded as one of the most common causative agents of foodborne disease outbreaks worldwide. Under Objective 4, researchers at Albany, California, used the human T-cell line, CCRF-CEM, to create a new detection assay specific to SEA. The CCRF-CEM cells, when paired with a human B-cell (Raji) line, responded to the presence of active SEA by secreting the cytokine interleukin-2 (IL-2). Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA but did not detect the related staphylococcal enterotoxin (SE) toxin subtypes B, D, and E. The combination of CCRF-CEM human T-cells and Raji cells also resulted in a significant decrease in expression of the T-cell receptor Vbeta 9 (Vß9) when in the presence of active SEA. Under Objective 4, researchers at Albany, California, developed a flow cytometry method to detect active SEA by measuring the corresponding decrease in TCR Vß9 levels and appearance of IL-2.
1. Development of novel antibodies and detection assay to screen food samples for colistin-resistant bacteria. The recent discovery and rapid spread of mobile colistin-resistant gene, MCR-1, in bacteria is undermining our ability to treat bacterial infections and threatening human health and safety. ARS researchers at Albany, California, developed novel polyclonal and monoclonal antibodies against MCR-1 and MCR-2. An enzyme-linked immunosorbent assay (ELISA) established using these antibodies was able to detect 0.01 nanogram per milliliter of MCR-1 in buffer and 0.4 colony forming units per gram of meat, including ground chicken, pork, and beef, demonstrating strong tolerance to complex food matrices. This immunoassay could be used for rapid and reliable screening of food samples contaminated with colistin-resistant bacteria, making this an important tool for reducing the risk of foodborne infections with antibiotic resistant bacteria.
2. Development of a sensitive activity assay for staphylococcus enterotoxin serotype A. Staphylococcal enterotoxins (SEs) are some of the most commonly found food contamination agents. Assays to detect and quantify SEA ideally respond only to the active form of the toxin and this usually means employing live animal testing. Researchers at Albany, California, developed a cell-based assay for active SEA quantification in which Staphylococcal enterotoxins A (SEA) is presented by human B-cells to T-cells. The assay measures a decrease in levels of the T cell receptor Vbeta 9 (Vß9) and an increase of the IL2 cytokine. This is the first demonstration of a sensitive alternative assay that completely eliminated the use of animals for the quantitative detection of active SEA.
3. Effects of food processing on abrin bioavailability. Abrin is a highly potent plant toxin and a potential bioterror weapon. ARS researchers at Albany, California, evaluated the effectiveness of common food processing and pasteurization conditions against abrin in the presence or absence of foods. Selected food processing and pasteurization parameters used by processors for whole milk, non-fat milk, and liquid egg were insufficient to fully inactivate abrin activity. These studies suggest that different food matrices can have significant effects on the biological activity of abrin, information which could be used for food adulteration risk assessments.
4. Identification of a positive regulator of Stx2a in the human circulatory system. Stx2 is the cause of diarrhea-associated hemolytic uremic syndrome (HUS). The toxin released in the intestine by bacteria enters target organs such as the brain and kidneys by interacting with blood components. Among them, human serum amyloid P component (HuSAP) is considered a negative regulator that specifically binds to Stx2 and impairs its toxic action. Through collaborations with scientists at the University of Bologna, Italy, ARS researchers at Albany, California, identified a Toll-like receptor (TLR4) that formed protein complexes with Stx2 and inhibited its capture by HuSAP, thus allowing the toxin to target and damage human cells. This research provides important new insights on a new target for the development of therapeutic strategies for Stx2 associated-HUS.
5. Development of novel antibodies to botulinum neurotoxin serotype E. Botulinum neurotoxin serotype E (BoNT/E) outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. ARS researchers at Albany, California, developed new mouse monoclonal antibodies for the sensitive detection of BoNT/E. Sandwich enzyme-linked immunosorbent assays (ELISAs) using these reagents detected as little as 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals. These reagents would be useful to kit manufacturing companies and regulatory agencies looking to improve BoNT/E detection.
Tam, C.C., Henderson II, T.D., Stanker, L.H., Cheng, L.W. 2018. Influence of food matrices on the stability and bioavailability of abrin. Toxins. 10(12):52. https://doi.org/10.3390/toxins10120502.
Tam, C.C., Flannery, A.R., Cheng, L.W. 2018. A rapid, sensitive, and portable biosensor assay for the detection of botulinum neurotoxin serotype A in complex food matrices. Toxins. 10(11):476. https://doi.org/doi:10.3390/toxins10110476.
He, X., Mavrici, D., Patfield, S.A., Rubio, F. 2018. Development of novel antibodies for detection of mobile colistin-resistant bacteria contaminated in meats. Scientific Reports. 8:16744. https://doi.org/10.1038/s41598-018-34764-2.
Brigotti, M., Arfilli, V., Carnicelli, D., Ricci, F., Tazzari, P., Ardissino, G., Scavia, G., Morabito, S., He, X. 2018. Soluble toll-like receptor 4 impairs the interaction of Shiga toxin 2a with human serum amyloid P component. Toxins. 10(9):379. https://doi.org/10.3390/toxins10090379.
Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2018. Alternative to animal use for detecting biologically active Staphylococcal enterotoxin type A. Toxins. 10(12):540. https://doi.org/10.3390/toxins10120540.
Bever, C.R., Scotcher, M., Cheng, L.W., Hnasko, R.M., Stanker, L. 2019. Development and characterization of monoclonal antibodies to botulinum neurotoxin type E. Toxins. 11:407. https://doi.org/10.3390/toxins11070407.
Rasooly, R., Bouton, J., Van Hecke, K., Van Calenbergh, S. 2018. Synthesis of pyrrolidine-based hamamelitannin analogues as quorum sensing inhibitors in Staphylococcus aureus. Beilstein Journal of Organic Chemistry. 14:2822-2828. https://doi.org/10.3762/bjoc.14.260.
Rasooly, R., Do, P.M., He, X., Hernlem, B.J. 2019. T cell receptor Vß9 in method to rapidly detect active staphylococcal enterotoxin type A without live animals. Toxins. 11:399. https://doi.org/10.3390/toxins11070399.