Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activate large numbers of T cells. The current method for detection of enterotoxins activity is an in vivo monkey or kitten bioassay; however, this method is not practical to test on a large number of samples. Several immunological assays have been developed however, but these assays cannot distinguish between active toxin which causes food poisoning and inactive toxin, which can bind antibody, but shows no toxicity. The current study demonstrates that short term ex vivo exposure of primary naïve CD4(+) T-cells or splenocytes to SEA induces differential expression and secretion of tumor necrosis factor (TNF) protein. We used immunomagnetic beads coated with anti-SEA antibody to specifically isolate SEA from food. After the eluted toxin was added to the cells SEA biological activity was measured by quantifying TNF protein expression or secretion.

Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of several SEs. However, these immunological methods cannot distinguish between active and inactive toxin. It is known that interferon-gamma (IFN-γ) expressed in response to stimulation by SEs contributes significantly to the pathogenesis of S. aureus infection. Nonetheless, the cellular source of IFN-γ is still unclear and the contributions of the specific splenocyte types. In our effort to understand the immunologic response to Staphylococcal Enterotoxin A (SEA) exposure, we studied IFN-γ production in mouse splenocytes. We demonstrated that short term ex vivo exposure of splenocytes or primary naïve CD4+ T-cells to biologically active SEA induces differential expression of IFN-γ mRNA in a time and dose dependent manner and the expression levels reflect the levels of IFN-γ secreted protein. Positive isolated CD4+ T-cells accounted for only 10% of IFN-γ production. We also demonstrate that expression of IFN-γ can be used for rapid quantitative analysis of active SEA with a detection limit of 1 ng/mL.

Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. Staphylococcal enterotoxin type A (SEA) is the predominant toxin produced by S. aureus strains isolated from food-poisoning outbreak cases. For public safety, assays to detect and quantify SEA ideally respond only to the active form of the toxin and this usually means employing disfavored live animal testing which suffers also from poor reproducibility and sensitivity. We developed a cell-based assay for SEA quantification in which biologically-active SEA is presented by Raji B-cells to CCRF-CEM T-cells resulting in internalization of Vβ9 within 2 hours with dose dependency over a 6-log range of SEA concentrations. This bioassay can discern biologically active SEA from heat-inactivated SEA and is specific to SEA with no cross reactivity to the homologically-similar SED or SEE. In this study, we terminated any ongoing biochemical reactions in accessory cells while retaining the morphology of the antigenic sites by using paraformaldehyde fixation and challenged the current model for mechanism of action of the SEA superantigen. We demonstrated for the first time that although fixed, dead accessory cells, having no metabolic functions to process the SEA superantigen into short peptide fragments for display on their cell surface, can instead present intact SEA to induce T-cell activation which leads to cytokine production. However, the level of cytokine secretion induced by intact SEA was statistically significantly lower than with viable accessory cells, which have the ability to internalize and process the SEA superantigen.

In this study, sixteen unique staphylococcal enterotoxin B (SEB)-reactive nanobodies (nbs), including ten monovalent and six bivalent nbs, were developed. All characterized nbs were highly specific for SEB and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb). The lowest limit of detection (LOD) reached 50 pg/mL in PBS. When applied to an ELISA to detect SEB-spiked milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the sixteen nbs, with a subset of nbs, SEB-5, SEB-9, and SEB-62, retaining activity even after exposure to 95 °C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat-labile properties. Several nbs demonstrated a long shelf-life, with one nb (SEB-9) retaining 93% of its activity after two weeks of storage at room temperature. In addition to their usage in toxin detection, eleven out of fifteen nbs were capable of neutralizing SEB's super-antigenic activity, demonstrated by their inhibition on IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs are relatively small, thermally stable, and easy to produce, making them useful in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products.

Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses through its production of a group of enterotoxins (SEs) which cause gastroenteritis and also function as superantigens to massively activate T cells. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which was detected in 17 of the 38 suspected staphylococcal food poisoning incidents in a British study and was the causative agent in outbreaks in France, UK and USA. The current method for detection of enterotoxin activity is an in vivo monkey or kitten bioassay; however, this expensive procedure has low sensitivity and poor reproducibility, requires many animals, is impractical to test on a large number of samples, and raises ethical concerns with regard to the use of experimental animals. The purpose of this study is to develop rapid sensitive and quantitative bioassays for detection of active SEE. We apply a genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC (major histocompatibility complex) class II to the engineered T cell line. Exposure of the above mixed culture to SEE induces differential expression of the luciferase gene and bioluminescence is read out in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 10⁸ times more sensitive than the monkey and kitten bioassay.

Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL-1 of SEA, which is 10⁷ times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4⁺ T-cell line, for the quantitative detection of SEA. 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, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA.