Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A

Abstract

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.

Development of Thermally Stable Nanobodies for Detection and Neutralization of Staphylococcal Enterotoxin B

“…Among them, SEA, SEB, SEC, SED, and SEE are the five major SEs commonly associated with SFP. These SAs are highly similar in structure [ 44 ]. To test if they are immunologically distinct, we investigated the cross-reactivity of the nbs to SEA, SEE, and SED (note: SEC was not tested because it was not available during this study).…”

Crocetin attenuating Urinary tract Infection and adherence of uropathogenic E. coli in NRK‐52E cells via an inflammatory pathway

“…Pro‐inflammatory cytokines, such as IL‐6, can aggravate the inflammatory reaction and are also generated by mononuclear macrophages. Furthermore, it encourages B cells to produce a significant quantity of immunoglobulins (Rasooly et al., 2018) Another pro‐inflammatory cytokine, IL‐8, is a chemotactic factor that is produced by mononuclear macrophages, fibroblasts, epidermal cells and mediates bacterial infections to cause inflammatory responses. Another pro‐inflammatory cytokine, IL‐10 is involved in immunologic modulation and inflammatory response, and it plays a dual role in immune regulation and inflammatory reaction.…”

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B

“…This preference between specific T-cell lines and specific SE subtypes has been noted before. When we applied SEA, SEB, SED, and SEE to the CCRF-CEM T-cell line in combination with the Raji B-cell line, the results show that this assay is very specific to SEA detection; only SEA activated the CCRF-CEM T-cell line [ 31 ]. Prior research has shown the relationship between super-antigenic activity and the emetic activity of Staphylococcal enterotoxin.…”