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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Foodborne Toxin Detection and Prevention Research » Research » Publications at this Location » Publication #323114

Research Project: Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins

Location: Foodborne Toxin Detection and Prevention Research

Title: Sensitive, rapid, quantitative and in vitro method for the detection of biologically active staphylococcal enterotoxin type E

Author
item Rasooly, Reuven
item Do, Paula
item Hernlem, Bradley - Brad

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2016
Publication Date: 5/13/2016
Publication URL: http://handle.nal.usda.gov/10113/63154
Citation: Rasooly, R., Do, P.M., Hernlem, B.J. 2016. Sensitive, rapid, quantitative and in vitro method for the detection of biologically active staphylococcal enterotoxin type E. Toxins. 8(5):150. doi: 10.3390/toxins8050150.

Interpretive Summary: Food poisoning caused by staphylococcal enterotoxins is among the leading causes of food-borne outbreaks with an average of 21 outbreaks annually. The largest outbreak in the US involved 1,300 people and in Japan 13,420 people. The current method for detection of enterotoxins 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. Several immunological assays have been developed, but these assays cannot distinguish between active toxin, which poses a threat to public health, and inactive toxin, which can bind to antibodies but shows no toxicity. Hence, there is a need for inexpensive alternative method for detection of active enterotoxins. In this study we used a cell based assay that makes quantification more robust. We utilized genetically engineered T cell-line expressing the luciferase reporter gene, combined with a B-cell line that presents the toxin to the engineered T cell line. Exposure of the above mixed culture to increasing active staphylococcal enterotoxin type E concentrations induces differential expression of luciferase gene and bioluminescence in a dose dependent manner over a 6-log range. The limit of detection of biologically active SEE is 1 fg/mL which is 104 times more sensitive than the spleen cell proliferation bioassay, 106 times more sensitive than a typical ELISA assay and 109 times more sensitive than the monkey and kitten bioassay.

Technical Abstract: Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of 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. Several SEs subtypes have been produced as an offensive biologic warfare agent. In the present study, we tested Staphylococcal enterotoxin type E (SEE), which has been detected in 17 of the 38 suspected staphylococcal food poisoning incidents and was the causative outbreaks in France, UK and USA. The current method for detection of enterotoxins 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. Several immunological assays have been developed, but these assays cannot distinguish between active toxin, which poses a threat to public health, and inactive toxin, which can bind to antibodies but shows no toxicity. Our previous work with mouse splenocytes, distinguishes between active and inactive toxin, and reduces the number of animals used. One mouse spleen can be used for 500 tests, yet this assay still requires the sacrifice of live animals. The current study utilizes the mechanism by which SEE, once presented by MHC II on B cells, is able to activate TCR on T cells and leads to the transcription activation of nuclear factor of activated T-cells (NFAT). We apply genetically engineered T cell-line expressing the luciferase reporter gene under the regulation of NFAT response element (NFAT-RE), combined with a Raji B-cell line that presents the SEE-MHC class II complex to the engineered T cell line. Exposure of the above mixed culture to increasing SEE concentrations induces differential expression of luciferase gene and bioluminescence 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 104 times more sensitive than the spleen cell proliferation bioassay, 106 times more sensitive than a typical ELISA assay and 109 times more sensitive than the monkey and kitten bioassay.