Non-animal testing of Iranian enterotoxemia vaccine: cell culture assay for Clostridium perfringens epsilon toxin.

Epsilon toxin produced by Clostridium perfringens type D is the third most potent clostridial toxin. It causes enterotoxemia in sheep and lambs. The clostridial vaccine has been used against clostridial disease, and its efficacy is evaluated using the serum neutralization (SN) assay as a gold standard. Researchers are concerned about replacing in vivo tests with in vitro tests. Our study aimed to evaluate the cell culture assay to measure the neutralizing antibodies against Clostridium perfringens epsilon toxin as an alternative SN assay. Madin-Darby canine kidney (MDCK) and African green monkey kidney (Vero) cell lines were used to monitor the cell line response after treatment with purified epsilon toxin through microscopic examination and 3-[4,5-imethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) staining. Antibodies were calculated in cell culture assays, and SN results were analyzed using Pearson's correlation. Based on our results, only MDCK was sensitive to the epsilon toxin. The cytopathic effect in this cell culture was rounded. The relationship between toxin concentration and cell viability showed that increasing toxin concentrations significantly decreased cell viability. Good correlation coefficients were obtained between SN and the in vitro assay (r = 0.987) (p < 0.01). The antibody titers obtained by SN were within the range of the cytotoxicity assay and had high reproducibility. Therefore, cell culture may be a suitable alternative for SN assays. Cell culture is one of the tools used in toxicity testing, resulting in consistent and reproducible results.