M Amini - Department of Research and Development, Razi Vaccine and Serum Research Institute, Kerman Branch.
M Shamsaddini Bafti - Department of Research and Development, Razi Vaccine and Serum Research Institute, Kerman Branch.
M Ezatkhah - Department of Research and Development, Razi Vaccine and Serum Research Institute, Kerman Branch.
M Alimolaeie - Department of Research and Development, Razi Vaccine and Serum Research Institute, Kerman Branch.
Z Ameri - Department of Research and Development, Razi Vaccine and Serum Research Institute, Kerman Branch.

Introduction and Objectives: The epsilon toxin is one of 20 protein toxins produced by Clostridium perfringens, a Gram-positive, spore-forming, anaerobic bacterium that is an important pathogen of humans and livestock.C. perfringens virulence is largely dependent upon toxin production, and epsilon toxin (ETX) is the most potent of all C. perfringens toxins that produced by type B and D and cause enterotoxemia and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX).Materials and Methods: Growth and epsilon toxin production by Clostridium perfringens strains was determined in Thioglycollate medium..The five C. perfringens type D isolates were cultivated in Thioglycollate medium and incubated at 37 °C for 6, 12, 18 and 24 hours .Toxinogenesis and presence of the toxin was demonstrated with a C. perfringens epsilon toxin enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instruction and Positive or negative readings were obtained according to the instructions, furthermore a Minimum lethal Dose test (MLD50/ml) did on samples too. Results: Results of ELISA test show that epsilon toxin production was positive in all four time and in MLD test results of isolates that was incubated 24 hours show higher result, so the overnight incubation is the best time for producing epsilon toxin. Conclusion: The amount of alpha toxin that produced may be influenced by the time of being held in incubator.

C. perfringens, Toxin, ELISA, MLD.