BACKGROUND: Clostridium perfringens is an important animal pathogen that causes severe loses to the livestock and poultry industries. Therefore, bacterial detection is believed to be of particular importance. OBJECTIVES: The present study aimed to identify Iranian isolates using conventional and molecular methods and to evaluate their toxicity. METHODS: In this work, 23 Clostridium perfringens type B isolates were examined via microbiological and biochemical tests. Subsequently, they were subjected to PCR technique for the final confirmation. After culturing of the isolates in specific medium, the minimum lethal dose test was performed. The most toxigenic isolate and reference strain was prepared the enterotoxaemia anaculture vaccine. Serum neutralization test was performed on the experimental inactive vaccines. RESULTS: The results revealed that etx and cpb gene could be found in all of the isolates, yet cpb2 gene was found in 65.2 % of the isolates. The minimum lethal dose ranges for these bacteria was less than 1/10 to more than 1/900. The results of serum neutralization in Iranian isolate and reference strains were 5 and 10 IU / ml, respectively. CONCLUSIONS: The findings herein implied that strain 1795 with high toxicity could be used in vaccine production. Of course, for use in production, further research on target animals is needed.

BACKGROUND: Clostridium perfringens (C. perfringens) is an anaerobic Gram-positive bacillus with spores, whose biotype A is responsible for a variety of diseases, including intestinal inflammation, bloody diarrhea, and gas gangrene, and hemorrhagic bowel syndrome. Genetic variety can explain the bacteria’s phenotypic diversity, geographic distribution, host specificity, pathogenicity, antibiotic resistance, and virulence. A molecular method using the pattern of DNA bands classifies bacteria based on the size of fragments produced by enzymatic digestion of the genome.OBJECTIVES: This study aims to standardize the polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) method in identifying the genetic diversity of C. perfringens biotype A isolates.METHODS: The genomic DNA of the investigated strains was extracted, and the complete sequence of the alpha toxin gene locus was synthesized using specific primers designed by PCR technique. Enzymatic cleavage of the synthesized amplicons was performed with the Mse l restriction enzyme, and the resulting fragments were separated by electrophoresis and analyzed by ImageJ and NTSYSPC software.RESULTS: The findings showed that the alpha toxin gene locus sequence may change and is not conserved. In this research, 4 different patterns were identified based on enzymatic cleavage. Mutations in this locus can lead to diversity in C. perfringens biotype A and the creation of new strains.CONCLUSIONS: The results of this research showed that the alpha toxin gene locus could be considered a DNA molecular marker in C. perfringens, and the PCR-RFLP technique can be used as a tool for typing this bacterium and estimating the phylogenetic relationships through comparative studies of nucleotide sequences.