Clostridium difficile (C. difficile) is known as the most important causative agent of antibiotic-associated pseudomembranous colitis in human. According to recent studies, food animals especially poultry are known as carriers for toxin harboring C. difficile. Since ostrich farming is a growing industry in Iran, the characterization and genotyping of C. difficile isolates in ostriches is very important for public health. A total of 100 fecal samples were collected from 10 ostrich farms located in Northeast of Khorasan Razavi province, Iran. The samples were cultured anaerobically according to standard procedures. Multiplex PCR was applied in order to detect tcdB, tcdA, cdtA and cdtB genes and 16S rDNA sequence, while single PCR was used to detect tcdC gene. Eleven fecal samples (11%) were suspected to contain C. difficile by the growth pattern, colony morphology and Gram stain. Ten of 11 suspected samples were later confirmed by 16s rDNA sequence PCR. In terms of toxin profile, five isolates (50%) were tcdA and tcdB positive, Two (20%) were tcdA negative and tcdB positive and the remaining three isolates were tcdA and tcdB negative. In addition, all isolates lacked CDT producing gene (cdtA and cdtB). The findings of this study showed that ostrich might be a potential reservoir of C. difficile infection for humans.

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