Detection of toxigenic Clostridium difficile in pig feces by PCR

Clostridium difficile is considered an important uncontrolled cause of neonatal diarrhea. Also, the presence of bacteria in the feces of the animal could represent a zoonosic risk for the contamination of meat products. Therefore, it is necessary to have procedures available for the early detection of C. difficile in animals. The current study describes a new semi-automated procedure for the recovery of C. difficile DNA from pig feces and subsequent amplification by polymerase chain reaction (PCR) of three different sequences: the triose phosphate isomerase gene tpi, specific for this bacterial species, and the tcdA and tcdB genes, which code for the A and B toxins of C. difficile, respectively. Twenty-two fecal samples microbiologically positive for C. difficile were used. The tpi and tcdA genes were amplified in all of them. The internal fragment of tcdB was detected from 21 of these extracts; the negative sample gave a positive result when a different primer pair was used. None of the 10 DNA extracts obtained from culture-negative samples gave a positive result. The method presented in this article eliminates the interference caused by the possible presence of PCR inhibitors. To the authors' knowledge, this is the first description of a PCR procedure for detection of C. difficile DNA from domestic animal feces.