Molecular Analysis and Diversity of Pathogenic Clostridium perfringens and Clostridium difficile Among Neonatal Pigs in the Midwest. (2006)

Enteric clostridial disease in swine is often caused by pathogenic Clostridium perfringens Types A and C as well as Clostridium difficile. Infection is most common in neonatal pigs and can lead to scouring, low weaning weights, and pre-weaning mortality. The focus of our study was to evaluate the diversity of C. perfringens Type A and C. difficile, although all types of C. perfringens were screened for. In this study, a total of 180 pigs were sampled from 16 locations across the Midwest region over a period of three months. Clinical pigs were swabbed rectally and samples plated on selective media for C. perfringens and C. difficile. Isolated colonies were picked from the plates and DNA was extracted for genetic analysis. Multiplex PCR was performed on the isolates targeting the four major toxins in C. perfringens: alpha, beta, iota, and epsilon and the toxin genes tcdA (Toxin A) and tcdB (Toxin B) in C. difficile to confirm identification. Multiplex PCR confirmed 479 of 502 isolates as C. perfringens Type A. Three isolates of C. perfringens Type C were identified at one regional site indicating a low incidence of Type C among the farms in this study. No other types of C. perfringens were found. A total of 102 C. difficile isolates were positive for Toxins A or B. RAPD PCR was used to characterize the toxigenic isolates of C. perfringens Type A and C. difficile. Isolates were constructed into dendrograms to evaluate genetic diversity among the isolates. The C. perfringens Type A dendrogram consisted of 479 isolates in 97 clusters at an 80% similarity coefficient. The C. difficile dendrogram was comprised of 102 isolates grouped into 51 clusters. Diversity indexes were calculated for each dendrogram using the formula: number of isolates/number of clusters. A low index indicates a high degree of diversity. Clostridium difficile isolates showed greater diversity than the C. perfringens Type A isolates with diversity indexes of 2.0 and 4.93 respectively. The largest cluster in the C. difficile dendrogram contained 20 isolates. Nineteen of the 51 clusters contained a single isolate. Conversely, the C. perfringens Type A dendrogram contained five large clusters with more than 25 isolates each, the largest cluster being 38 isolates. Overall, the incidence of C. difficile was found in the samples less frequently than C. perfringens Type A and seemed to be prevalent at only some farms. Many clostridia isolates were common to all farms evaluated in this study. However, the presence of some isolates that were unique to specific farm sites was also evident. These data indicate that enteric clostridial disease likely results from the presence of ubiquitous C. perfringens Type A and C. difficile strains. INTRODUCTION Enteric clostridial infections in swine occur predominantly in the neonatal period and C. perfringens Type A and C. difficile infections are now recognized with increasing frequency in neonatal pigs. The pathogenicity of C. perfringens is associated with several toxins and the species is divided into types based on the production of the four major toxins, alpha, beta, epsilon and iota. C. perfringens Type A (alpha toxin) and Type C (alpha and beta toxins)