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Abstract
The overall goals of this research were to: 1) determine prevalence of Clostridium difficile from healthy beef cattle fecal samples by comparing a single versus a double alcohol shock method using selective (cycloserine-cefoxitin fructose agar [CCFA]) and non-selective (blood agar) media; 2) determine and compare toxigenic profiles of C. difficile isolated from feces of healthy swine and cattle and from the dairy cattle environment using PCR targeting tcdA, tcdB, and cdtB genes; and 3) develop a rapid, sensitive, and specific PCR assay to detect C. difficile in enriched food and fecal samples. Healthy beef cattle were noted as minor carriers of C. difficile. The overall prevalence of C. difficile was 6.3% (188/2,965 samples) regardless of method or media used. The single ethanol shock method was significantly better (P < 0.0001) at recovery compared to the double shock method for each media tested and across both agars. There were no significant differences between media within each method. Healthy food animals and the dairy environment were sources of toxigenic C. difficile strains. C. difficile isolates (n=478) from the feces of swine, cattle, and the dairy cattle environment were examined. Toxin genes tcdA, tcdB, and cdtB were identified in 67.4%, 75.7%, and 26.6% of the samples, respectively. Three hundred (62.8%) of 478 isolates were positive for both tcdA and tcdB. Of those 300 isolates, 107 (35.7%) were also cdtB positive. Dairy (fecal and environmental) and swine isolates were significantly higher in tcdA and tcdB presence compared to beef isolates. Beef isolates were significantly higher in variant (tcdA-, tcdB+) strains. The PCR assay developed for C. difficile detection from enriched fecal (cattle, swine, broiler) and food (ground chuck, ground turkey, pork sausage) samples was rapid, specific, and sensitive. Detection was observed in ~ 32 h with as few as 20 C. difficile cells per 9 ml cycloserine-cefoxitin fructose broth with taurocholate (TCCFB) and at a level of 19 spores per 9 ml TCCFB without enrichment. The assay was specific to C. difficile only. This research provides a better understanding of the potential role that food animals play in C. difficile-associated disease.