Toxigenic Clostridium difficile in the bovine calf gut: Association with enteric disease in calves and significance to public health.

摘要

Clostridium difficile is an emerging pathogen in humans and animals. A real-time multiplex PCR assay was developed and standardized for the detection of genetic elements of toxigenic C. difficile. The assay targeted genes encoding TcdA, TcdB, and CDT toxins of C. difficile and was carried out in two duplex reactions. Reaction 1 targeted tcdA and tcdB while reaction 2 targeted cdtA and cdtB. The detection limit of the standardized real-time multiplex PCR assay for the toxin genes of C. difficile was 103cells/g and 101cells/g for non-enriched and enriched fecal and ground meat samples, respectively. When DNA is extracted directly from fecal and ground meat samples, toxin genes of C. difficile may be detected in as little as 4 hours, although, sample enrichment is recommended to increase sensitivity. The multiplex real-time PCR assay was used throughout the study.;The role of C. difficile in enteric disease in calves was investigated. The findings of the study revealed that young calves were nearly 11 times more likely to have enteric lesions when toxins TcdA and/or TcdB were detected in the feces. Toxigenic C. difficile was detected in 43% of calves with no enteric lesions or clinical symptoms of enteric disease. This observation confirms previous reports that calves could be asymptomatic carriers of C. difficile. Isolates collected from calves were characterized using genotypic and phenotypic methods. Genes encoding TcdA, TcdB, and CDT were detected in 86.6%, 85.1%, and 83.6% of isolates, respectively. Deletions (100 bp) in the tcdC gene, a negative regulator of toxin expression, were detected in 73.1% of toxigenic isolates. All isolates were resistant to cefoxitin, a third generation cephalosporin, and 52.2% of the isolates were resistant to tetracycline.;Nine representative toxigenic C. difficile isolates were examined for in vitro toxin production in the presence of sub-inhibitory concentrations of oxytetracycline, an antibiotic commonly administered to calves during the first two weeks of life. It was observed that exposure to oxyetracycline increased TcdA and/or TcdB production of one isolate. Exposure to oxytetracycline also resulted in TcdA and/or TcdB production by an isolate that did not produce toxin under control conditions.;A longitudinal study was conducted to determine the prevalence of toxigenic C. difficile in veal calves. Fifty veal calves from 4 herds (n=200) were followed for 18--22 weeks from the time of arrival on the veal farm to the time of slaughter. It was observed that 58 (29%) calves tested PCR positive for genetic elements of toxigenic C. difficile at least once over 18--22 week period. Calf age (p=0.011) and season (p=0.28e -6) influenced the prevalence of C. difficile toxin genes in calf fecal samples. Carcasses of calves (n=100) from two herds were sampled to determine the incidence of toxigenic C. difficile contamination. Carcass swabs were screened for toxigenic C. difficile contamination using culture and multiplex real-time PCR methods. Toxin genes of C. difficile were detected in 1 carcass swab by multiplex real-time PCR. Toxigenic C. difficile was detected by PCR and culture in 4 (8%) and 2 (4%) ground veal samples, respectively.;In summary, the findings of the study reveal that C. difficile is as an enteric pathogen in calves and calves could be asymptomatic carriers of toxigenic C. difficile. It is recommended that veterinary diagnostic laboratories should consider testing for C. difficile and TcdA/B toxins in fecal and necropsy samples from calves with suspected enteric disease. The study provided strong evidence to suggest that sub inhibitory concentrations of oxytetracycline could increase or induce production of toxins TcdA and TcdB. We have provided insight on the emergence of C. difficile in community acquired infections by identifying ground meat as a source of human exposure. Although the incidence of carcass contamination was low, viable toxigenic C. difficile was detected in finished ground veal product. Although other factors contributing to epidemiology of C. difficile-associated disease remain to be identified, this work has provided a basis for continued public health investigation. A full understanding of the changing epidemiology of C. difficile will provide valuable information needed for designing and implementing disease management and prevention strategies in veterinary and public health.