title: Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method.
abstract: Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1000 Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Avbersek
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1000 Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Cotman
authors.affiliation: Veterinary Faculty, University of Ljubljana, Gerbiceva 60, SI-1000 Ljubljana, Slovenia.
authors.forename: Matjaz
authors.initials: M
authors.lastname: Ocepek
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology; D016360:Clostridioides difficile / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000382:microbiology / Q000662:veterinary*; D046508:Culture Techniques; D003967:Diarrhea / Q000382:microbiology / Q000662:veterinary; D005243:Feces / Q000382:microbiology; D006734:Horse Diseases / Q000382:microbiology; D006736:Horses; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology