title: Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.
abstract: A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.
authors.affiliation: Queensland Department of Primary Industries and Fisheries, c/o Animal Research Institute, Locked Mail Bag No. 4, Moorooka, 4105 QLD, Australia. ala.lew@dpi.qld.gov.au.
authors.forename: Lyle
authors.initials: L
authors.lastname: McMillen
authors.forename: Geoffry
authors.initials: G
authors.lastname: Fordyce
authors.forename: Vivienne J
authors.initials: VJ
authors.lastname: Doogan
authors.forename: Ala E
authors.initials: AE
authors.lastname: Lew
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods* / Q000706:statistics & numerical data; D001483:Base Sequence; D002169:Campylobacter Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary; D002168:Campylobacter fetus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D002417:Cattle / Q000382:microbiology*; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes