Detection of Campylobacter fetus in bovine preputial scrapings using PCR and culture assays

Abstract

The traditional method for the diagnosis of bovine genital campylobacteriosis is the culture and identification of the causative organism, Campylobacter fetus subsp. venerealis (Cfv) from the genital tract. This approach is considered relatively insensitive due to the fragility of the bacteria, their specific nutritional and atmospheric requirements and their being easily overgrown by commensal bacteria. The identification of isolates is also problematic due to the limited biochemical activity of the bacteria. With the rapid advances made in the molecular field, assays have become more robust and cost-effective making them feasible for the diagnostic laboratory. The potential speed, sensitivity and specificity offered by these assays provide attractive alternatives for the identification of pathogens which are notoriously difficult to identify. The first part of this investigation was concerned with the implementation and evaluation of a polymerase chain reaction (PCR) assay for the direct detection of C. fetus in bovine preputial specimens. The specificity of a published C. fetus-specific primer pair was established by testing C. fetus reference and field isolates in addition to a collection of other Campylobacter species and organisms which may encountered in the genital tract of cattle. All C. fetus isolates tested yielded a single PCR amplicon of approximately 750 bp. No amplicons were generated when any of the other non-C. fetus isolates were tested. Following minor modifications to the assay, the sensitivity of the assay was determined using spiked Weybridge medium. A detection limit of 615 Cfv/mR Weybridge medium (or 6,15 cell equivalents per PCR assay) was obtained. Preputial material collected and submitted for laboratory testing may often be contaminated with faeces, urine, semen and/or blood. All of these components are known to be potential PCR inhibitors and the influence of each, on the sensitivity of the PCR assay, was subsequently evaluated. Faeces were identified as a potent inhibitor and contamination of specimens with as little as 1% (w/v) faeces reduced the sensitivity of the assay. Concentrations of up to 50% (v/v) of blood, urine and semen had no effect on the sensitivity of the assay. Preputial specimens, collected in Weybridge medium, were subsequently pooled and spiked and used to establish the sensitivity of both the PCR and culture methods as well as determine the influence of time on the sensitivity of the assays. Testing was carried out in triplicate on samples collected from different herds which were ascertained to be free of Cfv based on the use of specific selection criteria. The detection limit of the culture method was found to be better than that achieved using PCR only immediately after the samples were spiked. The detection limit of the culture method decreased with time whilst the detection limit of the PCR assay remain unchanged up to 72 hours post-inoculation. Ensuing field evaluation involved the testing of 212 clinical samples using both the culture method and the optimized PCR assay. Of the samples tested 4,2% were found to be positive using the PCR assay, whilst only 3,8% were found to be positive by culture. Based upon this evaluation the analytical specificity of the PCR assay was calculated to be 99% and the analytical sensitivity, 85,7%. The second part of this investigation was concerned with the subspeciation of C. fetus isolates. Currently the only test recommended by the Office International des Epizooties (OIE) for the subspeciation of isolates, is tolerance to 1% glycine. Doubts over the reliability of this test have led to alternative or supplementary tests being sought. Within the context of this investigation a collection of 40 South African field isolates were subspeciated using a previously described subspecies-specific primer set as well as the traditional 1% glycine tolerance phenotyping test. Additionally, other phenotyping tests (selenite reduction, growth at 42 °C and susceptibility to metronidazole and cefoperazone) were evaluated to determine their suitability for use as an aid in the subspeciation of C. fetus isolates. None of the field isolates yielded a Cfv-specific subspecies PCR amplicon using the published primer set suggesting that all of the isolates were Campylobacter fetus subsp. fetus (Cff). Based on tolerance to 1% glycine however, only 6 isolates were identified as Cff (glycine tolerant), whilst the remainder were classified as Cfv. The results of the ‘sensitive’ hydrogen sulphide test indicated that the Cfv isolates were specifically Cfv biovar intermedius. The lack of agreement between the PCR and the phenotyping subspeciation results concur with the findings reported by other researchers. It is consequently concluded that the published VenSF/VenSR subspecies-primer set is unsuitable for the subspeciation of South African field isolates.