The diagnosis of bovine genital campylobacteriosis (BGC) presents significant challenges, as traditional methods lack sensitivity when prolonged transport of samples is required. Assays of preputial samples by means of real-time polymerase chain reaction (PCR) provide good sensitivity and high throughput capabilities. However, there is limited information on the acceptable duration of transport and temperature during transport of samples. In addition, the use of pooled samples has proven to be a valuable strategy for the diagnosis of other venereal diseases in cattle. The objectives of the present study were to determine the effect of sample pooling and of transport time and temperature on the clinical sensitivity of a real-time quantitative PCR (qPCR) assay for Campylobacter fetus subsp. venerealis in preputial samples from beef bulls. Eight infected bulls and 176 virgin yearling bulls were used as the source of samples. The qPCR sensitivity was comparable for unpooled samples and pools of 5 samples, whereas sensitivity was decreased for pools of 10 samples. Sensitivity for the various pool sizes improved with repeated sampling. For shorter-term transport (2 and 48 h), sensitivity was greatest when the samples were stored at 4°C and 30°C, whereas for longer-term transport (96 h) sensitivity was greatest when the samples were stored at −20°C. The creation of pools of 5 samples is therefore a good option to decrease costs when screening bulls for BGC with the qPCR assay of direct preputial samples. Ideally the samples should be stored at 4°C and arrive at the laboratory within 48 h of collection, but when that is not possible freezing at −20°C could minimize the loss of sensitivity.

To test the efficacy of a polyvalent Tritrichomonas foetus vaccine, 130 nulliparous heifers were randomly assigned to either receive the test T. foetus vaccine or to serve as nonvaccinated controls. The polyvalent test vaccine consisted of a Campylobacter fetus/Leptospira canicola-grippotyphosa-hardjo-icterohaemorrhagiae-pamona bacterine containing 5 X 10(7) killed T. foetus/dose. The polyvalent control vaccine consisted of the aforementioned formulation without T. foetus. Heifers were administered 2 doses of control or experimental vaccine at 3-week intervals. Heifers were bred to T. foetus-infected bulls and their conception and pregnancy rates were determined throughout gestation. In addition, serum samples were analyzed to determine induced concentrations of antitrichomonal antibodies and vaginal secretions were sampled to determine T. foetus infection rates in control and vaccinated animals. One week after each of the 15-day breeding periods, 60% (6 of 10) of tested vaccinates and 80% (8 of 10) of tested control animals were T. foetus culture-positive. The mean duration of infection of vaccinates was 3.8 weeks (+/- 7.5 days), compared with 5.4 weeks (+/- 7.5 days) of infection for control heifers. All vaccinates developed increased immunofluorescence and serum neutralizing antibody titers following the first immunization, and had additional increases of at least fourfold in response to the second injection. In contrast, no consistent increase in immunofluorescence or serum neutralizing antibodies was observed in control animals. Conception rates were 89.2% for vaccinates and 85.9% for control animals 30 days after breeding and 80 to 90% of these remained pregnant 60 days after breeding. However, within the next 4 months, the pregnancy rate of control heifers decreased to 30% for those that had conceived. During the same 4-month period, more than 60% of vaccinated heifers remained pregnant. Significantly, 62.5% of heifers vaccinated against T. foetus produced calves, whereas only 31.5% of control heifers produced calves. These findings indicate that the polyvalent test vaccine induced an immune response that was effective in lowering the rate of T. foetus infection, decreasing the duration of infection, and reducing losses in calf production attributable to early fetal death and abortion caused by T. foetus infection.

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 10(3) CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test.