Bovine Genital Campylobacteriosis is a significant disease in Australian beef and dairy herds. The causative organism, Campylobacter fetus subsp. venerealis is a microaerophilic bacterium that often resides asymptomatically in bull penile sheaths and is passed on to females during mating. Virulent strains of the bacterium can cause subfertility and late term abortions in infected females. It has the potential to cause large economic losses and is often difficult to diagnose by culture due to the slow-growing, fastidious bacterium becoming overgrown by contaminants. The gold standard for detection is culture and biochemical testing for identification.
Isolation and identification methodologies for C. fetus subsp. venerealis have not been reviewed for many years partly due to the lack of an Australian culture collection. Determining the variation in transport media viability, culture phenotyping, addition of newer phenotypic tests or the evaluation of strain virulence relies on the availability of cultures for analysis. Therefore, the objective of this study was to use a shared culture collection to identify best practice for transport media and isolation methodologies, define the phenotypic profile through refined, standardised methods and develop a pregnant guinea pig model to facilitate future studies of the pathogenicity, abortion mechanism and vaccine efficacy of C. fetus subsp. venerealis.
This study biochemically characterised 152 Campylobacter-like isolates to determine the breadth of variability, and to test innovative diagnostic tools for phenotypic characterisations. Strains of C. fetus subsp. venerealis were used to determine viability in the commonly recommended Lander’s transport and enrichment medium (TEM) as well as the recently described Thomann transport and enrichment (TTE) medium in vitro and in vivo. The in vitro experiment also aimed to determine the efficacy of the two transport media with subsequent sub-culture onto: sheep blood agar (SBA) with and without filtration (0.45 µm) and Campylobacter selective agar. Incubation temperature effect was also studied, by comparing TEM enrichment at 25°C and 37°C. The in vivo cattle field trial found no significant difference in the isolation of C. fetus subsp. venerealis between Lander’s and TTE (p=0.1037), while a higher number of thermophilic Campylobacter cultures were isolated from TTE media. In vitro, both TTE and Lander’s media were able to sustain C. fetus subsp. venerealis growth for up to 7 days at either 25°C or 37°C, and allow enrichment in spiked mixed cultures with either P. vulgaris or C. jejuni. Campylobacter selective agar appeared to produce the most consistent positive results from spiked mixed cultures, while SBA was overgrown in most instances and 0.45 µm filtration produced inconsistent results and required a high starting concentration (105 CFU/ml). Therefore, diagnostically the use of either TTE or Lander’s TEM for transport of samples followed by plating onto Campylobacter selective agar was optimal for isolation of C. fetus subsp. venerealis from mixed cultures.
All cultures were phenotypically profiled with a selection of strains profiled using 27 different antibiotics. A subset of strains across different Campylobacter species were characterised using API Campy kits, Biolog GEN III and Biolog Phenotypic MicroArrays. It was found that automated technologies did not provide any identification advantages over culture phenotyping. API kits were unable to distinguish between Campylobacter species, while Biolog PMs had poor reproducibility. The GEN III microplate identified methyl pyruvate as a possible alternative source for H2S testing for the distinction of C. fetus subspecies biovars. Antibiotic profiling identified six antibiotics including ampicillin, cefuroxime, ceftiofur, cephalothin, novobiocin and penicillin which may differentiate between C. fetus subspecies, however further research is warranted to confirm these.
A pregnant guinea pig model was developed by testing dose (104, 107 and 109 CFU/ml), time to abortion (up to 168 h and 288 h), route of inoculation (intraperitoneal vs. intravaginal), and outcomes (culture, PCR and histology) using 7 groups (n=35) of pregnant dams. It identified suitable parameters for inoculation with reproducible results achieved via the intraperitoneal route with a dose of 107 CFU/ml to guinea pigs at 5-6 weeks of gestation. Animals were observed for up to 288 h before sampling of dams and foetuses for culture, PCR and histological analysis of placental and uterine tissue. A virulence variation study testing four C. fetus subsp. venerealis strains in groups of 10 guinea pigs (n=40) was then conducted with a protective dose of an experimental vaccine against bovine campylobacteriosis (determined in a pilot study) as a parameter for strain virulence. The study showed varying levels of virulence across strains, with between 17 – 100% abortions in non-vaccinated groups, 0 – 50% abortions in vaccinated. Histopathology showed placentitis, endometritis and vasculitis of varying severity correlating to time to abortion. Intraperitoneal administration of C. fetus subsp. venerealis to pregnant guinea pigs is a promising model for the investigation of C. fetus subsp. venerealis strain virulence variation, abortion mechanisms and potential vaccine efficacy studies.
This study has shown the level of biochemical variation and reliability of these test results for Australian Campylobacter isolates as well as optimal transport, isolation and identification methodologies for Australian C. fetus subsp. venerealis isolates. A small animal model for the assessment of C. fetus subsp. venerealis strains virulence was developed with the potential to be implemented in vaccine development or extrapolated to cattle infection studies.