Diagnosis of bovine venereal campylobacteriosis in New Zealand : a thesis presented in partial fulfilment of the requirements for the Master of Applied Science at Massey University, Palmerston North, New Zealand
Bovine venereal campylobacteriosis (BVC) is a venereal disease causing infertility in cows due to early embryonic loss. Caused by Campylobacter fetus subsp venerealis (Cfv), which lives in the preputial crypts of older asymptomatic carrier bulls and the vagina of carrier cows. Diagnosis of the disease is difficult as it is a fastidious organism to culture and the serological methods which have been developed to diagnose BVC, exhibit substantial cross-reactivity with Campylobacter fetus subsp fetus (Cff), due to the close genomic and phenotypic relationship of the two subspecies. Definitive differentiation between Cfv and Cff requires molecular biology techniques. In New Zealand, Cfv was last isolated in 1993 and suspected again in 2001/2002 after the use of a recently-developed IgA ELISA test that claimed to be 98.5% specific for Cfv. A nation wide study showed that there was no relationship between the test's results and the reproductive performance of the herds. Most of the positive results obtained were false probably due to cross-reactions with Cff The lack of positive isolations of Cfv raised questions about the sensitivity of the isolation methods used. Five experiments were undertaken. Experiments 1-3 examined the microbiological methods that are used to isolated Cfv. Results indicated that the optimal samples and culture conditions were preputial washes or scrapes from bulls, or vaginal washes (20 ml PBS) from cows, enriched in Lander's medium for three days before subculture onto blood agar. All cultures need to be undertaken under microaerophilic conditions. These experiments also addressed the reliability of the IgA ELISA for animals managed according to the husbandry conditions that prevail in New Zealand. Results showed that there was a great deal of within- and between- animal variation in ELISA values and that there was substantial cross reactivity with Cff. (ICC ranged from 0.29 to 0.03 depending on the Group and all heifers challenged with Cff became positive to IgA ELISA). In consequence, the test appears to be of limited diagnostic value in situations where cattle may be cross contaminated with Cff. Experiment 4 evaluated a PCR method for identification of Cfv in preputial washings that had been inoculated with small numbers of the organism, whilst Experiment 5 used the same method to attempt to identify Cfv in preputial washings or scrapings that had been collected from experimentally-infected bulls. Positive results were obtained with as little as 880 Cfv cells/ml of preputial wash after samples had been concentrated by centrifugation and filtration (5 μm and 0.8μm pore size filters). The PCR also detected the presence of Cfv in two bulls, 48 h after infection. It was concluded that the IgA ELISA test is unlikely to be suitable for use in New Zealand, due to the risk of cows being contaminated with Cff from the sheep with which they are co-managed. Microbiological identification, whether through PCR or culture and isolate, although difficult, remains the definitive diagnostic method. If the presence of the disease in New Zealand is to be confirmed or refuted, careful and methodical collection of samples from animals, whose history suggests the possibility of the condition, will be required.