Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates

INTRODUCTION Genital campylobacteriosis is a transmissible genital disease of cattle caused by the bacterium Campylobacter fetus subsp. venerealis (Cfv). The disease is characterised by temporary infertility of female cattle, early embryonic mortality, irregular oestrus cycles, delayed conception and infrequent abortions. Most cows recover from infection and oestrus cycles will recommence after a few months but the herd experiences a decreased pregnancy rate and a prolonged calving season. Infected bulls usually show no clinical signs of infection but become carriers and infect females at service. Campylobacteriosis is of considerable economic importance to the cattle industry worldwide and is considered to be one of the most important infectious causes of poor calving rates in southern Africa. Regional veterinary laboratories in South Africa and Namibia have reported between 0 and 12 % prevalences based on bacterial isolations made from bull sheath wash submissions. One particular study conducted on bulls which were communally grazed, reported a substantially higher prevalence of almost 29 %. It is believed that the availability of vaccines and artificial insemination practices have decreased the prevalence of the disease but many researchers still consider the prevalence of bovine venereal campylobacteriosis to be underestimated as a consequence of the lack of sensitive and reliable techniques for diagnosis. Currently diagnosis of genital campylobacteriosis relies on the cultivation and identification of the causative organism. The success hereof is dependent upon the collection and maintenance of the fragile C. fetus bacterium, which has limited viability outside the host. Most of the cattle-breeding farms in South Africa tend to be extensive in nature and located long distances from diagnostic laboratories. In many cases the consulting veterinarian may need to travel long distances to sample animals and the task of ensuring that samples reach the laboratory timeously becomes a logistical dilemma. In order to address these problems, alternative diagnostic methods have been sought. Polymerase chain reaction (PCR) assays present a number of advantages over more traditional methods and the technology is becoming increasingly more accessible to diagnostic laboratories. Apart from exhibiting high specificities and sensitivities, the ability of these types of assays to detect non-viable organisms could also potentially resolve problems encountered in the field with the preservation of organism viability for subsequent bacteriological culture. A number of C. fetus-speciesand subspecies-specific PCR assays have been described. Of these the multiplex PCR assay initially evaluated by Hum and colleagues has been described extensively. This assay, which is designed to identify and differentiate the 2 C. fetus subspecies, has been evaluated by various researchers and been shown to be both sensitive and specific. Good correlation of subspecies identification results, albeit not 100 %, was achieved when the PCR method was compared with traditional biochemical and genotyping methods, including random amplification polymorphic DNA (RAPD-PCR); pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The aim of this investigation was to assess the suitability of the C. fetus-specific primer set, initially evaluated by Hum and colleagues, to detect C. fetus directly in bovine preputial specimens. The multiplex assay was used in conjunction with traditional phenotyping tests to establish the suitability of the assay to assign South African C. fetus field isolates to subspecies.