Molecular diagnostic protocols for bovine genital campylobacteriosis using comparative genomics and virulence studies

Abstract

Bovine genital campylobacteriosis is difficult to identify due to problems with the isolation of its causative agent Campylobacter fetus subspecies venerealis. To optimise isolation methods, three selective culture and three transport and enrichment media (TEMs) were evaluated using a pure C. fetus subsp. venerealis culture, with and without simulated field contamination by Pseudomonas aeruginosa. For optimal C. fetus subsp. venerealis isolation and growth, TEMs should be returned from the field within 48h of collection and plated within 4h onto Campylobacter selective agar plates incubated for 48-72h at 37°C under microaerophilic conditions. Longer transport or TEM incubation resulted in overgrowth of P. aeruginosa limiting C. fetus subsp. venerealis recovery.In order to establish an isolate collection, the parA real-time PCR assay was used to screen bull preputial secretions prior to culturing positive samples. A more sensitive real-time PCR assay was designed from the ISCfe1 insertion element and used to assess additional preputial samples, compared to the parA real-time assay. A proportion of real-time negative samples was also cultured. Fourteen cultures were obtained from either ISCfe1 (8/43; 18.6%) or parA (6/51; 11.8%) negative samples. ISCfe1 and parA sequences were obtained from isolates not confirmed by conventional methods suggesting that both parA and ISCfe1 may be found in organisms other than C. fetus subsp. venerealis. Molecular testing of cultures (n=84) confirmed the presence of these sequences in isolates identified as other Campylobacter spp. or Arcobacter-like organisms, while they were not always detected in all biochemically confirmed C. fetus subsp. venerealis isolates. The lack of specificity of all methods highlights the need for more reliable C. fetus subsp. venerealis-specific gene targets.The draft genomes of two C. fetus subsp. venerealis biovars aligned to C. fetus subsp. fetus genome provided 26 unique C. fetus subsp. venerealis regions. In order to identify new diagnostic targets, 13 putative conventional and one real-time PCR assays designed from contigs common to both C. fetus subsp. venerealis biovars were developed to screen the Campylobacter-like culture collection (n=84). Isolates biochemically identified as organisms other than C. fetus subsp.venerealis were positive on the new real-time and six conventional PCR assays, indicating that they were not suitable subspecies-specific markers. Seven assays detected isolates phenotyped as C. fetus subsp. venerealis except for two aerotolerant isolates recognized as C. fetus subsp. venerealis by iii other published methods. These seven gene targets could be further investigated for real-time PCR assay development as in silico BLAST analysis confirmed their conservation in the genomes of five of our C. fetus subsp. venerealis isolates. However, further bioinformatics analysis revealed that they were found in several C. fetus subsp. fetus strains, underlining that a pan-genomics study including multiple C. fetus genomes is ne...