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Background   Campylobacter fetus  subsp.  venerealis  ( Cfv ) is the pathogen responsible for Bovine Genital Campylobacteriosis (BGC), a venereal disease of cattle associated with impaired reproductive performance. Although several PCR assays were developed to identify this pathogen, most of them are still poorly evaluated in clinical samples. This study evaluated real-time PCR assays for  Cfv  detection in preputial samples of bulls ( n  = 308).    Results  The detection at the subspecies level ( Cfv ) compared four assays: two targeting ISCfe1 and two targeting  parA  gene. The detection at the species level ( C. fetus ) considered an assay targeting the  nahE  gene and a commercial kit for  C. fetus  identification. At the subspecies level, assays directed either to different targets ( parA  and ISCfe1), or to the same target (ISCfe1 or  parA ), showed a high percentage of disagreeing results. All samples positive at the subspecies level ( n  = 169) were negative in  C. fetus  detection assays, which strongly suggests the horizontal gene transfer of ISCfe1 and  parA  to other bacterial species. This was confirmed by microbiological isolation of three  Campylobacter portucalensis  strains responsible for false positive results. Sequences with a high level of identity with ISCfe1 and  parA  gene of  Cfv  were identified in  C. portucalensis  genome.    Conclusions  Overall, this study reveals that PCR assays solely directed to a subspecies target originate a high rate of false positive results, due to the presence of  parA  and ISCfe1 homologous sequences in other bacterial species, namely of the  genus Campylobacter . Although the specificity of these methods may be higher if applied to bulls from herds with clinical features of BGC or in other geographical regions, current PCR diagnosis should couple subspecies and species targets, and further research must be envisaged to identify  Cfv  specific molecular targets.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12917-020-02634-7.

Assessment of Campylobacter fetus subsp. venerealis molecular diagnosis using clinical samples of bulls