AUTHOR=Silva Marta Filipa , Kienesberger Sabine , Pereira Gonçalo , Mateus Luísa , Lopes-da-Costa Luís , Silva Elisabete 

TITLE=Molecular diagnosis of bovine genital campylobacteriosis using high-resolution melting analysis

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.969825

DOI=10.3389/fmicb.2022.969825

ISSN=1664-302X

ABSTRACT=<p>Bovine Genital Campylobacteriosis (BGC) is a worldwide spread venereal disease of cattle caused by <italic>Campylobacter fetus</italic> subsp. <italic>venerealis</italic> (<italic>Cfv</italic>). Although several real-time PCR assays were developed for <italic>Cfv</italic> identification, most target mobile genetic elements, which may lead to false-positive diagnosis. In this study, a real-time PCR assay coupled with High-Resolution Melting analysis (HRM) was developed for the identification of <italic>Campylobacter fetus</italic> subspecies and application in BGC diagnosis. Two HRM assays targeting different single nucleotide polymorphisms were validated using 51 <italic>C. fetus</italic> strains, including 36 <italic>Cfv</italic> and 15 <italic>C. fetus</italic> subsp. <italic>fetus</italic> (<italic>Cff</italic>). The specificity was assessed in 50 preputial samples previously tested as negative for <italic>C. fetus</italic> and in 24 strains from other <italic>Campylobacter</italic> species. The analytical sensitivity was determined with ten-fold dilutions of <italic>Cfv</italic> genome copies and in preputial samples spiked with <italic>Cfv</italic> cells. Both HRM assays accurately identified the 51 <italic>C. fetus</italic> strains, showing 100% concordance with the previous identification. <italic>C. fetus</italic> subspecies identification by HRM showed concordant results with the glycine test in 98.0% of the isolates. No amplification was obtained in <italic>C. fetus</italic> negative preputial samples as well as in strains from other <italic>Campylobacter</italic> species. The assays were able to detect 10<sup>2</sup> genome copies of <italic>Cfv</italic>, while for preputial washing samples the limit of detection was 10<sup>3</sup> CFU/mL. These novel HRM assays represent a highly specific and sensitive tool for the identification of <italic>C. fetus</italic> subspecies and show potential for direct use in bull preputial samples for BGC diagnosis.</p>