Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain

Authors’ contributions: GA and ECF conceived the study and participated in its design. NPF performed the biochemical and PCR tests and interpreted the results with NK. NPF, MO and AH performed the whole genomes analysis and interpreted the results. MF isolated the Campylobacter spp. strains from bull preputial samples and identifed them by MALDI-TOF. NPF and NK wrote the manuscript, with interpretation of results, material and methods and discussion inputs from GA, ECF, MO, AH, MF and IP. All authors read and approved the fnal manuscript. Esther Collantes-Fernández and Gorka Aduriz contributed equally to this work and share last authorship.Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n=13), C. fetus feld isolates (n=64) and saprophytic isolates resembling Campylobacter (n=75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ=0.462; CI: 0.256–0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identifcation. For the diferentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concord‑ ance when H2S production was assessed after 5 days (72 h; κ=0.553, 0.329–0.778 CI vs. 5 days; κ=0.881, 0.631–1 CI), evidencing the efcacy of a longer incubation time. This study confrmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identifcation results were signifcantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identifcation of new C. fetus subspecies-specifc genes would help to improve BGC diagnosis.Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species

Author contributions: G.A. and A.H. conceived the study and participated in its design. N.P.F. and N.K. performed laboratory analysis. N.P.F., M.O., J.L.L. and L.G. participated in the bioinformatics analysis. N.P.F. wrote the manuscript, with interpretation of results and discussion inputs from GA, AH, E.C., M.O., N.K., J.L.L. and L.G. All authors read and approved the fnal manuscript.Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.Ministerio de Ciencia e Innovación (España)Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Prevalence of Bovine Genital Campylobacteriosis, Associated Risk Factors and Spatial Distribution in Spanish Beef Cattle Based on Veterinary Laboratory Database Records

Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease that causes early reproductive failure in natural breeding cattle that are managed extensively. The aim of this study was to assess the BGC prevalence in Spain from 2011 to 2019 using data collected cross-sectionally from the diagnostic reports issued by the SALUVET veterinary diagnostic laboratory from a total of 5,182 breeding bulls from 1,950 herds managed under "dehesa" systems (large herds within fenced pastures and all-year breeding season) or mountain systems (smaller herds with seasonal breeding management and grazing in communal mountain pastures). Infection was detected by PCR in 7.7 and 12.2% of the bulls and herds tested, respectively. The "dehesa" herd management system (OR = 2.078, P = < 0.001, 95% CI = 1.55-1.77), bovine trichomonosis status of the herd (OR = 1.606, P = 0.004, 95% CI = 1.15-2.22), and bulls ≥3 years old (OR = 1.392, P = 0.04, 95% CI = 1.01-1.92) were identified as risk factors associated with Campylobacter fetus venerealis infection. We also studied the high-risk areas for circulation of the infection in extensive beef cattle herds in Spain, showing four significant clusters in "dehesa" areas in the south-western provinces of the country and a fifth cluster located in a mountain area in northern Spain. The results obtained in the present study indicate that BGC is endemic and widely distributed in Spanish beef herds. Specifically, "dehesa" herds are at greater risk for introduction of Cfv based on relatively high local prevalence of the infection and the use of specific management practices.Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu