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

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

Occurrence of Campylobacter jejuni and Campylobacter coli in Cattle and Sheep in Northern Spain and Changes in Antimicrobial Resistance in Two Studies 10-years Apart

A cross-sectional survey was conducted in 2014–2016 in 301 ruminant herds to estimate C. jejuni and C. coli prevalence, and investigate their susceptibility to antimicrobials. Risk of shedding C. jejuni was higher in cattle than sheep (81.2% vs. 45.2%; ORadj = 5.22, p < 0.001), whereas risk of shedding C. coli was higher in sheep than in cattle (19.1% vs. 11.3%; ORadj = 1.71, p = 0.128). Susceptibility to six antimicrobials was determined by broth microdilution using European Committee for Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values. C. coli exhibited higher resistance (94.1%, 32/34) than C. jejuni (65.1%, 71/109), and resistance was more widespread in isolates from dairy cattle than beef cattle or sheep. Compared to results obtained 10-years earlier (2003–2005) in a similar survey, an increase in fluoroquinolone-resistance was observed in C. jejuni from beef cattle (32.0% to 61.9%; OR = 3.45, p = 0.020), and a decrease in tetracycline-resistance in C. jejuni from dairy cattle (75.0% to 43.2%; OR = 0.25, p = 0.026). Resistance to macrolides remained stable at low rates and restricted to C. coli from dairy cattle, with all macrolide-resistant C. coli showing a pattern of pan-resistance. Presence of the single nucleotide polymorphisms (SNPs) associated to quinolone and macrolide resistance was confirmed in all phenotypically resistant isolates. The increase in fluoroquinolone resistance is worrisome but susceptibility to macrolides is reassuring

Effects of dry whey powder and calcium butyrate supplementation of corn/soybean-based diets on productive performance, duodenal histological integrity, and Campylobacter colonization in broilers

Abstract Background Campylobacter is the main cause of gastroenteritis in humans in industrialized countries, and poultry is its principal reservoir and source of human infections. Dietary supplementation of broiler feed with additives could improve productive performance and elicit health benefits that might reduce Campylobacter contamination during primary production. The aim of this study was to assess the effect of dietary supplementation with whey (a prebiotic) and calcium butyrate (a salt of a short-chain fatty acid) on productive traits, duodenal histological integrity, and Campylobacter colonization and dissemination in broiler chickens during the 42-day rearing period. Results Six hundred one-day-old Ross-308 chickens were placed into 20 ground pens and assigned to one of 4 corn/soybean-based dietary treatments (5 replicates of 30 chicks per treatment) following a randomized complete block design: 1) basal diet with no supplementation as the control, 2) diet supplemented with 6% dry whey powder, 3) diet containing 0.1% coated calcium butyrate, and 4) diet containing 6% whey and 0.1% calcium butyrate. At age 15 days, 6 chickens per pen were experimentally inoculated with Campylobacter jejuni. The results showed that supplementation of the corn/soybean-based diet with 6% whey alone or, preferably, in combination with 0.1% coated calcium butyrate improved growth and feed efficiency, had a beneficial effect on duodenal villus integrity, and decreased mortality. These favourable effects were particularly significant during the starter period. Six days after oral challenge, Campylobacter was widespread in the flock, and the birds remained positive until the end of the rearing period. Although Campylobacter was not isolated from environmental samples, it was detected by real-time polymerase chain reaction (PCR) in dust, air filters, and drinkers while birds shed culturable C. jejuni cells. No differences (p > 0.050) in colonization or shedding levels that could be attributed to the diet were observed during the assay. Conclusions Beneficial effects on performance and intestinal health were observed, particularly during the starter period, when chickens were fed a diet supplemented with both whey and coated calcium butyrate. However, none of the tested diets provided the chicks any differential degree of protection against Campylobacter infection

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