Abstract
Subspecies of the species Campylobacter fetus are associated with specific host niches including mammals and reptiles. Campylobacter fetus subsp. fetus is a zoonotic pathogen infecting humans. Infections can vary from an acute intestinal illness to severe systemic infections, with sheep and cattle as major reservoirs. In contrast, Campylobacter fetus subsp. venerealis causes bovine genital campylobacteriosis, which leads to abortion in cattle and a high economic burden for the farmers. Therefore, high-quality molecular subtyping is indispensable for interventional epidemiology. We used whole-genome sequencing (WGS) data of 283 Campylobacter fetus strains from 18 countries and compared several methods for Campylobacter fetus subtyping, including WGS, multilocus sequence typing, PCR assays, and the presence of the insertion element ISCfe1. We identified a highly clonal clade (designated as clade 1) that harbors the insertion sequence ISCfe1. The presence of this insertion sequence is an essential diagnostic tool for the identification of the subspecies Campylobacter fetus subsp. venerealis, serving as a target for several PCR assays. However, we have found a high sequence variability for the ISCfe1 besides the presence of ISCfe1-paralogues in certain other genomes (n = 7) which may cause incorrect diagnostic results. Clade 1 seems to be the cattle-specific clade of this species. We propose that only this clade might be designated as Campylobacter fetus subsp. venerealis as it harbors the ISCfe1 marker sequence, which is a major target for molecular methods currently used for Campylobacter fetus subspecies identification. Fostering this proposal, we defined eleven stable nucleotide markers specific for this clade. Additionally, we developed a bioinformatics toolbox for the fast identification of this clade based on WGS data. In conclusion, our results demonstrate that WGS can be used for Campylobacter fetus subtyping overcoming limitations of current PCR and MLST protocols.
Highlights
The species Campylobacter (C.) fetus comprises three subspecies with distinct clinical significance: C. fetus subsp. fetus (Cff ) and C. fetus subsp. venerealis (Cfv) are both mammal-associated, and C. fetus subsp. testudinum (Cft) occurs in reptiles (Gilbert et al, 2017, 2018)
The data of Iraola et al, (n = 169 out of 182 genomes) were combined with 114 additional genomes from independent studies as well as sequence data from the NCBI (Table 1, details Supplementary Table S1). Based on this extended data set (n = 283 strains), we identified 18,793 high-quality SNPs that were present in a clonal frame in 281 genomes, while two genomes (GCF_000174675 [Azul94] and SRR6377517 [PNUSAC001504]) were unsuitable for recombination and phylogenetic analysis because of the high percentage of missing/ambiguous nucleotides (>25%)
As shown here and in previous studies (OIE; Van Der Graaf-Van Bloois et al, 2013, 2014), results from current polymerase chain reaction (PCR) protocols were not completely congruent and some of the ISCfe1-specific PCRs did not detect most of the group II ISCfe1 (Abril et al, 2007; Van Der GraafVan Bloois et al, 2013)
Summary
The species Campylobacter (C.) fetus comprises three subspecies with distinct clinical significance: C. fetus subsp. fetus (Cff ) and C. fetus subsp. venerealis (Cfv) are both mammal-associated, and C. fetus subsp. testudinum (Cft) occurs in reptiles (Gilbert et al, 2017, 2018). Cfv and Cff, on the other hand, are very similar at the DNA level (Iraola et al, 2017) These subspecies can be distinguished using biochemical testing based on 1% glycine tolerance and H2S production in cysteine media (Schulze et al, 2006; Silveira et al, 2018). The latter is valuable for detecting Cfv without culturing and includes Cfv-specific PCRs for detecting ISCfe (Abril et al, 2007; Mcgoldrick et al, 2013; Van Der Graaf-Van Bloois et al, 2013), a parA gene (Hum et al, 1997; Mcmillen et al, 2006) or others targets (Moolhuijzen et al, 2009; Iraola et al, 2012), and a Cff -specific PCR (Wang et al, 2002)
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