A new species of the Campylobacter genus is described, isolated from the preputial mucosa of bulls (Bos taurus). The five isolates obtained exhibit characteristics of Campylobacter, being Gram-negative non-motile straight rods, oxidase positive, catalase negative and microaerophilic. Phenotypic characteristics and nucleotide sequence analysis of 16S rRNA and hsp60 genes demonstrated that these isolates belong to a novel species within the genus Campylobacter. Based on hsp60 gene phylogenetic analysis, the most related species are C. ureolyticus, C. blaseri and C. corcagiensis. The whole genome sequence analysis of isolate FMV-PI01 revealed that the average nucleotide identity with other Campylobacter species was less than 75%, which is far below the cutoff for isolates of the same species. However, whole genome sequence analysis identified coding sequences highly homologous with other Campylobacter spp. These included several virulence factor coding genes related with host cell adhesion and invasion, transporters involved in resistance to antimicrobials, and a type IV secretion system (T4SS), containing virB2-virB11/ virD4 genes, highly homologous to the C. fetus subsp. venerealis. The genomic G+C content of isolate FMV-PI01 was 28.3%, which is one of the lowest values reported for species of the genus Campylobacter. For this species the name Campylobacter portucalensis sp. nov. is proposed, with FMV-PI01 (= LMG 31504, = CCUG 73856) as the type strain.

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.

Bovine Genital Campylobacteriosis (BGC) is a worldwide spread venereal disease of cattle caused by Campylobacter fetus subsp. venerealis (Cfv). Although several real-time PCR assays were developed for Cfv 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 Campylobacter fetus subspecies and application in BGC diagnosis. Two HRM assays targeting different single nucleotide polymorphisms were validated using 51 C. fetus strains, including 36 Cfv and 15 C. fetus subsp. fetus (Cff). The specificity was assessed in 50 preputial samples previously tested as negative for C. fetus and in 24 strains from other Campylobacter species. The analytical sensitivity was determined with ten-fold dilutions of Cfv genome copies and in preputial samples spiked with Cfv cells. Both HRM assays accurately identified the 51 C. fetus strains, showing 100% concordance with the previous identification. C. fetus subspecies identification by HRM showed concordant results with the glycine test in 98.0% of the isolates. No amplification was obtained in C. fetus negative preputial samples as well as in strains from other Campylobacter species. The assays were able to detect 102 genome copies of Cfv, while for preputial washing samples the limit of detection was 103 CFU/mL. These novel HRM assays represent a highly specific and sensitive tool for the identification of C. fetus subspecies and show potential for direct use in bull preputial samples for BGC diagnosis.

In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, which has profound detrimental effects on subsequent reproductive performance. To date, transcriptomic studies related to this condition were either based on biopsy-derived whole-endometrium tissue or endometrial swab or cytobrush samples, thus masking effects of disease on cell type-specific gene expression. This study tested the hypothesis that different endometrial health statuses are associated with distinct transcription profiles of endometrial stromal, glandular, and luminal epithelial cells. At 44 d postpartum (DPP), endometrial biopsies were taken from dairy cows (n = 24) classified as healthy, recovered from SCE, or affected by persistent SCE, according to endometrial cytology taken at 21 and 44 DPP. Stromal, glandular, and luminal epithelial cells were isolated from the whole-tissue biopsy by laser capture microdissection, and the cell-specific transcription profiles were determined by RNA sequencing. Differential gene expression was analyzed with DESeq2 (https: / / bioconductor .org/ packages/ release/ bioc/ html/ DESeq2 .html).Results demonstrated that global transcriptomic profiles and corresponding lists of differentially expressed genes between cows with different health statuses were distinct among cell types. Results also showed that although healthy and recovered cows presented similar endometrial clinically healthy phenotypes at 44 DPP, the prior presence of immune cells still affected the transcriptome of endometrial cells at this stage, delaying complete functional recovery. Recovery or persistence of inflammation was associated with gene expression patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity in a cell type-specific manner. Identifying these signatures may contribute to the development of novel diagnostic tools and therapeutic strategies. In addition, these results may help to define preventive measures or ways to stimulate recovery from endometrial inflammation, thus helping to restore the fertility of postpartum dairy cows.