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

BACKGROUND: 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 field 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. RESULTS: 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 identification. For the differentiation 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 concordance 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 efficacy of a longer incubation time. CONCLUSIONS: This study confirmed 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 identification results were significantly 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 identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Antimicrobial susceptibilities of Campylobacter fetus: report from a reference laboratory.

Campylobacter fetus is known to cause human disease, particularly in elderly and immunocompromised hosts. There are limited published data for antimicrobial susceptibility patterns with this organism, and no interpretive criteria are available. We reviewed antimicrobial susceptibilities of C. fetus isolates tested at a tertiary care center and reference laboratory over an 11-year period. C. fetus isolates from patients treated at Mayo Clinic and those sent as referrals for identification and susceptibility were included. Antimicrobial susceptibility testing was performed using agar dilution for ciprofloxacin, doxycycline, erythromycin, gentamicin, meropenem, and tetracycline. Geographic distribution, culture source, organism minimal inhibitory concentration (MIC) distributions, and MIC50 and MIC90 were examined. Excluding duplicates, 105 unique isolates were identified from 110 positive cultures. Blood cultures represented the most common source, followed by body fluids, skin and soft tissue, and central nervous system. Gentamicin and meropenem had favorable MIC50 and MIC90 of 1 µg/mL. Ciprofloxacin demonstrated an MIC50 of 1 µg/mL; however, the MIC90 was >2 µg/mL. Erythromycin demonstrated MIC50 and MIC90 of 2 µg/mL. Tetracycline and doxycycline were tested on a limited number of isolates and showed a wide range of MICs. Gentamicin and meropenem demonstrated favorable MICs in C. fetus isolates. These may represent therapeutic options for consideration in serious C. fetus infections, pending susceptibility results. Ciprofloxacin, which showed variable results, may be more appropriate for use only after susceptibility testing. C. fetus interpretive criteria are needed to aid clinicians in selection of both empiric and definitive therapies. IMPORTANCE: Our findings contribute to the scant literature on Campylobacter fetus antimicrobial susceptibility test results. We used a reference test method of agar dilution and provide MICs for a large number of organisms and antimicrobial agents.

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.

Effect of Campylobacter fetus on in vitro fertilization and embryonic development of preimplantation bovine embryos.

Campylobacter fetus is an important veterinary pathogen that causes campylobacteriosis. This disease causes decreased productivity of cattle by inducing reproductive losses. Although several virulence factors have been recognized in C. fetus, including a cytolethal distending toxin (CDT), the exact mechanism responsible for embryonic death remains unknown. The aim of this work was to evaluate the effect of C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv), and their toxin activity on the in vitro fertilization of bovine ova and early embryonic development. Two different experiments were performed. In Experiment 1, a total of 1524 cumulus-oocyte complexes (COCs) were inseminated, distributed into three groups: two of them infected with the microorganism (Cff, Cfv) and a control group. Percentages COCs cleaved were similar among groups (p = 0.1243); however, the embryonic development rate (blastocyst at day 7) in the control group was greater (p < 0.001) than those obtained in Cff and Cfv groups. In experiment 2, a total of 746 COCs were inseminated, divided into three groups: two of them treated with the bacterial-free culture filtrates to test toxin activity (Cff-CDT, Cfv-CDT) and a control group. Both cleavage and embryonic development rates were greater (p < 0.001) in the control group than those obtained in Cff-CDT and Cfv-CDT groups. This study provides evidence that both subspecies of C. fetus do not interfere with fertilization but do affect in vitro embryonic development. It is the first report on the biological effect of the CDT on bovine embryonic development.

Campylobacter fetus-induced primary psoas abscess in patient with gouty arthritis: A case report and literature review.

RATIONALE: Campylobacter fetus is rare pathogen with high mortality rate in immunosuppressive hosts. This study aimed to summarize clinical and pathological presentation of C fetus induced psoas abscess. PATIENT CONCERNS: A 66-year-old male patient with long medical history of poorly-controlled gouty arthritis and steroid intake complained of a severe low back pain. Physical examination showed tenderness in his psoas. DIAGNOSES: The patient underwent puncture biopsy to the lesion in the psoas under ultrasound guidance. The lesion was indicated as abscess by pathological examination, and its pathogen was indicated as C fetus by the next generation sequencing. INTERVENTIONS: Meropenem 1 g q8.h were administered intravenously for 10 days. Then the antibiotic treatment was switched to amoxicillin/clavulanate potassium 0.375g q.8.h and levofloxacin 0.5g q.d oral administration when discharge. OUTCOMES: The patient's fever and low back pain improved and infectious parameters declined. He was discharged in good general condition with advice for further monitoring and therapy. In the first month follow-up, the patient did not report recurrence or aggravation of his symptoms. LESSONS: C fetus should be noticed in immunosuppressive patient with exposure to livestock who present with rare systematic or local invasive infection. We advocated the meropenem for the first-line treatment against C fetus.

Prosthetic valve endocarditis caused by Campylobacter fetus: a case report and literature review.

Campylobacter fetus is a Gram-negative bacillus typically associated with bacteremia in immunocompromised patients. Prosthetic valve endocarditis (PVE) is a serious complication of prosthetic valve surgery, with a high mortality rate if not treated promptly. We present a rare case of PVE caused by C. fetus. A man in his mid-60s presented to the Emergency Department with a fever and showed elevated C-reactive protein concentrations. He had prosthetic mitral and aortic valve replacement surgery 15 years previously. Gram-negative rods were detected in a blood culture. These rods were identified as C. fetus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by 16S rRNA sequencing. The patient was treated with gentamicin and imipenem, and underwent valve replacement surgery. C. fetus was isolated in a left atrial appendage swab obtained during the surgery. Follow-up blood cultures were negative after treatment. However, after a cardiac arrest event, the patient's general condition deteriorated, and he died. To the best of our knowledge, this is the first case of PVE caused by C. fetus in Korea and the second fatality to date. This case highlights the importance of considering C. fetus as a potential cause of PVE, even in patients without known risk factors.

Campylobacter fetus Invasive Infections and Risks for Death, France, 2000-2021.

Campylobacter fetus accounts for 1% of Campylobacter spp. infections, but prevalence of bacteremia and risk for death are high. To determine clinical features of C. fetus infections and risks for death, we conducted a retrospective observational study of all adult inpatients with a confirmed C. fetus infection in Nord Franche-Comté Hospital, Trevenans, France, during January 2000-December 2021. Among 991 patients with isolated Campylobacter spp. strains, we identified 39 (4%) with culture-positive C. fetus infections, of which 33 had complete records and underwent further analysis; 21 had documented bacteremia and 12 did not. Secondary localizations were reported for 7 (33%) patients with C. fetus bacteremia, of which 5 exhibited a predilection for vascular infections (including 3 with mycotic aneurysm). Another 7 (33%) patients with C. fetus bacteremia died within 30 days. Significant risk factors associated with death within 30 days were dyspnea, quick sequential organ failure assessment score >2 at admission, and septic shock.

A cross-sectional study on Campylobacter fetus subsp. venerealis prevalence and associated factors in Brazilian southern cattle farms.

Cattle farming is a major livestock activity with economic relevance in Rio Grande do Sul (RS), Brazil. However, this activity is still considered of intermediate to low technological level, and in this region, there are few epidemiologic reports of Campylobacter fetus subsp. venerealis (Cfv), the causative agent of bovine genital campylobacteriosis (BGC). Thus, we designed a cross-sectional study to assess the prevalence and Cfv-associated factors in cattle farms in RS, Brazil. In total, 99 farms were randomly selected to participate in the survey. Preputial mucus samples from selected bulls were collected twice (within a 15-day interval) and subjected to Cfv molecular detection. A farm was considered positive when at least one sample was positive for Cfv. Our findings indicate that the farm-level Cfv prevalence in RS is 67.67%. On average, the chance of a farm using natural service to be Cfv-positive increased approximately twice compared to farms that do not use natural service. We also determined that Cfv routine tests reduce the chance of a farm being positive by 92%. Therefore, both Cfv detection tests and the reduction of natural services decrease the chance of a farm being positive for Cfv. Finally, we conclude that Cfv is widely spread in Southern Brazil cattle farms and it is urgent the implementation of control measures to reduce Cfv prevalence in the target population.

Campylobacter fetus seeding of a cavernous malformation resulting in brain abscess: case report and literature review.

Microbial seeding of a cerebral cavernous malformation is an extremely rare occurrence with only 3 cases reported in the literature thus far. Campylobacter fetus is an opportunistic pathogen that rarely causes neurological infection with only 3 cases of C. fetus cerebral abscesses and 38 cases of C. fetus meningitis reported in the literature. There have been no cases of cerebral cavernous malformation seeding by C. fetus reported to date. We report the first case of cerebral cavernous malformation seeding by C. fetus, a case occurring in a previously healthy 16-year-old female who presented with suspected left cerebellar cavernous malformation with subacute hemorrhage. She underwent a suboccipital craniectomy for the resection of the cavernous malformation with additional intraoperative findings suggestive of cerebral abscess. Following positive blood and CSF cultures and surgical pathology results, the patient was diagnosed with C. fetus meningoencephalitis with co-infected left cerebellar cavernous malformation. This is the fourth reported case of microbial seeding of a cerebral cavernous malformation, and to our knowledge, the first case of a C. fetus-infected cavernous malformation. Compared to previous reports, the clinical events of this case strongly support the presence of a preexisting lesion that was secondarily seeded versus de novo formation as a result of prior infection.

Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution.

Campylobacter fetus is a pathogen, which is primarily associated with fertility problems in sheep and cattle. In humans, it can cause severe infections that require antimicrobial treatment. However, knowledge on the development of antimicrobial resistance in C. fetus is limited. Moreover, the lack of epidemiological cut-off values (ECOFFs) and clinical breakpoints for C. fetus hinders consistent reporting about wild-type and non-wild-type susceptibility. The aim of this study was to determine the phenotypic susceptibility pattern of C. fetus and to determine the C. fetus resistome [the collection of all antimicrobial resistance genes (ARGs) and their precursors] to describe the genomic basis of antimicrobial resistance in C. fetus isolates over time. Whole-genome sequences of 295 C. fetus isolates, including isolates that were isolated in the period 1939 till the mid 1940s, before the usage of non-synthetic antimicrobials, were analysed for the presence of resistance markers, and phenotypic antimicrobial susceptibility was obtained for a selection of 47 isolates. C. fetus subspecies fetus (Cff) isolates showed multiple phenotypic antimicrobial resistances compared to C. fetus subspecies venerealis (Cfv) isolates that were only intrinsic resistant to nalidixic acid and trimethoprim. Cff isolates showed elevated minimal inhibitory concentrations for cefotaxime and cefquinome that were observed in isolates from 1943 onwards, and Cff isolates contained gyrA substitutions, which conferred resistance to ciprofloxacin. Resistances to aminoglycosides, tetracycline and phenicols were linked to acquired ARGs on mobile genetic elements. A plasmid-derived tet(O) gene in a bovine Cff isolate in 1999 was the first mobile genetic element observed, followed by detection of mobile elements containing tet(O)-aph(3')-III and tet(44)-ant(6)-Ib genes, and a plasmid from a single human isolate in 2003, carrying aph(3')-III-ant(6)-Ib and a chloramphenicol resistance gene (cat). The presence of ARGs in multiple mobile elements distributed among different Cff lineages highlights the risk for spread and further emergence of AMR in C. fetus. Surveillance for these resistances requires the establishment of ECOFFs for C. fetus.

Characterization of the cervicovaginal microbiota of female beef cattle harboring Campylobacter fetus subsp. venerealis using 16S rDNA gene sequencing.

Bovine genital campylobacteriosis (BGC) is a leading cause of return to estrus in cows. The etiologic agent, Campylobacter fetus subsp. venerealis (Cfv) is transmitted by venereal route. Hence, the surrounding reproductive tract microenvironment could play a role in return to estrus in cows. The presence of Cfv in cervicovaginal mucus of Angus breed females provide three experimental groups, which were subject to bacteriome analyses: 10 Cfv-positive cows (CVP), 10 Cfv-negative cows (CVN), and 10 nonsexually active heifers (NSA). Cows with return to estrus showed higher bacterial richness than NSA. Beta diversity analysis showed a significant difference (P = 0.006) in bacterial composition among the three groups analyzed (CVP, CVN, and NSA). However, no significant difference was found when comparing the CVP versus CVN groups. Ureaplasma and Pseudomonas were the genera most frequently observed in NSA, being Ureaplasma the predictor genus to that group, whereas Alistipes, Bacteroides, Rikenellaceae_RC9_gut_group, UCG-005, and UCG-10 were both significantly more abundant and predictors genera in cows with return to estrus. Our results provide an overview of the cervicovaginal bacterial microbiota in cows harboring Cfv and improve the knowledge of the pathogenesis of BGC.

Multicenter Retrospective Study of Vascular Infections and Endocarditis Caused by Campylobacter spp., France.

The incidence of campylobacteriosis has substantially increased over the past decade, notably in France. Secondary localizations complicating invasive infections are poorly described. We aimed to describe vascular infection or endocarditis caused by Campylobacter spp. We included 57 patients from a nationwide 5-year retrospective study on Campylobacter spp. bacteremia conducted in France; 44 patients had vascular infections, 12 had endocarditis, and 1 had both conditions. Campylobacter fetus was the most frequently involved species (83%). Antibiotic treatment involved a ß-lactam monotherapy (54%) or was combined with a fluoroquinolone or an aminoglycoside (44%). The mortality rate was 25%. Relapse occurred in 8% of cases and was associated with delayed initiation of an efficient antimicrobial therapy after the first symptoms, diabetes, and coexistence of an osteoarticular location. Cardiovascular Campylobacter spp. infections are associated with a high mortality rate. Systematically searching for those localizations in cases of C. fetus bacteremia may be warranted.

Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Investigation of Campylobacter fetus in breeding bulls of private farms in Bangladesh.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is a venereal disease caused by Campylobacter fetus that has a negative impact on animal reproduction. The bull is considered to be a symptomless carrier that spreads the disease agent to breeding cows, causing infertility and sporadic abortion. AIM: The study aims to estimate the prevalence, identify risk factors of Campylobacter fetus (C. fetus) infection and antimicrobial resistance pattern of the C. fetus isolates. METHOD: A cross-sectional survey was conducted in Mymensingh district of Bangladesh. Bull smegma samples (single sample from each bull) were collected from 300 bulls from four farms and tested via culture, biochemical identification and finally 16S rRNA and cdtA gene-based molecular assays (PCR) for herd and animal-level prevalence estimation. Herd- and animal-level data on risk factors were collected from the farmers using a pretested questionnaire and analysed by univariable and multivariable logistic regression models with a p value of <0.05 was taken statistically significant for both analyses. RESULTS: Among the surveyed farms, 75% (95% CI: 19.4%-99.4%) were confirmed to have bulls infected with Campylobacter fetus at herd level. However, animal-level occurrence of C. fetus was estimated to be 8.7% (26/300) (95% CI: 5.7%-12.4%). Natural service increases the odds of campylobacteriosis 38.18 times (95% CI: 13.89-104.94) in comparison to artificial insemination for C. fetus infection in bulls. Significantly, half of the isolates (50%, n = 13) were identified to be multidrug resistant (MDR) for three to five antimicrobial agents. CONCLUSION: This study highlights the need to develop official guidelines for C. fetus control and prevention in Bangladesh including mandatory artificial insemination in reproductive cows and heifers, routine screening of breeding bulls for C. fetus free status.

Genome analysis of clinical isolate of Campylobacter fetus subspecies fetus MMM01 from India reveals genetic determinants of pathogenesis and adaptation.

In this study we report the whole genome sequencing (WGS) based analysis of blood-borne Campylobacter fetus subsp. fetus MMM01 isolated from a diabetic patient to obtain deeper insights in to the virulence and host adaptability. The sequenced genome of C. fetus subsp. fetus MMM01 along with reference genomes retrieved from NCBI was subjected to various in-silico analysis including JSpecies, MLST server, PATRIC server, VFanalyzer, CARD, PHASTER to understand their phylogenetic relation, virulence and antimicrobial resistance profile. The genome had a size of 1,788,790 bp, with a GC content of 33.09%, nearly identical to the reference strain C. fetus subsp. fetus 82-40. The MLST based phylogenetic tree constructed revealed the polyphyletic branching and MMM01 (ST25) was found to be closely related to ST11, both belong to the sap-A serotype which are more common in human infections. VFanalyzer identified 88 protein-coding genes coding for several virulence factors including Campylobacter adhesion to fibronectin, flagellar apparatus, cytolethal distending toxin operons and Campylobacter invasion antigen proteins which enhance the virulence of bacteria along with resistance genes against antibiotics including fluoroquinolone, chloramphenicol, tetracycline, and aminoglycoside in MMM01, which points to enhanced survival and pathogenicity of this zoonotic pathogen. It was interesting to find that MMM01 lacked FGI-II island found in most of the clinical isolates, which encoded CRISPR Cas and prophage II regions. More details about the complexity and evolution of this zoonotic pathogen could be learned from future studies that concentrate on comparative genome analysis using larger genome datasets.

Campylobacter fetus bacteremia complicated by multiple splenic abscesses and multivisceral signs in a renal transplant recipient: a case report and review of the literature.

We report a rare case of Campylobacter fetus bacteremia in a 50-year-old woman following kidney transplantation. Bacteremia was complicated by multivisceral signs such as multiple splenic abscesses, bacterial hepatitis, erythema nodosum and reactive arthritis. Despite a prolonged diagnostic delay, the diagnosis was made on blood culture identification and the global outcome was favorable with adequate antibiotherapy. Reports in the literature describe a high rate of mortality for Campylobacter spp. septicemia, with most patients being immunocompromised. However, Campylobacter spp. has been rarely described in renal transplant patients. Moreover, a splenic septic localization due to Campylobacter spp. has been reported only once to our knowledge. Clinicians should be aware of the diagnostic difficulties related to the frequent negativity of stool samples in C. fetus septicemia, in order to implement a tailored medical strategy. Some data suggest that rapid introduction of adapted antibiotic therapy is associated with a reduction in mortality.

Placentitis and abortion caused by a multidrug resistant strain of Campylobacter fetus subspecies fetus in a sheep in Uruguay.

Campylobacter fetusfetus (Cff) is a major infectious cause of abortion in sheep worldwide, and an opportunistic human pathogen. Information on Cff as an ovine abortifacient in South America is limited. We describe a case of abortion caused by a multidrug resistant strain of Cff in a sheep in Uruguay. In August 2017, 3/57 pregnant ewes (5.3%) aborted whithin one week. Histopathologic examination of the placenta of an aborted ewe revealed severe neutrophilic and fibrinonecrotizing placentitis with vasculitis and thrombosis of the chorionic arterioles. Cff was isolated on microaerobic culture in Skirrow agar, and further confirmed by 16S rDNA PCR amplification and sequencing, and endpoint and real time PCR assays. Antimicrobial sensitivity testing revealed resistance to tetracyclines, nalidixic acid, telithromycin and clindamycin. Other abortifacients were not detected. Further studies are necessary to determine the geographic distribution, ecology, epidemiology, economic impact, and antimicrobial resistance of Cff in sheep flocks in Uruguay.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles.

The study of outer membrane vesicles (OMVs) became relevant because of their probable important role in the transfer of virulence factors to host cells. Campylobacter fetus is mainly a mammal pathogen whose virulence characterization is still limited. The aim of this study was to evaluate and to characterize the secretion of OMVs in this bacterium. By transmission electron microscopy, we confirmed the production of OMVs in all the strains assayed. Purified OMVs showed a spherical shape and variable size, although comparable to those of other gram-negative bacteria. We also confirmed the presence of the S-layer on the surface of the OMVs of all the strains assayed with the exception of those derived from the NTCC reference strain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C. fetus OMVs could contribute to the modulation of the host response and constitute a candidate to be evaluated as an adjuvant of current vaccines used in the veterinary field. This work represents a platform to drive future studies towards the role of these subcellular structures in C. fetus-host interaction.