Fetus Subsp Research Articles

Bovine campylobacteriosis in heifer: pathogenesis study and insights in the conventional and molecular diagnosis in an experimental bovine model and field cases.

Campylobacter fetus spp. is a bacterium associated to reproductive losses in cattle worldwide. It is a venereal infectious disease known as bovine campilobacteriosis, with high impact mainly in countries with extensive production systems. Here, we show pathogenesis and diagnostic methods for Campylobacter fetus detection in cervico-vaginal mucus (CVM) samples from heifers experimentally infected and field cases from herds with low reproductive performance by campylobacteriosis infection. Bacterial culture, direct immunofluorescence test and qPCR were used as diagnostic methods to evaluate detection of C. fetus. In the experimental model 30 Aberdeen Angus and crossbred heifers and 4 Aberdeen Angus bulls for natural mating were assigned to 3 groups experimentally challenged with C. fetus subsp. fetus (Cff), C. fetus subsps venerealis (Cfv) and C. fetus subsp venerealis biovar intermedius (Cfvi), respectively, and a negative control group, all followed for 9 months. Also, field samples of CVM and aborted fetuses were recollected from seven beef cattle farms. Bacteriological culture had the higher C. fetus detection rate in CVM being the most appropriate, followed by qPCR (with commercial extraction DNA kit), direct immunofluorescence test and qPCR (with in-house extraction DNA method), in both, experimental model and field cases. From experimental model after natural mating, 62.5% and 25% heifers got pregnant from Cff and Cfvi groups, respectively, while from Cfv no pregnancy was detected. The strain more frequently detected was Cfvi, followed by Cff and Cfv. Colonization of Cff in female genital tract with high number of carriers and presence in aborted fetuses was evidenced, suggesting a high risk to bovine reproductive health. Bacteriemia was not detected after genital infection. Given the low detection rate of either test, we suggest the use of both, PCR based methods and bacterial culture could result in higher detection rate in farms with endemic campylobacteriosis.

Heat shock transformation and expression of the plasmid containing cytolethal distending toxin of Campylobacter fetus subsp venerealis in Escherichia coli

The cytolethal distending toxins (cdt) is a multi-subunit toxin consisted of three subunit encoded cdtA, cdtB and cdtC. The cdt played an important role as a virulence factor of Campylobacter infection, including C. fetus subsp venerealis. The cdtA which responsible for binding the cdt to cell membrane, was cloned in plasmid expression and inserted into bacterial cells of Escherichia coli BL21(DE3). The research was conducted to evaluate the transformation using the heat shock method of a plasmid containing cdtA3 gene and the protein expression induced by various concentration of IPTG. Transformation was done using the heat shock method at 42oC for 90 second. Evaluation of the transformation was observed on the presence of E. coli BL21(DE3) colonies on Luria Bertani agar containing Ampicillin antibiotic with 100 µg/mL dosage. The recombinant protein was expressed using IPTG-induction with various concentration (0.1mM, 0.25mM, 0.5mM, 0.75mM and 1 mM). The result showed that the transformation and IPTG-induction 0.1 mM produced higher concentration of protein than other concentration applied. The protein characterization was observed with SDS PAGE and cdtA3 protein was detected on 23,4 kDa.

Pengembangan Media Transpor untuk Koleksi Sampel Preputium, untuk deteksi Bovine Genital Campylobacteriosis

Campylobacter fetus subsp. Venerealis (Cfv) is bacteria causing contagious genital diseases in cows called Bovine Genital Campylobacteriosis (BGC) or vibriosis. Isolation of Cfv is difficult, because the bacteria are fragile and need specific nutrients and oxygen (5-10%). The transport media is very important to maintain Cfv survival before culturing in laboratory. The aim of this study was to modify a new transport media as an alternative media for Cfv. Developed media capability was compared to Weybridge media, and Phosphate Buffered Saline (PBS). All transport media was contaminated by Cfv with concentrations of 105,104,103,102,101 (CFU/ml), and was stored for <6, 24, 48, 72, and 96 hours in each transport medium before culturing on blood agar, all in triplicate. The quality of transport media was analyzed based on bacterial growth on blood agar. PCR test was used as a confirmatory test of growing bacteria cultured on blood agar. Based on culture results, Cfv stored in three transport mediums for <6 hours, Cfv grew on blood agar from all concentration levels provided. Cfv stored for 24, 48, 72 and 96 hours on PBS did not grow, whereas on developed media and Weybridge media, the bacteria could grow and enrichment occurs at all concentration levels given. This study indicated the developed media can be used as an alternative transport medium for Cfv bacteria.

First reported detection of biofilm formation by Campylobacter fetus during investigation of a case of prosthetic valve endocarditis

AimsCampylobacter fetus subsp fetus (CFF) can cause intestinal illness, particularly in immunocompromised humans, with the potential to cause severe systemic infections. CFF is a zoonotic pathogen with a broad host...

전북 동부지역 한우에서의 유사산 유발 질병 항체가 조사

Reproductive losses in a herd can be huge. Fetal reabsorption or undefined infertility often remain undetected. Routine herds monitoring for exposure, controlling the introduction of potential agent carriers, appropriate biosafety procedures, and vaccination where possible are together the best security against abortion and stillbirth inducing disease. For biosecurity of local farms, we performed antibody titers of abortion and stillbirth related diseases such as bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), Neospora caninum, Toxoplasma gondii and Campylobacter fetus subsp venerealis. The blood samples were collected from 500 female Hanwoo over 1 year old of 100 farms in Jeonbuk eastern area. Champhylobater serological test was evaluated by the standard tube agglutination test (STAT) and other pathogen's antibodies were detected by indirect-enzyme linked immunoassay (I-ELISA). The seroprevalence of abortion and stillbirth inducing disease were BVDV 72.4%, IBRV 13.0%, N. caninum 1.2%, T. gondii 10.4% and C. venerealis 0.6%, irrespectively.

Clinical sensitivity and specificity of a real-time PCR assay for Campylobacter fetus subsp venerealis in preputial samples from bulls.

To determine clinical sensitivity and specificity of a quantitative real-time PCR (qRT-PCR) assay for Campylobacter fetus subsp venerealis (Cfv) in preputial samples of bulls. 313 beef bulls. Preputial samples were collected from 300 virgin bulls and 13 Cfv-infected bulls. Specificity of the qRT-PCR assay, determined on the basis of results for samples collected from virgin bulls, was compared with specificity of bacteriologic culture performed with transport enrichment medium (TEM). Sensitivity of the qRT-PCR assay, determined on the basis of results for multiple samples collected at weekly intervals from infected bulls, was compared with sensitivity of the direct fluorescent antibody test (DFAT), bacteriologic culture, and bacteriologic culture with TEM. Specificity was 85% for the qRT-PCR assay and 100% for bacteriologic culture; results were significantly different. Mean sensitivity was 85.4% for the qRT-PCR assay, 82.3% for direct culture in blood agar, 72.1% for the DFAT, 32.7% for direct culture in Skirrow agar, 30% for bacteriologic culture with TEM and blood agar, and 38.1% for bacteriologic culture with TEM and Skirrow agar. Differences in sensitivity among tests varied with ambient outdoor temperature. Repeated sampling significantly increased sensitivity of the qRT-PCR assay. Use of the qRT-PCR assay as a screening test on direct preputial samples had comparable sensitivity to bacteriologic culture, and repeated sampling improved sensitivity. Although improved performance of the qRT-PCR assay, compared with direct bacteriologic culture, was dependent on temperature, transport times that allow direct culture are unlikely under field conditions. The qRT-PCR assay would provide a fast and sensitive screening method for Cfv in bulls.

Isolation rates of Campylobacter fetus subsp venerealis from bovine preputial samples via passive filtration on nonselective medium versus selective medium, with and without transport medium

To compare the recovery rates of Campylobacter fetus subsp venerealis (Cfv) from preputial scrapings of infected bulls with passive filtration on selective medium versus nonselective medium, with and without transport medium. 217 preputial scrapings from 12 bulls (4 naturally and 8 artificially infected with Cfv). Preputial scrapings were collected in 2 mL of PBS solution and bacteriologically cultured directly on Skirrow medium or passively filtered through 0.65-μm filters onto blood agar, with or without 24 hour preincubation in modified Weybridge transport enrichment medium (TEM). After 72 hours, plates were examined for Cfv and bacterial and fungal contamination or overgrowth. Passive filtration of fresh preputial scrapings onto blood agar yielded significantly higher recovery rates of Cfv (86%) than direct plating on Skirrow medium (32%), whereas recovery from TEM was poor for both media (35% and 40%, respectively). Skirrow cultures without TEM were significantly more likely to have fungal contamination than were cultures performed with any other technique, and fungal contamination was virtually eliminated by passive filtration onto blood agar. Bacterial contamination by Pseudomonas spp was significantly more common with Skirrow medium versus passive filtration on blood agar, regardless of TEM use. The use of transport medium and the choice of culture medium had significant effects on Cfv recovery and culture contamination rates from clinical samples. Both factors should be considered when animals are tested for this pathogen.

Clinical sensitivity and specificity of a quantitative PCR assay for bovine genital campylobacteriosis in bulls

Bovine genital campylobacteriosis occurs worldwide wherever natural service is used. The disease is transmitted by carrier bulls and characterized by early pregnancy loss and infertility. Despite the challenge of maintaining viability of the Campylobacter fetus subsp venerealis (Cfv) during transport and avoiding overgrowth of contaminants, culture remains the "gold standard". Several PCR tests have been developed for the detection of Cfv, and primers VenSF/VenSR have been extensively tested and recognized worldwide. Recently, our research group at WCVM adapted these primers to a SYER Green qPCR platform. This assay presents a high analytical sensitivity and provides a fast and effective technique, suitable to be used as a screening test for the detection of carrier bulls. The objective of the present study was to determine the clinical sensitivity and specificity of the RT-PCR assay in direct preputial samples.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Bacteraemia due to Campylobacter fetus subsp. fetus in a liver transplanted patient

Campylobacter fetus is a mobile, curved or spiral-shaped Gram negative bacillus.We report a case of bacteraemia due to C. fetus subsp. fetus isolated from a 47-year-old male patient liver transplanted, with underlying cardiovascular disease and receiving immunosuppressive therapy with cyclosporine.This patient was admitted to the hospital with fever and pain to both the legs.After admission, the microorganism isolated from blood-culture was identified as Campylobacter fetus subsp fetus either by traditional methods or by 16S rRNA gene sequencing. Our observation emphasizes the possibility that such unusual microorganisms can be isolated from immunosuppressed patients and, therefore, particular attention should be paid to the strategies for their detection.

Expression of IL-8 and MIP-1α chemokines in HeLa cells infected with Campylobacter fetus subsp. Venerealis

Expression of IL-8 and MIP-1α chemokines in HeLa cells infected with Campylobacter fetus subsp. Venerealis

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. Venerealis

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. Venerealis

Investigation of bovine venereal campylobacteriosis in beef cow herds in New Zealand

AIMS: To determine regional prevalences of beef cow herds in New Zealand positive for Campylobacter fetus subsp venerealis antibodies in samples of vaginal mucus tested using an immunoglobulin (Ig) A enzyme-linked immunosorbent assay (ELISA), and to examine the suitability of the IgA ELISA for detecting infection with C. fetus subsp venerealis under field conditions in New Zealand. METHODS: Vaginal mucus samples (n=1,230) collected from beef cow herds (n=125) throughout New Zealand (approximately 10 samples/herd) were tested for antibodies to C. fetus subsp venerealis using an IgA ELISA. Test results were compared between herds classified as having low, medium and high fertility based on pregnancy test results interpreted in relation to the duration of the mating period used. In addition, a small number of samples were collected from dairy cows that were mated using artificial insemination (AI) and had no contact with breeding bulls. The influence of putative risk factors for the spread of venereal disease and the effect of sample quality on the status of herds according to test results was assessed using multivariate logistical regression. Preputial washings from 54 bulls from nine herds classified as low fertility in which mucus samples from ≥3 cows were IgA antibody-positive were cultured for the presence of Campylobacter spp, and isolates of C. fetus subspecies were characterised using a polymerase chain reaction (PCR) test. RESULTS: One or more mucus samples was positive to the IgA ELISA in 70% of all herds tested. The prevalence of IgA antibody-positive individuals was >20% in most regions of New Zealand and did not differ significantly for cows from herds classified as high, medium or low fertility (28%, 26% and 23%, respectively; p=0.39). No relationship was found between mucus antibody status and age of breeding group, herd size, herd fertility, number of herds that female replacements or breeding bulls were sourced from, whether a serving ability test (SAT) was used to assess bulls, or the quality of samples submitted to the laboratory. Campylobacter fetus subsp venerealis was not cultured from any of the 54 bulls sampled. Four other species of Campylobacter and related organisms were cultured, viz Arcobacter cryaerophilus, Campylobacter jejuni, Campylobacter fetus subsp fetus and Helicobacter cinaedi. CONCLUSIONS: The specificity of the IgA ELISA as a diagnostic test for C. fetus subsp venerealis was found to be unsatisfactory under New Zealand conditions. It is possible that an immunological response by cows to Campylobacter species other than C. fetus subsp venerealis caused cross-reactivity in the IgA ELISA. The results do not support the hypothesis that C. fetus subsp venerealis is widespread in New Zealand.

Bioserogroups of Campylobacter species isolated from sheep in Kaduna State, Nigeria

Sheep Campylobacter isolates from Kaduna State were characterized into their species and bioserogrouped. A total of 1100 samples were collected from Kaduna abattoir and National Animal Production Research Institute (NAPRI), Shika. The samples were from 250 gallbladder, 250 intestinal contents, 100 fetal stomach contents all from Kaduna abattoir while 250 rectal swabs and 250 vaginal swabs were from the NAPRI Small Ruminant Programme. Of a total of 1100 samples, 39 (3.54%) yielded Campylobacter organisms. The highest isolation rate (6.8%) was from samples of intestinal contents followed by those from gall bladders (4.0%). Samples from the vaginal and fetuses had the lowest isolation rates (2.80%) and (0%), respectively. Of the 39 Campylobacter isolates from all the sources, (79%) were characterized as C. fetus subsp jejuni, C. coli (13%) and C. laridis (8.0%). while C. coli and C. laridis were isolated from gall bladder and intestinal contents only. Campylobacter fetus subsp jejuni biotype 1 accounted for 40.3% of the total isolates. C. laridis biotypes I and II were also isolated and accounted for 5% and 3% of the isolates, respectively. 5% of the isolates were not typeable. The serogroups 4 (13%), 36 (10%), 9 (10%), 84 (8%), 29 (5%) and 20 (8%) were the commonest serogroups identified in sheep at two locations surveyed. The isolation of Campylobacter organisms from rectum, vagina, gallbladder, and intestinal contents is a clear indication that sheep serves as a reservoir of this organisms in Nigeria. Similarities between documented human Campylobacter isolates in Nigeria and those in the present study raised the possibility of cross-transmission between sheep and man. It is concluded that biotyping and serotyping can be used for epidemiological study of campylobacteriosis due to Campylobacter jejuni in sheep in Kaduna State of Nigeria.

Immunization and diagnosis in bovine reproductive tract infections

This chapter discusses immunization and diagnosis in bovine reproductive tract infections. Bovine genital tract infections that result in pregnancy loss are large economic problems in the cattle industry. Such infections can occur by either a hematogenous or an ascending route. Because the diseases acquired by the former route are primarily septicemic whereas local ascending infections are primarily sexually transmitted diseases (STDs), diagnosis and control may differ between these two categories. Systemic infection with localization in the gravid uterus occurs in leptospirosis, systemic campylobacteriosis (caused by Campylobacter fetus subsp, fetus), listeriosis, brucellosis, neosporosis, and Haemophilus sommus infection. In these syndromes, systemic immunity and serologic diagnosis are often reliable. Brucellosis is a good example of hematogenous infection with localization in the gravid uterus because systemic vaccination and serologic assays for diagnosis have been practiced for decades. Haemophilus sommus infection is also presented because it is a septicemic disease causing abortion and many other sequelae, thus immunity is complex.

Abdominal aortic aneurysm infected by Campylobacter fetus subsp. Fetus

Abdominal aortic aneurysm infected by Campylobacter fetus subsp. Fetus

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies. Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis. The results of identification of 99 bacterial strains as determined by conventional phenotyping or by polymerase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis; the remaining strains were field isolates putatively identified as C fetus. In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling. The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories. Our results indicate that misidentification of C fetus in routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.

Antigenic and restriction enzyme analysis of Campylobacter spp associated with abortion in sheep

Abstract Objective To determine the scope of strain variations among Campylobacter spp associated with abortion in sheep. Design To examine Campylobacter spp isolated from cases of abortion for biochemical, antigenic, and genetic differences. Sample Population 15 isolates of Campylobacter spp isolated from cases of abortion during a single lambing season. Procedure Isolates were examined, using biochemical tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins (OMP), and DNA restriction enzyme analysis (REA). Results Eight strain variants were detected among the 15 isolates. 14 of the isolates were C jejuni, 13 of which were biotype I and 1 biotype II, and the remaining isolate was identified as C fetus subsp fetus. Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis OMP patterns had distinctive profiles for C fetus subsp fetus and C jejuni biogroup-II isolates and 3 variants within the C jejuni bio-group-I isolates. Examination of REA patterns of DNA from the 15 isolates digested with Cfo I indicated clear differences correlating with species and biogroups and 4 REA variants among biotype-l isolates. Conclusions Marked antigenic and genetic heterogeneity of Campylobacter isolates were associated with ovine abortion within a defined geographic area. Clinical Relevance Representatives of differing OMP and REA profile groups should be considered for incorporation in vaccines to optimize protection in this region and possibly other geographic areas.

Diagnosis by ELISA of bovine abortion due to Campylobacter fetus

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antigen-specific secretory IgA antibody in bovine vaginal mucus after abortion due to Campylobacter fetus subsp venerealis. Abortions were diagnosed by isolating the organism from 8 foetuses and/or foetal membranes and by histopathology. Vaginal mucus was collected from 7 cows shortly after abortion. All showed a high level of IgA antibody in their vaginal mucus when they were compared with an uninfected control group. The new ELISA is simple and practical and provides a useful tool for diagnosis of bovine venereal campylobacteriosis.

Campylobacter infection associated with raw milk. An outbreak of gastroenteritis due to Campylobacter jejuni and thermotolerant Campylobacter fetus subsp fetus.

Raw milk is identified with increasing numbers of outbreaks of gastroenteritis and is an important vehicle for transmission of Campylobacter infection. Unlike Campylobacter jejuni, Campylobacter fetus subsp fetus has not been associated with common-source outbreaks of gastroenteritis. This report describes an outbreak of gastroenteritis involving C jejuni and a thermotolerant strain of C fetus subsp fetus associated with raw milk. Fifteen (39%) of 38 persons who attended a banquet in Wisconsin in June 1982 developed acute gastroenteritis. Stool specimens were obtained from nine ill guests; four yielded C jejuni and three yielded C fetus subsp fetus. The C fetus subsp fetus isolates were identified fortuitously, in part because of unusual thermotolerance (growth at 42 degrees C), permitting isolation at temperature appropriate for C jejuni. Survey results implicated raw milk as the source of the outbreak. Findings provide evidence of a potentially emergent milkborne pathogen contributing to the risk of raw milk consumption and suggest that current diagnostic laboratory techniques may fail to identify significant foodborne agents.