Bovine Genital Campylobacteriosis Research Articles

Polymerase chain reaction for the diagnosis of bovine genital campylobacteriosis

Bovine genital campylobacteriosis is a common venereal disease of cattle; the prevalence of this disease can be underestimated mostly because of the nature of the etiological agent, the microaerobic Campylobacter fetus subspecies venerealis. The purpose of the current study was to evaluate the utilization of polymerase chain reaction (PCR) in the diagnosis of genital campylobacteriosis in samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents of aborted fetuses, collected into enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lysis with proteinase K, lysis with guanidine isothiocyanate, lysis with DNAzol, and lysis with hexadecyltrimethylammonium bromide (CTAB). The specificity, sensitivity, and technical application of the PCR assay were also evaluated with clinical samples and compared to bacterial isolation by standard culture. DNA extraction by the CTAB protocol provided better results in PCR, and it was able to detect 63 colony-forming units per ml of C. fetus. Out of 277 clinical samples tested, 68 (24%) were positive for Campylobacter fetus using PCR, while only 8 (2.8%) of the samples were positive by bacterial isolation in solid medium, proving the superiority of the PCR technique when compared to the standard isolation method, and providing evidence for its usefulness as a better screening test in cattle for the diagnosis of bovine genital campylobacteriosis.

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.

Development of a loop-mediated isothermal amplification and PCR assays for rapid and simple detection of Campylobacter fetus subsp. venerealis

Campylobacter fetus is divided into CFV and CFF. Because CFV causes bovine genital campylobacteriosis, differentiation of the two subspecies is essential to the implementation of efficient CFV control and eradication programs. We have developed LAMP and duplex PCR assays for rapid and simple detection of CFV. The LAMP assay correctly detected 7 CFV strains and did not detect 53 CFF, 35 non-fetus Campylobacter and 25 non-Campylobacter strains. The PCR assay successfully differentiated the two subspecies. The LAMP and PCR assays were faster than conventional biochemical assays, requiring for detection less than 50 min and less than 4 hr, respectively, from the beginning of DNA extraction from a single colony on blood agar to final determination. Our LAMP and PCR assays are rapid and practical tools for detection of CFV.

PESQUISA DE Campylobacter fetus EM PREPÚCIO DE TOUROS, EM UMA PROPRIEDADE RURAL DO ESPÍRITO SANTO, BRASIL

Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease, which causes major economic losses due to reproductive failure in affected animals. It is not easily diagnosed because of the microaerophilic nature of its causative agent, Campylobacter fetus. This study assesses C. fetus in preputial bull fluid collected by scraping of smegma for isolation of the bacteria in selective medium. Animals were aged 42 to 60 months, were not sexually active, and were raised in the municipality of Piuma, Espirito Santo. C. fetus bacteria was not detected in any of the samples, although Pseudomonas aeruginosa (34,5%) and Klebsiella spp. (12,5%) strains increased. Some factors may have influenced these results, such as: age of animals, lack of sexual activity, confinement, healthy environments, difficulty to isolate bacteria, and interference of other microorganisms in culture. More sensitive and practical techniques are needed to better diagnose this venereal disease.

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

BackgroundCampylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes.ResultsEighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp.ConclusionThe comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus.

Discovery of insertion element ISCfe1: a new tool for Campylobacter fetus subspecies differentiation

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis (CFV) and C. fetus subsp. fetus (CFF). CFV is the causative agent of bovine genital campylobacteriosis, a highly contagious venereal disease that may lead to serious reproductive problems, including sterility and abortion. In contrast, CFF can be isolated from the gastrointestinal tract of a wide range of host species, is associated with abortion in sheep and cattle, and can also be isolated from local and systemic infections in humans. Despite differences in host and niche preferences, microbiological differentiation of the two subspecies of C. fetus is extremely difficult. This study describes the identification of a new insertion element, ISCfe1, which is present exclusively in CFV strains, with highly conserved specific ISCfe1 insertion sites. The results are useful for identification and differentiation of the two C. fetus subspecies and will help in understanding the evolution and pathogenesis of C. fetus.

Detección e identificación de Campylobacter fetus subesp. venerealis en el mundo

Bovine genital campylobacteriosis caused by Campylobacter fetus subsp. venerealis (Cfv) is a genital infection that threatens the cattle industry. Detection and identification of Cfv are key factors in control programmes. Trade regulations should be based on scientifically and internationally accepted methods of detection and identification of Cfv. Such methods are described in the World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. A study was conducted to determine which methods are in use in OIE Member Countries and to get an overview of new or improved tests. A questionnaire was sent to OIE Member Countries, and 26 out of 166 were returned. Globally, a diversity of methods for the detection and identification of Cfv are in use. The authors conclude that there is a lack of harmonisation that may have consequences for the description of the health status of countries and may lead to disputes with respect to trade regulations.

Clonal Nature of Campylobacter fetus as Defined by Multilocus Sequence Typing

Campylobacter fetus can be divided into the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. fetus causes sporadic infections in humans and abortion in cattle and sheep and has been isolated from a variety of sites in different hosts. C. fetus subsp. venerealis is host restricted, being isolated mainly from the genital tracts of cattle, and is the causative agent of bovine genital campylobacteriosis. Despite differences in niche preference, microbiological subspecies differentiation has proven difficult. Different typing methods divided C. fetus isolates into different subgroups, depending on the methods used. The relative value of these methods can be assessed by the evolutionary relationship of isolates belonging to the genus; therefore, we developed a multilocus sequence typing (MLST) scheme for C. fetus. This scheme was applied to 140 C. fetus isolates previously typed by amplified fragment length polymorphism (AFLP) analysis. A total of 14 different sequence types (STs) were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. These MLST data indicate that C. fetus is genetically homogeneous compared to the homogeneity of other Campylobacter species. The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which represents a "bovine" clone. The C. fetus subsp. fetus isolates studied were more diverse in terms of their STs, and the STs correlated with epidemiological relationships. Congruence was observed among C. fetus subspecies, sap type, and ST; therefore, MLST confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche.

Campilobacteriose genital bovina em rebanhos leiteiros com problemas reprodutivos da microrregião de Varginha - Minas Gerais

The frequency of bovine genital campylobacteriosis (BGC) in 157 cows from nine herds from the microregion of Varginha - Minas Gerais - Brazil was evaluated. Farmers use either artificial insemination and natural breeding after two insemination procedures or natural breeding. The diagnosis of BGC was performed by the direct fluorescent antibody test (DFAT) using vaginal mucus. All studied herds showed C. fetus infected animals and, of 157 animals, 40 (25.5%) were positive in DFAT: 7 (26.9%) from herds with natural breeding and 33 (25.1%) from herds with both artificial insemination and natural breeding after the second-to-third insemination. The high frequency of BGC found in this study shows that this disease is present among herds which have reproductive problems and the use of natural breeding after the second-to-third unsuccessful insemination could be a risk factor for the disease.

Campylobactériose génitale bovine dans le Pantanal, Etat du Mato Grosso do Sul, Brésil

La prévalence de la campylobactériose génitale bovine a été estimée chez des taureaux au Pantanal, Mato Grosso do Sul, Brésil. La recherche des anticorps par immunofluorescence directe a été effectuée avec les prélèvements des lavages des prépuces de 327 taureaux appartenant à 19 élevages. Ce test a révélé 170 taureaux (52,3 p. 100) positifs, provenant de 17 élevages (89,5 p. 100). Il n’y a pas eu de différence dans le nombre de taureaux positifs lorsque les différents groupes d’âge ont été considérés. En revanche, il y a eu une différence significative dans les élevages de plus de 15 000 ha (χ2 = 6,40 ; df = 1 ; P = 0,01), avec un risque relatif estimé de 1,33 (1,07 < RR < 1,66), par rapport aux élevages de 15 000 ha ou plus petits. Ces résultats ont indiqué que la campylobactériose génitale bovine était répandue dans la région étudiée et était probablement liée au mode de gestion des troupeaux.