Bovine Genital Campylobacteriosis is characterized by early pregnancy loss and temporary infertility in cattle. The purpose of this project was to compare diagnostic approaches to detect Campylobacter fetus subsp. venerealis and evaluate the efficacy of vaccination for Bovine Genital Campylobacteriosis. This thesis describes the results of two studies that compared different sample preparation methods for bovine vaginal mucus for real-time PCR and assessed a commercial vaccine in preventing infection and reproductive loss. The first study compared real-time PCR utilizing different bovine vaginal mucus sample preparation techniques to direct culture. The magnetic bead based protocol demonstrated higher sensitivity (48.4%, P=0.02) and lower specificity (78.9%, P=0.01) than the heat lysis protocol which involved an additional dilution step (Sens=29.4%, Spec=88.2%), but did not differ from the heat lysis protocol without sample dilution (Sens=35.0%, P=0.16; Spec=81.1%, P=0.62). The sample preparation method, designed for bovine preputial samples (Chaban et al. 2012. Can J of Vet Res; 76: 166), did not work well for vaginal mucus. All modifications of that method and magnetic bead based extraction technique had low sensitivity compared to culture probably due to the biophysical properties of vaginal mucus, which could cause loss of targeted DNA during processing, or repeated sample freezing and thawing. Release of DNA directly from vaginal mucus by a modified heat lysis protocol with consequent real-time PCR could be a promising rapid screening approach after validating on fresh samples. The second study compared the risk of infection and reproductive failure in heifers, vaccinated with a commercial multivalent vaccine containing C. fetus antigen, to heifers vaccinated with a comparable product without C. fetus, that were exposed to infected bulls. There was no significant difference between groups either in risk of Campylobacter fetus subsp. venerealis isolation (P>0.17) or in the proportion of heifers that cultured positive at least once (P=0.42), as well as in the median number times of cultured positive samples (P=0.24) and the time to first cultured positive (P=0.67). There was no difference by treatment in the weekly proportions of heifers diagnosed pregnant by either ultrasound (P>0.31) or serum concentration of pragnancy specific protein B (P>0.31) during the study, as well as in the time to first pregnancy for heifers ever diagnosed as pregnant (P=0.30) and those that remained pregnant at the end of the study (P=0.70). Similarly, the difference was not detected by treatment in the proportion of animals, ever detected pregnant during the study (P=0.57) and in pregnancy loss rates (P=0.28). However, heifers that aborted were 4 times more likely to be cultured positive than those that did not abort (P=0.01). Heifers that were not pregnant at the end of the study cultured positive 1.5 times more often than pregnant animals in treatment group (P=0.04), while in control group such difference was 4 times (P=0.01). Heifers that were not pregnant at the end of the study did not differ by treatment in the number of times cultured positive (P=0.14). In this study, the mean concentrations of ELISA antibodies to C. fetus after vaccination were more than 2 times higher in treatment group than in control group (P<0.02), but vaccination did not significantly reduce infection or improve pregnancy in heifers when exposed to Cfv-infected bulls. Sample preparation technique is important for successful real-time PCR; release of DNA directly from a CVM sample by a modified heat lysis protocol was easy to perform and could be promising as a rapid screening approach for Bovine Genital Campylobacteriosis after validating on fresh samples. Vaccinating of heifers with a polyvalent commercial vaccine, containing Campylobacter fetus antigen, according to the label, did not significantly reduce infection rate or improve reproductive performance when they were naturally challenged.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-02-07T13:04:40Z No. of bitstreams: 1 Jonas de Melo Borges.pdf: 1057256 bytes, checksum: 5a36d4a5894648ecc348d8146d19bca1 (MD5) / Made available in DSpace on 2017-02-07T13:04:40Z (GMT). No. of bitstreams: 1 Jonas de Melo Borges.pdf: 1057256 bytes, checksum: 5a36d4a5894648ecc348d8146d19bca1 (MD5) Previous issue date: 2016-07-20 / The objective of this study was to determine the occurrence of infection with Campylobacter fetus subsp. venerealis and Tritrichomonas foetus in buffaloes in the State of Pernambuco, Brazil and identify possible risk factors associated with infection. Biological samples were collected (cervico vaginal mucus and shaved prepucial) of 113 animals, coming from 8 properties in different regions of the state. The biological material collected was transferred into phosphate buffered saline (PBS) and inoculated in the specific transport, Lander for diagnosis of C. fetus subsp. venerealis and Diamond for T. fetus subsequently. For the diagnosis of infection by Campylobacter fetus subsp. venrealis and Tritrichomonas foetus the samples were submitted to Polymerase Chain Reaction ( PCR) grown in Columbia agar plus antibiotics and Diamond, respectively. There was an occurrence of 1.8% (2/113 ; I.C. 0.2 to 6.2 % ) of positive animals in the microbiological examination with confirmation by PCR, for C. fetus subsp. venerealis. It was observed that 100% of positive samples were from two (2) males from the same herd. No animals were positive for T. foetus. It was not possible to identify risk factors associated with infection. This is the first report of infection with C. fetus subsp. venerealis in buffaloes in Brazil. Despite the low occurrence it is recommended that control measures are adopted, in order to prevent the spread of the agent to other herds. / Objetivou-se com este estudo determinar a ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no Estado de Pernambuco, Brasil e identificar os possíveis fatores de risco associados às infecções. Foram coletadas amostras biológicas (muco cérvico-vaginal e raspado prepucial) de 113 animais, procedentes de 8 propriedades, de diferentes regiões do Estado. O material biológico coletado foi transferido para solução salina tamponada (PBS) e posteriormente inoculado nos meios de transporte específicos, Lander para diagnóstico de C. fetus subsp. venerealis e Diamond para T. foetus. Para o diagnóstico das infecções por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus as amostras foram cultivadas em meio ágar columbia acrescido de antibiótico e Diamond, respectivamente. Posteriormente as amostras de PBS foram submetidas à Reação em Cadeia da Polimerase (PCR). Para pesquisa de C. fetus subsp. venerealis, observou-se uma ocorrência de 1,8% (2/113; I.C. 0,2 – 6,2%) de animais positivos no exame microbiológico com confirmação pela PCR. Em relação à procedência, observou-se que 100% das amostras positivas pertenciam a dois machos do mesmo rebanho. Na pesquisa de T. foetus nenhum animal foi positivo. Não foi possível identificar os fatores de risco associados às infecções. Este é o primeiro registro da infecção por C. fetus subsp. venerealis em búfalos no Brasil. Apesar da baixa ocorrência recomenda-se que medidas de controle sejam adotadas, com o intuito de evitar a disseminação do agente para outros rebanhos.

The traditional method for the diagnosis of bovine genital campylobacteriosis is the culture and identification of the causative organism, Campylobacter fetus subsp. venerealis (Cfv) from the genital tract. This approach is considered relatively insensitive due to the fragility of the bacteria, their specific nutritional and atmospheric requirements and their being easily overgrown by commensal bacteria. The identification of isolates is also problematic due to the limited biochemical activity of the bacteria. With the rapid advances made in the molecular field, assays have become more robust and cost-effective making them feasible for the diagnostic laboratory. The potential speed, sensitivity and specificity offered by these assays provide attractive alternatives for the identification of pathogens which are notoriously difficult to identify. The first part of this investigation was concerned with the implementation and evaluation of a polymerase chain reaction (PCR) assay for the direct detection of C. fetus in bovine preputial specimens. The specificity of a published C. fetus-specific primer pair was established by testing C. fetus reference and field isolates in addition to a collection of other Campylobacter species and organisms which may encountered in the genital tract of cattle. All C. fetus isolates tested yielded a single PCR amplicon of approximately 750 bp. No amplicons were generated when any of the other non-C. fetus isolates were tested. Following minor modifications to the assay, the sensitivity of the assay was determined using spiked Weybridge medium. A detection limit of 615 Cfv/mR Weybridge medium (or 6,15 cell equivalents per PCR assay) was obtained. Preputial material collected and submitted for laboratory testing may often be contaminated with faeces, urine, semen and/or blood. All of these components are known to be potential PCR inhibitors and the influence of each, on the sensitivity of the PCR assay, was subsequently evaluated. Faeces were identified as a potent inhibitor and contamination of specimens with as little as 1% (w/v) faeces reduced the sensitivity of the assay. Concentrations of up to 50% (v/v) of blood, urine and semen had no effect on the sensitivity of the assay. Preputial specimens, collected in Weybridge medium, were subsequently pooled and spiked and used to establish the sensitivity of both the PCR and culture methods as well as determine the influence of time on the sensitivity of the assays. Testing was carried out in triplicate on samples collected from different herds which were ascertained to be free of Cfv based on the use of specific selection criteria. The detection limit of the culture method was found to be better than that achieved using PCR only immediately after the samples were spiked. The detection limit of the culture method decreased with time whilst the detection limit of the PCR assay remain unchanged up to 72 hours post-inoculation. Ensuing field evaluation involved the testing of 212 clinical samples using both the culture method and the optimized PCR assay. Of the samples tested 4,2% were found to be positive using the PCR assay, whilst only 3,8% were found to be positive by culture. Based upon this evaluation the analytical specificity of the PCR assay was calculated to be 99% and the analytical sensitivity, 85,7%. The second part of this investigation was concerned with the subspeciation of C. fetus isolates. Currently the only test recommended by the Office International des Epizooties (OIE) for the subspeciation of isolates, is tolerance to 1% glycine. Doubts over the reliability of this test have led to alternative or supplementary tests being sought. Within the context of this investigation a collection of 40 South African field isolates were subspeciated using a previously described subspecies-specific primer set as well as the traditional 1% glycine tolerance phenotyping test. Additionally, other phenotyping tests (selenite reduction, growth at 42 °C and susceptibility to metronidazole and cefoperazone) were evaluated to determine their suitability for use as an aid in the subspeciation of C. fetus isolates. None of the field isolates yielded a Cfv-specific subspecies PCR amplicon using the published primer set suggesting that all of the isolates were Campylobacter fetus subsp. fetus (Cff). Based on tolerance to 1% glycine however, only 6 isolates were identified as Cff (glycine tolerant), whilst the remainder were classified as Cfv. The results of the ‘sensitive’ hydrogen sulphide test indicated that the Cfv isolates were specifically Cfv biovar intermedius. The lack of agreement between the PCR and the phenotyping subspeciation results concur with the findings reported by other researchers. It is consequently concluded that the published VenSF/VenSR subspecies-primer set is unsuitable for the subspeciation of South African field isolates. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted