Resumo: Para verificar os fatores de risco e a viabilidade de métodos diagnósticos para a campilobacteriose genital bovina (CGB), amostras prepuciais de touros de 102 rebanhos leiteiros de Presidente Prudente, São Paulo, foram avaliadas por testes bacteriológicos, reação da polimerase em cadeia (PCR) e imunofluorescência direta (IFD). Durante a colheita de lavados prepuciais, um questionário sobre desordens reprodutivas nas fêmeas e condições sanitárias foi aplicado aos proprietários das fazendas. As amostras foram plaqueadas em ágar seletivo de Skirrow, ágar Brucella verde brilhante e ágar tioglicolato com 20% do sangue. As características seletivas dos três sistemas da cultura foram comparadas pela avaliação de escores de densidade de contaminantes bacterianos nas placas e a capacidade de suportar diferentes fontes de contaminantes. A PCR foi conduzida usando seqüências dos nucleotídeos que amplificam um fragmento com 835 pares das bases do DNA ribossomal bacteriano. A IFD foi executada usando o conjugado anti-C. fetus subesp. venerealis marcado com isotiocianato de fluoresceína. Resultados positivos foram obtidos para culturas microbianas, PCR e IFD de 7,8% , 5,8% e 1,9% das fazendas, respectivamente. Detectou-se associação significativa entre touros positivos e rebanhos cujas vacas apresentavam taxas de concepção abaixo de 85% (OR=7,6). A concordância entre a PCR e as culturas bacteriológicas foi considerada ótima (Kappa = 0,847), mas foi pobre entre a IFD e os outros testes. Apenas o ágar seletivo de Skirrow não foi influenciado por contaminantes do lavado prepucial. Conclui-se que a CGB está presente em rebanhos leiteiros de Presidente Prudente, onde é associada com os distúrbios reprodutivos. / Abstract: To verify the risk factors and viability of diagnostic methods for genital bovine campylobacteriosis (GBC), preputial samples from bulls from 102 dairy herds from Presidente Prudente, São Paulo State, were evaluated using bacteriological tests, polymerase chain reation (PCR) and direct immunofluorescence (DFA). During the preputial sampling, some questions were made about female reproductive disorders and sanitary conditions of the farms. The preputial samples were plated in Skirrowþs agar, Brucella Brilliant green agar and Thioglycolate agar with 20% of blood. The selective characteristic of the three systems of culture was compared by the evaluation of bacterial density scores in the plates and for the capacity to support bacterial contaminants of different sources. The PCR was conducted using sequences of nucleotides that amplify a fragment with 835 pairs of bases of the ribossomal DNA. Direct immunofluorescence was performed using anti-C. fetus subesp. venerealis conjugated with fluorescein isothiocyanate. Positive results were obtained by microbial cultures, PCR and DFA results of 7,8%, 5,8% and 1,9% of the farms, respectively. Statistical association between positive bulls and low fertility of the herd cows was detected (OR= 7,6). The overall agreement between the PCR and bacteriological cultures was excellent (Kappa = 0,847), but poor between DFA and the other tests. The Skirrowþs media was the only that was not influenced by contaminant characteristics of preputial fluid samples. It is concluded that GBC is present in dairy herds from Presidente Prudente, where was associated with reproductive disturbances. / Orientador: José Rafael Modolo / Coorientador: Andrey Pereira Lage / Banca: Marcelo George Mungai Chacur / Banca: Antônio Carlos Paes / Banca: Ary Fernandes Junior / Banca: Luis Carlos de Souza / Doutor

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-08-31Bitstream added on 2014-06-13T18:44:26Z : No. of bitstreams: 1 giuffrida_r_dr_botfmvz.pdf: 417414 bytes, checksum: 8eb3f229c45de0856b5cb7630e7c281a (MD5) / Outros / Para verificar os fatores de risco e a viabilidade de métodos diagnósticos para a campilobacteriose genital bovina (CGB), amostras prepuciais de touros de 102 rebanhos leiteiros de Presidente Prudente, São Paulo, foram avaliadas por testes bacteriológicos, reação da polimerase em cadeia (PCR) e imunofluorescência direta (IFD). Durante a colheita de lavados prepuciais, um questionário sobre desordens reprodutivas nas fêmeas e condições sanitárias foi aplicado aos proprietários das fazendas. As amostras foram plaqueadas em ágar seletivo de Skirrow, ágar Brucella verde brilhante e ágar tioglicolato com 20% do sangue. As características seletivas dos três sistemas da cultura foram comparadas pela avaliação de escores de densidade de contaminantes bacterianos nas placas e a capacidade de suportar diferentes fontes de contaminantes. A PCR foi conduzida usando seqüências dos nucleotídeos que amplificam um fragmento com 835 pares das bases do DNA ribossomal bacteriano. A IFD foi executada usando o conjugado anti-C. fetus subesp. venerealis marcado com isotiocianato de fluoresceína. Resultados positivos foram obtidos para culturas microbianas, PCR e IFD de 7,8% , 5,8% e 1,9% das fazendas, respectivamente. Detectou-se associação significativa entre touros positivos e rebanhos cujas vacas apresentavam taxas de concepção abaixo de 85% (OR=7,6). A concordância entre a PCR e as culturas bacteriológicas foi considerada ótima (Kappa = 0,847), mas foi pobre entre a IFD e os outros testes. Apenas o ágar seletivo de Skirrow não foi influenciado por contaminantes do lavado prepucial. Conclui-se que a CGB está presente em rebanhos leiteiros de Presidente Prudente, onde é associada com os distúrbios reprodutivos. / To verify the risk factors and viability of diagnostic methods for genital bovine campylobacteriosis (GBC), preputial samples from bulls from 102 dairy herds from Presidente Prudente, São Paulo State, were evaluated using bacteriological tests, polymerase chain reation (PCR) and direct immunofluorescence (DFA). During the preputial sampling, some questions were made about female reproductive disorders and sanitary conditions of the farms. The preputial samples were plated in Skirrowþs agar, Brucella Brilliant green agar and Thioglycolate agar with 20% of blood. The selective characteristic of the three systems of culture was compared by the evaluation of bacterial density scores in the plates and for the capacity to support bacterial contaminants of different sources. The PCR was conducted using sequences of nucleotides that amplify a fragment with 835 pairs of bases of the ribossomal DNA. Direct immunofluorescence was performed using anti-C. fetus subesp. venerealis conjugated with fluorescein isothiocyanate. Positive results were obtained by microbial cultures, PCR and DFA results of 7,8%, 5,8% and 1,9% of the farms, respectively. Statistical association between positive bulls and low fertility of the herd cows was detected (OR= 7,6). The overall agreement between the PCR and bacteriological cultures was excellent (Kappa = 0,847), but poor between DFA and the other tests. The Skirrowþs media was the only that was not influenced by contaminant characteristics of preputial fluid samples. It is concluded that GBC is present in dairy herds from Presidente Prudente, where was associated with reproductive disturbances.

Campylobacter spp., especially C. jejuni and C. coli, are the leading cause of bacterial gastroenteritis in Europe. There is a recognised need to develop detection tools which can be performed on farms to facilitate reducing the presence of Campylobacter in poultry. A similar application could be beneficial for detection of C. fetus, a veterinary pathogen which causes significant economic loss in the cattle industry. Campylobacter species perform protein N-linked glycosylation and in C. jejuni at least 150 proteins, many of which are surface-exposed, may be modified. Therefore, the first portion of this thesis investigated the feasibility of using N-linked glycans as targets for antibody-based detection of Campylobacter species. To do this, a His-tagged N-glycoprotein was expressed and purified from C. fetus and used as immunogen to raise an antiserum termed CfNgp. The Campylobacter N-glycan reactivity of this antiserum was characterised and it was shown to react with N-glycoproteins and cells of C. fetus and other emerging Campylobacter species such as C. concisus. Immunoblotting techniques and flow cytometry were used to characterise an antiserum (CjNgp) raised against a C. jejuni N-linked glycoprotein and demonstrated that it can specifically detect cells of C. jejuni, C. coli and other emerging Campylobacter species found in poulty. This thesis also describes the investigation of the relatively uncharacterised C. fetus N-linked glycosylation system. Functional analysis of C. fetus predicted glycosyltransferases was acheived by developing glycocompetent E. coli containing a hybrid C. jejuni/C. fetus pgl system. The N-glycan structures biosynthesised were analysed using mass spectrometry and this novel approach discovered the activity of two C. fetus glycosyltransferase enzymes. Finally, this work used a bioinformatics pipeline to produce a C. fetus predicted N-linked glycoproteome and experimentally verified a newly identified N-linked glycoprotein. This pipeline was also applied to investigate the putative conservation of N-linked glycoproteins throughout the Campylobacter genus and highlighted ‘core’ N-linked glycoproteins which are key targets for experimental investigation. Overall, this work demonstrates that Campylobacter N-linked glycans are attractive targets for antibody-based detection, expands our knowledge of C. fetus N-linked glycosylation and contributes to the broader understanding of this intriguing aspect of Campylobacter biology.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-05-05T15:00:33Z No. of bitstreams: 1 Junior Mario Baltazar de Oliveira.pdf: 1499654 bytes, checksum: 6880a06cd3c2f0f28c7d609803cb7e9e (MD5) / Made available in DSpace on 2017-05-05T15:00:33Z (GMT). No. of bitstreams: 1 Junior Mario Baltazar de Oliveira.pdf: 1499654 bytes, checksum: 6880a06cd3c2f0f28c7d609803cb7e9e (MD5) Previous issue date: 2014-08-08 / The objective of this study was to perform an epidemiological analysis of Campylobacter fetus subsp. venerealis and Tritrichomonas foetus infection in cattle from state of Pernambuco, Brazil. For this, 383 samples of cervical-vaginal mucus of cows from 21 properties distributed in the 19 counties that make up the microregion of Garanhuns and 105 samples of preputial smegma of males in the reproductive age, coming from 32 properties and four slaughterhouses were collected from state Pernambuco. The samples had their genomic materials extracted and subjected to polymerase chain reaction for diagnosis. To analyze the risk factors applied an investigative questionnaire on the hygienic, sanitary and reproductive management. The risk factors identified in this study was to bovine genital campylobacteriosis in female herds above 100 animals (OR=7.2, C.I. 1.3 to 38.4%, p=0.020) and bovine trichomoniasis was the use of natural mating (OR=2.4, C.I. 1.1 to 5.9%, p=0.041). In males, there was a frequency of 6.6% (2.7%-13.2%, 95% C.I.) for T. fetus and no sample was positive for C. fetus subsp. venerealis. Regarding the number of outbreaks, 21.8% of farms had animals positive for T. foetus. None analyzed variables was associated with infection. Regarding the samples from slaughterhouses, none was positive. It is concluded that infection by C. fetus subsp. venerealis and T. foetus are present in cattle in the state of Pernambuco and suggest that control measures such as diagnosis and prophylaxis, and sexual segregation and rest of infected females, using a program of artificial insemination with strict health care, elimination of infected bulls and replacement for young animals are implemented to prevent the spread of agents in herds. / Objetivou-se com este trabalho realizar uma análise epidemiológica da infecção por Campylobacter. fetus subsp. venerealis e Tritrichomonas foetus em bovinos do estado de Pernambuco, Brasil. Para isso, foram colhidas 383 amostras de muco cérvico-vaginal de vacas procedentes de 21 propriedades distribuídas nos 19 municípios que compõem a microrregião de Garanhuns e 105 amostras de esmegma prepucial de machos em idade reprodutiva, procedentes de 32 propriedades e quatro matadouros do estado de Pernambuco. As amostras tiveram seu material genômico extraído e submetido à reação em cadeia da polimerase para o diagnóstico. Para análise de fatores de risco aplicou-se um questionário investigativo com perguntas referentes ao manejo higiênico-sanitário e reprodutivo. Nas fêmeas, observou-se uma prevalência de 1,8% (0,8% - 3,9%; I.C. 95%) e 33,4% (28,7% - 38,4%; I.C. 95%;) para Campylobacter fetus subsp. venerealis e Tritrichomonas foetus, respectivamente. Em relação ao número de focos observou-se que 28,6% das propriedades apresentaram pelo menos um animal positivo para C. fetus subsp. venerealis e 90,5% para T. foetus. Os fatores de risco identificados neste estudo para campilobacteriose genital bovina em fêmeas foi rebanhos acima de 100 animais (OR=7,2; I.C. 1,3 - 38,4%; p=0,020) e para tricomonose bovina foi a utilização da monta natural (OR=2,4; I.C. 1,1 - 5,9%; p=0,041). Nos machos, observou-se uma frequência de 6,6% (2,7% - 13,2%; I.C. 95%) para T. foetus e nenhuma amostra foi positiva para C. fetus subsp. venerealis. Em relação ao número de focos, 21,8% das propriedades apresentaram animais positivos para T. foetus. Nenhuma das variáveis analisadas apresentou associação com a infecção. Quanto às amostras dos matadouros, nenhuma foi positiva. Conclui-se que as infecções por C. fetus subsp. venerealis e T. foetus estão presentes nos rebanhos bovinos do estado de Pernambuco e sugere-se que medidas de controle e profilaxia como o diagnóstico, separação e repouso sexual das fêmeas infectadas, utilização de um programa de inseminação artificial com cuidados sanitários rigorosos, descarte dos touros infectados e reposição por animais jovens devem ser implementadas a fim de evitar a disseminação do agente nos rebanhos.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-06-15T12:48:17Z No. of bitstreams: 1 Ruy Brayner de Oliveira Filho.pdf: 2028711 bytes, checksum: c66c5e9d041a3c1b673438d6c412bde8 (MD5) / Made available in DSpace on 2018-06-15T12:48:17Z (GMT). No. of bitstreams: 1 Ruy Brayner de Oliveira Filho.pdf: 2028711 bytes, checksum: c66c5e9d041a3c1b673438d6c412bde8 (MD5) Previous issue date: 2017-10-16 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The objective of this study was to determine the epidemiological situation of infection by Tritrichomonas foetus and Campylobacter fetus subsp. venerealis in cattle at Brejo Paraibano microregion, Northeastern Brazil, identifying the possible risk factors associated with T. foetus infection. For the study of T. foetus, 349 samples of cervico-vaginal mucus and smegma were collected in beef and dairy cattle (290 females and 59 males) in 31 farms and for C. fetus subsp. venerealis, 273 samples of cervico-vaginal mucus from dairy cows from 19 farms were collected. Polymerase Chain Reaction was used to identify the DNA of the agents and culture with Modified Diamond medium for isolation of T. foetus. For analysis of risk factors associated with Tritrichomonas foetus infection in bovine species, univariate analysis of the variables of interest was performed by the Pearson chi-squared test, or Fisher's exact test, when necessary. Subsequently, logistic regression was performed considering PCR (positive or negative) as the dependent variable for trichomoniasis. Thematic maps were prepared with prevalence distributions in the studied area. The prevalence of C. fetus subsp. venerealis infection in cows was 7.7% (21/273) (CI 95% 4.8% – 11.5%), and 31.6% (6/19) of the farms showed at least one positive animal. The prevalence of T. foetus infection was 3.7% (13/349) (CI 95%, 2.1 - 6.4%). Regarding the gender, a prevalence of T. foetus infection of 4.5% (females) and 0.0% (males) was observed. The percentage of farms that had at least one positive animal for T. foetus was 19.3% (6/31). In culture, no samples were positive for T. foetus. The risk factor associated with T. foetus infection identified in this study was contact of females with bulls of other farms (OR 5.9; CI 1.5 - 22.4). This is the first report of C. fetus subsp. venerealis infection in dairy cows in this region of Brazil and the first in Brejo Paraibano microregion that analyzed T. foetus infection in cattle. To reduce risk of infection, it is recommended to adopt an artificial insemination program on the farms with negative bull semen, as well as a vaccination program against Bovine Genital Campylobacteriosis to stimulate immunity to reduce the occurrence of infection and possible reproductive problems. Contact of females with males of other farms should be avoided, especially when the health status of these bulls is not known regarding these two infections. / Objetivou-se com este estudo determinar a situação epidemiológica das infecções por Tritrichomonas foetus e Campylobacter fetus subsp. venerealis em bovinos na microrregião do Brejo Paraibano, região Nordeste do Brasil, identificando os possíveis fatores de risco associados à infecção por T. foetus. Para pesquisa de T. foetus foram coletadas 349 amostras de muco cérvico vaginal e esmegma em bovinos de corte e leite (290 fêmeas e 59 machos) em 31 propriedades e para pesquisa de C. fetus subsp. venerealis foram coletadas 273 amostras de muco cérvico vaginal de vacas leiteiras procedentes de 19 propriedades. Para a pesquisa do DNA dos agentes utilizou-se a Reação em Cadeia da Polimerase e cultivo com meio Diamond Modificado para isolamento de T. foetus. Para análise dos fatores de risco associados à infecção por Tritrichomonas foetus na espécie bovina, foi realizada uma análise univariada das variáveis de interesse pelo teste qui-quadrado de Pearson, ou Exato de Fisher, quando necessário. Posteriormente foi realizada uma regressão logística considerando como variável dependente para Tricomonose a PCR (positivo ou negativo). Foram elaborados mapas temáticos com as distribuições das prevalências na área estudada. A prevalência da infecção por C. fetus subsp. venerealis em vacas foi 7,7% (21/273) (I.C. 95%; 4,8 - 11,5%) e 31,6% (6/19) das propriedades apresentaram ao menos um animal positivo. A prevalência da infecção por T. foetus foi 3,7% (13/349) (I.C. 95%; 2,1 – 6,4%). Em relação ao sexo, observou-se uma prevalência da infecção por T. foetus de 4,5% (fêmeas) e 0,0% (machos). A porcentagem de propriedades que apresentaram ao menos um animal positivo para T. foetus foi de 19,3% (6/31). No cultivo, nenhuma amostra foi positiva para T. foetus. O fator de risco associado à infecção por T. foetus identificado neste estudo foi contato de fêmeas com touros de outras propriedades (OR 5,9; I.C. 1,5 – 22,4). Este é o primeiro relato da infecção por C. fetus subsp. venerealis em vacas leiteiras nesta região do Brasil e o primeiro na microrregião do Brejo Paraibano a analisar a infecção por T. foetus em bovinos. Para diminuir os riscos de infecção, recomenda-se a adoção de um programa de inseminação artificial nas propriedades com sêmen de touros negativos, bem como um programa de vacinação contra a Campilobacteriose Genital Bovina para estimular a imunidade, com o intuito de reduzir a ocorrência da infecção e possíveis problemas reprodutivos. O contato das fêmeas com machos de outras propriedades deve ser evitado, principalmente quando não se conhece o status sanitário desses touros em relação a estas duas infecções.

The pathogen Campylobacter fetus (CF) is classified into two subspecies, Campylobacter fetus subspecies fetus (CFF) and Campylobacter fetus subspecies venerealis (CFV). Even though CFF and CFV are genetically closely related, they exhibit differences in their host adaptation; CFF inhabits the gastrointestinal tract of both humans and several animal species, while classical CFV is specific to the bovine genital tract and is of particular concern with respect to international bovine trade regulation. Traditionally, differentiation between the two subspecies has been achieved using a limited number of biochemical tests but more rapid and definitive genetic methods of discrimination are desired. A recent study suggested that the presence of a genomic island only in CFV could discriminate between the two sub- species but this hypothesis could not be confirmed on a collection of isolates originating in Canada. To identify alternative gene targets that would support accurate subspecies discrimination, this study has applied several approaches including suppression subtractive hybridization and whole genome sequencing supplemented with optical mapping. A subtractive hybridization screen, using a well-characterized CFV isolate recovered during routine screening of bulls in an Artificial Insemination center in western Canada and that lacked much of the genomic island and a typical Canadian CFF isolate, yielded 50 clones; characterization of these clones by hybridization screening against selected CF isolates and by nucleotide sequence BLAST analysis identified three potentially CFV-specific clones that contained inserts originating from a second genomic island. Further screening using a larger CF sample set found that only Clone #35 was truly CFV-specific. Optical maps (NcoI digest) of the Canadian CFF and CFV isolates used for the subtractive hybridization showed that certain regions of these genomes were quite distinct from those of two reference strains. Whole genome sequencing of these two isolates identified two target genes (PICFV5_ORF548 and CFF_Feature #3) that appear to be selectively retained in the two subspecies. Screening of a collection of CF isolates by PCRs targeting these three loci (SSH_Clone #35, PICFV5_ORF548 and CFF_Feature #3) supported their use for subspecies discrimination. This work demonstrates the complex genomic diversity associated with these CF subtypes and the challenge posed by their discrimination using limited genetic loci.

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

Campylobacter fetus subsp. venerealis ist der Erreger der bovinen genitalen Campylobacteriose, einer anzeigepflichtigen Tierseuche. Er hat sein natürliches Reservoir im Präputialsack klinisch gesunder Bullen und ist in der Lage, Aborte zu verursachen (enzooti-scher Abort). Davon abzutrennen sind Infektionen mit C. fetus subsp. fetus, welcher natürlicherweise im Intestinaltrakt des Rindes auftritt und ebenfalls Aborte auszulösen vermag (spo-radischer Abort). Während C. fetus subsp. venerealis einen ausgeprägten Tropismus für die Genitalschleimhaut des Rindes aufweist, handelt es sich bei der Subspezies fetus um einen Zoonoseerreger, der beim Menschen schwerwiegende Erkrankungen mit zumeist septikämischen Charakter verursachen kann. Die Übertragung von C. fetus subsp. venerealis erfolgt hauptsächlich durch den natürlichen Deckakt, es besteht aber auch die Gefahr der Verbreitung durch künstliche Besamung, da klinisch gesunde Bullen den Erreger im Samen enthalten können. Die Unterschiede in der Epidemiologie und die klinische Bedeutung der beiden C. fetus-Subspezies erfordern eine exakte Identifizierung und Differenzierung. Der erste Teil der Untersuchung befasst sich daher mit den Möglichkeiten des molekularen Erregernachweises. Im zweiten Teil wurden 50 Isolate aus den vergangenen fünf Jahren mit molekularen Methoden auf ihre genetische Ähnlichkeit untersucht. Eine Abgrenzung durch traditionelle mikrobiologische Methoden ist sehr problematisch, da sie im Wesentlichen auf nur zwei Reaktionen beruht: die Glycin-Toleranz und die Na-Selenit-Reduktion, wobei neueste Untersuchungen unter standardisierten Bedingungen nur die Glycin-Toleranz als eindeutig charakterische Reaktion für beide Subspezies übrigließe. Aus diesem Grunde wurden PCR-Untersuchungen zur Identifizierung und Differenzierung beider C. fetus-Subspezies eingeführt. In einem ersten Schritt wurde Campylobacter fetus spezifisch nachgewiesen. Danach erfolgte die Differenzierung der Subspezie durch eine weitere PCR, in der nur bei Vorliegen der DNS von C. fetus subsp. venerealis ein Amplikon erhalten wurde. Insgesamt wurden 103 C. fetus-Isolate untersucht, einschließlich der Typenstämme von C. fetus subsp. fetus und C. fetus subsp. venerealis. Auf Grund der Ergebnisse des Gly-cintoleranztests konnten 81 C. fetus subsp. venerealis (Glycin intolerant) und 22 C. fetus subsp. fetus (Glycin tolerant) identifiziert werden. Die Ergebnisse des Glycintoleranztests und der PCR stimmten bei allen 103 C. fetus Isolaten überein. Versuche zum Direktnachweis von C. fetus subsp. venerealis aus Bullensperma durch Anwendung von fünf unterschiedlichen DNS-Extraktionsmethoden waren in ihren Ergebnissen hinsichtlich ihrer Sensitivität nicht zufrieden stellend (104 KbE/ml). Daraus ergibt sich zwingend, dass die Kultivierung des Erregers vor seiner phäno- und genotypischen Charakterisierung weiterhin unverzichtbar bleibt. Durch Untersuchungen mittels PFGE wurde gezeigt, dass die Campylobacter fetus subsp. venerealis-Population, die in Deutschland vorkommt, genetisch nicht einheitlich ist. Eine strenge Gruppierung nach geografischen Regionen war nicht möglich. Innerhalb größerer Gruppen genetisch ähnlicher Stämme fielen Isolate mit identischen Mustern auf, was auf gemeinsame Infektionsquellen hindeutet. Die ERIC-PCR sollte in der vorliegenden Arbeit als zweite Methode der Analyse des Gesamtgenoms zur weiteren Stützung der Ergebnisse der Makrorestriktionsanalyse beitragen. Die Analysen mittels ERIC-PCR machte ein hohes Maß an Heterogenität innerhalb der einzelnen Spezies sichtbar, lässt jedoch keine Assoziation zwischen Bandenprofilen und einer Zuordnung zum Krankheitsbild oder zur geographischen Herkunft zu.

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