The overall objective of this thesis work was to develop new insights into the etiology of scleroderma, a human systemic autoimmune disease, by analyzing the autoantibodies to centrosome antigens that develop during the disease. Centrosomes are perinuclear organelles that form microtubule arrays, including mitotic spindles that ensure the faithful segregation of chromosomes during mitosis. These studies used a novel methodology to determine the prevalence of anti-centrosome autoantibodies in patients with scleroderma. Recombinant centrosome antigens were used to determine the antigenic specificity of anti-centrosome antibody subsets by immunoblotting. Centrosome marker antibodies were used in indirect immunofluorescence assays to distinguish centrosomes within the polymorphic staining pattern frequently given by scleroderma sera. We found that 43% of patients are autoreactive to centrosomes, a prevalence higher than has been reported for any other scleroderma autoantigen. Half of the centrosome-positive patients also had autoantibodies against other antigens used in scleroderma diagnosis. However, in the remaining half of these patients, anti-centrosome antibodies represented the sole class of autoantibodies that was detectable. Anti-centrosome antibodies were detected in only a small percentage of normal individuals and patients with other connective tissue diseases. These data suggest that anti-centrosome autoantibodies may represent a new diagnostic tool in scleroderma. Upon examination of anti-centrosome autoantibody development in an animal model, it appeared that this autoantibody specificity may develop in mice as a consequence of an infection. An infectious agent was isolated by plaque-formation from carrier mice. Further characterization of the infectious agent was undertaken to obtain information on its physical, morphological and cytopathological properties. The infectious agent was identified by sequence and unique antigenic properties to be homologous to the pig pathogen Mycoplasma hyorhinis. When reintroduced into naive mice, the murine mycoplasma triggered anti-centrosome autoantibody development. While anti-centrosome autoantibodies of IgM isotype are part of the repertoire of naive unimmunized mice, mycoplasma infection specifically triggered the development of anti-centrosome IgG. Moreover, centrosome autoreactivity was prevented by antibiotic treatment. The autoantibody response evolved to recruit additional specificities, having IgM isotypes, reactive to endoplasmic reticulum-associated autoantigens.

A suinocultura está amplamente difundida no Brasil, o quarto maior produtor mundial de carne suína. Seu efetivo está concentrado em maior parte na região Sul, com 48,8% dos suínos. Porém, a intensificação e o confinamento das criações expõem os animais a agentes patogênicos, como o Mycoplasma hyopneumoniae (Mhyo), causador da Pneumonia Enzoótica Suína (PES). A doença é caracterizada pela ocorrência de tosse seca não produtiva e lesões pulmonares de consolidação, denominadas hepatização. A vacinação de leitões é uma importante ferramenta empregada no controle da PES. Contudo, é relatado que a imunidade passiva adquirida pelos leitões através do colostro das matrizes suínas pode interferir na sua resposta à vacinação contra o Mhyo. Desta forma, este trabalho teve como objetivo avaliar a influência da imunidade materna na resposta à imunização de leitões contra Mhyo ao desmame. Dez matrizes foram divididas em dois grupos baseado na Razão S/P de um teste ELISA comercial , um com baixo nível de anticorpos (Ac) anti-Mhyo (BAc, Razão S/P <0,75) e outro com alto nível (AAc, Razão S/P ≥0,75). De cada fêmea, dois leitões eram controles (contr) e nove vacinados (vac) contra Mhyo. Estes grupos e tratamentos foram comparados entre si quanto a parâmetros de imunidade humoral, celular e comprometimento pulmonar ao abate. Após a ingestão de colostro, os leitões das fêmeas AAc mantiveram os maiores níveis de Ac, até os 56 dias de idade. Quando avaliados através do teste ELISA, dos 13 aos 99 dias depois de vacinados os leitões BAc-vac apresentaram níveis de Ac mais estáveis, com um aumento significativo aos 113 dias pós-vacinação. Os parâmetros celulares não diferiram entre grupos e tratamentos, com exceção dos linfócitos T CD4+CD8+, com menores percentuais entre os vacinados. Independente do grupo, os vacinados tiveram menor comprometimento pulmonar, porém os BAc-vac tiveram menos lesões que os AAc-vac. Os resultados demonstraram que a vacinação dos leitões contra Mhyo ao desmame os protege contra o agente, levando ao menor comprometimento pulmonar, principalmente quando a imunização ocorre na presença de baixos níveis de Ac passivos. / Swine production is widespread in Brazil, being the fourth largest producer of pork meat. Swinesare concentrated in the Southern region, comprising 48.8%of pig population. However, due to confinement system, pigs are exposed to pathogens such as the bacteria Mycoplasma hyopneumoniae (Mhyo), responsible for swine enzootic pneumonia (SEP). The disease is characterized by a dry, nonproductive cough, and macroscopic areas of consolidation in the lung. The piglet vaccination is an important practice to SEP control. However, it is reported that passive immunity acquired by the piglets through the colostrum of sows may affect their response to vaccination against Mhyo. The objective of this study was to evaluate the influence of maternal immunity in the pig vaccination against Mhyo after weaning. Ten sows were divide in two groups according with the ELISA’s S/P Ratio, with low (LAb, S/P Ratio <0.75) or high (HAb, S/P Ratio ≥0.75) level of antibodies (Ab). From each sow, two piglets were controls (contr) and nine vaccinated (vac) against Mhyo. Piglets' humoral and cellular immunity and pulmonary lesions were compared between the treatments and groups of sows.The Ab level after colostrum intake was higher in piglets from HAb group of sows until 56 days of age. When evaluated by ELISA, LAb-vac piglets showed a more stable Ab levels from 13 to 99 days post-vaccination, with a significant increase at 113 days post-vaccination. No differences were detected between groups and treatments according with cellular parameters, except T CD4+CD8+ lymphocytes percentages, that were lower in vaccinated piglets. Regardless of the group, vaccinated pigs had lower pulmonary lesions, but the LAb-vac piglets had less damage than the HAb-vac. These results demonstrated that piglet vaccination against Mhyo at weaning protects them against the pathogen and provides lower pulmonary lesions, especially when the immunization occurs in the presence of low levels of maternal Ab.

Orientador: Karin Werther / Banca: Aramis Augusto Pinto / Banca: Nilce Maria Soares Queiroz Gama / Resumo: Aves selvagens podem ser consideradas portadores/carreadores de agentes etiológicos patogênicos para galinhas de produção. O objetivo desse trabalho foi verificar, em aves selvagens, a presença de anticorpo contra: Mycoplasma gallisepticum (MG), Mycoplasma sinoviae (MS), Salmonella Pullorum (SP), Salmonella Gallinarum (SG), Vírus da Doença de Newcastle e Vírus da Bronquite Infecciosa; bem como a presença de Salmonella sp. Foram utilizadas 82 aves selvagens, e todas foram negativas na detecção de anticorpo anti-vírus da Doença de Newcastle e Bronquite Infecciosa, usando as técnicas de Inibição de Hemaglutinação e Soroneutralização, respectivamente. Para o diagnóstico de anticorpo anti Mycoplasma (MG e MS) foi utilizado inicialmente a técnica de Soroaglutinação Rápida em Placa, sendo sete aves positivas para MS e duas para MG. Amostras biológicas dessas aves foram posteriormente submetidas a técnicas de HI e/ou PCR, sendo todas negativas. Quanto ao diagnóstico de Salmonella sp., foi isolado Salmonella Muenchen da cultura de suabe de cloaca de uma curicaca; de uma seriema, foi isolado Salmonella Muenchen e Salmonella Saintpaul da cultura de órgãos e conteúdo intestinal, respectivamente; e do conteúdo intestinal de uma pomba foi isolado Salmonella Enteritidis. Apenas com os resultados dessa pesquisa, não foi possível concluir que as aves selvagens possam representar um risco sanitário para a avicultura. São necessários mais estudos envolvendo um número maior de aves e locais para elucidar o real potencial de transmissão e portador são das aves selvagens para os respectivos agentes pesquisados. / Abstract: Wild birds can be considered carrier/transmitter of patogenic etiologic agents for chickens. The objective of this work was to verify in wild birds the presence of antibody Mycoplasma gallisepticum (MG), Mycoplasma sinoviae (MS), Salmonella Pullorum (SP), Salmonella Gallinarum (SG), Newcastle Disease Virus, and Infectious Bronchitis Virus; as well as the presence of Salmonella sp. Eighty-two wild birds were sampled, and all was negative for antibody anti-virus Newcastle Disease and Infectious Bronchitis, using the techniques of Hemagglutination Inhibition and Seroneutralization, respectively. For the detection of antibodies anti Mycoplasma (MG e MS) were used initially the Fast Serum Agglutination on Plate method, seven birds were positive for MS and two for MG. Subsequently the positive sera and/or traqueal swab were submitted for HI and/or PCR techniques being all exams negative. Regarding the diagnosis of Salmonella sp., Salmonella Muenchen was isolated from a culture of cloacal swab of a buff-necked Ibis; from a red-legged seriema, Salmonella Muenchen and Salmonella Saintpaul were isolated from culture of organs and intestinal content, respectively; and Salmonella Enteritidis was isolated of intestinal content from a dove. It was not possible to conclude that the wild birds can represent a sanitary risk for aviculture considering only these results. More studies are necessary to elucidate the real potential of transmission and carrier of wild birds for the researched agents. / Mestre

A suinocultura está amplamente difundida no Brasil, o quarto maior produtor mundial de carne suína. Seu efetivo está concentrado em maior parte na região Sul, com 48,8% dos suínos. Porém, a intensificação e o confinamento das criações expõem os animais a agentes patogênicos, como o Mycoplasma hyopneumoniae (Mhyo), causador da Pneumonia Enzoótica Suína (PES). A doença é caracterizada pela ocorrência de tosse seca não produtiva e lesões pulmonares de consolidação, denominadas hepatização. A vacinação de leitões é uma importante ferramenta empregada no controle da PES. Contudo, é relatado que a imunidade passiva adquirida pelos leitões através do colostro das matrizes suínas pode interferir na sua resposta à vacinação contra o Mhyo. Desta forma, este trabalho teve como objetivo avaliar a influência da imunidade materna na resposta à imunização de leitões contra Mhyo ao desmame. Dez matrizes foram divididas em dois grupos baseado na Razão S/P de um teste ELISA comercial , um com baixo nível de anticorpos (Ac) anti-Mhyo (BAc, Razão S/P <0,75) e outro com alto nível (AAc, Razão S/P ≥0,75). De cada fêmea, dois leitões eram controles (contr) e nove vacinados (vac) contra Mhyo. Estes grupos e tratamentos foram comparados entre si quanto a parâmetros de imunidade humoral, celular e comprometimento pulmonar ao abate. Após a ingestão de colostro, os leitões das fêmeas AAc mantiveram os maiores níveis de Ac, até os 56 dias de idade. Quando avaliados através do teste ELISA, dos 13 aos 99 dias depois de vacinados os leitões BAc-vac apresentaram níveis de Ac mais estáveis, com um aumento significativo aos 113 dias pós-vacinação. Os parâmetros celulares não diferiram entre grupos e tratamentos, com exceção dos linfócitos T CD4+CD8+, com menores percentuais entre os vacinados. Independente do grupo, os vacinados tiveram menor comprometimento pulmonar, porém os BAc-vac tiveram menos lesões que os AAc-vac. Os resultados demonstraram que a vacinação dos leitões contra Mhyo ao desmame os protege contra o agente, levando ao menor comprometimento pulmonar, principalmente quando a imunização ocorre na presença de baixos níveis de Ac passivos. / Swine production is widespread in Brazil, being the fourth largest producer of pork meat. Swinesare concentrated in the Southern region, comprising 48.8%of pig population. However, due to confinement system, pigs are exposed to pathogens such as the bacteria Mycoplasma hyopneumoniae (Mhyo), responsible for swine enzootic pneumonia (SEP). The disease is characterized by a dry, nonproductive cough, and macroscopic areas of consolidation in the lung. The piglet vaccination is an important practice to SEP control. However, it is reported that passive immunity acquired by the piglets through the colostrum of sows may affect their response to vaccination against Mhyo. The objective of this study was to evaluate the influence of maternal immunity in the pig vaccination against Mhyo after weaning. Ten sows were divide in two groups according with the ELISA’s S/P Ratio, with low (LAb, S/P Ratio <0.75) or high (HAb, S/P Ratio ≥0.75) level of antibodies (Ab). From each sow, two piglets were controls (contr) and nine vaccinated (vac) against Mhyo. Piglets' humoral and cellular immunity and pulmonary lesions were compared between the treatments and groups of sows.The Ab level after colostrum intake was higher in piglets from HAb group of sows until 56 days of age. When evaluated by ELISA, LAb-vac piglets showed a more stable Ab levels from 13 to 99 days post-vaccination, with a significant increase at 113 days post-vaccination. No differences were detected between groups and treatments according with cellular parameters, except T CD4+CD8+ lymphocytes percentages, that were lower in vaccinated piglets. Regardless of the group, vaccinated pigs had lower pulmonary lesions, but the LAb-vac piglets had less damage than the HAb-vac. These results demonstrated that piglet vaccination against Mhyo at weaning protects them against the pathogen and provides lower pulmonary lesions, especially when the immunization occurs in the presence of low levels of maternal Ab.

A suinocultura está amplamente difundida no Brasil, o quarto maior produtor mundial de carne suína. Seu efetivo está concentrado em maior parte na região Sul, com 48,8% dos suínos. Porém, a intensificação e o confinamento das criações expõem os animais a agentes patogênicos, como o Mycoplasma hyopneumoniae (Mhyo), causador da Pneumonia Enzoótica Suína (PES). A doença é caracterizada pela ocorrência de tosse seca não produtiva e lesões pulmonares de consolidação, denominadas hepatização. A vacinação de leitões é uma importante ferramenta empregada no controle da PES. Contudo, é relatado que a imunidade passiva adquirida pelos leitões através do colostro das matrizes suínas pode interferir na sua resposta à vacinação contra o Mhyo. Desta forma, este trabalho teve como objetivo avaliar a influência da imunidade materna na resposta à imunização de leitões contra Mhyo ao desmame. Dez matrizes foram divididas em dois grupos baseado na Razão S/P de um teste ELISA comercial , um com baixo nível de anticorpos (Ac) anti-Mhyo (BAc, Razão S/P <0,75) e outro com alto nível (AAc, Razão S/P ≥0,75). De cada fêmea, dois leitões eram controles (contr) e nove vacinados (vac) contra Mhyo. Estes grupos e tratamentos foram comparados entre si quanto a parâmetros de imunidade humoral, celular e comprometimento pulmonar ao abate. Após a ingestão de colostro, os leitões das fêmeas AAc mantiveram os maiores níveis de Ac, até os 56 dias de idade. Quando avaliados através do teste ELISA, dos 13 aos 99 dias depois de vacinados os leitões BAc-vac apresentaram níveis de Ac mais estáveis, com um aumento significativo aos 113 dias pós-vacinação. Os parâmetros celulares não diferiram entre grupos e tratamentos, com exceção dos linfócitos T CD4+CD8+, com menores percentuais entre os vacinados. Independente do grupo, os vacinados tiveram menor comprometimento pulmonar, porém os BAc-vac tiveram menos lesões que os AAc-vac. Os resultados demonstraram que a vacinação dos leitões contra Mhyo ao desmame os protege contra o agente, levando ao menor comprometimento pulmonar, principalmente quando a imunização ocorre na presença de baixos níveis de Ac passivos. / Swine production is widespread in Brazil, being the fourth largest producer of pork meat. Swinesare concentrated in the Southern region, comprising 48.8%of pig population. However, due to confinement system, pigs are exposed to pathogens such as the bacteria Mycoplasma hyopneumoniae (Mhyo), responsible for swine enzootic pneumonia (SEP). The disease is characterized by a dry, nonproductive cough, and macroscopic areas of consolidation in the lung. The piglet vaccination is an important practice to SEP control. However, it is reported that passive immunity acquired by the piglets through the colostrum of sows may affect their response to vaccination against Mhyo. The objective of this study was to evaluate the influence of maternal immunity in the pig vaccination against Mhyo after weaning. Ten sows were divide in two groups according with the ELISA’s S/P Ratio, with low (LAb, S/P Ratio <0.75) or high (HAb, S/P Ratio ≥0.75) level of antibodies (Ab). From each sow, two piglets were controls (contr) and nine vaccinated (vac) against Mhyo. Piglets' humoral and cellular immunity and pulmonary lesions were compared between the treatments and groups of sows.The Ab level after colostrum intake was higher in piglets from HAb group of sows until 56 days of age. When evaluated by ELISA, LAb-vac piglets showed a more stable Ab levels from 13 to 99 days post-vaccination, with a significant increase at 113 days post-vaccination. No differences were detected between groups and treatments according with cellular parameters, except T CD4+CD8+ lymphocytes percentages, that were lower in vaccinated piglets. Regardless of the group, vaccinated pigs had lower pulmonary lesions, but the LAb-vac piglets had less damage than the HAb-vac. These results demonstrated that piglet vaccination against Mhyo at weaning protects them against the pathogen and provides lower pulmonary lesions, especially when the immunization occurs in the presence of low levels of maternal Ab.

La grippe porcine est une infection enzootique touchant 50% du cheptel français. Elle passe parfois inaperçue, mais peut également induire une forte morbidité au sein des lots d’animaux touchés, entraînant une baisse des performances zootechniques et des pertes économiques importantes. La sévérité de l’infection à virus influenza A chez le porc peut dépendre de divers facteurs, comme les virus eux-mêmes, les pratiques d’élevage, le statut immunitaire des animaux, les infections concomitantes par d’autres pathogènes respiratoires, etc. De la même manière, diverses formes épidémiologiques de la grippe existent en élevage. Ainsi, des infections peuvent se répéter à un âge déterminé, sur toutes les bandes successives d’un élevage, notamment chez des jeunes présentant une immunité passive. Afin de mieux comprendre cette diversité clinique et épidémiologique de la grippe porcine, et aider à l’élaboration de stratégies d’intervention adéquates pour le contrôle de la maladie, nous avons cherché à apporter de nouvelles connaissances quant à certains facteurs pouvant favoriser l’exacerbation du syndrome grippal et/ou son caractère récurrent, et plus généralement aux mécanismes sous-jacents à la pathogenèse des virus influenza A chez le porc, en relation avec les réponses de l’hôte infecté. Nous avons d’abord comparé, suite à inoculations expérimentales de porcs exempts d’organismes pathogènes spécifiés, la pathogénicité des deux virus influenza porcins les plus fréquemment rencontrés chez le porc en France, l’un du lignage européen « avian-like swine H1N1 » (H1avN1), l’autre du lignage européen « human-like reassortant swine H1N2 » (H1huN2), seuls ou en association avec Mycoplasma hyopneumoniae (Mhp), autre pathogène respiratoire répandu en élevage. Nous avons montré que l’infection H1huN2 induit une pathologie plus marquée que l’infection H1avN1, et que la pré-infection des porcs par Mhp induit une exacerbation de l’infection H1avN1, mais pas H1huN2. Nous avons utilisé le modèle de co-infection Mhp/H1avN1 pour évaluer deux approches alternatives qui permettraient de diminuer l’impact des infections grippales et de leurs complications : l’apport de composés aux propriétés antioxydantes via l’alimentation ; et la restriction alimentaire de courte durée. Dans ces deux cas nous avons montré des effets bénéfiques sur les paramètres zootechniques pendant les jours suivant l’infection grippale. Ce travail a également apporté de nouvelles connaissances quant aux modifications des marqueurs plasmatiques de stress oxydant, ainsi que sur les modifications métaboliques faisant suite à la co-infection Mhp/H1avN1. La sévérité des manifestations cliniques de la grippe étant liée à la qualité de la réponse immunitaire mise en place chez l’hôte infecté, nous avons entrepris d‘étudier les réponses immunitaires du porc touché par la grippe et d’évaluer l’impact de facteurs tels que la présence de Mhp ou d’anticorps d’origine maternelle sur ces réponses. Nous avons ainsi montré que l’infection virale induit une inflammation et une réponse interféron. La pré-infection par Mhp exercerait un effet additif sur cette inflammation et pourrait favoriser la persistance du virus dans le poumon. Nous avons également montré que la présence d’immunité passive protège cliniquement le porcelet mais n’empêche pas l’excrétion du virus, retarde la réponse lymphocytaire T et inhibe la réponse humorale post-infectieuse. Malgré la réponse immunitaire humorale défaillante, les animaux étaient totalement protégés d’une seconde infection homologue lorsque les anticorps maternels avaient disparus. Ces travaux ont permis d’apporter de nouvelles connaissances sur les facteurs influençant l’infection grippale en élevage porcin ainsi que sur les mécanismes sous-jacents, ce qui est une prérequis pour l’amélioration des mesures de lutte et de maitrise de la maladie. Ils permettent, d’envisager d’améliorer la santé des animaux en agissant sur leur régime alimentaire. / Swine influenza is an enzootic infection affecting 50% of the French livestock. The infection can be unnoticed but can also induce high morbidity among batches of affected animals, resulting in lower production performance and significant economic losses. The severity of influenza A virus in pig is influenced by many factors such as the virus strain, husbandry practices, the immune status of animals, concomitant infections with other respiratory pathogens, etc. In the same way, various epidemiological forms of influenza exist in farms. Thus, infections can be repeated in all successive batches within a farm, especially among young animals with passive immunity. In order to better understand the clinical and epidemiological diversity of the swine flu, and help develop appropriate strategies to control the disease, we tried to bring new knowledge about factors that promote the exacerbation of the flu syndrome and/or its recurrence, and more generally to give new information about the mechanisms underlying the pathogenesis of influenza viruses in pigs, in relation to the response of the infected host. Firstly, we compared, through experimental infections of specific pathogen free pigs, the pathogenicity of the two swine influenza viruses mostly detected in pigs in France, i.e. one from the European “avian-like swine H1N1” lineage (H1avN1) and the other one from the European “human-like reassortant swine H1N2” lineage (H1huN2), each one alone or in co-infection with Mycoplasma hyopneumoniae (Mhp), another respiratory pathogen widespread in French farms. We showed that the H1huN2 infection induced a more marked pathology than the H1avN1 infection, and that Mhp pre-infection induced the exacerbation of the H1avN1, but not the H1huN2, infection. Then, we used the Mhp/H1avN1 co-infection model to evaluate alternative approaches that could reduce the impact of influenza infections and their complications: firstly, a supply of compounds with antioxidant properties in food; and secondly, a feed restriction of short duration. In both cases, we showed beneficial effects on zootechnical parameters the days following influenza infection. This work has also brought new knowledge on modulation of oxidative stress markers in plasma, as well as metabolic changes following the co-infection with Mhp and H1avN1 in pigs. The severity of flu clinical manifestations being related, among other, to the quality of the immune responses developed by the infected host, we studied these responses in pigs experimentally infected by H1avN1 and assessed the impact of factors such as the presence of Mhp or maternal derived antibodies on these responses. We showed that the viral infection induced inflammation and interferon response. The Mhp pre-infection exerted an additive effect on inflammation of lung tissue and may promote the virus persistence in the lung. Finally, we have shown that the presence of maternally-derived immunity protected the piglets clinically but did not prevent viral shedding, delayed the T cell response and strongly inhibited the post-infectious humoral response. However, despite the failed humoral immune response, animals were completely protected from a second infection occurring when maternal antibodies had disappeared. Therefore, this work have brought new knowledge on factors influencing influenza infection in pig as well as the underlying mechanisms, which is a prerequisite for improving disease control. They allow, between-other, to consider improving the health and welfare of animals by acting on their diet.

Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by Chlamydia trachomatis or Neisseria gonorrhoeae, bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, Mycoplasma genitalium was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of M. genitalium infection became more feasible. The aim in paper I was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time Mycoplasma genitalium adhesin protein (MgPa) gene PCR) for detection of M. genitalium. The study also determined the prevalence of M. genitalium. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of M. genitalium and 213 of these specimens were used in the PCR comparative study. The prevalence of M. genitalium infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, M. genitalium DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of M. genitalium in urogenital specimens from men and women. The aim in paper II was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of Mycoplasma genitalium by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. M. genitalium was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of M. genitalium positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.

Tesis por compendio / [ES] En la actualidad, la avicultura es un pilar económico para diversos países a nivel mundial y sus productos, una pieza clave en la alimentación de muchos consumidores. El constante crecimiento de la industria y una mayor exigencia del consumidor en seguridad y calidad del producto ha llevado al sector avícola a enfrentarse a importantes retos. Desde el punto de vista de salud pública, encontramos la transmisión a través de los alimentos de origen avícola de varios patógenos zoonóticos causantes de infección gastrointestinal en humanos y la emergencia de microorganismos resistentes a antibióticos. El primer objetivo de esta Tesis Doctoral fue investigar la dinámica de las resistencias de Salmonella spp. en 3 orientaciones productivas (pollos, pavos y gallinas ponedoras) y evaluar los patrones de multiresistencia a los antibióticos utilizados actualmente en medicina veterinaria y terapia humana. Los resultados del estudio sugirieron que la reducción en el uso de antibióticos comienza a verse reflejada en la reducción del número de multirresistencias encontradas en las aves, mostrando una tendencia decreciente en las resistencias, especialmente en ponedoras desde que se implementaron los planes de reducción, junto a las mejores prácticas de bioseguridad de los avicultores. Sin embargo, el nivel de resistencias detectado en otras orientaciones productivas sugiere la necesidad de seguir trabajando en la reducción del uso de antibióticos en las aves para lograr una disminución significativa en las resistencias. La aparición de los microorganismos resistentes y las restricciones de uso de antibióticos han llevado a la búsqueda de soluciones alternativas a los antimicrobianos, como los compuestos derivados de productos naturales para controlar las infecciones en avicultura. El segundo objetivo fue evaluar la interacción del compuesto natural alicina in vitro junto a 14 antibióticos frente a algunos de los serotipos más relevantes de Salmonella spp. Los resultados obtenidos sugirieron que este compuesto junto a la ciprofloxacina y la gentamicina presentaban un efecto sinérgico estadísticamente significativo con reducción de las resistencias. Así mismo, también se observó una reducción estadísticamente significativa en ciertos serotipos de Salmonella. Dada la importancia en salud pública de Campylobacter spp., se estableció un criterio de higiene de proceso ¿1000 UFC/g en matadero para la vigilancia de Campylobacter spp. en canales de pollos de engorde a nivel europeo. En este contexto, el tercer objetivo fue investigar la presencia de este microorganismo a nivel de campo y reportar nuevos datos cuantitativos mediante recuentos en heces procedentes de granjas de pollos en edades cercanas a sacrificio durante 12 meses. Este estudio demostró alta presencia de Campylobacter spp. en lotes de pollos cercanos a edad de sacrificio, aunque se requieren más estudios para verificar si posteriormente se traducen en recuentos altos a nivel de matadero. Por otro lado, se encontraron diferencias estadísticamente significativas en la presencia de Campylobacter spp. durante los diferentes meses del año (junio a diciembre) coincidiendo con las estaciones de verano y otoño. Desde el marco de la sanidad animal y por su impacto económico, el cuarto y quinto objetivo fueron conocer las prevalencias y seroprevalencias de 2 microorganismos aviares, M. synoviae y el virus de la Bronquitis Infecciosa Aviar en una localización geográfica concreta (Comunidad Valenciana). Con ello, se pretendió evaluar la situación epidemiológica actual y proporcionar información para la toma de decisiones en la instauración de programas de control y estrategias profilácticas. Los hallazgos en ambos estudios demostraron la presencia de estos dos microorganismos en el campo y resaltaron la utilidad de la vigilancia y monitoreo de rutina en las manadas de aves para controlar su presencia y la de sus serotipos emergentes, permitiendo así ajustar los planes de vacunación. / [CA] En l'actualitat, l'avicultura és un pilar econòmic per a diversos països a nivell mundial i els seus productes, una peça clau en l'alimentació de molts consumidors. El constant creixement de la indústria i una major exigència del consumidor en seguretat i qualitat del producte ha portat al sector avícola a enfrontar-se a importants reptes. Des del punt de vista de salut pública, trobem la transmissió a través dels aliments d'origen avícola de diversos patògens zoonòtics causants d'infecció gastrointestinal en humans i l'emergència de microorganismes resistents a antibiòtics. El primer objectiu d'aquesta Tesi Doctoral va ser investigar la dinàmica de les resistències de Salmonella spp. en 3 orientacions productives (pollastres, titots i gallines ponedores) i avaluar els patrons de multiresistència als antibiòtics utilitzats actualment en medicina veterinària i teràpia humana. Els resultats de l'estudi van suggerir que la reducció en l'ús d'antibiòtics comença a veure's reflectida en la reducció del nombre de multiresistències trobades en les aus, mostrant una tendència decreixent en les resistències, especialment en ponedores des que es van implementar els plans de reducció, al costat de les millors pràctiques de bioseguretat dels avicultors. No obstant això, el nivell de resistències detectat en altres orientacions productives suggereix la necessitat de continuar treballant en la reducció de l'ús d'antibiòtics en les aus per a aconseguir una disminució significativa en les resistències. L'aparició dels microorganismes resistents i les restriccions d'ús d'antibiòtics han portat a la cerca de solucions alternatives als antimicrobians, com els compostos derivats de productes naturals per a controlar les infeccions en avicultura. El segon objectiu va ser avaluar la interacció del compost natural alicina in vitro al costat de 14 antibiòtics enfront d'alguns dels serotips més rellevants de Salmonella spp. Els resultats obtinguts van suggerir que aquest compost al costat de la ciprofloxacina i la gentamicina presentaven un efecte sinèrgic estadísticament significatiu amb reducció de les resistències. Així mateix, també es va observar una reducció estadísticament significativa en uns certs serotips de Salmonella. Donada la importància en salut pública de Campylobacter spp., es va establir un criteri d'higiene de procés ¿1000 UFC/g en escorxador per a la vigilància de Campylobacter spp. en canals de pollastres d'engreixament a nivell europeu. En aquest context, el tercer objectiu va ser investigar la presència d'aquest microorganisme a nivell de camp i reportar noves dades quantitatives mitjançant recomptes en femta procedent de granges de pollastres en edats pròximes a sacrifici durant 12 mesos. Aquest estudi va demostrar alta presència de Campylobacter spp. en lots de pollastres pròxims a l¿edat de sacrifici, encara que es requereixen més estudis per a verificar si posteriorment es tradueixen en recomptes alts a nivell d'escorxador. D'altra banda, es van trobar diferències estadísticament significatives en la presència de Campylobacter spp. durant els diferents mesos de l'any (juny a desembre) coincidint amb les estacions d'estiu i tardor. Des del marc de la sanitat animal i pel seu impacte econòmic, el quart i cinqué objectiu van ser conéixer les prevalences i seroprevalences de 2 microorganismes aviaris, M. synoviae i el virus de la Bronquitis Infecciosa Aviària en una localització geogràfica concreta (Comunitat Valenciana). Es va pretendre avaluar la situació epidemiològica actual i proporcionar informació per a la presa de decisions en la instauració de programes de control i estratègies profilàctiques. Les troballes en tots dos estudis van demostrar la presència d'aquests dos microorganismes en el camp i van ressaltar la utilitat de la vigilància i monitoratge de rutina en els ramats d'aus per a controlar la seua presència i la dels seus serotips emergents, permetent així ajustar els plans de vacunació. / [EN] Nowadays, poultry industry is an economic key for many countries worldwide and poultry products are a basic piece in the diet of many consumers. The industry constant growth and the high consumer demands for safety and quality products has imposed new challenges to poultry sector from the point of view of both public and animal health. From food safety point of view, we find the transmission of several zoonotic pathogens through food of poultry origin that cause gastrointestinal infection in humans and the emergence of bacterial resistance to antibiotics. The first objective of this Doctoral Thesis was to investigate the resistance dynamics of Salmonella spp. in 3 productive orientations (broilers, turkeys and laying hens) and evaluate the patterns of multi-resistance to antibiotics currently used in veterinary medicine and human therapy. Results shown in the present study suggest that reduction in the use of antibiotics begins to be reflected in the reduction of the number of multiresistances, since the implementation of resistance reduction measures. However, the level of resistances found in this study suggests the necessity of continuing working on the limitation of the use of antimicrobials in poultry to achieve the reduction in resistances. The appearance of resistant microorganisms and the restrictions on the use of antibiotics have motivated to the research for alternative solutions to antimicrobials, such as compounds derived from natural products to control infections in poultry. The second objective was to evaluate the interaction in vitro between allicin and 14 antibiotics against some of the most relevant serotypes of Salmonella spp. The results obtained suggested that this compound, together with ciprofloxacin and gentamicin, had a statistically significant synergistic effect with a reduction in resistance. Likewise, a statistically significant reduction in certain Salmonella serotypes was also observed. Campylobacter spp. is the most important zoonotic bacteria in public health. For this reason a process hygiene criterion of ¿1000 CFU/g in the slaughterhouse was established to monitor Campylobacter spp. in broiler carcasses at European level. In this context, the third objective was to investigate the presence of this microorganism at field level and to report new quantitative data through counts in feces from broilers farms at ages close to slaughter during 12 months. This study revealed a high presence of Campylobacter spp. in flocks of chickens close to slaughter age, although more studies are required to verify if they subsequently translate into high counts at the slaughterhouse level. On the other hand, statistically significant differences were found in the presence of Campylobacter spp. during the different months of the year (June to December) coinciding with the summer and autumn seasons. The fourth and fifth objectives were focused on two important diseases in animal health that have an economic impact, M. synoviae and the Avian Infectious Bronchitis virus. The aims were to determine the prevalence and seroprevalence of these microorganisms in a specific geographical location (Valencian Community). The current epidemiological situation was evaluated through 2 prevalence and seroprevalence studies to provide useful information for the establishment of control programs and prophylactic strategies. The findings in both studies demonstrate the presence of these two microorganisms in the field and revealed that routine surveillance and monitoring in poultry flocks to control the presence of both microorganisms and their emerging serotypes is useful in order to update vaccination plans. / Este trabajo fue financiado por el Centro de Calidad Avícola y Alimentación Animal de la Comunidad Valenciana (CECAV) y la Asociación Avícola Valenciana (ASAV). Agradecemos a Joaquín Girón (Merck Sharp & Dohme, MSD) y José Luis Balaguer (CEVA) por su asistencia técnica y soporte económico. / Cortés Moñiz, V. (2022). Epidemiología y control de las principales enfermedades avícolas de importancia en sanidad animal y salud pública [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/184126 / Compendio

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Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-15T16:51:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_OcorrenciaMicoplasmasHemotropicos.PDF: 1004565 bytes, checksum: 3c30858f52bad52050cef32a13457f12 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-05-19T12:48:03Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_OcorrenciaMicoplasmasHemotropicos.PDF: 1004565 bytes, checksum: 3c30858f52bad52050cef32a13457f12 (MD5) / Made available in DSpace on 2017-05-19T12:48:03Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_OcorrenciaMicoplasmasHemotropicos.PDF: 1004565 bytes, checksum: 3c30858f52bad52050cef32a13457f12 (MD5) Previous issue date: 2014-02-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Micoplasmas hemotrópicos compreendem um grupo de bactérias que podem causar anemia hemolítica e diversas doenças crônicas em animais. Em felinos três espécies infectantes de hemoplasmas são relatadas: Mycoplasma haemofelis, ‘Candidatus M. haemominutum’ e ‘Candidatus M. turicensis’, sendo o primeiro considerado mais patogênico, enquanto que os caninos são normalmente infectados por pelo menos duas espécies de micoplasmas: M. haemocanis e ‘Candidatus M. haematoparvum’. Com o objetivo de avaliar a ocorrência de micoplasmas hemotrópicos em população canina e felina da cidade de Belém, Estado do Pará, foi analisado através de PCR o material genético oriundo de 299 amostras sanguíneas de animais domiciliados e errantes. Em 8,03% (24/299) das amostras foi amplificado o DNA de micoplasmas hemotrópicos, sendo 1,78% (3/169) de cães e 16,15% (21/130) de gatos infectados. Entre os felinos as maiores taxas de infecção foram encontradas nos animais domiciliados 22,86% (16/70) e em animais machos 29,51% (18/61), sendo a espécie ‘Candidatus M. haemominutum’ 8,46% (11/130) a de maior ocorrência quando comparada a espécie M. haemofelis 6,92% (9/130). A infecção em caninos foi observada somente no grupo de animais errantes, sendo o ‘Candidatus M. haematoparvum’ a única espécie identificada com taxa de 1,78% (3/169). Foi possível avaliar que micoplasmas hemotrópicos circulam nas duas espécies estudadas, felinos domiciliados e machos formaram o grupo com maior risco para a infecção e que o bioagente ‘Candidatus M. haematoparvum’ parece ser o único em circulação na população canina. / Hemotrophic mycoplasmas comprise a group of bacteria that can cause hemolytic anemia and several chronic diseases in animals. In cats three infecting species of hemoplasmas are reported: M. haemofelis, ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’, the first being considered the most pathogenic, whereas the dogs are normally infected by at least two mycoplasma species M. haemocanis and ‘Candidatus M. haematoparvum’. In order to assess the occurrence of hemotrophic mycoplasmas in feline and canine populations of Belém city were analyzed, by PCR, the genetic material from blood samples of 299 domiciled and stray animals. In 8.03% (24/299) of DNA samples of hemotrophic mycoplasmas was amplified, 1.78 % (3 /169) from infected dogs and 16.15% (21 /130) from infected cats. Among the cats the highest rates of infection were found in domiciled animals 22.86% (16/70) and in males 29.51% (18/61), the species ‘Candidatus M. haemominutum’ 8.46 % (11/130) get the higher incidence when compared to species Mycoplasma haemofelis 6.92 % (9/130). The infection in dogs was only identified among stray animals, with the ‘Candidatus M. haematoparvum’ being the only species identified with a rate of 1.78 % (3/169). It was possible to assess that hemotrophic mycoplasmas circulate in both species in the present report, domiciled felines and males formed the group at highest risk for infection and the agent ‘Candidatus M. haematoparvum’ seems to be the only one in circulation in the canine population.