Les maladies diarrhéiques demeurent une cause majeure de mortalité infantile dans les pays en développement (PED). Du fait de l'insuffisance des plateaux techniques, les diagnostics étiologiques sont rarement réalisés et les traitements sont alors probabilistes. A Madagascar les données sur les diarrhées sont souvent parcellaires et anciennes. Le Réseau de surveillance sentinelle développé par l'Institut Pasteur de Madagascar à partir de 2007 nous a permis d'étudier la distribution spatio-temporelle des consultations pour diarrhée. Mais cette surveillance syndromique n'est pas couplée systématiquement à une surveillance biologique. Pour étudier les agents étiologiques des diarrhées, nous avons réalisé une enquête cas-témoins menée en 2008-2009 en milieu communautaire, chez les enfants de moins de 5 ans dans 14 districts. Nous avons pu identifier au moins un pathogène chez plus de la moitié des enfants (55%), avec une prédominance des étiologies parasitaires (37,2% des diarrhées), suivies par les bactéries (15%) puis les virus (6,7% de rotavirus). Les parasites ont été les seules étiologies pour lesquelles une pathogénicité a pu être mise en évidence. Parmi les étiologies bactériennes, l'infection à Campylobacter a été la plus fréquente (9,5%). Pour analyser le rôle de Campylobacter et les effets des facteurs environnementaux dans la survenue des diarrhées infantiles, nous avons initié et coordonné depuis 2010 une étude de cohorte dynamique d'enfants inclus avant l'âge de 24 mois et suivis jusqu'à l'âge de 36 mois à Moramanga, site où la prévalence de Campylobacter a été la plus élevée au cours de l'étude de 2008 (20,6%). Une surveillance des diarrhées a été menée 2 fois par semaine et les portages asymptomatiques évalués à l'inclusion et tous les 2 mois. Une étude de portage familial a été mise en œuvre ainsi qu'un suivi coprologique bi-annuel de la population avicole, des points d'eaux collectifs et de l'eau de boisson des familles. La recherche de Campylobacter chez les volailles portait sur les écouvillonnages rectaux. De janvier 2010 à mai 2012, 508 enfants correspondant à 256 346 enfant-jour ont participé à l'étude. La prévalence globale d'isolement de Campylobacter a été de 9,3%. Plus de 2/5 des enfants (43,3%) ont eu au moins un épisode d'infection à Campylobacter au cours de leur suivi. Les taux d'incidence annuelle des diarrhées ainsi que des infections symptomatiques ont été faibles, respectivement de 0,7 épisode /enfant et de 5,8 épisodes/100 enfant pouvant s'expliquer par le faible niveau d'exposition environnementale des enfants. Nous avons pu étudier l'importance des facteurs liés à l'hôte comme l'âge. Le pic d'infection à Campylobacter se situe entre 18 à 29 mois, celui des diarrhées entre 6 à 11 mois puis diminue ensuite. La 1ère infection à Campylobacter a été toujours pathogène chez les plus jeunes. Elle se situe vers le 8ème mois de la vie pour 10% d'entre eux. Les réinfections se font à des distances différentes de l'événement initial en fonction de l'âge. Ce profil d'infection pourrait traduire une compétence immunitaire différente selon l'âge et/ou une immunité acquise au cours du temps suite aux expositions répétées des enfants. L'environnement pourrait avoir un effet indirect dans l'entretien d'une immunité protectrice s'exprimant par un taux élevé d'infection asymptomatique. Il apparaît nécessaire de poursuivre des études de cohorte dans des zones à plus fort risque de transmission avec des données immunologiques car la compréhension actuelle des interactions entre l'hôte, le Campylobacter et l'environnement ne permet pas d'expliquer la variabilité de l'expression clinique de l'infection. / Diarrheal diseases remain a major cause of infant mortality in developing countries (DCs). Due to the lack of technical platforms, the etiologic diagnoses are rarely made and treatments are then probabilistic. In Madagascar data on diarrhea are often fragmented and old. The sentinel surveillance network developed by the Institut Pasteur of Madagascar from 2007 allowed us to study the spatial and temporal distribution of consultations for diarrhea. But this syndromic surveillance cannot be coupled to biological monitoring for many diseases. In this context, we have no information on the causative agents of diarrhea. To achieve the coupling of syndromic and etiologic data, we performed a case-control study conducted in 2008-2009 in children less than 5 years in 14 districts. We have identified at least one pathogen in more than half of the children (55%), with a predominance of parasitic etiologies (37.2% diarrhea), followed by bacteria (15%) and viruses (6.7% rotavirus). Parasites were the only etiologies for which pathogenicity has been demonstrated. Among the bacterial etiologies, Campylobacter infection was the most common (9.5%). To better understand the role of Campylobacter in the occurrence of diarrhea in children and analyze the effect of environmental factors, we initiated and coordinated a dynamical cohort study including of children before the age of 24 months and followed up till the age of 36 months in Moramanga site, where the prevalence of Campylobacter was highest during the 2008 study (20.6%). Diarrhea surveillance was conducted two times per week and asymptomatic carriers assessed at baseline and every 2 months. A family study has been implemented and bi-annual stool follow-up in poultry population, water points and community drinking water for families. Campylobacter monitoring in poultry focused on rectal swabs. From January 2010 to May 2012, 508 children - corresponding to 256,346 child days -participated in the study. The overall prevalence of Campylobacter isolation was 9.3%. More than two fifths of children (43.3%) had at least one episode of Campylobacter during follow-up. The annual incidence of diarrhea and symptomatic infections were low, respectively 0.7 episodes / child and 5.8 episodes /100 children, can be explained by the low level of environmental exposure of children. We have studied the role of host factors such as age. The peak of Campylobacter infection is between 18 to 29 months, the diarrhea between 6 to 11 months then decreases. The first Campylobacter infection was always pathogen in the youngest children. It happens to the eighth month of life for 10% of them. Reinfections are at different distances from the initial event according to the age. This pattern of infection may reflect a variation of the immune competence according the age and / or acquired immunity over time after repeated exposure of the children. The local environment may have an indirect impact on maintaining protective immunity expressed by a high rate of asymptomatic infection. However, it is necessary to continue cohort study with immunologic data in a high risk transmission area as the current understanding of the interactions between the host, the environment and Campylobacter does not explain the variability of the clinical expression of infection.

O uso de antimicrobianos de forma terapêutica, preventiva e promotora de crescimento trouxe inúmeras vantagens para a avicultura mundial, entretanto a utilização excessiva dos antimicrobianos e de maneira indevida tem estimulado um aumento no número de micro-organismos resistentes. Entre eles, destaca-se o Campylobacter jejuni, bactéria frequentemente associada a enterites em humanos, sendo o frango a principal reservatório e fonte de transmissão deste patógeno para o homem. A transmissão de bactérias resistentes entre animais e seres humanos pode resultar em infecções multirresistentes e insucesso no tratamento terapêutico, sendo a exposição continua destes micro-organismos a esses medicamentos o fator mais importante na origem da resistência. Diante desse cenário, objetivou-se nesse trabalho investigar e caracterizar, através de métodos fenotípico e genotípico, a susceptibilidade antimicrobiana de 54 isolados de C. jejuni coletados em diferentes etapas do processamento da carne de frango de matadouro-frigoríficos da região do Rio Grande do Sul. Para a determinação do MIC, os isolados foram testados frente aos seguintes antimicrobianos: ácido nalidíxico, ciprofloxacina, cloranfenicol, eritromicina, gentamicina e tetraciclina. Dos 54 isolados de C. jejuni, 94,4% foram resistentes a ciprofloxacina, 83,3% ao ácido nalidíxico, 51,8% a tetraciclina e 48% a eritromicina. Todos os isolados foram sensíveis a gentamicina e ao cloranfenicol. Doze isolados foram resistentes a três classes diferentes de antibióticos, sendo assim considerados multi-resistentes. Para verificar a presença da mutação gênica da Região Determinante de Resistência à Quinolona (RDRQ) no gene gyrA, foi realizado sequenciamento gênico de 31 isolados considerados resistentes por métodos fenotípicos. Todos os isolados possuíam a mutação Tre-86-Ile na RDRQ do gene gyrA, que confere resistência às fluoroquinolonas, confirmando a predominância dessa mutação em Campylobacter spp. resistentes a esses antimicrobianos. A ocorrência do gene de resistência à tetraciclina foi verificada por PCR. Dos 28 isolados considerados resistentes por métodos fenotípicos, 42,8% possuíam o gene tet(O), que confere resistência as tetraciclinas. Os resultados mostram um alto nível de resistência antimicrobiana em C. jejuni evidenciando a necessidade da implementação de políticas de uso prudente de antimicrobianos na medicina veterinária. / The use of antimicrobials in a therapeutic, preventive and growth promoting way has brought numerous advantages to the world poultry industry; however, the excessive and undue use of antimicrobials has stimulated an increase in the number of resistant microorganisms. Among them, we highlight Campylobacter jejuni, a bacterium frequently associated with enteritis in humans, with chicken being the main reservoir and source of transmission of this pathogen to man. The transmission of resistant bacteria between animals and humans can result in multi resistant infections and failure in therapeutic treatment, and the continued exposure of these microorganisms to these drugs is the most important factor in the source of resistance. Therefore, the aim of this study was to investigate and characterize, through phenotypic and genotypic methods, the antimicrobial susceptibility of 54 C. jejuni isolates collected at different stages of the processing of chicken meat from slaughterhouse in Rio Grande do Sul. For MIC determination, strains were tested against the following antimicrobials: nalidixic acid, ciprofloxacin, chloramphenicol, erythromycin, gentamicin and tetracycline. Of the 54 isolates of C. jejuni, 94.4% were resistant to ciprofloxacin, 83.3% to nalidixic acid, 51.8% to tetracycline and 48% to erythromycin. All isolates were sensitive to gentamicin and chloramphenicol. Twelve strains were resistant to three different classes of antibiotics, thus being considered multi resistant. To verify the presence of the gene mutation of the Quinolone Resistance Determinant Region (QRDR) in the gene gyrA, gene sequencing of 31 strains considered resistant by phenotypic methods was performed. All strains had the Tre-86-Ile mutation in the QRDR of the gyrA gene, which confers resistance to fluoroquinolones, confirming the predominance of this mutation in Campylobacter spp. resistant to these antimicrobials. The occurrence of tetracycline resistance gene was verified by PCR. Of the 28 strains considered resistant by phenotypic methods, 42.8% had the tet(O) gene. The results show a high level of antimicrobial resistance in C. jejuni that evidences the need for implementation of policies in the prudent use of antimicrobials in veterinary medicine.

Atualmente, a campilobacteriose representa uma importante zoonose tanto em países desenvolvidos quanto em países em desenvolvimento. No Brasil, vários estudos relatam a alta frequência de suínos eliminando o agente nas fezes, porém, a contaminação da carne suína comercializada tem sido pouco avaliada, principalmente no que diz respeito à presença de Campylobacter spp. A incidência deste agente em cortes de carne de aves é mais descrita, porém ainda faltam estudos que esclareçam a epidemiologia da doença. Os objetivos principais do presente estudo foram avaliar a presença de Campylobacter spp. em cortes de carne de frangos e suínos vendidos em mercados municipais, açougues e mercados de pequeno porte distribuídos nas cinco regiões do município de São Paulo. Das 115 amostras de origem suína avaliadas sete se mostraram positivas ao isolamento de Campylobacter coli e, das 105 amostras de carne de frango, 31 estavam contaminadas por Campylobacter jejuni ou Campylobacter coli. Dentre as 38 amostras de carne de frango e suíno positivas foram selecionadas 60 estirpes, as quais foram caracterizadas quanto à espécie, utilizando a reação em cadeia pela polimerase e a espectrometria de massa MALDI-TOF, foram ainda avaliadas quanto ao perfil de resistência a antimicrobianos e foram submetidas a genotipagem pelo polimorfismo de comprimento de fragmentos amplificados (AFLP). A partir dos resultados obtidos seis estirpes foram selecionadas para realização do sequenciamento do genoma completo e a partir da análise dos genomas foram identificados os STs, os genes de resistência e genes de virulência. / Currently, Campylobacteriosis represents an important zoonosis in both developed and developing countries. In Brazil, several studies report a high frequency of pigs excreting the agent in the feces, but the contamination of marketed pork has been insufficiently evaluated, especially with regard to the presence of Campylobacter spp. The incidence of this agent in cuts of poultry meat is more detailed; however, there are still studies lacking that clarify the epidemiology of the disease. The main aims of the present study were to evaluate the presence of Campylobacter spp. in chicken and pork cuts sold in municipal markets, butchers and small markets distributed in the five regions of the city of São Paulo. Of the 115 pork samples evaluated, seven were Campylobacter coli isolation positive and 31 of the 105 chicken samples were contaminated with Campylobacter jejuni or Campylobacter coli. Of the 38 chicken and pork positive samples, 60 strains were selected, which were characterized by species using polymerase chain reaction and MALDI-TOF mass spectrometry. They were also assessed for antimicrobial resistance profile and were genotyped by amplified fragment length polymorphism (AFLP). From the results obtained six strains were selected to carry out the complete genome sequencing and from the genome analysis the STs, the resistance genes and the virulence genes were identified.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:42Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-13T10:47:59Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) / Made available in DSpace on 2015-01-13T10:47:59Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Janaina Costa Feistel - 2013.pdf: 840570 bytes, checksum: 815615a485820497e44d61ee8a9f1f17 (MD5) Previous issue date: 2013-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Poultry products are recognized as sources of contamination by several pathogens. In the last decades the genus Campylobacter, particularly thermophilic species, stands out in developed countries, where it is considered the primary agent responsible for Foodborne Illnesses (DVA's). Due to the importance of this pathogen in public health and economics, researches are conducted in order to identify and characterize the isolated, as well as develop and improve techniques for the identification of the genus Campylobacter spp. with attention focused mainly to important thermophilic species related to food, allowing the determination of the prevalence of this pathogen in main placement sources, such as chicken. The objective of this study was to verify the occurrence of Campylobacter spp. and identify isolated thermophilic species from chicken carcasses in slaughterhouses in the state of Goiás submitted to the Federal Inspection Service which had proper authorization to internal and external trade. A total of 200 samples have been evaluated using the immunoenzymatic screening assay, with subsequent bacterial samples isolation and identification through biotyping. In this study it was observed that the employed methods allowed the isolation of Campylobacter spp. in 24% (48/200) of the assessed samples. Among these samples, 21 (10.5%) were demonstrably positive for genre, having as basis the set of analytical screening method, sequential bacterial isolation for positive samples and evidence of enzymatic and biochemical assimilation and antimicrobial profile. C.coli was the most frequently isolated species (58.6%), followed by C.jejuni ssp. jejuni (17.2%), C.lari (13.8%) and Campylobacter jejuni ssp. doylei (3.4%). By using a screening method, conventional isolation system and numerical biotyping it was immediately revealed the characterization of the isolated Campylobacter. It was observed that some suggestive isolated, due to its positivity in the screening test, growth on selective sources, incubation temperature, motility and morphology, characteristics of the genus assessed, have not been identified by numerical system and thus were considered negative. From the results, it can be concluded that the association of screening method to conventional isolation and biotyping numerical profile allowed a greater understanding of the isolated. It can also be concluded that Campylobacter spp. is present in chicken meat for human consumption and three thermophilic species and two subspecies have been identified. The pathogen presence attests public health risk. / Os produtos de origem avícola podem veicular diversos microrganismos patogênicos. Nas últimas décadas, o gênero Campylobacter, em especial as espécies termofílicas, destaca-se como um dos principais agentes responsáveis por Doenças Veiculadas por Alimentos (DVA’s). Em decorrência da importância deste patógeno na saúde pública e na economia, pesquisas são realizadas com o objetivo de identificar e caracterizar os isolados, bem como desenvolver e aprimorar as técnicas para a identificação do gênero, permitindo a determinação da prevalência deste patógeno nas principais fontes de veiculação, como a carne de frango. O presente estudo teve como objetivo verificar a ocorrência de Campylobacter spp. e identificar as espécies termofílicas isoladas de carcaças de frango de abatedouros do estado de Goiás submetidos ao Serviço de Inspeção Federal e que possuíam autorização para o comércio interno e externo. Um total de 200 amostras foi avaliado utilizando o ensaio imunoenzimático como triagem, com posterior isolamento bacteriano e identificação por meio da biotipificação. Observou-se que os métodos empregados permitiram a identificação de Campylobacter spp. em 24% (48/200) das amostras avaliadas. Destas, 21 (10,5%) foram comprovadamente positivas para o gênero, tendo por fundamentação o isolamento bacteriano de amostras positivas à triagem e testes de assimilação enzimática, bioquímicas e de perfil antimicrobiano. Campylobacter coli foi a espécie mais isolada (58,6%), seguida por Campylobacter jejuni ssp. jejuni (17,2%), Campylobacter lari (13,8%) e Campylobacter jejuni ssp. doylei (3,4%). Observou-se que alguns isolados sugestivos, por apresentar positividade no teste de triagem, crescimento em meios seletivos, temperatura de incubação, morfologia e motilidade características do gênero em questão, não foram identificados pelo sistema numérico empregado neste estudo e, portanto, foram considerados negativos. Pelos resultados, pode-se concluir que a associação do método de triagem ao isolamento convencional e perfil numérico para biotipificação permitiram melhor identificação dos isolados. Assim, é possível afirmar que Campylobacter spp. foi identificada em carne de frango destinada ao consumo, havendo a descrição de três espécies termofílicas e duas subespécies. A presença deste patógeno confere risco à saúde pública por ingestão de alimentos contaminados e decorrência de doenças gastroentéricas e extraintestinais e em casos mais graves, porém não raros, a síndromes reumatológicas, oftalmológicas e neurológicas. Especificamente as espécies Campylobacter coli e Campylobacter jejuni descritas como de maior interesse em alimentos foram as de maior frequência e relacionam-se à maioria dos casos de campilobacteriose de origem alimentos.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-04-11T18:05:11Z No. of bitstreams: 2 Dissertação - Maria Luiza Rezende Ribeiro - 2017.pdf: 1775886 bytes, checksum: 5582a3f36c670d7b39e468c2e09807d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-12T14:31:23Z (GMT) No. of bitstreams: 2 Dissertação - Maria Luiza Rezende Ribeiro - 2017.pdf: 1775886 bytes, checksum: 5582a3f36c670d7b39e468c2e09807d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-12T14:31:23Z (GMT). No. of bitstreams: 2 Dissertação - Maria Luiza Rezende Ribeiro - 2017.pdf: 1775886 bytes, checksum: 5582a3f36c670d7b39e468c2e09807d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The ingestion of foods contaminated with Campylobacter spp. results in campylobacteriosis in humans. This disease is the main cause of diarrhea in the United States and European Union. The most common reservoirs of this microorganism are chickens and the consumption of their meat, raw or undercooked, is the main source of contamination to humans. The most prevalent species involved in infections are Campylobacter jejuni, Campylobacter coli and Campylobacter lari that can contaminate carcasses during the slaughter and handling of chickens. The aim of this study was to determine the prevalence of thermotolerant Campylobacter in chilled and later frozen chicken carcasses commercialized in the city of Goiânia, Goiás. With the support of the Municipal Sanitary Surveillance Department and the Goiás Agency for Agricultural and Farming Defense, forty cooled and frozen carcasses samples were collected in abattoirs and its retail outlets, from May to September/2015. The microbiological analysis was performed according to the methods of the International Organization for Standardization 10272-1:2006. For molecular identification, the Polymerase Chain Reaction technique was performed to detect the hipO (Campylobacter jejuni) and glyA genes (Campylobacter coli and Campylobacter lari). It was found a contamination prevalence of 17.5 % (n=7) of Campylobacter spp.. Between the 24 samples collected from slaughterhouses, 12.5% (n = 3) were contaminated. Among the 16 collected in points of sale, 25.0% (n = 4) presented positivity for the bacterium. Campylobacter lari was not detected in the evaluated samples. The results show that the bacteria remains viable at all stages of the chicken production chain, representing a risk for outbreaks of gastroenteritis. The presence of bacteria in chicken from market reinforces the need for education of poultry farmers and traders regarding the public health risk that these products represent. Awareness raising is needed to improve management, as well as preventive and corrective measures in the production and marketing of these products. In addition, the prevalence found in the present study reinforces the need to establish legal standards that determine the research of the microorganism in this type of food. / A ingestão de alimentos contaminados por bactérias do gênero Campylobacter resultam em campilobacteriose em humanos. Esta é uma das causas mais comuns de diarreia nos Estados Unidos e União Europeia. Um dos maiores reservatórios deste micro-organismo são produtos avícolas, sendo sua carne, crua ou mal cozida, a principal fonte de contaminação para o homem. As espécies mais prevalentes envolvidas nas infecções são Campylobacter jejuni, Campylobacter coli e Campylobacter lari que podem contaminar as carcaças durante o abate e a manipulação de frangos. O objetivo desse estudo foi determinar a prevalência de Campylobacter termotolerantes em carcaças de frango refrigeradas e posteriormente congeladas comercializadas no município de Goiânia, Goiás. Com o apoio do Departamento de Vigilância Sanitária Municipal e Agência Goiana de Defesa Agropecuária, foram coletadas, entre os meses de maio e setembro de 2015, 40 amostras de carcaças refrigeradas e congeladas em abatedouros em Goiás e seus pontos de venda em Goiânia. A metodologia para isolamento e identificação obedeceu às normas da International Organization for Standardization 10272- 1:2006. Para identificação molecular, foi realizada a técnica da Reação em Cadeia da Polimerase para detecção dos genes hipO (Campylobacter jejuni) e glyA (Campylobacter coli e Campylobacter lari). Foi encontrada prevalência de 17.5 % (n=7) de contaminação de carcaças por Campylobacter spp.. Dentre as 24 amostras coletadas em abatedouros, 12,5 % (n=3) estavam contaminadas. Já entre as 16 coletadas em pontos de venda, 25,0 % (n=4) apresentaram positividade para a bactéria. Não foi detectado Campylobacter lari nas amostras avaliadas. Os resultados evidenciam que a bactéria permanece viável em todas as etapas da cadeia produtiva de frangos, representando um risco para a ocorrência de surtos de gastroenterites. A presença da bactéria em frangos aponta para a necessidade de educação de criadores de aves e comerciantes quanto ao risco à saúde pública que estes produtos representam. É necessária sensibilização para melhorar a gestão, além da realização de medidas preventivas e corretivas no sistema de produção e comercialização destes produtos. Além disso, a prevalência encontrada no presente estudo reforça a necessidade do estabelecimento de padrões legais que determinem a pesquisa do micro-organismo neste tipo de alimento.

Os procedimentos de limpeza e desinfecção usados na produção frangos de corte são fundamentais para a manutenção do alto nível de saúde do plantel, reduzindo o risco de ocorrência de enfermidades e melhorando o desempenho produtivo das aves. Existe a possibilidade de contaminação dos produtos desde a chegada das aves às instalações até o abate. Campylobacter é uma bactéria constantemente encontrada no trato gastrointestinal das aves e merece destaque na produção avícola devido a sua capacidade de provocar enfermidades em humanos. Para evitar a ocorrência de surtos de doenças transmitidas por alimentos como a campilobacteriose, para manutenção da saúde de plantéis avícolas e o aumento da produtividade de lotes de frangos de corte é necessário a realização das práticas preventivas como a limpeza e desinfecção. Desta maneira, o presente estudo teve como objetivos avaliar: o desempenho produtivo em lotes de frangos de corte desafiados com Campylobacter jejuni, a redução da pressão de infecção em instalações submetidas a diferentes protocolos de limpeza e desinfecção, e sua viabilidade econômico-financeira. Foi conduzido protocolo experimental no Laboratório de Pesquisa em Aves do departamento de Nutrição e Produção Animal, da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Campus Pirassununga. Foram realizados dois alojamentos cada um com 960 pintos de um dia de idade. No primeiro alojamento, todas as aves foram inoculadas com uma cepa conhecida de Campylobacter jejuni (atcc 33560) a fim de contaminar o ambiente criando desafio sanitário. No segundo alojamento foram realizados dois protocolos de limpeza e desinfecção, constituindo os dois tratamentos experimentais (Comum e Proposto). Neste lote, para avaliação da produtividade, foram mensurados os parâmetros: viabilidade, mortalidade, conversão alimentar, ganho de peso e consumo de ração. Para avaliação microbiológica do ambiente e das carcaças foi realizada a contagem total de microrganismos para ambiente e a identificação de Campylobacter spp.. As amostras positivas foram submetidas a um ensaio de PCR multiplex para identificação de espécies de Campylobacter spp. Para avaliação econômica financeira dos lotes de frangos de corte foram utilizados e adaptados os dados de custos de produção elaborados pela Embrapa Suínos e Aves às condições de criação e resultados obtidos no presente estudo. O tratamento Proposto demonstrou influências positivas no desempenho das aves, na redução da contagem total de microrganismos, na eliminação de Campylobacter spp. e viabilidade econômico-financeira / The procedures of cleaning and disinfection used in broiler production are necessary to maintaining a high standard of birds health, decreasing the risk of diseases, optimizing the animal performance, as well as to ensure sanitary quality of meat. There is possibility of contamination in all farming steps, since chicks arrival until the end of slaughter. Campylobacter is a bacteria constantly found at gastrointestinal tract of the birds, and so, a special concern must be given since it can cause human diseases. In order to avoid foodborne illness outbreaks such as campylobacteriosis, to ensure poultry flocks health maintenance and to improve broilers productivity, adopting preventive practices such as cleaning and disinfection is required. Thus, this study aimed to evaluate: the animal performance of broilers challenged with Campylobacter jejuni; the reduction of infection pressure in housing plants subjected to different cleaning protocols and disinfection, and its economic and financial viability. Experimental protocol was conducted at the Research Laboratory of Poultry of Nutrition and Animal Production Department, College of Veterinary Medicine and Animal Science, University of São Paulo, Campus Pirassununga. Two housings each with 960 day-old chicks of age were performed. In the first housing, all birds were inoculated with a known strain of Campylobacter jejuni (ATCC 33560) to contaminate the environment in order to create health challenge. In the second housing were performed two cleaning and disinfection protocols, featuring two experimental treatments (Common and Proposed). In this lot, to evaluate animal performance, the parameters were measured: feasibility, mortality, feed conversion, weight gain and feed intake. For microbiological evaluation of environment and of carcasses, was performed a total count of microorganisms for environmental and identification of Campylobacter spp. The positive samples were subjected to a multiplex PCR assay identification of species of Campylobacter spp. For economic and financial evaluation of batches, the data of husbandry costs elaborated by Embrapa Swine and Poultry were adapted to conditions and rsults obtained in this study. Based on results, the Proposed treatment has shown positive influences on bird performance, reduction of total count of microorganisms, on elimination of Campylobacter spp. and on economic and financial viability

Les campylobactérioses sont les infections intestinales bactériennes d’origine alimentaire les plus fréquemment rapportées au sein de l’Union Européenne et sont principalement associées à la consommation de viande de volailles. Une diminution de la colonisation intestinale des volailles par Campylobacter de 2 à 3 log10 UCF/g permettrait de réduire l’incidence des cas humains de 76 à 100 %. La vaccination aviaire constitue un moyen de lutte potentiel mais, malgré de nombreuses études, aucun vaccin commercial n’est actuellement disponible. L’objectif de ce projet a été d’identifier de nouveaux antigènes vaccinaux contre Campylobacter en appliquant la stratégie de la vaccinologie inverse et d’évaluer leurs pouvoirs immunogène et protecteur contre la colonisation intestinale des volailles. Sur la base de leur localisation subcellulaire, leur antigénicité, leur densité en épitopes B et leur homologie de séquence avec l’ensemble des souches de C. jejuni et C. coli, quatorze antigènes ont été sélectionnés. Six d’entre eux ont été produits et testés in vivo en appliquant un protocole vaccinal optimisé. Quatre antigènes ont montré des diminutions significatives de la charge intestinale des oiseaux de 2 à 4,2 log10 UFC/g associées à l’induction de réponses humorales spécifiques. L’immunogénicité de ces candidats vaccins et l’efficacité protectrice de deux antigènes ont été observées à nouveau. Ces premiers résultats montrent l’intérêt et la fiabilité de la vaccinologie inverse. L’évaluation du potentiel vaccinal de ces nouveaux antigènes doit être poursuivie et approfondie lors de futures expérimentations. / Campylobacteriosis is the most prevalent bacterial foodborne gastroenteritis reported in the European Union and is mainly associated to consumption of poultry meat. Reducing the intestinal colonization of broilers by Campylobacter from 2 to 3 log10 CFU/g could decrease human cases incidence by 76 to 100%. Vaccination of poultry could be a potential strategy but despite many studies, no efficient vaccine is available yet. The aim of this project was to identify new vaccine antigens against Campylobacter using the reverse vaccinology strategy and to assess their immune and protective powers against the avian intestinal colonization. Based on their sub-cellular localization, immunogenicity, B-epitopes density and their sequence conservation among C. jejuni and C. coli strains, fourteen antigens were selected. Six out of them were produced and in vivo tested according to an optimized avian vaccine protocol. Four antigens showed intestinal load decreases from 2 to 4.2 log10 CFU/g correlated with the induction of specific humoral responses. Vaccine candidates’ immunogenicity and the protective efficiency of two antigens were observed again. These first results highlight the interest and reliability of the reverse vaccinology. The assessment of these new antigens vaccine potential needs to be continued and deepened in next experiments.

Bovine genital campylobacteriosis is a disease of difficult diagnostic because of the microaerophilic nature of its etiologic agent, Campylobacter fetus, thereby causing the prevalence related to this disease to be underestimated. The purpose of this study was to evaluate the utilization of PCR for the diagnosis of genital campylobacteriosis, using samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents obtained from aborted fetuses, collected in transport and enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lyses with proteinase K, lyses with guanidine isothiocyanate, lyses with DNAzol? , and lyses with CTAB. The specificity, sensitivity and technical application of PCR assay were also evaluated with clinical samples. The PCR performance was compared to the culture technique for bacterial isolation. The CTAB was the most efficient extraction protocol; the pair of primers used was shown to be specific; and the limit of detection was of 63 CFU of Campylobacter fetus. PCR demonstrated that 24% (68/277) of the clinical samples were positive for Campylobacter fetus, while only 2.8% (8/277) of samples were positive by culture techinque. These results indicate that the PCR technique is specific and sensitivy, and is superior to the culture for the diagnosis of bovine genital campylobacteriosis. / A campilobacteriose genital bovina, caracterizada principalmente por infertilidade, é uma doença de difícil diagnóstico, devido à natureza microaerófila do seu agente etiológico, o Campylobacter fetus, tendo assim sua prevalência subestimada. Este trabalho teve por objetivo avaliar a utilização da técnica de PCR para o diagnóstico da campilobacteriose genital bovina, utilizando amostras de aspirado prepucial, muco cervical e conteúdo abomasal de fetos abortados, coletadas em meio de transporte e enriquecimento; bem como comparar seu desempenho com a técnica do isolamento bacteriano. Foram testados cinco diferentes protocolos de extração de DNA de Campylobacter fetus: termo extração, lise com proteinase K, lise com isotiocianato de guanidina, lise com DNAzol? e lise com brometo de cetiltrimetilamônio (CTAB). Também foram avaliadas a especificidade, a sensibilidade e a aplicação da técnica da PCR em amostras clínicas. Os resultados indicaram que o CTAB foi o protocolo de extração mais eficiente; o par de primers utilizado mostrou-se específico; e o limite de detecção foi 63 unidades formadoras de colônias (UFC) de C. fetus. A PCR encontrou 24% (68/277) das amostras clínicas positivas para C. fetus, enquanto a cultura encontrou 2,8% (8/277). A técnica da PCR é específica e sensível, e mostra-se superior a cultura no diagnóstico da campilobacteriose genital bovina.

Memoria para optar al Título Profesional de Médico Veterinario / La resistencia a los antibióticos es un problema de salud pública mundial, ya que complica y encarece el tratamiento de las enfermedades infecciosas. En el caso de Campylobacter spp., este es un problema emergente, dado que en los últimos años se ha observado un incremento en la resistencia a antibióticos principalmente a las fluoroquinolonas y macrólidos. Esto es de gran importancia dado que estos fármacos son utilizados como primera elección para el tratamiento de campilobacteriosis. El uso indiscriminado, no solo en producción animal, sino también en medicina humana se describe como unas de las principales causas de este fenómeno. En Chile, existen muy pocos estudios sobre la susceptibilidad antimicrobiana en cepas de Campylobacter spp. El objetivo de este trabajo fue evaluar la susceptibilidad a los antibióticos en 120 cepas de Campylobacter spp., provenientes de cerdos y 60 cepas aisladas de bovinos de carne. Los antibióticos analizados fueron ciprofloxacino, tetraciclina, eritromicina y gentamicina. Se utilizaron dos métodos, primero se realizó un screening con la técnica de Kirby Bauer y todas aquellas cepas que resultaron resistentes, fueron sometidas a determinación de concentración mínima inhibitoria en placa mediante el Método Etest. Se analizaron 180 cepas y se observó que el 10,5% de ellas fueron resistentes a gentamicina, el 57,9% a eritromicina, el 82,6% a ciprofloxacino y el 91,4% lo fue a tetraciclina, mientras que el 87,1% de las cepas fueron clasificadas como multiresistentes. Nuestros resultados indican que los niveles de resistencia a los antimicrobianos en las cepas de Campylobacter spp., son elevados especialmente para ciprofloxacino y tetraciclina. Esto hace necesario proponer y establecer sistemas de vigilancia de la resistencia en este patógeno, con un enfoque integral entre Medicina Veterinaria, Medicina Humana y en producción de alimentos, con el fin de resguardar la salud pública. / Antibiotic resistance is a public health problem, because it complicates and increases the cost of treatment of infectious diseases. As for Campylobacter spp., this is a relevant emerging problem, since in recent years it has seen an increase in antibiotic resistance mainly to fluoroquinolones and macrolides. This is of great importance since drugs are used as the first choice for the treatment of campylobacteriosis. Their indiscriminate use not only animal production but also in human medicine, is described as one of the main causes of this phenomenon. In Chile, there are only a few studies on the antimicrobial susceptibility of Campylobacter spp strains. This study was meant to evaluate the susceptibility to antibiotics in 120 strains of Campylobacter spp isolated from pigs and 60 strains isolated from beef cattle. The antibiotics analyzed were: ciprofloxacin, tetracycline, erythromycin and gentamicin. Two methods were used, the first, the Kirby Bauer screening technique and all of those strains that performed resistant on this test, were subjected to determination of minimum inhibitory concentration MIC`s through Etest Method. A total of 180 strains were evaluated, and of these 10.5% strains were resistant to gentamicin, 57.9% to erythromycin, 82.6% to ciprofloxacin and 94.1% to tetracycline. Also, a total of 87.1% from the tested strains were multiresistant. Our results indicate that levels of antimicrobial resistance in strains of Campylobacter spp., they are higher especially for ciprofloxacin and tetracycline. Making it necessary to propose and establish systems for monitoring and reporting of resistance in this pathogen, an integrated approach between Veterinary Medicine, Food production and Human Medicine, in order to protect public health. / Financiamiento: Proyecto Fondecyt no. 11110200.

Campylobacter est responsable de la majorité des gastro-entérites bactériennes dans le monde. Sa membrane externe contient des porines qui permettent les échanges entre la bactérie et le milieu extérieur. Elles sont une des voies principales d’entrée pour les antibiotiques et les nutriments. Le premier objectif de mon travail était de participer à la caractérisation des porines MOMP et Omp50. Nous avons purifié ces protéines dans le cadre de collaborations pour l’étude des propriétés biophysiques de ces porines (M. Wintherhalter, Université Jacob, Allemagne) et la détermination de leur structure par cristallisation (J. Naismith, Université St Andrews, Écosse). La deuxième partie de ma thèse a été consacrée à l’étude et la caractérisation du produit du gène Cj1169c qui est en opéron avec le gène codant Omp50. Nous avons cloné et exprimé le gène Cj1169c chez Escherichia coli, ce qui a permis de purifier la protéine et de produire des anticorps spécifiques. Ces anticorps ont été utilisés pour étudier la prévalence de Cj1169c chez les espèces les plus fréquentes. Nous avons montré qu’elle était présente uniquement chez C. jejuni et C. lari et absente chez C. coli. En deuxième lieu, nous avons caractérisé sa production en fonction de conditions de culture et montré qu’elle dépendait du pH et de la température. Nous avons ensuite démontré que cette protéine est située dans le périplasme de Campylobacter. Puis, en raison de la présence de deux résidus de cystéines dans sa séquence, nous avons étudié son comportement en présence et en absence d’agents réducteurs de ponts disulfures. De plus, des résultats préliminaires suggèrent que cette protéine interagit avec Omp50. / Campylobacter is a leading cause of bacterial gastroenteritis worldwide. Its outer membrane contains porins which are pore forming protein that transport hydrophilic compounds like nutrients and antibiotics into the bacteria. My first objective was to purify the two porins MOMP and Omp50 from C. jejuni, in order to allow the study of the biophysical properties of porins (Collaboration with Mr. Wintherhalter, Jacob University, Germany), and to determine the structure of these porins by crystallization (Collaboration with J. Naismith, St Andrews University, Scotland).The second part of my thesis was devoted to the study of the product of the gene Cj1169c which is operonic with the Omp50 coding gene. We cloned and expressed the gene Cj1169c in Escherichia coli, purified the protein and produced Cj1169c-specific antibodies. We studied the prevalence of this protein in the most frequent species of Campylobacter and found the protein only in C. jejuni and C. lari never in C. coli. Then, we also studied the Cj1169c production in the function of several growing conditions, and we found that it depended on the pH and temperature. Besides, we demonstrated by several methods that this protein is located in the periplasmic compartment of the bacteria. Furthermore, because it contains two cysteins residues in its primary sequence, we studied the Cj1169c behavior in the presence and absence of disulfide bond reducing agents. In addition, preliminary results suggest that this protein interacts with Omp50.