Salmonella Gallinarum (SG) et Salmonella Enteritidis (SE) sont deux sérotypes génétiquement proches. Pourtant, chez la volaille, SG induit une infection systémique létale tandis que SE est responsable d’une infection systémique transitoire et d’un portage intestinal asymptomatique. De plus, SE est un sérotype ubiquiste tandis que SG est spécifique d’hôte. Outre ces différences in vivo, ces deux sérotypes présentent des différences in vitro. Nous avons montré que SG est in vitro moins invasif que SE. Ce phénotype est indépendant de l’origine aviaire ou non-aviaire des cellules en dépit du tropisme de SG pour la volaille. LeT3SS-1, le facteur d’invasion majeur chez Salmonella, est composé d’un appareil de sécrétion et des effecteurs transloqués par celui-ci. Nous avons montré que les composants du T3SS-1 sont autant exprimés chez SG que chez SE et que tous deux possèdent un appareil de sécrétion fonctionnel. Pourtant, l’étude des capacités d’invasion dépendantes du T3SS-1 suggère que ce facteur est impliqué dans le défaut d’invasion de S. Gallinarum. L’analyse des gènes codant les effecteurs transloqués par le T3SS-1 a montré qu’il existe des mutations chez SG pouvant être à l’origine de ce défaut d’invasion. / Salmonella Gallinarum and Salmonella Enteritidis are genetically closed. However, whereas SG induces lethal systemic infection in poultry, SE is responsible for transient systemic infection and asymptomatic intestinal carriage. Moreover, SE is ubiquitous whereas SG is poultry specific. These serotypes also present in vitro differences. We have shown that SG is in vitro less invasive than SE whatever the avian or non-avian cell origin, in spite of SG’s tropism for avian species. T3SS-1, the main invasion factor in Salmonella, is composed of a secretion apparatus and its translocated effectors. We have shown that T3SS-1 components a expressed in a similar way by SE and SG and both harbor a functional secretion apparatus. However, study of T3SS-1 dependent invasion abilities suggested that T3SS-1 is involved in SG’s invasion defect. Sequence analysis has revealed that SG possesses several mutations in genes encoding main T3SS-1 effectors, that could explain the low invasive phenotype.

Com a expansão da avicultura, ocorreu um aumento no número de aves alojadas, concentrando as produções e, consequentemente, facilitando a disseminação de doenças. Entre as doenças transmitidas pelo consumo dos produtos avícolas, a salmonelose tem especial importância. Nos últimos anos tem ocorrido um aumento na prevalência do sorovar Salmonella Enteritidis em todo o mundo, sendo o mais isolado. A patogenia da Salmonella e a forma como ela interage com seu hospedeiro são fenômenos multifatoriais e complexos. Entre os principais fatores de virulência da Salmonella estão os genes de virulência. O objetivo deste trabalho foi a pesquisa de genes associados à virulência em cepas de S. Enteritidis oriundas de diferentes origens avícolas. Realizou-se a pesquisa de nove genes de virulência (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) em 84 amostras de S. Enteritidis. Os genes invA, hilA, sivH, sefA e avrA estiveram presentes em 100% das amostras (84/84); lpfA e sopE em 99% (83/84); agfA em 96% (81/84); e o gene spvC em 92% (77/84). Foi possível caracterizar as amostras em quatro diferentes perfis genéticos, sendo P1 positivo para todos os genes, P2 negativo apenas para spvC, P3 negativo apenas para agfA e P4 negativo para lpfA, spvC e sopE, sendo o grupo P1 o mais prevalente. Apesar de todas as cepas pertencerem a um mesmo sorovar, observou-se uma variação na presença dos genes de virulência. Os perfis estabelecidos e as frequências obtidas podem servir para a escolha de cepas a serem utilizadas para realização da PFGE e em futuros estudos in vivo. / The poultry industry has been characterized by an increase in the number of housed animals in recent years, concentrating the production and facilitating the spread of many diseases. Among all illness transmitted by food, including meat and eggs, salmonellosis is of particular importance. Currently, there has been an increase in prevalence of islament of Salmonella Enteritidis all around the world. The pathogenesis of Salmonella and how it interacts with the host are a complex and multifactorial phenomenon. Among virulence factors of Salmonella, the virulence genes are the most important. The aim of this survey was to search for genes associated with virulence in strains of S. Enteritidis isolated from different origins, all from poultry. We conducted a search of nine virulence genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) in 84 samples of S. Enteritidis. The genes invA, hilA, sivH, sefA e avrA were present in 100% of samples (84/84); lpfA and sopE in 99% (83/84); agfA in 96% (81/84); and spvC in 92% (77/84) of them. It was possible to characterize the samples in four different genetic profiles, P1 being positive for all genes, P2 negative only for spvC, P3 negative only for agfA and P4 negative for lpfA, sopE and spvC. The P1 was the most prevalent. Despite all the strains belong to the same serovar, it is possible to observe a variation in the presence of virulence genes. These established profiles and gene frequencies obtained are useful to select strains to be used in PFGE and in future in vivo studies.

Com a expansão da avicultura, ocorreu um aumento no número de aves alojadas, concentrando as produções e, consequentemente, facilitando a disseminação de doenças. Entre as doenças transmitidas pelo consumo dos produtos avícolas, a salmonelose tem especial importância. Nos últimos anos tem ocorrido um aumento na prevalência do sorovar Salmonella Enteritidis em todo o mundo, sendo o mais isolado. A patogenia da Salmonella e a forma como ela interage com seu hospedeiro são fenômenos multifatoriais e complexos. Entre os principais fatores de virulência da Salmonella estão os genes de virulência. O objetivo deste trabalho foi a pesquisa de genes associados à virulência em cepas de S. Enteritidis oriundas de diferentes origens avícolas. Realizou-se a pesquisa de nove genes de virulência (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) em 84 amostras de S. Enteritidis. Os genes invA, hilA, sivH, sefA e avrA estiveram presentes em 100% das amostras (84/84); lpfA e sopE em 99% (83/84); agfA em 96% (81/84); e o gene spvC em 92% (77/84). Foi possível caracterizar as amostras em quatro diferentes perfis genéticos, sendo P1 positivo para todos os genes, P2 negativo apenas para spvC, P3 negativo apenas para agfA e P4 negativo para lpfA, spvC e sopE, sendo o grupo P1 o mais prevalente. Apesar de todas as cepas pertencerem a um mesmo sorovar, observou-se uma variação na presença dos genes de virulência. Os perfis estabelecidos e as frequências obtidas podem servir para a escolha de cepas a serem utilizadas para realização da PFGE e em futuros estudos in vivo. / The poultry industry has been characterized by an increase in the number of housed animals in recent years, concentrating the production and facilitating the spread of many diseases. Among all illness transmitted by food, including meat and eggs, salmonellosis is of particular importance. Currently, there has been an increase in prevalence of islament of Salmonella Enteritidis all around the world. The pathogenesis of Salmonella and how it interacts with the host are a complex and multifactorial phenomenon. Among virulence factors of Salmonella, the virulence genes are the most important. The aim of this survey was to search for genes associated with virulence in strains of S. Enteritidis isolated from different origins, all from poultry. We conducted a search of nine virulence genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) in 84 samples of S. Enteritidis. The genes invA, hilA, sivH, sefA e avrA were present in 100% of samples (84/84); lpfA and sopE in 99% (83/84); agfA in 96% (81/84); and spvC in 92% (77/84) of them. It was possible to characterize the samples in four different genetic profiles, P1 being positive for all genes, P2 negative only for spvC, P3 negative only for agfA and P4 negative for lpfA, sopE and spvC. The P1 was the most prevalent. Despite all the strains belong to the same serovar, it is possible to observe a variation in the presence of virulence genes. These established profiles and gene frequencies obtained are useful to select strains to be used in PFGE and in future in vivo studies.

Orientador: Paulo Eduardo Martins Ribolla / Coorientador: Alexandre Secorun Borges / Banca: Vera Lúcia Mores Rall / Banca: João Pessoa Araújo Junior / Banca: Aline Maria da Silva / Banca: Mario Henrique de Barros / Resumo: Salmonella Enteritidis é um patógeno pertencente à Família Enterobacteriaceae que frequentemente está relacionada a infecções alimentares em humanos, principalmente em crianças, idosos e pacientes imunossuprimidos. A importância deste micro-organismo se dá pela sua prevalência significativa, com distribuição mundial, nos lotes de frangos de corte e de postura de ovos, levando a sérias implicações na saúde pública e ao mercado avícola. O controle eficaz da salmonelose é um desafio, pois envolve um complexo manejo dos animais e tratamento com uso de antibióticos que não garantem a eliminação da infecção. O uso de bacteriófagos líticos contra infecções bacterianas (fagoterapia) vem atender uma demanda crescente por alternativas ao tratamento antimicrobiano convencional. Todavia, a literatura tem relatado o surgimento de certa resistência bacteriana a alguns bacteriófagos líticos, como por exemplo, em Salmonella sp. Diversos são os mecanismos bacterianos de resistência aos fagos, tais como a prevenção de adsorção, o bloqueio da injeção do DNA do fago, restrição-modificação, infecção abortiva e o sistema CRISPR ("clustered regularly interspaced short palindromic repeats") /Cas ("CRISPR associated proteins") de imunidade adquirida. Assim sendo, o presente trabalho objetivou a caracterização molecular da resistência bacteriana de fagos líticos isolados contra cepas de Salmonella enterica subsp. enterica serovar Enteritidis (SE). Para tal, os mecanismos-alvos escolhidos foram o sistema CRISPR/Cas e o bloqueio da adsorção de fagos, via lipopolissacarídeo (LPS). Nas 22 cepas de estudo, um total de 72 CRISPRs foi identificado, com 14 diferentes domínios repetitivos (DR), apresentando um total de 551 sequências de espaçadores e os genes associados cas1, cas2 e cas3. Cas 9 e cas 10 não foram identificados. Foi observado que as cepas resistentes aos fagos líticos possuíam um maior número total... / Abstract: Salmonella Enteritidis is a pathogen that belongs to the Enterobacteriaceae family and is often related to infections transmitted by food in humans, especially in children, elderly and immunosuppressed patients. The importance of this microorganism is because it's significant prevalence with worldwide distribution in lots of poultry, leading to serious implications for public health and poultry industry. Effective control of salmonellosis is a challenging because involves a complex animal handling and treatment with antibiotics that do not guarantee the elimination of infection. The use of lytic bacteriophages against bacterial infections (phage therapy) meets a demand for alternatives to the conventional antimicrobial treatment. However, the literature has reported the emergence of bacterial resistance to some lytic bacteriophages, such as in Salmonella sp. There are several mechanisms of bacterial resistance to phages, such as prevention of adsorption, blocking the injection of phage DNA, restriction-modification, abortive infection and the CRISPR/Cas System acquired immunity. Thus, this research aims to study in a molecular level the bacterial resistance of lytic phages isolated from pathogenic strains of Salmonella enterica subsp. enterica serovar Enteritidis (SE). Two mechanisms have been chosen: the CRISPR/Cas system and blocking of phage adsorption via LPS. From 22 SE strains, a total of 72 CRISPRs was identified with 14 different repetitive domains (DR), presenting a total of 551 spacers' sequences and cas1, cas2 and cas3 CRISPR associated genes. Cas 9 and cas 10 genes weren't identified. It was observed that phage lytic resistant strains have a higher total number of spacers in the CRISPR locus in relation to sensitive strains, and this difference was statistically significant. Phylogenetic analysis of cas genes from CRISPR/Cas system, of rfaC, rfaH, cpsG, manB, manc, lpxA, lpxB and lpxC genes (related to LPS) and of... / Doutor

Com o aumento, a cada ano, dos alojamentos na cadeia de produção avícola, a disseminação de doenças ficou facilitada. Nos lotes de aves, a fase inicial tem principal importância no que tange à contaminação por Salmonella Enteritidis, devido a maior sensibilidade às infecções pela ausência de uma flora intestinal completa e a imaturidade do sistema imune. Avaliaram-se, neste estudo, os benefícios na progênie do uso de uma vacina inativada em adjuvante oleoso para Salmonella Enteritidis, em matrizes pesadas, através de coleta de ovos bicados e pintos natimortos no incubatório, bem como suabes de arrasto nos aviários de frangos de corte. Observou-se que a imunização das matrizes conferiu uma proteção para suas progênies, tanto em ovos bicados, pintos natimortos coletados no incubatório no dia do nascimento, bem como em granjas de frango de corte, onde foram coletados suabes de arrasto entre 21 e 30 dias. Em ovos bicados foram analisadas 850 amostras, sendo que nos lotes vacinados tiveram 10 amostras (1,18%) positivas, e, nos lotes não vacinados tiveram 288 amostras (33,88%) positivas. Em pintos natimortos foram analisados 850 amostras, sendo os lotes vacinados tiveram 16 amostras (1,88%) positivas e nos lotes não vacinados tiveram 210 amostras positivas (24,70%). Em suabe de arrasto, nas granjas de frangos de corte, foram analisadas 502 amostras para lotes não vacinados; sendo 22 amostras positivas (4,38%). Nos lotes vacinados foram analisados 475 amostras, sendo 6 amostras positivas (1,26%). Os resultados obtidos nos levam a concluir que a vacina de Salmonella Enteritidis inativada em adjuvante oleoso, propicia uma imunidade passiva para as progênies das matrizes de corte que foram vacinadas, proporcionando uma redução significativa a infecções precoces pela bactéria. / With the increase of households in the poultry production chain each year, the spread of diseases was facilitated. In lots of birds, the initial phase has primary importance regarding to contamination by Salmonella Enteritidis, due to a bigger sensitivity to the infections by the absence of a complete gut and the immaturity of the immune system. In this study it was evaluated, the benefits of using the progeny of an inactivated vaccine in oil adjuvant for Salmonella Enteritidis in the main weighted broilers, through collection of eggs pecked and hatchery stillbirth chicks, as well as drag swabs on poultry broiler. It was observed that immunization of matrices gave a protection to their progeny, both egg pecked, hatchery stillborn chicks collected on the day of birth, and in broiler farms, where the drag swabs were collected between 21 and 30 days. In pecked eggs were analyzed 850 samples, being that in the vaccinated lots 10 samples (1.18%) were positive and in non-vaccinated lots 288 samples (33.88%) were positive. In stillbirths chicks were analyzed 850 samples, and in the vaccinated lots were 16 samples (1.88%) positive and in the unvaccinated lots, 210 were positives samples (24.70%). In the drag swab in broiler farms, 502 samples were analyzed for non-vaccinated lots, with 22 positive samples (4.38%). In the vaccinated lots were analyzed 475 samples and six positive samples (1.26%). The results lead us to conclude that the Salmonella Enteritidis inactivated vaccine in oil adjuvant, provides passive immunity to the progeny of broiler breeder flocks were vaccinated, providing a significant reduction in early infections by bacteria.

Com a expansão da avicultura, ocorreu um aumento no número de aves alojadas, concentrando as produções e, consequentemente, facilitando a disseminação de doenças. Entre as doenças transmitidas pelo consumo dos produtos avícolas, a salmonelose tem especial importância. Nos últimos anos tem ocorrido um aumento na prevalência do sorovar Salmonella Enteritidis em todo o mundo, sendo o mais isolado. A patogenia da Salmonella e a forma como ela interage com seu hospedeiro são fenômenos multifatoriais e complexos. Entre os principais fatores de virulência da Salmonella estão os genes de virulência. O objetivo deste trabalho foi a pesquisa de genes associados à virulência em cepas de S. Enteritidis oriundas de diferentes origens avícolas. Realizou-se a pesquisa de nove genes de virulência (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) em 84 amostras de S. Enteritidis. Os genes invA, hilA, sivH, sefA e avrA estiveram presentes em 100% das amostras (84/84); lpfA e sopE em 99% (83/84); agfA em 96% (81/84); e o gene spvC em 92% (77/84). Foi possível caracterizar as amostras em quatro diferentes perfis genéticos, sendo P1 positivo para todos os genes, P2 negativo apenas para spvC, P3 negativo apenas para agfA e P4 negativo para lpfA, spvC e sopE, sendo o grupo P1 o mais prevalente. Apesar de todas as cepas pertencerem a um mesmo sorovar, observou-se uma variação na presença dos genes de virulência. Os perfis estabelecidos e as frequências obtidas podem servir para a escolha de cepas a serem utilizadas para realização da PFGE e em futuros estudos in vivo. / The poultry industry has been characterized by an increase in the number of housed animals in recent years, concentrating the production and facilitating the spread of many diseases. Among all illness transmitted by food, including meat and eggs, salmonellosis is of particular importance. Currently, there has been an increase in prevalence of islament of Salmonella Enteritidis all around the world. The pathogenesis of Salmonella and how it interacts with the host are a complex and multifactorial phenomenon. Among virulence factors of Salmonella, the virulence genes are the most important. The aim of this survey was to search for genes associated with virulence in strains of S. Enteritidis isolated from different origins, all from poultry. We conducted a search of nine virulence genes (lpfA, agfA, sefA, invA, hilA, avrA, sopE, sivH, spvC) in 84 samples of S. Enteritidis. The genes invA, hilA, sivH, sefA e avrA were present in 100% of samples (84/84); lpfA and sopE in 99% (83/84); agfA in 96% (81/84); and spvC in 92% (77/84) of them. It was possible to characterize the samples in four different genetic profiles, P1 being positive for all genes, P2 negative only for spvC, P3 negative only for agfA and P4 negative for lpfA, sopE and spvC. The P1 was the most prevalent. Despite all the strains belong to the same serovar, it is possible to observe a variation in the presence of virulence genes. These established profiles and gene frequencies obtained are useful to select strains to be used in PFGE and in future in vivo studies.

Com o aumento, a cada ano, dos alojamentos na cadeia de produção avícola, a disseminação de doenças ficou facilitada. Nos lotes de aves, a fase inicial tem principal importância no que tange à contaminação por Salmonella Enteritidis, devido a maior sensibilidade às infecções pela ausência de uma flora intestinal completa e a imaturidade do sistema imune. Avaliaram-se, neste estudo, os benefícios na progênie do uso de uma vacina inativada em adjuvante oleoso para Salmonella Enteritidis, em matrizes pesadas, através de coleta de ovos bicados e pintos natimortos no incubatório, bem como suabes de arrasto nos aviários de frangos de corte. Observou-se que a imunização das matrizes conferiu uma proteção para suas progênies, tanto em ovos bicados, pintos natimortos coletados no incubatório no dia do nascimento, bem como em granjas de frango de corte, onde foram coletados suabes de arrasto entre 21 e 30 dias. Em ovos bicados foram analisadas 850 amostras, sendo que nos lotes vacinados tiveram 10 amostras (1,18%) positivas, e, nos lotes não vacinados tiveram 288 amostras (33,88%) positivas. Em pintos natimortos foram analisados 850 amostras, sendo os lotes vacinados tiveram 16 amostras (1,88%) positivas e nos lotes não vacinados tiveram 210 amostras positivas (24,70%). Em suabe de arrasto, nas granjas de frangos de corte, foram analisadas 502 amostras para lotes não vacinados; sendo 22 amostras positivas (4,38%). Nos lotes vacinados foram analisados 475 amostras, sendo 6 amostras positivas (1,26%). Os resultados obtidos nos levam a concluir que a vacina de Salmonella Enteritidis inativada em adjuvante oleoso, propicia uma imunidade passiva para as progênies das matrizes de corte que foram vacinadas, proporcionando uma redução significativa a infecções precoces pela bactéria. / With the increase of households in the poultry production chain each year, the spread of diseases was facilitated. In lots of birds, the initial phase has primary importance regarding to contamination by Salmonella Enteritidis, due to a bigger sensitivity to the infections by the absence of a complete gut and the immaturity of the immune system. In this study it was evaluated, the benefits of using the progeny of an inactivated vaccine in oil adjuvant for Salmonella Enteritidis in the main weighted broilers, through collection of eggs pecked and hatchery stillbirth chicks, as well as drag swabs on poultry broiler. It was observed that immunization of matrices gave a protection to their progeny, both egg pecked, hatchery stillborn chicks collected on the day of birth, and in broiler farms, where the drag swabs were collected between 21 and 30 days. In pecked eggs were analyzed 850 samples, being that in the vaccinated lots 10 samples (1.18%) were positive and in non-vaccinated lots 288 samples (33.88%) were positive. In stillbirths chicks were analyzed 850 samples, and in the vaccinated lots were 16 samples (1.88%) positive and in the unvaccinated lots, 210 were positives samples (24.70%). In the drag swab in broiler farms, 502 samples were analyzed for non-vaccinated lots, with 22 positive samples (4.38%). In the vaccinated lots were analyzed 475 samples and six positive samples (1.26%). The results lead us to conclude that the Salmonella Enteritidis inactivated vaccine in oil adjuvant, provides passive immunity to the progeny of broiler breeder flocks were vaccinated, providing a significant reduction in early infections by bacteria.

O objetivo deste estudo foi o de avaliar a eficiência da aderência de produtos de exclusão competitiva e de probiótico sobre a tonsila cecal de frangos SPF, com idade de um a doze dias. Através da microscopia eletrônica de varredura, verificou-se a ação destes produtos comerciais com relação a proteção ao epitélio, quando desafiados com Salmonella Enteritidis. O delineamento experimental foi dividido em cinco (05) grupos, sendo o grupo T1 o controle negativo, o grupo T2 recebeu o produto de exclusão competitiva Aviguard, o grupo T3 recebeu o produto de exclusão competitiva Broilact, o grupo T4 recebeu o probiótico Biotop e o grupo T5 o controle positivo. Após 12 horas do tratamento com os produtos de exclusão competitiva ou probiótico as aves foram infectadas, por via oral, com S. Enteritidis. Os resultados obtidos mostraram que os produtos de exclusão competitiva e probiótico apresentaram uma maior eficiência em estabelecer uma microbiota protetora contra S. Enteritidis, e que a adição destes pode produzir um efeito de melhor índice zootécnico, quando comparado com as aves que foram desafiadas com S. Enteritidis e que apresentaram um ganho de peso inferior. / The aim of this study was to evaluate the ultra structure of the cecal tonsil in SPF chicks using scanning electron microscopy, determining the efficacy of competitive exclusion products and probiotic and its protection in cecal epithelium against an oral challenge with Salmonella Enteritidis. The experimental design included five groups: T1 negative control, T2 chicks treated with Aviguard, T3 chicks treated with Broilact, T4 chicks treated with Biotop, and, T5 positive control. After 12 hours of treatment with competitive exclusion products or probiotic the chicks were infected orally with S. Enteritidis. Results showed that treated groups had a protective effect stimulating the establishment of the resident microflora against pathogenic bacteria. Treated chicks showed high weight gain when compared to chicks challenged.

Com o aumento, a cada ano, dos alojamentos na cadeia de produção avícola, a disseminação de doenças ficou facilitada. Nos lotes de aves, a fase inicial tem principal importância no que tange à contaminação por Salmonella Enteritidis, devido a maior sensibilidade às infecções pela ausência de uma flora intestinal completa e a imaturidade do sistema imune. Avaliaram-se, neste estudo, os benefícios na progênie do uso de uma vacina inativada em adjuvante oleoso para Salmonella Enteritidis, em matrizes pesadas, através de coleta de ovos bicados e pintos natimortos no incubatório, bem como suabes de arrasto nos aviários de frangos de corte. Observou-se que a imunização das matrizes conferiu uma proteção para suas progênies, tanto em ovos bicados, pintos natimortos coletados no incubatório no dia do nascimento, bem como em granjas de frango de corte, onde foram coletados suabes de arrasto entre 21 e 30 dias. Em ovos bicados foram analisadas 850 amostras, sendo que nos lotes vacinados tiveram 10 amostras (1,18%) positivas, e, nos lotes não vacinados tiveram 288 amostras (33,88%) positivas. Em pintos natimortos foram analisados 850 amostras, sendo os lotes vacinados tiveram 16 amostras (1,88%) positivas e nos lotes não vacinados tiveram 210 amostras positivas (24,70%). Em suabe de arrasto, nas granjas de frangos de corte, foram analisadas 502 amostras para lotes não vacinados; sendo 22 amostras positivas (4,38%). Nos lotes vacinados foram analisados 475 amostras, sendo 6 amostras positivas (1,26%). Os resultados obtidos nos levam a concluir que a vacina de Salmonella Enteritidis inativada em adjuvante oleoso, propicia uma imunidade passiva para as progênies das matrizes de corte que foram vacinadas, proporcionando uma redução significativa a infecções precoces pela bactéria. / With the increase of households in the poultry production chain each year, the spread of diseases was facilitated. In lots of birds, the initial phase has primary importance regarding to contamination by Salmonella Enteritidis, due to a bigger sensitivity to the infections by the absence of a complete gut and the immaturity of the immune system. In this study it was evaluated, the benefits of using the progeny of an inactivated vaccine in oil adjuvant for Salmonella Enteritidis in the main weighted broilers, through collection of eggs pecked and hatchery stillbirth chicks, as well as drag swabs on poultry broiler. It was observed that immunization of matrices gave a protection to their progeny, both egg pecked, hatchery stillborn chicks collected on the day of birth, and in broiler farms, where the drag swabs were collected between 21 and 30 days. In pecked eggs were analyzed 850 samples, being that in the vaccinated lots 10 samples (1.18%) were positive and in non-vaccinated lots 288 samples (33.88%) were positive. In stillbirths chicks were analyzed 850 samples, and in the vaccinated lots were 16 samples (1.88%) positive and in the unvaccinated lots, 210 were positives samples (24.70%). In the drag swab in broiler farms, 502 samples were analyzed for non-vaccinated lots, with 22 positive samples (4.38%). In the vaccinated lots were analyzed 475 samples and six positive samples (1.26%). The results lead us to conclude that the Salmonella Enteritidis inactivated vaccine in oil adjuvant, provides passive immunity to the progeny of broiler breeder flocks were vaccinated, providing a significant reduction in early infections by bacteria.

6 Zusammenfassung In-vitro- und In-vivo-Studien zur Wirksamkeit von Eigelbantikörpern gegen Salmonella Enteritidis Michael Gürtler Institut für Lebensmittelhygiene der Veterinärmedizinischen Fakultät der Universität Leipzig Infektionen mit Salmonellen gehören weltweit zu den wichtigsten von Tieren auf den Menschen übertragbaren Erkrankungen. Anteilmäßig besitzen dabei die durch kontaminierte Lebensmittel, insbesondere Eier und Eiprodukte, hervorgerufenen Infektionen die größte Bedeutung. Dabei ist nach wie vor Salmonella Enteritidis (S. Enteritidis) in den Geflügelbeständen vorherrschend. Als Möglichkeit zur Bekämpfung von Salmonella-Infektionen bietet sich die passive Immunisierung der Legehennen durch orale Applikation von Eigelbantikörpern an. Bereits mehrfach wurde das Verfahren der Verabreichung von antikörperhaltigem Volleipulver zur Bekämpfung von bakteriellen Infektionskrankheiten, vor allem bei Kälbern und Ferkeln, getestet. Untersuchungen über die Einsatz solcher Eigelbantikörper mit dem Ziel der Zurückdrängung von Salmonellen in Legehennenbeständen sind bisher nicht bekannt geworden. Ziel dieser Arbeit war es, im Rahmen von In-vitro- und In-vivo-Studien den Einfluss von Eigelbantikörpern auf Vermehrung und Vorkommen von S. Enteritidis bei Legehennen zu untersuchen. Im Versuchskomplex A wurde in vitro ermittelt, ob Anti-S. Enteritidis-Antikörper im Eidotter in der Lage sind, die Vermehrung von Salmonellen zu hemmen. Im Versuchskomplex B wurde untersucht, ob es gelingt, durch die orale Verabreichung von Anti-S. Enteritidis-Eigelbantikörpern die Kontaminationsrate der gelegten Eier mit Salmonellen bei experimentell infizierten Legehennen zu beeinflussen. Im Versuchskomplex C sollte geprüft werden, ob Eigelbantikörper die Fähigkeit besitzen, das Adhäsions- und Invasionsvermögen von Salmonellen an bzw. in Epithelzellen einer permanenten Zelllinie zu beeinflussen. Im Rahmen von Versuchskomplex A wurden Legehennen mit einem inaktivierten S. Enteritidis-Stamm parenteral hyperimmunisiert, um hohe Konzentrationen an Antikörpern im Dotter zu induzieren. Die Eidotter wurden mit zwei S. Enteritidis-Stämmen in drei verschiedenen Kontaminationsdosen beimpft und das Vermehrungsverhalten durch Keimzählung als Generationszeit ermittelt. Im Versuchskomplex B wurden in zwei Infektionsversuchen Anti-S. Enteritidis-Eigelbantikörper in Form von Volleipulver in einer Menge von 3 g pro Tier und Tag über das Futter an experimentell mit S. Enteritidis infizierte Legehennen verabreicht und die Ergebnisse mit denen einer Kontrollgruppe ohne Volleipulver verglichen. Die gelegten Eier wurden getrennt nach Eischale, Eiklar und Eigelb auf den Infektionsstamm untersucht. Im Versuchskomplex C wurden die Adhäsivität und Invasivität verschiedener Salmonella-Stämme an bzw. in Epithelzellen bei An- bzw. Abwesenheit von Anti-S. Enteritids-Eigelb- bzw. -Serumantikörpern unter Verwendung einer permanenten Dünndarmepithelzelllinie von der Ratte (IEC-6) nach Inkubation durch Keimzählung überprüft. Die Ergebnisse in Versuchskomplex A zeigten, dass Eigelbantikörper nicht in der Lage sind, im Eigelb befindliche Salmonellen in ihrer Vermehrung zu hemmen oder gar abzutöten. Die Infektionsversuche im Versuchskomplex B ergaben, dass bei einer Infektionsdosis von 2x109 koloniebildenden Einheiten (kbE)/Tier die Salmonella-Kontaminationsrate der Eier durch das antikörperhaltige Eipulver nicht gesichert verändert wird. Dagegen konnte beim Einsatz einer solchen von 2x108 kbE/Tier eine Wirkung des antikörperhaltigen Eipulvers auf die Kontamination der einzelnen Eikompartimente nachgewiesen werden. Dabei wurden in der Versuchsgruppe mit 13,3 % gesichert weniger Eier als positiv detektiert als in der Kontrollgruppe mit 29,4 % (p ≤ 0,001), wobei mindestens in einem der drei Kompartimente Eischale, Eiklar oder Eigelb Salmonellen nachweisbar waren. In der Versuchsgruppe wies jedes Salmonella-positive Ei auch Keime auf der Eischale auf. In der Kontrollgruppe waren mit 29,9 % signifikant mehr Eischalen positiv als in der Versuchsgruppe mit 13,3 % (p ≤ 0,001). Beim Eiklar ließ die geringe Nachweisrate sowohl in der Versuchs- als auch in der Kontrollgruppe eine statistische Beurteilung des Eipulvereinflusses nicht zu. Aus dem Eidotter wurden in der Versuchsgruppe bei 0,6 % der Eier Salmonellen isoliert, während es in der Kontrollgruppe mit 2,4 % signifikant mehr waren (p ≤ 0,05). Die Ergebnisse des Versuchskomplexes C zeigten, dass Volleiantikörper und Serumantikörper die Invasion von S. Enteritidis in intestinale Epithelzellen serovarspezifisch vermindern können. Insgesamt lassen die Ergebnisse die Schlussfolgerung zu, dass bei einer oralen Verabreichung von Volleipulver mit S. Enteritidis-spezifischen Antikörpern an Legehennen bei einer Infektionsbelastung von 2x108 kbE/Tier mit einer gesicherten Reduzierung des Vorkommens von Salmonellen im Nahrungsmittel Ei im Vergleich zu Tieren ohne Zugabe von Eipulver zu rechnen ist. / In vitro and in vivo studies on the efficiency of egg yolk antibodies against Salmonella Enteritidis Michael Gürtler Institute of Food Hygiene, Faculty of Veterinary Medicine, University of Leipzig Infection of humans by Salmonella are among the most important zoonotic diseases. Contaminated food, especially eggs and egg products, represents the highest risk. Still Salmonella Enteritidis (S. Enteritidis) dominates, a serovar that occurs especially in poultry flocks. A possibility to combat Salmonella infection is the passive immunisation of laying hens by oral administration of egg yolk antibodies. The application of antibody-containing full-egg powder against bacterial diseases was tested previously especially on calves and piglets. The suitability of administration of egg-yolk antibodies to protect laying-hens flocks against Salmonella was not tested previously. It was the aim of the study to investigate the influence of egg-yolk antibodies of S. Enteritidis in egg laying hens by in-vivo and in-vitro studies. In part A, we investigated the influence of anti-S. antibodies in egg yolk on the multiplication of Salmonella in vitro. In part B, the impact of oral application of anti-Salmonella full-egg antibodies on the in-vivo Salmonella-contamination rate of eggs layed by experimentally infected hens was investigated. In part C the ability of egg-yolk antibodies and serum antibodies were tested with regard to the rate of adhesion and invasion of Salmonella on or in epithelial cells of a permanent cell culture line (two major pathogenic mechanisms). In part A, laying hens were hyper-immunised parenterally by an inactivated S. Enteritidis strain to induce a high concentration of antibodies in the egg yolk. Egg yolk was than inoculated with two S. Enteritidis strains in three different contamination doses. Multiplication of Salmonella cells was detected by cell counts and calculated as generation time. In part B, laying hens were immunised passively with anti-Salmonella egg yolk antibodies (by feeding 3 g of full egg powder per day and per animal) and the effects were compared with a control group without full-egg powder. The inoculation strain of S. Enteritidis was detected on egg shell, in egg white and in egg yolk separately. In part C, a suspension of S. Enteritidis was added to a permanent duodenal epithelial cell line of the rat (IEC-6). After incubation the adhesivity of Salmonella strains and their ability to invade these cells in the presence or in the absence of anti-S. Enteritidis egg yolk and serum antibodies, respectively, was detected by cell count. The results of part A demonstrated that egg-yolk antibodies are not able to inhibit multiplication of Salmonella in egg yolk. Experiments of part B showed that the Salmonella contamination rate of eggs could not be influenced by antibody-containing egg powder at a Salmonella infection dose of 2x109 colony forming units (cfu)/animal, whereas the antibody-containing egg powder at an Salmonella infection dose of 2x108 cfu/animal proved effective (i.e. contamination rate of egg components was reduced). In the experimental group 13.3 % of the eggs were Salmonella positive, which was significantly different from 29.4 % in the control group (p ≤ 0,001). At least in one of the three compartiments egg shell, egg white or egg yolk Salmonella was detectable. In the experimental group Salmonella could be isolated from the shell of every positive egg. The rate of Salmonella isolation from egg shells of the control group was significantly higher those in the experimental group (p ≤ 0,001). With regard to egg white, the low number of Salmonella detections in both groups did not allow statistical evaluation of the effect of full-egg powder. From egg yolk of the experimental group in 0.6 % of the eggs Salmonella could be isolated which was significantly lower than the detection rate in the control group with 2.4 % (p ≤ 0,05). The results of part C demonstrated that full-egg antibodies and serum antibodies can inhibit the serovar-specific invasion of S. Enteritidis into intestinal epithelial cells. As a whole, the results support the conclusion that oral administration of full-egg powder containing S. Enteritidis specific antibodies to egg-laying hens exposed to a administration of 2x108 cfu/animal reduce the occurrence of Salmonella in the egg compared to eggs of animals without egg powder administration.