A Salmonella é um dos mais importantes patógenos veiculados por alimentos. Dentre os sorotipos, a Salmonella Heidelberg (SH) vem se destacando nas monitorias dos plantéis avícolas devido ao fato de possuir um grande número de reservatórios, apresentar sorotipos inespecíficos quanto aos hospedeiros e possuir cepas multirresistentes aos antibacterianos. Aditivos Eubióticos, como probióticos e prebióticos vem sendo avaliados, não para substituir totalmente os antimicrobianos, mas para ser uma ferramenta estratégica para diminuir seu uso. O presente estudo teve como objetivo avaliar o efeito de dois probióticos e um prebiótico contra um desafio horizontal de Salmonella Heidelberg em frangos de corte, através da contagem de UFC/g de conteúdo cecal e sob os parâmetros zootécnicos. Foram utilizadas 224 aves, distribuídas em 16 boxes de 2m2 cada. As aves foram agrupadas em 4 tratamentos com 4 repetições de 14 aves por boxe, um total de 56 aves por grupo em um delineamento em blocos casualizado (DBC). Foi testado um prebiótico constituído por Mananoligossacarídeos (MOS) associado a um fermentado de Bacillus subtilis (Pre1) fornecido no 2º e 14º dias de idade, por gavagem oral, e mais dois probióticos, um constituído por 11 cepas de Lactobacillus (Pro1) fornecido no 1º, 17º e 18º dias, na água de bebida, e outro constituído de esporos de Bacillus subtilis (Pro2) fornecido na ração durante todo período experimental. A inoculação de SH foi aos 2 dias de idade usando (1,0x106 UFC/ave) por gavagem oral em 4 aves denominadas contaminadas por boxe, as demais 10 aves foram denominadas não contaminadas para avaliar a contaminação horizontal. Semanalmente as aves foram pesadas e calculado seu consumo de ração em cada grupo. Aos 28 dias de idade, 40 aves não contaminadas por grupo foram sacrificadas e avaliado a contagem de UFC/g do conteúdo cecal. Os resultados obtidos nas contagens de UFC/g de conteúdo cecal em cada tratamento Pre1, Pro1 e Pro2 não apresentaram diferenças significativas (P>0.05) em relação ao grupo controle. O resultado zootécnico apresentou apenas diferenças significativas (P<0.05) na conversão alimentar, com resultados favoráveis ao uso dos probióticos e prebiótico nos períodos finais de desenvolvimento das aves 14 a 21 e 21 a 28 dias de idade. / Salmonella is one of the leading foodborne pathogens. Among the serotypes, Salmonella Heidelberg (SH) has been standing out in the creation of chickens because of the fact of having a large number of reservoirs, presenting nonspecific serotypes and multidrug-resistant strains to antibacterial. Eubiótics additives such as probiotics and prebiotic are being evaluated not to fully replace antibiotics, but to be a strategic tool to reduce their use. This study aimed to evaluate the effect of two probiotics and prebiotic against a horizontal challenge Salmonella Heidelberg in broilers through count CFU/g of cecal contents and under the zootechnical parameters. 224 chickens were used, distributed in 16 boxes of 2m2 every. The chickens were grouped into 4 treatments with 4 replications of 14 chickens every boxe, a total of 56 chickens per group on a design in casualized blocks (DBC). It has been tested a prebiotic consisting mananoligossacarids (MOS) associated with a fermentation of Bacillus subtilis (Pre1) provided on the 2 and 14 days of age by oral gavage, and two probiotics, one composed of 11 Lactobacillus strains (Pro1) provided at 1, 17 and 18 days, in drinking water, and another composed of Bacillus subtilis spores (Pro2) supplied the feed all through the experimental period. Inoculation with SH was at 2 days of age using (1,0x106 UFC/chicken) by oral gavage for 4 chicks denominated contaminated by boxe, the other 10 chicks were denominated non contaminated to evaluate the horizontal contamination. The chickens were weighed a weekly basis, and feed consumption calculated from each group. At 28 days of age, 40 poultry non contaminated per group were sacrificed and we evaluated the counts CFU/g of cecal content. The results obtained for the count of CFU/g of cecal content in every Pre1 treatment, Pro1 and Pro2 no significant differences (P>0.05) compared to the control group. The zootechnical results presented only significant differences (P<0.05) feed conversion, with results favorable to the use of probiotics and prebiotics in late periods of growth of the poultry 14 to 21 and from 21 to 28 days.

Salmonella é uma das principais causas de Doenças Transmitidas por Alimentos em todo o mundo, sendo que no Estado do Rio Grande do Sul (RS) esse microrganismo tem sido apontado como um dos principais agentes de toxinfecções alimentares nos últimos anos. Neste trabalho foram caracterizados isolados de Salmonella envolvidas em salmoneloses ocorridas no RS, no período de 2007 a 2013. Entre os 163 isolados investigados, 138 (84,7%) foram sorotipificados com S. Enteritidis, enquanto os outros isolados foram S. Schwarzengrund (n = 9 – 5,5 %), S. Typhimurium (n = 6 – 3,7%), S. Infantis (n = 1 – 0,6 %), S. Agona (n = 1 – 0,6 %), S. Derby (n = 1 – 0,6 %), S. London (n = 1 – 0,6 %), S. Give (n = 1 – 0,6 %), S. Panama (n = 1 – 0,6 %) e S. enterica (n = 4 – 2,5 %). Os principais alimentos envolvidos nos surtos foram maionese caseira (17,39%), seguido dos produtos de confeitaria (15,94 %) e carnes (12,32 %). A resistência da S. Enteritidis a 12 agentes antimicrobianos também foi investigada. As maiores porcentagens de resistência foram encontradas em relação à nitrofurantoína (94,2 %) e ao ácido nalidíxico (89,1 %). A resistência para duas drogas foi verificada em 80,43 % dos isolados. Sendo que a multirresistência para três ou cinco antimicrobianos foi verificada em quatro e dois isolados, respectivamente. Quando os isolados foram submetidos à PCR-Ribotipificação, apenas um perfil de bandas foi identificado. Os resultados de PCR-Ribotipificação sugerem que uma mesma cepa de S. Enteritidis foi isolada a partir de alimentos envolvidos em salmoneloses ocorridas em diferentes municípios do Estado do RS no período de 2007 a 2013. Uma vez que o mesmo perfil de bandas foi identificado em S. Enteritidis causadoras de salmoneloses, durante 1999 a 2006, os resultados indicam que a mesma cepa de S. Enteritidis tem causado surtos alimentares no RS, durante o período de 1999 a 2013. / Salmonella is a major cause of Foodborne Diseases worldwide, and in the State of Rio Grande do Sul (RS) this microorganism has been identified as the main agent of foodborne diseases in last years. In this work, Salmonella isolates responsible for salmonellosis occurred in the State of RS, in the period 2007 to 2013 were characterized. Among the 163 isolates investigated, 138 (84.7 %) were serotyped as S. Enteritidis, whereas the other isolates were S. Schwarzengrund (n = 9 – 5.5 %), S. Typhimurium (n = 6 – 3.7 %), S. Infantis (n = 1 – 0.6 %), S. Agona (n = 1 – 0.6 %), S. Derby (n = 1 – 0.6%), S. London (n = 1 – 0.6 %), S. Give (n = 1 – 0.6 %), S. Panama (n = 1 – 0.6 %) and S. enterica (n = 4 – 2.5 %). The main food vehicles identified were homemade mayonnaise (17.39 %), followed by pastry products (15.94 %) and beef (12.32 %). The S. Enteritidis resistance to 12 antimicrobial agents was investigated. The highest percentages of resistance were found to nitrofurantoin (94.2 %) and nalidixic acid (89.1 %). The resistance to two different drugs was observed in 80.43 % of the isolates. Multidrug-resistance for three to five antimicrobials was observed in four and two isolates, respectively. When the isolates were analysed by PCR-Ribotyping, only one banding profile was identified. The results of PCR-Ribotyping suggest that the same strain of S. Enteritidis was isolated from foods involved in salmonelloses occurred in different municipalities of the State of RS in the period 2007-2013. Since the same banding pattern was found in strains involved in salmonellosis outbreaks of 1999 to 2006, results indicated that the same strain of S. Enteritidis has caused salmonellosis outbreaks in RS, during the period of 1999 to 2013.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-20T11:43:09Z No. of bitstreams: 2 Tese - Angélica Ribeiro Araújo Leonídio - 2018.pdf: 2065199 bytes, checksum: a1bb8ea110e5987fcdf43e15416a5c61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-20T14:32:14Z (GMT) No. of bitstreams: 2 Tese - Angélica Ribeiro Araújo Leonídio - 2018.pdf: 2065199 bytes, checksum: a1bb8ea110e5987fcdf43e15416a5c61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-11-20T14:32:14Z (GMT). No. of bitstreams: 2 Tese - Angélica Ribeiro Araújo Leonídio - 2018.pdf: 2065199 bytes, checksum: a1bb8ea110e5987fcdf43e15416a5c61 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-05-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of the present study was to evaluate the use of the ethanolic extract of of bark and seeds of Myrciaria cauliflora in the incubation of fertile eggs and in the feeding of broilers inoculated with Salmonella Heidelberg, observing possible beneficial and or protective effects of this phytogenic on health systemic effects of birds. Three experiments were carried out at the Experimental Center for Bird Diseases and Laboratory of Bacteriology of the Department of Veterinary Medicine of the Veterinary and Zootechnical School of the Federal University of Goiás. In the first experiment was evaluated the antimicrobial in vitro activity was evaluated and the minimum inhibitory concentration of ethanolic extract of M. cauliflora on Salmonella enterica different strains. In the second experiment, 320 fertile eggs distributed in randomized design were used in four treatments with 10 replicates each: CN - negative control group; EJ - group that received only the extract in the shell; SH - group that received only the bacterial inoculum in the shell; and SH + EJ - group that received the bacterial inoculum and the extract in the shell. The eggs incubated non-hatched were submitted to embryodiagnosis and processed for Salmonella in the embryos. The chicks born were housed until 10 days of age, during which time the collection and processing of samples for Salmonella research, serum biochemistry and hemogram were performed. In the third experiment, 336 one - day - old male chicks were distributed in randomized design in four treatments with seven replicates each: CN - negative control group; EJ - group that received only the vegetal extract in the diet; SH - group that received the inoculum bacterial in crop; and SH + EJ - group that received the bacterial inoculum in crop and the extract in the diet. Performance indexes, fecal excretion and presence of Salmonella in organs, organ biometry, intestinal histomorphometry, goblet cell counts and immune cells in organs and blood were evaluated. In the first experiment, the extract presented low inhibitory action on the strains of Salmonella enterica,, Salmonella Typhi, Salmonella Heidelberg and Salmonella Typhimurium. In the second experiment it was verified that the extract promoted less hatchability and higher embryonic mortality. The Salmonella isolation was higher in the embryo and meconium samples of the group that received the extract. At 10 days of age the extract reduced the excretion and colonization of the organs by Salmonella and the hematocrit and serum cholesterol level. In the third experiment it was verified that the extract increased the excretion of Salmonella in the inoculated birds and reduced the number of lymphocytes in the blood. It is concluded that the use of the ethanolic extract of bark and seeds of M. cauliflora presented weak antimicrobial activity in vitro for the strains of Salmonella enterica tested. In embryonated eggs its use is not feasible, however when added in the diet, it can stimulate the production of lymphocytes in lymphoid organs. / Objetivou-se com este estudo avaliar o uso do extrato etanólico de cascas e sementes de Myrciaria cauliflora na incubação de ovos férteis e na alimentação de frangos de corte inoculados com Salmonella Heidelberg, observando possíveis efeitos benéficos e/ou protetores do extrato sobre a saúde das aves. Foram conduzidos quatro experimentos no Núcleo Experimental de Doenças de Aves e Laboratório de Bacteriologia do Departamento de Medicina Veterinária da Escola de Veterinária e Zootecnia da Universidade Federal de Goiás. No primeiro experimento foi avaliada a atividade antimicrobiana in vitro e a concentração inibitória mínima do extrato sobre sorovares de Salmonella. No segundo experimento foram utilizados 320 ovos férteis distribuídos em delineamento inteiramente ao acaso em quatro tratamentos com 10 repetições cada: CN – grupo controle negativo; EJ – grupo que recebeu apenas o extrato na casca; SH – grupo que recebeu apenas o inóculo bacteriano na casca; e SH + EJ – grupo que recebeu o inóculo bacteriano e o extrato na casca. Os ovos incubados não eclodidos ao final da incubação foram submetidos ao embriodiagnóstico e processados para pesquisa de Salmonella. Os pintos nascidos foram alojados até os 10 dias de idade, período em que se realizou a coleta de amostras para pesquisa de Salmonella, bioquímica sérica e hemograma. No terceiro e quarto experimentos foram utilizados 336 pintos machos de um dia de idade distribuídos em delineamento inteiramente ao acaso em quatro tratamentos com sete repetições cada: CN – grupo controle negativo; EJ – grupo que recebeu apenas o extrato na ração; SH – grupo que recebeu o inóculo bacteriano via inglúvio; e SH + EJ – grupo que recebeu o inóculo bacteriano via inglúvio e o extrato na ração. Foram avaliados o desempenho, excreção fecal e presença de Salmonella em órgãos, biometria de órgãos, histomorfometria intestinal, contagem de células caliciformes e células imunológicas em órgãos e no sangue. No primeiro experimento, o extrato apresentou fraca ação inibitória sobre Salmonella enterica, Salmonella Typhi, Salmonella Heidelberg e Salmonella Typhimurium. No segundo experimento verificou-se que o extrato promoveu menor eclodibilidade e maior mortalidade embrionária. O isolamento de Salmonella foi maior nas amostras de embriões e mecônio do grupo que recebeu o extrato. Aos 10 dias de idade o extrato reduziu o hematócrito, o nível de colesterol sérico e a excreção e colonização dos órgãos. No terceiro experimento verificou-se que a suplementação com o extrato não influenciou o desempenho, aumentou a excreção de Salmonella nas aves inoculadas e reduziu o número de linfócitos no sangue das aves. Conclui-se que o uso do extrato etanólico de M. cauliflora apresentou fraca atividade antimicrobiana in vitro nas cepas de Salmonella testadas. Nos ovos embrionados o seu uso não é viável, entretanto quando acrescentado na ração, pode estimular a produção de linfócitos em órgãos linfoides.

Salmonella é uma das principais causas de Doenças Transmitidas por Alimentos em todo o mundo, sendo que no Estado do Rio Grande do Sul (RS) esse microrganismo tem sido apontado como um dos principais agentes de toxinfecções alimentares nos últimos anos. Neste trabalho foram caracterizados isolados de Salmonella envolvidas em salmoneloses ocorridas no RS, no período de 2007 a 2013. Entre os 163 isolados investigados, 138 (84,7%) foram sorotipificados com S. Enteritidis, enquanto os outros isolados foram S. Schwarzengrund (n = 9 – 5,5 %), S. Typhimurium (n = 6 – 3,7%), S. Infantis (n = 1 – 0,6 %), S. Agona (n = 1 – 0,6 %), S. Derby (n = 1 – 0,6 %), S. London (n = 1 – 0,6 %), S. Give (n = 1 – 0,6 %), S. Panama (n = 1 – 0,6 %) e S. enterica (n = 4 – 2,5 %). Os principais alimentos envolvidos nos surtos foram maionese caseira (17,39%), seguido dos produtos de confeitaria (15,94 %) e carnes (12,32 %). A resistência da S. Enteritidis a 12 agentes antimicrobianos também foi investigada. As maiores porcentagens de resistência foram encontradas em relação à nitrofurantoína (94,2 %) e ao ácido nalidíxico (89,1 %). A resistência para duas drogas foi verificada em 80,43 % dos isolados. Sendo que a multirresistência para três ou cinco antimicrobianos foi verificada em quatro e dois isolados, respectivamente. Quando os isolados foram submetidos à PCR-Ribotipificação, apenas um perfil de bandas foi identificado. Os resultados de PCR-Ribotipificação sugerem que uma mesma cepa de S. Enteritidis foi isolada a partir de alimentos envolvidos em salmoneloses ocorridas em diferentes municípios do Estado do RS no período de 2007 a 2013. Uma vez que o mesmo perfil de bandas foi identificado em S. Enteritidis causadoras de salmoneloses, durante 1999 a 2006, os resultados indicam que a mesma cepa de S. Enteritidis tem causado surtos alimentares no RS, durante o período de 1999 a 2013. / Salmonella is a major cause of Foodborne Diseases worldwide, and in the State of Rio Grande do Sul (RS) this microorganism has been identified as the main agent of foodborne diseases in last years. In this work, Salmonella isolates responsible for salmonellosis occurred in the State of RS, in the period 2007 to 2013 were characterized. Among the 163 isolates investigated, 138 (84.7 %) were serotyped as S. Enteritidis, whereas the other isolates were S. Schwarzengrund (n = 9 – 5.5 %), S. Typhimurium (n = 6 – 3.7 %), S. Infantis (n = 1 – 0.6 %), S. Agona (n = 1 – 0.6 %), S. Derby (n = 1 – 0.6%), S. London (n = 1 – 0.6 %), S. Give (n = 1 – 0.6 %), S. Panama (n = 1 – 0.6 %) and S. enterica (n = 4 – 2.5 %). The main food vehicles identified were homemade mayonnaise (17.39 %), followed by pastry products (15.94 %) and beef (12.32 %). The S. Enteritidis resistance to 12 antimicrobial agents was investigated. The highest percentages of resistance were found to nitrofurantoin (94.2 %) and nalidixic acid (89.1 %). The resistance to two different drugs was observed in 80.43 % of the isolates. Multidrug-resistance for three to five antimicrobials was observed in four and two isolates, respectively. When the isolates were analysed by PCR-Ribotyping, only one banding profile was identified. The results of PCR-Ribotyping suggest that the same strain of S. Enteritidis was isolated from foods involved in salmonelloses occurred in different municipalities of the State of RS in the period 2007-2013. Since the same banding pattern was found in strains involved in salmonellosis outbreaks of 1999 to 2006, results indicated that the same strain of S. Enteritidis has caused salmonellosis outbreaks in RS, during the period of 1999 to 2013.

Chapter I Introduction Salmonellae are facultative Gram-negative intracellular pathogens. Different serovars of it causes a variety of diseases in multiple hosts with different disease outcomes. Salmonella enterica serovar Enteritidis and Typhimurium (STM) can infect domestic animals causing gastroenteritis or typhoid like fever. Typhoid fever in humans which is actually caused by Salmonella enterica serovar Typhi still remains a significant health problem in many parts of the world with an estimated annual incidence of nearly 16 million cases and about 600,000 deaths. The infection begins via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18 macrophages present in the intestinal lumen or via a forced entry in the non-phagocytic enterocytes. Upon entry, Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV) and has several strategies to counteract the host defense mechanisms. Following phagocytosis and its compartmentalization into Salmonella containing vacuole (SCV), a series of defense mechanisms are initiated. These include toxic reactive oxygen species or super oxide production, nitric oxide production, phagosomal acidification and release of hydrolases and defensins through fusion of phagosome with lysosomes generating highly bactericidal environment. The SCV transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7 and resist killing by avoiding phagosomal maturation and vesicular trafficking of iNOS and NADPH oxidase vesicles. Moreover, Salmonella also uses acidic pH of the SCV (~ pH 4.5) to assemble the Salmonella Pathogenecity Island 2 (SPI-2) type three secretion system (TTSS) which is essential for survival inside the macrophages. Salmonella uses these hostile conditions inside the host as cues for regulating their virulence factors using global regulatory factors. Hfq is one such global regulator playing an important role in many physiological processes and stress responses. Understanding the importance of Hfq regulated genes which impart Salmonella survival advantage under hostile conditions for successful infection will be of particular significance. The host too recognizes pathogen using innate immune receptors present either on the cell surface like TLRs (Toll Like Receptors) or inside the cells like NLRs (Nod Like Receptors). Innate immune receptors recognize pathogen associated molecular patterns (PAMPs) such as Lipopolysacharide (LPS), peptidoglycon (PGN), or hypomethylated DNA or RNA. Recognition of PAMPs by innate receptors leads, via activation of transcription factors (NF-κB and IRF3), to the generation of pro and anti-inflammatory cytokines, chemokines. Vaccination has been practiced for many years and it is one of the most effective methods of controlling infectious diseases like typhoid. At present two licensed vaccines against Salmonella are in use globally namely, Vi polysaccharide subunit vaccine (Typhim Vi™) and live attenuated typhoid vaccine (Vivotif Berma™). Lack of immunological memory, low efficacy (55-75 % protection) and requirement of higher number of doses are the important practical shortcomings associated with the currently used vaccines. So there is a need for a safer and immunogenic vaccine to combat Salmonella infection. Chapter II Salmonella Typhimurium lacking hfq gene induces long term memory response and confers protective immunity Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new improved ones. Of the various strategies used for the generation of vaccine strains, focus is on manipulation of virulence regulator genes for bacterial attenuation. Hfq is a RNA chaperon which mediates the binding of small RNA to the mRNA and assists in post-transcriptional gene regulation in bacteria. Salmonella hfq deletion mutant is highly attenuated in vitro as well as in vivo implying its role in bacterial virulence. In this study, we have evaluated the efficacy of the Salmonella Typhimurium hfq deletion mutant as a candidate for live oral vaccine against Salmonella infection in murine salmonellosis model. The hfq deletion mutant is not only able to confer protection when administered orally to the mice against oral challenge with serovar Typhimurium virulent strain, but also elicits cross protective immune responses to other Salmonella serovars. The vaccine candidate appears to be safe for use in pregnant mice. This protection is partially mediated by the increase in the number of CD4+ T lymphocytes upon vaccination. STM hfq deletion mutant further exhibited significant increase in the lipopolysaccharide as well as outer membrane protein specific IgG in the serum as well as secretory S-IgA in the intestinal washes. In addition, vaccination led to an increased serum IFN-γ and IL-6. Taken together, our results suggest that the Salmonella Typhimurium hfq deletion mutant can be an excellent live oral vaccine candidate. Chapter III Acidic pH induced STM1485 gene governs intracellular replication and pathogenesis in Salmonella During the course of infection, Salmonella has to face several potentially lethal environmental conditions such as low pH both inside and outside the host. The ability to sense and respond to the acidic pH is crucial for survival and replication of Salmonella. Exposure to acidic pH results in the expression of large pool of virulence genes. One such gene highly up regulated inside the macrophage is STM 1485. In order to understand physiological role of STM 1485 in Salmonella pathogenesis, STM 1485 gene was deleted chromosomally and characterized in vitro and in vivo. In vitro the mutant did not show any growth defects at pH 4.5 and no difference in acid tolerance response. The 1485 deletion mutant was compromised in its capacity to proliferate inside the cells and is further lowered inside activated macrophages. We further showed that surface translocation of SPI-2 encoded translocon protein SseB was reduced at low pH in vitro in STM 1485 mutant and the mutant was found to colocalize with lysosomes higher than the wild type. In addition, the STM 1485 deletion mutant displayed decreased virulence in murine typhoid model when infected intragastrically. Based on our results, we hypothesize that the acid shock protein encoded by the STM 1485 might be involved in the formation of SPI-2 translocon at low pH and there by contributing to the virulence of Salmonella. Chapter IV Role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells Nod1 and Nod2 are the archetypal members of the Nod like receptor family (NLR) and they recognize distinct peptidoglycan motifs of Gram-negative and Gram-positive bacteria respectively. Role of Nod1 and Nod2 in sensing bacterial pathogens have been elucidated. However, the role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells is not understood. So in this study we investiged the role of Nod1 in the innate immune response against Salmonella in epithelial cells. We demonstrate that the recognition of Salmonella by Nod1 leads to NF-κB activation and this activation is diminished in epithelial cells expressing a dominant-negative Nod1 construct or Nod1 shRNA. Using a set of Salmonella mutants we show that the availability of ligand is higher when the bacteria were in cytosol rather than in vacuole. Further we also observed that the Nod1 mediated killing of Salmonella is mediated through the defensins. Based on our results we hypothesize that Salmonella uses its vacuolar niche to evade Nod1 mediated innate immune response.

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Huit cent trente et un troupeaux de vaches laitières répartis dans 5 états américains ont été enrôlés dans une étude de cohorte prospective. Un modèle d’équations d'estimation généralisées a été utilisé pour étudier l'association entre les signes cliniques et la détection de salmonelles dans les fèces des animaux soupçonnés de salmonellose clinique. La sensibilité et la spécificité de la culture bactériologique ont été estimées à l’aide d’un modèle de classes latentes. Dix-huit pour cent des 874 échantillons provenant de veaux et 29% des 1479 échantillons de vaches adultes étaient positifs pour Salmonella spp. Il n’a pas été possible d’établir une association claire entre les différents signes cliniques observés et la détection de salmonelles. Les 2 sérotypes les plus fréquemment isolés étaient Typhimurium et Newport. La probabilité de détecter des salmonelles était plus élevée chez les veaux où un autre agent entéropathogène était également détecté. La proportion d’échantillons positifs était plus élevée parmi les vaches ayant reçu des antibiotiques dans les jours précédant l’échantillonnage. La sensibilité de la culture a été estimée à 0,48 (intervalle de crédibilité à 95% [ICr95%]: 0,22-0,95) pour les veaux et 0,78 (ICr95%: 0,55-0,99) pour les vaches. La spécificité de la culture était de 0,94 (ICr95%: 0,87-1,00) pour les veaux et de 0,96 (ICr95%: 0,90-1,00) pour les vaches. Malgré une sensibilité imparfaite, la culture bactériologique demeure utile pour obtenir une meilleure estimation de la probabilité post-test de salmonellose clinique chez un bovin laitier, par rapport à la probabilité estimée suite au seul examen clinique. / Eight hundred and thirty-one dairy herds in 5 states in the northeast USA were enrolled in a prospective cohort study. A generalized estimating equations model including herd as a random effect was built to study the association between clinical signs and detection of Salmonella spp. in the faeces of animals suspected of clinical salmonellosis. The sensitivity and specificity of the bacteriological culture were estimated using a latent class model. Eighteen percent of the 874 samples from calves and 29% of the 1479 samples from adult cows were positive for Salmonella spp. It was not possible to establish a clear association between the various clinical signs and detection of Salmonella spp. in the faeces. The two most frequently isolated serotypes were Typhimurium and Newport. The probability of detecting salmonellas was higher for calves when another enteric pathogen was also detected. The proportion of positive samples was higher among cows that received antibiotics in the days preceding the sampling. The sensitivity of the bacteriological culture was estimated at 0.48 (95% credibility interval [95%CrI]: 0.22 to 0.95) for calves and 0.78 (95%CrI 0.55 to 0.99) for cows. The specificity of the culture was 0.94 (95%CrI: 0.87 to 1.00) for calves and 0.96 (95%CrI: 0.90 to 1.00) for cows. Despite imperfect sensitivity, bacterial culture remains useful to obtain a better estimate of the post-test probability of clinical salmonellosis in dairy cattle, compared to the estimated probability following the clinical examination alone.

Salmonellen spp. sind invasive, intrazelluläre Pathogene, die in einem membranumhüllten Kompartiment innerhalb der infizierten Wirtszelle überleben. Wie auch andere intrazelluläre Pathogene repliziert Salmonella in dieser intrazellulären Nische, obwohl es anscheinend von sowohl extra- als auch intrazellulären Nährstoffquellen isoliert ist. Wir zeigen hier, dass intrazelluläre Salmonella den Proteinabbau des Wirts ausnutzen, um Aminosäuren in Form von Peptiden zu erhalten. Dieser spezielle, auch als Chaperon-vermittelte Autophagie bekannte, Abbauweg spielt eine Rolle im Transport zytosolischer Proteine zum Abbau im Lysosom. Ein Salmonellenmutant, der nur in Anwesenheit von Peptiden im Medium als Aminosäurenquelle wächst, wies intrazellulär eine Wachstumsrate auf, die der des Wildtyps ähnlich war. Dies deutet darauf hin, dass Peptide intrazellulär für Salmonella zugänglich sind. Wir fanden heraus, dass die Salmonella-enthaltende Vakuole (SCV, Salmonella containing vacuole) die Wirtproteine LAMP-2A und Hsc73, Kernkomponenten von CMA, anzieht, jedoch nicht lysosomale Proteine wie LAMP-2B und LIMP-2. Im Gegensatz zum Salmonellawildtyp zeigte der peptidabhängige Mutantentstamm stark verringertes Wachstum, wenn die Wirtszellen mit CMA-Inhibitoren behandelt wurde. Diese Ergebnisse zeigen einen neuen Mechanismus auf, durch den ein intrazelluläres Pathogen vom membranumhüllten Kompartiment aus Zugriff auf Cytosol der Wirtzelle zur Beschaffung von Nährstoffen hat. Wir schlagen vor, dass diese Ergebnisse eine Erklärung für die Rückfälle von persistenten Salmonellainfektionen liefern können. Des Weitern schlagen wir diesen Mechanismus als moegliches Ziel antibakterieller Therapeutika zur Bekämpfung intrazellulärer Pathogene vor. / Salmonella spp. are invasive, intracellular pathogens which survive and proliferate within a membrane-bound compartment inside infected host cells. Like other intracellular pathogens, Salmonella replicates within this intracellular niche, despite its apparent isolation from both extra- and intracellular sources of nutrients. Here, we show that intracellular Salmonella acquire amino acids in the form of peptides by co-opting the host protein degradation pathway known as chaperone-mediated autophagy (CMA) involved in the transport of cytosolic proteins to the lysosome for degradation. A mutant of Salmonella strictly dependent upon peptides in growth media as a source of amino acids, showed intracellular growth similar to the wild-type strain in host cells, indicating intracellular access to peptides. We found that the Salmonella-containing vacuole (SCV) acquires the host cell proteins LAMP-2A and Hsc73, key components of CMA, but excludes lysosomal proteins such as LAMP-2B and LIMP-2. In contrast to wild-type Salmonella, the peptide-dependent mutant strain showed a severe reduction in growth when host cells were treated with inhibitors of CMA.. These results reveal a novel means whereby an intracellular pathogen can access the host cell cytosol to acquire nutrients from within its membrane-bound compartment. We suggest these results may provide an explanation for relapse infections resulting from persistent Salmonella infections, and suggest a possible means of targeting antibacterials against intracellular pathogens.

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No. of bitstreams: 2 Dissertação - Angelica Ribeiro Araujo - 2014.pdf: 1233873 bytes, checksum: 9b40d445937726aa9426d3b123936726 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-02-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The present study aimed to evaluate the encapsulated butyric acid in the control of Salmonella enterica serovar Enteritidis, performance and systemic health of broiler chickens experimentally inoculated. 576 one-day-old male chicks were distributed in a completely randomized design with eight treatments and six replications each. The poultries were inoculated orally at one-day-old with 0.3 ml of phosphate buffered saline, containing approximately 2.0 x 10 6 CFU/ml of Salmonella enterica serovar Enteritidis. Treatment 1 (T1) was the control group (Placebo); T2 - received butyric acid at a dose of 0.03%; T3 - received butyric acid at a dose of 0.075%; T4 - received the butyric acid at a dose of 0.15%; T5 -group inoculated orally (Positive Control SE); T6 - inoculated orally treated with 0.03%; T7 - inoculated orally treated with 0.075%; T8 - inoculated orally treated with 0.15%. At 22 days of age, the chickens were reinfected with 0.5 ml of phosphate buffered saline containing concentration greater than 1.0 x 10 9 CFU/ml of Salmonella. Butyric acid favored the performance up to 21 days. The acid reduced the intestinal population E. coli. The intestinal pH did not differ by butyric acid, however Salmonella caused a reduction. At 14 days, the acidification effected the growth of the villi of the jejunum and Salmonella caused a reduction in the villous and greater relation villi : crypt in the duodenum and jejunum. No Salmonella was isolated from cloacal swabs collected from poultries that received the dose of 0.03%. Salmonella reduced intestinal weight. The colonization of Salmonella in organs at eight and 28 days of age was reduced in the presence of acid. Liver weight was smaller in the inoculated poultries and larger in the non-inoculated poultries and treated with 0.03% at 15 days. The spleen was smaller than in inoculated poultries which received the dose of 0.03%, at eight days. Salmonella reduced the bursa weight at 15 days.Dietary acidification reduced the lymphoid depletion in the inoculated group. Salmonella influenced the antibody production against Newcastle Disease and Salmonella enterica serovar Enteritidis. The dose of 0.15% increased the antibody production against Newcastle Disease in the inoculated groups and antibodies against Salmonella enterica serovar Enteritidis in the non-inoculated group at 42 days. The mortality of poultries did not differ between treatments. We conclude that, among the investigated dosages, the inclusion of 0.03% yielded the best results in performance and beneficial effects on intestinal health and organ biometrics. The dose of 0.15% improved humor immunity of the poultries. / O presente estudo teve como objetivo avaliar o ácido butírico encapsulado no controle de Salmonella enterica sorovar Enteritidis, desempenho e saúde sistêmica de frangos de corte experimentalmente inoculados. Foram utilizados 576 pintos machos de um dia de idade distribuídos em delineamento inteiramente ao acaso com oito tratamentos e seis repetições cada. As aves foram inoculadas via oral ao primeiro dia de idade com 0,3 mL de solução salina tamponada, contendo aproximadamente 2,0 x 10 6 UFC/mL de Salmonella enterica sorovar Enteritidis. O tratamento 1 (T1) consistiu o grupo controle (Placebo); T2 – recebeu o ácido butírico na dose de 0,03%; T3 – recebeu o ácido butírico na dose de 0,075%; T4 – recebeu o ácido butírico na dose de 0,15%; T5 – grupo inoculado via oral (Controle positivo SE); T6 – inoculado via oral tratado com 0,03%; T7 - inoculado via oral tratado com 0,075%; T8 - inoculado via oral tratado com 0,15%. Aos 22 dias de idade, as aves foram re-inoculadas com 0,5 mL de solução salina tamponada, contendo concentração superior a 1,0 x 10 9 UFC/mL de Salmonella. O ácido butírico favoreceu o desempenho até os 21 dias. O ácido reduziu a população intestinal de E. coli. O pH intestinal não foi influenciado pelo ácido butírico, porém Salmonella promoveu redução. Aos 14 dias, a acidificação influenciou o crescimento das vilosidades do jejuno e Salmonella provocou redução nas vilosidades e maior relação vilo : cripta no duodeno e jejuno. Não foi isolado Salmonella dos suabes de cloaca coletados das aves que receberam a dose de 0,03%. Salmonella reduziu o peso do intestino. A colonização de Salmonella nos órgãos aos oito e 28 dias de idade foi reduzida na presença do ácido. Peso do fígado foi menor nas aves inoculadas e maior nas aves não inoculadas e tratadas com 0,03% aos 15 dias. O baço foi menor nas aves inoculadas que receberam a dose de 0,03%, aos oito dias. Salmonella reduziu o peso da bursa aos 15 dias. A acidificação da dieta reduziu a depleção linfoide no grupo inoculado. Salmonella influenciou a produção de anticorpos contra Doença de Newcastle e Salmonella enterica sorovar Enteritidis. A dose de 0,15% aumentou a produção de anticorpos contra Doença de Newcastle no grupo inoculado e de anticorpos contra Salmonella enterica sorovar Enteritidis no grupo não inoculado aos 42 dias. A mortalidade das aves não diferiu entre os tratamentos. Conclui-se que, dentre as dosagens pesquisadas, a inclusão de 0,03% promoveu os melhores resultados no desempenho e efeitos benéficos sobre a saúde intestinal e biometria de órgãos. A dose de 0,15% melhorou a imunidade humoral das aves.

Chapter-I Introduction The bacteria known as Salmonellae are gram-negative, rod-shaped intracellular pathogenic bacilli that belong to the family Enterobacteriacea and causes typhoid fever. Enteric fever or typhoid fever is a systemic infection caused by human specific enteric pathogen S.typhi. Another very similar but less severe disease, paratyphoid fever, is caused by another human pathogen S.paratyphi A, B and C and S.sendai. Typhoid fever is estimated to have caused 21.6 million illness and 1-4 % death worldwide in the year of 2000 effecting all ages and 90% of death occurs in Asia. In Asia, the incident of typhoid fever was highest with 274 cases per 100,000 persons worldwide, especially in Southeast Asian countries and the Indian subcontinent, followed by sub-Saharan Africa and Latin America with 50 cases per 100,000 persons. Transmission of the disease occurs through faecal-oral route upon ingestion of contaminated water and food. Salmonella can stay for long in ground and pond water. Typhoid fever can be fatal if left untreated and there are reports of 10-30 fatality in such cases and can persist for weeks. Prevention is better than cure. Same hold true even for typhoid fever also. The important and key preventive measures are clean and safe water, safe food, personal hygiene and appropriate sanitation. There are many antibiotics for typhoid fever but till now there are only two licensed vaccine recommended by the World Health Organization for the typhoid fever, one Vi polysaccharide subunit vaccine (sold as Typhim Vi by Sanofi Pasteur and Typherix by GlaxoSmithKline) which is administered through intramuscular route and another one is live oral attenuated vaccine Ty21a (sold as Vivotif Berna) for oral immunization. Both the vaccines are recommended to be used for the children above the age of 3-5 years. Both are between 50 to 80% protective and are recommended for travelers to areas where typhoid is endemic. Salmonella has evolved many strategies to survive inside host system especially during initial time of infection when bacteria counteract to host AMPs in intestine lumen. Salmonella has many pathogenesis island which help bacteria to invade the host system e.g. SPI-1(Salmonella pathogenicity island -1) and also help in intracellular survival as well proliferation e.g. SPI-2 (Salmonella pathogenicity island -2). Salmonella has many strategies to evade host immune system, one of them which is very important for bacteria is LPS modification. Salmonella is capable to modify its own LPS by increasing the +ve charge and increasing AMPs resistance. This modification and resistance is brought about by PhoP/Q and pmrA/B two different two-component system (TCS). These TCS regulate many genes like pmrD, pmrC, pmrG, pmrH-M operon, pmrE etc, which are important for LPS modification by adding 4-amino-arabinose and provide antimicrobial peptide resistance. Chapter-II Development of live attenuated Salmonella vaccine The superiority of live attenuated vaccines in systemic salmonellosis has been proven over killed and subunit vaccines, because of its ability to induce protective cell mediated immunity by CD8+ T cells. A live attenuated Salmonella enterica serovar Typhimurium vaccine has been developed by systematic site directed deletion of the pmrG-HM-D chromosomal genomic loci. This gene confers involved in antimicrobial peptide resistance and is involved in LPS modification, both of which are the major immune evasive mechanisms in Salmonella. The efficacy of the newly developed strain in inducing protection against mortality after challenge with the virulent wild type Salmonella typhimurium 12023 was evaluated in mice model of typhoid fever. Animals were immunized and then boosted on days 7 and 14. Following challenge with virulent S. typhimurium 12023, organ burden and mortality of vaccinated mice were less compared to non-immunized controls. The vaccine strain also induced elevated CD8+ T cells in the vaccinated mice. This multiple mutant vaccine candidate appears to be safe for use in pregnant mice and provides a model for the development of live vaccine candidates against naturally occurring salmonellosis and typhoid fever. Chapter -III A Safe and Efficient Vaccine against Salmonella Infection During Pregnancy Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before onset of pregnancy and followed the immune reaction against challenge with virulent S. typhimurium in pregnant mice. This vaccine strain gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards humoral immunity through Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. Chapter-IV Crosstalk between Salmonella genes involved in antimicrobial peptide resistance (pmrG, pmrD, pmr H-M) The pmr system of Salmonella consists of many genes and they are regulated by two component system (TCS), PmrA/B and PhoP/Q. These two component systems are activated at different Mg 2+and Fe3+ condition, low pH and the presence of antimicrobial peptides. Downstream genes like pmrD, pmrG, pmrH-M operon, pmrE, pmrC ect which are regulated by these TCS are involved in LPS modification and AMPs resistance. When these genes were deleted a highly attenuated strain with good vaccine potential was developed. The high degree of attenuation of the vaccine strain is a combined effect of the deletion of the all genes, when single mutation of the two single genes and the operon were created; the attenuation was not as good as the vaccine strain. When tried checking the cross-talk between these genes in vaccine strain and the single mutants of pmrD, pmrG and pmrH-M operon. In one of the previous report pmrH-M mutant was shown to be attenuated through oral route but not through intra-peritoneal route. However, pmrD-HM-G mutant (DV-STM-07) was attenuated when administered through both the routes of infection. To further explain the cross-talk and regulation of these genes, promoter analysis was done for all genes individually in different mutant background of pmrD, pmrG, pmrH-M and DV-STM-07. We hypothesize that the superior attenuation of the triple mutant is achieved because of transcriptional cross-regulation that exists between these genes which attenuates the bacteria when administered through the intra-peritoneal route.