Orientador: Luís Antonio Mathias / Banca: Angelo Berchieri Junior / Banca: Masaio Mizuno Ishizuka / Resumo: Esta pesquisa objetivou demonstrar a ocorrência de reações sorológicas para Salmonella sp, Mycoplasma gallisepticum e Mycoplasma synoviae nas populações de aves de explorações não tecnificadas e com finalidade de subsistência, em propriedades rurais localizadas em áreas geográficas próximas a granjas de reprodutoras (matrizeiros) do Estado de São Paulo. Foram selecionadas três granjas de matrizes, oficialmente reconhecidas como sendo livres de S. Pullorum (SP), S. Gallinarum (SG), S. Enteritidis (SE), S. Typhimurium (ST), M. gallisepticum (MG) e M. synoviae (MS), e quinze criações vizinhas, onde a população de aves existente foi amostrada, com colheita e processamento de 406 soros sanguíneos. O diagnóstico foi realizado pela técnica de soroaglutinação rápida em placa. As frequências encontradas foram 73%, 73% e 100% de propriedades com aves sororreagentes aos antígenos testados de SP (teste da pulorose), MG e MS, respectivamente. As ocorrências observadas de aves sororreagentes foram de 16,5%, 30,3% e 40,6% para os antígenos de SP, MG e MS, respectivamente. Os dados obtidos indicam que os agentes estudados estão amplamente difundidos nas criações informais de aves de "fundo de quintal", colocando em risco constante os criatórios de exploração industrial, os quais necessitam adotar e manter boas práticas de biosseguridade para preservar a integridade sanitária dos plantéis. / Abstract: This investigation aimed to demonstrate the occurrence of serological reactions to Salmonella spp., Mycoplasma gallisepticum and Mycoplasma synoviae in backyard domestic poultry for own consume located next to parent flocks in the State of São Paulo. Three parent flocks officially recognised as free from S. Pullorum (SP), S. Gallinarum (SG), S. Enteritidis (SE), S. Typhimurium (ST), M. gallisepticum (MG) and M. synoviae (MS) and 15 neighbour backyard flocks were selected. A sample of 406 chickens from the backyard flocks was bled, and the diagnostic was carried out by the plate agglutination test. The frequencies found were 73%, 73% and 100% of flocks with chickens reacting to the antigens of SP, MG and MS, respectively. The frequencies of reacting chickens were 16.5%, 30.3% and 40.6% for the antigens of SP, MG and MS, respectively. The results show that the aetiological agents studied are widespread among the backyard flocks, posing a constant risk for the commercial poultry flocks, that need to adopt and keep good biosecurity practices to preserve their sanitary status. / Mestre

Memoria para optar al título de Bioquímico / Salmonella enterica serovar Typhi (S. Typhi) es una patógeno exclusivo del ser humano y agente causal de la fiebre tifoidea. Esta enfermedad constituye un problema de salud pública a nivel mundial especialmente en países en vías de desarrollo. Nuestro trabajo se ha enfocado a comprender la regulación de la producción del lipopolisacárido (LPS) y su papel en la virulencia de esta bacteria. El LPS es el componente principal de la envoltura de las bacterias Gram negativas y, por lo tanto, es un importante mediador de las interacciones con el hospedero. Estudios anteriores de nuestro laboratorio han demostrado que la producción del antígeno O (AgO), el componente más externo de la molécula de LPS, varía durante el crecimiento bacteriano, aumentando en las fases exponencial tardía y estacionaria. Esta modulación es mediada por el factor RfaH, que regula positivamente la transcripción del operón wba encargado de la síntesis del AgO. La expresión del gen rfaH también aumenta en la etapa de transición a fase estacionaria y su activación requiere la función del factor sigma alternativo RpoN. En este trabajo se estudió el mecanismo de activación de la transcripción de rfaH mediado por RpoN. Se planteó como hipótesis que el factor sigma de estrés RpoN regula la expresión de rfaH en S. Typhi, mediante la unión directa a la región promotora de rfaH o indirectamente a través de proteínas accesorias. El hallazgo previo de la existencia de dos sitios de inicio de la transcripción con sus respectivos promotores, se confirmó mediante la construcción de vectores reporteros que contienen fusiones transcripcionales de los respectivos promotores P1 o P2 al gen lacZ. Se demostró que el promotor P1 es el responsable de la regulación fase-dependiente de la transcripción de rfaH, en cambio el promotor P2 contribuiría marginalmente a esta regulación. Se estudio la unión de RpoN a una probable secuencia de reconocimiento de este factor sigma en el promotor P1. Para esto se clonó el marco de lectura en el vector pET-21b(+) y se sobreexpresó y purificó la proteína recombinante RpoN-HisTag mediante cromatografía de afinidad a Ni. Se utilizó una fracción altamente purificada de esta proteína en ensayos de retardo en geles (EMSA). Se demostró que RpoN no se une al promotor P1 y por lo tanto no participa directamente en la regulación fase-dependiente de rfaH. La regulación mediada por este factor sigma es dependiente de su activación por proteínas activadoras, denominadas "enhancer binding proteins" (EBP). Para identificar la EBP que participa en la regulación de rfaH, se construyeron mutantes por deleción en 6 genes para las probables EBPs encontradas en el genoma de S. Typhi. De estas mutaciones, sólo aquella en ygaA, factor encargado de la transcripción de genes para detoxificar NO, presentó un efecto significativo sobre la transcripción de la fusión rfaH-lacZ, que consistió en un aumento de la actividad β-galactosidasa durante la fase exponencial temprana. Los resultados de este trabajo indican que la regulación transcripcional fase-dependiente de rfaH es mediada de forma indirecta por RpoN, a través de proteínas accesorias que serían reguladas por este factor sigma para ejercer su efecto sobre el promotor P1.

Memoria para optar al Título Profesional de Médico Veterinario / Salmonella spp. es uno de los principales agentes causantes de gastroenteritis en la población humana. Las fuentes de infección las constituyen alimentos y agua contaminada con este agente, siendo los de origen animal los más frecuentemente contaminados. El objetivo del presente estudio fue describir la situación de la problemática de salmonelosis humana y animal en Chile en el periodo 2000-2006 y comparar la aplicación de buenas prácticas con la incidencia de salmonelosis humana. Se recopilaron antecedentes sobre el agente en diversas instituciones de salud pública y de salud animal comparando así los datos de prevalencia. Al mismo tiempo, se realizó un cuestionario Delphi de 33 preguntas a expertos en el tema. Los casos de salmonelosis humana mostraron un constante aumento según los registros del Instituto de Salud Pública (ISP). Los alimentos de origen cárneo toman una gran importancia frente al resto de los alimentos en el contagio de esta bacteria. En los meses de altas temperaturas existe un mayor número de toxiinfecciones. Los casos de salmonelosis en mataderos de aves también mostraron un aumento en los años de estudio, no existiendo relación con los casos humanos. Los expertos plantean que la salmonelosis es un problema grave tanto en el ámbito nacional como internacional, cuya principal fuente de contagio se produce al interior del hogar. Se concluye que los casos de salmonelosis humana y animal son crecientes, lo cual hace notar que a pesar de los esfuerzos desplegados, Salmonella spp. sigue siendo un agente de preocupación. Salmonella Enteritidis es el principal serotipo responsable de toxiinfecciones alimentarias humanas en Chile, pero ahora los productos de origen cárnicos desplazaron a los derivados del huevo como principal fuente de contagio. Se concluye finalmente que es fundamental implementar esfuerzos en conjunto aplicando buenas prácticas en toda la cadena de producción y mejores programas de educación al consumidor.

Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its proton to the α-carbon to give cysteine and regenerate enzyme to start the second half reaction. In addition, substrate specificity, stereochemistry of the internal Schiff base at C4', and sequence around active site lysine of O-acetylserine sulfhydrylase-A have been determined. The [4'-^3H]pyridoxamine generated by reduction of the internal Schiff base with sodium [^3H]borohydride retained most of its tritium after incubation with apoaspartate aminotransferase. These results agree with the hypothesis put forth by Dunathan (Dunathan, 1971; Dunathan and Voet, 1974) that a single surface (Re face) of the active site PLP is accessible to solvent. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg.

Call number: LD2668 .T4 1933 S33 / Master of Science

Salmonella is an important food borne pathogen. Over 100,000 cases of human Salmonella infection are reported in the European Union each year, resulting in an economic burden estimated to be around 3 billion Euros per year (EFSA, 2012). In a European Food Safety Authority (EFSA) survey between 2006 and 2007 S. Typhimurium was the most common serovar of Salmonella isolated from pig carcasses (EFSA, 2008a). Pigs can be asymptomatic carriers of S. Typhimurium (Berends et al., 1996) and contaminated pork contributes significantly to the number of human infections. It has been estimated that the porcine Salmonella reservoir contributes between 10-20% of human salmonellosis cases per year (VLA, 2010). In addition to improvements in biosecurity and husbandry practices, immune-prophylaxis is an important method to reduce the prevalence of food borne pathogens such as Salmonella in reservoir species. An understanding of the molecular basis of bacterial colonisation and persistence in the reservoir host is crucial to rational vaccine design and targeting relevant species. S. Typhimurium expresses multiple surface factors involved in adherence and colonisation of gut epithelium in several host species. The aim of this project was to identify factors involved in S. Typhimurium colonisation of the porcine gut. The work presented here specifically focuses on the role of flagella in the colonisation of porcine gut epithelium. Flagella are motility organelles possessed by many bacterial species. Flagella can also function as surface adhesins, shown in Escherichia coli O157:H7 (Mahajan et al., 2009), and Pseudomonas (De Bentzmann et al., 1996, Lillehoj et al., 2002). Flagellin is the major flagellar filament structural protein approximately 50kDa in size. Salmonella enterica has the ability to switch between two alternate, antigenic forms of its flagellin filament protein, expressing either FliC or FljB (Macnab, 1996). The biological relevance of these two types of flagella filament protein is still not understood. It has been postulated that the presence of a second phase type of flagella may offer an advantage to the bacteria by avoiding recognition by the immune system. However, studies have shown that both FliC and FljB flagella activate Toll-like receptor-5 (TLR-5) mediated by nuclear factor (NF)-κB signalling (Simon and Samuel, 2007b). One specific objective of this research was to compare the role of flagellar phase types in S. Typhimurium adherence and colonisation of porcine gut. To this end a porcine colonic primary epithelial cell culture and ex vivo tissue explants were developed as in vitro infection models. Primary colonic cell cultures were phenotypically characterised using specific markers for epithelial and M cells. In addition to primary epithelial cell culture, porcine intestinal epithelial cell line, IPEC-J2, was also used for specific flagellar interaction studies. The role of flagella in interaction of S. Typhimurium to porcine intestinal epithelium was tested using S. Typhimurium strain SL1344 and flagella mutant derivative strains. Flagella mutant strains exhibited reduced binding to porcine intestinal epithelial cells. Purified flagella proteins were also shown to bind porcine intestinal epithelial cells. Moreover, flagella specific anti-sera suppressed S. Typhimurium adherence to both porcine intestinal epithelial cells as well as porcine colonic explants. The immuno-protective role of flagella as a potential S. Typhimurium vaccine candidate was tested during vaccine efficacy studies in pigs. Parenteral immunisation of pigs with purified FliC and FljB flagella proteins induced production of both IgG and IgA antibodies. The vaccination of pigs with Salmonella flagella provided some protection against challenge as fewer ileum tissue samples from the pigs in the vaccinated group tested positive for Salmonella. The intestinal contents from the vaccinated pigs tested for Salmonella post mortem appeared to also have lower levels of Salmonella compared to un-vaccinated controls, though these were not significantly different between groups. This project has identified flagella as one potential subunit of a multivalent subunit vaccine to help control salmonellosis in the porcine reservoir.

In the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella is far lower than that in Escherichia coli. In this study, I firstly improved the recombineering-based strategy by using linear DNA targeting cassettes that contain long flanking ?arms? of sequence (ca. 1,000 base pairs) homologous to the chromosomal target. This reliable and efficient method enables multiple gene targeting procedures to be performed on a single Salmonella enterica serovar typhi Ty21a (Ty21a) chromosome in a straightforward, sequential manner with high efficiency. Secondly, I applied this improved strategy in construction of Salmonella to be live attenuated oral vaccine and tumor targeting vector. In the first part of this thesis, I describe an improved method in Ty21a. Using this strategy, I inserted three different influenza antigen expression cassettes as well as a green fluorescent protein reporter gene into four different loci on the Ty21a chromosome with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. The immune response of this vaccine was also evaluated by ELISA and ELIspots. In the second part of this thesisi, I use this improved recombineering strategy to engineer bacteria of Salmonella typhimurium (S. typhimurium) as therapeutic agents against solid tumor. In current study, a major challenge for bacterial therapy of cancer is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. An alternative approach was developed here. By placing an essential gene under a hypoxia conditioned promoter, S. typhimurium strain SL7207 was engineered to generate strain YB1 that survives only in anaerobic conditions without otherwise affecting its functions. In breast and liver tumor bearing mice models, YB1 grew within tumor, retarding its growth, while being rapidly eliminated from normal tissues. Mice treated with SL7207 were killed by infection within short period time. Inhibition of tumor growth by YB1 was significant and was enhanced by the addition of 5-FU in breast cancer model. The development of an “obligate” anaerobic Salmonella provides a much safer bacterial vector for further development of anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do. In summary, I have developed an efficient, robust and versatile method in genome-wide Salmonella genetic manipulation. Furthermore, I used this method to construct a recombinant Ty21a antigen-expressing vaccine strain and a tumor targeting YB1 strain. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Salmonella enterica is a leading cause of food-borne gastrointestinal disease worldwide. A survey conducted in 2002-2003 showed that oysters may contain Salmonella and thus may also be a source of salmonellosis. Since oysters are commonly consumed raw, no amount of food safety education will prevent consumers from ingesting a possibly infectious dose from Salmonella contaminated oysters. The research in this dissertation employed a combination of traditional culture techniques as well as genomics-based molecular applications to explore Salmonella infection in oysters and the subsequent risk to consumers of raw oysters. A year-long survey of oysters served on the half-shell in local restaurants determined that overall 1.2% of oysters were contaminated with Salmonella. Oysters containing Salmonella were found in 7 of the 8 months surveyed and 7 of the 8 restaurants served contaminated oysters. Six different serovars were isolated, but one strain of S. Newport, as determined by matching pulsed field gel electrophoresis patterns, represented 43% of the positive samples. Interestingly, this is the same strain that was predominantly isolated in the earlier survey of oysters and was also resistant to at least 7 different antimicrobials. The remainder of this dissertation work was an exploration of why this particular strain is seen so often in oyster infections. A custom microarray was used to perform a transposon site hybridization (TraSH) assay to identify genes that are necessary for S. Newport survival in the oyster. In this way, a negative selection was able to determine the genes that were necessary for S. Newport to survive in oysters. A subset of the genes identified by TraSH was selected and site-directed mutagenesis was performed to knock those genes out of LAJ160311. Oysters were infected with those mutant strains to test for their ability to survive in oysters and thereby determine the role of those individual genes in pathogenesis. The conclusions of the TraSH assay were that virulence factors that are essential for survival of Salmonella in mammalian models, particularly the type three secretion systems, may not be important in the oyster model. Motility provided by flagella was identified as a major virulence factor in oyster colonization by S. Newport.

No description available.