Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Memoria para optar al Título Profesional de Médico Veterinario / Los serotipos de Salmonella tradicionalmente se han clasificado mediante métodos serológicos que determinan antígenos somáticos y flagelares específicos. Sin embargo, este método diagnóstico es caro y tarda mucho tiempo en dar un resultado ya que requiere implementar una batería de anticuerpos para detectar los más de 2.500 serotipos de Salmonella enterica que se han identificado. Por otra parte, la identificación molecular de los genes responsables de la expresión de antígenos flagelares son más rápidos y más sensibles que la identificación serológica. Es por esto que, en la presente memoria se implementaron pruebas de PCR para identificar S. enterica serotipos Typhimurium y Hadar, ambas incluidas en un plan nacional de control de Salmonella en establecimientos comerciales de aves. Se analizaron 135 cepas, 50 correspondientes a S. Typhimurium, 50 a S. Hadar y 35 enterobacterias como controles negativos. Estas cepas, previamente serotipificadas en el Instituto de Salud Pública (ISP), fueron sometidas a la prueba de PCR para determinar si existen diferencias de diagnóstico entre ambas técnicas. Del total de cepas analizadas, sólo 46 cepas de S. Typhimurium dieron positivas a la prueba de PCR y cuatro dieron negativas, mientras que las 50 cepas de S. Hadar dieron positivas a las dos pruebas de PCR. Las 35 enterobacterias dieron negativas a las 3 pruebas de PCR. De acuerdo a los resultados obtenidos en el estudio, la detección de antígenos mediante serotipificación y PCR para las cepas de S. Typhimurium fue menor al esperado (92%), a diferencia de lo que ocurrió con las cepas de S. Hadar en que hubo 100% de acuerdo entre ambas técnicas, sugiriendo que esta prueba de detección de Salmonella es posible reemplazarla por los métodos tradicionales de identificación y así poder acelerar el diagnóstico de esta bacteria de gran importancia para la salud pública

Manifiesta que el cuy es una especie de producción que se ve afectada principalmente por Salmonella Typhimurium, la cual expresa factores de virulencia codificados por diferentes genes, que, en conjunto, cumplen funciones coordinadas para llevar a cabo la patogenia y desarrollar la enfermedad. Por ello, el presente estudio evaluó la presencia de 10 genes codificantes de diversos factores de virulencia de importancia biológica de un total de 100 aislados de Salmonella Typhimurium, compuestos por 90 cepas procedentes de cuyes enfermos y 10 cepas procedentes de cuyes aparentemente sanos, confirmados en estudios previos. El ADN de los aislados fue extraído y analizado mediante la técnica de PCR múltiple, para evaluar la presencia de los genes spvB, spiA, cdtB, sipB, tolC, sitC, lpfC, sifA, sopB y pefA, obteniéndose un patrón genético similar, con frecuencias de detección variables mayores al 60%, tanto en aislados de cuyes sanos como enfermos, excepto el gen cdtB, el cual no fue detectado; concluyéndose que no existe diferencia entre los factores de virulencia presentes en cepas de Salmonella Typhimurium aisladas de cuyes enfermos y cuyes aparentemente sanos; sin embargo, debe tenerse en cuenta el potencial peligro que representan los animales portadores dentro de la producción. / Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado Perú. Ministerio de la Producción. Programa Nacional de Innovación para la Competitividad y Productividad (Innóvate Perú) / Tesis

Evalúa la inclusión de probióticos en la dieta sobre el desarrollo de vellosidades intestinales y criptas de Lieberkühn en cuyes de engorde desafiados a Salmonella Typhimurium. Se utilizaron 30 cuyes machos genéticamente mejorados, distribuidos en un sistema completamente al azar de tres tratamientos con 10 repeticiones cada uno. Los tratamientos fueron: Tratamiento T1: Cuyes alimentados con la dieta base y desafiados con Salmonella Typhimurium (control negativo). Tratamiento T2: Cuyes alimentados con la dieta base + antibiótico promotor de crecimiento desafiados con Salmonella Typhimurium (control positivo). Tratamiento 3: Cuyes alimentados con la dieta base suplementados con probiótico y desafiados con Salmonella Typhimurium. Todos los tratamientos fueron desafiados al duodécimo día de iniciada la investigación con dosis infectiva de 2 x 10 6 UFC/ml de 48 horas de incubación de Salmonella Typhimurium, administrados vía oral. Se tomaron muestras de las tres secciones del intestino de cada animal a los 63 días de edad y fueron remitidas para la elaboración de láminas histológicas para mediciones histomorfométricas. El grupo tratado con probiótico y APC mostró un efecto positivo (p<0.05) en la longitud y ancho de las vellosidades intestinales en la sección del íleon a los 63 días de edad con respecto al control. Con estos resultados se concluye que la dieta suplementada con probióticos tiene un efecto positivo sobre la morfometría intestinal en cuyes de engorde desafiados a Salmonella typhimurium llegando a ser estadísticamente significativa en la longitud y ancho de las vellosidades intestinales de íleon. / Tesis

Evalúa el efecto de la suplementación vía oral de una mezcla probiótica sobre los parámetros sanguíneos e índices productivos de cuyes (Cavia porcellus) de engorde desafiados con Salmonella Typhimurium. Los cuyes fueron distribuidos en tres tratamientos con diez (10) repeticiones cada uno. Los tratamientos fueron: Tratamiento T1: Cuyes alimentados con dieta base y desafiados con Salmonella Thyphimurium. Tratamiento T2: Cuyes alimentados con dieta base suplementados con probiótico y desafiados con Salmonella Thyphimurium. Tratamiento 3: Cuyes alimentados con la dieta base + antibiótico promotor de crecimiento desafiados con Salmonella Thyphimurium. Todos los tratamientos fueron desafiados al 12avo día de iniciada la investigación con una dosis infectiva de 2x106 UFC/ml de Salmonella Thyphimurium, administrados vía oral por única vez. Se registraron los datos de parámetros productivos: ganancia de peso, consumo de alimento e índice de conversión alimenticia. El periodo de engorde duró 49 días al término de los cuales se colectaron las muestras de sangre durante el sacrificio. Las muestras fueron remitidas para su posterior análisis. De los datos obtenidos se hizo un análisis de diferencia de medias por medio de la prueba no paramétrica Kruskal Wallis, y se realizó un análisis de pares a partir del método de Tukey para determinar diferencias entre grupos. En la evaluación de los resultados de parámetros productivos y hematológicos no se halló diferencia estadística significativa. / Tesis

Immunologic competence is correlated with resistance to infectious disease which may be affected by exposure to certain compounds. Methyl parathion (MP) and carbofuran (CF) have been reported to affect the development of active immunity in the rabbit and the present research was conducted to quantify the dosage relationships of these two pesticides to both host resistance and acquired resistance in the mouse. Preliminary study was made on the virulence of Salmonella typhimurium in mice. Mortality rates among infected animals fed Purina Laboratory Chow diet providing 0.08, 0.7 3.0 mg MP/kg/day or 0.1, 0.6, 1.0 mg CF/kg/day along with untreated controls were determined and protection afforded the animals by vaccination under the influence of chemical treatment was examined. The nature of dose response was further investigated with the study of various parameters of host defense against microbial infection. Measurements were made of the bacterial population in tissues of mice during and after the course of infection, differential leukocyte counts, in vivo and in vitro opsonic effect of serum, clearance of the reticulo-endothelial system, agglutination titers of serum, total serum gamma-globulin levels, serum immunoglobulin levels with classes and subclasses, and lymphocyte response to mitogen stimulation. Dosage-related increases in mortality were seen in unvaccinated mice under both chemical treatments, and protection by immunization was decreased. Pesticide treatment extending beyond two weeks was required to obtain significant increases in mortality. Increased mortality was associated with increased numbers of viable bacteria in blood, decreased total gamma-globulins and specific immunoglobulins in serum, and reduced splenic blast transformation in response to mitogens. Serum opsonic activity of CF-treated animals was slightly reduced. These results support those of other investigations indicating effects of environmental toxicants upon the resistance and immune competency of experimental animals.

<p>In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the <i>cob</i> operon and the <i>btuB</i> gene in <i>Salmonella typhimurium</i>. The <i>cob</i> operon encodes most of the 25 genes required for the <i>de novo</i> synthesis of vitamin B12, and the <i>butB</i> gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in <i>Salmonella typhimurium</i>. The regulation by vitamin B12 of the <i>cob</i> operon and the <i>btuB</i> gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.</p>

In this thesis I have studied the mechanism by which vitamin B12 regulates the expression of the cob operon and the btuB gene in Salmonella typhimurium. The cob operon encodes most of the 25 genes required for the de novo synthesis of vitamin B12, and the butB gene encodes the outer membrane protein needed for transport of exogenous vitamin B12 into the cell. Vitamin B12 is used as a cofactor in four enzymatic reactions in Salmonella typhimurium. The regulation by vitamin B12 of the cob operon and the btuB gene requires sequences in the long leader regions of the respective mRNAs. Proper folding of the reader mRNA is essential for normal repression, in particular a hairpin structure that sequesters the ribosomal binding site (RBS). The upstream leader region contains two conserved sequence elements that are required for the vitamin B12 regulation; the translational enhancer (TE) element element and the B12 box. The TE element confers its enhancer function by resolving the downstream inhibitory RBS hairpin through basepairing with nucleotides in the stem. In the presence of vitamin B12, either B12 itself, or a B12 regulatory factor binds to the upstream reader region and prevents the enhancer function. This will inhibit unfolding of the RBS hairpin and repress translation.

The secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.

The genotoxicity of energetic compounds (i.e., explosives) that are known to be present in contaminated soils at military training sites has not been extensively investigated. Thus, the Salmonella mutagenicity and Muta(TM)Mouse assays were employed as in vitro assays to examine the mutagenic activity of twelve explosive compounds, as well as three soil samples from Canadian Forces Base Petawawa. Salmonella analyses employed strains TA98 (frameshift mutations) and TA100 (base-pair substitution mutations), as well as the metabolically-enhanced YG1041 (TA98 background) and YG1042 (TA100 background), with and without exogenous metabolic activation (S9). For Salmonella analyses, the results indicate that ten of the explosive compounds were mutagenic, and consistently elicited direct-acting, base-pair substitution activity. All three soil samples were also observed to be mutagenic, eliciting direct-acting, frameshift activity. Mutagenic potencies were significantly higher on the metabolically-enhanced strains for all compounds and soil samples. For Muta(TM)Mouse analyses on FE1 cells, the results indicate that the majority of explosive compounds did not exhibit mutagenic activity. All three soil samples elicited significant positive responses (PET 1 and PET 3 without S9, and PET 2 with S9), and although there is some evidence of a concentration-related trend, the responses were weak. Correspondence of the mutagenic activity observed with the two assay systems, for both the explosive compounds and soil samples, was negligible. The differential response is likely due to differences in metabolic capacity between the two assay systems. Furthermore, it is likely that there are unidentified compounds present in these soil samples that are, at least in part, responsible for the observed mutagenic activity. Additional testing of other explosive compounds, as well as soil samples from other military training sites, using a variety of in vitro and in vivo assays, is warranted in order to reliably estimate mutagenic hazard and subsequently assess risk to human health.