Orientador: Tomomassa Yano / Dissertação (mestrado) - Universidade Estadual de Campinas,. Instituto de Biologia / Made available in DSpace on 2018-08-14T11:00:37Z (GMT). No. of bitstreams: 1 Thome_JacquelineDarcSilva_M.pdf: 1223668 bytes, checksum: 17699f260f82fb8a09136004fcc1e28a (MD5) Previous issue date: 2006 / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

This study was conducted to determine the extent in which Campylobacter jejuni colonized live birds would survive evisceration and contaminate the processed carcasses. Birds were infected with a marker strain of Campylobacter jejuni and allowed to grow to market age. Cloacal and fecal samples were analyzed to determine the level of Campylobacter jejuni present in the live bird. Prior to slaughter, birds were selectively subjected to two different temperatures (21 and 32°C) and three different times of feed withdrawal for chickens (3, 6,and 9 hours and turkeys 0, 4, and 8 hours). Birds were then slaughtered and the carcasses were sampled to determine the level of Campylobacter jejuni that survived. Results indicated a difference between chickens and turkeys, especially regarding the infective dose and bacterial survival rates. No significant differences in carcass contamination due to feed withdrawal times at either temperature were noted. The correlation of fecal samples with cloacal samples was significant for year 2 with r = .53 (p .04). For turkeys, the correlations were not significant. A longitudinal study of turkeys showed that the percentage of birds infected with Campylobacter jejuni peaked when the birds were 5-7 weeks old. The amount of Campylobacter contamination in each turkey peaked when the birds were 5 weeks old and then dropped off quickly. / Master of Science

Résumé : Depuis plusieurs années, la géomatique occupe une place de plus en plus importante dans la compréhension des phénomènes complexes ou la dimension spatiale est requise. De nombreuses données sont nécessaires pour faire l’étude de la distribution du Campylobacter dans l’environnement. La bactérie Campylobacter jejuni (CJ ) est un pathogène entérique responsable d'environ 300 0 cas de campylobacteriose humaine par année au Québec . Le principal moyen de transmission de C J serait la consommation ou la manipulation de volaille crue ou mal cuite ainsi que la consommation d'eau contaminée ou de produits laitiers non pasteurises. L'objectif principal du projet CampyloGIS est d'utiliser un système d'information géographique (SIG ) pour représenter spatialement les cas de campylobacteriose humaine acquis en Estrie en vue d'obtenir des informations d'aide à la décision dans la lutte contre la maladie. Les objectifs particuliers à la présente recherche sont de créer le modèle conceptuel du SIG. Le site d'étude choisi dans le cadre du projet CampyloGIS est la région de l’Estrie, au Québec. Le modèle conceptuel de l’annexe 1 a été construit à partir des hypothèses de recherche de CampyloGIS. II a permis de bien agencer les besoins thématiques en fonction des hypothèses de recherche de CampyloGIS. Donc, la construction du modèle conceptuel a permis de mieux cerner les différents thèmes inclus dans chaque hypothèse de recherche en plus de faciliter le travail d'identification des données nécessaires a chaque thème. Aussi, de par la hiérarchisation et les regroupements de concepts effectues dans le modèle conceptuel, une partie de la modélisation spatiale du SIG a été faite en amont du modèle logique. Par ce travail, les données nécessaires à la création du SIG ont pu être identifiées précisément regroupées en fonction des questions soulevées par le projet. Lors d'un stage sur le terrain au cours de l’hiver 2007 , nous avons observé que les sources potentielles de contamination des cours d'eau en Estrie par la bactérie C J étaient : l’activité agricole, les sorties d'égout en milieu fortement urbanise (Sherbrooke) , les fosses septiques et/ou champs d'épuration en milieu inondable ou près des cours d'eau ainsi que les rassemblements importants d'oiseaux situes près des points de collecte. // Abstract : Over recent years, geomatics has taken increasing importance place in the understanding of complex phenomena where a spatial dimension is requested. According to previous studies, the analysis of the distribution of campylobacter in the environment requires considerable data. Campylobacter jejuni (CJ) is an enteric pathogen that causes 3000 cases of human campylobacteriosis per year in Québec only. The main transmission mechanism for CJ is probably the consumption or the manipulation of raw or insufficently cooked poultry or the consumption of contamined water and unpasteurised dairy products. The main objective of the CampyloGIS project is to use a GIS to spatially represent human campylobacteriosis cases in the Eastern Townships area, to obtain information that will aid decision-making by Public Health authorities for disease control. The particular objectives of the present research are to create the conceptual model for the GIS. The study area chosen for the CampyloGIS project is the Eastern Townships area in Québec. The conceptual model presented in annex 1 has been built from the CampyloGIS research hypotheses. The conceptual model has allowed us to match the thematic needs according to the research hypotheses of CampyloGIS. Accordingly, the construction of the conceptual model has allowed us to better delineate the themes included in each of the research hypotheses and to facilitate data identification work for each theme. Thanks to the concept grouping and hierarchy established in the conceptual model, a part of the GIS spatial modelisation was already made possible by the logical model. From this work, the data needed for the creation of the GIS was precisely identified and grouped according to the questions the project wanted to answer. During field work done during the winter of 2007, it has been possible to observe that possible sources of river contamination by CJ included agricultural activities, sewers pipes in urban areas (Sherbrooke), septic pits in floodable areas or near rivers, and the gathering of numerous birds near the water collecting points.

Bovine Genital Campylobacteriosis is characterized by early pregnancy loss and temporary infertility in cattle. The purpose of this project was to compare diagnostic approaches to detect Campylobacter fetus subsp. venerealis and evaluate the efficacy of vaccination for Bovine Genital Campylobacteriosis. This thesis describes the results of two studies that compared different sample preparation methods for bovine vaginal mucus for real-time PCR and assessed a commercial vaccine in preventing infection and reproductive loss. The first study compared real-time PCR utilizing different bovine vaginal mucus sample preparation techniques to direct culture. The magnetic bead based protocol demonstrated higher sensitivity (48.4%, P=0.02) and lower specificity (78.9%, P=0.01) than the heat lysis protocol which involved an additional dilution step (Sens=29.4%, Spec=88.2%), but did not differ from the heat lysis protocol without sample dilution (Sens=35.0%, P=0.16; Spec=81.1%, P=0.62). The sample preparation method, designed for bovine preputial samples (Chaban et al. 2012. Can J of Vet Res; 76: 166), did not work well for vaginal mucus. All modifications of that method and magnetic bead based extraction technique had low sensitivity compared to culture probably due to the biophysical properties of vaginal mucus, which could cause loss of targeted DNA during processing, or repeated sample freezing and thawing. Release of DNA directly from vaginal mucus by a modified heat lysis protocol with consequent real-time PCR could be a promising rapid screening approach after validating on fresh samples. The second study compared the risk of infection and reproductive failure in heifers, vaccinated with a commercial multivalent vaccine containing C. fetus antigen, to heifers vaccinated with a comparable product without C. fetus, that were exposed to infected bulls. There was no significant difference between groups either in risk of Campylobacter fetus subsp. venerealis isolation (P>0.17) or in the proportion of heifers that cultured positive at least once (P=0.42), as well as in the median number times of cultured positive samples (P=0.24) and the time to first cultured positive (P=0.67). There was no difference by treatment in the weekly proportions of heifers diagnosed pregnant by either ultrasound (P>0.31) or serum concentration of pragnancy specific protein B (P>0.31) during the study, as well as in the time to first pregnancy for heifers ever diagnosed as pregnant (P=0.30) and those that remained pregnant at the end of the study (P=0.70). Similarly, the difference was not detected by treatment in the proportion of animals, ever detected pregnant during the study (P=0.57) and in pregnancy loss rates (P=0.28). However, heifers that aborted were 4 times more likely to be cultured positive than those that did not abort (P=0.01). Heifers that were not pregnant at the end of the study cultured positive 1.5 times more often than pregnant animals in treatment group (P=0.04), while in control group such difference was 4 times (P=0.01). Heifers that were not pregnant at the end of the study did not differ by treatment in the number of times cultured positive (P=0.14). In this study, the mean concentrations of ELISA antibodies to C. fetus after vaccination were more than 2 times higher in treatment group than in control group (P<0.02), but vaccination did not significantly reduce infection or improve pregnancy in heifers when exposed to Cfv-infected bulls. Sample preparation technique is important for successful real-time PCR; release of DNA directly from a CVM sample by a modified heat lysis protocol was easy to perform and could be promising as a rapid screening approach for Bovine Genital Campylobacteriosis after validating on fresh samples. Vaccinating of heifers with a polyvalent commercial vaccine, containing Campylobacter fetus antigen, according to the label, did not significantly reduce infection rate or improve reproductive performance when they were naturally challenged.

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

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

Memoria para optar al Título Profesional de Médico Veterinario / Aunque la presencia de Campylobacter jejuni ha disminuido considerablemente en productos avícolas, esta bacteria sigue siendo un riesgo importante para la salud humana. Es por ello que se analizó la presencia de C. jejuni en carne de pollo congelado obtenido de una planta procesadora en Santiago, Chile. Las muestras provinieron de dos plantas faenadoras (A y B) ubicadas en la Región Metropolitana, pertenecientes a la misma planta procesadora. Un total de 300 muestras fueron analizadas, siendo la unidad de muestra una bolsa sellada de 500 gramos, la cual contenía distintos cortes de ave congelada. Las bolsas fueron transportadas manteniendo la cadena de frío y procesadas inmediatamente después de su descongelación. Submuestras de cada bolsa fueron sembradas en caldo de enriquecimiento (caldo Exeter modificado), y luego se determinó la presencia de C. jejuni utilizando paralelamente dos medios de cultivo (mCCDA y Skirrow). Todos los cultivos se observaron macroscópicamente, siendo aquellas colonias sospechosas, verificadas con tinción de gram (bacilo curvo negativo). La determinación de la especie se realizó mediante el esquema de bio-tipificación descrita por Lior (Nachamkin y Blaser, 2000). Según los análisis realizados, la presencia de C. jejuni en carne de ave congelada alcanzó un 12% (36/300). Los porcentajes de presencia en cada planta faenadora fue de 15,15% (20/132) y 9,52% (16/168), para A y B, respectivamente. De acuerdo con los análisis estadísticos realizados, la diferencia entre estas proporciones no fue significativa (p>0,05). Estos valores son menores a los encontrados por Figueroa et al. (1996), quienes determinaron un 37% para muestras similares, lo que podría deberse a un mejor manejo sanitario en el faenamiento de las aves u otros procedimientos realizados a las muestras evaluadas, como por ejemplo en la crianza o transporte. Es posible entonces concluir, según los resultados obtenidos, que C. jejuni estuvo presente en las muestras de carne de ave analizadas, en similar nivel e independientemente de la fuente muestreada (planta faenadora A y B). Los 2 resultados obtenidos muestran la urgente necesidad de determinar la situación actual nacional y específicamente definir los puntos de contaminación dentro del faenamiento, para establecer las posibles medidas de control.

Memoria para optar al Título Profesional de Médico Veterinario / Campylobacter es un patógeno entérico, Gram negativo altamente diseminada en el medio ambiente provocando en el humano la campylobacteriosis, que es una de las enfermedades gastrointestinales más frecuente a nivel mundial. Las especies involucradas son C. jejuni y C. coli. Se considera el consumo de carne de ave la principal fuente de contagio. Para determinar su prevalencia en cepas aisladas desde carne de ave (n=40), heces de ave (n=40) y deposiciones pacientes humanos infectados (n=20), se escogieron once genes que determinan mecanismos de virulencia cómo flaA, cadF, racR y dnaJ relacionados con adherencia y colonización, virB11, ciaB y pldA relacionados con la invasión celular y los genes cdtA, cdtB y cdtC relacionados con la expresión de citotóxinas y el gen wlaN relacionado con el síndrome de Guillian Barré (GBS). Tanto flaA como cadF obtuvieron la mayor prevalencia en C. jejuni con 79,7%, mientras la menor la obtuvo virb11 con 7,6%. La determinación de su distribución en distintas fuentes permitirá conocer la situación del país y contribuir a la creación de métodos de prevención de esta enfermedad / Financiamiento: Proyecto Fondecyt 11110200 y Proyecto FIV 2011