Pasteurella multocida é um importante patógeno para suínos, causando rinite atrófica progressiva, pneumonia, pleurite, e septicemia. Para implantação de estratégias de controle e prevenção desta infecção, torna-se necessário o conhecimento a respeito do perfil de disseminação do agente em condições naturais e em diferentes tipos de sistema de produção. Tendo em vista a inexistência de técnicas sorológicas padronizadas para esta finalidade na espécie suína, o presente estudo teve por objetivos avaliar em um grupo de 90 animais a influência de diferentes sítios de coleta de amostra na detecção de animais portadores de P. multocida através da PCR e comparar estes dados a detecção de anticorpos na espécie suína através de um kit de ELISA comercial registrado para detecção de anticorpos contra P. multocida em aves. Foram coletados suabes de cavidade nasal, suabes de tonsila e sangue de 90 animais em idade de abate provenientes de dois sistemas de produção de suínos. Dentre os 90 animais, 14 (15,55%) foram positivos para detecção de P. multocida em suabes nasais e nenhum foi positivo em suabe de tonsila. Através do ELISA, 25 (27,77%) animais apresentaram anticorpos contra o agente. Através da análise de comparação de proporção, houve diferença significativa em relação à metodologia de diagnóstico nos diferentes testes realizados considerando-se valor de p < 0,0001 e intervalo de confiança de 95%. Ou seja, a freqüência de positivos foi significativamente maior no teste de ELISA, seguido pela reação em cadeia pela polimerase em suabes nasais, sugerindo que a cavidade nasal é o sitio primário de colonização dos animais por este agente e que o ELISA testado pode ser facilmente adaptado para avaliação do perfil sorológico do agente na espécie suína. / Pasteurella multocida is an important pathogen for pigs, causing progressive atrophic rhinitis, pneumonia, pleurisy, and septicemia. For implementation of strategies to control and prevent infections, it is necessary to know about the profile of agent spread in natural conditions and in different types of production system. Given the lack of standardized serological techniques for this purpose, this study aims to evaluate the influence of different methods and sites of sample collection in the detection of animals with P. multocida by polymerase chain reaction (PCR) and ELISA. We evaluated different pairs of primers specific for the agent and the reaction that have lower detection threshold will be used in the evaluation of the study sites. Swabs were collected from the nasal cavity, tonsil and also blood of 90 animals. Among these, 14 (15.55%) were positive for Pasteurella multocida by polymerase chain reaction (PCR), all of which were nasal swabs. There are no positive animals among the tonsil swabs. In the ELISA, 25 (27.77%) were positive, and of these, three (3.33%) were positive in both the polymerase chain reaction and the ELISA and the remaining 22 (24.44%) was positive by ELISA and negative by polymerase chain reaction. Using comparison of proportion analysis, was observed a significant differences in the methodology of diagnosis in different tests considering p-value <0.0001 and a confidence interval of 95%. Concluding, the frequency of positives was significantly higher in ELISA than by the polymerase chain reaction.

O objetivo deste estudo foi o de verificar a ocorrência de quatro genes de virulência em Salmonella Enteritidis de acordo com o fagotipo e ribotipo, assim como a virulência “in vivo”. Os genes estudados foram invA, spvC, sefC e pefA em 120 amostras isoladas de várias fontes, pertencentes a diferentes fagotipos e ribotipos provenientes de sete estados brasileiros. Para a verificação da presença dos genes, as amostras foram examinadas pela técnica de Reação em Cadeia da Polimerase (PCR) individualmente e em multiplex. A ocorrência para o gene invA foi de 100% nas amostras (120/120), spvC em 94% (113/120), sefC em 97,5% (117/120) e pefA em 97% (116/120) das amostras. Foi possível caracterizar as amostras em cinco diferentes perfis. O primeiro, P1, foi positivo para os genes invA, spvC, sefC e pefA, P2 para invA, spvC e pefA, P3 para invA, sefC e pefA, P4 para invA e sefC e o último P5 para os genes invA e spvC. Para as amostras PT4RT1 obteve-se o perfil P1 em 94% (64/68), P2 em 1,5% (1/68), P3 em 3% (2/68) e P4 em 1,5% das amostras (1/68). Para as amostras PT4RT2, 86% (18/21) pertenceram ao perfil P1, 5% (1/21) ao P3 e 9% (2/21) das amostras ao perfil P4. Nas amostras PT4RT3 80% (4/5) pertenceram ao perfil P1 e 20% (1/5) ao P2. Nas amostras PT4TR9, 91% (10/11) pertenceram ao perfil P1 e 9% (1/11) ao P3. Todas as amostras PT4RT5, PT7RT1 e PT4RT10 pertenceram ao perfil P1 e apenas a amostra PT1 apresemtou o perfil P5. A virulência foi avaliada desafiando as aves via oral e subcutânea, através da colonização do ceco e invasão do fígado e baço. O gene spvC está relacionado com a sobrevivência e aumento da média de crescimento da bactéria no fígado e baço, mas neste estudo não demonstrou diferença na porcentagem de aves com reisolamento positivo para a bactéria nestes orgãos. Os genes sefC e pefA foram importantes para promover a colonização cecal, pois somente quando estes dois genes simultaneamente estiveram presentes no perfil, obteve-se o reisolamento cecal quando as aves foram desafiadas oralmente sendo esta via a principal rota de infecção deste patógeno. / This study has the intention of verify the four virulence genes event in Salmonella Enteritidis according with phage type and ribotype and the virulence “in vivo”. The genes studied were invA, spvC, sefC and pefA in 120 Salmonella Enteritidis isolates from different origins, belongs at different ribotypes and phage types of seven Brazilian states. To verify the genes presence the strains were examined by Polimerase Chain Reaction (PCR) technique, singly and multiplex. The event of invA gene was in 100% (120/120) of strains, the spvC gene was in 94% (113/120) of strains, the sefC gene was in 97.5% (117/120) of strains and the pefA gene was in 97% (116/120) of strains. There were discovered five different profiles. The pattern one, P1, was positive for invA, spvC, sefC e pefA. The P2 was positive for invA, spvC and pefA. The P3 was positive for invA, sefC e pefA. The P4 was positive for invA and sefC and the last one, P5, was positive for invA and spvC. For the PT4RT1 strains, the P1 profile was present in 94% (64/68) of strains; P2 profile was in 1.5% (1/68); P3 in 3% (2/68); and P4 in 1.5% (1/68) of strains. For the PT4RT2 strains, the P1 profile was present in 86% (18/21) of strains; P3 profile was in 5% (1/21); and P4 in 9% (2/21) of strains. For the PT4RT3 strains, the P1 profile was present in 80% (4/5) of strains, and P2 in 20% (1/5) of strains. For the PT4RT9 strains, the P1 profile was present in 91% (10/11) of strains, and P3 in 9% (1/11) of strains. The strains: PT4RT5, PT7RT1 e PT4RT10 belongs at the P1 profile, and only the PT1 strain belongs at the P5’s profile. The virulence was evaluated challenging the birds orally and subcutaneous, through the colonization of the caecum, liver and spleen invasion. The gene spvC was related with the survivance and the increase of the average grow of the bacteria in the liver and spleen, but this study doesn’t demonstrate the difference in the percentage of positive birds for the bacteria in their organs. The sefC and pefA genes were important to promote the caecum colonization, because only when both genes were present simultaneously in the profile, obtain the caecum isolation when the birds were been by orally challenged this way the main route of the infection of this pathogen.

As doenças respiratórias estão entre as maiores causas de prejuízo para a indústria suinícola, seja pelo retardo no crescimento e ganho de peso, mortalidade de animais ou pelos gastos com vacinas, medicamentos e assistência veterinária. Neste contexto os quadros de rinite têm apresentado uma contribuição significativa. O presente estudo propõe a determinação dos perfis de eliminação de agentes envolvidos em rinite nos suínos avaliando diferentes faixas etárias em nove propriedades de ciclo completo com histórico de lesão em cornetos e que utilizem diferentes formas de prevenção e controle destas manifestações. Foram avaliados suabes de tonsilas de 12 animais, nas seguintes faixas etárias: matrizes, leitões de 20, 40 e 60 dias, suínos de 90, 110 e 140 dias, totalizando 84 animais por propriedade e 756 amostras em todo o estudo. As amostras foram submetidas à pesquisa de P. multocida tipo capsular A e D, gene codificador de toxina dermonecrótica de P. multocida, B. bronchiseptica e cytomegalovirus suíno através da reação em cadeia pela polimerase (PCR). Apesar do histórico de lesões em corneto em todas as propriedades apenas um animal foi positivo para presença de P. multocida tipo A e todos foram negativos para a presença do gene codificador da toxina dermonecrótica. Dentre os 756 animais 22 (2,9%) foram positivos para presença de B. bronchiseptica e 198 (26,1%) para detecção cytomegalovirus suíno. A presença B. bronchiseptica apresentou associação estatisticamente significativa com as fases de maternidade e terminação. A maior freqüência de cytomegalovirus suíno apresentou associação estatisticamente significativa com a fase de creche. Observaram-se matrizes eliminando B. bronchiseptica nos três tipos de granjas avaliadas, indicando que a fêmeas tem participação ativa na infecção dos leitões pelo agente. O mesmo não foi detectado na disseminação do cytomegalovirus suíno. Maiores estudos devem ser realizados para esclarecer a baixa eliminação de P. multocida e o verdadeiro impacto do cytomegalovirus nos rebanhos suínos. / Respiratory diseases are one of the largest cause of economic losses in swine industry, it is related with grown and weight gain reduction, mortality, vaccines and medicaments costs, veterinary assistance. In that context, rinithis cases have been a major contribution. The present study propose the determination of elimination profile of agents related with rhinitis evaluating different ages in nine swine herds with history of cornet lesions and that uses different ways to control and prevent this problem. There were examined tonsils swabs from 12 animals in the following ages: sows, piglets of 20, 40 60 days and pigs of 90, 110 and 140 days, totalizing 84 pigs for farm. The swabs were searched to P. multocida capsular type A and D, dermonecrotic toxin gene from P. multocida, B. bronchiseptica and porcine cytomegalovirus through polymerase chain reaction (PCR). Despite de turbinate bones lesions present in all herds P. multocida type A was detected in only one pig and none were positive to dermonecrotic toxin gene. From 756 animals, 22 (2.9%) were positive to B. bronchiseptica and 198 (26.1%) to porcine cytomegalovirus detection. The presence of B. bronchiseptica presented statistical association with the farrowing and finishing times. Larger number of animals positive to cytomegalovirus show statistical association with the post weaning pigs. Sows carrying B. bronchiseptica in the three types of herds examined, suggesting that sows have an active participation in piglet infection by this agent. The same was not observed in porcine cytomegalovirus spread. More projects were need to clarify the low detection of P. multocida and to understand the impact of cytomegalovirus in swine production.

As espécies do gênero Arcobacter eram classificadas até a década de 1990 como pertencentes ao gênero Campylobacter. Atualmente três das cinco espécies deste gênero são consideradas potencialmente zoonóticas, podendo ser transmitidas por alimentos de origem animal. O presente estudo teve como objetivo isolar e caracterizar geneticamente amostras de Arcobacter spp., isoladas a partir de 120 amostras de carcaças, 120 amostras de fezes e 24 de amostras de músculo suíno coletadas em dois abatedouros localizados no Estado de São Paulo. Os isolados obtidos foram submetidos ao PCR-Multiplex para a determinação das espécies do gênero e analisados através da eletroforese em campo pulsado. O agente foi isolado de 71,6% das carcaças, 4,16% das amostras de fezes e 8,3% das amostras de músculo. As espécies mais prevalentes foram A. butzleri e A. cryaerophilus. A análise dos dados obtidos no PFGE (SmaI) revelou 51 perfis distintos, com um índice discriminatório de 0,98 e grande diversidade genotípica entre as amostras . O sítio de isolamento mais frequente para o agente foram as carcaças e o PFGE mostrou-se um bom instrumento para a caracterização e discriminação de cepas deste gênero / The species of Arcobacter spp. genus have been classified until 1990 as species belonging to the genus Campylobacter. Nowadays, three of five species of this genus are consider as potencially zoonotic, and can be transmitted from food of animal origin. The present study goal was to isolate and characterize genetically strains of Arcobacter spp. isolated from 120 carcasses, 120 faeces and 24 swine muscle sampled in two swine slaughterhouses located in São Paulo State. Isolates were submitted to multiplex-PCR to identify species of the genus and analyzed by pulsed field gel electrophoresis. The agent was isolated from 71,6% of carcasses, 4,16% of faeces and 8,3% of muscles samples. A. butzleri and A. cryaerophilus were the most prevalent species. The analysis of data obtained in PFGE showed 51 distinct profiles, the discriminatory index of 0,98 and it demonstrated large genotipic diversity among the strains. The main isolation site were the carcasses and the PFGE was a good tool to characterize and discriminate Arcobacter spp. strains.

The etiological agent of rabies encephalitis belongs to the genus Lyssavirus in the Rhabdoviridae family. Lyssaviruses are negative sense, single stranded RNA viruses and cause an estimated 55 000 human deaths per year with 44% of these deaths occurring in Africa (WHO, 2005). With intense research effort and increased sequence information it is becoming evident that the Lyssavirus genus is much more diverse than initially thought and therefore diagnostic methods need to be modified accordingly. The African continent sustains a diverse variety of lyssaviruses, however, most countries in Africa do not have active surveillance or necessary diagnostic tools and therefore rabies-related lyssaviruses are underreported. Previous studies have indicated that real-time PCR has improved sensitivity and rapidity over conventional molecular diagnostic methods with the added advantage of allowing accurate estimations of viral load in a wide variety of samples. Several realtime PCR assays have been developed; however, none were specifically aimed at detection of lyssaviruses present on the African continent. This study was therefore aimed at evaluating certain molecular diagnostic methods for the detection of African lyssaviruses. Furthermore, the application of real-time PCR for various fields in lyssavirus research i.e. diagnostics, surveillance and pathogenicity studies were evaluated. This study revealed two different hemi-nested PCR assays capable of detecting representatives of African lyssaviruses. A real-time PCR was developed that was successful for the detection of African lyssaviruses. In addition, a quantitative assay and internal control was successfully employed for confirming ante-mortem human rabies diagnosis as well as post-mortem animal rabies diagnosis in formalin fixed brain material. As such the real-time PCR assay developed in this study could therefore be routinely used for ante-mortem diagnosis and as a confirmatory test for post-mortem diagnosis. The ability of this assay to detect and quantify all currently known African lyssaviruses not only offers improved surveillance capacity, but offers unique potential as a sensitive tool to track virus movement in pathogenicity studies. These aspects are important in our search for a better understanding of the complex epidemiological and viral characteristics of African lyssaviruses. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.

The aim of this thesis is the isolation and identification of yeasts obtained from the wine berries and the characterization of the collection yeast by using processes of PCR - RFLP. The type yeasts were obtained from the collection of yeasts of CCY in Bratislava, yeasts from the wine berries were collected from the species of Hibernal wine from the wineries of Štěpán Maňák. Identification of individual yeast is then based on analysis of the DNA segment in the area of 5,8S - ITS using primers ITS1 and ITS4. The restriction analysis was performed using restriction endonucleases HaeIII, HinfI, HhaI a TaqI(a). Restriction analysis is used to chopp the DNA to specific sections that are characteristic for each microorganism. For the assesment of the genetic similarity analyzed yeasts the BioNumerics software has been used. BioNumerics processes the results using cluster analysis using Jaccard´s coefficients.

The PCR-based genome walking method has been commonly used to isolate upstream regions from known cDNA sequences. The limitation of this technique is based on the location of the restriction site upstream to the gene-specific primer in the genome; hence, different restriction enzymes have to be used to isolate larger upstream fragments. In this paper, we present the advantageous use of partial and size-selected DNA as templates for genome walking, in isolating larger upstream fragments. We have successfully tested this approach to isolate larger upstream fragments using the FailSafe™ PCR System. Use of partial digestion and size selection can provide better chances in obtaining larger flanking regions of known DNA sequence, when compared to use of total digested DNA.

Le Campylobacter est le plus important agent d'entérites bactériennes dans les pays industrialisés et en voie de développement. Des études récentes suggèrent que l'eau non-traitée est une source sous-estimée d'infections sporadiques chez l'humain. Dans le cadre du volet environnemental du projet CampyloGIS, mon projet principal consistait en l'étude de la prévalence et de la quantité du Campylobacter retrouvé dans les eaux environnementales de l'Estrie. Trente-deux sites d'échantillonnage d'eau ont été sélectionnés dans les 7 MRC de l'Estrie pour être échantillonnés hebdomadairement du 17 juillet 2005 au 08 juillet 2007. Globalement, 1071/2481 (43%), 1481/2471 (60%) et 1463/2471 (59%) échantillons d'eau étaient respectivement positifs pour Campylobacter spp., les coliformes thermotolérants et E. coli . Il y avait une faible corrélation entre la prévalence hebdomadaire du Campylobacter spp. et des coliformes thermotolérants (rho de Spearman = 0,27; P = 0,008) et entre la quantité de ces deux microorganismes (tau-b de Kendall = 0,233; P < 0,0001). Également, plus de 150 échantillons d'eau de puits privés situés en Estrie ont été gracieusement analysés au cours de ce projet. Cinq échantillons d'eau de puits de surface sur 53 étaient positifs pour C. jejuni et seulement deux d'entre eux étaient aussi positifs pour les coliformes thermotolérants. Ces résultats suggèrent que les indicateurs de pollution fécale, comme les coliformes thermotolérants, ne sont pas suffisants pour correctement évaluer la présence et/ou la quantité du Campylobacter dans l'eau environnementale. Mon deuxième projet consistait en le développement d'une approche moléculaire d'identification à l'espèce de C. jejuni, C. coli, C. lari, C. upsaliensis et C. fetus basée sur la PCR hippurate, la PCR 16S et la PCR-RFLP. Présentement, l'approche moléculaire permettrait d'éviter la mauvaise identification de 526/1950 isolats (27%) préalablement mal identifiés par les tests biochimiques seuls. Jusqu'à présent, l'approche moléculaire ne permettrait toutefois pas d'identifier correctement à l'espèce 35/1950 isolats (1,8%) de Campylobacter. Cette approche moléculaire a permis de confirmer la nécessité d'utiliser plus d'une technique afin de s'assurer de l'identification à l'espèce adéquate du Campylobacter. Finalement, la présence soutenue et la forte concentration occasionnelle du Campylobacter détecté dans les eaux environnementales de l'Estrie, ont porté à se questionner sur l'utilité de développer une méthode efficace de détection et de quantification du Campylobacter dans l'eau. De cette réflexion et des difficultés d'exécution qu'entraîne l'utilisation de la méthode MPN comme outil quantitatif, est né mon troisième projet, soit un protocole de RT-PCR semi-quantitative précédée d'un enrichissement pour la quantification de C. jejuni, C. coli et C. lari dans l'eau. Ce projet a permis de constater qu'un enrichissement de 16 h permettait d'augmenter, par un facteur relativement constant, le nombre de C. jejuni contenu dans un échantillon d'eau naturellement contaminé et ce, peut importe la condition cellulaire initiale occasionnée par un séjour dans l'eau. De plus, les résultats obtenus via le protocole de RT-PCR semi-quantitative sont comparables à la quantification obtenue via la méthode MPN (rho de Spearman = 0,84; P < 0,001), ce qui permet de croire que ce protocole simple pourrait éventuellement être un choix envisageable pour la détection et la quantification du Campylobacter dans l'eau.

Les Chlamydiaceae sont une famille de bactéries relativement récente dans la systématique microbienne. Deux bactéries sont principalement associées à des pathologies de l'homme, soit Chlamydia trachomatis et Chlamydophila pneumoniae. C. trachomatis est la première maladie à déclaration obligatoire au Québec et l'infection sexuellement transmissible (ITS) le plus fréquemment rencontré dans le monde. Mon projet consistait au développement d'une méthode de"Multi Locus Sequence Typing" (MLST) pour C. trachomatis à partir d'isolats cliniques, sans passer par la culture cellulaire, et ce grâce au PCR (réaction en chaîne de la polymérase) multiplex niché. L'objectif ultime de cette technique est de mieux connaître l'épidémiologie de la bactérie, pour pouvoir comprendre la résistance et la persistance aux antibiotiques et l'origine géographique commune, par exemple. Outre les 15 isolats de référence, 115 isolats d'origines diverses (38 ITS de l'Estrie, 54 ITS de l'Afrique et 22 trachomes de l'Afrique) ont été testés par PCR multiplex niché puis séquencés. Globalement, les 130 isolats se séparent en 29 séquences-types différentes, dont 17 ne sont retrouvées qu'une seule fois. Ces STs se regroupent en 4 complexes clonaux distincts. De plus, les isolats sont séparés selon le type d'infection qu'ils causent, soit l'ITS, le trachome ou la lymphogranulomatose vénérienne. Dans son ensemble, l'évaluation de la pertinence et de la qualité de la nouvelle technique MLST montre une force discriminatoire de 90,1%, qui est dans les normes de qualité pour une technique d'épidémiologie moléculaire. Un index d'association de 3,809 pour le schéma complet est trouvé, et un de 2,447 lorsque le calcul est refait avec un exemplaire de chaque ST seulement, indiquant une population clonale forte. Finalement, un ratio d[indice inférieur N]/d[indice inférieur S] variant entre 0.145 et 0.773 pour les gènes choisis pour le schéma MLST démontre que les gènes sélectionnés ne sont pas soumis à une pression de sélection positive. Toutes ces données tendent à prouver que le nouveau schéma MLST est une technique discriminante, qui va permettre de faire des liens épidémiologiques intéressants pour C. trachomatis. De plus, au cours de ce projet, des analyses de certains isolats de C. trachomatis ont montré des caractéristiques nouvelles au niveau du génotype ompA.Les 22 isolats de trachome de la Tanzanie (Afrique) ainsi que 5 isolats ITS des Îles Comores ont cette particularité. Il s'agit selon la séquence d'un génotype A variant. Des analyses supplémentaires restent à faire pour caractériser complètement le génotype.