PhD, Faculty of Health Sciences, University of the Witwatersrand / The South African gold-mining workforce has an unusually high incidence of Mycobacterium kansasii disease, yet little is known about the possible sources of M. kansasii infection, genetic diversity and the basis for this organism’s pathogenicity. The purpose of this study was to investigate these issues in a gold-mining environment. Five M. kansasii isolates and 10 other potentially pathogenic mycobacteria were cultured mainly from showerhead biofilms. PCR-restriction analysis (PRA) of the hsp65 gene on 191 clinical and on the 5 environmental M. kansasii isolates revealed 160 subtype I (157 clinical and 3 environmental), 8 subtype II (clinical) and 6 subtype IV (5 clinical and 1 environmental) strains. Twenty-two isolates (21 clinical and 1 environmental) did not show the typical M. kansasii PRA patterns. After confirmation by DNA sequencing as belonging to the M. kansasii species, the results suggested that these isolates were probably new subtypes of M. kansasii. In contrast to the clonal population structure found amongst the subtype I isolates from studies in other countries, DNA fingerprinting of 114 subtype I clinical and environmental isolates showed genetic diversity amongst the isolates. One of the 2 environmental isolates showed 100% identity with a clinical isolate, suggesting that water distribution systems are the possible sources of M. kansasii infection for the miners. An investigation into the genetic differences between clinical (subtype I) and environmental (III, IV and V) isolates, using Hybridisation Monitored Differential Analysis (HMDA), identified 45 open reading frames (ORFs) encoding predominantly membrane-associated proteins that include six potential virulence factors, two family members of transcription regulators for drug and xenobiotic metabolism, three family members of multidrug efflux systems, a number of proteins associated with lipid and carbohydrate metabolism and transport, and a number of hypothetical proteins with unknown function. Immunological analysis of M. kansasii isolates, using the Lymphocyte Transformation and Cytometric Bead Array assays, showed that M. kansasii modulates immune responses through suppression of lymphocyte blastogenesis and by altering the expression of Th1/Th2/Th17 cytokines by human lymphocytes in vivo for its own survival. This study demonstrated for the first time that water distribution systems in South Africa are possible sources of M. kansasii infection, and showed that subtype I strains of M. kansasii from the study region display genetic diversity and have unique or divergent genes not found in other subtypes. It also demonstrated that immunosuppression is one of the pathogenic mechanisms employed by M. kansasii.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-11-25T15:10:49Z No. of bitstreams: 1 Dissertação - Marla Jalene Alves.pdf: 2258783 bytes, checksum: 2a65889894741102692a4ac397f43a07 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-11-25T15:11:05Z (GMT) No. of bitstreams: 1 Dissertação - Marla Jalene Alves.pdf: 2258783 bytes, checksum: 2a65889894741102692a4ac397f43a07 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2016-11-25T15:11:18Z (GMT) No. of bitstreams: 1 Dissertação - Marla Jalene Alves.pdf: 2258783 bytes, checksum: 2a65889894741102692a4ac397f43a07 (MD5) / Made available in DSpace on 2016-11-25T15:11:19Z (GMT). No. of bitstreams: 1 Dissertação - Marla Jalene Alves.pdf: 2258783 bytes, checksum: 2a65889894741102692a4ac397f43a07 (MD5) Previous issue date: 2015-10-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The melanized fungi are widely distributed in the environment and has been commonly related in human diseases such as Chromoblastomycosis, Phaeomycosis and Eumycetoma, but in the Amazon region, which have a favorable weather for the proliferation of microorganisms, there are few studies about these fungi and little is known about their distribution in this environment and its pathogenic potential. Thus, in this study were studied melanized fungi in environmental samples from rural areas of the Amazon and the relationship of these environmental isolates with melanized fungi that cause mycoses. Environmental samples were obtained in rural areas of Iranduba and Presidente Figueiredo – Amazonas (Brazil). Also entered in the study, clinical specimens deposited in Fungal Collections from Amazonas. Two hundred thirteen samples of various environmental substrates were collected: soil, parts of living plants, decaying plant material and drinking water. Of these samples, 45 isolates were subjected to identification by sequencing the ITS region, 7 species of melanized fungi that were identified have been described in the literature as etiological agents of mycoses in humans: Cladophialophora immunda, Cladosporium sphaerospermum, Cladosporium cladosporioides, Exophiala oligosperma, Exophiala jeanselmei, Hortaea werneckii and Curvularia lunata. Most isolates were obtained from thorns. Some isolates grown at 37°C temperature. The molecular identification of clinical isolates showed similarity with Fonsecaea pedrosoi, Cladosporium sphaerospermum and Knufia epidermidis. Of these clinical specimens, only the species C. sphaerospermum was isolated from the environment. This study shows that the studied environmental substrates are potential sources of infection of melanized fungi that may cause mycoses. / Os fungos melanizados estão amplamente distribuídos no ambiente e têm sido comumente relacionados à doenças em seres humanos tais como a Cromoblastomicose, a Feomicose e o Eumicetoma, porém na região Amazônica, que possui clima favorável para a proliferação de microrganismos, há raros estudos realizados sobre estes fungos e pouco se sabe a respeito da sua distribuição neste ambiente e do seu potencial patogênico. Desta forma, na presente pesquisa foram estudados fungos melanizados em amostras ambientais de áreas rurais do Amazonas e a relação desses isolados ambientais com os fungos melanizados causadores de micoses. As amostras ambientais foram obtidas em áreas rurais dos municípios de Iranduba e Presidente Figueiredo, no Amazonas (AM). Também entraram no estudo amostras clínicas depositadas em Coleções Fúngicas do AM. Foram coletadas 213 amostras de diferentes substratos ambientais: solo, partes de plantas vivas, material vegetal em decomposição e água de consumo humano. Dessas amostras, 58 isolados foram submetidos a identificação por sequenciamento da região ITS, 7 espécies de fungos melanizados que foram identificados já foram citadas na literatura como agentes etiológicos de micoses em humanos: Cladophialophora immunda, Cladosporium sphaerospermum, Cladosporium cladosporioides, Exophiala oligosperma, Exophiala jeanselmei, Hortaea werneckii e Curvularia lunata. A maioria dos isolados foi obtida de espinhos. Alguns destes isolados apresentaram crescimento à temperatura de 37°C. A identificação molecular dos isolados clínicos mostrou similaridade com Fonsecaea pedrosoi, Cladosporium sphaerospermum e Knufia epidermidis. Dessas espécies clínicas somente a espécie C. sphaerospermum foi isolada do ambiente. Este trabalho evidencia que os substratos ambientais estudados são fontes potenciais de infecção de fungos melanizados que podem vir a causar micoses.

Salmonella est une bactérie zoonotique qui représente un problème majeur de santé publique. Le porc est identifié comme l'un des principaux réservoirs de Salmonella. L'objectif de ces travaux de recherche était d'identifier les mesures prioritaires à mettre en place afin de limiter la présence de Salmonella dans la filière porcine réunionnaise. Près de 80% des élevages de porcs investigués étaient infectés par Salmonella en fin d'engraissement. Cette infection asymptomatique est associée à plusieurs facteurs de risque : les conditions géo-climatiques (altitude et pluviométrie), la production de volailles sur le site, la taille de l'élevage et la présence de salmonelles dans les salles avant l'entrée du lot. Le suivi longitudinal a d'ailleurs mis en évidence que les salmonelles résiduelles étaient la principale source d'infection, que ce soit pendant l'élevage, le transport et l'attente. Ces résultats soulignent l'importance d'améliorer l'efficacité des protocoles de nettoyage et de désinfection. La faune sauvage semble également impliquée dans l'épidémiologie de Salmonella dans la filière porcine. Une forte similarité génétique a été observée entre les souches isolées de rongeurs et de cafards et celles isolées des porcs. Par ailleurs, une prévalence en Salmonella de 16,5% dans les saucisses à base de porc démontre que Salmonella dans la filière porcine peut représenter un risque pour la santé humaine à La Réunion. Ces travaux de recherche démontrent la nécessité d'agir aux différents maillons de la filière porcine. En outre, une lutte globale doit être envisagée, incluant les différentes filières, les industriels, les services vétérinaires et les pouvoirs publics. / Salmonella is a zoonotic bacterium that represents a public health burden all over the world. Pig is considered as one of the main reservoir of this pathogen. The goal of this research project was to identify the main measures that would limit the presence of Salmonella in the Reunionese pig production. Nearly 80% of the pig farms investigated were infected with Salmonella at the end of the fattening period. This asymptomatic infection was associated with several risk factors: geo-climatic conditions (altitude and rainfall), poultry production on the same site, the size of the farm and the presence of residual Salmonella in rooms before the loading of the pig batch. Furthermore, the longitudinal investigation highlighted that environmental residual Salmonella were the main source of infection whether at farm, during the transport and the lairage of pigs. These results strengthen the importance of improving efficiency of cleaning and disinfection procedures. The wild fauna of Reunion Island seems to be involved in the epidemiology of Salmonella in the pig production, since high genetically relationships were observed between Salmonella strains isolated from rodents and cockroaches and those isolated from pigs. Otherwise, our study also highlighted Salmonella prevalence of 16.5% in pork sausages demonstrating that Salmonella in the pig production may represent a risk for public health in Reunion Island. These works stress the need to act at each stage of the pig production. Moreover, a global effort has to be considered, including professionals of the different animal production, industrials, veterinary and health services and authorities.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Toxoplasma gondii is an intracellular parasite that infects virtually all warm-blooded animals, including humans. In human beings, the infection is usually asymptomatic in immunocompetent individuals, but can cause severe clinical manifestations in immunocompromised individuals and in cases of congenital toxoplasmosis. The diagnosis of this infection is usually performed by serological methods that detect IgG, IgM and IgA antibodies in biological samples. The conventional serological assays currently available detect only the exposure to parasite, and there is no serological techniques to accurately estimate the source of infection (oocyst or cyst), which hinders the implementation of prevention and control procedures of this infection. In addition, the serological differentiation between recent and chronic phases of the infection is difficult to achieve in the laboratory routine, making difficult the correct diagnosis of toxoplasmosis, mainly in immunocompromised individuals and pregnant women. In the present study, two recombinant proteins (CCp5A and OWP1) from oocyst/sporozoite of T. gondii were evaluated in serological tests to differentiate infections occurring by ingestion of oocysts or tissue cysts. The reactivity of these two recombinant proteins was assessed, in parallel with soluble Toxoplasma antigen (STAg), against panels of serum samples from animals (chickens, pigs and mice) naturally or experimentally infected by different infective stages of the parasite. Also, we tested sera from humans who have been infected by oocyst during a well-characterized toxoplasmosis outbreak, as well as sera from pregnant women tested IgM+/IgG+ for T. gondii, which source of infection was unknown. Only the sporozoite-specific CCp5A protein was able to differentiate the parasite stage that infected chickens, pigs and mice, with specific reactivity for sera from oocyst-infected animals. Furthermore, this protein showed a preferential reactivity for recent infection by oocyst/sporozoite in pigs and mice. In humans, CCp5A showed higher reactivity with serum samples from the outbreak, compared with serum from pregnant women. Altogether, these findings demonstrate the usefulness of the CCp5A protein as a new tool to identify the parasite stage of T. gondii responsible for the infection (oocyst or tissue cyst). Also, in order to evaluate an alternative antigenic preparation to differentiate the phases of T. gondii infection, whether acute or chronic, a synthetic peptide from the microneme 8 protein (pMIC8) was tested, in parallel with STAg in immunoassays, using serum samples from individuals in different infection phases. Initially, this peptide was used to evaluate the kinetics of IgG antibodies in mice experimentally infected with T. gondii. After, immunoassays using pMIC8 and STAg were conducted to detect IgM, IgA and IgG antibodies in 124 human serum samples divided into five groups, according to the phase of T. gondii infection: Group I (up to 4 months of infection); Group II (5 to 8 months of infection); Group III (9 to 12 months of infection); Group IV (over 12 months of infection); and Group V (seronegative individuals). In the murine model, pMIC8 showed to be a potential marker of recent infection with strong detection of IgG antibodies in the early phase of infection. In humans, IgM and IgA to pMIC8 showed better characterization of the time of T. gondii infection in serum samples from recent phase (up to 12 months of infection), when compared to those tested against STAg. The percentage of IgG detection to pMIC8 was higher in sera from Group I (early acute phase) and lower in sera from Group IV (chronic phase). This pattern was the opposite of those observed to STAg, that showed lower detection percentage in Group I). To underline the differences in IgG detection using pMIC8 and STAg, it was determined a ratio between the ELISA index obtained from both antigenic preparations (STAg/pMIC8), which showed an accurate parameter to differencialte the phases of infection. Overall, these findings suggest that pMIC8 could be a valuable tool to differentiate recent from chronic T. gondii infection. / Toxoplasma gondii é um parasito intracelular que infecta virtualmente todos os animais de sangue quente, incluindo a espécie humana. Nestes hospedeiros, a infecção geralmente é assintomática em indivíduos imunocompetentes, mas em indivíduos imunocomprometidos e em casos de toxoplasmose congênita, as manifestações podem ser graves. O diagnóstico da toxoplasmose é usualmente realizado por métodos sorológicos, com detecção das imunoglobulinas IgG, IgM e IgA em amostras biológicas. As técnicas sorológicas atuais detectam a exposição ao parasito, mas não apresentam capacidade de diferenciar as vias de infecção, as quais podem ocorrer por ingestão de oocistos ou de cistos teciduais, dificultando a implementação de medidas preventivas para controlar e reduzir a infecção por T. gondii. Além disso, tais técnicas sorológicas não apresentam a possibilidade de diferenciar infecção aguda de infecção crônica, o que limita a determinação da fase da infecção, principalmente em indivíduos imunocomprometidos e em gestantes, além dos casos de toxoplasmose congênita. No presente trabalho, as proteínas recombinantes CCp5A e OWP1 de oocistos/esporozoítos de T. gondii foram utilizadas em testes sorológicos com o objetivo de diferenciar infecções via ingestão de oocistos ou cistos teciduais do parasito em amostras de soros de animais e humanos. A reatividade destas proteínas foi analisada, em paralelo com o antígeno solúvel de Toxoplasma (STAg), utilizando-se de um painel de amostras de soros de animais (galinhas, porcos e camundongos) infectados naturalmente ou por meio de protocolos de infecção experimental, a partir de diferentes estágios infecciosos do parasito. Em adição, estas proteínas foram testadas em amostras de soros de pessoas infectadas por via hídrica (via oocisto) em um surto de toxoplasmose e de gestantes soropositivas (IgM+/IgG+) para T. gondii, cuja via de infecção era desconhecida. Em animais, somente a proteína CCp5A foi capaz de diferenciar o estágio infeccioso de T. gondii responsável pela infecção, com reatividade específica para soros de indivíduos infectados por oocistos. Além disso, esta proteína também apresentou maior reatividade em amostras de soros na fase recente de infecção em porcos e camundongos. Em humanos, CCp5A apresentou reatividade preferencial com soros do surto de toxoplasmose, em comparação com soros das gestantes. Esses resultados indicam que a proteína CCp5A pode ser uma nova ferramenta para identificar o estágio infeccioso de T. gondii responsável pela infecção (oocisto ou cisto tecidual). Em uma segunda parte deste trabalho, com o intuito de avaliar outras preparações antigênicas visando a determinação das fases da infecção por T. gondii, se infecção aguda ou crônica, o peptídeo sintético pMIC8 foi testado, em paralelo com STAg, em imunoensaios utilizando-se amostras de soros de indivíduos em diferentes fases da infecção. Inicialmente, tal peptídeo foi utilizado para avaliar a cinética de anticorpos IgG em camundongos experimentalmente infectados com T. gondii. Posteriormente, imunoensaios utilizando pMIC8 e STAg foram realizados para a detecção de anticorpos IgM, IgA e IgG em 124 amostras de soros humanos divididos em 5 grupos, de acordo com a fase da infecção por T. gondii: Grupo I (infecção até 4 meses); Grupo II (infecção entre 5 e 8 meses); Grupo III (infecção entre 9 e 12 meses); Grupo IV (infecção acima de 12 meses); e Grupo V (indivíduos soronegativos). No modelo murino, pMIC8 mostrou-se como um marcador em potencial para a caracterização da infecção recente, demonstrando forte reação com anticorpos IgG na fase precoce da infecção. Em humanos, anticorpos IgM e IgA contra pMIC8 apresentaram melhor caracterização do tempo de infecção em amostras de soros de fase aguda (até 12 meses de infecção), quando comparados aos anticorpos dirigidos contra STAg. Por outro lado, a porcentagem de detecção de IgG contra pMIC8 foi maior nas amostras de soros do Grupo I (fase aguda precoce) e menor nas do grupo IV (fase crônica). Este padrão foi inverso ao observado com STAg, que apresentou menor porcentagem de detecção de IgG no grupo I. Visando caracterizar as diferenças quanto a detecção de IgG quando são utilizadas as preparações antigênicas STAg e pMIC8, foi calculada a razão entre os valores dos Índices ELISA de IgG obtidos nas reações com estes dois antígenos (Razão STAg/pMIC8), que demonstrou ser um parâmetro importante na diferenciação sorológica da fase da infecção. Em síntese, os resultados obtidos neste estudo sugerem que pMIC8 pode ser uma ferramenta relevante na diferenciação entre infecção recente e infecção distante na infecção por T. gondii. / Doutor em Imunologia e Parasitologia Aplicadas