Thesis (M.Sc. (Public Health))--Mahidol University, 1979.

Thesis--University of Chicago. / Bibliography: leaves 53-56.

Crohn's disease (CD) is an idiopathic, chronic, relapsing inflammatory disorder, which is most commonly involved terminal ileum and colon. The incidence and prevalence of CD has dramatically increased during the last 50 years; however, the etiology and mechanism of this disorder remain unveiled. Besides genetic susceptibility, recent integrated researches investigated the role of environmental triggers such as microflora, measles viruses and mycobacteria in the pathogenesis of CD. The association between M. avium subsp paratuberculosis (MAP) and CD has been heightened because of clinical resemblance to Johne's disease (JD), a granulomatous enteritis in ruminants caused by MAP. Isolation of MAP from tissue and milk samples from CD patients and from commercial pasteurized milk and dairy products from JD-infected animals implies a possible re-classification of CD as zoonotic disorder. Clinical signs and symptoms of CD are often non-specific and a challenge to distinguish it from other disorders. Current methods for inflammatory bowel disease diagnosis, especially for CD are highly invasive, distressing and expensive. In this study, the recombinant clone pB11 containing 1.1 kb insert, identified from a MAP genomic library constructed in E. coli, expressed a 20 kDa (p20) antigen encoded on 549 bp partial MAP gene with an ORF cloned in frame within pBAD/His cloning vector. Immunoreactivity of p20 was confirmed by Immunoblot. Purified p20 antigen was then used in the development of an enzyme-linked immunosorbent assay (ELISA) for possible serodiagnosis of Inflammatory Bowel Disease (IBD) associated with MAP infection. All variables associated with ELISA test with regard to concentrations, washes and incubations were optimized using hyper immune rabbit t-anti-MAP polyclonal IgG antibodies and sera from CD and non-CD subjects. The cut-off value for positive response was established as 0.3 following the analysis of statistically formulated samples from normal and non-CD subjects. The developed ELISA test was then used to test a blindly coded 2 17 clinical sera. All sera samples were tested in duplicates and in both p20-coated and uncoated micro titer plates. Consequently, 116/134 (87%) CD sera were positive compared to 24/83 (33%) non-CD sera (P<0.05). Specifically anti-MAP IgG was detected in 8/22 (36%) Ulcerative colitis and 16/61 (26%) non-IBD sera. p20-ORF encoding sequence was recloned (pB11/B6) and the expressed protein reactivity remained consistent. Moreover, the full length of the cloned gene was also identified through blast and alignment analysis and predicted to encode 346 amino acids with unknown function and no identity with other known proteins. The latter supports the clinical data, which reflect on the unique characteristics of this antigen. The result so far suggests that the recombinant clone and its subclone derivative may have potential role in serodiagnosis of CD

Orientador: Márcia Aparecida Silva Graminha / Banca: Pio Colepicolo Neto / Banca: Flávio Henrique da Silva / Resumo: As leishmanioses são um grupo de doenças causadas pelo gênero Leishmania e colocam em risco 350 milhões de pessoas no mundo. Após o sequenciamento do genoma de algumas espécies de Leishmania spp., estudos moleculares de suas formas celulares (amastigotas e promastigotas) trouxeram informações importantes sobre sua biologia e potencial aplicação no aperfeiçoamento ou desenvolvimento de técnicas diagnósticas e terapêuticas. Nesse contexto, este projeto visou avaliar o gene LinJ.36.419 de Leishmania infantum (syn. Leishmania chagasi) e seu produto quanto a potencial aplicação no dsenvolvimento de novas abordagens diagnósticas. Ensaios de PCR quantitativa mostraram que a expressão deste gene é maior em formas amastigotas, estágio responsável pela manifestação da doença, podendo corresponder a um potencial marcador diagnóstico. Um fragmento de 255 aminoácidos da proteína Lc36 foi caracterizado quanto ao seu potencial antigênico utilizando ensaio imunoenzimático (ELISA). Os resultados mostraram especificidade de 74% e sensibilidade de 78% (99% de confiança), com acurácia de 76%, em uma população escolhida randomicamente. Análises adicionais mostraram reação cruzada pouco significativa para soro de cães infectados com Leptospira interrogans e Toxoplasma gondii, e revelaram reação cruzada contra soro de cães infectados por Babesia canis e Ehrlichia canis, todos organismos infecciosos comuns em cães. Análises de bioinformática mostraram que a proteína Lc36 possui um domínio denominado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leishmaniasis are diseases caused by Leishmania genus protozoan and endanger 350 million people worldwide. After genomic sequencing of Leishmania spp., molecular studies of their cycle forms (amastigotes and promastigotes) brought important information, which can contribute to the development of new diagnostic approaches and treatments. In this study we evaluated the potencial application of the Leishmania infantum (syn. Leishmania chagasi) gene Lc.36.4190 and its encoded protein for development of new diagnostic approaches. Quantitative PCR assays showed that the gene is more expressed in intracellular amastigotes, the stage related to the clinical manifestation of these diseases, thus presenting potential to be a diagnostical target. We evaluated antigenic features of a 255 aminoacids fragment of the Lc36 protein using immunoenzimatic assay (ELISA). The results showed specificity of 74% and sensibility of 78% (99% of confidence) and accuracy of 76%, in a randomically chosen population. Additional analysis showed low cross-reaction against Leptospira interrogans and Toxoplasma gondii, and showed cross-reaction against Babesia canis and Ehrlichia canis, all infective organisms common in dogs. Bioinformatics analysis showed that the protein Lc36 contains one DNA polymerase sigma domain (COG5260) found on several organisms, being related to nucleic acid replication, recombination and repair, and might be related to the crossreactions observed. Thus, another fragment (297 aminoa... (Complete abstract click electronic access below) / Mestre

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

 The emergence of the pandemic H1N1 2009 virus of swine-origin and its transmission back to swine highlighted the need for global surveillance of swine influenza. Serology can help to address the epidemiological situation of influenza infection. Since typical serology tests such as hemagglutination inhibition or microneutralization assays are subtype and partially virus-lineage specific, it is important to select appropriate viral antigens for such studies. A poorly chosen panel of antigens will lead to underestimation of the seroprevalence. The choice of well-matched antigen is difficult if there is no prior virological surveillance in that area and even if there was virological surveillance data, transient infections may go undetected. Hence an universal influenza A type reactive serological test is needed. While such tests are available for poultry, there is little published data on the performance of these commercial influenza ELISA assays for serology on swine sera. In this study we evaluated 3 commercially available competitive ELISA assays, IDEXX? Influenza A Ab test, IDEXX? AI MultiS-Screen Ab Test and IDVet ID Screen? Influenza A Antibody Competition ELISA kit for detecting influenza type A reactive antibodies in swine. The virus antigens and the serum samples were obtained from a 14-year systematic abattoir-based virological and serological surveillance for swine influenza in southern China. The performance was evaluated by ROC curve and scatter plot, together with other statistical parameters including the Youden index to optimize the cut-off levels. Using the optimized cut-off levels, sensitivity and specificity of the IDEXX? Influenza A Ab test was 86% and 89% respectively; for IDEXX? AI MultiS-Screen Ab Test was 91% and 87% and for IDVet ID Screen? Influenza A was 95% and 79%, respectively. These findings help to provide different cut-off levels to maximize the sensitivity or specificity to suit different purposes. We found that the ELISA assay was useful in detecting serum samples that may be positive for influenza antibody but missed in the serology screening tests due to limitations in the chosen antigen panel. The ELISA assay maybe helpful in global swine influenza surveillance programs. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Thesis (M.Sc. (Clinical Pathology))--Mahidol University, 1984.