Thesis (Ph.D. (Tropical Medicine)) -- Mahidol University, 1999.

The Gram-negative bacterium Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases including bacillary angiomatosis which is characterized by vasoproliferative tumor-like lesions on the skin and internal organs of some immunosuppressed individuals. Several virulence factors associated with Bartonella-induced pathogenesis have been characterized. However, the study of those virulence factors has been limited to in vitro cell culture systems due to the lack of a practical animal model. Therefore, we wanted to investigate whether the zebrafish embryo (Danio rerio) could be used to model human infection with Bh. We investigated if Bh can mount an infection in zebrafish embryos during their early stage of development. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. This was evident by plating of zebrafish homogenates, quantitative PCR, and confocal microscopy analysis. We assessed the interaction of Bh with EC and the phagocytic cells in live embryos by microscopy. Our data showed that aggregates of Bh interact with the endothelium of the embryo vasculature. Evidence showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. We also wanted to determine the response to infection with Bh. Infected embryos showed evidence of a Bh-induced angiogenic phenotype as well as an increase in expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. A deletion mutant for the entire VirB type IV secretion system (ΔvirB2-11 supported bacterial replication although to a lesser degree compared to the wild type control. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished pro-angiogenic and pro-inflammatory host response compared to wild type Bh, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical animal model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

Bacillary angiomatosis (BA), one of the clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Endothelial cells have been identified as host cells for this pathogen and are presumed important for pathogenesis as lesions contain bacteria in direct contact with the endothelium. Lesions also contain infiltrating macrophages, which contribute to the angiogenic process during B. henselae infection by secreting vascular endothelial growth factor (VEGF). While virulence factors have been characterized, and the role for macrophages in B. henselae infection has been established, endothelial cell mediators of angiogenesis have not been well defined. We investigated three potentially important pathways that are triggered by the bacterial interactions with endothelial cells. We examined the ability of endothelial cells to upregulate the chemokines monocyte-macrophage chemoattracta nt protein-1 (MCP-1) and CXCL8 and the mechanism by which B. henselae secreted proteins (BHSP) induce endothelial cell proliferation. We determined that MCP-1 production is upregulated in response to B. henselae infection, which very likely contributes to angiogenic lesion formation by recruiting the VEGF-secreting macrophage. The chemokine CXCL8 is an important mediator of angiogenesis which can cause endothelial cell survival, proliferation, and capillary tube formation. We determined that CXCL8 is secreted from B. henselae-infected cells and contributes to B. henselae-induced angiogenesis in an autocrine manner. We also investigated the role of Ca2+ signaling during B. henselae infection. We determined that BHSP induce a robust intracellular Ca2+ response in HUVEC which originates from intracellular Ca2+ pools. Additionally, endothelial cell proliferation in response to BHSP required Ca2+ activity, indicating a role for intracellular Ca2+ pools during B. henselae-induced angio genesis. Endothelial cell proliferation during B. henselae infection possibly indicates a mechanism by which a pathogen induces proliferation of its host cell in order to propagate its own survival. Numerous factors culminate in the vascular lesions that are characteristic of BA. B. henselae infection represents an important and unique model for pathogen-triggered angiogenesis, and studies into the specific mechanisms of this process may elucidate host cell-pathogen interactions as well as pathways of pathogenic angiogenesis.

Dissertation (M.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 71-84).

Source: Masters Abstracts International, Volume: 47-01, page: 0319. Advisers: Irina Cech; Michael Smolensky. Includes bibliographical references (leaves xx-xx).

Les maladies diarrhéiques sont la deuxième cause de mortalité chez les enfants de moins de cinq ans. Les entérobactéries Shigella flexneri sont les principales responsables de la forme endémique de la shigellose, une maladie diarrhéique importante dans les pays en développement et pour laquelle de nombreuses stratégies vaccinales sont à l’étude. La partie polysaccharidique (antigène O, Ag-O) du lipopolysaccharide de surface est l’une des cibles majeures d'immunité protectrice contre la réinfection. Une grande variété d’Ag-Os, reflétant la diversité sérotypique, a été identifiée. De façon intéressante ces Ag-Os se différencient par la nature des substituants portés par le tétrasaccharide ABCD qui définit leur squelette commun. Afin de développer un vaccin issu de sucres de synthèse à large couverture sérotypique contre S. flexneri, des stratégies de synthèse hautement convergente d’analogues orthogonalement protégés du tétrasaccharide ABCD ont été explorées. Elles prennent en compte les sites de -D-glucosylation et de O-acétylation spécifiques de sérotypes. Les approches mises en place s’appuient sur la synthèse d’une diversité de précurseurs en série L-rhamnopyranose et 2-N-acétyl-2-désoxy-D-glucopyranosamine et leurs combinaisons optimisées. Quelques exemples de glucosylation 1,2-cis régiosélective, chimique et/ou enzymatique, valident le concept. / Diarrhoeal diseases are the second cause of death among children under five. Shigella flexneri enterobacteria are the main causative agents of the endemic form of shigellosis, a diarrhoeal disease of high prevalence in developing countries and one for which numerous vaccine strategies are under studied. The polysaccharide part (O-antigen, O-Ag) of the bacterial lipopolysaccharide is a major target of protective immunity against reinfection. A large variety of O-Ags, expressing serotypic diversity, has been identified. Interestingly, these O-Ags differ by the nature of the substitutions occurring on the ABCD tetrasaccharide, which defines their common backbone. In order to develop a synthetic carbohydrate-based vaccine with broad serotype coverage against S. flexneri, highly convergent synthetic strategies towards orthogonally protected analogs of tetrasaccharide ABCD were investigated, while taking into account serotype-specific -D-glucosylation and O-acetylation sites. The selected approaches feature the synthesis of a variety of suitable L-rhamnopyranose and 2-N-acetyl-2-deoxy-D-glucopyranosamine precursors and their optimized combinations. The concept is supported by selected examples of 1,2-cis chemical and/or enzymatic glucosylation.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-28T09:47:26Z No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-01-28T09:49:33Z (GMT) No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-28T09:49:33Z (GMT). No. of bitstreams: 2 Tese - Bruna Daniella de Souza Silva - 2015.pdf: 3849700 bytes, checksum: 267d28b0d91fc274ddb5bfd83ed08e5b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Millions of people die every year due to tuberculosis (TB), an infectious disease that have effective treatment, can be prevented and is curable. One of the greatest problems faced by this disease is the latent infection (LTBI), where individuals do not manifest clinical symptoms, is a reservoir of the causing agent, Mycobacterium tuberculosis (Mtb), and can reactive the disease at any time during their life span. Moreover, TB can affect any organ in the body, such as the skin, causing extrapulmonary TB, a rare form of TB, the TB skin. Some of these forms may be more severe than pulmonary TB, causing serious consequences to the patient, contributing to the high mortality rate of this disease. In this context, understanding the immunological events related to the interaction between pathogen and host the development of active disease or latent infection is a crucial point that can contribute to the control of TB. Thus, the objectives of this study were to evaluate the specific immune response of CD8+ T cells and cytokines in cutaneous tuberculosis, latent and active pulmonary TB. Thirty six patients with pulmonary TB, one patient with cutaneous TB and 36 healthy controls, classified as LTBI (N = 13) or negative (TST-, N = 23) by the tuberculin skin test were recruited and the peripheral blood mononuclear cells, plasma and sera from those individuals were collected to perform flow cytometry, ELISA and multiplex bead array analysis. It was observed that patients with active pulmonary TB presented TCD8+ cells with a regulatory profile, expressing IL-10 and TGF-β in a direct relation to the bacillary load. The same profile was observed in the individual with cutaneous TB, an extra-pulmonary form of TB. The findings observed in this studyco nclude that Mtb can modulate CD8+ T cell response in lung and skin tuberculosis, demonstrating the importance of studies assessing the immune interaction between the pathogen and the host. / A tuberculose (TB) é uma doença causada por Mycobacterium tuberculosis, principal agente etiológico da TB humana. Milhões de pessoas morrem todo ano em decorrência da TB, doença infecciosa que tem prevenção, tratamento e cura. Um dos maiores problemas enfrentados com essa doença é a infecção latente (TBIL), onde o indivíduo não manifesta os sintomas clínicos e constitui um reservatório da bactéria, podendo desenvolver a doença ativa em qualquer momento. Além disso, a TB pode afetar qualquer órgão do corpo, como por exemplo, a pele, causando uma forma rara de TB extrapulmonar, a TB cutânea. Algumas dessas formas podem ser mais severas que a TB pulmonar, trazendo consequências graves ao paciente, contribuindo para o alto índice de mortalidade dessa enfermidade. Nesse contexto, entender os eventos imunológicos relacionados à interação entre patógeno e o hospedeiro no desenvolvimento da doença ativa ou da infecção latente é um ponto crucial que pode contribuir para o controle da TB. Diante disso, os objetivos desse trabalho foram avaliar a resposta imune específica de células TCD8+ e citocinas na tuberculose cutânea, latente e pulmonar ativa. Para isso, foram recrutados 36 pacientes com TB pulmonar ativa, 01 paciente com TB cutânea e 36 controles sadios classificados quanto à prova tuberculínica em indivíduos com infecção latente (TBIL = 13) ou não (PT-=23). Foram obtidas, de todos os pacientes, as células mononucleares do sangue periférico, o plasma e o soro para realização dos ensaios de citometria de fluxo e ELISA. Foi observado que os pacientes com TB pulmonar ativa apresentam um perfil regulador de células TCD8+ específicas, com expressão de IL-10 e TGF-β relacionados com a carga bacilar quando comparado aos indivíduos com TBIL e controles sadios PT negativa. Esse mesmo perfil também foi observado e descrito no caso clínico do paciente com TB cutânea. Diante de todos os achados observados nesse trabalho podemos concluir que Mtb pode modular a resposta de células TCD8+ na tuberculose pulmonar e cutânea, demonstrando a importância de estudos que avaliem a interação imunológica entre o patógeno e o hospedeiro tais como este.

Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is considered to be the most serious disease occurring in farmed striped catfish (Pangasianodon hypophthalmus) in Vietnam. This disease has had an increasing impact over the last ten years and has been reported to cause 50-90% mortality of stocks during a single outbreak. Data obtained from natural outbreaks of E. ictaluri in striped catfish showed the role of environmental factors in the establishment and progression of this disease. At present, factors affecting the virulence and transmission of E. ictaluri in striped catfish are poorly understood. The central hypothesis of this thesis focuses on the complex picture of the environmental factors and infectivity of E. ictaluri in striped catfish. In this study, 80 isolates of E. ictaluri recovered from natural clinical disease outbreaks occurring in striped catfish farms between 2002 and 2011 located in 4 distinct geographical areas within Vietnam were characterised using a variety of methods. The biochemical profiles showed that E. ictaluri isolates from striped catfish in Vietnam have similar phenotypic characteristics to other E. ictaluri isolates from other infected fish species. These data showed high levels of phenotypic homogeneity between the E. ictaluri isolates investigated. The status of isolates recovered from natural infections over time and from geographically distinct farms was evaluated using pulsed-field gel electrophoresis (PFGE), plasmid profile identification and antibiotic sensitivity tests. The PFGE results showed 6 main groups with a similarity of 82% and the corresponding genotypes of the prevalent isolates illustrated annual differences. Three plasmid groups were identified distributed among the isolates investigated, in which high molecular weight plasmids of approximately 35 and 140 kb were found in two of the groups. Plasmid profiles of the present study did not show any trend of geographical region or year of isolation. The 140 kb plasmid has been considered as a multi-antibiotic resistance plasmid which confers resistance to tetracycline, trimethoprim and sulphonamides. All Vietnamese isolates showed a high level of resistance to Oxolinic acid, Sulfadimethoxine/Ormetoprim (Romet), Oxytetracycline and Amoxicillin. A reproducible bacterial immersion challenge model was developed and the LD60 estimated prior to performing subsequent experimental challenge studies. Fish were exposed to 107 cfu ml-1 of E. ictaluri by immersion for up to 30 seconds, resulting in a cumulative percentage mortality of 63%. Edwardsiella ictaluri was recovered and identified from all the dead and moribund fish during these experiments and affected fish showed similar clinical signs and pathology to those reported from natural E. ictaluri infections. The present study resulted in a successful experimental immersion challenge model for E. ictaluri infection in healthy striped catfish. Cohabitation challenges were also developed and produced 15-40% mortality, typical clinical signs and pathology, and successful recovery of the challenge organism demonstrating horizontal transmission of E. ictaluri in striped catfish. Experimental studies were then conducted to investigate the association between pH or salinity of water and susceptibility to E. ictaluri infection in striped catfish. The first experiments were performed in in vitro conditions in which E. ictaluri isolates were cultured in a variety of pH and salt concentrations. In vivo experiments were then designed where striped catfish were exposed to 107 cfu ml-1 of E. ictaluri for 30 seconds and then held at 4 different water pHs (5.5, 6.5, 7.5 and 8.5) or NaCl concentrations (0, 0.5, 1 and 1.5%). The results of in vitro experiments showed that a pH value between 5.5 to 6.5 and salt concentration between 0-0.5% were optimal for the growth of E. ictaluri. The in vivo experiments demonstrated that the cumulative mortality of striped catfish in water at pH 5 and pH 6 was significantly higher than that of fish maintained in more alkaline water (p<0.05). By contrast, the cumulative mortality of the striped catfish maintained in 0.5% salt concentration was significantly lower than those kept in 0%, 1% and 1.5% salt concentration (p<0.05). Clinical signs, lesions and histopathological changes in the affected fish were consistent with those reported in natural infections. This study highlighted the use of pH 8.5 and salinity of 0.5% NaCl as a means of decreasing the susceptibility of striped catfish to E. ictaluri. In conclusion, this study used a variety of methods in order to enhance the understanding of the biochemical, biophysical characteristics, plasmid profile and antibiotic resistance as well as the relatedness of E. ictaluri isolates recovered from farmed striped catfish in Vietnam. This study provided two reliable and reproducible bacterial challenge models (immersion and cohabitation) and emphasised the link between pH and salinity with the infectivity and pathogenicity of E. ictaluri in striped catfish.

Made available in DSpace on 2014-08-20T14:38:01Z (GMT). No. of bitstreams: 1 dissertacao_pedro_quevedo.pdf: 852940 bytes, checksum: 646d10b8df90285825807c9a0da507ab (MD5) Previous issue date: 2010-12-20 / A retrospective study of clostridial diagnosed in ruminants from 1978-2008 in southern Rio Grande do Sul, was conducted in the influence area of the Regional Diagnostic Laboratory of the Federal University of Pelotas. Out of 5.446 bovine specimens sent by practitioners or from necropsies performed 91 (1,67%) were diagnosed as clostridiosis. Of those, 30 (0.55%) were diagnosed as by black leg, 15 (0.27%) as bacillary hemoglobinuria, 9 (0.16%) as botulism, 9 (0.16%) as malignant edema, and 28 (0.51%) as tetanus. Out of 860 sheep specimens 49 (5.69%) were bacterial diseases and 14 (28,57%) of those were diagnosed as clostridiosis, being 10 (1.16%) cases of tetanus, 3 (0.34%) of malignant edema, and 1 (0.11%) %) of enterotoxemia. It was concluded that some clostridiosis cause important losses in livestock in Rio Grande do Sul, mainly black leg. The vaccination of young stock against black leg is recommended in the whole state. Vaccination against botulism is recommended in areas of native pastures where phosphorus deficieny occurs, and vaccination against bacillary hemoglobinuria is recommended in low lands where fasciolosis is enzootic. In cattle, tetanus may occur as outbreaks with significant economic losses, recommending also the vaccination of animals. In sheep clostridioses are less important being tetanus the more frequent. Good hygienic practices, mainly during castration or tail are recommended to prevent the disease. Enterotoxemia is a rare disease in the region, because most sheep are raised in native pastures with limited grass production; vaccination is only recommended in sheep grazing in cultivate pastures or supplemented with concentrates. / Foi realizado um estudo retrospectivo dos diagnósticos de clostridioses ocorridos na região sul do Rio Grande do Sul correspondente a área de influência do Laboratório Regional de Diagnóstico da Faculdade de Veterinária da Universidade Federal de Pelotas entre janeiro 1978 e janeiro de 2008 e acompanhados os casos de clostridioses diagnosticados nos anos 2009 e 2010. De um total de 5.446 materiais de bovinos provenientes de necropsias ou órgãos enviados 91 (1,67%) correspondiam a enfermidades causadas por bactérias do gênero Clostridium sendo 30 (0,55%) de carbúnculo sintomático, 15 (0,27%) de hemoglobinúria bacilar, 9 (0,16%) de botulismo, 9 (0,16%) de edema maligno e 28 (0,51%) de tétano. De 860 materiais de ovinos 49 (5,69%) diagnósticos corresponderam a doenças bacterianas e desses 14 (28,57%) foram de surtos de clostridioses. Dos 14 surtos de clostridioses observados em ovinos 10 (1,16%) foram de tétano, três (0,34%) de edema maligno e um (0,11%) de enterotoxemia. Concluiu-se que algumas clostridioses causam prejuízos econômicos à bovinocultura da região, principalmente o carbúnculo sintomático, recomendando-se a vacinação sistemática dos bovinos jovens contra esta clostridiose. Recomenda-se, também, a vacinação contra botulismo, em áreas de campo nativo onde ocorre carência de fósforo e contra hemoglobinúria bacilar em áreas de campos baixos onde ocorre infecção por Fasciola hepatica. Em bovinos o tétano pode ocorrer como surtos com prejuízos econômicos importantes, recomendando-se, também, a vacinação dos animais. Em ovinos as clostridioses têm pouca importância, sendo o tétano a mais frequente. Para a profilaxia desta doença em ovinos recomenda-se utilizar práticas de manejo, como descola e castração, em condições adequadas de higiene. Enterotoxemia é uma enfermidade rara na região por que a maioria dos ovinos permanece em campos nativos com limitada produção de foragem; recomenda-se a vacinação somente em animais em pastagens cultivadas ou suplementados com concentrados.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.