Culture-independent methodologies to assess diversity and dynamics of Aeromonas communities

Mestrado em MicrobiologiaAs Aeromonas são bactérias autóctones em ambientes aquáticos e constituem um grave problema em sistemas de aquacultura devido à sua capacidade de provocar doença em peixes. Estas bactérias são actualmente consideradas patogéneos emergentes em humanos. Os surtos de doença podem estar associados à introdução de estirpes virulentas que evoluíram a partir de outros nichos ou da aquisição de determinantes de virulência do mesmo nicho, ou ainda como resultado de um desequilíbrio na comunidade local de Aeromonas. Assim, torna-se essencial desenvolver métodos fiáveis e reprodutíveis para seguir rotineiramente a dinâmica de comunidades indígenas de Aeromonas de forma a permitir uma detecção precisa de alterações na sua estrutura, antecipando potenciais riscos. Neste estudo, foram desenhados três conjuntos de primers para amplificar especificamente os genes gyrB, rpoD e sodB de Aeromonas. Métodos de PCR-DGGE (Denaturing Gradient Gel Electrophoresis) foram desenvolvidos com base nestes primers para obter perfis das comunidades. A especificidade dos primers foi testada utilizando como molde DNA de 27 estirpes de Aeromonas pertencentes a 17 espécies previamente descritas e também de 13 estirpes de 11 espécies não alvo. Simultaneamente a especificidade dos primers foi também avaliada in silico através da pesquisa de homologia com sequências depositadas nas bases de dados. Ambas as metodologias permitiram confirmar a total especificidade dos três conjuntos de primers e apenas para os primers RpoD não foi obtida amplificação de duas estirpes de Aeromonas. Para determinar a consistência da informação filogenética fornecida pelos fragmentos amplificados, árvores filogenéticas construídas com base nas sequências dos genes gyrB, rpoD e sodB de Aeromonas depositadas na base de dados GenBank e com base nas sequências dos fragmentos amplificados foram comparadas. Verificou-se que os fragmentos alvos fornecem informação filogenética consistente. Os conjuntos de primers foram testados em DNA de amostras de água da Ria de Aveiro e os produtos foram separados por DGGE. Para todas as amostras, e com todos os conjuntos de primers, obtiveram-se perfis complexos e muito estáveis ao longo do gradiente de salinidade. Resultados obtidos com as três metodologias indicam uma maior variabilidade sazonal das comunidades de Aeromonas. A clonagem e sequenciação dos fragmentos obtidos a partir das amostras ambientais confirmam a especificidade dos primers e revelam que os filotipos dominantes nestas comunidades apresentam elevada similaridade com estirpes de várias espécies de Aeromonas comuns em ambientes aquáticos como sejam A. alossacarophila, A. veronii e A. sobria. Segundo o nosso conhecimento, este estudo apresenta a primeira tentativa de optimização e validação de métodos independentes do cultivo específicos para Aeromonas. Os sistemas desenvolvidos apresentam várias vantagens e consequentemente constituem valiosas ferramentas para avaliar a diversidade e seguir a dinâmica de comunidades de Aeromonas. A utilização de mais de um conjunto de primers pode ser útil para obter uma representação mais clara e real da comunidade em estudo. Os métodos desenvolvidos poderão ainda ser adaptados de forma a serem aplicados em outro tipo de amostras ambientais o que poderá ser importante devido à ampla distribuição das Aeromonas e às suas capacidades patogénicas. ABSTRACT: Aeromonas are bacteria autochtonous in aquatic environments, and they constitute a serious problem in aquaculture systems because of their capability to cause disease in fishes. Aeromonads are also nowadays considered as emerging pathogens in humans. Disease outbreaks may be associated with the introduction of virulent Aeromonas strains that evolved in other niches, acquisition of virulence determinants in the same niche, or as a result of disequilibrium in the local Aeromonas community. Thus, it becomes essential the development of reliable and reproducible methods to routinely follow the dynamics of indigenous Aeromonas communities and to allow an accurate detection of alterations in their structure, anticipating potential risks. In this study 3 primer sets were designed to specifically amplify fragments of the genes gyrB, rpoD and sodB from Aeromonas. A PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method based on such primers was established to obtain community specific profiles. Primers specificity was tested by using as template DNA from 27 Aeromonas strains belonging to 17 previously described species, and also from 13 strains from 11 non-target species. Specificity was also assessed in silico using the BLAST tool, by checking sequence matches against the GenBank database. Results obtained confirmed the specificity of all primer sets and only primer set RpoD failed to amplify from two Aeromonas strains. To determine the phylogenetic information contained in the amplified fragments, gyrB, rpoD and sodB sequences available in the GenBank database from Aeromonas species were downloaded and submitted to phylogenetic analyses. A phylogenetic analysis following the same procedures was performed using the PCR target fragments and trees were compared. The phylogenetic information contained in the target fragments was confirmed to be consistent. Primer sets were tested in total DNA from estuarine water samples. A DGGE assay was optimized to separate the PCR products. Community specific profiles were obtained from each sample, for each primer pair. Profiles were very complex and rather stable along the salinity gradient. Aeromonas communities varied essentially in a seasonal basis. Cloning and sequencing of the fragments obtained from environmental DNA confirmed the specificity of the primers and revealed that the dominant phylotypes affiliated with strains included in species common in aquatic environments such as A. alossacarophila, A. veronii e A. sobria. To our knowledge, this study presents the first attempt to optimize and validate Aeromonas-specific culture-independent methodologies. Results indicate that all the developed systems present several advantages and therefore constitute valuable tools to assess diversity and follow the dynamics of Aeromonas communities. The utilization of more than one set of primers may be useful for providing a more reliable and clear representation of the community. The developed methods may be adapted to apply in other types of samples, which may be important due to the wide distribution of aeromonads in the environment and to their pathogenic properties

Balance of IL-10 and Interferon-γ plasma levels in human visceral leishmaniasis: Implications in the pathogenesis

BACKGROUND: Leishmaniasis remains a serious public health problem in several parts of the developing world. Effective prophylactic measurements are hampered by imprecise comprehension of different aspects of the disease, including its immunoregulation. A better comprehension of immunoregulation in human VL may be useful both for designing and evaluating immunoprophylaxis. METHODS: To explore immunoregulatory mechanisms, 20 visceral leishmaniasis (VL) patients were evaluated during active disease and at different periods up to one year after treatment determining their plasma cytokine levels, clinical parameters (palpable spleen and liver) and antibody levels. RESULTS: Elevated plasma levels of IFN-γ and of IL-12 p40 were observed during active disease, significantly decreasing after treatment whereas in vitro Leishmania antigen-stimulated IFN-γ production by PBMC exhibited an inverse pattern being low during disease and increasing steadily thereafter. Absence of IFN-γ activity is a hallmark of VL. The main candidate for blunting IFN-γ activity is IL-10, a cytokine highly elevated in plasma with sharp decrease after treatment. Activity of IL-10 is inferred by high levels of anti-Leishmania specific IgG1 and IgG3. TGF-β had elevated total, but not of active, levels lessening the likelihood of being the IFN-γ counterpart. Spleen or liver size presented a steady decrease but return to normal values at only 120 days after treatment. Anti-Leishmania IgG (total and subclasses) levels and DTH or Leishmania-stimulated lymphocyte proliferation conversion to positive also present a slow decrease after treatment. IL-6 plasma levels were elevated in only a few patients. CONCLUSION: Taken together our results suggest that IFN-γ and IL-10 are the molecules most likely involved in determining fate of disease. After treatment, there is a long delay before the immune profile returns to normal what precludes using plasma cytokine levels as criteria of cure as simpler clinical evaluations, as a palpable spleen or liver, can be used

Improved functionalization of oleic acid-coated iron oxide nanoparticles for biomedical applications

Superparamagnetic iron oxide nanoparticles can providemultiple benefits for biomedical applications in aqueous environments such asmagnetic separation or magnetic resonance imaging. To increase the colloidal stability and allow subsequent reactions, the introduction of hydrophilic functional groups onto the particles’ surface is essential. During this process, the original coating is exchanged by preferably covalently bonded ligands such as trialkoxysilanes. The duration of the silane exchange reaction, which commonly takes more than 24 h, is an important drawback for this approach. In this paper, we present a novel method, which introduces ultrasonication as an energy source to dramatically accelerate this process, resulting in high-quality waterdispersible nanoparticles around 10 nmin size. To prove the generic character, different functional groups were introduced on the surface including polyethylene glycol chains, carboxylic acid, amine, and thiol groups. Their colloidal stability in various aqueous buffer solutions as well as human plasma and serum was investigated to allow implementation in biomedical and sensing applications.status: publishe

Enzyme-linked immunosorbent assay for the detection of Bothrops Jararaca Venom

Manoel, Barral Netto. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-05-09T18:22:07Z No. of bitstreams: 1 Barral MN Enzyme-linked immunosorbent assay .....pdf: 526126 bytes, checksum: 164bb2224de40d38cc2b2188124b13c9 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-05-09T19:14:21Z (GMT) No. of bitstreams: 1 Barral MN Enzyme-linked immunosorbent assay .....pdf: 526126 bytes, checksum: 164bb2224de40d38cc2b2188124b13c9 (MD5)Made available in DSpace on 2018-05-09T19:14:21Z (GMT). No. of bitstreams: 1 Barral MN Enzyme-linked immunosorbent assay .....pdf: 526126 bytes, checksum: 164bb2224de40d38cc2b2188124b13c9 (MD5) Previous issue date: 1990CNPq's (Conselho National de Desenvolvimento Cientifico e Tecnologico) scientific initiation fellowship . This project was supported by Financiadora de Estudos e Projetos (FINEP).Universidade Federal da Bahia. Hospital Universitário Professor Edgard Santos. Serviço de Imunologia. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário Professor Edgard Santos. Serviço de Imunologia. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário Professor Edgard Santos. Serviço de Imunologia. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário Professor Edgard Santos. Serviço de Imunologia. Salvador, BA, BrasilEnzyme-linked immunosorbent assay for the detection of Bothrops jararaca venom. Toxicon 28, 1053-1061, 1990.-This study reports an enzyme-linked immunosorbent assay for detecting Bothrops jararaca venom in fluids, employing the sandwich method with biotin/avidin amplification. The assay exhibits high accuracy in correlating optical densities with venom concentrations (r = 0.98), high reproducibility, low background and limited crossreactivity with venom from other snake genera . Nevertheless, it was unable to distinguish among venoms from different bothropic species. Using this method we evaluated the serum kinetics of Bothrops jararaca venom in C57BL/6 mice. High concentrations were found in serum just 15 min after injection (151 f41 ng/ml; mean±S.D.), followed by a progressive fall (102 f46, 74 f39 and 50±22ng/ml after 1, 3 and 6 hr respectively), being undetectable by 24 hr. Such serum kinetics indicates a pattern of a rapid absorption of venom from the inoculation site, followed by a slow and progressive drop in its serum levels . This ELISA was a reliable tool in the determination of Bothrops jararaca venom levels in mouse serum, and may become useful in other fields of bothropic venom researc

PERFIL DO AMBULATÓRIO DE PRÉ NATAL (CIS E TRANS) DE PESSOAS GESTANTES QUE VIVEM COM HIV (PGVHIV) OU PESSOAS GESTANTES DE PARCERIAS SORODIFERENTES (PGPSD), INCLUINDO HOMENS TRANSEXUAIS NO CRT DST-AIDS SP E SEU IMPACTO SOBRE A TRANSMISSÃO VERTICAL

Introdução: O ambulatório de pessoas gestantes (PG) do CRT AIDS SP foi fundado em 1998 dado o aumento da demanda de PG e a necessidade de atendimento multidisciplinar especializado para esse fim. É composto por médicas: obstetra, infectologista e infecto-pediatra, enfermeira, técnica de enfermagem, psicóloga, assistente social e doula voluntária. Esse serviço visa ao atendimento de PGVHIV ou de PGPSD do pré Natal ao puerpério, contracepção e acompanhamento do concepto. Objetivo: descrever o funcionamento do ambulatório de pré Natal e o perfil das PG acompanhadas no CRT DST-AIDS SP durante 6 anos. 2. Métodos: análise sistemática, retrospectiva de dados dos prontuários das gestantes do ambulatório de pré Natal do CRT DST-AIDS SP no período de 2017 a 2022. 3. Resultados: foram acompanhadas 114 PG, incluindo 2 homens transgênero HIV negativos (1 deles PGPSD). A maioria (85%) proveio do ambulatório do CRT AIDS SP e 15% eram PGPSD. Apenas 10 (8,7%) PGVHIV provieram de outro serviço, sendo que 7 (70%) eram angolanas. Quanto ao status sorológico, 98 (85,2%) PG eram PGVHIV e 16 (14,8%) PGPSD. Em relação às ISTs, somente 2 gestantes (1,7%) tinham sífilis. Nenhuma delas teve COVID 19. Quanto ao desfecho da gestação, ocorreram 102 partos; sendo 76 (74,5%) cesáreas, 21(20,5%) partos normais, 1 parto fórceps e 4 casos sem essa informação. Ocorreram 12 perdas gestacionais, sendo duas não espontâneas. As 10 perdas espontâneas ocorreram entre 7 e 37 semanas. Apenas obtivemos dados de 3 desses casos. Dois ocorreram em primigestas: uma gestação gemelar em uso de Tenofovir (TDF) + Lamivudina (3TC) + Raltegravir (RTG) e carga viral (CV) indetectável (indet); a outra apresentava saco gestacional ístmico com 7 sem e 5 dias, CV de 5072 cópias/mL (log 3,7), uso de Zidovudina (AZT) + TDF + RTG. A terceira era uma gestação ectópica, CV indet, uso de TDF + 3TC + EFZ com 13 sem e 5 dias. Sobre contracepção, 70 de 96 puérperas (73%) optaram por um método pós gestação. O mais utilizado (77,1%) foi o implante subdérmico de etonogestrel, seguido por laqueadura tubária (7,1%) e demais métodos como preservativo, DIU e coito interrompido (30%). Nossa Taxa de transmissão vertical foi zero. 4. Conclusão: a existência do ambulatório de assistência às PGVHIV e PGPSD é fundamental para a condução adequada de suas intercorrências e cuidados no pré e pós concepção para a eliminação da transmissão vertical

Risk factors associated with asymptomatic infection by Leishmania chagasi in north-east Brazil.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-12-12T13:06:18Z No. of bitstreams: 1 Caldas A J M Risk factors....pdf: 898418 bytes, checksum: dc1f0bac263e1c43111433454490ad38 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2014-12-12T13:06:27Z (GMT) No. of bitstreams: 1 Caldas A J M Risk factors....pdf: 898418 bytes, checksum: dc1f0bac263e1c43111433454490ad38 (MD5)Made available in DSpace on 2014-12-12T13:23:21Z (GMT). No. of bitstreams: 1 Caldas A J M Risk factors....pdf: 898418 bytes, checksum: dc1f0bac263e1c43111433454490ad38 (MD5) Previous issue date: 2002Federal University of Maranhão. Nucleus of Tropical Pathology and Social Medicine. São Luis, MA, BrasilFederal University of Maranhão. Nucleus of Tropical Pathology and Social Medicine. São Luis, MA, BrasilFederal University of Maranhão. Public Health Department. São Luis, MA, BrasilFederal University of Bahia. School of Medicine. Immunology Service. Salvador, BA, BrasilFederal University of Bahia. School of Medicine. Immunology Service. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilVarious factors have been associated with a predisposition to the development of clinical American visceral leishmaniasis (AVL). However, little information is available about the factors that predispose to asymptomatic infection. To identify the risk factors associated with asymptomatic infection, a study was carried out between July 1997 and June 1998 on children aged 0-5 years in the districts of Vila Nova and Bom Viver in the municipality of Raposa in the island of São Luís, State of Maranhão, Brazil. A questionnaire containing socioeconomic, demographic and epidemiological data was used. The delayed-type hypersensitivity (DTH) test was carried out on 639 children in the first phase, and on 572 in the second, 7 months after the first survey, using Leishmania amazonensis antigen. Infection was determined by enzyme-linked immunosorbent assay (ELISA) in 638 children during the first phase, and in 572 during the second. Six outcome measures were used: initial prevalence, final prevalence and incidence, each determined by DTH and ELISA. The incidence of infection was 10.8% when determined by DTH and 28.5% when determined by ELISA. After adjustment for confounding variables using Cox regression, infection by L. chagasi was associated with child's age (> or = 2 years), location of the dwellings (Vila Nova) and reporting of relatives with AVL. Bathing outside the house and playing outdoors between 18:00 and 20:00 were identified as risk factors in some analyses but not in others. Presence of intra- and peridomestic Lutzomyia sandflies and animals such as dogs or chickens in the house or in the neighbourhood appeared as risk factors in some analyses but in others they unexpectedly seemed to protect from infection. Malnutrition was not found to be associated with infection

Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-03-17T17:40:44Z No. of bitstreams: 1 Vinhas V Human anti-saliva....pdf: 347167 bytes, checksum: 54a24ccf2c3aab33b0ff6aebd5613dc7 (MD5)Made available in DSpace on 2014-03-17T17:40:44Z (GMT). No. of bitstreams: 1 Vinhas V Human anti-saliva....pdf: 347167 bytes, checksum: 54a24ccf2c3aab33b0ff6aebd5613dc7 (MD5) Previous issue date: 2007Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, BrasilNational Institutes of Health NIAID. National Institutes of Health. Immunobiology Section. Bethesda, USAFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. / Salvador, BA, Brasil / Instituto de Investiga ção em Imunologia. Instituto do Milênio. SAlvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. / Salvador, BA, Brasil / Instituto de Investiga ção em Imunologia. Instituto do Milênio. SAlvador, BA, BrasilExperiments in animals verified that phlebotomine saliva enhances Leishmania infection, and vaccination with saliva prevents disease. We have shown that individuals from an endemic area of visceral leishmaniasis displayed robust antibody responses to saliva from the vector Lutzomyia longipalpis, which correlated with anti-parasite cellmediated immunity. Here, we explored human anti-saliva responses following exposure to sand flies, using an in vivo bite model in which normal volunteers were exposed four times to 30 laboratory-reared Lu. longipalpis. Following the third exposure, normal volunteers developed diverse dermatological reactions at the site of insect bite. Serum from normal volunteers displayed high levels of anti-salivary gland sonicate IgG1, IgG4 and IgE as well as several salivary gland proteins. Furthermore, following in vitro stimulation with salivary gland sonicate, there was an increased frequency of CD4+CD25+ and CD8+CD25+ T cells as well as IFN-c and IL-10 synthesis. Strikingly, 1 year after the first exposure, PBMC from the volunteers displayed recall IFN-c responses that correlated with a significant reduction in infection rates using a macrophage-lymphocyte autologous culture. Together, these data suggest that human immunization against sand fly saliva is feasible and recall responses are obtained even 1 year after exposure, opening perspectives for vaccination in man

Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-10-02T20:35:03Z No. of bitstreams: 1 Barral A Human immune response ....pdf: 73439 bytes, checksum: 5354b8fbac841b0e77b6db52195eea1b (MD5)Made available in DSpace on 2012-10-02T20:35:03Z (GMT). No. of bitstreams: 1 Barral A Human immune response ....pdf: 73439 bytes, checksum: 5354b8fbac841b0e77b6db52195eea1b (MD5) Previous issue date: 2000Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Hospital Universitário. Salvador, BA, BrasilUniversidade Federal do Maranhão. São Luiz, MA, BrasilWalter Reed Army Institute of Research. Department of Entomology. Washington, District of ColumbiaNational Institutes of Allergy and Infectious Diseases. Bethesda, MarylandFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilAntibody (IgG) responses to salivary gland homogenate and to a recombinant salivary protein from the sand fly Lutzomyia longipalpis were investigated using sera from children living in an endemic area of visceral leishmaniasis in Brazil. We classified children into four groups according to their responses to Leishmania antigen: (Group I) positive serology and positive delayed type hypersensitivity (DTH), (Group II) positive serology and negative DTH, (Group III) negative serology and positive DTH, and (Group IV) negative serology and negative DTH. A highly significant correlation was found between anti-salivary gland IgG levels and DTH responses. An L. longipalpis salivary recombinant protein used as an antigen in an enzyme-linked immuno sorbent assay (ELISA) gave a significant but different result. A positive correlation was found between anti-Leishmania IgG and anti-recombinant protein IgG titers. The results indicate that sand fly salivary proteins may be of relevance to the study the epidemiology of leishmaniasis