Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Clinical, epidemiological and transmission cycle aspects of leishmaniasis urbanization in Barreiras, Bahia, Brazil.

Leishmaniasis remains one of the world's leading infectious diseases and a public health problem for Bahia and Brazil. We made a retrospective cohort study of leishmaniasis cases reported between 2007 and 2016 in the Barreiras city, an important agribusiness city whose urbanization process was recent. Leishmaniasis presented epidemiological data with similar characteristics of the disease in other regions of the Brazil, that is, with the highest prevalence of visceral leishmaniasis found in children, while cutaneous leishmaniais was found in adults. The disease presented an urban configuration with wide distribution in the Barreiras city. We found the vectors of the different clinical forms of the disease occurring in the urban area of Barreiras city, particularly the sand fly of the Lutzmyia longipalpis, Evandromyia sallesi and Nyssomyia intermedia species. More studies will be necessary to indentify whether these species are really incriminated in the urban transmission of the parasites in the urban areas. This study indicates the places of the spatio-temporal greatest occurrence and transmission of the disease in the urban zones of Barreiras city.

Increased capture of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) by removing one ADULTRAP component.

INTRODUCTION: Aedes aegypti is the main vector responsible for the transmission of numerous arboviruses. Adultrap® has been developed to catch these insects. METHODS: We tested the effectiveness of capturing adults with and without one of the components of Adultrap®. RESULTS: The mean number of insects caught by the original trap was 1.25 (standard deviation = 1.28), while the average obtained with the modified trap was 8.88 (standard deviation = 3.44). The medians were statistically different (p = 0.001) according to the Mann-Whitney test. CONCLUSIONS: The modification of Adultrap® increased the average catch of Ae. aegypti by up to seven times.

Increased capture of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) by removing one ADULTRAP component

Abstract INTRODUCTION: Aedes aegypti is the main vector responsible for the transmission of numerous arboviruses. Adultrap® has been developed to catch these insects. METHODS: We tested the effectiveness of capturing adults with and without one of the components of Adultrap®. RESULTS: The mean number of insects caught by the original trap was 1.25 (standard deviation = 1.28), while the average obtained with the modified trap was 8.88 (standard deviation = 3.44). The medians were statistically different (p = 0.001) according to the Mann-Whitney test. CONCLUSIONS: The modification of Adultrap® increased the average catch of Ae. aegypti by up to seven times.

Seroepidemiological survey of canine visceral leishmaniasis in a continental island of northeast Brazil / Investigação soroepidemiológica da leishmaniose visceral canina em uma ilha continental do Nordeste do Brasil

Introduction: Visceral leishmaniasis (VL) is endemic disease in the neighboring municipalities of the continental island Ilha de Maré, Salvador, Bahia, Brazil. Nevertheless, VL has not been reported in the island itself. Objective: the present study aimed to investigate the seroprevalence and clinical signs of Canine Visceral Leishmaniasis (CVL) and to identify the sand fly population present in the village of Botelho, Ilha de Maré. Methodology: sera of local dogs were tested for anti-Leishmania IgG by immunoassays (screening with TR DPP ™ rapid test and confirmatory with indirect ELISA, Bio-Manguinhos/Fiocruz) and an entomological survey was conducted to estimate and identify the phlebotomine fauna of the region. Results: seven out of 106 samples (6.6%) were positive using rapid test. These positive samples were sent to the Central Laboratory of Bahia for confirmation by indirect ELISA. However, all samples presentednegative results. Nine specimens of Pressatia choti, subfamily Phlebotominae were identified, being this species frequently found in areas with cutaneous leishmaniasis transmission in Brazil. Conclusion: although this work did not confirm the presence of CVL in Ilha de Maré, new serological and entomological studies in a larger area are required for the maintenance of the epidemiological surveillance in the emphasized insular area.

Introdução: Leishmaniose Visceral (LV) é uma doença endêmica em municípios vizinhos à Ilha de Maré, situada na plataforma continental do município de Salvador, Bahia, Brasil. Entretanto, casos de LV não tem sido notificados nesta Ilha. Objetivo: O presente trabalho objetivou investigar a soroprevalência e sinais clínicos de Leishmaniose Visceral Canina (LVC) e identificar a população de flebótomos presentes no povoado de Botelho, Ilha de Maré. Metodologia: soro de cães locais foram testados para IgG anti-Leishmania por imunoensaios (triagem com teste rápido TR DPP™ e confirmatório com Elisa Indireto, Bio-Manguinhos/Fiocruz) e uma investigação entomológica foi conduzida para estimar e identificar a fauna flebotomínea da região. Resultados: sete de 106 amostras (6,6%) foram positivas usando o teste rápido. As amostras positivas foram encaminhadas ao Laboratório Central da Bahia para a confirmação por ELISA indireto. Entretanto, essas amostras apresentaram resultado negativo. Foram encontrados nove exemplares da espécie Pressatia choti, subfamília Phlebotominae, espécie frequentemente encontrada em áreas de transmissão de leishmaniose cutânea no Brasil. Conclusão: apesar deste trabalho não ter confirmado a presença de LVC na Ilha de Maré, novos inquéritos sorológicos e analises entomológicas em uma maior área são necessários para a manutenção de uma vigilância epidemiológica na região insular em destaque.

Experimental study on the action of larvicides in Aedes aegypti populations collected in the Brazilian municipality of Itabuna, Bahia, under simulated field conditions. / Estudo experimental sobre a ação de larvicidas em populações de Aedes aegypti do município de Itabuna, Bahia, em condições simuladas de campo.

OBJECTIVE: to evaluate, under simulated field conditions, the efficacy of pyriproxyfen (juvenile hormone), novaluron (chitin inhibitor) and spinosad (biolarvicide) in controlling Aedes aegypti. METHODS: periodic exposition of Ae. aegypti larvae collected in Itabuna, BA, Brazil, to recipients treated with larvicides and comparison of residual effect of treatment with the Rockefeller strain. RESULTS: the inhibitory effect on adult emergence after 60 days was spinosad 89.5%, novaluron 96.5% and pyriproxifen 75.4% for Itabuna larvae, with no statistical difference (p=0.412) between treatments; spinosad and novaluron had a higher percentage of mortality in the larval stage, 98.8% and 97.9% respectively; pyriproxyfen showed higher mortality (95.1%) in the pupal stage. CONCLUSION: the three larvicides demonstrated similar control; however, pyriprofyxen might give a false impression of breeding ground positivity as it acts at the pupal stage, compromising the indicators of infestation that are strategic parameters for control actions.

Estudo experimental sobre a ação de larvicidas em populações de Aedes aegypti do município de Itabuna, Bahia, em condições simuladas de campo / Estudio experimental sobre la acción de larvicidas en poblaciones de Aedes aegypti del municipio brasileño de Itabuna, BA, en condiciones simuladas de campo / Experimental study on the action of larvicides in Aedes aegypti populations collected in the Brazilian municipality of Itabuna, Bahia, under simulated field conditions

Objetivo: avaliar, em condições simuladas de campo, a eficácia do pyriproxyfen (hormônio juvenil), do novaluron (inibidor de quitina) e do spinosad (biolarvicida) no controle do Aedes aegypti. Métodos: exposição periódica de larvas de Ae. aeypti obtidas em Itabuna a recipientes tratados com os larvicidas, e comparação do efeito residual do tratamento com cepa Rockfeller. Resultados: o efeito inibitório na emergência de adultos, após 60 dias, foi de 89,5% spinosad, 96,5% novaluron e 75,4% pyriproxifen para larvas de Itabuna, não havendo diferença estatística (p=0,412) entre os tratamentos; spinosad e novaluron apresentaram maior percentual de mortalidade na fase larval, 98,8% e 97,9% respectivamente; pyriproxifen apresentou mortalidade maior na fase pupal, 95,1%. Conclusão: os três larvicidas apresentaram controle semelhante; no entanto, o pyriproxifen pode deixar a falsa impressão de positividade dos criadouros, por agir em fase pupal, comprometendo os indicadores de infestação que são parâmetros estratégicos para as ações de controle.

Objetivo: evaluar en condiciones simuladas de campo la eficacia del piriproxifén (hormona juvenil), novaluron (inhibidor de quitina) y spinosad (biolarvicida) en el control del Ae. Aegypti. Métodos: exposición periódica de larvas obtenidas de Itabuna, BA, Brasil, a recipientes tratados con larvicida y comparación del efecto residual con cepa Rockefeller. Resultados: el efecto inhibidor en la emergencia de adultos, después de 60 días, fue de 89,5% spinosad, 96,5% novaluron y 75,4% piriproxifén para larvas de Itabuna, no habiendo diferencia estadística (p=0,412) entre los tratamientos; spinosad y novaluron presentaron mayor mortalidad en la fase larval, 98,8% y 97,9% respectivamente; piriproxifén presentó mayor mortalidad en la fase de pupa, 95,1%. Conclusión: los tres larvicidas fueron eficaces en el control de larvas de Ae. Aegypti; sin embargo, piriproxifén puede dejar falsa positividad de los criaderos, por actuar en fase de pupa, comprometiendo los indicadores de infestación, parámetros estratégicos para las acciones de control.

Objective: to evaluate, under simulated field conditions, the efficacy of pyriproxyfen (juvenile hormone), novaluron (chitin inhibitor) and spinosad (biolarvicide) in controlling Aedes aegypti. Methods: periodic exposition of Ae. aegypti larvae collected in Itabuna, BA, Brazil, to recipients treated with larvicides and comparison of residual effect of treatment with the Rockefeller strain. Results: the inhibitory effect on adult emergence after 60 days was spinosad 89.5%, novaluron 96.5% and pyriproxifen 75.4% for Itabuna larvae, with no statistical difference (p=0.412) between treatments; spinosad and novaluron had a higher percentage of mortality in the larval stage, 98.8% and 97.9% respectively; pyriproxyfen showed higher mortality (95.1%) in the pupal stage. Conclusion: the three larvicides demonstrated similar control; however, pyriprofyxen might give a false impression of breeding ground positivity as it acts at the pupal stage, compromising the indicators of infestation that are strategic parameters for control actions.

Leishmania amastigotes as targets for drug screening.

Direct drug screening against the mammalian stage of Leishmania has been hampered by cost and the time consuming effort required to accomplish it. The ability to derive transgenic Leishmania expressing reporter genes opened up new possibilities for the development of drug screening tests. Further developments to standardize and gather multiple informations could now be envisionned. We will discuss on such available methodologies that could improve sensitivity, reliability, versatility and the rapidity, of the screen based on intracellular model.

Trypanosoma cruzi: sequence polymorphism of the gene encoding the Tc52 immunoregulatory-released factor in relation to the phylogenetic diversity of the species.

We have previously identified a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin family involved in thiol-disulfide redox reactions. Gene targeting strategy and immunological studies allowed showing that Tc52 is among T. cruzi virulence factors. Taking into account that T. cruzi has a genetic variability that might be important determinant that governs the different behaviour of T. cruzi clones in vitro and in vivo, we thought it was of interest to analyse the sequence polymorphism of Tc52 gene in several reference clones. The DNA sequences of 12 clones which represent the whole genetic diversity of T. cruzi allowed showing that 40 amino-acid positions over 400 analysed are targets for mutations. A number of residues corresponding to putative amino-acids playing a role in GSH binding and/or enzymatic function and others located nearby are subject to mutations. Although the immunological analysis showed that Tc52 is present in parasite extracts from different clones, it is possible that the amino-acid differences could affect the enzymatic and/or the immunomodulatory function of Tc52 variants and therefore the parasite phenotype.

Targeted disruption of cytosolic SIR2 deacetylase discloses its essential role in Leishmania survival and proliferation.

Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/-) but achievement of null chromosomal mutants (LiSIR2-/-) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/-) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target.

Consideraçöes sobre pulga (Siphonaptera) da raposa Cerdocyon thous (Canidae) da área endêmica de leishmanioe visceral de Jacobina, Bahia, Brasil / Notes on fleas (Siphonaptera) of the fox Cerdocyon thous (Canidae) from the visceral leishmaniasis endemic area of Jacobina, Bahia, Brazil

No período de julho a setembro de 1998 foram coletadas 152 pulgas em 18 exemplares da raposa Cerdocyon thous capturados na área endêmica de leishmaniose visceral de Jacobina, Estado da Bahia. As pulgas foram identificadas como: 136 Rhopalopsyllus lutzi lutzi, 11 Pulex irritans, 2 Ctenocephalides canis, 1 Ctenocephalides felis felis e 2 Xenopsylla