Estudo epidemiológico de fontes de alimentação sangüínea dos triatomíneos da fazenda aroeira (Catolé do Rocha, Paraíba) e circunvizinhanças

Analisamos a fonte de alimentação sangüínea de 94 triatomíneos (51 Triatoma brasiliensis, 42 T. pseudomaculata e um Rhodnius neglectus) da Fazenda Aroeira, no Município de Catolé do Rocha, no Estado da Paraíba, e de quatro localidades próximas. Observamos positividade para marsupial, homem e ave, respectivamente, de 39,7 , 6,2 e 23,0%. Três insetos da Fazenda Aroeira, positivos para Trypanosoma cruzi, foram positivos para marsupial, só um deles também para outro mamífero. Os marsupiais são a mais importante fonte de T. cruzi para os insetos da área e estes têm pouco contato com o homem.

Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)

RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos. Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies. In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.

Biodemes and zymodemes of Trypanosoma cruzi strains: correlations with clinical data and experimental pathology

With the objective of establishing biological and biochemical characteristics of a significant number of Trypanosoma cruzi strains from different geographical areas, 138 strains isolated from naturally infected humans, triatomine or vertebrate hosts were studied; 120 were isolated from different areas of Brazil and 18 from other South and Central American countries. Inocula from triatomine or culture forms were injected into suckling Swiss mice, followed by passages into mice 10 to 12 g. Biological characters and histopathological study permitted the inclusion of the strains into three Types or biodemes: I, II, III. Isoenzymic analysis confirmed a correspondence between the biodemes and zymodemes : Type I and Z2b, Type II and Z2, Type III and Z1. Results showed the ubiquitary distribution of the several types of strains. The predominance of the same Type and zymodeme in one geographical area was confirmed : Type II strains among the human cases from eastern Bahia and east of Goiás; Type III strains from humans of north Brazil and Central America and from silvatic vectors or vertebrates from other geographical areas. The biological types of strains correlate with different histopathological lesions considering cardiac involvement and neuronal lesions. These findings suggest that the biological behavior together with isoenzymes patterns and pathological pictures in the vertebrate host can be an important tool for establishing correlations between strains behavior and clinico-pathological manifestations of Chagas' disease in different geographical areas.

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Vetores selváticos de doença de Chagas na área urbana de Manaus (AM): atividade de vôo nas estações secas e chuvosas

Adultos de Rhodnius prolixus, R. pictipes e Panstrongylus geniculatus encontrados em casas em Manaus apresentam altas taxas de infecção por Trypanosoma cruzi. Rhodnius spp não apresentavam sazonalidade acentuada, porém os encontros de machos de P. geniculatus eram muito mais freqüentes na estação seca.

Purification of complex biopharmaceuticals with new processes, advanced analytics and computer-aided process design tools

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Nota sobre tipos e espécimes de triatomíneos depositados na Coleção Entomológica do Instituto Butantan, São Paulo (Hemiptera:Reduviidae)

Esta nota relata a descrição cronológica da Coleção Entomológica do Instituto Butantan e relaciona o registro de 343 espécimens e oito exemplares tipo pertencentes à Subfamilia Triatominae depositados nesta Coleção. São representados nesta listagem 5 espécies do Gênero Panstrongylus, 18 espécies de Triatoma, 2 espécies de Psamolestes e Rhodnius

Excito-repellency effect of deltamethrin on triatomines under laboratory conditions

This work demonstrates that deltamethrin in low doses produces an excito-repellency effect on triatomines, as already observed for mosquitoes. A wooden box covered with a cloth impregnated with deltamethrin at doses of 2.5 and 5mg ai/m² was utilized for the experiment. The triatomine species studied were Triatoma infestans, Panstrongylus megistus, Rhodnius neglectus and Triatoma sordida. Adults were released in one of the sides of the box and their position was noted in subsequent periods. The observations were realized on the day the cloth was impregnated and subsequently repeated at 30 and 60 days for T. sordida; on day 120, the remaining species were included. Insect mortality and attempts at flight from the box were also observed. Excito-repellency was evident for all species and doses up to day 120. The only species that attempted to fly was P. megistus. The excito-repellency effect may be considered as an additional advantage to the insecticide power, as it should be able to prevent the installation of new colonies by females that fly into the homes, and at the moment of the spraying, it should promote the flushing out of triatomines from the wall crevices and from other shelters.

Evaluation of the impact of chemical control measures and entomological surveillance on Chagas' disease in the counties of Mambaí and Buritinópolis, Goiás State, Brazil

Epidemiological surveillance activities were implemented in 1980 in Mambaí and Buritinópolis counties, Goiás State. Twenty years later the authors evaluated the impact of these vector control measures on Chagas' disease transmission, based on entomological indicators. Entomological investigation was conducted using the man-hour technique and covering all domiciles. In order to study vector food sources the stomach contents of triatomines were analyzed using the modified precipitins technique. Triatomines were shown to be present in 48 (71.6%) of the 67 locations. Peridomiciliary infestation rates in Mambaí and Buritinópolis were 8.7% and 12.1%, respectively, while intradomiciliary rates were 0.7% and 1.2%. Triatoma sordida was the species identified in 97.3% of all captured specimens. It was also the only species found to be naturally infected with Trypanosoma cruzi. Birds were the most frequent food source (45%) for Triatoma sordida. The most significant result was the complete absence of Triatoma infestans in the two counties.

Ampliação da distribuição geográfica de Triatoma deaneorum Galvão, Souza & Lima 1967, nova denominação para Triatoma deanei (Hemiptera, Reduviidae)

A distribuição geográfica de Triatoma deaneorum Galvão, Souza & Lima, 1967, nova denominação para Triatoma deanei, é ampliada baseada em um espécime recentemente encontrado no município de Chapada de Guimarães, próximo a Cuiabá, Mato Grosso, Brasil. A nova denominação é baseada no Código Internacional de Nomenclatura Zoológica.

Software for pattern recognition of the larvae of Aedes aegypti and Aedes albopictus

Software for pattern recognition of the larvae of mosquitoes Aedes aegypti and Aedes albopictus, biological vectors of dengue and yellow fever, has been developed. Rapid field identification of larva using a digital camera linked to a laptop computer equipped with this software may greatly help prevention campaigns.

Environmental sanitation conditions and health impact: a case-control study

This epidemiological investigation examines the impact of several environmental sanitation conditions and hygiene practices on diarrhea occurrence among children under five years of age living in an urban area. The case-control design was employed; 997 cases and 999 controls were included in the investigation. Cases were defined as children with diarrhea and controls were randomly selected among children under five years of age. After logistic regression adjustment, the following variables were found to be significantly associated with diarrhea: washing and purifying fruit and vegetables; presence of wastewater in the street; refuse storage, collection and disposal; domestic water reservoir conditions; feces disposal from swaddles; presence of vectors in the house and flooding in the lot. The estimates of the relative risks reached values up to 2.87. The present study revealed the feasibility of developing and implementing an adequate model to establish intervention priorities in the field of environmental sanitation.

Integrated functional genomics for improved manufacture of recombinant enveloped virus

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences-Biotechnology

Using epidemiological survey data to infer geographic distributions of leishmaniasis vector species

An important aspect of tropical medicine is analysis of geographic aspects of risk of disease transmission, which for lack of detailed public health data must often be reduced to an understanding of the distributions of critical species such as vectors and reservoirs. We examine the applicability of a new technique, ecological niche modeling, to the challenge of understanding distributions of such species based on municipalities in the state of São Paulo in which a group of 5 Lutzomyia sandfly species have been recorded. The technique, when tested based on independent occurrence data, yielded highly significant predictions of species' distributions; minimum sample sizes for effective predictions were around 40 municipalities.

Triatoma rubrovaria (Blanchard 1843): tábua de vida das ninfas, duração das formas e oviposição das fêmeas

Os autores acompanharam a evolução de 150 ovos de Triatoma rubrovaria o que permitiu a construção de uma tábua de sobrevida das ninfas. A forma alada foi atingida por 94,64% dos exemplares, sendo 79 deles machos e 65 fêmeas. O tempo médio dos alados machos foi de 115 dias e das fêmeas, 99 dias. Parte desses exemplares formaram 30 casais, mantidos isolados, o que possibilitou o levantamento da postura de cada fêmea. Outro lote, também formado por 30 machos e 30 fêmeas, foi mantido em um único cristalizador de vidro, o que permitiu constatar sua maior postura (7.832 ovos) em relação aquela das fêmeas acasaladas por único macho (5.167). O bom desenvolvimento dos exemplares no processo de evolução e de reprodução da espécie, nas condições do experimento, mostrou a boa capacidade de manutenção das colônias do Triatoma rubrovaria, em condições de laboratório.