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Previous issue date: 2005 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / As cepas do Trypanosoma cruzi são constituídas por populações multiclonais, heterogêneas, diplóides e apresentam polimorfismo nos fragmentos de restrição no minicirculo do kDNA e nos cromossomos do nDNA. Esta variabilidade genética intraespecífica das cepas é correlacionada ao seu comportamento biológico, tropismo tissular e à resposta aos quimioterápicos. A cepa Colombiana do T. cruzi, protótipo do Biodema Tipo III, Zimodema Z1, classificada como T. cruzi I é altamente resistente ao benzonidazol e ao nifurtimox, No presente estudo, investigou-se a composição clonal desta cepa. Foram estudadas em camundongos o comportamento biológico, o histotropismo, caracteres bioquímicos e genéticos e a resposta ao benzonidazol de 12 clones. Foram isolados sete clones no 30° dia de infecção, no pico da parasitemia (fase aguda avançada) e cinco clones no 10° dia de infecção (fase aguda precoce). Os clones isolados foram submetidos a passagens sucessivas em camundongos albinos Suíços para a obtenção do inóculo de 1x10® tripomastigotas, para infecção dos animais dos grupos experimentais. O comportamento biológico foi avaliado pela parasitemia, estudo morfológico dos parasitos e mortalidade dos animais infectados. Os padrões isoenzimáticos para os diversos clones foram obtidos pelas enzimas ALAT, ASAT, PGM e GPI. Os animais infectados com cada clone e com a cepa parental foram submetidos ao tratamento com benzonidazol durante 90 dias na dose de 100 mg/kg/dia. O estudo genéíco foi realizado pela amplificação por POR de fragmentos de 330 pares de bases das porções variáveis do minicirculo do kDNA e por RFLP. Os fragmentos foram digeridos com as enzimas de restrição Rsa I, Eco RI e Hinfl No geral o estudo biológico mostrou homogeneidade entre os clones e entre os clones e a cepa parental. Houve variação na parasitemia sendo que foi mais elevada em quatro dos sete clones isolados no 30° dia da infecção e em três dos cinco clones isolados no 10° dia. A caracterização isoenzimática incluiu os clones no mesmo Zimodema Z1, idêntico á cepa Colombiana parental. O estudo genético mostrou que os clones e a cepa parental apresentaram idêntico padrão de bandas nos fragmentos de restrição gerados pelas enzimas de restrição. O histotropismo dos clones foi ubiquitário e não restritivo, foram mais encontrados no músculo esquelético e miocárdio, seguido pela presença de parasitos na parede intestinal, células de Kupffer e macrófagos do baço, plexo de Auerbach e adipócitos, Todos os clones apresentaram-se altamente resistente ao benzonidazol com índices de cura de 0% a 16, 7% (para os clones isolados no 30° dia) e de 0% a 36,3% (para os clones isolados no 10° dia). A cepa Colombiana do T. cruzi apresenta uma grande homogeneidade populacional, sugerindo a predominância de um clone principal responsável pelas características da cepa. / Strains of T. cruzi are heterogeneous multiclonal, diploid populations, presenting high polymorphic restriction fragments of the kDNA minicircles and nDNA chromosomes. This intra-specific genetic variability is correlated with the biological behavior, tissue tropism and responses to chemotherapy. The Colombian strain of T. cruzi, prototype of Type III biodeme and Z1 zimodeme , classified as T. cruzi I. is highly esistant to benznidazole and nifurtimox. In the present study it was investigated the clonal composition of this strain to identify whether there are an homogeneity or heterogeneity of the isolated clones, considering different aspects: biological, biochemical and genetic, and the clonal histotropism, as well as the susceptibility to chemotherapy with benznidazole. The Clones have been isolated in two different phases of the acute infection in mice; seven clones on the 30‘^ day of infection, corresponding to the parasitemic peaks (advanced acute phase) and five clones on the 10**^ day of infection (early acute phase) and inoculated into newborn mice. Successive passages into Swiss albino mice were done, to obtain the inoculum of 1 x 10® trypomastigotes for the infection of the experimental groups of mice. Biological behavior was evaluated by the parasitemic profiles, morphology of parasites in the blood and mortality indices. Isoenzymic profiles of ALAT, ASAT, PGM and GPI were established for the several clones. Treatment with Benznidazole was performed in experimental groups infected with each clone and the parental strain, in the dose of 250mg/kg/day, during 90 days. Genetic characterization was performed by the analyses of the kinetoplast DNA (kDNA) of parental strain and the clones. Schizodeme was established by comparative study of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles and by RFLP. Fragments have been digested by restriction endonucleases Rsa I, Hinf I and Eco Rl. In general, bological characterization showed an homogeneity of the several clones and amongst the clones and the parental strain, although with variations in the parasitemia that was higher for four of the seven clones isolated in the 30*^ day and for three of five clones isolated in the 10*^ day. Isoenzymic characterization, confirms the clones into the zymodeme Z1, identical to the Colombian parental strain. Genetic characterization demonstrated an homology of the bands profiles of the restriction fragments from the minicircles of kDNA (RFLP) between the parental strain and the 12 clones. Histotropism of the several clones was ubiquitous and not restrictive, predominant for the skeletal muscles and myocardium also present in the intestinal wall, Kupffer cells, macrophages of the spleen, cells of the Auerbach' plexus and adipocytes. All the clones were highly resistant to chemotherapy with benznidazole with cure indices of 0% to 16% (for the isolated in the 30*^ day) and 0% to 36,3% (for the isolated in the 10*^ day) .The overall analyses of the several parameters (genetic, biological, biochemical and response to chemotherapy) have shown phenotypic and genotypic homogeneity, of the Colombian strain and its clones, suggesting the predominance of a “principal clone” responsible for these characteristics.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/5887 |
| Date | January 2005 |
| Creators | Camandaroba, Edson Luiz Paes |
| Contributors | Reis, Mitermayer Galvão dos, Andrade, Sonia Gumes |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | Edson Luiz Paes Camandaroba, info:eu-repo/semantics/openAccess |
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