Caracterização biológica e molecular de populações clonais de cepas do Trypanososma cruzi: 21SF - biodema tipo II e colombiana - biodema tipo III, isolados de camundongos infectados, tratados com benzonidazol e não curados / Biological and molecular characterization of clonal populations Trypanososma cruzi strains: 21SF - biodeme type II and Colombian - biodeme type III, isolated from infected mice treated with benznidazole and uncured

As cepas e clones do Trypanosoma cruzi apresentam diferentes aspectos de resistência e susceptibilidade aos quimioterápicos. Vários estudos vem sendo desenvolvidos para avaliar a resposta de diversas drogas em diferentes cepas protótipos dos Biodemas tipos I, II e III, de acordo com a caracterização biológica. Resultados vêm demonstrando que cepas protótipos do Biodema Tipo I (cepas Y e Perunana) apresentam uma alta susceptibilidade ao tratamento com Benzonidazol e Nifurtimox; cepas do Biodema Tipo II (protótipo: cepa 21SF) demonstram média susceptibilidade; as cepas do Biodema Tipo III (cepa Colombiana) são altamente resistentes. Considerando que as cepas do T.cruzi são populações multiclonais complexas, que diferem nas suas características genéticas e biológicas, clones de duas cepas do T.cruzi foram avaliadas com o objetivo de investigar se o uso da quimioterapia anti–T.cruzi poderia estar levando à seleção de clones resistentes que poderão ou não diferir nos seus caracteres biológicos e moleculares. No presente trabalho investigamos os caracteres biológicas e moleculares de clones da cepa Colombiana (Biodema Tipo III) e 21SF (Biodema Tipo II) do T. cruzi, isolados de animais tratados e não curados em comparação com clones isolados de animais não tratados, com o intuito de investigar se estes clones resistentes à quimioterapia apresentavam diferenças em suas características que pudessem estar justificando tal resistência. Para isto, 18 clones foram isolados de camundongos infectados da cepa Colombiana e 8 clones foram isolados de camundongos infectados com a cepa 21SF. Os resultados mostraram que as características biológicas dos clones isolados da cepa Colombiana foram mantidas; clones da cepa 21SF mostraram diferentes níveis de parasitemia quando comparados com a cepa parental. As características moleculares foram avaliadas a partir dos fragmentos do k-DNA de cada clone isolado, que foram submetidos à técnica de RFLP, utilizando as enzimas de restrição RSA I, HINF I e ECO RI. A análise dos fragmentos de restrição das cepas parentais e dos respectivos clones demonstrou grande similaridade entres os mesmos. A possibilidade de investigar a estrutura molecular utilizando outras técnicas moleculares, poderá contribuir para demonstrar diferenças na resistência dos clones isolados de animais tratados e não não curados, como visto na discussão

Different strains and clones of Trypanosoma cruzi present differents degrees of susceptibility to treatment with chemotherapic drugs. Several studies have been developed to evaluate the response to different drugs concerning the strains prototypes of the Biodemes Types I, II and III according to the biological characterization. Results have shown that the strains prototypes of the Biodeme Type I (Y and Peruvian strains) disclosed a high susceptibility to treatment with Benznidazole and Nifurtimox; strains of the Biodeme Type II (prototype: the 21SF strain) showed a medium susceptibility; the strains of the Biodeme Type III (Colombian strain) were highly resistant. However a variability was detected according to the phase of infection in which the clones were isolated, and varied from 0% to 23,5% for the clones isolated in an early phase and 0% to 16.0% for those isolated in a late phase of infection. This indicates that the clonal populations could differently respond in different phases of treatment. In the present study we investigated the biological and molecular characters of clones of the 21SF strain (Biodeme Type II ) and of the Colombian strain (Biodeme Type III) isolated from mice treated with Benznidazole, but not cured , in comparison with clones isolated from untreated mice, with the objective of to investigate possible differences in the biological and molecular characteristics of these resistant clones. For that 18 clones were isolated from mice infected with the Colombian strain and 08 clones isolated from those infected with the 21SF strain. Results have shown that the biological characteristics were maintained in the Colombian strain; clones of the 21SF strain showed different levels of parasitemia as compared with the parental strain. This behavior is peculiar to clones isolated from the 21SF as shown in previous studies with clones isolated from untreated mice. The molecular characteristics were evaluated through the restriction fragment length polymorphism (RFLP) of the k DNA for each isolate clone, using restriction enzymes RSA I, HINF I and ECO RI...

Cinética de crecimiento in vitro de dos cepas de Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) en células miocárdicas de roedores con diferente susceptibilidad / In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility

In vitro growth kinetics of two Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae) clones in myocardial cells from rodents of different susceptibility. Two Trypanosoma cruzi isolates, TCR-4 from Costa Rica and UES-1 from El Salvador, were studied in vitro to compare their infectivity or resistance and intracellular replication in myocardial cells in three strains of mice and rats: NGP white mice, C3 H mice and Sprague Dowley rats. Myocardial cells were cultured on coverslips at 37 degrees C in a humid 10% CO2 atmosphere and then infected at a ratio of one tripomastigote per cell. Samples were studied after 24, 72, 96 and 120 h of infection to determine parasite infection capacity and intracellular multiplication. Both parasites had the highest infection capacity in C3 H mice, followed by NGP mice cells with a very low infection rate. Lastly, almost no Trypanosoma cruzi multiplication was observed in Sprague Dowley rats, suggesting a strong natural resistance in this animal to both strains of the parasite. The UES-1 isolate presented higher multiplication and greater invasion than the TCR-4 strain, showing greater virulence of UES-1 in heart cells, at least in vitro.

Cryptic infections in mice with the Trypanosoma cruzi CL-14 clone

A infectividade do clone CL-14 do Trypanosoma cruzi para camundongos foi revista utilizando-se como inoculo metaciclicos de cultura em NNN+LIT, pre-incubados ou nao com complemento de cobaio. Nos animais inoculados nao observamos parasitemia patente, mas a presenca do parasito foi confirmada em 30 por cento deles (9/30) atraves de hemocultivo ou xenodiagnostico, este examinado aos 100 dias. A positividade...

Comparative analysis by polymerase chain reaction amplified minicircles of kinetiplast DNA of a stable strain of Trypanosoma cruzi from Säo Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of Säo Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodene 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reation (PCR) from the variable regions of the minicicles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment lenght plymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100 per cent. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone.

Contrasting genomic variability between clones from field isolates and laboratory populations of Schistosoma mansoni

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using ramdomly amplified polymorphic DNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43 per cent of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analysis of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates.