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1.
Proc Natl Acad Sci U S A ; 106(25): 10224-9, 2009 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-19497885

RESUMO

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 +/- 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy-Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.


Assuntos
Leishmania braziliensis/genética , Animais , Bolívia , Heterozigoto , Humanos , Desequilíbrio de Ligação , Repetições de Microssatélites/genética , Peru , Polimorfismo Genético , Reprodução/genética
2.
Am J Trop Med Hyg ; 70(6): 607-12, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15211000

RESUMO

The southernmost limit of the distribution of endemic Andean cutaneous leishmaniasis (CL), commonly known as Uta, is localized in the western Andean valleys of Ayacucho, Peru. This area is completely isolated from other regions endemic for this disease. Identification of the insect vector for Andean CL was carried out by combining entomologic and parasitologic approaches. Two Lutzomyia species were captured: Lutzomyia ayacuchensis and Lu. noguchii. The former species was considered responsible for transmission of Leishmania because 1) there was a coincidence in space and time between the presence of this insect and the distribution of Andean CL, 2) it was shown to be highly anthropophilic, 3) Leishmania parasites of the subgenus Viannia were detected by a specific polymerase chain reaction assay, 4) promastigotes isolated from this insect were shown by multilocus enzyme electrophoresis and molecular karyotyping to belong to the same deme of Leishmania (Viannia) peruviana as the one circulating in humans living in the study area, and 5) the complete cycle of L. (V.) peruviana was observed in experimental infections of Lu. ayacuchensis. Parasite and vector homogeneity found in Ayacucho contrasted with the heterogeneity reported for other areas endemic for Andean CL. The potential influence of ecologic determinants on this geographically isolated area is discussed.


Assuntos
Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Psychodidae/parasitologia , Altitude , Animais , Feminino , Humanos , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/transmissão , Peru/epidemiologia , Reação em Cadeia da Polimerase
3.
Mem Inst Oswaldo Cruz ; 98(4): 477-80, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12937757

RESUMO

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.


Assuntos
DNA de Protozoário/genética , Biblioteca Gênica , Leishmania infantum/genética , RNA de Protozoário/genética , Animais , Primers do DNA/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Trans R Soc Trop Med Hyg ; 97(1): 80-7, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12886810

RESUMO

Accurate identification of Leishmania species is important for monitoring clinical outcome, adequately targeting treatment, and evaluation of epidemiological risk in tegumentary leishmaniasis. This is especially the case in regions where several species coexist and for travel medicine where the geographical source of infection is not always obvious. Species identification presently depends on parasite isolation, which is not very sensitive and not necessarily representative of parasites actually present in human tissues. We evaluated a polymerase chain reaction (PCR) assay combining amplification of the gp63 genes and restriction fragment length polymorphism (RFLP) analysis (gp63 PCR-RFLP) for direct Leishmania species-identification in tissues collected from Peruvian patients in 1999. By comparison with a kinetoplast DNA-based PCR, our PCR assay showed a detection sensitivity of 85%. Three species were encountered among patient samples, Leishmania (Viannia) braziliensis, L. (V.) peruviana and L. (V.) guyanensis, and their frequency and geographical distribution corresponded to earlier epidemiological studies of leishmaniasis in Peru. However, unexpected results raised questions about (i) the contribution of human migration to the emergence of new foci of given species, (ii) the pathogenicity of some species, and (iii) the frequency of mixed or hybrid infections.


Assuntos
Leishmania braziliensis/isolamento & purificação , Leishmaniose/diagnóstico , Animais , Sondas de DNA , DNA de Protozoário , Humanos , Leishmania braziliensis/classificação , Leishmaniose/parasitologia , Parasitologia/métodos , Parasitologia/normas , Peru , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade
5.
Mem. Inst. Oswaldo Cruz ; 98(4): 477-480, June 2003. ilus, tab
Artigo em Inglês | LILACS | ID: lil-344238

RESUMO

We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen). We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired


Assuntos
Animais , DNA de Protozoário , Biblioteca Gênica , Leishmania infantum , RNA de Protozoário , Primers do DNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro
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