RESUMEN
The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.
Asunto(s)
Leishmania major/clasificación , Leishmania major/aislamiento & purificación , Leishmaniasis Cutánea/parasitología , Animales , Brasil , Células Cultivadas , Análisis por Conglomerados , Medios de Cultivo , Dermatoglifia del ADN , ADN Protozoario/genética , Femenino , Genotipo , Humanos , Isoenzimas/análisis , Leishmania major/patogenicidad , Leishmania major/fisiología , Leishmaniasis Cutánea/patología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Protozoarias/análisis , Técnica del ADN Polimorfo Amplificado Aleatorio , Índice de Severidad de la Enfermedad , Piel/patología , VirulenciaRESUMEN
A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html 'Microsatellite Locus Extractor', and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1.6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.
Asunto(s)
Leishmania braziliensis , Leishmania donovani , Leishmania major , Repeticiones de Microsatélite/genética , Parasitología/métodos , Animales , Biología Computacional/métodos , Cartilla de ADN , ADN Protozoario/análisis , Humanos , Irán , Leishmania braziliensis/clasificación , Leishmania braziliensis/genética , Leishmania donovani/clasificación , Leishmania donovani/genética , Leishmania major/clasificación , Leishmania major/genética , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/parasitología , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadAsunto(s)
Sondas de ADN , Leishmania/clasificación , Animales , Secuencia de Bases , ADN de Cinetoplasto , Leishmania donovani/clasificación , Leishmania infantum/clasificación , Leishmania major/clasificación , Leishmania tropica/clasificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/clasificaciónRESUMEN
Leishmania parasites isolated from two patients with cutaneous leishmaniasis from geographically different localities in Paraguay have been characterized by enzyme electrophoresis (zymodeme) and digestion profiles of kinetoplast DNA with restriction enzymes (schizodeme). Both Paraguayan isolates showed identical zymodeme profiles to each other using 14 enzymes (glutamic pyruvate transaminase, glutamic oxaloacetic transaminase, enolase, fumarate hydratase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, malic enzyme, mannose phosphate isomerase, nucleoside phosphorylase, peptidase-D, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and pyruvate kinase). Although two Paraguayan isolates showed different zymodeme profiles from those of six Leishmania reference strains of Old and New World Leishmania species, they showed identical zymodeme profiles to those of an L. major-like parasite from Ecuador. These observations were confirmed by schizodeme analysis using three restriction endonucleases (Msp I, Hae III, and Taq I). These results indicate that Leishmania parasites isolated in Paraguay are identified as an L. major-like parasite, and it is necessary to consider the existence of L. major-like parasites when classifying Leishmania isolates from the New World.