Molecular diversity in the Leishmania subgenus Viannia

The aim of this work was to provide further insight into the relationships between species of the Leishmania subgenus Viannia by examining inter- and intra-specific genetic diversity. Diversity among Viannia strains and stocks was investigated using biochemical and molecular techniques. Isoenzyme and microsatellite analyses were found to be the most discriminatory and generated results which could be interpreted genotypically. These techniques were used to study diversity in Viannia populations consisting of i) Brazilian L V. braziliensis stocks, ii) Nicaraguan stocks belonging to different Viannia species complexes, including putative species-complex hybrids, and iii) uncharacterized stocks from a new epidemic focus in Huanuco, Peru. LEA identified all stocks to at least the species level. Microsatellite analyses using Genescan® / Genotyper® and direct sequencing were found to be more discriminatory than EEA for all populations. The application of Genescan® / Genotyper® to the identification of alleles at these microsatellite loci has not been described previously. Phylogenetic analysis was carried out for each population using enzyme and microsatellite sequence data: phylogenies constructed from multilocus enzyme data were most accurate. Putative species heterozygotes between/,. V. braziliensis/L. V. panamensis and/,. V. braziliensis / L. V. peruviana were identified in the Nicaraguan and Huanuco populations, respectively, using IEA. Microsatellite analysis identified heterozygous stocks in all 3 populations. This approach also supported the hybrid status of the Nicaraguan and Huanuco stocks. Population genetic analysis of stocks from Huanuco provided statistical evidence for a limited degree of genetic recombination between stocks in this population. Results indicated, however, that clonal expansion was the predominant mode of replication. To explore the possibility of the occurrence of genetic recombination between species, genetic transformation experiments were initiated using putative parental stocks from the Nicaraguan population