INTERGENIC REGION TYPING (IRT) - A RAPID MOLECULAR APPROACH TO THE CHARACTERIZATION AND EVOLUTION OF LEISHMANIA

In the New World, Leishmania of the Viannia subgenus cause both cutane ous and mucocutaneous disease. These parasites show considerable intra -species genetic diversity and variation, which complicates taxonomic classification and epidemiology. We have used the variability of the t ranscribed noncoding regions between the small and large subunit rRNA genes to examine relationships in this group. In a method termed inter genic region typing (IRT), PCR amplification products were obtained fo r the rapidly evolving 1-1.2-kb internal transcribed spacers (ITS) bet ween the SSU and LSU rRNAs, from 50 parasites isolated from different hosts and geographic areas. Amplified DNAs were digested with 10 diffe rent enzymes, and fragment patterns compared after acrylamide gel elec trophoresis. High levels of intra- and inter-specific variation were o bserved, and quantitative similarity comparisons were used to associat e different lineages. A complex evolutionary tree was obtained. Some s pecies formed tight clusters (L. equatorensis, L. panamensis, L. guyan ensis, L. shawi), while L. bratiliensis was highly polymorphic and L. naiffi showed intraspecific distances comparable to the largest obtain ed within all Viannia. L. colombiensis, L. equatorensis and L. lainson i clearly represent distinct lineages. Good agreement was obtained wit h molecular trees based upon isoenzyme or mini-exon repeat sequence co mparisons. Overall, IRT appears to be a superb method for epidemiologi cal and taxonomic studies of Leishmania, being sensitive, rapid and qu antitative while simultaneously revealing considerable molecular diver sity. IRT could also be applied to other nonconserved intergenic regio ns, including those separating protein-coding genes.