Amplified fragment length polymorphisms reveals high intraspecific variability in field isolates of Leishmania panamensis

Abstract

Leishmania parasites cause leishmaniasis, a potentially deadly re-emergent disease that affects millions throughout the world. In Panama, the disease is showing an increasing trend, with estimates of thousands of new cases every year. The main manifestations are the cutaneous and mucocutaneous forms. Genetic variability studies in Leishmania are extremely important to define key elements of the eco-epidemiology of the disease. However, few studies have addressed this issue in Panama, and these have been based mainly on kinetoplastid DNA RFLP. The amplified fragment length polymorphisms (AFLP) is a very efficient technique for rapid detection of genetic variability, particularly useful on organisms without sequenced genomes. Although this technique has been used successfully on many species, including several protozoa, its use for studying genetic variability in Leishmania parasites is just in its beginnings. We have optimized and used AFLP to address genetic diversity in Leishmania panamensis, a poorly studied member of the Viannia subgenus. We have found that this technique is able to generate high numbers of peaks when low selective EcoRI and MseI primers were used (+0, +1, +2 series). Additionally, we have found that an important proportion of those alleles, up to 57% for some primer combinations, are polymorphic. Some of these alleles are potentially useful to rapidly distinguish L. panamensis and L. guyanensis, the two most genetically similar species of the subgenus. The AFLP was an efficient technique to screen the Leishmania panamensis genome for polymorphisms, allowing the rapid detection of hundreds of polymorphic alleles.