In this study, we were able to identify hypervariable microsatellite loci and compile a set of markers usable for future epidemiological and population genetic studies for strains of L. aethiopica. Since this method is rapid and reproducible, we believe that it can be used for the reliable identification and characterization of L. aethiopica parasites. Of the 22 markers developed in this study, 5 polymorphic markers and one species specific marker were identified. MLMT has an advantage over other molecular techniques as results are reproducible and exchangeable between laboratories . It has proved to be a powerful tool for population genetic investigations, as well as epidemiological investigations, of Leishmania species . These short sequence repeats are highly polymorphic, codominant, and dispersed throughout the parasite genome. It has been shown that microsatellite loci of the family Trypanosomatidae are stable under laboratory conditions and can be detected directly in biological samples containing low amounts of parasitic DNA [21, 28]. In addition, the results of microsatellite analysis are much easier to compare between laboratories and store in databases .
This present study using MLMT divided the available isolates of L. aethiopica into four clusters. Previous studies conducted using L. aethiopica isolated from the skin of patients indicated genetic variation within the species; Multilocus Enzyme Electrophoresis (MLEE) separated strains into two different genetic groups . However, the techniques used suffer from poor reproducibility . In addition polymorphic repeats are not conserved between different species of Leishmania. Recently, analysis of length polymorphisms of microsatellite-containing regions has become an important tool for population and genetic studies for many different species [13, 30]. Microsatellites are tandemly repeated stretches of short nucleotide motifs which mutate at rates of five to six orders of magnitude higher than that of the bulk of DNA and present high variability mainly due to allelic repeat length variation. The length variation of individual loci can easily be screened after amplification with primers that anneal specifically to their flanking regions . These microsatellites are used for mapping genes in the genome because of their abundant distribution [31, 32].
A number of researchers have developed microsatellite markers for different species of Leishmania including L. major[17, 18], L. tropica[19, 20] and L. donovani, as well as other organisms such as Penicillium marneffei, which are now available and used for MLMT. To our knowledge, this is the first report demonstrating the use of MLMT for the molecular characterization of L. aethiopica, a parasite which is highly prevalent in Ethiopia and the major cause of LCL, MCL and DCL, accounting for an annual incidence of 50,000 cases .
Feasibility of high-throughput MLMT requires the optimization of PCR product analysis. In this study we show that MetaPhor gel electrophoresis and sequencing both produced analogous and reproducible results. Sequencing was used to determine the number of repeats. This is indispensable for the analysis of large fragments containing more than one microsatellite. However, this method is expensive and sequences containing small tandem repeats may be difficult to process. We demonstrate in this study that using MetaPhor agarose gel electrophoresis to screen for polymorphisms produces sufficient resolution to distinguish between L. aethiopica strains and could identify short tandem repeats .
All of the L. aethiopica isolates tested showed exclusive multilocus microsatellite patterns using the five identified markers. Thus MLMT could potentially enable researchers to potentially track strains of this parasite, making this an effective epidemiological tool. Even with these promising results, however, more isolates will need to be processed to confirm the spatial clusters or subdivisions of L. aethiopica in Ethiopia. Studies conducted on L. aethiopica are few compared to other species of Leishmania possibly due to its prevalence restricted to the eastern part of Africa. This study provides tools that will enable further molecular epidemiological and population genetic research on CL caused by L. aethiopica.
Primers previously developed for L. aethiopica were unable to identify the different Leishmania species and isolates in our study. We therefore utilized the previously characterized ITS1 regions to identify and confirm the identity of the Leishmania species used in this study. Sequencing of PCR products generated using ITS1 specific primers and performing sequence alignments against the Leishmania genome database enabled us to identify these isolates. The species specific primers developed in our current study could provide a quicker, cost effective and highly useful tool for the typing/diagnosis of L. aethiopica on clinical samples. This would be useful for case detection, determination of appropriate therapeutic regimens as well as implementation of control measures. Further, since this method does not require a restriction enzyme digestion step as in restriction fragment length polymorphism (RFLP), it provides an added advantage in accelerating species identification.