Genetic markers and evolution of Sudanese Leishmania parasites

Abstract

Leishmania donovani caused different clinical forms of Leishmaniasis constituting major health problems in several countries. This study is aimed to study the taxonomy and evolution of Leishmania parasite isolated from eastern Sudan using molecular and genetic approaches. The isoenzyme profiles of 27 Sudanese Leishmania isolates were determined. Isoenzyme analysis showed the presence of three main zymodemes; MON82 (L.archibaldi) the most common, followed by MON30 (L.infantum) and MON18 (L.donovani). Four genetic markers were applied to study the genetic structure of L.donovani complex: kDNA-PCR, restriction analysis of the amplified ITS1 locus (ITS1-RFLP), RAPD and Microsatellite. PCR amplification of kDNA of Leishmania isolates resulted in amplification of 800bp in extract of 46 isolates and 700bp amplicon in 3 isolates .Compared with the reference strain the 46 isolates were identified as L.donovani complex isolates and the later 3 isolates were identified as L.major. Molecular typing of the isolates using kDNA-PCR technique agreed with the results obtained by isoenzyme profiles. To investigate the evolutionary relationships among the studied isolates the ribosomal DNA internal transcribed spacer (ITS) sequences of 49 Leishmania strains of different zymodemes were analyzed using PCR-RFLP. Amplification of ITS sequence of the studied isolates produced DNA fragment of 320bp, the amplicon was of similar size in all tested isolates. The results of the ITS1-RFLP showed a clear difference between L.donovani and L.major complexes. V Random amplified polymorphic DNA (RAPD) was applied using seven decamer primers to detect genetic diversity of Sudanese Leishmania donovani isolates. Four primers out of the 7 tested gave discriminative profiles among L.donovani complex. Two primers A10 & B12 were able to discriminate between the three main zymodemes of Sudanese L.donovani complex (MON82, MON18 & MON30). To determine the phylogenetic relationships of species of the Leishmania donovani complex microsatellite analysis was performed using 16 polymorphic markers. The topology of the NJ and UPGMA trees was similar and both trees differentiated the Sudanese parasite into three main clusters with high heterogeneity. Leishmania parasites isolated before 1995 from Sudan were compared with recent isolates from the same endemic area in eastern Sudan. The constructed NJ tree proved the replacement of the old isolates (clone) by new clones during the last 10 years. In order to compare the microsatellite evolution in multiple geographical areas comparing with Sudan different Leishmania isolates from different countries (Sudan, Kenya, India, Ethiopia, Spain and China) were included in this study. Two main phylogenetic groups were identified: 1.The Sudanese, Ethiopian and Chinese isolates 2. The Kenyan and Indian isolates. The program structure was used in this study to investigate Sudanese Leishmania population structure. The results showed the existence of three to four phylogenetic groups and these findings were in agreement with the microsatellite results.