Search

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa.

The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)—extensively used for bacteria—has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes—glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)—from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences. [ABSTRACT FROM AUTHOR]

Abstract: To understand phylogenetic relationships of species and strains within the Leishmania donovani complex, we have analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 27 Leishmania infantum, 2 Leishmania chagasi, 18 L. donovani and 5 Leishmania archibaldi strains of different zymodemes and geographical origin. Eight ITS sequence types were found. All detected sequence variation within ITS1 and ITS2 was based on 12 polymorphic microsatellites. The L. infantum strains from the Mediterranean region, China and L. chagasi from the New World formed a phylogenetic group well separated from the second main group including all strains from East Africa and India. Within the latter group three distinct phylogenetic subgroups could be differentiated: (1) L. donovani (Sudan/Ethiopia, China)+ L. archibaldi (Sudan), (2) L. donovani (Sudan/Ethiopia)+ L. infantum (Sudan)+ L. archibaldi (Sudan/Ethiopia), and (3) L. donovani (Kenya, India). These groups are not consistent with previous species definitions based on isoenzyme analyses, e.g. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Two groups of Indian strains could be differentiated, one of which has an identical sequence type to the strains from Kenya. Three main lineages of strains can thus be differentiated in East Africa: two quite distantly related groups of strains from Sudan/Ethiopia, and a third group including all strains from Kenya, which is more closely related to part of the Indian strains than to any of the Sudanese/Ethiopian groups. The ITS sequence analysis presented here supports the need for revision of the taxonomy of the L. donovani complex. [Copyright &y& Elsevier]

Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.