Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas

Abstract

Background: Parasites' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease.

Methodology/principal findings: Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system.

Conclusions/significance: This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated.

Figure 1. Map of Ethiopia.

The VL foci in NE and SE are indicated. The NE foci are bordering the VL foci in Sudan, whereas the SE foci are bordering Kenya.

Figure 2. Populations, subpopulations and clusters for 123 L. donovani strains as inferred by STRUCTURE.

The peak at ΔK represents the most probable number of populations and subpopulations. Presence of smaller peaks in ΔK plot for the subpopulations indicated the presence of further clustering. A) Three main populations: North Ethiopia/Sudan (NE/SD), India, and South Ethiopia/Kenya (SE/KE) were identified in the whole data set of 123 strains. B) Within each East African population, two subpopulations were identified, A and B in the NE/SD population, and Konso and NB/KE in the SE/KE population. C) Five clusters were apparent, three (A1, A2 and A3) in the NE/SD-A subpopulation and two (B1 and B2) in the NE/SD-B subpopulation. Four clusters were detectable in the SE/KE population, named Konso, Kenya, Negele Borena and KO+ KE+NB. Each strain is represented by each vertical line in the STRUCTURE bar plot. Strains with mixed membership to the different populations, subpopulations or clusters are represented by different colored segments of the vertical bar which is proportional to the membership coefficient. The maximum membership coefficient is 1 meaning that a strain is a member in only one population, subpopulation or cluster.

Figure 3. Phylogenetic network (unrooted) for 123 L. donovani strains constructed with the Neighbor-Net algorithm.

STRUCTURE, NJ tree and network analyses detected the same three main populations as well as the two subpopulations (A and B) in the North Ethiopia/Sudan population and the four clusters; Konso, Negele Borena, Kenya and KO+KE+NB; in the South Ethiopia/Kenya population. The reticulate patterns seen in the network indicates either hybridization or recombination events between similar or closely related genotypes. The putative hybrid genotypes are indicated by a red bar line on the top. They are located in between their hypothetical parental strains in NE/SD-A subpopulation A and in cluster B1 of the NE/SD subpopulation B.