Differentiation and gene flow among European populations of Leishmania infantum MON-1

Abstract

Background: Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1.

Methodology/principal findings: We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups-MON-1 and non-MON-1-could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands-Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles.

Conclusion: In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.

Figure 1. Population structure of Mediterranean L. infantum as inferred by STRUCTURE on the basis of data for 14 microsatellite markers obtained for 141 strains.

(A) Barplot for K = 4-each of the strains is represented by a single vertical line divided into K colors, where K is the number of populations assumed. Each color represents one population, and the length of the colors segment shows the strain's estimated proportion of membership in that population. (B) Distribution of Mediterranean L. infantum MON-1 strains belonging to populations 1–3 in the respective endemic foci. Pie-charts show the proportion of each population sampled in the respective geographical region. Colors correspond to the population specific ones in Figure 1A.

Figure 2. Neighbor-joining tree inferred from Dps-distances calculated for the data of 14 microsatellite markers and all 141 L. infantum strains.

Geographical origins, zymodemes and clinical manifestation are shown. Phlebotomus strains are marked by grey rectangles, relapses by white rectangles. Four strains with mixed MON-1/non-MON-1 ancestry are marked by an asterix. Populations as inferred by STRUCTURE K = 2, K = 3 and K = 4 are indicated by bars in different colors next to the tree. Midpoint rooting was applied for the NJ tree, no outgroup has been used.

Figure 3. Traces of gene flow between the L. infantum populations shown by a triangle plot.

The triangle plot of proportion of ancestry from three sources designated population 1 (Greece), population 4 (non-MON-1) and combined populations 2 and 3 (Majorca/Ibiza and mainland Spain and Portugal) as inferred from K = 4 in STRUCTURE analysis shows traces of gene flow between the populations. Each data point corresponds to a single strain whose proportion of ancestry from each of the three sourcesis represented by its proximity to the corresponding corner of the triangle. Strains of mixed MON-1/non-MON-1 ancestry and mosaic genotypes are labeled in grey (rectangles-most of the loci are heterozygous; ellipses-few loci are heterozygous). ES16(I) is homozygous for all loci, however representing a combination of alleles typical for different populations.

Figure 4. Factorial correspondence analysis (FCA) of L. infantum strains from the Mediterranean region.

(A) All MON-1 and non-MON-1 strains included (141 strains). (B) Analysis of the three MON-1 populations (113 strains). TR–Turkey, TN–Tunisia, IL–Israel; Population 1 (Greece)–yellow squares, population 2 (Majorca+Ibiza)–blue squares, population 3 (mainland Spain+Portugal)–white squares, population 4 (non-MON-1)–grey squares.