Practical approach for typing strains of Leishmania infantum by microsatellite analysis

Abstract

Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.

FIG. 1.

Complete nucleotide sequences of genomic DNA regions containing microsatellites from strains of L. infantum. Microsatellites are shown in bold type. The superscripts indicate the minimum and maximum numbers of repeat units found for each microsatellite. (A) Nucleotide sequence of PCR product determined with primers A2 and B1. (B) Nucleotide sequence of PCR product determined first with primers Lm2A1 and Lm2B1 and then with primers Lm2A3 and Lm2B3. The internal primers, Lm2A3 and Lm2B3, are underlined. (C) Nucleotide sequence of PCR product determined with primers Lm4A1 and Lm4B1.

FIG. 2.

Dendrogram calculated with fastDNAml (version 1.2.2), which is based in part on Felsenstein's nucleic acid sequence maximum likelihood method (version 3.3), and elaborated with TREEVIEW tree drawing software. Each genotype is characterized by its microsatellite combination (ITS-Lm2-Lm4), its zymodeme, and its geographical origin. C, Catalonia; M, Madrid; P, Provence; I, Israel; #, the zymodeme is not available for all of the samples of the genotype (Table 2).