Multilocus microsatellite typing as a new tool for discrimination of Leishmania infantum MON-1 strains

Abstract

The Leishmania donovani complex, which consists of L. donovani, L. infantum-L. chagasi, and L. archibaldi, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of Leishmania. For L. infantum, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of L. infantum, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the L. donovani complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between L. donovani sensu stricto, a non-MON-1 group of L. infantum isolates, and a MON-1 group of L. infantum isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.

FIG. 1.

Fragment length analysis by PAGE (A) and MetaPhor agarose gel electrophoresis (B) of PCR products amplified with marker TubCA. Lanes (repeat numbers are in parentheses): 1, PP75 (n = 9); 2, WR317 (n = 9); 3, Lombardi (n = 11); 4, LEM75 (n = 9); 5, LPN114 (n = 13); 6, PM1 (n = 9); 7, LSL29 (n = 9); 8, BCN16 (n = 9); 9, IMT260 (n = 9); 10, LRC-L47 (n = 12); 11, RM1 (n = 9); 12, LEM3249 (n = 11); 13, LEM2298 (n = 9); 14, BUCK (n = 10); 15, LEM189 (n = 11); M, 10-bp ladder.

FIG. 2.

(A) Bootstrap analysis of all isolates analyzed (D_SA, UPGMA). Bootstrap values >50 are shown above the branches. (B) Population inference by model-based analysis. Populations are as inferred by STRUCTURE for K equal to 3 and K equal to 6. Strains that could not be clearly assigned to a population because of admixture were excluded. For the strain code, see Table 1.

FIG. 3.

Model-based analysis of the MON-1 corresponding cluster (K = 3). The lengths of the bars within boxes represent joint assignment to defined populations. The origin is the code defined by World Health Organization nomenclature (Table 1). PT, Portugal; FR, France; BR, Great Britain; ES, Spain; GR, Greece; TK, Turkey; IL, Ireland; TN, Tunisia.