Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil

Abstract

Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations of Leishmania (Viannia) braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity of L. (V.) braziliensis strains from different regions where they are endemic. The results reinforce the clonal theory for Leishmania parasites showing the genetic diversity of this pathogen and an association of L. (V.) braziliensis genotypes with specific transmission cycles, probably reflecting an adaptation of different clones to the vector species involved.

FIG. 1.

(A) MLEE phenogram showing the similarity between groups of L. (V.) braziliensis zymodemes. Unless indicated otherwise by the number in parentheses, IOC/Z contains a unique isolate. (B) Molecular tree showing the clustering of L. (V.) braziliensis genotypes classified in this work (PCR-RFLP ITSrDNA). The bootstrap values (italic numbers) are shown for the principal clusters. Details about strains are given in Table 1. The three main clusters represent parasites collected from different hosts and geographic areas in Brazil (i.e., the Atlantic coast [A and B] or the Amazon region [C]). Isolates characterized as IOC/Z-27 (outlined with continuous and dashed lines) presented genotypes in either group A and B. A, Strains collected in rural localities (Espírito Santo and Pernambuco) where different sandfly and animal species are involved in the transmission cycle; B, strains from urban areas (Espírito Santo and Rio de Janeiro) where transmission is related with a cycle involving domestic animals and preidomestic sandfly species; and C, strains from the Amazon region (in this case, the parasites are maintained in an enzootic cycle involving various wild animals and sylvan sand flies).

FIG. 2.

Acrylamide (12%) gel electrophoresis comparison of ITS fragment patterns generated with the restriction enzymes BstUI (A) and HhaI (B), among selected strains of L. (V.) braziliensis from Brazil. The codes in each lane represent the genotypes and the respective zymodemes (IOC/Z). The genotypic groups correspond to those described in Fig. 1. (A) Differences among groups A, B, and C; (B) differences among isolates from the same zymodeme.

FIG. 3.

Map of Brazil showing the areas from which the L. (V.) braziliensis isolates originated. A schematic and summarized dendrogram showing the relationship among the genotypic groups (A, B, and C as defined in Fig. 1) was superimposed. The level of genetic diversity found among the isolates from each area where the parasites were endemic was estimated by using the average of similarities as follows (averages ± the standard deviations of MLEE and IRT, respectively): Amazonia (0.75 ± 0.082 and 0.76 ± 0.095), Pernambuco (0.92 ± 0.01 and 0.83 ±0.082), Espírito Santo A (1.0 and 0.89 ± 0.05), Espírito Santo B (1.0 and 0.83 ± 0.11), and Rio de Janeiro (1.0 and 0.93 ± 0.035). The Brazilian states where the isolates were collected included Amazonia, Rondônia, Pernambuco, Espírito Santo, and Rio de Janeiro (see Table 1).