Dominique Le Ray

Extreme inbreeding in Leishmania braziliensis

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 ± 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy−Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.

Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru

During an outbreak of tegumentary leishmaniasis that developed in the 1990s in the Eastern Andean valley of Huanuco, Peru, the coexistence of Andean (uta) and sylvatic leishmaniases was suspected for ecological and geographical reasons, and sympatric sampling was carried out. Seven human isolates of Leishmania were characterized by multilocus enzyme electrophoresis, random amplification of polymorphic DNA and molecular karyotyping. The three methods identified 3 isolates as L. braziliensis, and 4 isolates as putative hybrids with characters of L. braziliensis and L. peruviana. Data from Huanuco are compared to previous results from other areas endemic for uta. Biological and epidemiological implications are discussed.

RELATION BETWEEN VARIATION IN COPY NUMBER OF RIBOSOMAL RNA ENCODING GENES AND SIZE OF HARBOURING CHROMOSOMES IN LEISHMANIA OF SUBGENUS VIANNIA

Chromosomal size polymorphism in Leishmania of subgenus Viannia has been correlated with eco-geography. The sizes of chromosomes bearing rDNA genes were determined in 69 isolates. A considerable size-variation was observed, ranging from 1100 to 1500 kb. Chromosomes of L.(V.). braziliensis, L.(V.)guyanensis and L.(V.) peruviana from northern Peru were significantly larger (200 kb) than those of L.(V.) peruviana from southern Peru. In addition, 31 out of 69 isolates presented each two different-sized homologues of the rDNA chromosome. Long range restriction mapping of three different-sized rDNA chromosomes from L.(V.)braziliensis M2903 and L.(V.)peruviana HB31 (north) and LC106 (south) each revealed three fragments delimited by PmeI restriction sites: two constant in size (the centre and one extremity of the chromosome) and one variable (the other extremity, containing a single cluster of rDNA genes). Further analysis of the M2903 rDNA chromosome allowed the localization of its 140 kb rDNA cluster at 85 kb from the telomeric end. Two arguments indicated that size-variation of the rDNA chromosome is partially due to amplification/deletion of the clustered rDNA genes: (i) size-variation of the cluster-containing fragment was proportional to the size-variation of the whole chromosome, and (ii) hybridization signal intensity of the rDNA chromosome with a small subunit rDNA probe strongly correlated with chromosomal size. Nevertheless, DNA sequences present between the rDNA cluster and the telomere might also play a role in chromosomal size polymorphism. In addition, our data suggest that rDNA gene copy number (20-40 copies cell(-1) under a diploid hypothesis) in subgenus Viannia is lower than reported previously.

Is Leishmania (Viannia) peruviana a distinct species? A MLEE/RAPD evolutionary genetics answer

Abstract A set of 38 Leishmania stocks from the Andean valleys of Peru was characterized by both Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA (RAPD). Data were analyzed in terms of taxonomy and evolutionary genetics. Synapomorphic MLEE and RAPD characters, clear-cut clustering, and strong agreement between the phylogenies inferred from either MLEE or RAPD supported the view that Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis correspond to two closely related, but distinct monophyletic lines (clades) and can therefore be considered as “discrete typing units” (DTUs). The question whether the L. (V.) peruviana DTU deserves species status is dependent upon the desirability of it, in terms of epidemiological and medical relevance. A previous Orthogonal Field Alternating Gel Electrophoresis (OFAGE) analysis of the same L. (V.) peruviana isolates was published by Dujardin et al. (1995b). The data from the different markers (i.e. MLEE, RAPD and OFAGE) were compared by population genetics analysis. RAPD and OFAGE provided divergent results, since RAPD showed a strong linkage disequilibrium whereas OFAGE revealed no apparent departure from panmictic expectation. MLEE showed no linkage disequilibrium. Nevertheless, contrary to OFAGE, this is most probably explainable by the limited variability revealed by this marker in L. (V.) peruviana (statistical type II error). RAPD data were consistent with the hypothesis that the present L. (V.) peruviana sample displays a basically clonal population structure with limited or no genetic exchange. Disagreement between RAPD and OFAGE can be explained either by accumulation of chromosomal rearrangements due to amplification/deletion of repeated sequences, or by pseudo-recombinational events.

Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis

Accurate identification of Leishmania species is important for monitoring clinical outcome, adequately targeting treatment, and evaluation of epidemiological risk in tegumentary leishmaniasis. This is especially the case in regions where several species coexist and for travel medicine where the geographical source of infection is not always obvious. Species identification presently depends on parasite isolation, which is not very sensitive and not necessarily representative of parasites actually present in human tissues. We evaluated a polymerase chain reaction (PCR) assay combining amplification of the gp63 genes and restriction fragment length polymorphism (RFLP) analysis (gp63 PCR-RFLP) for direct Leishmania species-identification in tissues collected from Peruvian patients in 1999. By comparison with a kinetoplast DNA-based PCR, our PCR assay showed a detection sensitivity of 85%. Three species were encountered among patient samples, Leishmania (Viannia) braziliensis, L. (V.) peruviana and L. (V.) guyanensis, and their frequency and geographical distribution corresponded to earlier epidemiological studies of leishmaniasis in Peru. However, unexpected results raised questions about (i) the contribution of human migration to the emergence of new foci of given species, (ii) the pathogenicity of some species, and (iii) the frequency of mixed or hybrid infections.

Latent class analysis permits unbiased estimates of the validity of DAT for the diagnosis of visceral leishmaniasis

Summary background Substantial uncertainty surrounds the specificity of the Direct Agglutination Test (DAT) for visceral leishmaniasis (VL) in clinical suspects, since no good gold standard exists for unequivocally identifying diseased subjects. We explored the Latent Class Analysis (LCA) modelling technique to circumvent this problem.

Chromosome rearrangement in Leishmania mexicana M379

Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.