Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain

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Abstract

Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.

Introduction

Human leishmaniasis, caused by protozoan parasites of the genus Leishmania, constitutes a serious public health problem in several countries, according to the World Health Organization (W.H.O., 1997). In human hosts, the clinical profile of different Leishmania species can vary from a single cutaneous lesion, which may undergo spontaneous cure, to mucocutaneous lesions that can become grossly disfiguring. Severe diffuse cutaneous lesions, that is, extremely difficult to treat, can also occur. Moreover, the disease can evolve to visceral forms that are lethal in most cases (Ferreira et al., 2003).

Clinicians are confronted with steadily higher numbers of leishmaniasis patients not only in countries where the disease is endemic but also in countries where these parasites are not endemic. This increased incidence is partly due to geographical expansion of the disease, changing patterns of international travel and population migration, non-immune people moving into endemic regions, or infected people moving into non-endemic regions (Desjeux, 2001).

Leishmania species are morphologically very similar and species identification is possible using standard biochemical methods (lectin agglutination, isoenzyme analysis, analysis of kDNA restriction fragment using different restriction endonuclease, etc.). As has been demonstrated in many works where these techniques have been satisfactorily used to characterize Leishmania isolates (Andrade and Saraiva, 1999, Belhadj et al., 2003, Sampali et al., 2003, Shamsuzzaman et al., 2000), the ability to distinguish between Leishmania species is crucial when prescribing treatment as well as when determining possible control measures in epidemiological studies. Frequently, Leishmania species are identified based on their geographical distribution and on clinical manifestations of the resulting disease. However, geographical origin is an inadequate criterion in non-endemic areas, as well as endemic regions where multiple species of Leishmania may co-exist. Identification of the infecting species based on clinical symptoms can be problematic, since several species cause both cutaneous and mucocutaneous disease, while others cause visceral and cutaneous disease (Schönian et al., 2003).

In the present work, we characterized eight Leishmania isolates from different areas of Latin America (Peru and Mexico) as well as from the Mediterranean region (Spain), using interaction of the parasites with lectins together with electrophoretic analysis of their isoenzyme profiles and analysis of kDNA restriction fragment. Morphologically, all these have been considered to be members of the genus Leishmania. For comparison, we used four isolates from human cases characterized as: L. (L.) donovani, two strains belonging to Leishmania (L.) infantum and another characterized as Leishmania (Viania) peruviana. In addition, we made a comparative study of the major end-products excreted into the culture medium by the parasites.

Section snippets

Parasite isolation and in vitro culture

The eight Leishmania were isolated from different areas of Peru, Mexico, and Spain. Three of these isolates were from cutaneous cases in the central zone (LP1 and LP2) and north-eastern part (LP3) of the department of La Libertad (Peru); a fourth isolate (LP4) from a mucosal lesion was isolated in the zone of Cajamarca, in north-eastern Peru (near the border of Ecuador and Colombia). Three isolates (LM1, LM2, and LM3) were from cutaneous lesions in Campeche (Mexico) during the period 2000–2002.

Results

Leishmania isolates were cultured in vitro, assaying different liquid media. In general, all the isolates grew satisfactorily in MEM (Fig. 1), reaching cell densities on the order of 6.5 × 106–1 × 107 cells/ml, depending on the isolate.

Table 1 presents the results for lectin-agglutination tests. All the isolates showed agglutination with the lectins Con A, but with different degrees of agglutination and minimum concentrations required to agglutinate. The reference strain L. (V.) peruviana weakly

Discussion

To prevent variability derived from the culture medium, all the isolates were cultured in the same type of medium. The isolates were cultured in different cultured media having been assayed to identify the most adequate one. The MEM gave the highest growth density for most of the isolates and was selected to grow the isolates.

After determining the most suitable medium, we characterized all the isolates which identified to the genus Leishmania, by agglutination tests with lectins, by

Acknowledgment

This work was supported by “ATP 2002/30: Circulation of Trypanosomatidae” CIRAD (France) project.

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