"Leishmania infantum" : molecular analysis for identification of potential virulence factors and genes of diagnostic use

Leishmania infantum is the causative agents of zoonotic visceral leishmaniasis, a fatal disease if left untreated. In Southern Europe, the number of HIV/Leishmania infantum co-infections is increasing and becomes an emerging concern for the public health. Dogs represent the main reservoir of this parasite. In order to control canine leishmaniasis, an early diagnosis system is needed because dogs are capable to transmit the parasite, before showing any symptoms of the disease. Here fore, cDNA expression libraries were screened with sera of infected dogs. We selected several antigens reacting with the sera. These were different histones, ribosomal and mitochondrial proteins. Four of them were subcloned into expression plasmids, and recombinantly expressed in E. coli. The purified proteins were sent to a collaborating laboratory for further examination. Although several diagnostic polymerase chain reactions (PCRs) for Leishmania species causing human leishmaniasis are published, none of them is able to detect all different species and distinguish among them at the same time. Therefore, a diagnostic PCR was developed using the mini-exon gene (encoding the spliced leader) as template. A restriction fragment length polymorphism (RFLP) scheme was elaborated in order to identify different Leishmania species causing human leishmaniasis. The PCR and RFLP scheme is now in routine use at the STI. In order to identify potential virulence factors, vaccine candidates and drug targets of Leishmania infantum, two cDNA subtractions were performed: (1) A none-infective L. infantum strain was subtracted from a highly infective strain. Several genes which were higher or exclusively expressed in the infective strain were identified. However, association with virulence could not be shown, since the two strains differed not only in virulence but also in growth rate. (2) Non-infective early promastigote stages were subtracted from late promastigote stages (metacyclic transmission stage) of the same strain. Several genes showing differential expression were selected including genes encoding nutrient transporters, cytochrome b5 and unknown proteins. One of them was the previously described metacyclic specific gene MAT-1. Three of the genes were recombinantly expressed in E. coli in order to perform immunization and preliminary localization studies.