Leishmaniasis in Turkey: Visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey

Abstract

In Turkey, the main causative agents are Leishmania tropica (L. tropica) and Leishmania infantum (L. infantum) for cutaneous leishmaniasis (CL) and L. infantum for visceral leishmaniasis (VL). In this study, we investigated leishmaniasis cases caused by L. donovani and established animal models for understanding its tropism in in vivo conditions. Clinical samples (lesion aspirations and bone marrow) obtained from CL/VL patients were investigated using parasitological (smear/NNN) and DNA-based techniques. For species identification, a real time ITS1-PCR was performed using isolates and results were confirmed by hsp70 PCR-N/sequencing and cpb gene PCR/sequencing in order to reveal Leishmania donovani and Leishmania infantum discrimination. Clinical materials from CL and VL patients were also inoculated into two experimental groups (Group CL and Group VL) of Balb/C mice intraperitoneally for creating clinical picture of Turkish L. donovani strains. After 45days, the samples from visible sores of the skin were taken, and spleens and livers were removed. Measurements of the internal organs were done and touch preparations were prepared for checking the presence of amastigotes. The strains were isolated from all patients and amastigotes were seen in all smears of the patients, and then isolates were immediately stored in liquid nitrogen. In real time ITS1-PCR, the melting temperatures of all samples were out of range of L. infantum, L. tropica and L. major. Sequencing of hsp70 PCR-N showed that all isolates highly identical to previously submitted L. donovani sequences in GenBank, and cpb gene sequencing showed five isolates had longer cpbF allele, whereas one isolate contained a mixed sequence of both cpbF and cpbE. All mice in both experimental groups became infected. Compared to controls, the length and width of both liver and spleen were significantly elevated (p<0.001) in both groups of mice. However, the weight of the liver increased significantly in all mice whereas the weight of spleen increased only in VL group. Amastigotes were also seen in all touch preparations prepared from skin sores, spleen and liver. L. donovani strain was isolated from autocutaneous a VL patient first time in Turkey. Animal models using clinical samples were successfully established and important clinical differences of the isolated strains were observed.