The present study aimed to assess the population genetic structure of L. donovani complex strains from eastern Mediterranean focusing on southeastern (SE) Europe, where leishmaniasis represents a major public health problem. L. infantum zymodeme MON-1 is the main causative agent of human (HVL) and canine visceral leishmaniasis (CanL), and occasionally of human cutaneous leishmaniasis (HCL) in this area. The evident re-emergence of cases in classical endemic foci and reports of imported or autochthonous cases in formerly non-endemic regions calls for new epidemiological studies using highly discriminating markers.
Leishmania strains were isolated from HVL, CanL and HCL cases in six countries ranging from Croatia to Turkey, and from Phlebotomus tobbi sand flies in Turkey. These strains were initially typed by the K26-PCR assay, which amplifies the HASPB region, and multilocus enzyme electrophoresis (MLEE). Both methods corroborated the predominance of L. infantum MON-1 in SE Europe. MLEE exposed also a considerable number of zymodeme MON-98 strains in Turkey and Greece. All human isolates from Cyprus were identified as L. donovani sensu stricto MON-37. The Turkish strains from Cukurova and one HVL isolate from Kusadasi represented two novel zymodemes, MON-308 and MON-309.
Multilocus microsatellite typing (MLMT) was performed to exploit genetic diversity and population structure of the SE L. donovani complex strains. It assigned the SE and southwestern (SW) European strains to clearly distinct genetic populations. Croatian strains were divided in-between reflecting the geographic position of this country. All Cypriot canine isolates constituted a monophyletic group suggesting the circulation of a homogenous MON-1 population among dogs. MLMT did not support the differentiation between MON-1 and MON-98 zymodemes. Strains of these zymodemes from Turkey, Bulgaria, Greece and Albania grouped together in different highly heterogeneous populations. Our data point to recombination events and substantial gene flow among them. MLMT showed that strains of zymodeme MON-37 are paraphyletic. The Cypriot MON-37 strains formed a unique genetic group and were clearly distinct from MON-37 strains from the Indian subcontinent, Middle East and East Africa. Thus, the Cypriot MON-37 strains could possibly be autochthonous. When the Turkish MON-308 and MON-309 strains and the Cypriot MON-37 were compared to other L. donovani complex strains, the Turkish and Cypriot strains formed a new monophyletic group, clearly distinct from all other previously identified groups. Our observations suggest the existence of hybrids, aneuploidies or mixed infections for these Cypriot and Turkish strains thus raising concerns about their spreading to neighboring Mediterranean countries.
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