Genetic Polymorphism Analysis And Molecular Typing Of Leishmania Isolates From China

Objective:The purpose of this study was to elucidate the genetic relationships of Leishmania isolates from different endemic regions of China based on sequences of several leishmanial molecular markers and perform molecular typing on these isolates.Furthermore suitable molecular markers will be selected for the identification of Leishmania in China.Methods:The K26,mini-exon,cpa and 7SL RNA sequences of Leishmania isolates were amplified and sequenced using specific primers.The sequences obtained were aligned by MEGA7.0 software using Clustal W method.The sequences of K26,mini-exon,cpa and 7SL RNA of related Leishmania isolates were downloaded from GenBank,and the phylogenetic trees of corresponding sequences were constructed by MEGA7.0 software using Neighbor-Joining method.Results:The K26 sequence of Leishmania isolates from China were amplify by specific primers.Type from anthroponotic type visceral leishmaniasis(AVL)endemic area three isolates were amplified 920 bp single fragment,from the zoonotic deserts type visceral leishmaniasis(DT-ZVL)endemic area of 4 isolates were amplified 491 bp single fragment,from the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area 4 isolates were amplified 404 bp single fragment,from cutaneous leishmaniasis(CL)endemic area 5 isolates were amplified 449 bp single fragment.The sequence identity between isolates within each endemic area was 100%,while the sequence identity of isolates between endemic area was 40.2%～91.4%.Phylogenetic tree analysis showed that 16 isolates of Leishmania in China clustered into 2 groups and 3 subgroups,among which isolates from cutaneous leishmaniasis(CL)endemic area and zoonotic deserts type visceral Leishmaniasis(DT-ZVL)endemic area clustered into A subgroup,isolates from the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area and L.infantum clustered into B subgroup,and the clusters of A and B into Ⅰ group.Isolates from anthroponotic type visceral leishmaniasis(AVL)endemic area clustered with the L.donovani as subgroups C,which further clustered with the international reference isolate DD8 into Ⅱ group.The mini-exon sequence of Leishmania isolates from China were amplify by specific primers.Type from anthroponotic type visceral leishmaniasis(AVL)endemic area three isolates were amplified 434 bp single fragment,from the zoonotic deserts type visceral leishmaniasis(DT-ZVL)endemic area of 4 isolates were amplified 385 bp single fragment,from the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area 4 isolates were amplified 395 bp single fragment,from cutaneous leishmaniasis(CL)endemic area 5 isolates were amplified 425 bp single fragment.The sequence identity between isolates within each endemic area was 100%,while the sequence identity of isolates between endemic area was 87.4%～97.5%.Phylogenetic tree analysis showed that 16 isolates of Leishmania in China clustered into 1 groups and 2 subgroups,among which isolates from cutaneous leishmaniasis(CL)endemic area,zoonotic deserts type visceral leishmaniasis(DT-ZVL)endemic area and the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area and L.infantum clustered into A subgroup.Isolates from anthroponotic type visceral leishmaniasis(AVL)endemic area clustered with the L.donovani as subgroups B,and the clusters of A and B into I group.The cpa sequence of Leishmania isolates from China were amplify by specific primers.A single fragment of 1058 bp was amplified from 16 Leishmania isolates in China.The sequence identity between isolates within each endemic area was 100%,while the sequence identity of isolates between endemic area was 99.3%～99.9%.Phylogenetic tree analysis showed that 16 isolates of Leishmania in China clustered into 1 groups and 2 subgroups,among which isolates from cutaneous leishmaniasis(CL)endemic area,zoonotic deserts type visceral leishmaniasis(DT-ZVL)endemic area and the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area and L.infantum clustered into A subgroup.Isolates from anthroponotic type visceral leishmaniasis(AVL)endemic area clustered with the L.donovani as subgroups B,and the clusters of A and B into I group.The 7SL RNA sequence of Leishmania isolates from China were amplify by specific primers.All 16 Leishmania isolates in China amplified 119 bp single fragment,and the sequence identity among the isolates was 100%.Phylogenetic tree analysis showed that 16 isolates of Leishmania in China clustered into 1 groups,which isolates from cutaneous leishmaniasis(CL)endemic area,zoonotic deserts type visceral leishmaniasis(DT-ZVL)endemic area,the zoonotic mountain type visceral leishmaniasis(MT-ZVL)endemic area and anthroponotic type visceral leishmaniasis(AVL)endemic area clustered with the L.donovani and L.infantum into I group.Conclusion:Leishmania isolates from the same leishmaniasis endemic area in China were completely homologous,while Leishmania isolates from different endemic areas showed high genetic polymorphism.The K26 sequence is an ideal molecular marker for the identification of Leishmania in China.The Leishmania isolates of China can be divided into four molecular types according to K26 sequence.