Molecular Typing And Study On Infectivity Of Leishmania Isolates From Three Endemic Areas Of Kala-azar In China

ObjectiveTo analyze and evaluate the differences of genetic background and infectivity of Leishmania isolates from three endemic areas of kala-azar in ChinaMethodsThe hsp70 and ITS1 sequences of Leishmania isolates from different endemic areas of leishmaniasis in China were amplified based on specific primers,respectively,and relative phylogenetic trees were constructed based on these sequences to identify the isolates.Three representative isolates from three different VL epidemic areas were selected and cultivated in NNN medium,M199+20%fetal bovine serum medium.M199+20%horse serum medium and brain heart medium respectively,promastigotes were counted every day to construct the growth curves.Aappropriate medium was selected to culture Leishmania,promastigotes in a stable growth period were collected to infect BALB/c mice.The infected mice were killed and sampled including liver,spleen and blood were collected at 1 week.2 weeks,4 weeks,6 weeks.8 weeks,10 weeks,12 weeks after infection,respectively.qPCR absolute quantitative method was used to detect parasitic load in mice liver and spleen,ELISA was used to detect cytokines IL-2,IL-10,IL-12,IFN-γ,TGF-β and specific antibodies IgM.IgG,IgG1.IgG2a in serum.Results:A 1442 bp of fragment was amplified from all 10 Leishmania isolates from China based primers specific to hsp70 gene sequences.These sequences showed 100%agreement among isolates JIASHI-1,JIASHI-5,XJ771,KXG-Xu,KXG-Lliu,927,Cy and SC6 isolates,and 100%agreement of 801 and KS-2 isolates,there was only one base difference among the isolates.Amplification of ITS1 sequences of Leishmania isolates generated a fragment of 318 bp from SC6 and Cy isolates from the MST-ZVL endemic area,a fragment of 312bp from KXG-Xu,KXG-Liu and 927 isolates from the endemic area of cutaneous leishmaniasis in Karamay,Xinjiang,a fragment of 313bp from JIASHI-1,JIASHI-5 and XJ771 isolates from the DST-ZVL endemic area in Xinjiang,a fragment of 316bp from 801 and KS-2 isolates from the AVL endemic area in Kashi,Xinjiang.Alignment analysis showed that the ITS1 sequences of Leishmania isolates from the same endemic area were identical.The phylogenetic tree showed that 10 isolates were clustered into two groups and three subgroup:Subgroup A and B were closely related to L.infantum reference as group Ⅰ,subgroup C was fallen into group Ⅱ of L.donovani.The representative isolates KS-2,Cy and JIASHI-5 from three endemic areas of visceral leishmaniasis,respectively,can grow and reproduce in NNN medium,M199+20%fetal bovine serum medium and M199+20%horse serum medium,KS-2 isolates proliferated fastest in the corresponding medium.BALB/c mice were respectively infected with KS-2,Cy and JIASHI-5 isolates.In the liver,the parasitic load was the highest in KS-2 infected mice,peaked at 4 weeks,then gradually decreased to a lower level,and was the lowest at 12 weeks.In JIASHI-5 infected mice,the parasitic load peaked at 1 week,then gradually decreased,and was the lowest at 10 weeks after infection.In Cy infected mice,the parasitic load was the least,which reached the peak at 4 weeks,then decreased slowly,and was the least at 8 weeks.With the prolongation of the infection period,the parasitic load in the liver was maintained at a relatively low level.In general,the parasitic load was highest in KS-2 infected mice in each period of infection,and the second in JIASHI-5 infected mice.In the spleen,the parasitic load was the highest in KS-2 infected mice,which was low at the first 2 weeks,peaked at 6 weeks,and then decreased.In JIASHI-5 infected mice,only a small amount of the parasitic load was detected in the spleen at the first 2 weeks,peaked at 4 weeks,and then gradually decreased.In Cy infected mice,the parasitic load was the lowest,reached a peak at 6 weeks,and then gradually decreased.The parasitic load was highest in KS-2 infected mice in each period of infection,and the second in JIASHI-5 infected mice.The concentrations of IFN-γ,TGF-β,and IL-10 were increased in serum of three Leishmania isolates infected mice,and the three cytokines increased the most in KS-2 infected mice.The concentrations of IL-2 and IL-12 were decreased.Specific antibodies IgM,IgG,IgGl and IgG2a can be detected in serum of three Leishmania isolates infected mice,and the levels of antibodies increased with the progression of infection.The levels of IgM,IgG and IgG2a of Cy isolate infected mice were lower than those of KS-2 and JIASHI-5 isolates mice during the whole observation period.Conclusion:1.There were genetic differences among leishmania isolates from three endemic areas of kala-azar.2.There were differences in the growth and reproduction of leishmania isolates from three endemic areas in different culture media.3.The infection of leishmania isolates from three endemic areas on BALB/c mice and the induced cellular and humoral immunity of mice were also different.