| Schistosomiasis is a widespread and serious zoonotic parasitic diseases.In China,the distribution area of snails in the epidemic area of schistosomiasis is still relatively large,and the objective factors of epidemic and spread of schistosomiasis as well as the risk factors of repeat and relapse still exist.However,the treatment of praziquantel can not solve the problem of repeated infection.The development of new drugs and vaccines is still the focus of the disease research.Microtus fortis is the only mammal found to date that has natural resistance to Schistosoma japonicum.The study of its resistance mechanism may provide a new approach for the development of schistosomiasis control techniques.In this paper,thoracic aorta perfusion and tissue culture were used to collect schistosomula from M.fortis and BALB/c mice infected with Schistosoma japonicum cercariae at 1,3,8,12 and 15 days,and the recovery rate of worm of skin-stage,lung-stage and liver-stage were respectively calculated and compared.The results showed that the total recovery of worms in Microtus fortis were 15.4%,7.0%,8.33%,0.48%,0.77% at different time points after infestation of cercariae,which were significantly lower than those in mice 39.24%,38.56%,46.17%,19.02%,43%,suggested that Schistosoma japonicum schistosomula died out in Microtus fortis mainly at skin-stage and liver-stage after infection.The pathological appearance indicated that white cysts appeared in the surface of liver in M.fortis at day 10,remained at day14,and disappeared day 21 post infection.Histopathological examination showed that the residual body of parasite and inflammatory cells infiltration can be observed in the blood vessels of liver from day 10 to day 14,which formed eosinophilic abscesses,and multiple local necrosis of hepatocytes.The results of blood routine test revealed that the number of leukocytes increased significantly at 1-16 d after infection,mainly due to the increase of neutrophils revealed by the results of the 5-classification analysis.Serum biochemical analysis showed that ALB and HDL-C increased post infection.The serum complement activity CH50 of Microtus fortis were 512 U/ml.The expression levels of the complement-associated molecules C3,C9,CD59,and DAF in Microtus fortis and BALB/c mice were detected by RT-PCR and ELISA.The results showed that the transcriptional level of C3,C9 and CD59 in Microtus fortis was significantly increased on the 8th days after infection,and the transcriptional level of DAF in Microtus fortis was significantly increased on the 12 th days after infection,whereas that in mice did not change significantly.The results of worm incubation test in vitro showed that the mortality of schistosomula incubated with normal and inactivated complement serum of the vole was 85.50% ± 7.15% and 60.99% ± 8.61%respectively,the difference was significant(p <0.001),and there are differences in the completeness of surface between the two collected larvae revealed by scanning electron microscopy,indicating that the complement of Microtus fortis has a significant killing effect against Schistosoma japonicum.The mortality of schistosomula incubated with sera in which C1 q was inhibited by C1 qBP protein,was significantly lower than that of normal serum(P<0.001).However,there was no significant change in mortality of larvae incubated with serum in which complement factor B was inactivated at 50°C.The results suggested that theclassical pathway of complement may play an important role in the killing effect of vole on larvae.In the vivo trial,the recovery rates of the worms of MF at the 8 days after administration with CVF and PBS were23.20% and 8.54%,respectively,and at 15 th days were 1.18% and 0.42%.The results also suggested that complement plays an important role in the resistance mechanism.Three RNA interference specific target sequences to the C5 gene of Microtus fortis were designed and screened,and the corresponding recombinant RNA interference lentivirus was constructed.The LV2 recombinant lentivirus induced a decrease in C5 gene transcription of 25.76% in vole peritoneal macrophage cultured in vitro,providing a basis for RNA interference experiment in vivo in Microtus fortis.In conclusion,the results indicated that Schistosoma japonicum schistosomula died out in Microtus fortis mainly at skin-stage and liver-stage after infection.Complement plays an important role in natural resistance,and the classical pathway maybe the important mechanism of complement against worms.The results provide data for the elucidation of the role and mechanism of complement against the Schistosoma japonicum in Microtus fortis. |
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