A Comparative Study Of Irradiation And Excretion And Secretion Of Larvae Of Haemonchus Contortus

Haemonchus contortus is a parasitic nematode that seriously endangers ruminants such as sheep and goats.This nematode usually inhabits in the abomasum of ruminants and causes haemonchosis in the hosts.Having been infected with H.contortus,sheep/goats not only produce anemia,emaciation and weakness,but also may be damaged to death,which brings about a great loss to the production of sheep industry.At present,the preventive measure against haemonchosis is mainly to apply anthelmintic.However,with the emergence of increasing problems such as resistant strains and anthelmintic residues,immunoprophylaxis is urgently needed to control haemonchosis.So more and more scholars dedicated their works to development of vaccine against H.contortus.Previous studies have shown that high levels of protective immunity can be induced by different kinds of radiation-attenuated larvae vaccines against different species of parasites.But there are such problems as limitation of getting a lot of parasite materials and uncertain repeatability of protective effects to be solved for the use of RA H.contortus larvae.So if we can combine advantages of molecular vaccine with that of RA vaccine,we may achieve breakthrough for the successful development of efficient and safe vaccine against H.contortus.To this end,it is urgently needed to understand the cellular and molecular mechanisms of protective immunity induced by RA H.contortus larvae.Previous studies have shown that immunization with RA H.contortus larvae can induce a higher level of protective immunity in sheep/goats;whereas no protective effect can be observed in hosts infected with normal H.contortus larvae.Furthermore,in a recent investigation,we have also observed that normal and RA H.contortus larvae/cells cultured in vitro could show apoptotic and necrotic phenotypes,respectively.These investigations suggest that immunobiologic functions and properties of the cells/molecules derived from RA H.contortus larvae are different from that of cells/molecules from normal larvae.However,cellular and molecular mechanisms responsible for this kind of difference were not very clear as yet.We speculated that components,expression levels and immunobiologic functions of excretory-secretory proteins?ESP?released from RA H.contortus larvae are different from that of ESP from normal larvae.That is to say,the ESPs from RA larvae might contain more immunogenic molecules,whereas the ESPs from normal larvae contain more tolerogenic antigens.Therefore,we carried out a preliminary study on identification and immunolgic functions of ESPs from both RA and normal H.contortus larvae L₃?the third stage larvae?.We firstly analyzed molecular component and expression levels of ESPs from RA and normal H.contortus L₃ larvae groups.Radiation-attenuated and normal L3larvae were cultured for 5d,10d,13d and 16d in vitro,respectively,the supernatants were collected from cultures at different time points mentioned above,and the proteins were concentrated by ultrafiltration and freeze-drying.After that,iTRAQ technique and liquid chromatography tandem mass spectrometry were used to compare the differences of proteomes of ESPs between RA and normal L₃ larvae groups.We identified 186 differentially expressed proteins;the results of further bioinformatics analysis revealed that,in early stage,molecules/pathways related to apoptosis present more frequently in normal group,and in RA larvae group more molecules/pathways are relative to immuogenic necroptosis;in late stages,molecules/pathways related to necrosis or cell death occur more frequently in both RA and normal groups.We also found that expression levels of danger signal molecule HSP90 and ATPase related to immunogenic necrosis are higher in RA larvae group than in normal group;while expression levels of Glutathione S-transferase and Cytochrome-c related to apoptosis are higher in normal group than RA larvae group.These suggest that the ESP from RA larvae might contain more immunogenic molecules,whereas the ESP from normal larvae contain more proteins related to apoptosis.Furthermore,we preliminarily investigated the immunolgic functions of ESPs from both RA and normal H.contortus larvae L₃.The peripheral blood mononuclear cells?PBMC?from goats were incubated with RA and normal larvae by using Transwell co-culture system for 24h.The mRNA expression level of cytokines in PBMC were detected by using RT-PCR method.NO secretions by PBMCs stimulated with ESPs from both RA and normal H.contortus larvae L₃ were detected by Griess method.The effects of ESPs from RA and normal larvae L3 groups on the migration of PBMCs from goats were assayed by using Transwell cell migration system.We found that mRNA expression levels of cytokines?including pro-inflammatory IFN?and IL-4,anti-inflammatoryIL-10 and regulatory cytokine1L-17?were significantly higher in PBMCs stimulated with RA larvae than those in PBMCs incubated with normal larvae;the numbers of migratory PBMCs incubated with ESPs from RA larvae are greater than those from normal larvae.We also found that more NO could be produced by PBMCs stimulated with ESPs from RA larvae group.These investigations suggest that ESPs from RA larvae might have function of immune enhancement and regulation,and ESPs from normal larvae are of properties of immunosuppression.In summary,in this study,we found the ESPs from RA larvae might have more immunogenic molecules and immune-enhancement function,whereas the ESPs from normal larvae contain more tolerogenic antigens and might have properties of immunosuppression.It provided the basis for further elucidating molecular mechanisms of high protective immunity induced by the RA H.contortus larvae vaccine.