Preliminary Study On The Protective Immune Effect And Mechanism Of The L3 Larvae Of Haemonchus Contortus Induced By Irradiation In Sheep

Haemonchus contortus is an important cattle and sheep parasite,which is widespread in large pastoral areas around the world.It mostly parasitizes the abomasum of young small ruminants,causing extreme weight loss in young cattle and sheep.The peak period of infection can be given to pastoral areas.The aquaculture industry causes huge economic losses.At present,the prevention and treatment of the disease is mainly through anthelmintics,but as more and more antibiotics are used for the prevention and treatment of the disease,drug-resistant strains of H.contortus have also appeared,which poses new challenges for the prevention and treatment of the disease.Related studies have pointed out that high-dose UV irradiation can effectively reduce the development rate of H.contortus larvae and the rate of adult egg laying.Inoculation of infectious larvae treated with high-dose UV irradiation can induce the host to produce a high level of protection.Immune effect,which is not observed in sheep hosts inoculated with normal larvae.Research in this laboratory shows that appropriate intensity of ultraviolet radiation can make H.contortus stress molecules such as calreticulin and heat shock protein 70 highly expressed on the surface of the body cells,which may play a key role in inducing the host's highly protective immune response.Therefore,this experiment comparatively studied the attenuation effect of different irradiation doses on the L3 stage larvae of H.contortus;preliminary evaluation of the effect of the radiation attenuation of the L3 stage larvae of H.contortus on the induction of high protective immunity of the host;The radiation-induced weakening of H.contortus larvae ESP and recombinant worm proteins(Hc CRT,Hc HSP70)induce the immune response of sheep immune cells to explore.The aim is to further clarify the cellular and molecular mechanisms of the L3 stage larvae of H.contortus that are weakened by irradiation to cause the host's highly protective immune response.First,we analyzed the attenuation effect of different irradiation doses on the L3larvae of H.contortus.The larvae of different groups were irradiated 0-10 min and cultured in vitro 0-16d for light microscope observation.It was found that there was a significant difference in the survival rate of the larvae with irradiation time of 0 min and 3 min,and the survival rate was higher than 60%;the sheep host was immunized with 3 min irradiation and normal larvae respectively,and the eggs were counted after immunization 18-22d,22d necropsy for adult count.The results showed that the number of fecal worm eggs in the irradiated group was significantly reduced compared with the normal group,and the larval development rate decreased by more than 99%,indicating that the irradiation for 3 minutes had a significant weakening effect on the L3 larvae of H.contortusSecondly,we verified whether irradiation of L3 larvae of H.contortus can induce the host to produce highly protective immunity.We challenged the animals 25 days after the initial immunization,and calculated the egg reduction rate 23 days after the challenge infection;26 days after the challenge infection,the sheep of each group were necropsied at the same time to calculate the abomasum reduction rate.At the same time,the sera of each group of sheep after the initial immunization 2d,7d,13d,18d and after the challenge infection 2d,5d,13d were separated for antibody level detection.It was found that after challenge infection,the egg reduction rate of the irradiated group was75%,and the insect reduction rate was 97%;after the initial immunization,the specific antibodies of each group of Ig G and typing antibodies Ig G₁,Ig G₂,Ig A,Ig M were up-regulated in the early stage of immunization.The total antibody content of the irradiated group was significantly higher than that of the normal group.After challenge infection,the serum levels of parasite-specific antibody Ig G and various types of antibodies in the irradiated group showed a trend of continuing to increase;indicating that the radiation-induced weakening of H.contortus L3 larvae can cause a higher host humoral immune response.In addition,we used the Transwell method to preliminarily study the immune function of irradiated and normal H.contortus L3 larvae ESP,collected the PBMC of the sheep host after the initial immunization and challenge infection and co-cultured with the normal larvae for 24 hours,and used the ELISA method to detect the expression level of cytokines of PBMC.The result showed that the worms of the irradiated group and the normal group co-incubated with negative PBMC can up-regulate the expression of IL-4,IL-13,IFN-?,TNF-?IL-17 and other cytokines of PBMC,but the expression level of cytokines IL-4,IL-13 in the irradiated group is higher than that of the normal group,and the expression level of cytokines TNF-?,IL-17 in the normal group is higher than that of the irradiated group;After irradiated and immunized sheep with normal worms,PBMC was taken and incubated with the larvae of the normal group.Both the TH1/TH2 cytokines IL-4,IL-10,IL-13,IFN-?,TNF-?of each group and the regulatory factor IL-17 were up-regulated.The irradiated group can induce a TH1/TH2 mixed immune response tends to TH2 immunity,while the normal group tends to TH1 immunity.At the same time,we preliminarily studied the mechanism of stress proteins Hc CRT,Hc HSP70 inducing the immune response of sheep immune cells,and took the PBMCs of each group at different time periods after the initial immunization and challenge infection and the mesenteric lymph node cells in each group for stimulated.It was found that the nematode recombinant protein Hc CRT can up-regulate the expression of TNF-?,IFN-?,IL-4,IL-10 and other cytokines of sheep-negative PBMC,and down-regulate the expression of negative lymphocyte cells,and down-regulate the expression of IL-4,IL-13,TNF-?,IL-10,IL-17 of PBMC/lymph node cells in each group after the initial immunization and challenge infection;Nematode recombinant protein Hc HSP70 can up-regulate the expression of TNF-?,IFN-?,IL-4,IL-10 and others of sheep PBMC/lymphocytes,producing a mixed immune response similar to the irradiated worms stimulationIn conclusion,this study found that the appropriate dose of UV irradiation can not only ensure the survival rate of the larvae,but also inhibit the development rate of the L3 stage larvae of H.contortus.It was also found that irradiation weakened the L3 stage larvae of H.contortus can induce the host to produce highly protective humoral and cellular immune effects.Irradiation attenuates the L3 stage larva of H.contortus ESP and the candidate stress protein Hc HSP70 can induce sheep immune cells to produce a TH1/TH2 mixed immune response that tends to TH2-type cellular immune response Type immune response.This study provides experimental evidence for confirming the immune effect of irradiated vaccines and elucidating the molecular and cellular mechanisms.