Live-Attenuated ME49Δcdpk3 Strain Of Toxoplasma Gondii Protects Against Acute And Chronic Toxoplasmosis

Background:Toxoplasma gondii is a specialized intracellular opportunistic pathogenic protozoan parasite.It can infect mammals,including humans,and is widely spread worldwide and approximately 30%of the population is serologically positive.Toxoplasmosis,caused by Toxoplasma gondii,is now a common disease.It also causes significant economic losses to livestock such as cattle and sheep in the agricultural sector.Toxoplasma gondii CDPK3,a calcium-dependent protein kinase,is localized in the periphery of the parasite and plays a crucial role in parasite escape.However,the role of CDPK3 in the regulation of host cell immunity is still unclear,genetic and biological methods were used to study the protective effect of CDPK3 knockout strain as a live attenuated vaccine against acute and chronic Toxoplasma gondii infection.Objective:Study of the protective immunity of Toxoplasma gondii CDPK3 knockout strains as live-attenuated vaccines against acute and chronic Toxoplasma gondii infectionMethods:(1)Virulence assay of wild-type and ME49Δcdpk3 in mice:Intraperitoneal injection of10 ~6wild-type and ME49Δcdpk3 strains to observe the death rate of each mouse within35 days,the formation of brain cyst,the viability of mice and the level of Ig G in the serum of mice,and the burden of Toxoplasma gondii in the liver,spleen,eyes,heart,brain and lungs of mice infected with Toxoplasma gondii for 7 days.(2)Evaluation of ME49Δcdpk3 strain as a vaccine:Immunized mice were re-infected with various wild-type strains,including type I RH,type II ME49,Chinese1 WH3 and WH6,and ME49,WH6 cysts.The levels of IFN-γ,TNF-α,IL-12p70 and IL-10 in splenocyte supernatants and the proportion of CD3+CD8+Tlymphocytes CD3+CD4+Tlymphocytes in splenocytes were measured in mice after 75 days of immunization.The levels of Ig G,Ig G1 and Ig G2a in the sera of mice immunized for 35,75 and 125days were measured.To detect the effect of vaccine immunization of 125 days mice sera against parasites infection.Results:(1)Mouse virulence assay showed that mice injected intraperitoneally with 10~6ME49Δcdpk3 hardly died,while mice infected with the wild-type strain began to die on day 8,with a mortality rate of 70%;The load of Toxoplasma gondii in the liver,spleen,eyes,heart,brain and lungs of mice infected with the wild-type strain for 7 days was significantly higher than that of mice infected with the ME49Δcdpk3 strain.(2)ME49Δcdpk3 vaccine-immunized mice were able to resist reinfection including type I RH,type II ME49,Chinese1 WH3 and WH6,and ME49,WH6 cysts;Compared with unimmunized mice,the levels of IFN-γ,TNF-α,IL-12p70 and IL-10 in splenocyte supernatants and the proportion of CD3+CD8+Tlymphocytes and CD3+CD4+T lymphocytes in splenocytes were significantly higher in mice 75 days after vaccine immunization;The levels of Ig G,Ig G1 and Ig G2a in the serum of mice immunized for35,75 and 125 days were significantly higher and the level of Ig G2a was significantly higher than Ig G1;The serum of mice immunized with ME49Δcdpk3 for 125 days was able to help normal mice resist Toxoplasma gondii infection.Conclusions:ME49Δcdpk3 vaccination can trigger cellular and humoral immunity and protects mice from Toxoplasma gondii infection.