Safety Evaluation And Immune Protection Of ΔCDPK2 Deletion Mutants Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular protozoa that can infect most endotherms.Human or animal infection with T.gondii can lead to hydrocephalus,brain malformation,abortion,malformation,stillbirth and other serious symptoms,brought great hidden dangers to economic development and public health safety.Cats are the only final host of T.gondii.Cats infected with toxoplasma will release a large number of oocysts into the environment,which is an important factor causing the widespread spread of T.gondii.Vaccination of cats against T.gondii infection is of great significance to control the spread of toxoplasma and is an important measure for the prevention and control of toxoplasmosis.However,there are few studies on toxoplasmosis vaccine.Studies have shown that CDPK2 is a key regulator of amylopectin metabolism in T.gondii,and the virulence of T.gondii is decreased without CDPK2.PRUΔCDPK2 attenuated vaccine has a good protective effect on acute,chronic and congenital toxoplasmosis in mice.The purpose of this study was to investigate the feasibility of RHΔCDPK2 and PRU ΔCDPK2 deletion strains as T.gondii vaccines,and to explore their potential value as T.gondii vaccines by evaluating their safety,immunogenicity and immunoprotection in cats.First,to explore the safety of the two deleted strains in cats,physiological indicators of the cats inoculated with the deleted strains were monitored.It was found that there was no significant increase in body temperature and only mild clinical symptoms,but the cat recovered quickly.The cats inoculated with PRUΔCDPK2 deficient strain produced oocysts,while those inoculated with RHΔCDPK2 deficient strain did not produce oocysts.Therefore,we believe that the RHΔCDPK2 deficient strain is not seriously pathogenic to cats,and cats do not produce oocysts,so it is safe and may be a vaccine candidate strain.Although the PRUΔCDPK2 deficient strain is not pathogenic to cats,it can’t be used as T.gondii vaccine due to the release of oocysts after inoculation.Then,to evaluate the protective effect of the vaccine,we simulated the natural infection of T.gondii to cats and established an infection model of T.gondii.Cats infected naturally with T.gondii exhibit mild clinical symptoms and a large number of oocysts in their faeces.We treated cats with high and low doses of wild strains of tachyzoites and tissue cysts,and evaluated clinical symptoms and oocysts output.No obvious clinical symptoms and no oocysts were found in the faeces of cats treated with low-dose tachyzoites.Cats treated with high doses of tachyzoites showed severe clinical symptoms and less fecal oocysts than normal infection;no obvious clinical symptoms were observed in cats that were attacked with appropriate tissue cysts,and a large number of oocysts were excreted in feces.Therefore,we believe that attack with tissue cyst can better simulate the natural infection of T.gondii in cats,and the construction of this infection model will lay a foundation for evaluating the immune protection of RHΔCDPK2 deficient strains in cats.Next,in order to investigate the immunogenicity of RHΔCDPK2 deficient strain as a vaccine candidate strain,we immunized cats twice on day 0 and day 28 respectively,and detected the changes of blood routine,humoral immunity and cellular immunity indexes during this period.The results showed that the expression of antibody and IFN-y in serum of the immunized cats increased significantly,and the proliferation of neutrophils,eosinophils,lymphocytes and monocytes in the blood of the immunized cats showed that the deficient strain had good immunogenicity and could induce good humoral and cellular immunity.Finally,in order to explore the protection of RHΔCDPK2 deficient strain on cats,we conducted an challenge experiment on the 56th day after immunization according to the constructed infection model.The results showed that the cats immunized with RHΔCDPK2 deficient strain produced 89.15%fewer oocysts after challenge than the control group,and the patent period was significantly shortened.These results indicated that the deficient strain had better immune protection in cats and could reduce large number of oocysts produced after toxoplasma infection.In conclusion,RHΔCDPK2 deficient strain was screened out for cat toxoplasmosis,and the infection model was successfully established to simulate the natural infection of T.gondii.The potential value of RHΔCDPK2 deficient strain as T.gondii vaccine was preliminally studied,and it was found that RH ΔCDPK2 deficient strain had certain protective effect against T.gondii.It laid a good foundation for the development of cat toxoplasma vaccine and promoted the research process of prevention and control of toxoplasmosis transmission.