Construction Of A △bag1 △ku80 Strain And Evaluation Of Its Safety, Immunogenicity And Efficacy As A Vaccine Candidate Against Cat Toxoplasmosis

Toxoplasmosis usually occurs in cats by orally consuming food containing Toxoplasma gondii (T. gonii) cysts, oocysts or pseudocysts. Cats are the definitive hosts of this parasite. T. gondii completes its sexual life cycle in the intestine of primarily infected cats, which shed millions of oocysts to the environment, resulting in the spreading of toxoplasmosis to humans and animals. Cats are also the intermediate hosts of T. gondii. Tissue cysts are often observed in the infected cats, which increases the possibility for the transmission of this parasite. Thus, it is of great significance to develop a vaccine with high efficacy to prevent oocyst shedding and cyst formation. Previous studies showed that the ability of cyst and oocyst formation for T. gondii has decreased significantly after long-term maintenance in vitro. Besides, disruption of the T. gondii bradyzoite-specific gene BAG1 leads to the decrease of cyst formation in vivo. What’s more, △ku80 strain fails to complete meiotic crosses to produce infective oocyst to the environment.In this study, we transfected the gene targeting plasmid pTgBAG1-HXGPRT-KI/KO into the recipient △ku80 strain, and compared the virulence and proliferation of the △bag1△ku80 strain and △ku80 strain by cultivating them on HFF (Human foreskin fibroblast), plaque assay and mouse infectivity study. Next, to evaluate the immunogenicity of the vaccine candidate, interferon gamma release assay (IGRA) was developed to detect T. gondii infection in mice and cats after oral infection with cysts. Besides, the immunogenicity of tachyzoites would be compromised if immunization was conducted orally. To overcome this disadvantage, we evaluated all-trans retinoic acid (ATRA) as an adjuvant during T. gondii subcutaneous (s.c.) infection. Next, cats were immunized s.c. with △bag1△ku80 tachyzoites together with ATRA, and immune responses were detected before challenge. Then, to evaluate the safety of this strain, we inoculated cats with different doses of △bag1 △ku80 tachyzoites. Finally, we detected whether △bag1 △ku80 tachyzoites could protect cats against oocyst production on being challenged with cysts.Results showed that there was no difference between the △bag1 △ku80 strain and △ku80 strain on the virulence and proliferation. IGRA was an easy-operating and low-cost method to measure cell-mediated immunity (CMI) in cats. Immunized s.c. with T. gondii together with ATRA upregulated the expression of α4β7 and CCR9 on intestinal intraepithelial lymphocytes (IELs), and enhanced the gut humoral and cellular immune responses following s.c. inoculation. High levels of systemic humoral and cellular immune responses were elicited after immunization of cats with △bag1 △ku80 strain. Moreover, s.c. ATRA-supplementation stimulated higher levels of gut humoral and cellular immune responses in cats, and enhanced the systemic cellular immune responses. Besides, we inoculated cats with different doses of △bag1 △ku80 tachyzoites, and no cysts were detected in animals’ brain, which proved that this strain was quite safe to be conducted in cats. Furthermore, the prepatent period of cats vaccinated with △bag1 △ku80 strain was reduced from 7.7 to 1.6 days. Oocyst preventable fraction was 98%, which proved that the △bag1△ku80 strain had a high efficiency as a vaccine candidate again cat toxoplasmosis.In conclusion, △bag1△ku80 strain lost the ability to form cysts and exhibited quite high safety in cats. More importantly, △bag1 △ku80 strain immunized cats together with ATRA significantly shortened the prepatent period and prevented most of the oocysts output, which reduced the risk for the transmission of T. gondii. Our study provided new ideas for the development new vaccines against cat toxoplasmosis.