Vaccination with live-attenuated Toxoplasma gondii mutants RHΔtkl1 and PruΔpp2a-c induces protective immunity in...

Toxoplasma gondii is a globally distributed intracellular parasitic protozoan. It infects nearly all warm-blooded animals and causes a zoonotic disease of worldwide significance. Currently, the only commercially available vaccine, Toxovax^®, is solely used for the prevention of Toxoplasma-induced abortion in sheep, but it has limitations such as a short shelf life and the potential of reversion to virulence. This study evaluated the safety and immune-protective efficacy of two live-attenuated strains RHΔtkl1 and PruΔpp2a-c in sheep.

Sheep were immunized via intramuscular injection in the neck with 1 × 10⁷ tachyzoites of RHΔtkl1 or PruΔpp2a-c. Sheep were challenged orally with 5 × 10⁵ type II Pru oocysts at 28 days post-vaccination (dpv), followed by a second challenge on day 70 with 1 × 10⁷ type II Pru tachyzoites injected intramuscularly at 70 dpv. Safety and immuno-protection were evaluated by monitoring clinical symptoms and body temperatures, T. gondii-specific IgG antibody levels, histopathological changes, immunohistochemistry, brain cysts, parasite load, and mouse bioassay results.

The results demonstrated that both knockout strains induced only transient fever. Following immunization and subsequent challenge with Pru oocysts, the T. gondii-specific IgG antibody levels in sheep increased rapidly and remained elevated for an extended period. Histopathological analysis indicated mild organ lesions in heart, liver and lung tissues among immunized infected sheep, whereas non-immunized infected sheep exhibited severe widespread inflammation. Immunohistochemical analysis of brain tissue revealed significantly lower values for four parameters (positive cell ratio, density, histochemistry score, immunoreactive score) in immunized groups (P < 0.01). A significant reduction in brain cysts was observed in immunized and challenged sheep (P < 0.01) compared with unimmunized and challenged sheep. The parasite burden of T. gondii in heart tissue was significantly reduced (P < 0.01). Compared with mice inoculated with sheep brain tissue from unimmunized groups challenged with T. gondii Pru oocysts and tachyzoites, mouse bioassay results showed that the mice inoculated with sheep brain tissue from groups immunized with RHΔtkl1 or PruΔpp2a-c tachyzoites and subsequently challenged with T. gondii Pru oocysts and tachyzoites exhibited a significantly lower proportion of positive genomic T. gondii DNA in the brain (P < 0.001), as well as significantly reduced levels of T. gondii-specific antibody IgG in the serum (P < 0.0001). Similarly, mice inoculated with sheep visceral tissue from the same immunized and challenged groups also showed a significantly reduced proportion of positive genomic T. gondii DNA in the brain (P < 0.0001) and significantly reduced levels of T. gondii-specific antibody IgG in the serum (P < 0.0001).

The gene knockout strains RHΔtkl1 and PruΔpp2a-c showed a certain degree of safety in sheep, and they induced strong humoral and cellular immune responses in sheep, significantly mitigating acute infection symptoms and tissue damage. Notably, PruΔpp2a-c showed greater potential in suppressing cyst formation. Both strains are potential attenuated candidates against sheep toxoplasmosis.