Live attenuated RHΔtkl1 and PruΔpp2a-c mutants of Toxoplasma gondii are promising vaccine candidates conferring protection in pigs

Abstract Background Toxoplasma gondii is an apicomplexan parasite which infects nearly all warm-blooded animals and humans, causing zoonotic toxoplasmosis. Pork infected with T. gondii is considered a significant source of human infection. Currently, no commercial vaccines are available for porcine toxoplasmosis globally, thus a safe and effective vaccine is urgently needed. This study evaluated the immuno-protective effects of two T. gondii gene knockout attenuated strains, RHΔ tkl1 and PruΔ pp2a-c , in pigs. Methods Pigs immunized by intramuscular injection with 1 × 10 7 tachyzoites of attenuated RHΔ tkl1 or PruΔ pp2a-c strains were challenged orally with 1.000 sporulated oocysts of the Pru strain at 28 days post-vaccination (dpv), followed by a secondary challenge via intraperitoneal injection of 1 × 10 7 Pru strain tachyzoites at 70 dpv. Clinical signs were monitored. T. gondii -specific antibody levels were examined by enzyme-linked immunosorbent assay. The protective efficacy was evaluated by analyzing pathological lesions, pathological score, parasite load, brain cyst burden, and by mouse bioassay. GraphPad Prism software was employed to perform the log-rank (Mantel Cox) test, Student’s t test and one-way ANOVA. Results Pigs immunized with either RHΔ tkl1 or PruΔ pp2a-c exhibited only low-grade fever. Combined with pathological changes and pathological scores, these findings support the moderate safety of both strains. Pigs immunized with either RHΔ tkl1 or PruΔ pp2a-c and subsequently challenged with T. gondii Pru oocysts and tachyzoites exhibited a sharp increase in T. gondii -specific antibodies, which remained high for 5 weeks ( P < 0.01). Pathological lesions were alleviated after immunization, with a significant reduction in parasite load observed in tissues including the brain, heart, gastrocnemius, longissimus dorsi, psoas major, and diaphragm ( P < 0.01). Furthermore, a marked decrease in brain cyst burden was recorded ( P < 0.0001). Mouse bioassay results confirmed a significant reduction in the proportion of mouse brain tissues positive for T. gondii genomic DNA in the group immunized and then challenged, compared to the non-immunized challenged group ( P < 0.0001). Conclusions The two live attenuated RHΔ tkl1 and PruΔ pp2a-c mutants of demonstrated moderate safety, immunogenicity, and protective efficacy in pigs, identifying them as potential candidate vaccines against porcine toxoplasmosis. Graphical Abstract