A newly characterized dense granule protein (GRA76) is important for the growth and virulence of Toxoplasma gondii

Immunization with Live-Attenuated RHΔhad2a Strain Confers Partial Protective Immunity against Acute and Chronic Infection of Toxoplasma gondii in Mice

“…To examine whether RHΔ had2a vaccination induces the production of anti- T. gondii -specific IgG, Kunming mice were immunized with 5 × 10 4 RHΔ had2a tachyzoites ( Figure 3 A) [ 25 ]. The sera of RHΔ had2a -vaccinated and unvaccinated mice were isolated at 30 days post-infection.…”

“…To assess the protective efficacy of RHΔ had2a vaccination against tachyzoite infection, Kunming mice were immunized with 5 × 10 4 RHΔ had2a tachyzoites ( Figure 3 A) [ 25 ]. After 30 days post-immunization, the vaccinated mice and unvaccinated mice were infected by 10 3 wild-type RH or PYS tachyzoites ( Figure 3 C).…”

“…To generate the had2a gene knockout CRISPR plasmid pSAG1::CAS9-U6::Sg HAD2a, the SgRNA of had2a gene was used to replace the SgRNA of the uracil phosphoribosyl transferase ( uprt ) gene in the pSAG1::CAS9-U6::SgUPRT template plasmid [ 24 ]. The 5′ and 3′ homologous arms of the had2a gene (1000~1300 bp) were fused to the DHFR fragment and the pUC19 backbone fragment to construct the homologous DHFR-selective plasmid [ 25 ]. The sequence-validated plasmid was used as a template to amplify the homologous DHFR fragment.…”

“…Confluent HFF monolayers grown in 12-well plates (Thermo Fisher Scientific, USA) were infected with 300 freshly egressed tachyzoites of wild-type RH or mutant RHΔ had2a strains for 7 days. After the culture medium was removed, samples were washed with PBS, fixed with 4% paraformaldehyde for 20 min and stained with 0.2% crystal violet for 20 min, which was followed by two washes with PBS [ 25 ]. Three independent replicates were performed.…”

“…Mice were euthanized immediately when reaching the humane endpoint. Brain cysts of all chronically survived mice were counted as previously [ 20 , 25 ]. For the detection of brain cyst burden, the brains of the mice that survived for 30 days post-chronic challenge were collected, homogenized, and used to count brain cyst number, as described previously [ 15 ].…”