Fig. 4

Infectivity potential of NcGRA23. a Schematic illustration of the replacement of the entire NcGRA23 coding region with the chloramphenicol resistance gene (CmR) and red fluorescence protein gene (RFP) for the generation of the NcGRA23 knockout strain. b Diagnostic PCRs on two Δncgra23 clones (1 and 2). (F1-R1) and (F2-R2) check the correct integration of the NcGRA23 gene locus, whereas (F3-R3) examines the deletion of the NcGRA23 gene. c Western blotting with NcGRA23 anti-mouse antibodies with NcGRA23 of 23 kDa in the parent Nc1 strain. No NcGRA23 protein was detected in the Δncgra23 polyclonal strains (1, 2, 3, and 4). NcActin was used as a loading control. d Plaque assay comparing the growth of Nc1 and Δncgra23. Each well (HFF cell) was infected with 200 parasites, and plaques were stained 9 days later. e The plaque areas were counted by randomly selecting at least 20 plaques and using the pixel point in Photoshop C6Ssoftware (Adobe, USA). The data were compiled from two independent experiments. Analysis of the plaque area was performed using one-way ANOVA with Tukey’s post hoc comparison. n.s. indicates no difference (p > 0.05). f Mouse survival after infection with 5 × 10⁶ or 8 × 10⁶ doses of Δncgra23 and Nc1. There were five female mice in each group. Statistical analysis was performed using the LIFETEST (life test data = surv) SAS software (SAS Institute Inc., USA). The data are representative of two experiments with similar outcomes