Fig 3. T. brucei impairs early liver stage of P. berghei infection.

(A) P. berghei liver infection load quantified by qRT-PCR 30 min, 2 h, 6 h, 12 h, 24 h and 46 h after injection of 3 x 10⁴ P. berghei sporozoites into naïve mice (Pb–blue bars) or mice infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of three to five independent experiments and error bars indicate the SEM. The two-way ANOVA with post-test Bonferroni was employed to assess the statistical significance of differences between experimental groups. *** P < 0.001 and **** P < 0.0001. (B) Number of P. berghei-infected hepatocytes per square millimeter of liver section quantified by immunofluorescence microscopy 6 h, 12 h, 24 h and 46 h after injection of 3 x 10⁴ P. berghei sporozoites into naïve mice (Pb–blue bars) or mice infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of one representative experiment out of two independent experiments and error bars indicate the SEM. The two-way ANOVA with post-test Bonferroni was employed to assess the statistical significance of differences between experimental groups. *** P < 0.001 and **** P < 0.0001. (C) Representative confocal microscopy images of EEFs at 12 h, 24 h and 46 h after injection of 3 x 10⁴ P. berghei sporozoites into naïve mice or mice infected 5 days earlier with T. brucei. White: Hoechst—nuclear staining; green: P. berghei GFP labeling showing the parasite heat shock protein 70; purple: P. berghei UIS4 labeling showing the parasitophorous vacuole membrane. Scale bars, 10 μm. (D) EEF area at 12 h, 24 h and 46 h after injection of 3 x 10⁴ P. berghei sporozoites into naïve mice (Pb—blue dots) or mice infected 5 days earlier with T. brucei (Tb/Pb—green dots), assessed by immunofluorescence microscopy. Results are expressed as the mean values of one representative experiment out of two independent experiments and error bars indicate the SEM.