Analysis of cerebral complications in mice infected with wt⁺ and Δpm4⁺ parasites. A: Histological analysis of the brains of C57BL/6 mice with asynchronous infections at day 7 after injection of wt⁺ or Δpm4⁺ parasites. Longitudinal sections of the brain, stained with H&E, show extensive hemorrhagic areas in wt⁺-infected animals (black arrows), which are absent in Δpm4⁺-infected mice. In brains of wt⁺-infected mice, infected erythrocytes, recognized by pigment granules, were found as infiltrates in brain tissue (red arrow). B: Representative digital images of Evans Blue dye extrusion analysis of brains of wt⁺ or Δpm4⁺-infected C57BL/6 mice at day 7 postinfection. The blue staining of the brains shows vascular leakage in wt⁺-infected animals, which is absent in the brains of Δpm4⁺-infected mice. C: Bioluminescent images of brain isolated from C57BL/6 mice at day 7 after infection with 10⁵ wt⁺ or 10⁵ to 10⁶ Δpm4⁺ parasites showing a significant higher parasite load (see D) in brains of wt⁺ than in brains of Δpm4⁺-infected mice as a result of differences in accumulation of infected erythrocytes in brain tissue. Percentages shown represent the parasitemias at the time of collection of the brains. D: Differences in luminescence signal between the brains of mice infected with either wt⁺ or Δpm4⁺ parasites (n = 6) showing the differences in parasite load. *P < 0.05.

The course of infection of Δpm4 parasites in different mouse lines/parasite strains combinations. Cumulative death and parasitemia levels are shown in the left and right panels, respectively. A: BALB/c mice infected with 10⁷ wild-type (wt) (n = 30) or Δpm4 parasites of lines Δpm4cl1 (n = 50) and Δpm4cl6 (n = 50). All wild-type infected mice died within the 3rd week after infection with high parasitemia, whereas all mice infected with Δpm4 parasites survived infection. B: NIH Swiss mice infected with 10⁷ wild-type (cl15cy1) (n = 10) or Δpm4 parasites of line 688cl2 (n = 10). All NIH Swiss mice infected with wild-type (cl15cy1) parasites developed high parasitemia, and experiments were terminated on day 15 after infection at a parasitemia 80%. All mice infected with Δpm4 parasites after the peak of parasitemia were able to control the infection and completely cleared the parasites. C: C57BL/6 mice infected with 10⁵ wild-type (n = 15) or Δpm4cl1 (n = 15) and Δpm4cl6 (n = 50) parasites Ninety percent of wild-type-infected C57BL/6 mice developed ECM at day 6 to 8 after infection. None of the mice infected with Δpm4 parasites showed signs of cerebral complications. Most Δpm4-infected mice (80%) developed high parasitemia and died in the 3rd week after infection. The surviving mice cleared the parasites. D: Swiss-OF1 mice infected with either 10⁵ wild-type (cl15cy1) (n = 10) or Δpm4 parasites of lines 688cl2 (n = 13) or line 688cl3 (n = 6). All Swiss-OF1 mice developed ECM at day 6 to 8 after infection at a parasitemia ranging from 15 to 20%, whereas the mice infected with Δpm4 parasites did not show cerebral complications. These mice developed high parasitemia, and experiments were terminated between day 14 and 16 after infection at a parasitemia >30%. ***P < 0.0001.

The role of the spleen in parasite clearance and protective immunity. A: Macroscopic observation of spleens from BALB/c mice infected with either 10⁷ wild-type or Δpm4cl6 parasites. This analysis revealed that at 21 days postinfection the spleens of Δpm4-infected mice (lower panel) were much bigger than those from wild-type infected animals (upper panel). This observation was confirmed by comparing the spleen weight in either wild-type or Δpm4cl6-infected mice at different time points (7, 14, 21, and 40 days) after infection. Numbers represent the mean values ± SD of spleen weight of eight animals. B: Cumulative death (left panel) and levels of parasitemia (right panel) of either intact (gray) or splenectomized (black) BALB/c mice infected with 10⁷ Δpm4cl6 parasites. ***P < 0.0001. C: Cumulative death (left panel) of splenectomized Δpm4 convalescent BALB/c mice (convalescent splenectomized [c.s.]) after a challenge with wild-type parasites. As a control Δpm4 convalescent intact BALB/c mice (convalescent not splenectomized [c.n.s.]) and naïve intact BALB/c mice (naïve not splenectomized [n.n.s]) were challenged with the same dose of wild-type parasites. Right panel: Time course of parasitemia in eight individual convalescent splenectomized BALB/c mice (M1–M8) after a challenge with wild-type parasites. None of the Δpm4 convalescent immune mice was able to control the parasitemia when challenged after splenectomy. All mice showed oscillating levels of parasitemia that increased sharply in a few days, leading to the death of the animals. ***P < 0.0001.

Distribution of luciferase expressing Δpm4⁺ and wt⁺ parasites in live mice as visualized by real time in vivo imaging through measurement of luciferase activity. A: A time course of an atypical distribution pattern of schizonts in mice with synchronous infections of Δpm4⁺ and wt⁺ parasites at different time points (hours) after the injection of purified merozoites. Schizont sequestration at 19 and 21 hours in wt⁺-infected mice (belly fat tissue, lungs, and spleen) and reinvasion of ring forms at 23 and 25 hours, resulting in bioluminescence in the whole body. In Δpm4⁺ parasites schizont sequestration and invasion start later (21 and 23 hours), but the distribution remains similar. B: Bioluminescence in organs of mice infected either with wt⁺ or Δpm4⁺ and isolated at 21 and 23 hours after infection, respectively. C: Quantification of luciferase signals in organs of mice infected with wt⁺ and Δpm4⁺ (six mice per group) showed no significant differences in parasite distribution. D: Parasite load and distribution in mice treated with IgG obtained from Δpm4 convalescent mice and infected with wt⁺⁺ parasites. IgG purified from the serum of animals infected with Δpm4cl6 parasites at day 18 or 40 after injection were given to BALB/c mice at days −1, 0, and + 1 relative to the time of i.v. inoculation with wt⁺⁺ parasites. As a control, mice were treated with IgG from wild-type infected mice obtained at day 18 postinfection. Nontreated: BALB/c mice infected with the wt⁺⁺ parasite line only. Parasites were visualized at day 2 after infection of the mice with the wt⁺⁺ parasites. E: Quantification of luciferase signals in organs of mice (six mice per group) treated with the different IgG samples shows a significant increase in parasite accumulation in the spleen of mice treated with IgG from convalescent animals 40 days postinfection. F: The course of the parasitemia in mice treated with the different IgG samples shows a significant reduction of the parasite load in mice treated with IgG that was collected from convalescent animals (day 40 after infection).*P < 0.05. RLU, relative light unit.