Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice

Abstract

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.

Figure 1. Hepatic development of P. falciparum in LH TK-NOG mice.

Representative pictures of P. falciparum schizonts at days 5 and 7 post-infection. Sixteen-μm thick frozen and 5-μm thick deparaffinized liver sections were stained by indirect immunofluorescence with antibodies specific to P. falciparum and DAPI to stain host cell and parasite nuclei. (a). Day-5 and day-7 schizonts stained for Plasmodium HSP70, scale bar, 20 μm. (b). Maximal diameter and area of day-5 and day-7 schizonts; n=32 at day 5 and n=27 at day 7, both from 3 different lobes, mean±s.d. ***P<0.0001, Mann–Whitney U-test. (c). Day-7 schizonts stained for EXP-1 and SERP. (d). Schizonts were stained for MSP-1, AMA-1 and RON-4 at day 5 and day 7. Scale bar, 20 μm. (e). Merosome-like structures stained for HSP70 and DAPI observed in day-7 schizonts, left picture, Scale bar, 20 μm; central picture, magnification of left picture showing merozoites nuclei in the merosome-like structure, Scale bar, 10 μm; right picture, Scale bar, 50 μm.

Figure 2. Asexual and sexual blood stages of P. falciparum in sporozoite-infected TK-NOG mice engrafted with human hepatocytes and RBC.

(a). Experimental procedure for the double engraftment of TK-NOG mice with human hepatocytes and RBC before infection with P. falciparum sporozoites. (b). In vitro asexual blood stage culture: at day 8 of infection, 200 ul of venous blood was put in culture to follow emergence of parasitaemia. Results of one experiment with three mice are expressed as the number of parasites within 2 μl of blood for each mouse. Results are representative of three independent experiments performed with a total of eight mice. Representative pictures of a trophozoite and a schizont observed on Giemsa-stained thick blood smears at day 0 of in vitro culture (= day 8 of infection) and of trophozoites observed on Giemsa-stained thin blood smears at day 4 of culture. (c). In vivo asexual blood stage development: individual parasitaemias were determined using Giemsa-stained thin blood smears from a total of 4 mice from two independent experiments. Representative pictures of schizonts observed on Giemsa-stained thick blood smears during the course of infection, scale bar, 5 μm. (d). In vivo sexual blood stage development: representative pictures of gametocytes at different stages of their development as observed in Giemsa-stained thick blood smears from two mice.

Figure 3. Hepatic development of P. ovale in LH-TK-NOG mice.

Representative pictures of P. ovale schizonts and hypnozoites at days 8 and 21 post-infection. 50-μm thick frozen (a,b) or 5-μm thick deparaffinized liver sections (d) were stained by indirect immunofluorescence with immune serum specific to Plasmodium HSP70 and DAPI to stain host cell and parasite nuclei. (a). Day-8 schizonts, scale bar, 20 μm. (b). Upper panel: day-8 schizont and—hypnozoite (white arrow), scale bar, 20 μm. Lower panel: higher magnification of the hypnozoite showing the single nucleus within the 5 μm round-shape parasite, scale bar, 5 μm. (c) Maximal diameter and area of day-8 schizonts; n=35, from three different lobes, mean±s.d. (d). Day-21 schizont, scale bar, 20 μm.