A. Pal, Isaline Renard, Pankaj Singh
Dec 1, 2021
Hematozoa are a subclass of protozoan parasites that invade and develop within vertebrate red blood cells to cause the pathological symptoms associated with diseases of both medical and veterinary importance such as malaria and babesiosis. A major limitation in the study of the most prominent hematozoa, Plasmodium spp, the causative agents of malaria, is the lack of a broadly accessible mouse model to evaluate parasite infection in vivo as is the case for P. falciparum or altogether the lack of an in vitro culture and mouse models as is the case for P. vivax, P. malariae and P. ovale. Similarly, no in vitro culture system exists for Babesia microti, the predominant agent of human babesiosis. In this study, we show that human red blood cells infected with the human pathogen Babesia duncani continuously propagated in culture, as well as merozoites purified from parasite cultures, can cause lethal infection in immunocompetent C3H/HeJ mice. Furthermore, highly reproducible parasitemia and survival outcomes were established using specific parasite loads and different mouse genetic backgrounds. Using the combined in culture-in mouse (ICIM) model of B. duncani infection, we demonstrate that current recommended combination therapies for the treatment of human babesiosis, while synergistic in cell culture, have weak potency in vitro and failed to clear infection or prevent death in mice. Interestingly, using the ICIM model, we identified two new endochin-like quinolone prodrugs, ELQ-331 and ELQ-468, that alone or in combination with atovaquone are highly efficacious against B. duncani and B. microti. The novelty, ease of use and scalability of the B. duncani ICIM dual model make it an ideal system to study intraerythrocytic parasitism by protozoa, unravel the molecular mechanisms underlying parasite virulence and pathogenesis, and accelerate the development of innovative therapeutic strategies that could be translated to unculturable parasites and important pathogens for which an animal model is lacking. Author Summary Use of model organisms is vital to the understanding of virulence and pathogenesis of a large number of human and animal pathogens. In case of hematozoan parasites that invade and develop within vertebrate erythrocytes, the studies are challenging because of the dearth of small animal model systems and the lack of continuous parasite growth in in vitro culture conditions. Here, we report a small animal model of lethal infection of Babesia duncani, one of the causative agents of human babesiosis. We show that in vitro cultured parasites and as well as parasites propagated in vivo can establish highly reproducible parasitemia which is dependent on the parasite load and also defined by different mouse genetic backgrounds. We further use this combined in culture-in mouse (ICIM) model of B. duncani infection to demonstrate the anti-babesial efficacy of two novel endochin like quinoline compounds. We propose that this ICIM dual model of B. duncani is an ideal system to get insights into protozoan intraerythrocytic parasitism, virulence, pathogenesis, and therapeutics and will open the vista to other important pathogens which are unculturable or lack an animal model.