The ecology and epidemiology of malaria parasitism in wild chimpanzee reservoirs.

Chimpanzees (Pan troglodytes) harbor rich assemblages of malaria parasites, including three species closely related to P. falciparum (sub-genus Laverania), the most malignant human malaria parasite. Here, we characterize the ecology and epidemiology of malaria infection in wild chimpanzee reservoirs. We used molecular assays to screen chimpanzee fecal samples, collected longitudinally and cross-sectionally from wild populations, for malaria parasite mitochondrial DNA. We found that chimpanzee malaria parasitism has an early age of onset and varies seasonally in prevalence. A subset of samples revealed Hepatocystis mitochondrial DNA, with phylogenetic analyses suggesting that Hepatocystis appears to cross species barriers more easily than Laverania. Longitudinal and cross-sectional sampling independently support the hypothesis that mean ambient temperature drives spatiotemporal variation in chimpanzee Laverania infection. Infection probability peaked at ~24.5 °C, consistent with the empirical transmission optimum of P. falciparum in humans. Forest cover was also positively correlated with spatial variation in Laverania prevalence, consistent with the observation that forest-dwelling Anophelines are the primary vectors. Extrapolating these relationships across equatorial Africa, we map spatiotemporal variation in the suitability of chimpanzee habitat for Laverania transmission, offering a hypothetical baseline indicator of human exposure risk.

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.