The apicomplexan intraerythrocytic parasite Babesia microti is an emerging human pathogen and the primary cause of human babesiosis, a malaria-like illness endemic in the United States. The pathogen is transmitted to humans by the tick vector, Ixodes scapularis, and by transfusion of blood from asymptomatic B. microti-infected donors. Babesiosis is a major public health concern because the population at risk for severe disease (people over the age of 50 and of any age with compromised immune systems) is increasing. Presently, drug combinations of azithromycin+atovaquone or clindamycin+quinine are recommended therapies. These drug combinations were first evaluated for their anti-babesia activity because of their known antimalarial activity. With these drugs and other supportive measures such as exchange transfusion, the mortality rate is 9% in infected patients requiring hospitalization and approaches 28% in immunocompromised hosts. Patients who survive may experience recrudescent parasitemia for more than a year, requiring more prolonged therapy. The reasons that Babesia can persist despite drug therapy remain unknown. A better understanding of the parasite metabolism, diversity, virulence and tissue distribution are critical for the development of more effective therapies for treatment of human babesiosis. Towards this goal, we have completed genomic and transcriptomic analyses and initiated thorough annotation of the genomes and reconstruction of the metabolic machineries of seven B. microti clinical isolates. Our preliminary findings indicate that B. microti exhibits genotypic variations among strains. These variations may account for their notable differences in rate of proliferation and host selectivity (mice vs hamsters). We have also identified two key metabolic pathways, the non-mevalonate (MEP) pathway and the folate biosynthesis pathway (FBP), which can be targeted by drugs already approved by the FDA. In order to fully characterize the efficacy of these drugs against B. microti infection in vivo, a mouse model of B. microti infection must be more clearly defined in terms of sites of parasite persistence that may lead to recrudescence of parasitemia. Accordingly, this proposal aims to (1) define in mice the kinetics of parasitemia and sites of persistence of two prototype B. microti strains isolated from non-immunocompromised patients who experienced mild or severe disease; and (2) assess the efficacy of drugs that target the MEP and FBP pathways of B. microti to resolve parasitemia and eliminate infection. Successful completion of these exploratory studies will help define mechanisms underlying B. microti pathogenesis and set the stage for future clinical studies to evaluate the efficacy of these drugs for treatment of human babesiosis in situations when current therapies fail.