title: Continuous in vitro propagation of the malaria parasite Plasmodium vivax.
abstract: The difficulty in controlling Plasmodium vivax, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy's 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% AB+ human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36-40 hr, when reticulocytes were added and cultures transferred to a shaker for 10-12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of P. vivax, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture P. vivax opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing.
authors.affiliation: Department of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
authors.forename: C F
authors.initials: CF
authors.lastname: Golenda
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: R
authors.initials: R
authors.lastname: Rosenberg
mesh_terms: D000818:Animals; D000882:Haplorhini; D006801:Humans; D016780:Malaria, Vivax / Q000469:parasitology*; D010274:Parasitology / Q000379:methods*; D010966:Plasmodium vivax / Q000254:growth & development* / Q000472:pathogenicity