Abstract
Plasmodium coatneyi is an important model for severe malaria due to its P. falciparum-like sequestration, yet research has been limited by the lack of in vitro culture systems. Recently, molecular evidence of human P. coatneyi infections (3.5%, 3/85 of identified simian malarias in Malaysian archival samples), suggested a zoonotic potential. We report the first continuous in vitro culture of P. coatneyi and characterize its zoonotic potential using a dual-colour flow cytometry assay. Using CellTracker Deep Red-labelled target cells, we quantified invasion across primate species, revealing potential sample-specific plasticity. Robust continuous culture was established in Macaca fascicularis erythrocytes for over 40 days, with parasitaemia reaching 3% following cryopreservation recovery. While P. coatneyi successfully invaded human erythrocytes, asexual development was arrested post-invasion, with parasitaemia falling below detection limits by day 9. Analysis by microscopy confirmed a failure to complete the 48-hour developmental cycle in human cells, identifying a putative biological barrier to sustained zoonotic transmission. Ultimately, the cultivation protocol provides a sustainable in vitro platform for functional investigations, particularly severe malaria pathogenesis, and for monitoring host promiscuity in zoonotic malaria parasites.