Continuous in vitro propagation of the malaria parasite Plasmodium vivax

Claudia F. Golenda, Jun Li, Roland Rosenburg

            The control of Plasmodium vivax, one of the more severe strains of the devastating disease Malaria, has been very complicated due to its potential drug resistance factors. The culturing of P. vivax not only will give scientist a broader scope into the morphology of the parasite, but it will also help develop vaccines, test drugs, and clone parasites for genome sequencing. The cultivation of the P, vivax parasite was done by using Chesson strains of the parasite from owl monkeys, and passages them to human reticulocytes. The Chesson strain was isolated shortly after WWII from an American soldier, which has been maintained in Aotus monkeys since 1994.  Peripheral blood from a hemochromatosis patient was sued to separate the human reticulocytes by differential centrifugation (Golenda, Li, Rosenburg 1997).

 The parasites were grown in 48 hour durations (considered one cycle) in a static candle jar, until the beginning of schizogony. At about hour 36-40 human reticulocytes were added and the cultures were transferred to a shaker for 10-12 hours. The success of the human reticulocytes binding to the susceptible red blood cells and causing an infection was merely based on the availability of sufficient red blood cell numbers. Malaria treatment usually includes the drug hloroquine, but P. vivax has recently become more resistant in Southeast Asia (Golenda, Li, Rosenburg 1997).

 Two karyotypes of the Aotus monkeys were used: 1. Autos nancymai and 2. Aotus lemurinus griseimembra. The A. nancymai species was inoculated with the Chesson strain intravenously and blood was tested and every other day until the monkey self- cured or was treated with Chloroquine. The A. griseimembra species was inoculated with the ring, and young trophozoite stage of the parasite. Both species were monitored the same. The reticulocytes used were separated from the positive Duffy Antigen group from a patient undergoing hemochromatosis. Reticulocytes were enriched by differential centrifugation in homologous plasma. The results of the experiment showed a successful passage of owl monkey to human erythrocytes, when recycled in a culture for 6-8 generation. The addition of human reticulocytes during schizogony provided merozoites with the targets cells needed for invasion of the cell. In both cultures (both monkey species) the parasite density increased at least 2-fold by cycle 3 (Golenda, Li, Rosenburg 1997).

            By alternating between the static candle jar and the shaker, as well as periodically adding reticulocytes, P. vivax was maintained in a culture for 6-8 cycles. The parasites were mostly in the ring and young trophozoite stage and were confirmed by the analysis of the 18S rRNA gene, a extremely sensitive indicator for Plasmodium. Although the asexual in vitro recycling of P. vivax has been achieved, it leads to some limitations. For one the lack of gametocyte formation in the cultures prohibits the infection of mosquitoes which is directly related to the control of the parasite. Secondly the procedure is labor and time sensitive, and you need access to patients that are suffering from hemochromatosis to do the experiment (Golenda, Li, Rosenburg 1997).

            Exposure to P. vivax is strongly correlated with poverty and low- income living. Those in third world countries at most risk are working aged men. In regions where P. vivax is the most prevalent, effective immunity to the parasite is usually never achieved because it occurs at low incident rates in certain regions (Mendis et. Al 2001). The cultivation of P. vivax will hopefully expose the biochemistry, immunology, biology, physiology, and pharmacology of the parasite for the ultimate regulation of the parasite (Golenda, Li, Rosenburg 1997).

Literature Cited

1.      Golenda CF, Li J., Rosenberg, R. 1997. Continuous in vitro propagation of the malaria parasite plasmodium vivax. Developmental Biology. 94: 6786-6791.

2.      Mendis, K., Sina BJ, Marchesini P, Carter R. 200. The neglected burden of plasmodium vivax malaria. The American Society of Tropical Medicine and Hygien. 64:97-106.