James L. Leef

RAPID, LARGE-SCALE ISOLATION OF PLASMODIUM BERGHEI SPOROZOITES FROM INFECTED MOSQUITOES*

The discontinuous gradient technique for recovery of malarial sporozoites from mosquitoes (Beaudoin et al., 1977) has been modified to speed up recovery and prevent sensitization of mice by components of the gradient which contaminate the sporozoites used as antigen. Mouse serum was substituted for BSA in the gradient because the latter produced hypersensitivity. Best results were obtained with gradients consisting of Medium 199, Renografin and mouse serum. Heavy and light solution of gradient components are layered in a centrifuge tube. Centrifugation of comminuted, infected mosquitoes applied to the top of the discontinuous gradient concentrates sporozoites at the interface. Sporozoites recovered from the gradient were infective, immunogenic, and relatively free of mosquito tissue. This improved method enables recovery of 100,000 sporozoites from each Anopheles stephensi infected with the ANKA strain of Plasmodium berghei. As many as 2,800 mosquitoes have been processed in 2 hr without a significant decrease in yield.

THE INFLUENCE OF CELL TYPE AND CULTURE MEDIUM ON THE IN VITRO CULTIVATION OF EXOERYTHROCYTIC STAGES OF PLASMODIUM BERGHEI

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.

In vitro infectivity of cryopreserved Plasmodium berghei sporozoites to cultured cells

Plasmodium berghei sporozoites frozen in MEM (Eagle) medium supplemented with 10% hydroxyethyl starch and 50% normal mouse serum retained 0.5% infectivity to cultured hepatoma cells, compared to 6.8% before freezing. This demonstrates that frozen-thawed sporozoites can be used in in vitro investigations of the exoerythrocytic malarial parasite and means that when large numbers of sporozoites are available, they may be frozen and preserved for later use.

Free-flow electrophoretic separation of Plasmodium berghei sporozoites.

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.