Initial extracellular forms of Plasmodium falciparum: their ultrastructure and their definition with monoclonal antibodies.

Merozoites of the erythrocytic stage of the human malaria parasite Plasmodium falciparum, when placed under appropriate conditions in a culture medium with erythrocyte extract, differentiate into early trophic forms. These forms have much the same ultrastructure as rings of the same age that have developed intracellularly and have then been freed from their host cells by immune lysis. However, these forms differ in two respects: the extracellular forms have only their single plasma membrane, whereas the forms freed from host cells have, in addition, a surrounding parasitophorous vacuole membrane; the forms that develop extracellularly have fewer ribosomes. Five monoclonal antibodies against the ring stage have been prepared and characterized. Their pattern of immunofluorescence localization differs in merozoites as compared with rings, but their pattern is identical in rings developed extracellularly and those developed intracellularly. These results and the observations on fine structure demonstrate biochemical and morphological differentiation in the extracellular forms.

Clones of the malaria parasite Plasmodium falciparum obtained by microscopic selection: their characterization with regard to knobs, chloroquine sensitivity, and formation of gametocytes.

A culture line of Plasmodium falciparum (FCR-3/Gambia) was used to select and place in culture cells containing a single parasite. The method depends on examination of minute droplets of dilute cell suspensions with oil immersion phase-contrast microscopy. Droplets found to contain a single parasite were maintained under appropriate culture conditions until detectable numbers of parasites were present (generally by day 21). Of nine clones that grew up, seven were knobless and two were knobby. The clones differed somewhat in chloroquine sensitivity. Their 50% inhibition point under one set of conditions in vitro ranged from 0.02 to 0.06 micrograms of base per ml, compared with only 0.003 micrograms for the highly sensitive line FCR-8/West Africa. All three tested clones formed gametocytes under appropriate in vitro conditions.

An electron microscopic study of Babesia microti invading erythrocytes.

Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of the other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.