Susan G. Langreth

Plasmodium falciparum: Loss of knobs on the infected erythrocyte surface after long-term cultivation

After long-term in vitro cultivation in human erythrocytes, variants of three strains of the malaria parasite Plasmodium falciparum no longer produce the “knob” alterations on the host erythrocyte surface. The time in continuous culture before knobs failed to appear ranged from 18 months for the Gambian strain FCR-4 to 33 months for the Vietnamese strain FCR-1. The loss of knobs is correlated with the inability to concentrate trophozoites, schizonts, and segmenters from these variant lines by the use of gelatin-containing media. This is the first report of a change in Plasmodium falciparum or its host cell as a consequence of long-term culture.

Evidence for a polysaccharide surface coat in the developmental stages of Leishmania donovani: A fine structure-cytochemical study

SummaryA polysaccharide surface coat was demonstrated in the various developmental stages of Leishmania donovani at the fine structural level by several cytochemical methods. During the course of in vitro cultivation, amastigote forms which had lost the outer membrane gave positive surface coat staining reactions following Alcian blue-lanthanum nitrate treatment. Coat matrix deposition appeared to increase during cultivation of the transforming amastigotes, reaching a maximum thickness at ∼7 hr characteristic of that observed with the promastigote stages. Differences were observed in the appearance of the surface coat materials in amastigotes treated with Alcian blue and lanthanum nitrate at 0°C and 24°C. Heavy deposits of coat-like material were observed in the flagellar tip region of promastigotes. Results of growth studies indicated that total rosette formation increased during the first 5 days of the promastigote growth phase. No differences were observed, however, in the appearance of the surface coat in promastigotes obtained from culture after various periods. Surface coat materials also were observed in promastigotes treated with purified ruthenium red and violet fractions. It was suggested that the surface coat might have a role in the adhesion of the organisms to various substrates and other cells in culture.

Plasmodium falciparum: merozoite invasion in vitro in the presence of chloroquine.

Abstract The fine structure of invasion of human erythrocytes by merozoites of the malaria parasite Plasmodium falciparum was observed in vitro. The invasion process is similar to that described for P. knowlesi. Merozoites enter apical end first by invagination of the erythrocyte membrane. At the rim of the invagination, where merozoite and erythrocyte are in closest contact, the erythrocyte membrane is thickened. The brushy cell coat of the P. falciparum merozoite appears to be lost at this attachment zone. The part of the merozoite within the erythrocyte invagination has no visible coat. The coat on the portion outside is unaltered. Merozoites can successfully invade erythrocytes after 3 hr in the presence of a concentration of chloroquine harmful to feeding stages.

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis.

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.

Immunization of owl monkeys to Plasmodium falciparum with merozoites from cultures of a knobless clone

Summary Aotus trivirgatus monkeys of karyotype 2 were treated as follows. Three received two injections of purified merozoites of a knobless (K‐) clone of Plasmodium falciparum with muramyl dipeptide as adjuvant; three received similar injections but with merozoites of a wild‐type knobby (K+) strain; three controls received MDP with human erythrocytes in the amounts estimated to be present as contaminants in the merozoite preparations. A month after the second injection all nine monkeys were inoculated with parasites of the wild‐type knobby strain from another infected Aotus. The monkeys that had received the K‐ material developed only extremely low infections markedly different from the infections in the controls. Of those that received K+ material, one died early with low parasitaemia, one was protected and one had the same level of infection as the controls. In the combined group of immunized animals, four out of six were protected.