Host cell specificity and schizogony of Plasmodium berghei under different in vitro conditions.

Invasion and intra-erythrocytic growth of two strains of Plasmodium berghei (ANKA and K173) were studied under different in vitro conditions. Some important limiting factors for the mass cultivation of this rodent malaria parasite were reconsidered. Parasites of both strains developed normally from ringforms into mature schizonts in RPMI1640 supplemented with Fetal Calf Serum (FCS). At a temperature of 37 degrees C the duration of the schizogonic cycle was comparable to that of the same parasites developing under in vivo conditions. At 27 degrees C, however, the asexual cycle took 60-72 h. In medium supplemented with mouse serum instead of FCS the growth of the parasites was severely inhibited. Parasites of both strains showed a strict preference for reticulocytes. Red blood cells from rats, mice and hamsters were readily invaded by merozoites from both strains. Erythrocytes from rabbits and guinea pigs were resistant to invasion by P. berghei. It is concluded that host cell specificity technically limits the possibilities for mass cultivation of P. berghei. The validity of recent publications, describing alternative culture systems for this rodent parasite, is discussed.

Plasmodium berghei: gametocyte production, DNA content, and chromosome-size polymorphisms during asexual multiplication in vivo.

In this study the DNA content and the karyotype of clones of Plasmodium berghei, which differed in the capability to produce gametocytes, were determined. The DNA content per haploid genome was established by cytofluorometric methods after staining of the haploid merozoites with DNA-specific fluorescent dyes. Field inversion gel electrophoresis was used to establish the number and size of the chromosomes. Parasites of a high gametocyte producer clone (original HP) and a low producer clone (original LP) contained 13 to 14 chromosomes in the size range of 0.5-3.8 megabase. In four independent experiments parasites of the original HP clone were maintained in mice and were mechanically transmitted for prolonged periods of time (up to 90 weeks). During the transmission period the capability to produce gametocytes decreased in all four lines. After mosquito transmission of parasites from these low producer lines, the gametocyte production returned to the level of the original HP clone. The total DNA content per haploid genome of low producer parasites was not significantly different from that of HP parasites. During prolonged periods of asexual multiplication of the HP clone in vivo, changes in the relative size of several chromosomes were detected. Mosquito transmission of the original HP clone did not result in a change of the karyotype. However, novel karyotypes were readily observed after mosquito transmission of parasites of the low producer lines. The decrease of the capability to produce gametocytes did not necessarily involve detectable changes in DNA content or in karyotype.

Synchronized erythrocytic schizogony and gametocytogenesis of Plasmodium berghei in vivo and in vitro.

Both asexual and sexual development of Plasmodium berghei was synchronized without chemical intervention using in vitro culture techniques. Combined in vivo and in vitro experiments were performed on the relationship between age, morphology and maturity of gametocytes. Schizogony took 22-23 h in the experiments. At 26 h post-invasion (p.i.) the first males became capable of exflagellation. By 20 h p.i. the first gametocytes were recognizable in Giemsa-stained smears but the sex was hardly distinguishable until maturity (26 h p.i.). Survival time of gametocytes was estimated at 26 h in vitro (half-life 13 h) and the same survival time was suggested for gametocytes in vivo. Schizonts of P. berghei apparently disappeared from the peripheral circulation upon maturity, rupturing almost immediately. Mature schizonts in vitro persisted up to 48 h p.i. in non-agitated cultures. No evidence was collected for sequestration of any sub-population of gametocytes.