An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca²⁺) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca²⁺ imaging showed that LZ treatment completely abolished Ca²⁺ oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP₃–Ca²⁺ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.     

Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro

Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41 °C. Febrile episodes typically have a deleterious effect on parasites and probably benefit the host by aiding parasite clearance; however, the parasite may also gain advantage from limiting its burden on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Programmed cell death (PCD) may provide the parasite with a mechanism of self-limitation, although the occurrence and phenotype of PCD in the erythrocytic stages remain controversial due to conflicting data. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress. A variety of biochemical markers of PCD, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation, as well as morphological studies of Giemsa-stained thin smears and real-time microscopy were utilised to characterise the phenotype. Heat stress decreased P. falciparum growth and development in vitro. Late-stage parasites were more susceptible, although early stages were more affected than expected. Early-stage parasites exposed to 41 °C exhibited markers of an apoptosis-like PCD phenotype, including DNA fragmentation and mitochondrial depolarisation. Heat-stressed late-stage parasites showed no significant DNA fragmentation or mitochondrial dysregulation; however, cytoplasmic vacuolisation was suggestive of an autophagy-like form of PCD. Our results therefore showed that biochemical and morphological markers of PCD varied with intra-erythrocytic parasite development and that P. falciparum exhibited facets of both apoptosis- and autophagy-like phenotypes after exposure to febrile temperatures, which may reflect a unique PCD phenotype.     

Plasmodium falciparum Dynein Light Chain 1 Interacts with Actin/Myosin during Blood Stage Development

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein γ heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.     

Adaptation of the Plasmodium falciparum FCB strain for in vitro and in vivo analysis in squirrel monkeys (Saimiri sciureus)

The asexual blood stages of the Plasmodium falciparum parasite are responsible for inducing the clinical symptoms and the most severe presentations of malaria infection that causes frequent mortality and morbidity in tropical and subtropical areas of the world, making the blood stages of infection a key target of new malaria treatment and prevention strategies. Progress towards the development of more effective treatment and prevention strategies has been hindered by the limited availability of infection models that permit the sequential analysis of blood stage parasites in vitro followed by in vivo analysis to confirm therapeutic benefits. To advance a model for in vitro and in vivo analysis of blood stage parasites, we examined nine laboratory strains of P. falciparum to determine which strains could adapt to growth in vivo in splenectomized squirrel monkeys (Saimiri sciureus). Only one of the nine laboratory strains tested, the FCB strain, adapted to in vivo growth. Morphological analysis show that the adapted ring-stage parasites have a different morphology from original parasites cultured in vitro, and more often they were found to localize at the edge of the infected red blood cell. No remarkable differences were observed for both trophozoites and schizonts. The adapted strain can be cultured back in vitro similar to the original parasite, indicating that the adapted parasite can develop both in vitro and in vivo. This squirrel monkey-adapted P. falciparum parasite is expected to be suitable and is advantageous for the research and development of vaccines and antimalarial drugs.     

The accumulation of lactic acid and its influence on the growth ofPlasmodium falciparum in synchronized cultures

Summary Synchronization ofPlasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66±0.55 μmol LA per 12 h per 10⁷ parasitized cells (n=26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72±0.62 μmol LA per 12 hours per 10⁹ red blood cells) (n=41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth ofPlasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield. J. W. Z. was supported during part of this study by a long-term fellowship of the European Molecular Biology Organization, Heidelberg, West Germany, followed by a Research Associateship from the National Research Council, Washington, D.C. The project was supported by grants from the Medical Research Council to J. G. S. and by the Naval Research and Development Command, Work Unit No. 3M 162 770 A871 AE 312. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large.     

Stress,drugs and the evolution of reproductive restraint in malaria parasites

Life-history theory predicts that sexually reproducing organisms have evolved to resolve resource-allocation trade-offs between growth/survival versus reproduction, and current versus future reproduction. Malaria parasites replicate asexually in their vertebrate hosts, but must reproduce sexually to infect vectors and be transmitted to new hosts. As different specialized stages are required for these functions, the division of resources between these life-history components is a fundamental evolutionary problem. Here, we test how drug-sensitive and drug-resistant isolates of the human malaria parasite Plasmodium falciparum resolve the trade-off between in-host replication and between-host transmission when exposed to treatment with anti-malarial drugs. Previous studies have shown that parasites increase their investment in sexual stages when exposed to stressful conditions, such as drugs. However, we demonstrate that sensitive parasites facultatively decrease their investment in sexual stages when exposed to drugs. In contrast to previous studies, we tested parasites from a region where treatment with anti-malarial drugs is common and transmission is seasonal. We hypothesize that when exposed to drugs, parasites invest in their survival and future transmission by diverting resources from reproduction to replication. Furthermore, as drug-resistant parasites did not adjust their investment when exposed to drugs, we suggest that parasites respond to changes in their proliferation (state) rather the presence of drugs.     

Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death

The ATP-dependent ClpQY protease system in Plasmodium falciparum is a prokaryotic machinery in the parasite. In the present study, we have identified the complete ClpQY system in P. falciparum and elucidated its functional importance in survival and growth of asexual stage parasites. We characterized the interaction of P. falciparum ClpQ protease (PfClpQ) and PfClpY ATPase components, and showed that a short stretch of residues at the C terminus of PfClpY has an important role in this interaction; a synthetic peptide corresponding to this region antagonizes this interaction and interferes with the functioning of this machinery in the parasite. Disruption of ClpQY function by this peptide caused hindrance in the parasite growth and maturation of asexual stages of parasites. Detailed analyses of cellular effects in these parasites showed features of apoptosis-like cell death. The peptide-treated parasites showed mitochondrial dysfunction and loss of mitochondrial membrane potential. Dysfunctioning of mitochondria initiated a cascade of reactions in parasites, including activation of VAD–FMK-binding proteases and nucleases, which resulted in apoptosis-like cell death. These results show functional importance of mitochondrial proteases in the parasite and involvement of mitochondria in programmed cell death in the malaria parasites.     

Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc^-, phosphoenolpyruvate carboxylase and pbmdh^-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt^-, orotate phosphoribosyltransferase and pbompdc^-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc^-, pboprt^- and pbompdc^- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Purinergic signalling is involved in the malaria parasite <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca²⁺ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca²⁺ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca²⁺ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca²⁺ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca²⁺]_c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca²⁺]_c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.     

Effects of Mitochondrial Protein Synthesis Inhibitors on the Incorporation of Isoleucine into Plasmodium falciparum In Vitro1

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [³H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [³H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids

Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC₅₀ between 20–87 µM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment.     

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.     

Differentiation of Gametocytes in Microcultures of Human Blood Infected with Plasmodium falciparum*

SYNOPSIS. Gametocytes differentiated from ring-stage parasites in microcultures of human blood infected with Plasmodium falciparum. Immature gametocytes could be distinguished morphologically from late asexual trophozoites after ～ 40 h of culture. Differentiation into crescentic forms took several days and the incorporation of [³H]-isoleucine by developing gametocytes was demonstrated. About 1% of red cells contained gametocytes at the maximum densities attained. Differentiation of gametocytes occurred either directly from rings placed in culture or from the progeny of subsequent cycles of schizogony and invasion in vitro. The latter occurrence was confirmed by the development of gametocytes in marker fetal red cells added to cultures, although fetal red cells provide a less favorable environment than those with HbA for growth of the parasites.     

Giardia muris: Correlation between oral dosage,course of infection,and trophozoite distribution in the mouse small intestine

Oral inoculations of Giardia muris cysts to CD-1 Swiss mice resulted in a reproducible pattern of infection measured by cyst excretion and the number of trophozoites in the small intestine. Housing of animals together or individually did not alter the pattern of cyst release for the first 30 days of infection. Administration of 10 to 10⁵ cysts/mouse resulted in a similar rate of cyst excretion from Day 10 to the end of the infection period. There was a direct correlation between the size of inoculum and the length of the latent period. Mice which received larger doses (10³, 10⁴, 10⁵) had significantly higher numbers of cysts in feces during the first week of infection, compared to those which received lower doses (10 or 100). The extent of trophozoite colonization and their distribution in the small intestine was not related to the size of inoculum. The location of trophozoites in the small intestine varied during the infection. In the first 3 to 8 days of infection the trophozoites were situated in the upper 25% of the small intestine, but moved posteriorly (upper 40%) during the period of peak cyst output. The number of trophozoites in the small intestine and cyst excretion were found to be proportional at all stages of infection.     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488 nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.