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Plasmodium falciparum, the Apicomplexan parasite that causes the most severe form of human malaria, divides via schizogony during the asexual blood stage of its life cycle. In this method of cell division, multiple daughter cells are generated from a single schizont by segmentation. During segmentation, the basal complex forms at the basal end of the nascent daughter parasites and likely facilitates cell shape and cytokinesis. The requirement and function for each of the individual protein components within the basal complex remain largely unknown in P. falciparum. In this work, we demonstrate that the P. falciparum membrane occupation and recognition nexus repeat-containing protein 1 (PfMORN1) is not required for asexual replication. Following inducible knockout of PfMORN1, we find no detectable defect in asexual parasite morphology or replicative fitness. IMPORTANCE Plasmodium falciparum parasites cause the most severe form of human malaria. During the clinically relevant blood stage of its life cycle, the parasites divide via schizogony. In this divergent method of cell division, the components for multiple daughter cells are generated within a common cytoplasm. At the end of schizogony, segmentation partitions the organelles into invasive daughter parasites. The basal complex is a ring-shaped molecular machine that is critical for segmentation. The requirement for individual proteins within the basal complex is incompletely understood. We demonstrate that the PfMORN1 protein is dispensable for blood stage replication of P. falciparum. This result highlights important differences between Plasmodium parasites and Toxoplasma gondii, where the ortholog T. gondii MORN1 (TgMORN1) is required for asexual replication.

The transcription factor (TF) AP2-G is essential for gametocytogenesis in the malaria parasite; however, it remains unclear if AP2-G determines commitment to sexual stage development fate in the schizont stage, or whether AP2-G directly initiates sexual stage differentiation and development beginning in the late-trophozoite stage. In this study, we addressed this issue by investigating the expression profile of AP2-G and determining genome-wide target genes in Plasmodium berghei. Fluorescence microscopy showed that AP2-G expression was first observed in the parasite 12 h after erythrocyte invasion and peaked at 18 h when sexual features were first manifested in early gametocytes. Expression of AP2-G decreased with manifestation of sex-specific features. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed at peak AP2-G expression and identified over 1000 binding sites in the genome. The main binding motif of the TF predicted from the binding sites was GTACNY. Predicted targets contained a number of genes related to protein biogenesis, suggesting that AP2-G plays a role in establishing a cellular basis required for sexual differentiation. AP2-G binding sites also existed upstream of gametocyte-specific TFs, namely AP2-G2, AP2-FG, and AP2-G itself. Furthermore, the target contained two AP2 TF-related genes. Disruption of these genes resulted in the arrest of ookinete development. These results suggest another role of AP2-G: activating a transcriptional cascade to promote conversion into early gametocytes. Taken together, AP2-G is involved not in establishing sexual commitment of schizonts, but rather in triggering the initiation of differentiation and the early development of gametocytes in the late trophozoite stage., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Malaria parasites cause disease through repeated cycles of intraerythrocytic proliferation. Within each cycle, several rounds of DNA replication produce multinucleated forms, called schizonts, that undergo segmentation to form daughter merozoites. Upon rupture of the infected cell, the merozoites egress to invade new erythrocytes and repeat the cycle. In human malarial infections, an antibody response specific for the Plasmodium falciparum protein PF3D7_1021800 was previously associated with protection against malaria, leading to an interest in PF3D7_1021800 as a candidate vaccine antigen. Antibodies to the protein were reported to inhibit egress, resulting in it being named schizont egress antigen-1 (SEA1). A separate study found that SEA1 undergoes phosphorylation in a manner dependent upon the parasite cGMP-dependent protein kinase PKG, which triggers egress. While these findings imply a role for SEA1 in merozoite egress, this protein has also been implicated in kinetochore function during schizont development. Therefore, the function of SEA1 remains unclear. Here, we show that P. falciparum SEA1 localizes in proximity to centromeres within dividing nuclei and that conditional disruption of SEA1 expression severely impacts the distribution of DNA and formation of merozoites during schizont development, with a proportion of SEA1-null merozoites completely lacking nuclei. SEA1-null schizonts rupture, albeit with low efficiency, suggesting that neither SEA1 function nor normal segmentation is a prerequisite for egress. We conclude that SEA1 does not play a direct mechanistic role in egress but instead acts upstream of egress as an essential regulator required to ensure the correct packaging of nuclei within merozoites. IMPORTANCE Malaria is a deadly infectious disease. Rationally designed novel therapeutics will be essential for its control and eradication. The Plasmodium falciparum protein PF3D7_1021800, annotated as SEA1, is under investigation as a prospective component of a malaria vaccine, based on previous indications that antibodies to SEA1 interfere with parasite egress from infected erythrocytes. However, a consensus on the function of SEA1 is lacking. Here, we demonstrate that SEA1 localizes to dividing parasite nuclei and is necessary for the correct segregation of replicated DNA into individual daughter merozoites. In the absence of SEA1, merozoites develop defectively, often completely lacking a nucleus, and, consequently, egress is impaired and/or aberrant. Our findings provide insights into the divergent mechanisms by which intraerythrocytic malaria parasites develop and divide. Our conclusions regarding the localization and function of SEA1 are not consistent with the hypothesis that antibodies against it confer protective immunity to malaria by blocking merozoite egress., (Copyright © 2021 Perrin et al.)

The eukaryotic cell cycle is typically divided into distinct phases with cytokinesis immediately following mitosis. To ensure proper cell division, each phase is tightly coordinated via feedback controls named checkpoints. During its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The multi-nucleated schizont is then subjected to a single round of cytokinesis that produces dozens of daughter cells called merozoites. To date, no cell cycle checkpoints have been identified that regulate the Plasmodium spp. mode of division. Here, we identify the Plasmodium homologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP), which co-purified with the Mini-Chromosome Maintenance (MCM) complex, a replicative helicase required for genomic DNA replication. By conditionally depleting PfMCMBP, we disrupt nuclear morphology and parasite proliferation without causing a block in DNA replication. By immunofluorescence microscopy, we show that PfMCMBP depletion promotes the formation of mitotic spindle microtubules with extensions to more than one DNA focus and abnormal centrin distribution. Strikingly, PfMCMBP-deficient parasites complete cytokinesis and form aneuploid merozoites with variable cellular and nuclear sizes. Our study demonstrates that the parasite lacks a robust checkpoint response to prevent cytokinesis following aberrant karyokinesis., (© 2020 John Wiley & Sons Ltd.)

The progression to schizont formation of individual activated hypnozoites has been observed in vitro for the first time by Voorberg-van der Wel et al. Green-fluorescent protein-positive hypnozoites turned red-fluorescent (mCherry) upon activation. Thus, we now have empirical parasitological proof that supports the 40-year-old hypnozoite theory of relapse in malaria., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis., Methods: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed., Results: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca 2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation., Conclusions: Serum proteins were taken up by parasites under culture conditions with sufficient Ca 2+ levels. This uptake phenomenon was associated with their pathogenicity.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recent concepts suggest that both Plasmodium falciparum factors and coagulation contribute to endothelial activation and dysfunction in pediatric cerebral malaria (CM) pathology. However, there is still limited understanding of how these complex inflammatory stimuli are integrated by brain endothelial cells. In this study, we examined how mature-stage P. falciparum infected erythrocytes (IE) interact with tumor necrosis factor α (TNFα) and thrombin in the activation and permeability of primary human brain microvascular endothelial cell (HBMEC) monolayers. Whereas trophozoite-stage P. falciparum-IE have limited effect on the viability of HBMEC or the secretion of pro-inflammatory cytokines or chemokines, except at super physiological parasite-host cell ratios, schizont-stage P. falciparum-IE induced low levels of cell death. Additionally, schizont-stage parasites were more barrier disruptive than trophozoite-stage P. falciparum-IE and prolonged thrombin-induced barrier disruption in both resting and TNFα-activated HBMEC monolayers. These results provide evidence that parasite products and thrombin may interact to increase brain endothelial permeability.

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.

Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1-deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Background: Evaluation of local Plasmodium falciparum malaria transmission has been investigated previously using the reversible catalytic model based on prevalence of antibody responses to single antigen to estimate seroconversion rates. High correlations were observed between seroconversion rates and entomological inoculation rates (EIR). However, in this model, the effects of malaria control interventions and clinical episodes on serological measurements were not assessed. This study monitors the use of antibody responses to P. falciparum crude extracts for assessing malaria transmission, compares seroconversion rates estimated from longitudinal data to those derived from cross-sectional surveys and investigates the effects of malaria control interventions on these measures in an area of declining malaria transmission. In addition, the validity of this model was evaluated by comparison with the alternative model., Methods: Five cross-sectional surveys were carried out at the end of the wet season in Dielmo, a malaria-endemic Senegalese rural area in 2000, 2002, 2008, 2010 and 2012. Antibodies against schizonts crude extract of a local P. falciparum strain adapted to culture (Pf 07/03) were measured by ELISA. Age-specific seroprevalence model was used both for cross-sectional surveys and longitudinal data (combined data of all surveys)., Results: A total of 1504 plasma samples obtained through several years follow-up of 350 subjects was used in this study. Seroconversion rates based on P. falciparum schizonts crude extract were estimated for each cross-sectional survey and were found strongly correlated with EIR. High variability between SCRs from cross-sectional and longitudinal surveys was observed. In longitudinal studies, the alternative catalytic reversible model adjusted better with serological data than the catalytic model. Clinical malaria attacks and malaria control interventions were found to have significant effect on seroconversion., Discussion: The results of the study suggested that crude extract was a good serological tool that could be used to assess the level of malaria exposure in areas where malaria transmission is declining. However, additional parameters such as clinical malaria and malaria control interventions must be taken into account for determining serological measurements for more accuracy in transmission assessment.

Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs., Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels., Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.

Background: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility., Methods: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector ® electroporation technology., Results: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherry Hsp70 -expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination., Conclusion: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.

Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection 1 . DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly 2,3 . However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

Palmitoylation is the post-translational reversible addition of the acyl moiety, palmitate, to cysteine residues of proteins and is involved in regulating protein trafficking, localization, stability and function. The Aspartate-Histidine-Histidine-Cysteine (DHHC) protein family, named for their highly conserved DHHC signature motif, is thought to be responsible for catalysing protein palmitoylation. Palmitoylation is widespread in all eukaryotes, including the malaria parasite, Plasmodium falciparum, where over 400 palmitoylated proteins are present in the asexual intraerythrocytic schizont stage parasites, including proteins involved in key aspects of parasite maturation and development. The P. falciparum genome includes 12 proteins containing the conserved DHHC motif. In this study, we adapted a palmitoyl-transferase activity assay for use with P. falciparum proteins and demonstrated for the first time that P. falciparum DHHC proteins are responsible for the palmitoylation of P. falciparum substrates. This assay also reveals that multiple DHHCs are capable of palmitoylating the same substrate, indicating functional redundancy at least in vitro. To test whether functional redundancy also exists in vivo, we investigated the endogenous localization and essentiality of a subset of schizont-expressed PfDHHC proteins. Individual PfDHHC proteins localized to distinct organelles, including parasite-specific organelles such as the rhoptries and inner membrane complex. Knock-out studies identified individual DHHCs that may be essential for blood-stage growth and others that were functionally redundant in the blood stages but may have functions in other stages of parasite development. Supporting this hypothesis, disruption of PfDHHC9 had no effect on blood-stage growth but reduced the formation of gametocytes, suggesting that this protein could be exploited as a transmission-blocking target. The localization and stage-specific expression of the DHHC proteins may be important for regulating their substrate specificity and thus may provide a path for inhibitor development., (© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry., (© 2016 International Society for Advancement of Cytometry.)

The intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division., (© 2016. Published by The Company of Biologists Ltd.)

This study describes the effect of increasing exposure dose on Ichthyophonus prevalence and infection intensity in experimentally infected rainbow trout, Oncorhynchus mykiss. Specific-pathogen free trout were exposed per os to increasing numbers of Ichthyophonus schizonts obtained from naturally infected donor fish, then sampled after 30 and 60 days post-exposure. Both in vitro explant culture and histology revealed that as the number of schizonts per dose increased there was a proportionate increase in the number of infected fish, as well as an increase in the number of infected organs; parasite density in individual infected organs also increased with dose. Explant culture revealed that all fish exposed to the highest dose (≥2,080 schizonts) became infected, while only 67% of those exposed to the intermediate dose (1,040-1,153 schizonts) were Ichthyophonus-positive after 60 days; Ichthyophonus was not detected in fish exposed to the 2 lowest doses (≤280 schizonts). Histologic examination of individual infected organs also revealed increasing infection prevalence and parasite density in response to exposure to increasing numbers of Ichthyophonus schizonts.

Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing., (© 2015 John Wiley & Sons Ltd.)

Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion.

There is now a growing body of evidence that challenges the current view that Plasmodium vivax-infected erythrocyte (Pv-iE) are unable to sequester. Here we used ex vivo adhesion assays with Pv-iE before and after maturation to demonstrate a higher binding potential of schizonts compared to other asexual stages. These experimental results are correlated with our observations in a panel of 50 vivax malaria patients where schizonts were completely absent in 27 isolates, and few schizonts were observed in the remaining patients. These observations prompt a paradigm shift in P. vivax biology and open avenues to investigate the role of Pv-iE sequestration.

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

Theileria annulata, the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. In vivo, parasitized cells undergo clonal expansion and infiltrate both the lymphoid and non-lymphoid tissues of the infected host. To determine whether the small ruminants and their red blood cells (RBCs) were invaded by T. annulata schizonts or not, T. annulata schizonts were used to infect bovine, ovine and caprine RBCs in vitro, and sheep and goats in vivo. The results showed that the schizonts infected bovine, ovine and caprine RBCs in vitro, but not sheep and goats, which showed only an increase in body temperature and no development of piroplasms. To our knowledge, this is the first report of infection of small ruminants and their RBCs by T. annulata schizonts., (Copyright © 2014. Published by Elsevier Inc.)

Low reticulocytosis, indicating reduced red blood cell (RBC) output, is an important feature of severe malarial anemia. Evidence supports a role for Plasmodium products, especially hemozoin (Hz), in suppressed erythropoiesis during malaria, but the mechanism(s) involved remains unclear. Here, we demonstrated that low reticulocytosis and suppressed erythropoietin (Epo)-induced erythropoiesis are features of malarial anemia in Plasmodium yoelii- and Plasmodium berghei ANKA-infected mice, similar to our previous observations in Plasmodium chabaudi AS-infected mice. The magnitude of decreases in RBC was a reflection of parasitemia level, but low reticulocytosis was evident despite differences in parasitemia, clinical manifestation, and infection outcome. Schizont extracts and Hz from P. falciparum and P. yoelii and synthetic Hz suppressed Epo-induced proliferation of erythroid precursors in vitro but did not inhibit RBC maturation. To determine whether Hz contributes to malarial anemia, P. yoelii-derived or synthetic Hz was administered to naive mice, and the development of anemia, reticulocytosis, and RBC turnover was determined. Parasite-derived Hz induced significant decreases in RBC and increased RBC turnover with compensatory reticulocytosis, but anemia was not as severe as that in infected mice. Our findings suggest that parasite factors, including Hz, contribute to severe malarial anemia by suppressing Epo-induced proliferation of erythroid precursors.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Background and Purpose: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria., Experimental Approach: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed., Key Results: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts., Conclusions and Implications: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria., (© 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.)

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

This study investigated the occurrence of the protozoan Perkinsus in the oyster Crassostrea rhizophorae on the coast of Bahia State, Brazil. The oysters (n = 900) were collected in February-March and July-August 2010. The Ray's fluid thioglycollate medium (RFTM) analysis of gills and rectum revealed hypnospores of Perkinsus sp. with a high mean prevalence (63%). The infection intensity varied from very light to advanced. The polymerase chain reaction confirmed Perkinsus in 87.2% of the RFTM-positive oysters. Histological analysis showed trophozoites and schizonts phagocytized by hemocytes, mainly in the intestine and the stomach epithelium., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Eimeria tenella is recognized worldwide as a significant pathogen in the poultry industry. However, a lack of methods for isolating developing schizonts has hindered the use of transcriptome analyses to discover novel and developmentally regulated genes. In the present study, we characterized the long-term successive development of E. tenella in infected chicken caeca and assessed the utility of laser microdissection (LMD) for the isolation of schizont RNA. Developmental stages, including those of the first, second, and third-generation schizonts and gametocytes, were synchronous. Using LMD, only the mature second-generation schizonts were successfully excised from the lamina propria, and non-degraded RNA was purified from the schizonts. E. tenella-specific genes were amplified by reverse transcription polymerase chain reaction (RT-PCR). These results augment our understanding of the E. tenella life cycle, and reveal LMD as a potentially useful tool for gene expression analyses of the intracellular stages of E. tenella.

It is becoming clear that, as is the case with many human diseases, targeting protein phosphorylation in strategies aimed at developing the next generation of anti-malarials is likely to bear considerable fruit. A major barrier to this development, however, is the paucity of information regarding the role of protein phosphorylation in malaria. A major step has recently been taken in this area with the publication of the first analyses of the phospho-proteome of the most virulent species of human malaria Plasmodium falciparum. Here, we discuss these studies., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Cytoadherence of infected red blood cells to brain endothelium is causally implicated in malarial coma, one of the severe manifestations of falciparum malaria. Cytoadherence is mediated by specific binding of variant parasite antigens, expressed on the surface of infected erythrocytes, to endothelial receptors including, ICAM-1, VCAM and CD36. In fatal cases of severe falciparum malaria with coma, blood vessels in the brain are characteristically congested with infected erythrocytes. Brain sections from a fatal case of knowlesi malaria, but without coma, were similarly congested with infected erythrocytes. The objective of this study was to determine the binding phenotype of Plasmodium knowlesi infected human erythrocytes to recombinant human ICAM-1, VCAM and CD36., Methods: Five patients with PCR-confirmed P. knowlesi malaria were recruited into the study with consent between April and August 2010. Pre-treatment venous blood was washed and cultured ex vivo to increase the proportion of schizont-infected erythrocytes. Cultured blood was seeded into Petri dishes with triplicate areas coated with ICAM-1, VCAM and CD36. Following incubation at 37°C for one hour the dishes were washed and the number of infected erythrocytes bound/mm2 to PBS control areas and to recombinant human ICAM-1 VCAM and CD36 coated areas were recorded. Each assay was performed in duplicate. Assay performance was monitored with the Plasmodium falciparum clone HB3., Results: Blood samples were cultured ex vivo for up to 14.5 h (mean 11.3 ± 1.9 h) to increase the relative proportion of mature trophozoite and schizont-infected red blood cells to at least 50% (mean 65.8 ± 17.51%). Three (60%) isolates bound significantly to ICAM-1 and VCAM, one (20%) isolate bound to VCAM and none of the five bound significantly to CD36., Conclusions: Plasmodium knowlesi infected erythrocytes from human subjects bind in a specific but variable manner to the inducible endothelial receptors ICAM-1 and VCAM. Binding to the constitutively-expressed endothelial receptor CD36 was not detected. Further work will be required to define the pathological consequences of these interactions.

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.

The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.

Background: Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of Plasmodium falciparum. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a head-to-head comparison of the in vitro invasive capabilities of parasites isolated by either of the two methods., Methods: The P. falciparum laboratory strain isolate 7G8 was grown in vitro using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes., Results: In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation (95% CI, p = 0.00067) A paired t test with two tails was used for these comparisons., Conclusions: In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation.

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Intracellular leukoproliferative Theileria are unique as eukaryotic organisms that transform the immune cells of their ruminant host. Theileria utilize the uncontrolled proliferation for rapid multiplication and distribution into host daughter cells. The parasite distribution into the daughter cells is accompanied by a tight association with the host cell mitotic apparatus. Since the molecular basis for this interaction is largely unknown, we investigated the possible involvement of the immunodominant Theileria annulata surface protein, TaSP, in the attachment of the parasite to host cell microtubule network. Confocal microscopic analyses showed co-localization of the TaSP protein with alpha-tubulin and reciprocal immuno-co-precipitation experiments demonstrated an association of TaSP with alpha-tubulin in vivo. In addition, the partially expressed predicted extracellular domain of TaSP co-localized with the mitotic spindle of dividing cells and was co-immunoprecipitated with alpha-tubulin in transiently transfected Cos-7 cells devoid of other T. annulata expressed proteins. Pull-down studies showed that there is a direct interaction between TaSP and polymerized microtubules. Analysis of the interaction of TaSP and host microtubulin during host cell mitosis indicated that TaSP co-localizes and interacts with the spindle poles, the mitotic spindle apparatus and the mid-body. Moreover, TaSP was demonstrated to be localized to the microtubule organizing center and to physically interact with gamma-tubulin. These data support the notion that the TaSP-microtubule interaction may be playing a potential role in parasite distribution into daughter host cells and give rise to the speculation that TaSP may be involved in regulation of microtubule assembly in the host cell.

Background: Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary., Methods: A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from Plasmodium falciparum cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates., Results: In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures., Conclusion: The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either small interfering RNA-mediated suppression or genetic deletion of calpain activity and could be restored by genetic complementation.

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.

Measurement of parasite proliferation in cultured red blood cells underpins many facets of malaria research, from drug sensitivity assays to assessing the impact of experimentally altered genes on parasite growth, virulence and fitness. Pioneering efforts to grow Plasmodium falciparum in cultured red blood cells revolutionised malaria research and spurred the development of semi-high-throughput growth assays using radio-labelled hypoxanthine (Hx), an essential nucleic acid precursor, as a reporter of whole-cycle proliferation [Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673-675; Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710-718]. The isotopic Hx assay remains the standard quantitative growth assay with which newer non-radioactive procedures based on fluorescent DNA dyes or ELISA are compared. All of these readouts are surrogate reporters of changes in bulk parasitemias, reflecting proliferation over entire asexual reproductive cycles. While quantitatively robust and amenable to semi-high-throughput applications, these methods are blind to the underlying developmental and cellular events of growth in human red blood cells. Modern whole-genome tools including gene knockouts, mutagenesis and small molecule screens promise to reveal much about basic parasite biology; however methods to precisely quantify the within-cycle growth process are needed. Here we elaborate on the classical growth index, i.e. changes in parasitemia, by quantifying sub-phenotypes of a rapid proliferator, the multi-drug resistant clone Dd2, and a standard wild-type clone, HB3. These data illustrate differences in cycle duration, merozoite production, and invasion rate and efficiency that underpin Dd2's average 2-fold proliferation advantage over HB3 per erythrocytic cycle. The ability to refine growth phenotypes will inform the search for molecular determinants of differential parasite growth rates and broaden our understanding of killing mechanisms and cellular targets of antimalarial drugs.

Background: Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release., Method and Results: To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint, composed of 1) separated parasites, 2) a digestive vacuole with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37 degrees C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release., Conclusion: In this study, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion.