Abstract
BackgroundIntracellular 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 resultsTo 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°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.ConclusionIn 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.
Highlights
Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication
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
Assay description The unexpected finding of preserved "sites of parasite release" from ruptured infected erythrocytes in sealed chambers filled with synchronized cultures of P. falciparum undergoing cycle transition [2] is the basis of this method
Summary
Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. The asexual erythrocyte cycle of the human malaria parasite Plasmodium falciparum causes severe forms of disease [1]. Invasion of an individual parasite into a red blood cell initiates the cycle; approximately 48 hours later release of. 16 – 32 daughter parasites terminates the cycle to spread the infection. These two space-time coupled events, parasite release and invasion, termed cycle transition, is the shortest stage of the plasmodial erythrocyte cycle. An increased proportion of the ring stage-infected erythrocytes with higher parasitaemia in synchronized cultures indicate the end of the previous cycle and the beginning of the new one. The quantification of parasite release without any contribution of parasite invasion is the prerequisite for studying parasite release from host cells, a largely unexplored aspect of parasite biology
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