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Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

ABSTRACT: Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a single-step Percoll gradient. Importantly, we demonstrate that the enriched ring-stage parasites develop synchronously regardless of the parasite strain or species used. Using a flow cytometry assay with Hoechst and ethidium or MitoTracker dye, we show that parasite development is easily and rapidly monitored. Finally, we demonstrate that this approach can be used to screen antimalarial drugs.

SUBMITTER: Chang ZW

PROVIDER: S-EPMC5075096 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

Chang Zi Wei ZW Malleret Benoit B Russell Bruce B Rénia Laurent L Claser Carla C

Antimicrobial agents and chemotherapy 20161021 11

Ex vivo assay systems provide a powerful approach to studying human malaria parasite biology and to testing antimalarials. For rodent malaria parasites, short-term in vitro culture and ex vivo antimalarial susceptibility assays are relatively cumbersome, relying on in vivo passage for synchronization, since ring-stage parasites are an essential starting material. Here, we describe a new approach based on the enrichment of ring-stage Plasmodium berghei, P. yoelii, and P. vinckei vinckei using a s ...[more]

PMID: 27600050

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Project description:BackgroundMalaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay.MethodsExperiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion.ResultsThe staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice.ConclusionsThe invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.

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Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

Publications

Ex Vivo Maturation Assay for Testing Antimalarial Sensitivity of Rodent Malaria Parasites.

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