Abstract
Background: Malaria 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. Methods. Experiments 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. Results: The 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. Conclusions: The 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.
Original language | English |
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Article number | 100 |
Pages (from-to) | 1-9 |
Number of pages | 9 |
Journal | Malaria Journal |
Volume | 13 |
Issue number | 1 |
DOIs | |
Publication status | Published - 17 Mar 2014 |
Bibliographical note
This version is archived for private and non-commercial use under the terms of this BioMed Central open access license ("license") (see http://www.biomedcentral.com/about/license). The work is protected by copyright and/or other applicable law. Any use of the work other than as authorized under this license is prohibited. For further rights please check the terms of the license, or contact the publisher.Keywords
- Flow cytometry
- In vivo. JC-1
- Invasion
- Malaria
- Merozoite
- Parasitaemia
- Plasmodium berghei
- Plasmodium chabaudi