Abstract
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.
Highlights
Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites
A novel flow cytometric method to detect parasitized red blood cell (RBC) in in vivo samples In preliminary studies using uninfected mice, a flow cytometric cell staining and analysis protocol was tested that took advantage of the DNA-specific dye, Hoechst 33342, which distinguishes DNA-containing blood cells [33], in conjunction with fluorescently-labelled antibodies raised against the leukocyte common antigen (CD45) and a red cell progenitor marker (CD71) to exclude leukocytes and non-parasitized nucleated blood cells
HJ-RBCs were observed at similar frequencies in both SJL/J and C57BL/6 strains of mice, and were detected when other nucleic acid-specific dyes, including SYTO-16, Dihydroethidium, and Thiazole Orange were used (Additional file 1)
Summary
Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. The symptomatic stage of infection occurs when the merozoite form of the Plasmodium parasite invades circulating red blood cells (RBCs), and undergoes development and replication. Interfering with merozoite invasion is regarded a potentially useful and novel anti-malarial approach, and in vitro cultured parasites [5,8,17,18,19], and there is a need for methods to test and translate these findings in vivo. This is of particular importance when considering the interaction between the host’s immune system and invading merozoite. The development of vaccines against merozoite antigens, or other invasion blocking therapies, may, benefit from an in vivo invasion assessment, which would account for the role of the immune system in this interaction
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