A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

dc.contributor.authorLelliott, Patrick M
dc.contributor.authorLampkin, Shelley
dc.contributor.authorMcMorran, Brendan J
dc.contributor.authorFoote, Simon J
dc.contributor.authorBurgio, Gaetan
dc.date.accessioned2016-01-20T00:38:53Z
dc.date.available2016-01-20T00:38:53Z
dc.date.issued2014-03-17
dc.date.updated2016-02-24T08:07:48Z
dc.description.abstractBACKGROUND 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.
dc.description.sponsorshipWe acknowledge funding support from the National Health and Medical Research Council (grant APP605524, 490037 and 1047082), the Australian Research Council (grant DP12010061), the National Collaborative Research Infrastructure Strategy of Australia and the Education investment fund from the Department of Innovation, Industry, Science and Research. PML is a recipient of an Australian Postgraduate award.en_AU
dc.identifier.issn1475-2875en_AU
dc.identifier.urihttp://hdl.handle.net/1885/95525
dc.publisherBioMed Central
dc.relationhttp://purl.org/au-research/grants/nhmrc/605524
dc.relationhttp://purl.org/au-research/grants/nhmrc/490037
dc.relationhttp://purl.org/au-research/grants/nhmrc/1047082
dc.relationhttp://purl.org/au-research/grants/arc/DP12010061
dc.rights© 2014 Lelliott et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
dc.sourceMalaria Journal
dc.subjectanimals
dc.subjecterythrocytes
dc.subjectflow cytometry
dc.subjectmalaria
dc.subjectmice
dc.subjectmice, inbred c57bl
dc.subjectparasitology
dc.subjectplasmodium chabaudi
dc.subjectrodent diseases
dc.titleA flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo
dc.typeJournal article
local.bibliographicCitation.issue1en_AU
local.bibliographicCitation.lastpage9
local.bibliographicCitation.startpage100en_AU
local.contributor.affiliationLelliott, Patrick, Macquarie University, Australiaen_AU
local.contributor.affiliationLampkin, Shelley, Macquarie University, Australiaen_AU
local.contributor.affiliationMcMorran, Brendan, College of Medicine, Biology and Environment, CMBE John Curtin School of Medical Research, JCSMR General, The Australian National Universityen_AU
local.contributor.affiliationFoote, Simon, College of Medicine, Biology and Environment, CMBE John Curtin School of Medical Research, Emerging Pathogens and Vaccines, The Australian National Universityen_AU
local.contributor.affiliationBurgio, Gaetan, College of Medicine, Biology and Environment, CMBE John Curtin School of Medical Research, Immunology and Infectious Disease, The Australian National Universityen_AU
local.contributor.authoremailbrendan.mcmorran@anu.edu.auen_AU
local.contributor.authoruidu5267721en_AU
local.description.notesImported from ARIESen_AU
local.identifier.absfor060499en_AU
local.identifier.absfor110803en_AU
local.identifier.absfor111203en_AU
local.identifier.ariespublicationa383154xPUB600en_AU
local.identifier.citationvolume13en_AU
local.identifier.doi10.1186/1475-2875-13-100en_AU
local.identifier.essn1475-2875en_AU
local.identifier.scopusID2-s2.0-84899111276
local.identifier.uidSubmittedByu3488905en_AU
local.publisher.urlhttp://www.biomedcentral.com/en_AU
local.type.statusPublished Versionen_AU

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