title: Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.
abstract: The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.
authors.affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre (RUNMC), Nijmegen, The Netherlands.
authors.forename: Ivo H J
authors.initials: IH
authors.lastname: Ploemen
authors.forename: Miguel
authors.initials: M
authors.lastname: Prudêncio
authors.forename: Bruno G
authors.initials: BG
authors.lastname: Douradinha
authors.forename: Jai
authors.initials: J
authors.lastname: Ramesar
authors.forename: Jannik
authors.initials: J
authors.lastname: Fonager
authors.forename: Geert-Jan
authors.initials: GJ
authors.lastname: van Gemert
authors.forename: Adrian J F
authors.initials: AJ
authors.lastname: Luty
authors.forename: Cornelus C
authors.initials: CC
authors.lastname: Hermsen
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sauerwein
authors.forename: Fernanda G
authors.initials: FG
authors.lastname: Baptista
authors.forename: Maria M
authors.initials: MM
authors.lastname: Mota
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Waters
authors.forename: Ivo
authors.initials: I
authors.lastname: Que
authors.forename: Clemens W G M
authors.initials: CW
authors.lastname: Lowik
authors.forename: Shahid M
authors.initials: SM
authors.lastname: Khan
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Janse
authors.forename: Blandine M D
authors.initials: BM
authors.lastname: Franke-Fayard
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D003952:Diagnostic Imaging / Q000379:methods; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000469:parasitology; D006801:Humans; D008099:Liver / Q000469:parasitology*; D049449:Luminescence; D008288:Malaria / Q000469:parasitology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010962:Plasmodium berghei / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D034101:Sporozoites / Q000378:metabolism
chemical_list: D049452:Green Fluorescent Proteins