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-Luccon, 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.

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development

“…Pb infection of Huh7 cells after down-modulation of ApoH expression was then compared with that of control cells, transfected with scrambled siRNA (Neg siRNA). Initially, cells were infected with either luciferaseexpressing or wild-type (WT) Pb sporozoites, and infection was measured by bioluminescence (44) or qRT-PCR, respectively ( Fig. 2 A and B).…”

Advances in Antimalarial Drug Evaluation and New Targets for Antimalarials

“…A high-throughput in vitro screening of drug activity on Plasmodium liver stages was developed based on a sophisticated infrared fluorescence scanning system, which allows rapid, automatic counting of infected hepatocytes (Gego et al, 2006). The recent development of bioluminescent parasites allows now a non-invasive real time monitoring and quantitative analysis of liver stage development in vitro and in vivo in rodents that offers new tools for drug evaluation (Mwakingwe et al, 2009, Ploemen et al, 2009) (see section 4.3.2). Automated visual assay was also set up to follow the extracellular cell death of sporozoites (Hegge et al, 2010).…”

“…A technique such as real time PCR allows quantification of the parasite charge in liver and thus, the inhibitory effect of the molecule tested (Carraz et al, 2006). Recently, the use of a transgenic P. berghei parasite expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells in live mice using real-time luminescence imaging was reported (Ploemen et al, 2009). The applicability of real time imaging to assess parasite drug sensitivity in the liver was demonstrated by analysing the effect of primaquine and tafenoquine in vivo.…”

Towards genome-wide experimental genetics in thein vivomalaria model parasitePlasmodium berghei

“…The introduction of a number of bioluminescence-based methods enables faster and less subjective quantification of the P. berghei burden when compared with traditional microscopy-based approaches. [123][124][125][126] These methods, however, do not permit the comparison of multiple mutants simultaneously. A flow cytometry-based in vivo competition assay has been developed that enables the analysis of blood-stage development of three differently coloured fluorescent parasite lines within a single mouse.…”