Malaria pp 353-368 | Cite as

Bioluminescence Imaging of P. berghei Schizont Sequestration in Rodents

  • Joanna Braks
  • Elena Aime
  • Roberta Spaccapelo
  • Onny Klop
  • Chris J. JanseEmail author
  • Blandine Franke-Fayard
Part of the Methods in Molecular Biology book series (MIMB, volume 923)


We describe a technology for imaging the sequestration of infected red blood cells (iRBC) of the rodent malaria parasite Plasmodium berghei both in the bodies of live mice and in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of sequestered iRBC is performed by measuring bioluminescence produced by the enzymatic reaction in parasites between the luciferase enzyme and its substrate luciferin injected into the mice several minutes prior to imaging. The bioluminescence signal is detected by a sensitive I-CCD photon-counting video camera. Using a reporter parasite that expresses luciferase under the control of a schizont-specific promoter (i.e., the ama-1 promoter), the schizont stage is made visible when detecting bioluminescence signals. Schizont sequestration is imaged during short-term infections with parasites that are synchronized in development or during ongoing infections. Real-time in vivo imaging of iRBC will provide increased insights into the dynamics of sequestration and its role in pathology, and can be used to evaluate strategies that prevent sequestration.

Key words

Malaria Plasmodium berghei Schizonts Sequestration Adherence In vivo imaging Luminescence Luciferase Luciferin 



This work was supported by The Netherlands Organization for Scientific Research (ZonMw TOP grant number 9120_6135) and the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 201222.


  1. 1.
    Ho M, White NJ (1999) Molecular mechanisms of cytoadherence in malaria. Am J Physiol 276:C1231–C1242PubMedGoogle Scholar
  2. 2.
    Sherman IW et al (2003) Cytoadherence and sequestration in Plasmodium falciparum: defining the ties that bind. Microbes Infect 5:897–909PubMedCrossRefGoogle Scholar
  3. 3.
    Rogerson SJ et al (2007) Malaria in pregnancy: pathogenesis and immunity. Lancet Infect Dis 7:105–117PubMedCrossRefGoogle Scholar
  4. 4.
    Desai M et al (2007) Epidemiology and burden of malaria in pregnancy. Lancet Infect Dis 7:93–104PubMedCrossRefGoogle Scholar
  5. 5.
    Beeson JG, Duffy PE (2005) The immunology and pathogenesis of malaria during pregnancy. Curr Top Microbiol Immunol 297:187–227PubMedCrossRefGoogle Scholar
  6. 6.
    Mackintosh CL et al (2004) Clinical features and pathogenesis of severe malaria. Trends Parasitol 20:597–603PubMedCrossRefGoogle Scholar
  7. 7.
    Rasti N et al (2004) Molecular aspects of malaria pathogenesis. FEMS Immunol Med Microbiol 41:9–26PubMedCrossRefGoogle Scholar
  8. 8.
    Clark IA et al (2004) Pathogenesis of malaria and clinically similar conditions. Clin Microbiol Rev 17:509–539PubMedCrossRefGoogle Scholar
  9. 9.
    van der Heyde HC et al (2006) A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction. Trends Parasitol 22:503–508PubMedCrossRefGoogle Scholar
  10. 10.
    Miller LH et al (2002) The pathogenic basis of malaria. Nature 415:673–679PubMedCrossRefGoogle Scholar
  11. 11.
    Idro R et al (2005) Pathogenesis, clinical features, and neurological outcome of cerebral malaria. Lancet Neurol 4:827–840PubMedCrossRefGoogle Scholar
  12. 12.
    Schofield L, Grau GE (2005) Immunological processes in malaria pathogenesis. Nat Rev Immunol 5:722–735PubMedCrossRefGoogle Scholar
  13. 13.
    Mishra SK, Newton CR (2009) Diagnosis and management of the neurological complications of falciparum malaria. Nat Rev Neurol 5:189–198PubMedCrossRefGoogle Scholar
  14. 14.
    Franke-Fayard B et al (2005) Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration. Proc Natl Acad Sci USA 102:11468–11473PubMedCrossRefGoogle Scholar
  15. 15.
    Mons B et al (1985) Synchronized erythrocytic schizogony and gametocytogenesis of Plasmodium berghei in vivo and in vitro. Parasitology 91:423–430PubMedCrossRefGoogle Scholar
  16. 16.
    Janse CJ, Waters AP (1995) Plasmodium berghei: the application of cultivation and purification techniques to molecular studies of malaria parasites. Parasitol Today 11:138–143PubMedCrossRefGoogle Scholar
  17. 17.
    Spaccapelo R et al (2010) Plasmepsin 4-deficient Plasmodium berghei are virulence attenuated and induce protective immunity against experimental malaria. Am J Pathol 176:205–217PubMedCrossRefGoogle Scholar
  18. 18.
    Franke-Fayard B et al (2006) Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice. Nat Protoc 1:476–485PubMedCrossRefGoogle Scholar
  19. 19.
    Amante FH et al (2007) A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria. Am J Pathol 171:548–559PubMedCrossRefGoogle Scholar
  20. 20.
    Hearn J et al (2000) Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature. Infect Immun 68:5364–5376PubMedCrossRefGoogle Scholar
  21. 21.
    Nie CQ et al (2009) IP-10-mediated T cell homing promotes cerebral inflammation over splenic immunity to malaria infection. PLoS Pathog 5:e1000369PubMedCrossRefGoogle Scholar
  22. 22.
    Neres R et al (2008) Pregnancy outcome and placenta pathology in Plasmodium berghei ANKA infected mice reproduce the pathogenesis of severe malaria in pregnant women. PLoS One 3:e1608PubMedCrossRefGoogle Scholar
  23. 23.
    Amante FH et al (2010) Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria. J Immunol 185:3632–3642PubMedCrossRefGoogle Scholar
  24. 24.
    Avril M et al (2010) Immunization with VAR2CSA-DBL5 recombinant protein elicits broadly cross-reactive antibodies to placental Plasmodium falciparum-infected erythrocytes. Infect Immun 78:2248–2256PubMedCrossRefGoogle Scholar
  25. 25.
    Rowe JA et al (2009) Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications. Expert Rev Mol Med 11:e16PubMedCrossRefGoogle Scholar
  26. 26.
    Franke-Fayard B et al (2010) Sequestration and tissue accumulation of human malaria parasites: can we learn anything from rodent models of malaria? PLoS Pathog 6:e1001032PubMedCrossRefGoogle Scholar
  27. 27.
    Engwerda CR et al (2005) The importance of the spleen in malaria. Trends Parasitol 21:75–80PubMedCrossRefGoogle Scholar
  28. 28.
    Claser C et al (2011) CD8+ T cells and IFN-gamma mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria. PLoS One 6:e18720PubMedCrossRefGoogle Scholar
  29. 29.
    Haque A et al (2010) CD4+ natural regulatory T cells prevent experimental cerebral malaria via CTLA-4 when expanded in vivo. PLoS Pathog 6:e1001221PubMedCrossRefGoogle Scholar
  30. 30.
    Janse CJ et al (2006) High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol 145:60–70PubMedCrossRefGoogle Scholar
  31. 31.
    Kirchgatter K, Del Portillo HA (2005) Clinical and molecular aspects of severe malaria. An Acad Bras Cienc 77:455–475PubMedCrossRefGoogle Scholar
  32. 32.
    Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124:755–766PubMedCrossRefGoogle Scholar
  33. 33.
    Gilks CF et al (1989) Host diet in experimental rodent malaria: a variable which can compromise experimental design and interpretation. Parasitology 98:175–177PubMedCrossRefGoogle Scholar
  34. 34.
    Sadikot RT, Blackwell TS (2005) Bioluminescence imaging. Proc Am Thorac Soc 2:537–542PubMedCrossRefGoogle Scholar
  35. 35.
    Welsh DK, Kay SA (2005) Bioluminescence imaging in living organisms. Curr Opin Biotechnol 16:73–78PubMedCrossRefGoogle Scholar
  36. 36.
    Ntziachristos V et al (2005) Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 23:313–320PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Joanna Braks
    • 1
  • Elena Aime
    • 2
  • Roberta Spaccapelo
    • 2
  • Onny Klop
    • 1
  • Chris J. Janse
    • 1
    Email author
  • Blandine Franke-Fayard
    • 1
  1. 1.Center of Infectious Diseases, Leiden University Medical CenterLeidenThe Netherlands
  2. 2.Department of Experimental MedicineUniversity of PerugiaPerugiaItaly

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