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Background  Genetic engineering with luciferase reporter genes allows monitoring  Trypanosoma brucei  ( T.b. ) infections in mice by  in vivo  bioluminescence imaging (BLI). Until recently, luminescent  T.b.  models were based on  Renilla  luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for  in vivo  BLI in a set of diverse  T.b.  strains of all three subspecies, including some recently isolated from human patients.    Methodology/Principal findings  We transfected  T.b. brucei ,  T.b. rhodesiense  and  T.b. gambiense  strains with either RLuc, click beetle red (CBR) or  Photinus pyralis  RE9 (PpyRE9) luciferase and characterised their  in vitro  luciferase activity, growth profile and drug sensitivity, and their potential for  in vivo  BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of  T.b. brucei  AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged  T.b. gambiense  LiTaR1,  T.b. rhodesiense  RUMPHI and  T.b. gambiense  348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during  in vivo  BLI.  Ex vivo  BLI on the brain confirmed central nervous system infection by all luminescent strains of  T.b. brucei  AnTaR 1,  T.b. rhodesiense  RUMPHI and  T.b. gambiense  348 BT.    Conclusions/Significance  We established a genetically and phenotypically diverse collection of bioluminescent  T.b. brucei ,  T.b. gambiense  and  T.b. rhodesiense  strains, including drug resistant strains. For  in vivo  BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity  in vivo  and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models