Double Transfection Report

Results
Double transfection do not change Ld1S parasites’ performance
The double transfection approach generated bioluminescent and fluorescent Ld1S parasites as virulent as Ld1S wild-type ones. Indeed, the virulence of the transfected parasites was verified at each stage of the process. Hamsters infected with Ld1S_luci presented the same characteristics than hamsters infected with Ld1S wild-type parasites, such as a regular weight gain until 30 days post-infection, with a subsequent progressive weight loss. By measuring the bioluminescent signals in the liver and the spleen of the infected hamster, we noticed a gradual increase of the parasite load in both organs (Figure 1A).
With the second transfection, we inserted the E2-crimson gene aiming

In both liver and spleen, the first bioluminescent signals were detected as soon as 7 days p.i., then rose slightly to reach a peak at day 30 p.i. (Figure 2A-C). After this, the bioluminescence signal declined slightly up to 90 days pi. To determine the absolute number of Leishmania in the whole organs, parasite transcripts were quantified by RT-qPCR. L. donovani transcript abundance and bioluminescence kinetics recorded from the liver and the spleen had a similar profile, indicating a good correlation between RT-qPCR and bioluminescence (Figure

IFN-γ was up-regulated in both organs, throughout the study, from 7 to 90 days p.i. The response in the liver seemed to be more intense than in the spleen, with up-regulation of IL-1β, IL-2 and TNF-α, along with high levels of IL-10. The spleen, on the other hand, presented a delayed and discreet overexpression of IL-1β and TNF-α. The weight of both organs considerably increased during the infection (Figure 3C), and at histology at 90 days p.i., the liver presented mild multifocal clusters of mononuclear cells and parasites (Figure 3D) whereas the spleen presented remarkable alterations in its architecture, with important amounts of mononuclear cells infiltration and parasites (Figure