<i>Ex vivo</i> organs of infected mice confirming the systemic dissemination of the parasite and the brain commitment in late phases of infection.

<p>Detection of bioluminescence <i>ex-vivo</i> in organs isolated from mice injected intraperitoneally with 10²<i>Tv</i>LrDNA-luc BSF. Organs were rinsed in PBS and D-Luciferin was added before light detection. Organs from a representative mouse out of 3 for each time point are depicted. Color scale to the right of the pictures encodes for signal intensity (photons/second). The values of each animal have been taken into consideration to obtain the arithmetic means of bioluminescence ×10⁵ ph./s ± the Standard Deviations of the means and are indicated underneath the pictures. (<b>A</b>) spleen, (<b>B</b>) liver, (<b>C</b>) lungs and heart (white arrows) and (<b>D</b>) brain. Even if individual variations between the organs pertaining to the same group of mice are observed for each time point, they do not influence the data interpretation.</p

Proliferation of <i>T. vivax</i> in the skin during the prepatent period after sub-cutaneous injection.

<p><b>A</b>. Ventral and dorsal view of a representative Outbred mouse out of two experimental groups of 12 and 14 mice injected subcutaneously with 10² BSF expressing luciferase (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001976#pntd-0001976-g003" target="_blank">Figure 3</a>). Bioluminescence was measured at days 9 (left panel) and 10 (right panel). <b>B</b>. Lateral view of the mouse on day 10. <b>C</b>. The mouse was sacrificed on day 10 and bioluminescence measured in the subcutaneous compartment. Color scale to the right of the pictures encodes for signal intensity (photons/second).</p

Characterization of bioluminescence production by epimastigotes and bloodstream evolutive forms of <i>Tv</i>LrDNA-luc strain.

<p><b>A</b>. Serial dilutions of <i>Tv</i>LrDNA-luc epimastigotes (EPI, black squares) or bloodstream forms purified from blood (BSF, gray squares) were measured <i>in vitro</i> for bioluminescent activity expressed in RLU (Relative Light Unit); the results are representative of more than three independent experiments. <b>B</b>. Bioluminescence emission per <i>Tv</i>LrDNA-luc BSF without drug pressure selection; the results are expressed in RLU/parasite and correspond to arithmetic means +/− SD of the means; p>0,05.</p

Mice injected by the subcutaneous route show a longer prepatent period than those injected intraperitoneally.

<p>Parasitemia (empty symbols) and total bioluminescence (full symbols) were determined in groups of mice injected with 10²<i>Tv</i>LrDNA-luc parasites by (<b>A</b>) the intraperitoneal route (squares) or (<b>B</b>) the subcutaneous route (diamonds). The graphs depict the arithmetic means of values obtained individually for at least 3–6 mice per time point ± SD of the means. The results are representative of two independent experiments that used respectively 16 and 18 mice for the intraperitoneal route and 12 and 14 mice for the subcutaneous route. For a better comparison, the dashed vertical line that crosses the figures A and B illustrates the onset of microscopically-detectable parasitemia (10⁴/ml blood) for the IP injected group. <b>C</b>. Survival rates for the different infection routes. The cumulative mortality was recorded over time and survival curves are the result from the combination of the two independent experiments and Kaplan-Meir survival estimate curves were plotted. Only 3–6 mice (20–30%) are alive by the end of each of the two experiments but all die at day 28 of infection. Comparison between the curves was performed using the non parametric Mantel-Cox log-rank test. No significant difference was observed between the plots; p>0.5.</p

Correlation between parasitemia and total bioluminescence recorded from the entire infected animal.

<p>Male Outbred mice were injected intraperitonneally with 10²<i>Tv</i>LrDNA-luc BSF. <b>A</b>. Total light emission kinetics following D-Luciferin IP injection in a mouse with 1.10⁶ parasites/mL. The figure depicts the data from 1 out of more than 34 independent mice analyzed individually <b>B</b>. Bioluminescence over a Region of Interest (ROI) including the entire animal body surface measured 10 minutes after D-Luciferin i.p. injection. The results are expressed individually for each measurement resulting from two groups of 17 mice. Parasitemia was determined by optical microscopy concomitantly with each bioluminescence measurement. Correlation factor was calculated using Spearman's non parametric correlation test r = 0.9365, p<0.0001.</p

Bioluminescent <i>T. vivax</i> validates the presence of the parasite in the central nervous system in late infection phases.

<p>Detection of bioluminescence <i>ex-vivo</i> in brains isolated from mice injected intraperitoneally with 10²<i>Tv</i>LrDNA-luc BSF analyzed at days 20, 22, 25 and 28 of infection as compared with a brain from a non infected mouse. Brains were rinsed in PBS and D-Luciferin was added before light detection. One brain from a representative mouse out of 3 for each time point is depicted. Color scale to the right of the picture encodes for signal intensity (photons/second). The values for each brain are indicated underneath the pictures (×10⁵ ph./s).</p

<i> In vivo</i> bioluminescence imaging of <i>Leishmania</i> in the C57BL/6 ear model.

<p>Simultaneous follow up of parasite load in the ear dermis and of lesion onset, features and cure in mice inoculated with luciferase-transgenic <i>Leishmania major</i>. 10⁴ luciferase-expressing NIH 173 metacyclic promastigotes were inoculated into the dermis of the right ear of C57BL/6 mice (day 0) and followed for more than 80 days. The bioluminescent signal is displayed as a pseudo-colour image representing light intensities over the body surface area. Red represents the most intense signal while blue corresponds to the weakest one. A, B: Individual follow up of a representative mouse left without any ointment application. Clinical features (A) were simultaneously monitored with parasite load fluctuations assessed by the bioluminescence of luciferase-expressing parasites (B). (C) Bioluminescence quantification of the parasite load (left panel; photons/sec/ear; grey area = background measurement) and “lesion” area (right panel; mm²) were followed for 7 mice left without any ointment and depicted as medians +/−sd. Note the detection of a bioluminescence signal before any significant clinically detectable features. Of note, the so called “lesion” area measured between day 11 and day 15 was still made up of inflammatory processes free of any leukocyte infiltrates.</p

<i>Trypanosoma vivax</i>-specific luciferase vectors.

<p>Schematic representation of p<i>Tv</i>LUC vector constructs (A); <i>Tv</i>PRAC5′UTR, upstream of the <i>Tv</i>PRAC region and containing spliced leader acceptor site; Tubαβ, intergenic region between alpha and beta tubulin genes; NeoR, neomycin phosphotransferase gene; Tubβα, intergenic region between β and α tubulin genes. Representation of the ribosomal promoter region cloned into p<i>Tv-</i>Luc and associated luminescence in Relative Light Units (RLU) detected in the supernatant 48 h after epimastigote transfection with p<i>TvS</i>rDNA-Luc and p<i>TvL</i>rDNA-Luc (B).</p

C57BL/6 mice given an application of WR279396 formulation under an occlusive dressing.

<p>10⁴ luciferase-expressing <i>L. major</i> metacyclic promastigotes were inoculated into the dermis of the right ear of C57BL/6 mice (day 0). (a, b) From day eleven post-<i>L. major</i> inoculation, the topical ointment WR279396 was applied directly to parasite-loaded ears and (c) then covered with an adhesive polyurethane dressing (arrow). (d,e) Two independent leaflets of surgical tape were applied directly on the occlusive dressing. This surgical tape permitted maintenance of the dressing and kept the formulation in contact with the parasite-loaded site for two days.</p

Parasite culture media.

<p>Parasite culture media.</p