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

<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

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

Obtaining the stably transfected <i>T. vivax TvLrDNA-Luc</i> strain.

<p>(A). <i>T. vivax</i> axenic epimastigotes were transfected with circular or <i>AfeI</i> linearized p<i>TvL</i>rDNA-Luc using the ×006 Amaxa program and luciferase light emission (RLU) was measured 24 h post-transfection in the supernatants. (B) Schematic representation of p<i>Tv</i>LrDNA-Luc linearized with <i>AfeI</i> and its expected recombination in the parasite ribosomal DNA region. Hatched boxes show ribosomal promoter-containing regions; Black crosses represent recombination events; grey arrows indicate primers used to verify plasmid integration into <i>T. vivax</i> genome. 18 S, 5.8 S and 28 S, rRNA genes. (C) Verification of the 5′- and 3′- integration of the vector. PCR using respectively upF/upR (upper panel) and downF/downR (lower panel) primers. PCR was performed on genomic DNA from 2 independent cultures stably transfected with p<i>TvL</i>rDNA-Luc (1, 2) or from WT strain (3). (D) Changes in luminescence (RLU) emitted per p<i>TvL</i>rDNA-Luc parasite after G418 selection. (E) Graphic representation of bioluminescence expressed as luciferase light emission (RLU) of increasing numbers of <i>TvLr</i>DNA-luc parasites.</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

Mutant <i>Tv</i>LrDNA-Luc strain displays the same levels of <i>in vivo</i> infectivity and virulence as WT <i>T. vivax</i>.

<p>Four to six Outbred mice were injected intraperitoneally with 10² WT (⋄) or <i>Tv</i>LrDNA-Luc (▪) BSF <i>T. vivax</i> parasites and parasitemia were followed individually every 5 days. Continous and dotted lines stand respectively for parasitemias and cumulative survival rates, as determined by Kaplan-Meier methodology. Parasitemias are expressed as arithmetic means ± SD. No significance was observed between the curves, as ascertained by the Mantel-Cox log-rank test where <i>p</i>>0.5.</p

Kinetics of <i>T. vivax</i> growth <i>in vitro</i>.

<p>Microscopic examination of adherent cells 2 hours (A), 7 days (B), 14 days (C) and 21 days (D) after inoculation with 1.5.10⁷ parasites. 100× magnification. Parasite numbers in ongoing cultures (E). Results are expressed as arithmetic means ± SD.</p