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Scheme 1. Timeline of in vivo characterisation of Chagas disease on BALB/c mice infected with 5.0×105bloodstream trypomastigotes of T. cruzi Arequipa strain. dpi, days post-infection.
Scheme 2. Timeline of BNZ activity assays on BALB/c mice infected with 5.0×105 bloodstream trypomastigotes ofT. cruzi Arequipa strain. *Oral treatment at 20 mg·kg-1 per day for 5 consecutive days; dpi, days post-infection.
Figure 1. Representative images of the different morphologic forms of Trypanosoma cruzi Arequipa stained with Giemsa. A, Epimastigote forms; B, Metacyclic trypomastigote forms; C, Vero cells infected by intracellular amastigote forms; D, Culture-derived trypomastigote forms infecting Vero cells; E, Mouse peripheral blood infected by bloodstream trypomastigotes (1:100 dilution in PBS); F, Mouse peripheral blood infected by bloodstream trypomastigotes (blood smear); G, Mouse peripheral blood infected by bloodstream trypomastigotes (after enrichment buffer for trypomastigote forms).
Figure 2. Dose-response curves and inhibitory concentrations (IC) 50 and 90 of benznidazole against the three morphological forms ofT. cruzi Arequipa using GraphPad Prism 6 software.
Figure 3. (A) Percentage of infected Vero cells and average number of intracellular amastigote forms of Trypanosoma cruziArequipa per Vero cell of benznidazole-treated infected cultures. (B) Infectivity index (average number of amastigote forms in infected cells multiplied by the percentage of infected cells) of benznidazole-treated infected cultures. (C) Representative images of Vero cells infected, benznidazole-treated and Giemsa stained.
Figure 4. (A) Acute-phase parasitaemia (blue) and tropism (red) in BALB/c mice after intraperitoneal inoculation of 5×105 T. cruzi Arequipa bloodstream trypomastigotes. Values constitute means of 5 mice ± standard deviation. Tropism bars represent the percentage of organs/tissues with nested amastigote forms with respect to the 9 target organs/tissues. (B) Tropism evaluation by PCR analyses of 14 potential target organs/tissues with the Spliced Leader (SL) intergenic region sequence of T. cruzi . Table show the positive mice for each organ/tissue; n = 5 for each experimental time.
Figure 5. (A) Parasitaemia profiles of untreated and benznidazole(BNZ)-treated mice during the acute-phase of Chagas disease over 60 days, and parasitaemia reactivation after immunosuppression of untreated and BNZ-treated mice during the acute- and chronic-phase of Chagas diseae. Treatment days are represented in grey. Values are the means of five mice ± standard deviation. (B) Tropism evaluation by PCR analyses of the target organs/tissues with the Spliced Leader (SL) intergenic region sequence of T. cruzi of untreated mice and benznidazole(BNZ)-treated mice during both the acute- and chronic-phase of Chagas disease, Lanes: (M), base pair marker; (-), PCR negative control; (+), PCR positive control; (1-9), target organs/tissues: (1), adipose; (2), bone marrow; (3), brain; (4), esophagus; (5), heart; (6), lung; (7), muscle; (8), spleen; (9), stomach. * 4/5 of the corresponding organs/tissue PCR products showed 300 bp band on electrophotesis; 3/5 of the corresponding organs/tissue PCR products showed 300 bp band on electrophotesis.
Figure 6. Organomegaly of spleens and hearts of uninfected, untreated and BNZ-treated mice at the euthanasia day. Values are the means of five mice ± standard deviation.