Fig 1.
Evaluation of in vivo infection of two L. infantum strains.
(A) Spleen weights of BALB/c and Swiss Webster mice infected by L. infantum strains. (B) Parasite load in the spleens of infected mice. (C) Nitrite levels (NOS activity) of spleen cell cultures. (D) Urea levels (ARG activity) of spleen cell cultures. BALB/c and Swiss Webster mouse infections were maintained for 30 or 60 dpi. The number of parasites/mg of tissue was estimated based on the total weight of the spleen removed and the parasite load in the serial dilution. The nitrite and urea levels were measured by spectrophotometry at 540 nm. Control: noninfected mice; LII (L. infantum infantum); LIC (L. infantum chagasi). *p ≤ 0.05; **p ≤ 0.0009; ***p < 0.0001. The values are represented by mean ± standard deviation of 3 independent experiments with 5 animals in each group.
Fig 2.
In vitro proliferation of promastigote forms.
Promastigotes were obtained from amastigotes freshly isolated from the four infected experimental groups and cultivated for 7 days. The parasites were counted daily, and the growth profile was evaluated. LIC.B (L. infantum chagasi isolated from BALB/c mice); LII.B (L. infantum infantum isolated from BALB/c mice); LIC.S (L. infantum chagasi isolated from Swiss Webster mice); and LII.S (L. infantum infantum isolated from Swiss Webster). *p ≤ 0.05; **p ≤ 0.009; ***p < 0.0001. The values are represented by mean ± standard deviation of 3 independent experiments realized in experimental triplicate.
Table 1.
Evaluation of the metacyclogenesis for the four Leishmania isolates.
Fig 3.
Workflow of in vitro infection.
Four L. infantum isolates were used to infect peritoneal macrophages from BALB/c and Swiss Webster mice, at a ratio of 5:1 (parasite/macrophage). The infections were maintained for 24 or 72 h. MØ (macrophage); LIC.B (L. infantum chagasi isolated from BALB/c mice); LII.B (L. infantum infantum isolated from BALB/c mice); LIC.S (L. infantum chagasi isolated from Swiss Webster mice); and LII.S (L. infantum infantum isolated from Swiss Webster mice).
Fig 4.
Infectivity of the four Leishmania isolates to peritoneal macrophages.
(A) Infection index in macrophages from BALB/c mice (top) and percentage of infected macrophages (bottom). (B) Infection index in macrophages from Swiss Webster mice (top) and percentage of infected macrophages (bottom). Peritoneal murine macrophages were infected (overnight) by stationary-phase promastigotes (MOI: 5:1), and cultures were maintained for 24 or 72 h. On each coverslip, at least 200 cells were randomly counted, and infection index was calculated using the following formula: % infected macrophages x number of amastigotes)/total number of macrophages. MØ (macrophage); LIC.B (L. infantum chagasi isolated from BALB/c mice); LII.B (L. infantum infantum isolated from BALB/c mice); LIC.S (L. infantum chagasi isolated from Swiss Webster mice); and LII.S (L. infantum infantum isolated from Swiss Webster mice). *p ≤ 0.05; *p < 0.0001. The values were represented by mean ± standard deviation of 3 independent experiments realized in experimental triplicate.
Fig 5.
In vitro NOS and ARG activities.
(A) NOS activity of promastigotes. (B) ARG activity by urea production by promastigotes. (C) Relative expression of the arginase gene. The intracellular NO production was detected by DAF-2DA labeling. RAW 264.7 macrophages were used as the positive control. The urea production was measured by spectrometry at 540 nm. The relative expression of the arginase gene was evaluated by qRT-PCR. The alfa-tubulin gene was used as the reference gene. LIC.B (L. infantum chagasi isolated from BALB/c mice); LII.B (L. infantum infantum isolated from BALB/c mice); LIC.S (L. infantum chagasi isolated from Swiss Webster mice); and LII.S (L. infantum infantum isolated from Swiss Webster mice). *p ≤ 0.05; **p ≤ 0.009; ***p ≤ 0.0009; ****p < 0.0001. The values are represented by mean ± standard deviation of 3 independent experiments realized in experimental triplicate.