Figure 4.

The protective effect of laminarin (LAM) against Neospora caninum infection is dependent on reactive oxygen species (ROS). For in vivo experiments, wild-type (WT) and/or NADPH oxidase isoform 2 (NOX2)^−/− mice were treated or not with LAM (1 mg/kg/day), during seven consecutive days. In the fourth day of treatment, mice were infected with non-lethal doses (1 × 10⁶) of N. caninum tachyzoites. For the in vitro assay, bone marrow-derived macrophages (BMDMs) (10⁶ cells/mL) were infected with live Nc-1 tachyzoites (0.5:1, parasite:cell ratio) for 1 h, pretreated or not with LAM (500 μg/mL) for 3 h. (A) Percentage of ROS and NO positive cells obtained from the peritoneal cavity of WT mice, determined by flow cytometry. (B) Liquid production of ROS (RFU reading of infected cells subtracted of each individual control) of BMDMs generated from WT and Dectin-1^−/− mice, treated or not with LAM. (C) Peritoneal acute phase parasitism in WT and NOX2^−/− mice after 3 days of infection with 1 × 10⁶ N. caninum tachyzoites stained with CFSE, analyzed by flow cytometry, and expressed as mean intensity of fluorescence (MIF). (D) In a similar experiment, WT and NOX2^−/− mice were treated or not with LAM, and the acute phase parasite burden was again assessed through MIF of CFSE⁺ cells. (E) IL-12p40 concentration was also determined in the peritoneal fluid of WT and NOX2^−/− mice, under the same infection and treatment protocol. Results were expressed as mean ± SEM and are representative of three independent experiments. *Statistically significance differences (p < 0.05), assessed by analysis of variance followed by Bonferroni multiple comparison posttest.