Figure 1.
Different routes of administration of Neospora caninum and effect on inhibition of tumor development. B16F10 cells (1×105 in 100 µL of phosphate buffered saline) were inoculated intravenously in C57Bl/6 mice and 2×106 NC1 tachyzoites were injected subcutaneously, intravenously, or intranasally 7 days later (A). Twenty-eight days after tumor cell inoculation, mice were euthanized to determine macroscopic observation of lung metastases implantation (B) and tumor size as a percentage of lung tissue using ImageJ software (C). Lungs were collected and dissociated, tumor cell suspensions were centrifuged and cells were resuspended for staining and flow cytometry analysis (D). Sera were collected at day 9, 14, 21 and 28 after tumor cell injection, and levels of IFN-γ (E), anti-B16F10 cells IgM and IgG (F) and anti-NC1 IgM and IgG (G) were assayed. Sections of lung from control mice (H) and intranasally treated (I) mice were stained by immunofluorescence using an anti-CD68 antibody to reveal macrophages (green) and Hoechst for cell nucleus staining (blue), whereas B16F10 cells are visualized due to mCherry expression (red). n=10 mice per group. *p<0.05; ****p<0.0001. IFN, interferon; i.v., intravenous; i.n., intranasally; NK, natural killer; s.c., subcutaneously.