The high resistance to Toxoplasma gondii in Microtus fortis is associated with the activation of the complement lectin pathway

Trypan blue can penetrate the cell membrane of dead cells and stain the cytoplasm blue. The tachyzoites that are stained blue can be considered dead under the optical microscope. To further distinguish between dead and surviving tachyzoites, a fluorescent strain of T. gondii (RH-GFP-TgAtg8) was used, which was fluorescently labelled with a cytoplasmic protein [34]. It was observed that the fluorescence disappeared upon death of tachyzoites. The mortality of tachyzoites was assessed using a combination of fluorescent strains and trypan blue staining. Tachyzoites were prepared as described previously, and the concentration was adjusted to 2×107/ml with PBS. The tachyzoite suspension was then incubated for 2 hours with M. fortis serum (30%) or KM serum (30%) at 4 ℃ in 200 μl volumes. After centrifugation at 3000 rpm for 8 min to remove the reaction buffer, the parasites were resuspended in 50 μl PBS and stained with trypan blue (0.5%, Sigma Chemical Co, USA). The viability of the tachyzoites was immediately assessed using a fluorescence microscope (Carl Zeiss, Suzhou, China). Parasites were randomly counted in 5 microscopic fields, and the mortality rate of tachyzoites was calculated using the following formula: mortality (%) = (number of dead parasites (tachyzoites stained blue)/number of total parasites (tachyzoites stained blue plus fluorescent tachyzoites)) × 100%. These assays were performed in triplicate, with serum derived from three distinct animals for each of the three replicates within each group.In some experiments, M. fortis serum was pretreated before being mixed with the tachyzoites suspension. The treatments included heat inactivation (56 °C for 30 minutes), 10 mM EDTA, 10 μg/ml CVF, 5 mM EGTA with 10 mM MgCl₂, and 100 nM Narsoplimab (Aladdin, China) [35, 36]. All pre-treatments, except for heat inactivation of complement, were mixed and incubated at 4 °C for 10 minutes.Tachyzoites co-incubated with M. fortis serum or PBS were centrifuged to collect the precipitate, which was then resuspended in 50 μL of PBS. A 10 μL aliquot of the tachyzoite suspension was used to prepare smears. After drying, the smears were fixed with methanol for 10 minutes, washed three times with PBS for 5 minutes, incubated with 2% (w/v) BSA at 37 °C for 30 minutes, and washed three times with PBS for 5 minutes. They were then stained with MASP2 recombinant rabbit polyclonal antibody (Affinity Biosciences, China) or MAC recombinant rabbit polyclonal antibody (Bioss, China) at 37 ℃ for 1 hour, washed three times with PBS for 5 minutes. Stained with AF488 fluorescein conjugated sheep anti-rabbit antibody (Servicebio, China) at 37 ℃ for 1 hour under light-protected conditions and washed three times with PBS for 5 minutes. The slides were sealed with an anti-fluorescence quencher containing the fluorescent dye DAPI, and subsequently photographed under a fluorescence microscope.