Fig. 2

The non-effect of luciferase expression on the growth of N. caninum in vitro. A Plaque formation assay; 300 tachyzoites were inoculated into HFF cells, cultured for 9 days, then fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The total number of plaques in each well was manually counted, and at least 10 plaques per well were randomly selected to measure the pixel size of each plaque using pixels in Photoshop CC software (Adobe, USA) to represent its plaque size. Three independent experiments were performed, and the plaque number represented the average number of plaques for the three wells. B Invasion assay. The numbers of PVs and HFF cells were calculated by randomly selecting five visual fields. The percentages of invaded parasites were based on the number of PVs divided by the number of HFF cells in one field. C Intracellular proliferation assay. A total of 100 PVs were randomly selected, and the number of tachyzoites per PV was counted. D Egress assay. The egress ability of the parasites was assessed after treatment with 5% absolute ethanol. A total of 100 PVs were randomly counted, and the number of ruptured PVs was determined. Egress ratio = (ruptured PVs/total PVs) × 100%. E Morphological characteristics of Nc1-Luc and Nc1 detected by IFA. Shapes of parasites were visualized using anti-NcSRS2 (red), and nuclear DNA was stained using Hoechst (blue). Scale bar = 5 μm. The results of plaque formation, invasion, and egress assays were analyzed by t-test. The intracellular proliferation assay was analyzed by two-way ANOVA in GraphPad Prism (San Diego, CA, USA). ns: no difference, P > 0.05