Fig. 1

Generation of a luciferase-expressing N. caninum strain. A Schematic illustration of the replacement of the NcUPRT locus with a luciferase expression cassette. The homologous donor sequence contains the luciferase expression cassette driven by the Tgtubulin promoter and flanked by regions of homology to NcUPRT. B Diagnostic PCRs of Nc1-Luc strains. The position of the primers is shown in A. Primers F1/R1 and F2/R2 were designed to identify homologous integration based on the products amplified between the luciferase cassette and the external regions of the NcUPRT amplicon. The primer F5/R5 was designed to verify the integration of luciferase. Primers F3/R3 and F4/R4 confirm deletion of the NcUPRT locus. The Nc5 gene served as a N. caninum-specific gene. C Expression of luciferase in Nc1-Luc strains. HFF cells were infected with N. caninum tachyzoites (2 × 10⁴), and luciferase activity expressed in relative light units (RLU) was measured at 72 h post-infection. Control: parental Nc1 strain. The assay was performed in triplicate. Statistical analysis was performed by t-test in GraphPad Prism (San Diego, CA, USA). ****P < 0.0001