Semiquantitative real-time PCR analyses were performed to determine parasite loads in brains as described previously [66 (link)]. FastStart Essential DNA Green Master (Roche, Grenzach-Wyhlen, Germany) was used with 90 ng genomic DNA in a reaction volume of 20 μL. Triplicate reactions were developed in a LightCycler 480 Instrument II (Roche, Grenzach-Wyhlen, Germany). After an initial activation step (95 °C for 10 min), 45 amplification cycles were run, comprising of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and elongation at 72 °C for 15 s. The primers manufactured by Tib MolBiol (Berlin, Germany) were used at a final concentration of 0.3 μM. The specific primer pairs are listed in Table 1 (T. gondii B1 gene, murine argininosuccinate lyase/Asl as a reference). For normalization against Asl, target–reference ratios were calculated with the LightCycler 480 Software release 1.5.0 (Roche, Grenzach-Wyhlen, Germany). The resulting data were further normalized against a control group, i.e., the ileum of WT animals.