Trypanosome load in the brain was determined using Taqman real-time PCR as described previously , . Mice were euthanased at 7, 14, 21 and 28 days -post-infection and perfused transcardially with 120 mL of sterile saline to remove peripheral blood from the CNS. The brains were then excised, immediately placed in dry ice and stored at −70 °C until required. DNA was prepared from a 25 mg sample of whole brain homogenate (DNeasy Tissue kit; Qiagen) and Taqman real-time PCR performed , . Briefly, Taqman PCR was carried out in a 25 μL reaction volume comprising 1 x Taqman Brilliant II master mix (Agilent), 0.05 pmol/μL forward primer (CCAACCGTGTGTTTCCTCCT), 0.05 pmol/μL reverse primer (GAAAAGGTGTCAAACTACTGCCG), 0.1 pmol/μL probe (FAM-CTTGTCTTCTCCTTTTTTGTCTCTTTCCCCCT-TAMRA) (Eurofins MWG Operon) and 100 ng template DNA. The primers and probe were specifically designed to detect the trypanosome Pfr2 gene. A standard curve, constructed using a serial dilution of pCR®2.1 vector containing 1 × 106–1 × 101 copies the cloned Pfr2 target sequence (Eurofins MWG Operon), was include in each PCR plate. The amplification was performed using a MxPro 3005 (Agilent) with a thermal profile of 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 1 s. The trypanosome load within the brain samples was extrapolated from the standard curve using the MxPro qPCR software (Agilent).