Induction and clinical evaluation of EAN
EAN was induced by immunizing mice twice (days 0 and 8) by subcutaneous injection inoculum containing 150 ug P0 peptide 180-199 (GenScript, USA)) and 0.5 mg Mycobacterium tuberculosis (strain H37 RA; Difco, Franklin Lakes, NJ, USA) in 25 ml saline, and 25 ml Freund’s incomplete adjuvant (FIA, ICN Biomedicals, Costa Mesa, CA, USA) into the back of mice. All mice were injected 400, 300, and 300 ng pertussis toxin (PTX, Merck, Whitehouse Station, NJ, USA) via tail veins on days -1, +1 and +3, respectively. Using a blinded protocol, the clinical signs of EAN were scored immediately before immunization (day 0) by two different examiners as follows until day 30: 0 = normal; 1 = less lively, reduced tonus of the tail; 2 = flaccid tail; 3 = abnormal gait; 4 = gait ataxia; 5 = mild paraparesis; 6 = moderate paraparesis; 7 = severe paraparesis; 0.5= intermediate clinical signs. The control group is treated in the same way as mentioned above, but without adding P0 peptide 180-199.