Begin by grinding 10 milligrams of freeze-dried Mycobacterium paratuberculosis to a fine powder using a mortar and pestle. Add five milliliters of Incomplete Freund's adjuvant to obtain a 10 milligram per milliliter stock solution. Store the stock at minus four degrees Celsius.
Before immunization, dilute the stock solution. Then, dilute the MOG 35-55 peptide to two milligrams per milliliter and store it at minus 20 degrees Celsius. Mix equal volumes of the MOG 35-55 with the adjuvant in a five milliliter tube with a homogenizer until a thick emulsion is formed.
After every 10 seconds of mixing, place the solution on ice for 20 seconds and spin the tube to recover all the solution. Transfer the emulsion into a one milliliter syringe, ensuring it is free of air bubbles. Then, fit it with a 27-gauge needle.
Subcutaneously, inject 200 microliters of the emulsion into the lower back of an anesthetized mouse. Then, intraperitoneally inject a dose of pertussis toxin on day zero and two after immunization. All mice manifested an acute monophasic disease, characterized by a single peak of disability observed at 14 to 17 days, followed by a partial recovery of symptoms over the next 10 days.
Mice immunized with M.paratuberculosis showed an earlier onset after immunization and greater severity in the acute phase. Both groups demonstrated similar body weights. Proliferation assay with titrated thymidine incorporation performed on the EAE mice spleen cells showed a strong proliferative response to the peptide MOG 35-55, but not to ovalbumin.
Cytofluorometric analysis showed increased T-lymphocytes, dendritic cells, and monocytes in the spleen of M.paratuberculosis-immunized EAE mice compared to CFA-immunized mice. Hematoxylin and and eosin tissue analysis revealed typical paravascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord.
