Animal experiments.

Infection of 8- to 12-week-old female BALB/c mice (Jackson Laboratories) was initiated by the i.v. or i.p. injection of 10³ to 10⁴ bloodstream form T. b. brucei parasites (respectively) or 10⁵ bloodstream form T. b. rhodesiense parasites suspended in 100 μl of PBS plus 1% glucose at 37°C. Mice intended as amplification hosts for T. b. rhodesiense were treated for 24 h prior to and 5 days following infection with immunosuppressive cyclophosphamide at 300 mg/kg i.p. daily to prevent VSG-clone switching, harvested between days 5 and 6 postinfection, and frozen aliquots were stored in 14% glycerol at −80°C until use (41). Forty animals were used in two experiments to establish the effectiveness of i.p. hydroxyurea, and 65 animals were used in three experiments supporting oral hydroxyurea therapy. We employed 105 animals in two experiments to support the effectiveness of hydroxyurea against T. b. rhodesiense. Serial parasitemia and WBC counts were measured by diluting 2.5 μl of blood obtained by tail snip every 1 to 3 days in a red blood cell lysis buffer and manual counting on a hemocytometer under phase-contrast microscopy at ×20 to ×40 magnification. Animal health was monitored frequently during infection for grooming, fighting, or weight loss. Animals receiving hydroxyurea alone experienced a 10 to 15% weight loss while on therapy, with recovery following termination of therapy, and there were no unexpected deaths during hydroxyurea therapy alone. Animals were euthanized if they were clinically moribund, demonstrated neurologic disability (seizures, hind limb paralysis, weakness), or had a parasite count of ≥10⁹/ml. All experimental procedures were approved by the Northwestern University IACUC and carried out in the Center for Comparative Medicine at Northwestern University.