Isolation and Quantification of Parasites from Infected Ear Tissue

Ear tissue was prepared as previously described [40 ]. Briefly, the two sheets of infected ear dermis were separated, deposited in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp.), and incubated for 1.5 h at 37°C. Digested tissue was processed in a tissue homogenizer (Medimachine; Becton Dickinson) and filtered through a 70 μm cell strainer (Falcon Products). Parasite titrations were performed as previously described [41 (link)]. Briefly, tissue homogenates were serially diluted in 96-well flat-bottom microtiter plates containing biphasic medium, prepared using 50 μl NNN medium containing 20% of defibrinated rabbit blood and overlaid with 100 μl M199/S. The number of viable parasites in each ear was determined from the highest dilution at which promastigotes could be grown out after 7–10 days of incubation at 26°C.