In vitro cultivation of Babesia bigemina

A strain of Babesia bigemina was isolated from an infected calf and propagated in vitro. Culture conditions included washing of infected and normal bovine erythrocytes in a special solution, and the use of a 5% to 10% (v/v) erythrocyte suspension in medium 199 (with 20% to 50% fresh normal bovine serum) at a depth of 4 mm in a 5% CO2, 2% O2, 93% N2 atmosphere. After 36 days in vitro and 9 subcultures, the cultured organism was inoculated into a susceptible calf. This calf developed clinical signs of disease and recovered when treated with 1% trypan blue solution. The strain was also reisolated from the second calf. The original isolate had been maintained in continuous in vitro cultivation for more than 99 days.