Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.