In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei, T. rhodesiense, and T. gambiense1

Abstract

A series of new in vitro systems for the cultivation of bloodstream forms of T. (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense, and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast-like cells derived from embryos of New Zealand white rabbits (REF) or a mountain vole, Microtus montanus (MEF), with HEPES-buffered Minimum Essential Medium (MEM), with Earle''s salts, supplemented with 15% inactivated rabbit serum. These 2 and other feeder layers were cross-checked with different sera to test for growth support of bloodstream forms of the 3 trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be used as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some of the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by EM. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieved with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.