Isolation of blood and intracellular forms ofTrypanosoma cruzifrom rats and other rodents and preliminary studies of their metabolism

Abstract

Summary: Isolation of blood and intracellular forms ofTrypanosoma cruziwas made mainly from rats (90–110 g) which had received 580 rad of whole-body γ-irradiation not more than 24 h before subcutaneous inoculation with 10⁷trypomastigotes of the Sonya strain ofT. cruzi. Unirradiated chinchillas (250–350 g) were, however, used for some experiments. Blood forms were isolated using a technique involving differential centrifugation to remove most of the erythrocytes and DEAE–cellulose chromatography to remove the remaining blood cells. Overall recoveries were usually in the range 30–70%. Parasites were mainly (approximately 98%) broad forms and were motile, metabolically active (as judged by respiratory and radio-tracer incorporation studies) and had lost none of their infectivity for mice. Intracellular forms were isolated from hind-limb muscle tissue. This was disrupted in an MSE tissue homogenizer and the homogenate incubated with DNase, collagenase and trypsin. Parasites, contaminated only by a few blood cells, were then obtained by differential centrifugation. For purer preparations, a terminal sucrose gradient step was used. Recoveries ranged between 40 and 70%. About 1–3% of the parasites isolated were epimastigotes and trypomastigotes; the remainder are probably best collectively termed ‘amastigotes’, though they were pointed and most had a short, free flagellum. They were undamaged as judged by light and electron microscopy and metabolically active as judged by respiratory and radio-tracer incorporation studies. However, the infectivity for mice of both these purified preparations and the initial cell homogenates could be accounted for by the epimastigotes and trypomastigotes present in them. Preliminary biochemical studies with isolated parasites have shown that blood, intracellular and culture forms ofT. cruzihave a respiratory system which is in part sensitive to CN- and that all forms synthesize nucleic acids and proteins when incubatedin vitro. There appears, however, to be a lack of DNA synthesis in blood stages, and thus it is not surprising that these forms do not divide.