Study on Trypanosoma evansi-derived haemolytic activity
Anaemia and tissue damage are characteristic of African trypanosomiases. The underlying pathophysiologic mechanisms are still obscure. It is, however, known that lysed African trypanosomes generate haemolytic activity (HA). During the course of this investigation it was shown by other workers that pathogenic trypanosomes possess phospholipase A^ activity. This investigation attempts to define the basis of T.evansi HA. It has been shown, in this investigation, that purified and bloodstream T.evansi parasites possess haemolytic, lymphocytolytic and phospholipases A^ and B activities. The HA was present only in the particulate fraction of T.evansi lysate (i.e. the fraction with HA was completely sedimented at 100,000xg) while the lymphocytolytic activity was present in both the crude lysate and soluble 100,000xg supernatant. All the three fractions: the crude lysate, the 100,000xg pellet and the 100,000xg supernatant possessed both phospholipases A2 and B activities. The HA and phospholipases and B were completely inactivated by heating a freshly prepared parasite lysate at 89°C for fifteen minutes. However, the HA of the parasite lysate that was first incubated at 37°C for sixteen hours and then heated at 89°C/15 min was not inactivated by the heat. This HA was regenerated when commercial phospholipase A2 or 100,000xg T.evansi lysate supernatant was added to the heat inactivated fresh parasite lysate. The HA was present only in the chloroform extract of the parasite lysate that had been incubated at 37°C for sixteen hours. However, the chloroform extracts of non-incubated and heat-inactivated parasite lysate or fresh unheated parasite lysate had no HA. The HA and phospholipase B (but not phospholipase A^) were inhibited by 5mM KCN or E.D.T.A. HA and phospholipase B (but not phospholipase A^ ) were detected in heavily parasitaemic plasma from rats infected with T.evansi. Anaemia could be induced in mice by a single intravenous dose of T.evansi lysate extracts, commercial phospholipase A^, lysolecithin or palmitic acid. However, a similar injection of heat-treated (89°C, 15 min) T.evansi lysate failed to cause anaemia. An antiserum (RaTe) raised in rabbits against crude T.evansi lysate blocked HA. Neither phospholipase nor B activity was blocked by this antiserum. It is concluded that T.evansi contains phospholipases A2 and B activities. The T.evansi derived HA is generated by phospholipase B enzymatic reaction on the parasite endogenous substrates. The haemolysis itself is caused by heat-resistant, chloroform soluble products (probably free fatty acids) of phospholipase action on parasite phospholipids. Phospholipases released in vivo by lysed trypanosomes may be responsible for the anaemia and tissue damage characteristic of African trypanosomiases. Part of this study: "Haemolytic Activity of Trypanosomes" has been published in the East African Medical Journal, 58, (12), 907-911, (1981).