Studies 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 Al activity. This investigation attempts to define the basis of T.evansi HA. It has been shown, ~n this investigation, that purified and bloodstream T.evansi parasites possess haemolytic, lymphocytolytic and phospholipases A2 and B activities. The HA was present only.>'i.n·-the particulate fraction of T.evansi lysate (i. e. the fraction witJ'r"'1fA--was completely sedimented at -;.~ IOO,OOOxg) while the lymphocyto~~ic activity was present ~n ./ both the crude lysate and soluble IOO,OOOxg supernatant. All the three fractions: the crude lysate, the IOO,OOOxg pellet and the IOO,OOOxg supernatant possessed both phospholipases A2 and B activities. The HA and phospholipases A2 and B were completely inactivated by heating a freshly prepared parasite lysate at 89 0 C for fifteen minutes. However, the HA of the parasite lysate that was first incubat~d at 37 0 C for sixteen hours and then heated at 89 0 C/15 min was not inactivated by the heat. This HA was regenerated when commercial phospholipase A2 or IOO,OOOxg T.evansi lysate supernatant was added to the heat inactivated fresh parasite lysate. The HA was present - 2 - only 1n the chloroform extract of the parasite lysate that had been incubated at 37 0 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 2 ) were inhibited by 5mM KCN or E.D.T.A. HA and phospholipase B (but not phospholipase A2 ) were detected in heavily parasitaemic plasma from rats infected with T.evansi. Anaemia could be induced 1n m1ce 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 0 C, 15 min) T.evansi lysate failed to cause anaem1a. An antiserum .•..(Rii'Te) raised in rabbits against crude T.evansi lysate blocked HA. ~~~her phospholipase A2 nor B -,,~ activity was blocked by this antiserum. . ~ .,. - It is concluded that T.evanS1 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 1n V1VO by lysed trypanosomes may ~e 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)