Immunochemical Studies on East Coast Fever: II. Partial Segregation and Characterization of the Theileria parva Piroplasm Antigen

Abstract

The piroplasm antigens of Theileria parva were isolated from infected bovine erythrocytes either by sonication or by French pressure cell disruption. A soluble bovine erythrocyte antigen was also isolated from both infected and noninfected blood. Both piroplasm and erythrocyte antigens were readily detected by either complement fixation or immunodiffusion tests. Partial characterizations were achieved by Sephadex G200 and Sepharose 2B chromatography, density gradient centrifugation, and precipitation by ammonium sulfate solution. Results indicated that the piroplasm antigen occurred in a wide molecular weight range of 4 to 20 million with an approximate sedimentation coefficient range of 7 to 15S. The erythrocyte antigen had a molecular weight range of 20 to 40 million with sedimentation coefficient ranging from 3 to 9S. The piroplasm antigens were partially separable from the erythrocyte antigens by differential ammonium sulfate precipitations. The problems inherent in the preparation of soluble specific antigens from parasites that normally develop intracellularly have been recently reviewed by Fife (1972). These problems most often relate to the inability to separate completely the parasites from remnants of the host cell, particularly red cell stromata. Residual contaminants often give rise to preparations of low specificity and sensitivity, and if CF tests are used for assay, the antigens are often anticomplementary as well. Recent work by D'Antonio et al. (1966) on Plasmodium knowlesi-infected monkey blood, and Amerault and Roby (1968) on Anaplasma marginale-infected bovine blood has shown that passage of parasitized blood through a French pressure cell selectively fragments the erythrocyte membranes without apparent damage to the parasites. Resultant antigen preparations in both instances have Received for publication 29 October 1973. * Project supported by the United Nations Development Program, with the Food and Agriculture Organization of the United Naitons as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Overseas Development Administration of the United Kingdon (Research Projects R 2396 and R 2494), the United States Department of Agriculture, the Rockefeller Foundation, the International Atomic Energy Agency, and the Pfizer Corporation. been found highly specific and sensitive and without anticomplementary activity. Ultrasonication has also been used for the preparation of semipurified Babesia antigens (Goodger, 1971). Recently, work in this laboratory with the French pressure cell has resulted in the isolation and partial characterization of T. parva schizont antigens, using infected bovine lymphoblasts in cell culture as the source of material (Wagner et al., 1974). Similar techniques have been applied to T. parva-infected bovine blood (piroplasm stage). A comparison of the piroplasm antigens isolated from French pressure cell extracts and by sonication is the subject of this report. MATERIALS AND METHODS Steers of Bos taurus type were provided by the FAO project at EAVRO, Muguga, Kenya, and were infected with the Aitong strain of T. parva (Irvin et al., 1972) by the subcutaneous inoculation of a supernate extracted from infected adult Rhipicephalus appendiculatus ticks (Purnell et al., 1973). The inoculum used to infect the animals, popularly termed stabilate (Lumsden and Hardy, 1965) was preserved in liquid nitrogen (Cunningham et al., 1973). The animals were selected at a time when the level of parasitized erythrocytes rose above 50%. Five liters of infected blood were collected by venesection into 200 ml 0.1 MI phosphate-buffered 0.15 M saline solution (PBS) pH 7.2, containing 150,000 units of heparin. The parasitized erythrocytes were washed 4 times