The Properties of Antigens from Trypanosoma gambiense

The results of this study suggest that there is an agglutinating antigen and a separate protective antigen in Trypanosoma gambiense. These two antigens are believed to give rise to separate antibodies in laboratory rabbits, one responsible for protection and a second responsible for agglutination. The relationship of these two antigens to the immunology of trypanosome infections is discussed. Various investigators have shown that serum from animals infected with Trypanosoma gambiense contains antibody capable of (1) passively protecting animals (Seed, 1963) and (2) agglutinating trypanosomes (Inoki, Kitaura, Nakabayasi, and Kurogochi, 1952; Soltz, 1957; and Seed, 1963). In the course of characterizing antigens from various trypanosomes the question arose as to whether the properties of agglutination and protection were due to two antigens; one giving rise to agglutinating antibody and a second to antibody capable of passively protecting animals, or to a single antigen which elicited antibody that has both properties. The work to be described in this paper suggests that two separate antigens are responsible for these properties. In a previous publication (Seed, 1963) it was suggested that the trypanosome enzyme hexokinase might be involved in the respiratory inhibition test of Desowitz (Desowitz, 1961). It has also been considered that antibody to hexokinase might possibly be involved in protection. This hypothesis has also been examined. METHODS OF PROCEDURE Strains of trypanosomes Three strains of the blood form of Trypanosoma gambiense were used in these experiments. Figure 1 shows their interrelationships. The TS strain of T. gambiense has been previously described (Seed, Baquero, and Duda, 1965). These strains were maintained by syringe passage every 2nd day through 20-g mice. Large quantities of blood trypanosomes (BT) were obtained by infecting white (male) rats with 0.25 ml infected mouse blood at peak parasitemia. The BT were harvested 2 days following inoculation. The methods used to harvest the BT have been previously described (Seed and Baquero, 1965). Five-kilogram rabbits were infected by inoculating them intraperitoneally with 0.25 ml infected mouse blood at peak parasitemia. Rabbits were bled by heart puncture at periodic intervals. Received for publication 15 October 1965. Isolation of trypanosomes Mice were bled by heart puncture at peak parasitemia. One per cent sodium citrate (w/v) was used as the anticoagulant. The suspension was centrifuged at 500 g in an International Clinical Centrifuge for 4 min. After centrifugation the serum and trypanosome layers were removed from the underlying red cell layer with a Pasteur pipette. The serum and trypanosome suspension was then centrifuged at 1,000 g for 4 min in a clinical centrifuge, and the serum layer removed. The trypanosomes were resuspended in Ringer's phosphate (0.1 M NaCl, 0.06 M KC1, 0.02 M MgSO4, and 0.025 M phosphate buffer, pH 7.2) containing (0.01 M) glucose, and centrifuged again for 4 min at 1,200 g in a clinical centrifuge. The trypanosomes were then resuspended in glucose-Ringer's phosphate at the appropriate concentrations.