[Serological studies on rabbits inoculated with Toxoplasma gondii]

Serological studies on toxoplasmosis were conducted with rabbits sera that were immunized with RH strain or infected with Beverley strain of Toxoplasma gondii. Complement fixation tests, agar-gel double diffusion tests and agar-gel immunoelectrophoresis were performed. Toxoplasma crude antigen was prepared from the organisms in mice peritoneal fluids, which were infected with RH strain of Toxoplasma gondii. The organisms were suspended in saline volume originally exudated and counted in hemocytometer for purity of the organisms over 99%. These suspended organisms were prepared by sonication, and the solution was centrifuged for 30 min. at 10,000 rpm in 4 degrees C. These supernatant fluids were used as crude antigen. On the other hand, purified antigens were fractionated on Sephadex G-200 gel filtration. A Sephadex G-200 column, 80 by 4 cm, equilibrated with Tris-HCl-(0.1M)-NaCl (1.0M) buffer, pH 8.0 was used. The eluate fractions were collected in 3 ml per hour and the absorbance at 280 nm was measured with a Beckman Du-2 spectrophotometer. Each tube is pooled into 6 fractions by protein density graph. For immunization of rabbits, crude antigen of RH strain was emulsified with an equal amount of incomplete Freund's adjuvant and l ml of mixture was injected subcutaneously into them once a week for 5 successive weeks. Antisera were obtained at an interval of a week, beginning the first week after the last immunization, while several rabbits were infected with Beverley strain of Toxoplasma gondii by inoculating about 200 cysts and antisera were obtained from them serially at a week interval. The results were as follows: 1. The sera from the rabbits immunized with the RH strain or infected with Beverley strain of Toxoplasma gondii againist the crude antigen showed the first positive reactions in 2 or 3 weeks after the administration or immunization in complement fixation tests. Maximum titers appeared in 4 or 5 weeks after immunization with RH strain and in 7 or 9 weeks after infection with Beverley strain respectively. 2. Complement fixation tests showed the positive reactions in the rabbits sera immunized with RH strain against the purified antigens II, III, IV, V and VI: moreover, antigen IV fraction showed the highest titer. On the other hand in the rabbits sera infected with Beverley strain against the purifed antigens II, III and IV fractions showed the positive reaction; especially, antigen fraction IV showed the highest titer. 3. In immuno-diffusion tests, the sera from the rabbits immunized with RH strain and infected with Beverley strain, against the crude antigen appeared the precipitin bands 2 weeks after the immunization or infection. And the former showed the 2 or 5 precipitin bands after 5-8 weeks and the latter showed the l or 2 precipitin bands after 6 weeks. 4. The sera from the rabbits immunized with RH strain against the purified antigens II, III, IV,V and VI showed the precipitin bands, and the sera from the rabbits infected with Beverley strain against the purified antigens II, III and IV showed the precipitin bands in the immuno-diffusion tests. Especially antigen IV was the strongest reaction against the sera from RH strain and Beverley strain. 5. In agar-gel immunoelectrophoresis, the immunized sera against the crude antigen showed 8 arcs. But the infected sera against the crude antigen showed 4 or 5 arcs. 6. The immunized sera against the fractionated antigens II, III, IV, V, VI showed arcs, but against the fractionated antigen IV showed 6 arcs and in the antigens II, III, V, VI showed l or 2 arcs only. On the other hand, the infected sera against the fractionated antigens IV showed 4 arcs, II and III showed the l arcs, which was the most weak of all.