To clarify the cellular components of Yersinia enterocolitica related to toxicity and antigenic characteristics, studies were made on toxicity and chemical and antigenic characteristics of the saline extract (Fr. I) and hot phenol-water extract (Fr. II) of the bacterial cells, as well as of sugar components (Fr. III) obtained by hydrolysis of Fr. II with 0.1 N acetic acid. When determined by thin-layer chromatography and liquid chromatography the sugar composition was essentially the same among Fr. I, II, and III. It consisted of galactose, glucose, xylose, and an unidentified sugar that appeared to be 6-deoxy-L-altrose reported by Ellwood and Kirk. In the double gel-diffusion precipitation test against anti- Y. enterocolitica whole cell antiserum, Fr. I, II, and III formed one fused precipitation line, and Fr. I and II an additional precipitation line. Even heating at 100°C for 1 hour or treatment with chloroform did not alter the precipitation pattern of Fr. I. When 250 μg (as sugars) of Fr. II was injected intravenously into 5 mice, 2 mice died within 72 hours. When 5 mice were injected with 1, 500 μg of Fr. II, all of them died. In contrast, no mice died when injected intravenously with 2, 000 μg of Fr. III.
The populations of T- and B-lymphocytes in the spleen were examined in mice of the AKR strain by the cytotoxicity test with rabbit anti-mouse-specific bone marrow-derived lymphocyte antigen serum or rabbit anti-mouse thymocyte serum plus complement. Some of these mice had been injected intravenously with live organisms of BCG, Salmonellaty-phimurium TV148, or Staphylococcus aureus 209 P, and others with heat-killed organisms of BCG or TV148. Those two prepared antisera had been confirmed in several ways to have specific cytotoxicity against T- and B-lymphocytes, respectively, in the presence of complement. The T cell population in the spleen showed a slight decrease in mice in the early stage of infection with 1× 107 organisms of BCG, but a slight increase in the same mice 3 to 5 weeks later. During the experimental period of infection with BCG no obvious change could be found in the B cell population in the spleen in these mice. A significant increase of the T cell population in the spleen was also observed in mice which were infected with 1× 103 to 1× 107 organisms of TV 148 3 weeks before. The B cell population in the spleen, however, decreased gradually in these mice after infection. Mice injected with heat-killed organisms of BCG or TV148, instead of live organisms, showed no increase of the T cell population in the spleen. In mice infected with 2× 108 organisms of 209P, there was a slight decrease of both T- and B-lymphocyte populations in the spleen. This result suggested an expansion of the population of some other cells in the spleen.
Staphylococcal enterotoxin A was isolated in a highly purified form by means of ionexchange resin adsorption, gel filtration, trypsinization, and ultrafiltration. Amberlite CG-50 ion-exchange resin adsorption is a convenient way to concentrate crude toxin from a large volume of culture fluid. Ultrafiltration with Amicon UM-10 is another useful means to concentrate. The molecular sieve is an efficient tool to concentrate the toxin from diluted fluid. When used in an insolubilized form, trypsin was proved to be significantly effective for eliminating α-hemolysin selectively from crude enterotoxin A. The purified sample appeared to be a single entity when examined by immunoelectrophoresis and CM-cellulose column chromatography. The specific activity of the purified sample was 356 times as high as that of the starting material when determined on protein concentration. The yield of the sample was 40% of the starting material.