Antigens of Brucella abortus I . Chemical and Immunoelectrophoretic Characterization

Extracts of Brucella abortus 2308S, prepared either by aqueous extraction of sonically ruptured cells or by phenol-water extraction of whole cells, were subjected to various fractionation procedures and then analyzed to determine their immunoelectrophoretic patterns and chemical properties. Fraction A, prepared from sonic extracts, contained at least nine precipitable components when analyzed by immunoelectrophoresis. Of these, five components gave reactions of nonidentity with each other and, hence, represented separate antigens having unrelated determinant groups. Antigenic component IX, found in both the phenol and sonic extracts, did not form a precipitin line in the presence of serum that had been adsorbed with whole cells and was therefore tentatively identified as a "surface" antigen. From several lines of evidence, component IX was thought to be a lipopolysaccharide similar to the AP substance described by Miles and Pirie and shown by them to carry the "abortus" and "melitensis" determinant groups. There is a striking lack of precise knowledge concerning the identity and biological significance of the antigens of the brucellae. The present investigation was undertaken in an effort to provide information about the number and types of antigens present in Brucella abortus and, if possible, to bring about their separation by chemical or physical means so as to allow eventually for a study of their individual biological properties. The starting material was obtained either by water extraction of cells ruptured by sonic oscillation or by the Westphal phenol-water extraction procedure. Only the fractionation procedures employed and the chemical and immunoelectrophoretic characterization of the various antigens isolated will be dealt with in this communication. The biological significance of these antigens, as determined by their toxicity for monocytes in culture and ability to evoke skin reactions in guinea pigs, will be reported separately. MATERIALS AND METHODS Source of microorganisms. All antigenic fractions were prepared from cultures of B. abortus 2308S. This strain is a subculture of the one originally obtained from the Agricultural Research Service, U.S. Department of Agrlculture. Lyophilized stock cultures of the organism in skim milk (18) were prepared and used as needed to avoid problems arising from I Present address: Department of Bacteriology, University of Wisconsin, Madison. population changes. Evidence of smoothness was obtained by the acriflavine test (4) and the crystal violet test (19). Methods of culture. Subcultures were grown on brucella agar (Albimi) slants. After incubation for 12 hr at 37 C, the growth from each tube was suspended in sterile saline and was used as the inoculum for each flask containing 500 ml of brucella broth (Albimi). The flasks were placed on a rotary shaker at 37 C for 24 hr, after which phenol was added to the flasks to give a final concentration of 0.5% (w/v). The flasks were returned to the shaker for an additional 12 hr at 37 C to insure killing. The cells were sedimented by centrifugation, suspended in saline, and again collected by centrifugation. The washing procedure was repeated three more times by use of distilled water. A sufficient quantity of distilled water was added to 50 g of cells (wet weight) to give a final volume of 60 ml prior to making the sonic extracts. Cells to be used for the phenol extraction procedure were harvested in the same manner, suspended in a minimal quantity of distilled water, and lyophilized. Sera for immunological studies. The immune serum used for the immunoelectrophoretic studies came from a single pooled harvest, obtained from six cows which had been artificially infected with B. abortus 2308S. The agglutination titer of the serum was 1:10,240 when tested by the tube method, with U.S. Department of Agriculture B. abortus standard tube test antigen. Phenol was used as the preservative in a final concentration of 0.5% (w/v). Absorbed serum was prepared by mixing 0.4 g of dry, lyophilized B. abortus 2308S with 25 ml of immune serum, incu544 on A uust 8, 2017 by gest http/jb.asm . rg/ D ow nladed fom CHARACTERIZATION OF B. ABORTUS ANTIGENS bating in a water bath at 37 C for 30 min, and collecting the supernatant fluid after centrifugation. The procedure was repeated three more times, the dry bacteria being mixed with the supernatant fluid of the previous tube in each case. The final absorbed serum had an agglutination titer of less than 1:20 when tested by the tube method with the standard tube test antigen. Sonic extracts. Cell breakage was achieved in 1 hr at 3 C by use of a 10-kc, 250-w sonic oscillator. Immediately after this procedure, the suspension was centrifuged at 18,400 X g for 30 min at 4 C. The supernatant fluid was set aside, and the cellular debris was resuspended in 60 ml of distilled water and centrifuged in the same manner. The two supernatant fluids were combined and filtered through Whatman no. 1 filter paper. The resulting filtrate was lyophilized and labeled fraction A. Phenol-water extracts. These were prepared by the Westphal procedure as modified by Redfearn (Ph.D. Thesis, Univ. Wisconsin, Madison, 1960), and were subsequently described in a paper by Baker and Wilson (1). The material recovered from the phenol phase was designated as fraction 5. Zone electrophoresis in starch. The starch block was made of powdered potato starch (technical grade) and tris(hydroxymethyl)aminomethane-acetic acid buffer adjusted to pH 8.2 (ionic strength, 0.02). The starch trough was 35 cm long with a radius of 2.75 cm. Continuous-flow buffer chambers similar to those described by Kunkel and Slater (7) were used. The technique employed was essentially that described by Bodman (3). Immunoelectrophoresis. The immunoelectrophoretic studies were carried out in a buffered gel consisting of 1.5% agar (Difco) and sodium diethylbarbituratehydrochloric acid buffer (pH 8.2; ionic strength, 0.385). The gel was supported on 4 by 5 inch (10.2 by 12.7 cm) photographic plates, and the antigens were incorporated into melted buffered agar before being deposited in the antigen wells. Chemical determinations. Nitrogen was determined by micro-Kjeldahl analysis (6), and the percentage of protein was calculated by multiplying the percentage of nitrogen by a factor of 6.25. The protein content of eluates obtained from starch segments after zone electrophoresis was determined by the Folin-Ciocalteu tyrosine method as modified by Lowry et al. (9). Crystalline bovine serum albumin was used to prepare a standard curve. Carbohydrate was estimated by the method described by Loewus (8), with glucose as a standard. The qualitative test for the presence of polysaccharide in immunoelectrophoretic plates was performed as described by Uriel and Grabar (17), except that the initial oxidation with periodic acid was lengthened to 3 hr.