In an attempt to obtain pure and well characterized smooth lipopolysaccharide (S-LPS) and rough lipopolysaccharide (R-LPS), smooth and rough strains of Brucella abortus were extracted by two different modifications Qf the phenolwater method. S-LPS was obtained in the phenol phase, and R-LPS was obtained in the aqueous phase. Further purification was accomplished by treatment with enzymes, detergents, NaI as a chaotropic agent to separate non-covalently bound contaminants, and by gel filtration. The degree of purity of the molecules was determined by chemical and immunological analysis and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Lipid identification by gas-liquid chromatography showed seven major fatty acids. Palmitic acid accounts for about 50%, stearic acid accounts for about 10%, and hydroxylated fatty acids account for less than 5% of total fatty acids. 2-Keto-3-deoxyoctonate but not heptose was detected in the sugar analysis. Protein was found to be firmly bound to S-LPS but not to R-LPS. Several investigators have shown that the crude endotoxin from smooth Brucella spp. is isolated from the phenol phase when cells are extracted with hot phenol-water (2, 3, 6, 18, 22, 23, 30). This fraction, containing 2-keto-3-deoxyoctonate (KDO), sugars, and lipids, presents both similarities and differences when compared with lipopolysaccharides (LPS) from Enterobacteriaceae. Examples of such similarities and differences include observations on toxicity, pyrogenicity, and hybrid formation with Escherichia coli LPS. Baker and Wilson (1, 2) found that endotoxin preparations of smooth Brucella abortus were toxic for mice, but were less toxic than those from E. coli. Leong et al. (30) described qualitative differences in biological activity but not structural differences between brucella endotoxins and enterobacterial endotoxins. Jones et al. (22) reported that saline-extracted rough LPS (R-LPS) from B. ovis was toxic for mice and had limulus lysate gelation activity (LLGA) comparable to that of E. coli LPS. Munoz et al. (36) showed that an endotoxin preparation of smooth B. abortus had comparable LLGA to LPS from several different strains of Salmonella and E. coli, but did not increase sensitivity of mice to histamine. Because these experiments were performed with crude brucelia endotoxin preparations rather than with purified LPS, it was not possible to establish any relationship between chemical composition and biological activity that could help to explain the differences in behavior of these LPS molecules. The work reported here describes preparation and analytical data for purified smooth LPS (S-LPS) and R-LPS from B. abortus which is essential to experiments on biological activity. These, in turn, may contribute to the understanding of the relationship of chemical composition to activity. MATERIALS AND METHODS Bacterial cultures. The bacterial strains used, their characteristics, and the conditions of culture were described previously (22). Extraction of crude LPS. The crude S-LPS (fM) was extr...

Purified lipopolysaccharide (LPS) extracted with phenol-water from smooth Brucella abortus was hydrolyzed with 1% acetic acid at 1000C. The degraded polysaccharide (AH) released gave reactions of identity with the native polysaccharide hapten (NH) in phenol-wateror trichloroacetic acid-extracted endotoxin preparations of B. abortus and with the polysaccharide (poly B) extracted by trichloroacetic acid from rough B. melitensis strain B115. Poly B was present in the soluble cytoplasmic fraction but not in the membrane fraction, of disrupted B115 cells. It could not be extracted from three rough mutants of B. abortus or from B. canis or B. ovis cells. Both AH and NH shared determinants present on smooth LPS and missing from poly B. Sugars found in purified LPS, NH, and AH included mannose, glucose, quinovosamine, glucosamine, and 2-keto-3-deoxyoctonate. Poly B contained only a trace amount of quinovosamine and no 2keto-3-deoxyoctonate detectable by the thiobarbiturate assay. Sera from some rabbits immunized with pure smooth LPS and some, but not all, cows infected with field strains of B. abortus recognized the determinants missing from poly B. A subclass-specific enzyme-linked immunoassay showed that most of the antibody in sera from infected cows which binds to smooth LPS and to NH is of the immunoglobulin Gl subclass.

A Brucella antigen containing polysaccharide but lacking smooth lipopolysaccharide was employed in a rapid radial immunodiffusion test. With this serological test, cattle infected with Brucella abortus could be identified in recently vaccinated herds which had high numbers of reactors to standard diagnostic tests.

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily. At least 24 antigenic components were demonstrated in a water extract from Brucella suis

Lipopolysaccharides (LPS) were extracted from rough strains of Brucella abortus and Brucella melitensis and from strains of the naturally occurring rough species Brucella ovis and Brucella canis. Brucella rough lipopolysaccharides (R-LPS) were readily distinguished from Brucella smooth lipopolysaccharides (S-LPS) and enterobacterial R-LPS, by their chemical, physical, and serological characteristics. B. ovis R-LPS was differentiated from B. abortus, B. melitensis, and B. canis R-LPS by its reaction of partial identity in immunodiffusion. Monospecific mouse sera against B. ovis R-LPS agglutinated only the homologous bacteria but not R cells of other species ofBrucella. B. ovis R-LPS contained more 2-keto, 3-deoxyoctonate, and glucosamine as a percentage of dry weight than any other R-LPS tested. B. abortus R-LPS was identified by the absence of an unidentified sugar present in the other R-LPS molecules, and B. melitensis R-LPS could be differentiated from B. canis R-LPS by its higher content of fatty acids. In contrast to S-LPS, all of the R-LPS studied lacked quinovosamine. In electron micrographs, Brucella R-LPS had a granular appearance, in contrast to typical lamellar structures formed by Brucella S-LPS and Escherichia coli R-LPS.

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