Biomedical subjects
Publications and source records attributed to R Diaz.
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Hot phenol-water extraction of smooth Brucella abortus and B. melitensis cells yielded a toxic fraction which was recovered from the phenol phase (fraction 5). Chemically, fractions 5 from both Brucella species were lipid-carbohydrate-protein-2 keto-3-deoxyoctulosonic acid complexes which were stable to heat and resistant to Pronase digestion. Electron micrographs of the Brucella toxins were morphologically indistinguishable from those of enterobacterial endotoxins. Biologically, Brucella toxins were lethal for mice and immunogenic for rabbits. An intravenous injection of Brucella toxin induced severe leukopenia with subsequent leukocytosis in mice. Cross-tolerance experiments with mice demonstrated that pretreatment with B. abortus toxin lessened the hypoferremia produced by challenge with Escherichia coli endotoxin. Furthermore, fractions 5 from B. abortus and B. melitensis were able to form hybrids with E. coli and Salmonella enteritidis endotoxins and also with each other. Although Brucella toxins possess many structural and biological properties in common with endotoxins from the Enterobacteriaceae, some quantitative differences in their biological potencies were observed. Brucella toxins were relatively innocuous in tests for pyrogenicity in rabbits and lethality for chick embryos. In nonspecific protection tests, Brucella toxin had only 1/75 the potency of E. coli endotoxin in protecting mice against challenge with virulent S. typhi. However, on the basis of the data presented and on the work done previously, we concluded that the heat-stable toxins of B. abortus and B. melitensis were endotoxins.
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The evidence obtained from immuno-gel diffusion, centrifugation, and toxicity studies employing the serum iron assay proved that most of the toxicity in an ether-water brucella endotoxin preparation lies in the slow-diffusing component identified as the biologically active endotoxin. Subsequent destruction of the slow-diffusing component by acid hydrolysis resulted in a corresponding loss of toxicity. Chromate-51 was found to attach almost entirely on the slow-diffusing biologically active component and, hence, is a valid label for endotoxin derived from smooth Brucella abortus by the ether-water method.
The use of whole-cell antigens in agglutination and agglutinin-absorption tests showed that the organism causing abortion in dogs is similar to rough Brucella abortus, B. melitensis, and B. ovis, but different from smooth Brucella cultures. Water-soluble antigens obtained by ultrasonic treatment and examined by immuno-electrophoresis and gel diffusion show extensive cross-reactions within the genus Brucella, but little or no cross-reaction with similar antigens from other gram-negative genera in the family Brucellaceae. The dog organism showed near identity with rough and smooth Brucella cultures on the basis of immuno-gel diffusion tests with water-soluble antigens, but it lacked the lipopolysaccharide-endotoxin associated with the agglutinogen of smooth brucellae. These findings support the proposal of Carmichael and Bruner for the designation of a new species, "Brucella canis."
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.
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Immune sera were prepared in rabbits by the injection of living and acetone-killed cells of Brucella ovis and smooth and rough B. melitensis. The use of whole-cell antigens in agglutination and agglutinin-absorption tests revealed little relationship between B. ovis and smooth B. melitensis, although there was extensive cross-agglutination between B. ovis and rough B. melitensis. The use of water-soluble antigens prepared from ultrasonically treated cells of the three strains revealed extensive cross-reactions in indirect hemagglutination, agar gel precipitation, and immunoelectrophoresis tests, as well as in allergic skin tests in rabbits. The most definitive results were obtained with the immunoelectrophoresis technique. B. ovis antigen produced at least 11 lines with its homologous serum. All were removed by absorption of the serum with rough B. melitensis antigen. All but three were removed by absorption with smooth B. melitensis antigen. Smooth B. melitensis antigen produced 11 lines with its homologous serum, and all but 3 were removed by absorption with B. ovis antigen. Rough B. melitensis produced nine lines with its homologous serum, and eight were removed by B. ovis antigen. The extensive cross-reactions between soluble antigens of B. ovis and B. melitensis are added evidence that B. ovis belongs in the genus Brucella.