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Fatty acid compositions of 7 strains of Rickettsia were analyzed by a on-line GC/MS system. These strains were R. prowazekii E, R. conorii Simko, R. rickettsii R, R. sibirica Barbash and 246, R. sinkiangensis Jinghe, and R. heilungkiangensis 54. The samples were purified by means of the concentrated salt-ether method. There were about 50 peaks in the fatty acid profiles, and 16 of these peaks were determined, i.e. C22:0, C19:0, C18:0, C18:1, C18:2, C17:0, C16:0, C16:1, 3-OH-C14:0, C15:0, C14:0, C13:0, 2-OH-C12:0, C12:0, 2-OH-C10:0, and C11:0. The major fatty acids were the saturated straight chain acids (e.g. C16:0, C18:0, C14:0) and the unsaturated straight chain acids (e.g. C18:1, C18:2, C16:1). Similarities of fatty acid profiles of tested strains were discriminated by the improved Kulik-Vincent method. The result showed that the KV's coefficient of strains Jinghe and 246 was 97.0%, and the KV's coefficient of strains 54 and the others was 81.6-94.6%.

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The fatty acid compositions of selected strains of rickettsiae were studied by gas-liquid chromatography. The profiles of all the rickettsiae except Coxiella burnetii were qualitatively similar. The fatty acid composition of C. burnetii was similar to that of certain Legionella species.

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Eight strains of spotted fever group rickettsiae were studied to gain insight into the extent of variation of their properties. Two standard strains of Rickettsia rickettsii and one strain of Rickettsia conorii were included among the eight for comparison. The molar percentage of guanine plus cytosine for each strain did not differ significantly from that for R. rickettsii, 32.6 +/- 0.7%. Two strains caused extended fever in guinea pigs, one strain caused fever of short duration, and the other strains induced little or no fever. Polyacrylamide gel electrophoresis of the detergent-solubilized rickettsial proteins indicated that the protein content of all strains, except the two strains of R. rickettsii, were different, particularly in the molecular weight range of 40,000 to 60,000. Virulent strains produced large clear plaques in Vero cells monolayers; the strains of low virulence generally produced smaller or more turbid, or both, plaques. On the basis of agglutination reactions with rabbit antisera, the eight strains were placed into five serotypes. These results indicate considerable heterogeneity in properties of spotted fever group rickettsiae in the United States.

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Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

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Purified radioactive rickettsiae were obtained from irradiated and cycloheximide-inhibited L cells, and their proteins were analyzed by polyacrylamide gel electrophoresis. Rickettsial species could be distinguished by comparing the relative mobilities of constituent proteins after migration of two differentially labeled preparations in a single gel. Distinct differences were observed in gel patterns of rickettsiae from the typhus and spotted fever groups, as well as with different species within a group. Rickettsial organisms causing murine and epidemic typhus were clearly distinguished, as were the causative agentsof boutonneuse fever and rickettsialpox. The use of both internal and external molecular weight standards allowed molecular weight estimates for 19 proteins from both Rickettsia prowazekii and Rickettsia conorii. A flexible system for designating rickettsial proteins is proposed that lends itself to modification as more detailed analysis progresses.

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Rickettsia rickettsii was treated with ether and examined by negative-contrast electron microscopy. Group-specific complement-fixing antigen was seen to be originating from the cell wall. The antigen was composed predominately of round particles 10 to 60 nm in diameter. Intact R. rickettsii and antigen from ether-treated organisms were purified by density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. The whole rickettsial cell was composed of a minimum of 30 proteins which ranged in molecular weight from about 23,000 to 155,000. The "soluble" antigen contained nine proteins ranging in molecular weight from about 28,000 to 150,000.

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There is a small but distinct difference in DNA base composition between the typhus and spotted fever groups of rickettsiae. The molar percentages of guanine plus cytosine for Rickettsia prowazeki, R. typhi, and R. canada are approximately 30, for R. rickettsi, R. conori, and R. akari they are about 32.5. The percentage for trench fever rickettsia, Rochalimaea quintana, is 38.6.

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