Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects.
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Most juvenile periodontitis patients respond to infection by Actinobacillus actinomycetemcomitans by producing serum antibodies. Specific antigens inducing the humoral immune response have not been identified, nor has the role of the resulting antibodies in disease progression been determined. Adsorbed and unadsorbed sera from juvenile periodontitis patients and normal subjects were analyzed by enzyme-linked immunosorbent assay and Western blots (immunoblots), using digested and undigested bacterial sonicates and French pressure cell fractions to determine the biochemical class, cross-reactivity, and cellular location of the antigens in different A. actinomycetemcomitans serotypes. Antigens detected by using high-titer sera included the following: (i) serotype-specific nonprotein material located on the cell surface, (ii) soluble-fraction proteins showing highly variable antibody binding, (iii) cross-reactive proteins, and (iv) a protein present in soluble and cell wall fractions and immunopositive for all sera tested. In addition, one apparently nonprotein component that was enriched in the cell wall fraction was observed. Sera with high immunoglobulin G titers to one, two, three, or none of the three A. actinomycetemcomitans serotypes were observed. There was a high degree of variation from one patient to another in the humoral immune response to serotype-specific and cross-reactive antigens. As demonstrated by whole-cell adsorption experiments, the serotype-specific surface antigen accounted for approximately 72 to 90% of the total antibody-binding activity for sera with titers greater than 100-fold above background, while cross-reactive antigen accounted for less than 28%. Antibody binding the whole-cell sonicate for high-titer sera was inhibited 90% by lipopolysaccharide from the same serotype, strongly suggesting that lipopolysaccharide is the immunodominant antigen class. (+info)
Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.
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A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism. (+info)
The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.
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Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS. (+info)
Emended description of Actinobacillus capsulatus Arseculeratne 1962, 38AL.
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The taxonomic position of Actinobacillus capsulatus, a member of the family Pasteurellaceae found in rabbits, hares and hamsters, has been challenged. 16S rRNA gene (rrs) sequence data show the species to be heterogeneous. Using a polyphasic approach, 23 strains that were identified previously as belonging, or closely related, to A. capsulatus were analysed. Eighty characters were included in the phenotypic analysis. Phylogenetic analysis was done based on rrs, rpoB, infB and recN sequences. In addition, the recN sequence similarities were used to calculate the whole-genome sequence relatedness of all strains investigated as well as that with other members of the family Pasteurellaceae. The phenotypic analysis allowed identification of five groups. The major group of 17 strains could be classified as A. capsulatus. Two hamster isolates were closely related to A. capsulatus but differed in a few characters. Single isolates from a rabbit and snowshoe-hare were phenotypically related to Actinobacillus suis. One rabbit isolate was related to the genus Mannheimia, while another isolate could not be classified phenotypically with known taxa. The phylogenetic analysis confirmed the phenotypic grouping. In contrast to the rrs-based tree, the A. capsulatus strains clustered unambiguously with the type species and related species of the genus Actinobacillus in the rpoB-, infB- and recN-based trees. Genome similarity comparison using recN finally confirmed the high genomic relationship of the A. capsulatus strains with the type species and related species of the genus Actinobacillus and allowed a clear assignment of the other unrelated strains to the phenotypic and phylogenetic clusters outlined. The present findings allow the description of A. capsulatus to be emended and separate it more clearly from other species, both phenotypically and genotypically. The type strain of A. capsulatus is CCUG 12396(T) (=Frederiksen 243(T)=ATCC 51571(T)=NCTC 11408(T)=CIP 103283(T)). (+info)
Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.
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Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes. (+info)
Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria.
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Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 x 10(6) and 7.1 x 10(6) transformants/mug DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria. (+info)
Actinobacillus spp. and related bacteria in infected wounds of humans bitten by horses and sheep.
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We describe the isolation of Actinobacillus lignieresii and an A. equuli-like bacterium from an infected horse-bite wound in a 22-year-old stable foreman and A. suis from a bite injury in a 35-year-old man who had been attacked by a horse. A. lignieresii was also isolated in pure culture from an infected sheep-bite wound in a rural worker. These species of the genus Actinobacillus are primarily associated with animals and animal diseases and are rarely isolated from humans. The purpose of this report is to raise awareness of the possible occurrence of Actinobacillus spp. in bite wounds inflicted by farm animals and to discuss the difficulties encountered in the identification of species of Actinobacillus and related bacteria. (+info)
Effect of adoptive transfer of cloned Actinobacillus actinomycetemcomitans-specific T helper cells on periodontal disease.
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Previously we isolated several Actinobacillus actinomycetemcomitans-specific T-cell clones from the spleens and lymph nodes of immunized Rowett rats. These clones were characterized as W3/13+, W3/25+, OX8-, and OX22-, suggesting a T helper (Th) phenotype. In the current experiments, 10(6) cells from a single A. actinomycetemcomitans-specific clone (A3) were adoptively transferred to a group (AaTh; n = 13) of normal heterozygous rats (rnu/+) at 28 days of age. A second group received no T cells (AaNT; n = 15), and a third group also received no T cells (NAaNT, n = 11). Beginning 1 day after transfer, the first and second groups were infected orally with A. actinomycetemcomitans for 5 consecutive days. The presence of infection was confirmed immediately after challenge and after 5 months, when the experiments were ended. Significantly higher numbers of lymphocytes were recovered from the gingival tissues of the first group than from those of either of the other groups. Also, this group showed significantly elevated (P less than 0.01) serum immunoglobulin G and immunoglobulin M antibody to A. actinomycetemcomitans in an enzyme-linked immunosorbent assay when compared with both other groups. Bone loss was significantly lower (P less than 0.01) in recipients of A. actinomycetemcomitans-specific cloned cells when compared with the other infected group and was approximately equal to the bone loss of the uninfected group. These results are consistent with the hypothesis that T-cell regulation can affect periodontal disease. In this regulation, T helper cells appear to interfere with periodontal bone loss. (+info)