Murine macrophage interleukin-1 release by capsularlike serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans.

Serotype-specific polysaccharide antigens (SPAs) were extracted from whole cells of Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a), Y4 (serotype b), and NCTC 9710 (serotype c) by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. Y4 SPA induced interleukin-1 (IL-1) release by P388D1 murine macrophages. Polymyxin B had virtually no effect on the release of IL-1. Rabbit anti-murine IL-1 serum strongly suppressed the proliferation of C3H/HeJ mouse thymocytes induced with the culture supernatants of Y4 SPA-stimulated P388D1 cells and a submitogenic dose of concanavalin A. Gel filtration of the culture supernatants of Y4 SPA-stimulated macrophages on Sephacryl S-200 showed that an IL-1 peak at a point corresponding to approximately 16.5 kDa was eluted. The ability of SPAs from strains ATCC 29523 and NCTC 9710 to induce the release of IL-1 was lower than that of Y4 SPA. The IL-1-releasing ability of serotype a and c antigens was enhanced by deacetylation of both polysaccharides, suggesting that acetyl groups of these antigens might hinder the interaction between the antigens and macrophages.     

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.     

Cross-reactivity and antigenic heterogeneity among Actinobacillus pleuropneumoniae strains of serotypes 4 and 7.

Actinobacillus pleuropneumoniae strains of serotypes 4 and 7 were studied for their antigenic properties by means of agglutination, coagglutination, indirect hemagglutination, immunodiffusion, and counterimmunoelectrophoresis tests. Strains of serotype 4 showed cross-reactivity with those of serotype 7 in various serological tests. Serotype 7 strains were antigenically heterogeneous and shared common antigens with several other serotypes. By using boiled whole-cell saline extract as the antigen in the immunodiffusion test, serotype 7 strains could be divided into four subgroups. Subgroup I strains did not have antigens in common with other serotypes, whereas subgroup II strains had antigens in common with serotype 4; subgroup III strains had antigens in common with serotype 10, and subgroup IV had antigens in common with serotypes 1, 9, and 11. The indirect hemagglutination test using unheated whole-cell saline extract as the antigen detected serotype-specific activity. Quantification of serotype-specific and group-specific antigens by coagglutination and immunodiffusion tests was found useful for identifying strains that belonged to serotype 4 or 7.     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.     

Novel apoptosis-inducing activity in Bacteroides forsythus: a comparative study with three serotypes of Actinobacillus actinomycetemcomitans

Bacteroides forsythus, which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that B. forsythus produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from B. forsythus, the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with B. forsythus, the same analyses were applied to two strains with different serotypes of Actinobacillus actinomycetemcomitans, serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of A. actinomycetemcomitans serotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did B. forsythus, while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the A. actinomycetemcomitans serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while B. forsythus induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from B. forsythus and the A. actinomycetemcomitans serotype a strain which induces apoptotic cell death.     

Heterogeneity of Actinobacillus actinomycetemcomitans strains in various human infections and relationships between serotype, genotype, and antimicrobial susceptibility

Actinobacillus actinomycetemcomitans, an oral pathogen, only occasionally causes nonoral infections. In this study 52 A. actinomycetemcomitans strains from 51 subjects with nonoral infections were serotyped and genotyped by arbitrarily primed PCR (AP-PCR) to determine whether a certain clone(s) is specifically associated with nonoral infections or particular in vitro antimicrobial susceptibility patterns. The promoter structure of leukotoxin genes was additionally investigated to find the deletion characteristic of highly leukotoxic A. actinomycetemcomitans strains. The nonoral A. actinomycetemcomitans strains included all five known serotypes and nonserotypeable strains, the most common serotypes being b (40%) and c (31%). AP-PCR distinguished 10 different genotypes. A. actinomycetemcomitans serotype b strains were more frequently found in blood samples of patients with bacteremia or endocarditis than in patients with focal infections. One AP-PCR genotype was significantly more frequently found among strains originating in focal infections than in blood samples. Resistance to benzylpenicillin was significantly more frequent among A. actinomycetemcomitans serotype b strains than among strains of other serotypes. No differences in the leukotoxin gene promoter region or benzylpenicillin resistance between nonoral and oral A. actinomycetemcomitans strains were observed. Nonoral A. actinomycetemcomitans strains showed great similarity to the oral strains, confirming that the oral cavity is the likely source of nonoral A. actinomycetemcomitans infections. The predominance of serotype b strains in endocarditis and bacteremia supports the hypothesis of a relationship between certain A. actinomycetemcomitans clones and some nonoral infections. The mechanisms behind the exceptionally high rate of occurrence of benzylpenicillin resistance among A. actinomycetemcomitans serotype b strains are to be elucidated in further studies.     

Antigenically diverse reference strains and autologous strains of Actinobacillus actinomycetemcomitans are equally efficient antigens in enzyme-linked immunosorbent assay analysis

Actinobacillus actinomycetemcomitans is a major pathogen in periodontitis. Data on the clinical relevance of serum immunoglobulin G (IgG) antibody levels against this species are controversial. The aim of the present study was to elucidate how different strains used as antigens in enzyme-linked immunosorbent assay (ELISA) influence the detection of individuals with elevated serum IgG levels against A. actinomycetemcomitans. We hypothesized that the highest antibody levels are targeted to the autologous strains. A total of 19 strains-six antigenically diverse reference strains (serotypes a through e and a nonserotypeable strain) and 13 serotyped autologous strains-were used as whole-cell antigens in ELISA. Serum samples were from 26 untreated adult patients with periodontitis, whose subgingival bacterial samples were either culture positive (n = 13) or culture negative (n = 13) for A. actinomycetemcomitans, and from 10 culture-negative nonperiodontitis subjects. The highest individual (P < 0.05) IgG levels against the reference strains were most commonly against serotypes a and b in patients and against serotype c in nonperiodontitis subjects. The culture-positive patients had the highest (P < 0.05) IgG antibody levels against their autologous strains and against the reference strains of the same serotype. On the contrary, for these patients the levels of antibody against the reference strains of other serotypes were comparable to those of the nonperiodontitis subjects. The results indicated that the serum IgG antibody levels against A. actinomycetemcomitans strongly depend on the strains used as antigens in the ELISA. Elevated serum IgG levels against A. actinomycetemcomitans can be detected equally well using either the autologous strains or a variety of antigenically diverse reference strains as antigens.     

Characterization of an inducible bacteriophage from a leukotoxic strain of Actinobacillus actinomycetemcomitans.

A bacteriophage, designated phi Aa17, was isolated by mitomycin C induction from leukotoxic Actinobacillus actinomycetemcomitans strains 651. Electron microscopy of the virus revealed particles with regular, nonelongated, polyhedral heads, and tails consisting of a contractile sheath and core. Spikes emanated from the base of the tail. The head had a diameter of 70 nm. The fully extended tail sheath had a length of 127 nm and a diameter of 22 nm. In its contracted form, the tail sheath measured 47 nm in length and 25 nm in diameter. The phage had a buoyant density of 1.370 in CsCl, and its genome was found to be double-stranded DNA. A single-cycle growth curve revealed that the phage had a latent period of 30 min and a burst size of 435 PFU per cell. The host range of the phage was examined, and A. actinomycetemcomitans strains ATCC 29523 and ATCC 29524 were found to be phage sensitive, whereas strains Y4, ATCC 29522, 2043, 652, 651, 627, 2097, N27, 2112, and 511 were resistant. The host range of this virus does not suggest any association between the phage and leukotoxin production.     

Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients

The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.     

Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA.

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.     

Identification of Actinobacillus actinomycetemcomitans serotypes by multiplex PCR

Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify Actinobacillus actinomycetemcomitans serotypes a to e using the multiplex PCR. This method may be useful for serotype-specific genotyping rapidly and directly from clinical samples containing various organisms.     

Characterization of the O-polysaccharide structure of lipopolysaccharide from Actinobacillus actinomycetemcomitans serotype b.

We previously reported that the serotype b antigen of Actinobacillus actinomycetemcomitans is a constituent of the polysaccharide region of lipopolysaccharide (LPS) and contains significant amount of the neutral sugars rhamnose and fucose (M. Wilson and R. Schifferle, Infect. Immun. 59:1544-1551, 1991). In the present study, we determined the structure of the O antigen of A. actinomycetemcomitans Y4 (serotype b) LPS. Aqueous phase LPS was obtained from a phenol-water extract of A. actinomycetemcomitans Y4. This material was found to react with rabbit polyclonal antiserum to serotype b but not with antisera specific for other A. actinomycetemcomitans serotypes. Analyses revealed that the O polysaccharide of Y4 LPS consists of a polymer of trisaccharide repeating units composed of D-Fuc, AL-Rha, and D-GalNAc residues. An identical structure was obtained for the O polysaccharide of LPS from A. actinomycetemcomitans JP2, another serotype b strain. These results indicate that the serotype b antigen of A. actinomycetemcomitans is defined by a trisaccharide repeating unit present in the O polysaccharide of LPS.     

Cloning of the Gene Encoding the Actinobacillus actinomycetemcomitans Serotype b OmpA-Like Outer Membrane Protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

Serotyping of Canadian isolates of Treponema hyodysenteriae and description of two new serotypes.

A total of 30 isolates of Treponema hyodysenteriae collected in the Saint-Hyacinthe (Quebec, Canada) area were serotyped by agar gel double immunodiffusion by using extracted lipopolysaccharide and hyperimmune rabbit antisera. Only 17% (5 of 30) of the isolates were typed with antisera specific for each of the seven known serotypes of T. hyodysenteriae. Antisera raised against 11 untypeable local isolates were then produced and tested against each lipopolysaccharide extract. Results showed two serologically distinct groups among 21 of the 25 untypeable isolates. The isolates in each group shared identical antigens. No detectable reactions could be observed between antisera raised against these 11 isolates and the antigens extracted from 7 reference serotype strains. On the basis of these results, two new serotypes of T. hyodysenteriae, serotypes 8 and 9, are proposed. We also propose isolate FM 88-90 as the reference strain for serotype 8 and isolate FMV 89-3323 as the reference strain for serotype 9. These two new serotypes, which represented 70% of the isolates tested, seem to be the major serotypes found in the province of Quebec.     

Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects.

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.     

Molecular genotyping of isolates of Actinobacillus actinomycetemcomitans by pulsed-field gel electrophoresis after separate digestion with Sse8387I and XhoI

Genomic DNA from 18 Japanese clinical isolates of Actinobacillus actinomycetemcomitans was obtained from six periodontitis patients and analyzed using pulsed-field gel electrophoresis (PFGE) after separate digestion with Sse8387I and with XhoI. Three isolates from an identical patient were found to share an identical PFGE profile, and isolates from distinct patients were found to have PFGE profiles distinctly different from each other. Consequently, the 18 Japanese clinical isolates were discriminated into six distinct genotypes by means of PFGE.The genomic DNA from the other six reference strains (ATCC33384, ATCC43717, ATCC 43718, JCM2434, JCM2435 and Nig-1) was discriminated into six genotypes by the same PFGE methodology, and these six genotypes were found to be distinctly different from the six genotypes of the 18 Japanese clinical isolates described above.Serotyping demonstrated three PFGE genotypes in the serotype a strains, four the serotype b strains and three the serotype c strains.The present results clearly suggest that the PFGE procedure after separate digestion with Sse8387I and with XhoI has an excellent discriminatory power amongst strains and has a good genotypability for A. actinomycetemcomitans.     

Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.

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.     

Serum antibody in Actinobacillus actinomycetemcomitans-infected patients with periodontal disease

This study was designed to (i) delineate the characteristics of serum antibody responses to Actinobacillus actinomycetemcomitans in patients with periodontitis who are infected with A. actinomycetemcomitans; irrespective of disease classification; (ii) assess the relationship of the elevated antibody levels to colonization of the oral cavity by A. actinomycetemcomitans; and (iii) describe the serotype distribution of A. actinomycetemcomitans and antibodies to the microorganism in infected patients with various clinical classifications. To compare the levels of various isotype-specific antibodies to the different antigens, studies were performed that allowed quantitation of each isotype-specific antibody in a human reference standard. By using this reference standard, it was shown that the levels of immunoglobulin G (IgG), IgM, and IgA responses to A. actinomycetemcomitans were similar among the infected patients, irrespective of disease classification. Also, we demonstrated that the serum antibody response to serotype b was quantitatively greater in all isotypes. Our findings indicate that b was the most frequent A. actinomycetemcomitans serotype detected in the patients and appears to be capable of initiating a substantial serum IgG antibody response that may contain cross-reactive antibodies to other serotypes of A. actinomycetemcomitans. Generally, in cases in which the response to a single serotype was elevated, only that type of A. actinomycetemcomitans was detected in the plaque. Individuals exhibiting elevated antibodies to multiple serotypes were most consistently colonized by the serotype b microorganism. This study represents the first report detailing the distribution of IgG subclass antibodies to A. actinomycetemcomitans in periodontal disease. The results demonstrated that the primary responses of patients with periodontitis to A. actinomycetemcomitans were of the IgG1 and IgG3 subclasses, which is consistent with elicited responses to protein antigens. In contrast, the primary subclass response in normal subjects was limited to the IgG2 subclass and may represent broader cross-reactivity to polysaccharide antigens-lipopolysaccharide from the bacteria.     

Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies.     

Aggregatibacter actinomycetemcomitans serotypes infections and periodontal conditions: a two-way assessment

This study investigated a large population of individuals positive for A. actinomycetemcomitans and performed a two way analysis assessing the relation between the different serotypes of the bacterium and periodontal conditions. The serotypes analysis (serotypes a, b, c, d, e, f) showed that out of the 204 selected individuals positive for A. actinomycetemcomitans, 152 were positive for a single serotype, 27 showed a variable mixed infection and 25 individuals were not positive for any of the serotypes tested. Serotypes a, b and c were largely found (98%), and serotype c was the most prevalent. Serotypes d, e, and f were either not detected or relatively rare. It was also verified that in non-periodontitis individuals, serotypes a and c were more prevalent (p<0.05); in individuals with mild or moderate/severe chronic periodontitis serotype c was also more common (p<0.05); and aggressive periodontitis subjects showed high prevalence of both serotypes b and c (p<0.05). In conclusion, our study showed that serotype c was the most prevalent in both diseased and healthy subjects. Aggressive periodontitis subjects were not exclusively associated with A. actinomycetemcomitans serotype b. Non-typeable strains were either not detected or were relatively infrequent, and serotypes d and f were not detected in the examined Brazilian population.