Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.     

Protective immunization against experimental Bacteroides (Porphyromonas) gingivalis infection.

The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.     

Immunoglobulin G response to subgingival gram-negative bacteria in human subjects

Serum and gingival crevicular fluid from normal healthy adults and patients with periodontitis were screened for immunoglobulin G antibodies to antigens from Bacteroides gingivalis 381, Bacteroides intermedius 24, Bacteroides loescheii ATCC 15930, Fusobacterium nucleatum ATCC 25586, Eikenella corrodens 1073, Actinobacillus actinomycetemcomitans ATCC 29522, and Capnocytophaga sp. strain M-12. Immunoglobulin G antibody titers to the antigens were measured by an enzyme-linked immunosorbent assay. The antibody levels to B. gingivalis in serum and gingival crevicular fluid were significantly higher in the samples from patients with periodontitis than in samples from healthy individuals. Although there were individual differences within patient groups, a positive correlation (P less than 0.01) was found between the serum immunoglobulin G levels to B. gingivalis and the development of periodontitis. The antibodies to F. nucleatum (P less than 0.05), E. corrodens (P less than 0.05), and A. actinomycetemcomitans were slightly higher in patients with periodontitis than in normal subjects. There were no remarkable differences between the two groups in titers to B. intermedius, B. loescheii, and Capnocytophaga sp.     

Production of monoclonal antibodies that recognize specific and cross-reactive antigens of Bacteroides gingivalis

Four monoclonal antibodies directed against Bacteroides gingivalis were established by hybridoma technology. Their reactivity against B. gingivalis, Bacteroides intermedius, and Bacteroides melaninogenicus was detected by the enzyme-linked immunosorbent assay. Three monoclonal antibodies specifically reacted with B. gingivalis. One recognized antigens that were cross-reactive between B. gingivalis and B. intermedius. These monoclonal antibodies provide new tools for antigenic analysis of B. gingivalis.     

Serological identification of oral Bacteroides spp. by enzyme-linked immunosorbent assay

A rapid method for identifying black-pigmented oral Bacteroides spp. is described. Species-specific rabbit antisera to Bacteroides gingivalis, B. intermedius, and B. melaninogenicus were used in an enzyme-linked immunosorbent assay to identify clinical isolates of black-pigmented Bacteroides spp. from humans. The results showed excellent agreement with biochemical identification of B. gingivalis and B. intermedius. Only 36% of the B. melaninogenicus isolates were identified with the enzyme-linked immunosorbent assay, suggesting that this group of black-pigmented Bacteroides spp. is made up of more than one serotype. The serological enzyme-linked immunosorbent assay should enable rapid identification of black-pigmented Bacteroides spp. isolated from sites of oral diseases and may also be used to identify the presence of these organisms in complex bacterial mixtures from oral sites.     

Long-wave UV light fluorescence for identification of black-pigmented Bacteroides spp.

Black-pigmented Bacteroides strains were grown on blood agar, and the colonies were evaluated for fluorescence from long-wave UV light. Most test strains of Bacteroides melaninogenicus subsp. intermedius exhibited a brilliant red fluorescence. B. melaninogenicus subsp. melaninogenicus fluoresced mostly red-orange. Bacteroides asaccharolyticus showed a yellow or red fluorescence. The intensity of the Bacteroides fluorescence weakened when the black pigment of the colonies developed. In contrast, neither young nor old colonies of the oral species Bacteroides gingivalis displayed fluorescence. Since B. gingivalis can produce severe oral infections and also can seed to nonoral sites, awareness of the inability of this organism to fluoresce is important for microbiologists utilizing UV light fluorescence to screen for black-pigmented Bacteroides spp. The present data also indicate that UV light fluorescence may be a rapid method of distinguishing some black-pigmented Bacteroides spp.     

Antigenic heterogeneity of Bacteroides intermedius as recognized by monoclonal antibodies.

Four hybrid cell lines secreting monoclonal antibodies against antigens of Bacteroides intermedius were generated by fusing murine NSI cells with splenocytes from a rat immunized with B. intermedius strain OMZ248. An enzyme-linked immunosorbent assay was used to analyze the distribution of the recognized antigens on 39 strains from various Bacteroides species and on 5 strains from other genera. Only Bacteroides species B. intermedius, B. loescheii, B. melaninogenicus, and B. corporis were found to express at least one of the recognized antigens. Strains of the two asaccharolytic black-pigmenting Bacteroides species were negative. Among the strains capable of binding to one or more of the monoclonal antibodies, five groups with different reactivity patterns could be distinguished. Two of the monoclonal antibodies were specific for B. intermedius. The B. intermedius strains were metabolically almost identical, expressed at least three of the recognized antigens, and fell into three distinct antibody reactivity groups, suggesting a tentative separation of this species into three new serogroups. Oral and nonoral isolates of B. intermedius were, however, not distinguished by the monoclonal antibodies. One monoclonal antibody was directed against an antigen strongly expressed on all saccharolytic black-pigmenting Bacteroides strains tested so far, thus confirming the previously noted antigenic relationship between the species which had emerged from the former B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus groups.     

The use of monoclonal antibodies to detect Bacteroides gingivalis in biological samples.

Hybridomas were established which produce monoclonal antibodies specific for Bacteroides gingivalis, a pathogen associated with human periodontal disease. Spleen cells from BALB/c mice immunized with formalinized B. gingivalis were fused with Sp2/0-Ag14 myeloma cells. Of 1,050 wells with positive growth, 60 contained antibody reactive with the immunizing strain of B. gingivalis by enzyme-linked immunosorbent assay. Expansion of these cultures and cloning by limited dilution resulted in 28 clones which reacted with B. gingivalis but not with other orals and nonoral black-pigmented Bacteroides species or any of 29 representative strains of other oral bacteria. Of these 28 clones, 14 were also specific for B. gingivalis by indirect immunofluorescence microscopy. One clone, BBG-12 producing immunoglobulin G2b(kappa), was chosen to identify B. gingivalis in subgingival plaque because of its high reactivity in indirect immunofluorescence assays. This antibody reacted strongly with all 17 representative B. gingivalis strains obtained from diverse sources. Furthermore, when this reagent was applied to subgingival plaque samples, B. gingivalis was stained with high specificity and low background fluorescence, indicating that it may be useful for clinical identification of this organism.     

Monoclonal antibody-coated latex agglutination assay for identification of Actinobacillus actinomycetemcomitans

Three monoclonal antibodies (MAbs) to lipopolysaccharide of Actinobacillus actinomycetemcomitans strain Y4 (serotype b) and eight MAbs to a serotype b-specific polysaccharide antigen of strain Y4 were obtained. Latex particles sensitized with an MAb to the Y4 lipopolysaccharide produced a positive agglutination with whole cells of all three serotypes of A. actinomycetemcomitans, but not with Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Porphyromonas (Bacteroides) gingivalis, "Bacteroides" intermedius, Fusobacterium nucleatum and Escherichia coli. On the other hand, latex particles sensitized with an MAb to the serotype b-specific polysaccharide antigen agglutinated with whole cells of serotype b A. actinomycetemcomitans and P. gingivalis, but not with heated and trypsinized cells of P. gingivalis. The simple and rapid latex agglutination assay using MAbs may be useful for the identification of A. actinomycetemcomitans.     

Antigenic characteristics and serological identification of 10 black-pigmented Bacteroides species.

Strains of 10 black-pigmented Bacteroides species were serologically characterized using absorbed and unabsorbed rabbit antisera. An agglutination test using intact cells or heated cells (100 degrees C for 60 min) from each species and unabsorbed antisera revealed only homologous reactions with little or no reactivity in heterologous assays. Immunodiffusion tests using sonicated antigen demonstrated that Bacteroides gingivalis, B. endodontalis, B. asaccharolyticus, B. macacae, and B. levii are antigenically distinct. Strains of B. gingivalis, B. endodontalis, and B. asaccharolyticus were also clearly identified by the indirect immunofluorescent antibody method. B. intermedius, B. corporis, B. loescheii, B. melaninogenicus, and B. denticola possessed common antigens; however, species-specific antigens detectable with immunoabsorbed antisera were also demonstrated. B. intermedius strains isolated from the human oral cavity included at least two serogroups. In each black-pigmented Bacteroides species, lipopolysaccharide constituted one of the species-specific antigens.     

Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis

Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin.     

Application of monoclonal antibodies to the detection of black-pigmented Bacteroides spp. in subgingival plaques by immunoslot blot assay

The aim of the present study was to assess the application of monoclonal antibodies to the detection of black-pigmented Bacteroides spp. in subgingival plaques by immunoslot blot assay. Subgingival plaque samples from adult periodontal patients were examined by immunoslot blot assay with monoclonal antibodies that specifically recognize Bacteroides gingivalis, Bacteroides intermedius serogroups I and II, and Bacteroides melaninogenicus. The assay can detect specifically these Bacteroides spp. in the subgingival plaques. Therefore, we investigated the distribution of these Bacteroides spp. in the subgingival plaques of patients classified by Russell's periodontal index. Reactivities of their plaques with monoclonal antibodies toward B. gingivalis and B. intermedius serogroup I were clearly related to the severity of the periodontal disease, but this was not the case with B. intermedius serogroup II and B. melaninogenicus. These results indicate that this immunoslot blot assay using monoclonal antibodies toward these Bacteroides spp. provides simple detection and monitoring of these organisms in periodontal patients.     

Histopathological changes in the hind foot of the mouse induced by black-pigmented Bacteroides strains

The pathogenic potential of black-pigmented Bacteroides strains was studied in an animal model in which the effect on bone tissue could be determined. Bacteria suspended in agar were injected subcutaneously in the left hind paw of a mouse. After 3-5 days, B. gingivalis strain HG 66 had caused a massive infiltration with polymorphonuclear cells, destruction of the periosteum on the metatarsals and bone resorption by osteoclasts. After 7 days only a few osteoclasts remained and reactive bone formation was observed. In a comparative study with strains of several black-pigmented Bacteroides species, differences in bone resorbing potential were seen. B. gingivalis strains caused severe inflammation which resulted in bone resorption. Strains of B. asaccharolyticus, B. endodontalis, B. intermedius, B. melaninogenicus and B. loeschei caused less inflammation and less bone resorption. Killed bacteria or agar alone caused a relatively mild inflammation and no bone resorption.     

Humoral immune response to oral microorganisms in periodontitis.

Serum antibody titers from patients with periodontitis were compared with those from periodontally healthy subjects. With the micro-enzyme-linked immunosorbent assay, immunoglobulin G (IgG), IgA, and IgM antibody titers to isolates of Streptococcus sanguis, Actinomyces viscosus, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides ochraceus, and Fusobacterium nucleation were determined. Antibody titers of the IgG and IgA classes to B. melaninogenicus, B. ochraceus, F. nucleatum, and S. sanguis were found to be significantly higher in the controls than in the patients. No correlations were found with serum IgM titers. These findings indicate that periodonitis may be associated with depressed antibacterial serum antibody titers of the IgG and IgA classes.     

Inhibition of gingival fibroblast growth by Bacteroides gingivalis.

Human gingival fibroblasts were exposed in culture to cell extracts of different black-pigmented Bacteroides species, and their growth was monitored by determining thymidine uptake and counting cells. Of the Bacteroides species tested (B. gingivalis, B. asaccharolyticus, and B. intermedius), B. gingivalis gave the extract with the strongest inhibitory effect on fibroblast thymidine uptake. Linear inhibition reaching 80% of the control level was obtained with a dose of 100 micrograms of B. gingivalis extract protein per ml. The effect of B. asaccharolyticus resembled that of B. gingivalis, but even at the highest dose tested B. intermedius had only a slight inhibitory effect. When fibroblasts were counted after 2- and 4-day exposures to B. gingivalis extracts, a clear depression in the number of fibroblasts was found. The effects of extracts obtained from early and late growth phases of B. gingivalis cultures were similar. A fraction of B. gingivalis consisting essentially of lipopolysaccharides (LPSs) was obtained by degrading the extract proteins with proteinase K. Silver staining of polyacrylamide gels revealed a LPS pattern with a molecular mass ranging from 37 to 60 kilodaltons. This LPS-rich fraction caused inhibition of thymidine uptake by gingival fibroblasts similar to that caused by the native extract alone. Thus, the inhibition of gingival fibroblast growth by B. gingivalis appeared to be LPS mediated. This inhibitory effect of B. gingivalis on oral fibroblast growth may be a virulence factor of this bacterium.     

Ability of oral bacteria to degrade fibronectin.

The fibronectin-degrading ability of 116, mainly oral, strains was assayed by using plasma-derived fibronectin adsorbed to a polystyrene surface. Ability to degrade fibronectin was revealed in strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii, Staphylococcus aureus, Staphylococcus epidermidis, Peptococcus prevotii, Clostridium sporogenes, and Propionibacterium acnes. The fibronectinolytic activity of subgingival bacteriological samples was found to be related to the presence of B. gingivalis and B. intermedius. In addition, strains of the nonoral Bacteroides species B. asaccharolyticus and B. fragilis showed fibronectin-degrading ability. No such ability was detected in the oral strains tested of Streptococcus, Veillonella, Actinomyces, Lactobacillus, Actinobacillus, Capnocytophaga, Fusobacterium, or Haemophilus species.     

Effects of estradiol and progesterone on Bacteroides melaninogenicus and Bacteroides gingivalis.

Bacteroides melaninogenicus subsp. intermedius increases in the subgingival microflora during pregnancy. These studies evaluated direct interactions between hormonal steroids and oral Bacteroides species. Resting cell suspensions of pure cultures of plaque organisms were incubated anaerobically with [14C]estradiol and [14C]progesterone. Uptake of labeled compound per microgram of bacterial protein was determined by thin-layer chromatography and liquid scintillation counting. B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus took up 2.6 x 10(-4) to 5.4 x 10(-4) mumol of estradiol or progesterone per microgram of cell protein. Minimal steroid uptake was observed with B. gingivalis and five other organisms. Uptake of steroids by B. melaninogenicus subsp. intermedius was temperature dependent and resulted in a labeled product as detected on thin-layer chromatography. Growth curves indicated that intermedius and melaninogenicus subspecies of B. melaninogenicus but not B. gingivalis could substitute progesterone or estradiol for vitamin K, an essential growth factor. Growth of B. melaninogenicus subsp. intermedius in steroids was concentration dependent. Addition of fumarate to resting cells of B. melaninogenicus subspecies as well as B. gingivalis increased steroid uptake by 70 to 500% and resulted in the gas-liquid chromatographic detection of succinate. Cultures given fumarate alone or steroids alone produced no succinate. Steroids appeared to directly interact with the fumarate reductase system and foster the growth of B. melaninogenicus subsp. intermedius. This interaction may be of ecological significance.     

Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components.

Selected cell envelope components of Porphyromonas gingivalis were tested in a BALB/c mouse model in an attempt to elucidate further the outer membrane components of this putative oral pathogen that might be considered as virulence factors in host tissue destruction. Lipopolysaccharide (LPS), outer membrane, and outer membrane vesicles of P. gingivalis W50, ATCC 53977, and ATCC 33277 were selected to examine an immunological approach for interference with progressing tissue destruction. Mice were actively immunized with heat-killed (H-K) or Formalin-killed (F-K) whole cells or with the outer membrane fraction, LPS, or outer membrane vesicles of the invasive strain P. gingivalis W50. The induction of invasive spreading lesions with tissue destruction and lethality were compared among different immunization groups in normal, dexamethasone-treated (dexamethasone alters neutrophil function at the inflammatory site), and galactosamine-sensitized (galactosamine sensitization increases endotoxin sensitivity) mice after challenge infection with the homologous strain (W50) and heterologous strains (ATCC 53977 and ATCC 33277). Enzyme-linked immunosorbent assay analyses revealed significantly elevated immunoglobulin G and M antibody responses after immunization with H-K or F-K cells or the outer membrane fraction compared with those of nonimmunized mice. The killed whole-cell vaccines provided significantly greater protection against challenge infection in normal mice (decreased lesion size and death) than did either the outer membrane fraction or LPS immunization. The lesion development observed in dexamethasone-pretreated mice was significantly enhanced compared with that of normal mice after challenge with P. gingivalis. Immunization with P. gingivalis W50 provided less protection against heterologous challenge infection with P. gingivalis ATCC 53977; however, some species-specific antigens were recognized and induced protective immunity. Only viable P. gingivalis induced a spreading lesion in normal, dexamethasone-treated, or galactosamine-sensitized mice; F-K or H-K bacteria did not induce lesions. The F-K and outer membrane vesicle immunization offered greater protection from lesion induction than did the H-K immunogen after challenge infection simultaneous with galactosamine sensitization. The H-K cell challenge with galactosamine sensitization produced 100% mortality without lesion induction, suggesting that LPS or LPS-associated outer membrane molecules were functioning like endotoxin. Likewise, P. gingivalis W50 LPS (1 micrograms per animal) administered intravenously produced 80% mortality in galactosamine-sensitized mice. In contrast to the effects of immunization on lesion development, immunization with H-K or F-K cells or LPS provided no protection against intravenous challenge with LPS; 100% of the mice died from acute endotoxin toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey.

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.