Original Article
Identification of Spirochetes Related to Treponema pallidum in Necrotizing Ulcerative Gingivitis and Chronic Periodontitis
N Engl J Med 1991; 325:539-543August 22, 1991
- Abstract
- Abstract
Background.
Spirochetes are commonly asciated with periodontal disease, but it is not known whether these treponemes are pathogenic or merely opportunistic. We sought to determine whether spirochetes present in periodontal disease share antigens thought to be unique to spirochetes that are known pathogens.
Methods.
We examined dental plaque from 24 healthy subjects, from ulcerative sites in 17 patients with ulcerative gingivitis, and from areas of involvement in 19 patients with chronic periodontitis, using an immunocyto-chemical technique with monoclonal antibodies against pathogen-specific determinants on 47-kd and 37-kd molecules from Treponema pallidum subspecies pallidum. Serum was tested against T. pallidum by immunoblotting and by serologic assays for syphilis.
Results.
Spirochetes with a pathogen-specific epitope on a 47-kd molecule were not found in plaque samples from any of the 24 healthy subjects, but they were identified in plaque samples from 11 of 17 patients with ulcerative gingivitis (P<0.001) and from 10 of 19 patients with periodontitis (P<0.01). Monoclonal antibodies directed against a 37-kd molecule reacted with spirochetes in plaque samples from 1 of 14 controls, from all 11 patients with gingivitis from whom samples could be obtained (P<0.001), and from 14 of 19 patients with periodontitis (P<0.001). Five of 18 normal subjects had IgG against 47-kd and 37-kd molecules, but none had IgG against 14-kd or 12-kd molecules from T. pallidum subspecies pallidum. Among 19 patients with ulcerative gingivitis, IgG was identified against 47-kd molecules in 15, against 37-kd molecules in 12, against 14-kd molecules in 4, and against 12-kd molecules in 15.
Conclusions.
Media in This Article
Figure 1
Spirochetes in Plaque Samples from Patients with Gingivitis after Identification with Monoclonal Antibodies C2–1 (Panels A and C) and B1A1 (Panels B and D).Figure 2
Immunoblots of Human Serum with IgG against Antigens of T. pallidum Subspecies pallidum.Article Activity
- Article
SPIROCHETES are among the most common bacteria found in subgingival plaque associated with periodontal diseases, but many species that have been observed directly have not been isolated, cultivated, or characterized.1 Patients with acute necrotizing ulcerative gingivitis2 or chronic adult periodontitis3 4 5 have higher levels of serum antibodies to treponemes cultivable from dental plaque than do persons with healthy periodontal tissues, and it is possible that noncultivable spirochetes also stimulate antibodies in patients with these periodontal diseases.
Perhaps the best known group of noncultivable spirochetes are the pathogens associated with the treponematoses: Treponema pallidum subspecies pallidum (venereal syphilis), T. pallidum subspecies pertenue (yaws), T. pallidum subspecies endemicum (endemic syphilis), and T. carateum (pinta). The subspecies of T. pallidum share unique, pathogen-restricted antigens6 7 8 against which monoclonal antibodies have been developed.9 , 10 The purpose of this study was to determine whether spirochetes in dental plaque associated with ulcerative gingivitis or chronic periodontitis share pathogen-restricted antigens with pathogenic treponemes.
Methods
Subjects
Ulcerative gingivitis and periodontitis were diagnosed according to established clinical criteria.11 , 12 All subjects were in good health except for having gingivitis or periodontitis. All subjects in the study reported no history of any spirochetal disease. None had clinical evidence of syphilis, and no subject was considered to be at high risk for syphilis.
Plaque was collected above and below the gingival crest (pooled supragingival and subgingival plaque) from 10 subjects with no evidence of inflammatory periodontal disease (5 women and 5 men; mean [±SD] age, 22.8±3.1 years), and subgingival plaque was collected from another 14 subjects with no clinical evidence of periodontal disease at any site (7 women and 7 men; mean age, 34.1±10.3). Subgingival plaque was collected from 17 patients at sites affected by ulcerative gingivitis (9 women and 8 men; mean age, 27.2±5.7) and from 19 patients at sites affected by moderate-to-severe chronic adult periodontitis (5 women and 14 men; mean age, 47.0±15.0). Although an effort was made to collect uniform samples of plaque, they were not standardized according to weight or other physical characteristics. The samples were dispersed gently in phosphate-buffered saline (pH 7.0), and 20-μl aliquots were deposited on glass slides and air-dried.
Serologic Analysis and Immunoblotting
For serum analysis, blood was collected from 18 subjects with clinically healthy gingiva (11 women and 7 men; mean age, 35.9±11.8 years) and 19 patients with ulcerative gingivitis (8 women and 11 men; mean age, 27.7±7.2). Serum samples from five patients with untreated syphilis (D-51–81, D-112–82, D-208–83, D-275–86, and D-218–88) were obtained from the Centers for Disease Control (Atlanta) and were used as positive controls. Serum samples were not available from the patients with periodontitis.13
Serum samples from 19 patients with ulcerative gingivitis, 18 subjects with healthy gingiva, and 5 persons with syphilis were examined for reactivity with use of the Venereal Disease Research Laboratory test and the fluorescent treponemal antibody absorption test according to recommended procedures.13
Serum samples were also evaluated for IgG directed against antigens of T. pallidum subspecies pallidum (Nichols strain) by immunoblotting as previously described.14
Monoclonal Antibodies
Monoclonal antibody H9–1 reacts with a pathogen-specific epitope on a 47-kd molecule of T. pallidum subspecies pallidum and pertenue.9 , 10 Monoclonal antibody B1A1, also directed against a pathogen-specific determinant on a 47-kd molecule of T. pallidum subspecies pallidum, reacts on immunofluorescence assay and enzyme-linked immunosorbent assay with T. pallidum subspecies pallidum and pertenue but not with T. phagedenis biotype Reiter (Lukehart S: unpublished data). Monoclonal antibody H9–2 reacts with a pathogen-specific epitope on the 37-kd endoflagellar-sheath molecule of T. pallidum.9 , 15 Monoclonal antibody C2–1 reacts with all types of spirochetes tested, including leptospira, borrelia, and cultivable and noncultivable treponema, but not with nonspirochetal bacteria (Neisseria gonorrhoeae or Chlamydia trachomatis).9 , 16
Each monoclonal antibody was tested in the immunocytochemical assay for reactivity with T. phagedenis biotype Reiter (gift of J.N. Miller, UCLA), T. scoliodontum (gift of J.N. Miller), T. vincentii (gift of J.N. Miller), T. refringens (gift of R. George, Centers for Disease Control), Fusobacterium periodonticum (American Type Culture Collection [ATCC] 33693), T. denticola (ATCC 33520 and 33521), T. pectinovorum (ATCC 33763), and T. socranskii (ATCC 35536).
Immunocytochemical Assay
Samples of dried plaque and of dried control bacteria were examined on slides as previously described with biotinylated rabbit antimouse second antibodies and streptavidin—alkaline phosphatase.16 Slides were viewed at a magnification of 1000× by an observer who was unaware of the source of the specimen and monoclonal antibody used. For each specimen, 10 nonoverlapping high-power fields (1000×) were examined for stained spirochetes. A sample was considered positive if one or more stained spirochetes were observed.
Statistical Analysis
The significance of differences in the frequency of positive observations between normal subjects and subjects with gingivitis or periodontitis was determined by chi-square analysis.
Results
Specificities of Monoclonal Antibodies
B1A1, H9–1, and H9–2 reacted only with T. pallidum (Table 1Table 1
Reactivity of Monoclonal Antibodies.*). G2–1 reacted with all treponemes tested.Identification of Spirochetes in Plaque
As shown in Table 2Table 2
Frequency with Which Spirochetes Were Detected in Dental Plaque by Monoclonal Antibodies., all 10 control samples of supragingival plaque but only 1 of 14 control samples of subgingival plaque contained spirochetes detectable with monoclonal antibody C2–1 (this sample was also reactive with pathogen-restricted monoclonal antibody H9–2). By contrast, all 17 patients with gingivitis (P<0.001) and 14 of the 19 patients with periodontitis (P<0.001) had spirochetes in subgingival plaque. No subject with healthy gingiva had spirochetes in plaque that were reactive with monoclonal antibodies B1A1 or H9–1, as compared with 11 of 17 subjects with gingivitis (P<0.001) and 10 of the 19 subjects with periodontitis (P<0.01). Similarly, only 1 of the 14 normal subjects had spirochetes in plaque that were reactive with monoclonal antibody H9–2, as compared with all 11 subjects with gingivitis for whom samples were available (P<0.001) and 14 of the 19 subjects with periodontitis (P<0.001).Figure 1Figure 1
Spirochetes in Plaque Samples from Patients with Gingivitis after Identification with Monoclonal Antibodies C2–1 (Panels A and C) and B1A1 (Panels B and D). shows representative treponemes in plaque obtained from areas of ulcerative gingivitis and identified by means of monoclonal antibodies C2–1 (which reacts with all spirochetes) and B1A1 (which reacts only with oral spirochetes related to T. pallidum).Presence of Specific Antibodies in Serum
The results of serologic analyses are summarized in Table 3Table 3
Serum Antibody Analyses.. None of the serum samples from normal subjects or from the subjects with gingivitis were reactive in standard serologic tests for syphilis, whereas the positive control serum samples from patients with untreated syphilis were uniformly reactive. Serum samples from patients with periodontitis were not available for these tests.Immunoblot analyses of serum samples from patients with gingivitis revealed IgG directed against 47-kd, 37-kd, 14-kd, and 12-kd molecules (Table 2, Fig. 2Figure 2
Immunoblots of Human Serum with IgG against Antigens of T. pallidum Subspecies pallidum.). More patients with gingivitis had IgG directed against 47-kd molecules (15 of 19) and 12-kd molecules (15 of 19) from T. pallidum than against other antigens. Fourteen of these patients had IgG to both 47-kd and 12-kd molecules. Twelve of these patients had antibodies to 37-kd molecules, always in association with IgG to 47-kd molecules and usually in association with antibodies to 12-kd molecules (11 of 12 patients). Thus, 11 patients had antibodies to 47-kd, 37-kd, and 12-kd molecules, and 3 had IgG to all four molecules containing pathogen-specific determinants of T. pallidum. In contrast, serum samples from 5 of the 18 normal healthy subjects had weak reactivity against 47-kd molecules (P<0.01 for the comparison with samples from patients with gingivitis) and 37-kd molecules (P<0.05) and no activity against 14-kd or 12-kd molecules (P<0.05 and P<0.001, respectively). All five control patients with syphilis had serum IgG directed against all four molecules containing pathogen-specific determinants of T. pallidum. Serum samples were not available from patients with periodontitis for immunoblotting.Discussion
Necrotizing ulcerative gingivitis is a recurrent infection of the gingiva surrounding the teeth and is usually associated with rapid onset, spontaneous bleeding, pain, fetor oris, and necrotic interdental papillae; it can progress to periodontitis (Fig. 3Figure 3
Acute Necrotizing Ulcerative Gingivitis.). Chronic periodontitis, characterized by swollen gingiva, the formation of pockets between gingiva and tooth, and loss of alveolar bone, may be found as early as adolescence and usually continues as long as teeth are present. Periodontitis is common after the third decade of life and is a frequent cause of tooth loss in older persons. Spirochetes found in plaque are commonly associated with ulcerative gingivitis and periodontitis.1 , 17 , 18 They are found on the surface of plaque below the gum line, in direct contact with gingival epithelium,19 and they have also been found in healthy connective tissue around ulcerated gingiva.20 21 22 23 However, it is not known whether oral spirochetes are pathogenic or merely opportunistic.Two independent observations support the conclusion that a spirochete closely related to T. pallidum subspecies pallidum infects humans with necrotizing ulcerative gingivitis and chronic adult periodontitis. First, spirochetes were identified in plaque with the use of monoclonal antibodies specific for pathogenic subspecies of T. pallidum 9 , 10 , 15 (and Lukehart S: unpublished data). Second, in the current study serum samples from the majority of patients with ulcerative gingivitis reacted strongly with the 47-kd and 37-kd molecules from T. pallidum that are known to contain pathogen-specific epitopes,8 9 10 whereas only five of the serum samples from healthy subjects reacted weakly with antigens at these two sites. The weak reactivity of normal serum to the 37-kd and 47-kd molecules of pathogenic treponemes on immunoblotting14 , 24 , 25 is thought to be due to the presence of public antigens (antigens common to all treponemes) on these or similar-sized molecules.26 , 27
A more clear distinction with respect to reactivity against 14-kd and 12-kd pathogen-specific antigens13 , 24 was made between serum samples from normal subjects and those from patients with gingivitis or with syphilis. All five serum samples from the patients with syphilis reacted to both antigens, but none of the samples from healthy subjects reacted to either of the low-molecular-weight molecules. Fifteen of the 19 patients with gingivitis had IgG directed against the 12-kd antigen, and 4 of 19 had IgG directed against the 14-kd antigen. Since pathogen-restricted antigens have not been found on bacteria other than subspecies of T. pallidum and T. carateum, the finding of antibodies to these molecules in patients with ulcerative gingivitis is a strong indication that these patients had been infected with an organism closely related to T. pallidum. It may be that this organism has not been observed before because it is one of the uncultivated spirochetes known to exist in dental plaque.1 If it has resisted cultivation in vitro, this characteristic also links it to T. pallidum and distinguishes it from other, more common and readily cultivable oral spirochetes. It is extremely unlikely, however, that T. pallidum subspecies pallidum was present in the mouths of these patients, since two serologic tests for syphilis were negative, there were no indications of current infections with pathogenic treponemes, and they were not considered to be at risk for syphilis. It is also unreasonable to conclude that ulcerative gingivitis or any other form of periodontal disease is a manifestation of syphilis or other kinds of venereal disease. Rather, we suggest that a heretofore unrecognized oral spirochete contains antigens previously thought to be restricted to known pathogenic treponemes. Further research is needed, including molecular and genetic comparisons, to determine whether this spirochete is a new species of treponeme or a new subspecies of T. pallidum. Additional research is also needed to determine whether this organism contributes to the pathogenesis of periodontal diseases.
Supported in part by an award from the University of Missouri Weldon Spring Endowment Fund (to G.R.R.), a contract (N00014–89-C-0033) from the Naval Medical Research and Development Command (to G.R.R.), and a National Institutes of Health grant (AI-18988) (to S.A.L. and S.A.B.-Z.).
Portions of this work were presented at the American Society for Microbiology Annual Meeting in Anaheim, California, on May 15, 1990.
We are indebted to Drs. R. George and J.N. Miller for their generous gifts of treponeme cultures, to Dr. C. Cobb for serum and plaque samples, to Dr. Brenda Bohaty and Ms. Sherelene Pratt for assistance with analysis of plaque samples, and to Paul Miller for photomicroscopy.
Source Information
From the Departments of Pediatric Dentistry (G.R.R., M.A.W., K.S.W.) and Periodontology (D.F.A.), School of Dentistry, Oregon Health Sciences University, Portland; the Department of Medicine, School of Medicine, University of Washington, Seattle (S.A.B.-Z, S.A.L.); and the Naval Dental Research Institute, Great Lakes, III. (L.S.). Address reprint requests to Dr. Riviere at the Department of Pediatric Dentistry, School of Dentistry, Oregon Health Sciences University, 611 S.W. Campus Dr., Portland, OR 97201–3097.
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