Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer's disease.

The purpose of this investigation was to use molecular and immunological techniques to determine whether oral Treponema infected the human brain. Pieces of frontal lobe cortex from 34 subjects were analyzed with species-specific PCR and monoclonal antibodies. PCR detected Treponema in 14/16 Alzheimer's disease (AD) and 4/18 non-AD donors (P < 0.001), and AD specimens had more Treponema species than controls (P < 0.001). PCR also detected Treponema in trigeminal ganglia from three AD and two control donors. Cortex from 15/16 AD subjects and 6/18 controls contained Treponema pectinovorum and/or Treponema socranskii species-specific antigens (P < 0.01). T. pectinovorum and/or T. socranskii antigens were also found in trigeminal ganglia and pons from four embalmed cadavers, and 2/4 cadavers also had Treponema in the hippocampus. These findings suggest that oral Treponema may infect the brain via branches of the trigeminal nerve.

Isolation of oral spirochetes from dogs and cats and provisional identification using polymerase chain reaction (PCR) analysis specific for human plaque Treponema spp.

Identification of plaque spirochetes from dogs is rare and no studies to date report cultivation of canine or feline plaque spirochetes. Plaque samples obtained from canine and feline patients were cultured in broth media. Spirochetes cultured were subjected to microscopic evaluation and were cloned on a solid medium. The clones were provisionally identified using species-specific PCR for treponema isolated from human plaque. Canine spirochete clones included Treponema denticola, T. socranskii ssp., T. vincentii, T. maltophilum, T. medium, and T. pectinovorum. Feline clones included T. maltophilum and T. socranskii. Non-amplified clones may represent novel treponemes. Future studies will be aimed at comparison of the spirochetes present in dogs and cats with or without periodontal disease.

Phenotypic and genotypic heterogeneity among cultivable pathogen-related oral spirochetes and Treponema vincentii.

Recent findings challenge the assumption that pathogen-related oral spirochetes (PROS) are related to Treponema pallidum. Treponema vincentii, grown in OMIZ-Pat media, cross-reacted with monoclonal antibody H9-2 against T. pallidum, and cultivable PROS had 16S rRNA gene sequences similar to those of T. vincentii (C.-B. Choi, C. Wyss, and U. B. Göbel. J. Clin. Microbiol. 34:1922-1925, 1996). Aims of the present study were to determine whether antigen phenotypes of oral treponemas were influenced by growth conditions and to evaluate the genetic relatedness of cultivable PROS to T. pallidum and T. vincentii. Results show that three T. pallidum monoclonal antibodies (H9-1, H9-2, and F5) cross-reacted with whole cells from four Treponema species grown in modified OMIZ-Pat medium, but not with treponemas grown in NOS medium. Only H9-2 reacted in immunoblots with reduced proteins from cultivable PROS and T. vincentii. Three of five PROS isolates were amplified by T. vincentii-specific PCR, and one was amplified by Treponema medium-specific PCR. None were amplified by T. pallidum-specific PCR. Three of five PROS isolates had 16S ribosomal DNA restriction fragment length polymorphism patterns identical to that of T. vincentii, and the patterns of two isolates resembled that of T. medium. Arbitrarily primed-PCR profiles from whole genomic DNA were distinct among five PROS isolates and two T. vincentii strains. Thus, PROS isolates represent a heterogeneous group of treponemas that share some 16S rRNA gene sequences with T. vincentii and T. medium, but not with T. pallidum. It is proposed that the PROS nomenclature be dropped.

Identification of seven Treponema species in health- and disease-associated dental plaque by nested PCR.

Species-specific nested PCR was used to detect Treponema amylovorum, Treponema denticola, Treponema maltophilum, Treponema medium, Treponema pectinovorum, Treponema socranskii, and Treponema vincentii in dental plaque. Subjects with periodontitis harbored all species, but T. pectinovorum and T. vincentii were not found in plaque from disease-free subjects.

Association of oral spirochetes from periodontally healthy sites with development of gingivitis.

The purpose of this investigation was to determine whether the presence of selected disease-associated bacteria in health-associated plaque correlated with future gingivitis. Sites of periodontal health were identified in 65 adults. Six months later (recall 1) plaque was collected from sites that remained in periodontal health, and 5 species of specific bacteria and pathogen-related oral spirochetes were detected using monoclonal antibodies in a microscopic assay. Members of the spirochete morphogroup were also identified by phase contrast microscopy. The relationship between site-specific detection of bacteria at recall 1 and development of gingivitis at recall 2 or 3 was evaluated by means of logistic regression using generalized estimating equations, from which odds ratios (OR) were estimated. Significance was conservatively defined as OR > 2.0 and P < 0.05. We found that 488 of 1,424 healthy sites developed gingivitis over the 12-month interval between recall 1 and 3. Only the spirochete morphogroup (OR =2.04; P=0.002) was significantly associated with the transition from health to gingivitis. The association of Treponema socranskii with future gingivitis was higher than expected (OR=2.27), but the relationship was not statistically significant (P=0.163). Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, and pathogen-related oral spirochetes did not correlate well with gingivitis (OR < 2.0). Health-associated plaque from 5 sites contained Treponema denticola, and all 5 sites progressed to gingivitis. An OR could not be calculated because T. denticola was not detected in health-associated plaque from stable healthy sites. These findings indicated that the presence of T. denticola and unidentified spirochetes in health-associated plaque was associated with increased susceptibility to gingival inflammation. Future studies assessing a larger panel of dental plaque microorganisms, with shorter intervals between baseline and follow-up assessment, are necessary to more fully evaluate the association between detection of specific organisms at healthy sites and risk for gingivitis.

Differential attachment of oral treponemes to monolayers of epithelial cells.

This in vitro study describes the attachment properties of several oral treponemes to monolayers of epithelial cells and the effect of epithelial cell confluence on treponeme attachment. Four serotypes of Treponema denticola, Treponema scoliodontum, three subspecies of Treponema socranskii, and Treponema vincentii were tested with monolayers of epithelial cells of human and canine origin. Attachment of oral treponemes were compared to attachment by T. pallidum subsp. pallidum, and by the non-pathogen Treponema phagedenis. Results indicated that different serotypes of T. denticola had similar abilities to attach to epithelial cells. However, subspecies of T. socranskii differed in their ability to attach to epithelial cells. The proportion of epithelial cells susceptible to attachment by oral spirochetes was strongly related to the confluence level of the monolayer. In contrast, T. pallidum attached equally well to both epithelial cell lines at all confluence levels. T. phagedenis attached to < 1% of all epithelial cells. In general, attachment of oral treponemes to canine cells was lower than to human cells, suggesting species-specificity for adherence. Attachment of oral treponemes to epithelial cells may promote colonization of the periodontal pocket, as well as retention of treponeme colonies within plaque. The preference of oral treponemes to attach to cells of low confluence fields may translate in vivo to an increased ability to attach to cells which are actively dividing. Such cells are found in areas of repair, a common status within inflamed periodontal pockets. Furthermore, attachment of oral treponemes to epithelial cell barriers may promote or potentiate cytopathic processes.

Association of oral spirochetes from sites of periodontal health with development of periodontitis.

The purpose of this investigation was to determine whether the presence of disease-associated bacteria in health-associated plaque correlated with susceptibility to periodontitis over time. Sites of periodontal health were identified in 65 adults. Six months later (recall 1), plaque was collected from sites that remained in periodontal health, and specific bacteria were detected using monoclonal antibodies in a microscopic assay. The spirochete morphogroup was identified by phase contrast microscopy. The relationship between detection at recall 1 and development of periodontitis over two successive 6-month intervals (recalls 2 and 3) was evaluated by means of logistic regression using generalized estimating equations (GEE), from which odds ratios (OR) were estimated and tested for significance. Significant relationships were defined as those having ORs with P < 0.05. Ninety-three of 1,032 sites developed signs of early periodontitis over the 12-month interval between recall 1 and recall 3. The spirochete morphogroup (OR = 3.13, P < 0.001) and pathogen-related oral spirochetes (PROS) (OR = 3.68, P < 0.001) were significantly associated with healthy sites that developed periodontitis. The association of Treponema socranskii was not significant (OR = 3.62, P = 0.0918). Odds ratios for Campylobacter rectus, Eikenella corrodens, and Porphyromonas gingivalis were less than 2.0 and not significant. Treponema denticola was not detected in health-associated plaque from stable health sites and was detected in only three sites that progressed to periodontitis. These findings indicate that the presence of PROS and some unidentified spirochetes in health-associated plaque is associated with increased susceptibility to periodontitis.

Detection of pathogen-related oral spirochetes, Treponema denticola, and Treponema socranskii in dental plaque from dogs.

Spirochetes have been observed in dental plaque from dogs, but specific spirochetes have not been identified. In particular, it is not known whether treponemes associated with periodontal diseases in humans also occur in dogs, and whether, like in humans, detection of specific treponemes correlates with periodontal status of dogs. Forty-two dogs were grouped according to the worst periodontal condition in the mouth, as determined by overt signs of inflammation and pocket probing depths. A representative specimen of dental plaque was obtained by pooling subgingival plaque collected from three uniform reference sites, irrespective of periodontal status at selected sites. The presence of pathogen-related oral spirochetes. Treponema denticola, and T. socranskii was determined using specific monoclonal antibodies in an immunocytochemical microscopic assay. All three treponemes were detected in all groups, but a significantly greater proportion of dogs with pocket probing depths > or = 5 mm had detectable treponemes, compared to dogs that were in periodontal health.

Associations between Porphyromonas gingivalis and oral treponemes in subgingival plaque.

Colonization and/or proliferation of Treponema denticola may depend on the presence of Porphyromonas gingivalis. The aims of this study were to confirm this synergistic relationship, to determine whether other oral bacteria were similarly associated with P. gingivalis and to relate coinfection to the periodontal status of plaque donors. Subgingival plaque was collected from every tooth except third molars in 106 subjects who were grouped by their worst periodontal condition. In addition to P. gingivalis, monoclonal antibodies were used to identify Campylobacter rectus, Eikenella corrodens, T. denticola, Treponema socranskii and pathogen-related oral spirochetes. Associations of these bacteria with coinfection by P. gingivalis were assessed by estimated odds ratios. The results indicate that coinfection with P. gingivalis is linked to all tested bacteria, but each pair was associated with distinct periodontal conditions. The distribution of coinfected sites suggests biased colonization of facial surfaces over lingual surfaces.

Periodontal status and detection frequency of bacteria at sites of periodontal health and gingivitis.

It is generally recognized that bacteria in dental plaque at sites of periodontal diseases are not commonly found at sites of periodontal health. One hypothesis to explain the etiology of periodontitis is that pathogenic bacteria from diseased sites infect healthy sites. It has been suggested that gingival inflammation may predispose sites to colonization by bacteria associated with periodontal diseases. The purpose of this cross-sectional study was to determine whether the detection frequency of selected bacteria at sites of periodontal health or gingivitis differed between subjects who were in good periodontal health, subjects who had gingivitis, or subjects with periodontitis. The clinical status of every tooth (except third molars) from 106 subjects was characterized by means of clinical attachment level, probing depth and by signs of inflammation. Subgingival plaque was collected from mesio-facial and disto-lingual surfaces. Specific monoclonal antibodies were used in an immunocytochemical assay to identify Campylobacter rectus, Eikenella corrodens, Porphyromonas gingivalis, pathogen-related oral spirochetes (PROS, using Treponema pallidum subspecies pallidum monoclonal antibodies), T. denticola (serotypes A-D), T. socranskii subspecies buccale and T. socranskii subspecies socranskii. Differences in detection of bacteria between groups of subjects were measured using odds ratios (OR). Results of this study indicate that PROS was the only identified bacterium at sites of both health and gingivitis that demonstrated a significant positive relationship with the presence of periodontitis. These findings do not prove that bacteria spread from periodontitis sites, nor do they imply that disease necessarily results from infection. However, these data do suggest that some bacteria associated with periodontitis are more likely than others to tolerate conditions at healthy sites and that the presence of periodontitis increases risk of infection at healthy sites.

Subgingival distribution of Treponema denticola, Treponema socranskii, and pathogen-related oral spirochetes: prevalence and relationship to periodontal status of sampled sites.

Aims of this study were to comprehensively describe the intraoral distribution of the spirochete morphogroup and of 7 antigenically distinct oral treponema, and to relate their presence to periodontal status. Periodontal tissues were evaluated at 4 sites on every tooth except third molars and 76 subjects were classified according to the worst periodontal condition at any one site: Group 1, gingivitis (n = 13); Group 2, early periodontitis (n = 38); and Group 3, advanced periodontitis (n = 25). Subgingival plaque was collected from each half of every tooth evaluated clinically. Spirochetes were identified with phase contrast microscopy and specific treponema were detected immunochemically using monoclonal antibodies to Treponema denticola serovars A-D, T. socranskii subspecies bucalle, T. socranskii subspecies socranskii, and T. pallidum (pathogen-related oral spirochetes, PROS). The counting protocol was conservative and probably underestimated the actual presence of organisms. Spirochetes were found at one or more sites in approximately 60% of subjects in all groups. PROS were found in approximately 40% of subjects in all groups while T. denticola (predominantly serotype B) and T. socranskii (exclusively T. socranskii subsp. buccale) were more frequently observed in Group 2 (roughly 25% for both treponema) than in Groups 1 or 3. Overall, spirochetes were detected in less than 15% of the 4,040 sites examined. Spirochetes were found at more sites of periodontitis (group mean range 20 to 40%) than of gingivitis (6 to 20%), and were only infrequently found at sites of periodontal health (4 to 10%). Spirochetes were identified most often in plaque from around molars and they were usually found in only one of two samples from individual teeth. Results of this study suggest that although spirochetes are most often found associated with periodontitis, their distribution is restricted and most periodontitis sites do not harbor spirochetes.

HIV-associated periodontal disease: new oral spirochete found.

This study examines plaque for the presence of a recently described oral spirochete, tentatively called pathogen-related oral spirochete. This investigation found PROS in plaque of patients with HIV-associated periodontal disease.

Glycosaminoglycans and periodontal disease: analysis of GCF by safranin O.

The purpose of this study was to quantify glycosaminoglycans (GAG) released into the gingival crevicular fluid (GCF) during health, gingivitis, and adult periodontitis. The investigation tested the hypothesis that increased amounts of GAG can be measured in GCF associated with gingivitis and adult periodontitis as compared to health. An individual patient's sampling sites were assigned to either a health (control) group or 1 of 3 experimental groups, gingivitis, periodontal "maintenance" (perio-M), or periodontal "non-maintenance" (perio-NM) according to standard clinical criteria of pocket probing depth, bleeding on probing, and radiographic evidence of bone loss. The perio-M group was defined as a periodontal patient who had received a dental prophylaxis and/or root planning within 6 months prior to GCF collection. The perio-NM group had received no periodontal therapy during the previous 6 months. Subsequent to air-drying and isolation, GCF was collected by a microcapillary pipette held at the gingival margin. All fluid samples were digested overnight at 37 degrees C with 25 micrograms of papain and analyzed for GAG content using a chondroitin-4-sulfate standard. Data generated from safranin "O" dye binding assays of GAG revealed 4.41 +/- 9.82 ng GAG from the health (control) group (n = 23); the gingivitis group (n = 13) showed 15.23 +/- 11.85 ng GAG/sample; perio-M group (n = 11) showed 23.64 +/- 12.98 ng GAG/sample and the perio-NM group (n = 12) exhibited 119.08 +/- 33.14 ng GAG/sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic peroral immunization of conventional laboratory rats with mutans streptococci leads to stable acquired suppression of salivary antibodies.

Prior investigations have demonstrated that salivary antibody responses to mutans streptococci are dose-dependent and temporary. The purpose of this study was to evaluate the stability of antibody suppression established by mutans streptococci. Streptococcus mutans 6715-15 was provided in food to conventional rats for 18 weeks. Antigen was withdrawn for 10 weeks and then resumed for an additional 6 weeks. Saliva and serum from nonimmunized controls and from experimental rats were tested with a quantitative enzyme-linked immunosorbent assay for IgA and IgG antibodies to whole bacterial cells and to soluble antigen. Results show that salivary antibodies were stimulated by primary peroral immunization, that IgA was the dominant isotype and that IgA antibodies were primarily directed against soluble antigen. This study also shows that immunity is not maintained, even while challenge continues, and that once suppression is established, immunized animals do not recover their ability to respond, even if exposure is stopped for 10 weeks before re-exposure.

Relative proportions of pathogen-related oral spirochetes (PROS) and Treponema denticola in supragingival and subgingival plaque from patients with periodontitis.

The purpose of this study was to use monoclonal antibodies to enumerate spirochetes in dental plaque, including the newly recognized pathogen-related oral spirochete (PROS) and specific serovars of Treponema denticola. Plaque was collected from control subjects with no apparent periodontal disease and from sites of moderate to severe chronic periodontitis in patients with inflammatory periodontal disease. Individual monoclonal antibodies were used to determine whether spirochetes were present and then a double-staining protocol was employed to count total spirochetes and specific treponemes in individual microscopic fields. Results indicate that spirochetes are more common at diseased sites and in subgingival plaque than at healthy sites or in supragingival plaque. Together PROS and T. denticola comprised the majority of all spirochetes in all samples and PROS and T. denticola serovars "B" and D were most numerous in plaque from patients with periodontitis. PROS were the majority of all spirochetes in supragingival plaque (76.2% +/- 23.8%) and subgingival plaque (60.9% +/- 19.1%) from periodontitis patients, significantly larger than the percentage of T. denticola serovar "B" (P less than .001 for both supragingival and subgingival plaque) and serovar D (P less than .01 for supragingival and P less than .001 for subgingival plaque). These observations indicate that PROS are the predominant spirochete in plaque from sites of patients with periodontitis, but other analytical approaches are necessary to determine if PROS or T. denticola are pathogenic.

Pathogen-related oral spirochetes from dental plaque are invasive.

Spirochetes that share pathogen-restricted antigens with Treponema pallidum subsp. pallidum have been identified in dental plaque and diseased gingival tissues, but it is not known whether these spirochetes possess virulence characteristics. In this study, plaque spirochetes were able to transmigrate a tissue barrier in vitro and were identified on the other side by using monoclonal antibodies specific for pathogen-restricted determinants from T. pallidum subsp. pallidum. This invasive capability is shared with T. pallidum subsp. pallidum, but cultured oral and intestinal treponemes did not perforate the tissue barrier. Cocultures indicated that invasive treponemes do not create opportunities for cultivable oral treponemes to cross the barrier. These findings indicate that gingival tissues may be a port of entry for previously unrecognized invasive spirochetes in humans.

Pathogen-related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis.

The purpose of this investigation was to determine whether monoclonal antibodies against pathogen-restricted antigens of Treponema pallidum subsp. pallidum could be used as probes for spirochetes in diseased gingival tissue from subjects with acute necrotizing ulcerative gingivitis. A biotin-streptavidin system was used to identify spirochetes bound by monoclonal antibodies in cryostat sections of tissue. Twelve of 16 tissue samples from diseased sites, but none of 8 tissue specimens from healthy sites, reacted with pathogen-restricted antibodies. Organisms were found in intact epithelium and connective tissues adjacent to ulcers. Staining intensity was often high in perivascular locations and around vesicular spaces. Monoclonal antibodies to Bacteroides gingivalis and Treponema denticola were each reactive with diseased gingival tissues, but staining was usually restricted to ulcerated areas. These studies extend recent observations that showed that subjects with acute necrotizing ulcerative gingivitis had both pathogen-related spirochetes in dental plaque and serum immunoglobulin G to pathogen-restricted antigens on T. pallidum subspecies, suggesting that pathogen-related spirochetes may be associated with the pathogenesis of certain periodontal diseases.

Identification of spirochetes related to Treponema pallidum in necrotizing ulcerative gingivitis and chronic periodontitis.

BACKGROUND: Spirochetes are commonly associated 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 less than 0.001) and from 10 of 19 patients with periodontitis (P less than 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 less than 0.001), and from 14 of 19 patients with periodontitis (P less than 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: The spirochetes found in dental plaque from patients with ulcerative gingivitis or chronic periodontitis have antigens that are thought to be unique to pathogenic treponemes. This close antigenic relation suggests that T. pallidum or a closely related organism may be involved in the pathogenesis of periodontal disease.

Use of monoclonal antibodies to enumerate spirochetes and identify Treponema denticola in dental plaque of children, adolescents and young adults.

Identification of spirochetes in dental plaque is difficult. Not all spirochetes can be cultured and microscopic analysis based on darkfield or phase optics cannot determine the genus and species of individual bacterial cells. The purpose of this study was to use monoclonal antibodies in an immunoenzyme technique to stain spirochetes in dental plaque. Separate mAb were used to estimate total spirochetes and relative numbers of 2 distinct types of Treponema denticola. Plaque samples were collected from 40 subjects grouped by age. Results showed that older subjects are more likely to have spirochetes, to have more spirochetes and to have more diverse populations or spirochetes than younger subjects. Our studies suggest that T. denticola may be the first treponeme to colonize the primary dentition, that T. denticola appears to comprise a major proportion of all spirochetes at all ages and that two distinct serotypes of T. denticola are found to coexist in plaque from most children.

Chronic peroral administration of Streptococcus sobrinus to conventional laboratory rats produces cycling levels of salivary antibodies.

Conventional outbred rats were fed Streptococcus sobrinus for 24 weeks and ELISA was used to identify isotypes of antibodies against bacteria in saliva and serum. Quantities of antibodies from experimental rats were compared with values derived from the control population. Saliva IgM and IgA anti-S. sobrinus from experimental rats were greater than controls at week 3, were much less at week 9, but normal levels were found by week 13. IgG antibodies in saliva peaked at weeks 5 and 9 but fell to control levels by week 13. Relative levels of antibodies in saliva of experimental animals continued to cycle during weeks 13-24 but did not differ greatly from controls. Serum IgM and IgG antibodies to S. sobrinus were essentially like controls throughout the experiment. Serum IgA increased briefly during the first 12 weeks then returned to normal levels. The results suggest that prolonged peroral exposure to cariogenic bacteria ultimately leads to modulation of antibody around unimmunized control levels even though antigenic stimulation persists.