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Treponema hyodysenteriae was established in the ceca of gnotobiotic mice in the absence of other organisms. Superficial mucosal lesions characteristic of swine dysentery were present in the ceca of mice inoculated with T. hyodysenteriae in combination with viable Bacteroides vulgatus. Deep crypt necrosis was detected in the ceca of mice inoculated with T. hyodysenteriae alone.
Soluble sonic extracts of several strains of Treponema denticola and Treponema vincentii were examined for their abilities to alter proliferation of both murine and human fibroblasts. We found that sonic extracts of all tested strains of T. denticola caused a dose-dependent inhibition of murine and human fibroblast proliferation when assessed by both DNA synthesis ([3H]thymidine incorporation) and direct cell counts. T. vincentii had only a minimal inhibitory effect at comparable doses. No inhibition was observed when sonic extracts were added simultaneously with [3H]thymidine, indicating that suppression was not due to the presence of excessive amounts of cold thymidine in the extract, nonspecific effects on thymidine utilization by the cells (transport and incorporation), or degradation of label. RNA ([3H]uridine incorporation) and protein ([3H]leucine incorporation) synthesis were similarly altered after exposure to the T. denticola sonic extracts. There was no effect on cell viability as measured by trypan blue exclusion. Inhibition could be reversed by extensive washing of the cells within the first few hours of exposure to sonic extracts. Preliminary characterization and purification indicated that the inhibitory factor(s) is not endotoxin since it is heat labile, and elutes in a single, well-defined peak on a Sephadex G-150 chromatography column corresponding to a molecular weight of approximately 50,000. Since oral spirochetes have been implicated in the pathogenesis of periodontal disorders, it is possible that they contribute to the disease process by inhibition of fibroblast growth and therefore may, at least in part, account for the loss of collagen seen in diseased tissue.
Sonicated preparations of Treponema hyodysenteriae and Treponema innocens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Treponemal proteins were electrophoresed on a 10% polyacrylamide slab gel in a discontinuous Tris-glycine system and either stained with Coomassie blue dye or transferred electrophoretically at 20 mA for 16 h and 30 mA for 3 h to nitrocellulose paper. Staining of the gels revealed at least 42 distinct T. hyodysenteriae and T. innocens proteins, with molecular sizes ranging from greater than 100 to 14 kilodaltons (kDa). Each species contained 12 to 16 major protein bands; five of the proteins were common to both species. Fourteen major antigens were identified in T. hyodysenteriae isolate B204 by using serum specimens from pigs in the acute stage of swine dysentery. Twelve additional antigens were detected in isolate B204 when convalescent-phase serum specimens were reacted to the blot. A wide band at 16 kDa was identified with convalescent-phase serum specimens in T. hyodysenteriae but not in T. innocens. This 16-kDa antigen was also identified in T. hyodysenteriae with colonic secretions from convalescent pigs.
The Treponema denticola content of plaque was quantitatively estimated for samples taken from periodontitis patients as well as periodontally healthy subjects among two separate human populations. The populations studied included military volunteers and civilians at a university dental clinic. The plaque samples from each population were grouped according to pocket depth measurements at the collection site. A biotin-avidin enzyme-linked immunosorbent assay procedure was developed with a monoclonal antibody specific for a serovariety of T. denticola. T. denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to the healthy controls and a group with moderate disease. The ratio of T. denticola content per milligram of plaque in the deep pocket groups to that of the other two groups was about 2:1 for both populations. This is the first quantitative evidence of a positive relationship between a specific spirochete species and severe periodontitis.
Eleven strains of Treponema hyodysenteriae isolated from pigs with swine dysentery were examined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. T. hyodysenteriae strains formed a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. However, immunoblotting with antiserum from rabbits immunized with T. hyodysenteriae CN8368 revealed heterogeneity among the lipopolysaccharide complexes of different strains. Polypeptides of molecular weights between 30,000 and 36,000 were the predominant T. hyodysenteriae polypeptides detected by porcine immune serum. In contrast, Treponema innocens did not form a homogeneous group with respect to sodium dodecyl sulfate-soluble proteins. Adsorption studies and immunoblotting identified polypeptide antigens present on cells of T. hyodysenteriae which were not detected on cells of T. innocens. These unique antigens may play a role in the virulence of T. hyodysenteriae.
The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.
The etiologic role of oral treponemes in human periodontitis is still under debate. Although seen by dark-field microscopy in large numbers, their possible role is still unclear since they comprise some 60 different phylotypes, most of which are still uncultured. To determine their status as mere commensals or opportunistic pathogens, molecular epidemiological studies are required that include both cultured and as-yet-uncultured organisms. Here we present such data, comparing treponemal populations from chronic periodontitis (CP) or generalized aggressive periodontitis (GAP) patients. As a periodontitis-resistant (PR) control group, we included elderly volunteers with more than 20 natural teeth and no history of periodontal treatment and no or minimal clinical signs of periodontitis. Almost every treponemal phylotype was present in all three groups. For most treponemes, the proportion of subjects positive for a certain species or phylotype was higher in both periodontitis groups than in the PR group. This difference was pronounced for treponemes of the phylogenetic groups II and IV and for Treponema socranskii and Treponema lecithinolyticum. Between the periodontitis groups the only significant differences were seen for T. socranskii and T. lecithinolyticum, which were found more often in periodontal pockets of GAP patients than of CP patients. In contrast, no difference was found for Treponema denticola. Our findings, however, strengthen the hypothesis of treponemes being opportunistic pathogens. It appears that T. socranskii, T. lecithinolyticum and group II and IV treponemes may represent good indicators for periodontitis and suggest the value of the respective probes for microbiological diagnosis in periodontitis subjects.
Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantiiT (90.1%). The spirochete, here named "Candidatus Treponema suis," was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.
Three isolates of pathogenic Treponema hyodysenteriae, B8044, B6933, and Ack 300/8, were serotyped by double immunodiffusion using lipopolysaccharide antigen and rabbit antiserum. The precipitation reactions of these three isolates were then compared with precipitin reactions from the reference strains of serotypes 1 through 4. The data indicated that B8044, B6933, and Ack 300/8 share antigenic determinants with either serotype 1 or 2 or both. However, when the cross-reacting antibodies were removed by adsorption, all reactions were found to be monospecific. Based on these results, three new serotypes of pathogenic T. hyodysenteriae are proposed: B8044 serotype 5, B6933 serotype 6, and Ack 300/8 serotype 7.
Specimens of colonic mucosa from two pigs with diarrhea were examined by light and electron microscopy. The epithelial surfaces of both pigs were extensively colonized by large spirochetes morphologically compatible with Treponema hyodysenteriae or Treponema innocens. The microorganisms were intimately attached end-on to the luminal cells. A weakly beta-hemolytic spirochete similar to T. innocens was isolated from the colon of one of the pigs.
Oligodeoxynucleotide probes (17 and 28 bases long) complementary to a unique region of Treponema hyodysenteriae 16S rRNA were developed. These probes bound specifically to partially purified rRNA and whole-cell rRNA of T. hyodysenteriae. No binding to partially purified rRNA or whole-cell rRNA of Treponema innocens, Treponema succinifaciens, Treponema bryantii, or Escherichia coli occurred under stringent conditions. The 28-base probe was 5 to 10 times more sensitive than the 17-base probe when hybridized with T. hyodysenteriae rRNA. The 28-base probe detected T. hyodysenteriae in the feces of experimentally inoculated pigs exhibiting clinical signs of swine dysentery.
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.
Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone. Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role. Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable. To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. Subgingival plaque specimens taken from diseased sites (n = 200) and healthy control sites (n = 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. Spirochetes were found in all patients, but their distributions varied considerably. Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency. These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated. All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g., Treponema maltophilum, or uncultivable phylotypes. Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.
We examined 26 atherosclerotic lesions and 14 nondiseased aorta specimens to detect the periodontopathogenic part of the bacterial 16S rRNA locus by PCR. Treponema denticola sequence of the 16S rRNA locus was found in 6 out of 26 DNA samples (23.1%) from the formalin-fixed, paraffin-embeded atherosclerotic lesions obtained during surgery but not in any of the 14 nondiseased aorta samples from deceased persons. Utilizing immunofluorescence microscopy, we observed aggregated antigenic particles reacting with rabbit antiserum against T. denticola in thin sections of the PCR-positive samples, but we could not detect any reacting particles in the PCR-negative thin sections.
Pure cultures of six pathogenic isolates of Treponema hyodysenteriae, the colonic mucosal scrapings of seven pigs with acute swine dysentery, and feces from seven unaffected pigs were diluted in phosphate-buffered saline and plated on Trypticase soy agar with 5% citrated bovine blood (TSA) and TSA with various levels of spectinomycin (TSA-S). The plates were incubated at 42 degrees C in a vented GasPak jar with a cold palladium catalyst and either 80:20 H2-CO2 by evacuation and refilling or a H2-CO2 generator envelope. Viable cell counts of the six pathogenic isolates were not altered by plating on TSA-S with 400 mug of spectinomycin per ml (TSA-S400) as compared with TSA alone. Dilutions of colonic mucosal scrapings from seven pigs with acute swine dysentery showed numbers of T. hyodysenteriae to be unchanged when plated on TSA-S400. Flora other than T. hyodysenteriae present in acute swine dysentery was inhibited, on the average, by 99.99%. Plating of dilutions of feces of unaffected pigs on TSA-S400 showed inhibition of flora that averaged more than 99.9%. Pathogenicity of T. hyodysenteriae was not altered by isolation or serial passage on TSA-S400.
Oral treponemes have been associated with periodontal diseases. We developed a highly sensitive and specific method to detect and quantify cultivable oral treponemes (Treponema denticola, Treponema vincentii, and Treponema medium) in 50 subgingival plaque samples from 13 healthy subjects as well as 37 patients with periodontal diseases using real-time PCR assays with specific primers and a TaqMan probe for each 16S rRNA sequence. The specificity for each assay was examined by using DNA specimens from various treponemal and other bacterial species. The TaqMan real-time PCR was able to detect from 10(3) to 10(8) cells of the oral treponemes, with correlation coefficients as follows: T. denticola, 0.984; T. vincentii, 0.991; and T. medium, 0.984. The frequencies of occurrence of these three oral treponemes in subgingival plaque samples were as follows: T. denticola, 68.0%; T. vincentii, 36.0%; and T. medium, 48.0%. In addition, the number of T. denticola, T. vincentii, and T. medium cells in plaque samples detected by real-time PCR ranged from 3 to 15,184, 1 to 447, and 1 to 7,301 cells/pg of plaque DNA, respectively. Increased numbers of T. denticola cells were detected in plaque samples from deep periodontal pockets, and T. medium was also detected in deep pockets. On the other hand, T. vincentii was mainly found in shallow pockets. These results suggest that various oral treponemes are associated with the formation of each stage of periodontal disease.
Papillomatous digital dermatitis (PDD), an emerging infectious disease of cattle, is characterized by painful, ulcerative foot lesions. The detection of high numbers of invasive spirochetes in PDD lesions suggests an important role for these organisms in the pathogenesis of PDD. PDD-associated spirochetes have phenotypic characteristics consistent with members of the genus TREPONEMA: Partial 16S ribosomal DNA (rDNA) sequence analysis of clonal isolates from California cattle showed that they comprise three phylotypes which cluster closely with human-associated Treponema spp. of the oral cavity (T. denticola and T. medium/T. vincentii) or genital area (T. phagedenis). The goal of our study was to apply 16S-23S rDNA intergenic spacer region (ISR) sequence analysis to the molecular typing of U.S. PDD-associated Treponema isolates. This methodology has potentially greater discriminatory power for differentiation of closely related bacteria than 16S rDNA analysis. We PCR amplified, cloned, and sequenced the ISRs from six California PDD-associated Treponema isolates and, for comparative purposes, one strain each of T. denticola, T. medium, T. vincentii, and T. phagedenis. Two ISRs that varied in length and composition were present in all the PDD-associated Treponema isolates and in T. denticola, T. medium, and T. phagedenis. ISR1 contained a tRNA(Ala) gene, while ISR2 contained a tRNA(Ile) gene. Only a single ISR (ISR1) was identified in T. vincentii. Comparative analyses of the ISR1 and ISR2 sequences indicated that the California PDD-associated Treponema isolates comprised three phylotypes, in agreement with the results of 16S rDNA analysis. PCR amplification of the 16S-tRNA(Ile) region of ISR2 permitted rapid phylotyping of California and Iowa PDD-associated Treponema isolates based on product length polymorphisms.
Four spirochete strains were isolated from papillomatous digital dermatitis (PDD) lesions in Iowa dairy cattle and compared with two previously described spirochete strains isolated from dairy cattle in California. These six strains shared an identical 16S ribosomal DNA sequence that was 98% similar to Treponema phagedenis and 99% similar to the uncultivated PDD spirochete sequence DDLK-4. The whole-cell protein profiles resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these six strains were similar. However, these strains showed differences in the antigenic diversity of lipopolysaccharide (LPS). Genetic diversity was also detected by pulsed-field gel electrophoresis of genomic DNA digests, revealing differences among five of the six strains. Serum immunoglobulin G antibodies from dairy cattle with active PDD lesions reacted with the LPS of all but one PDD spirochete strain. Likewise, peripheral blood mononuclear cells from cattle with active PDD lesions produced blastogenic responses to one of the two California isolates. Both antibody and lymphocyte blastogenic responses were reduced in convalescent dairy cattle, suggesting the immune response to these spirochetes has short duration. These results demonstrate genetic and antigenic diversity among T. phagedenis-like treponemes and provide further evidence for the involvement of these spirochetes in the pathogenesis of PDD.