Human Oral Treponemes Research Articles

The occurrence of Treponema spp. in gingival plaque from dogs with varying degree of periodontal disease.

Periodontal disease is common in dogs and is initiated by gingival plaque composed of several hundred bacterial species. Some of these species have specifically been pointed out as potential periodontal pathogens, such as Treponema spp. Treponema spp. are difficult to culture and therefore the majority have been detected by culture-independent methods, such as PCR (Polymerase Chain Reaction). This leaves many Treponema spp. uncharacterized and unnamed. In this study, treponemes were investigated in gingival plaque from dogs with varying degree of periodontal disease with the aim to describe their occurrence and diversity in dogs. The methods used were culture, phase-contrast microscopy, PCR targeting the 16SrRNA-tRNAIle intergenic spacer region (ISR2), sequencing of the ISR2 and phylogenetic analysis. Treponema spp. were detected in samples from 10 out of 11 dogs and isolates were obtained from six dogs. Both healthy and periodontitis affected dogs were Treponema positive. Phylogenetic analysis, based on ISR2 sequences, revealed a large diversity of treponemes in the study population that were found to be distributed mainly in two groups, corresponding to the human oral treponeme phylogroups II (Treponema denticola) and IV (Treponema maltophilum) genetic groups. They were generally more distantly related to other treponemes in these groups. Treponemes from dogs with periodontitis and dogs with mild gingivitis without periodontitis did not differ in any obvious way. The results indicate that several phylotypes of oral treponemes are common in dogs regardless of periodontal status.

In-depth snapshot of the equine subgingival microbiome

This study explored the range of bacterial taxa present within healthy subgingival (below the gum-line) niches in the horse oral cavity using 16S rRNA gene amplicon pyrosequencing. Pooled subgingival plaque samples were collected from approximately 200 sulcus sites from two horses (EQ1, EQ2) for analysis. A total of 14,260 quality-filtered pyrosequencing reads were obtained, which were assigned to 3875 operational taxonomic units (OTUs; 99% identity cut-off); 1907 OTUs for EQ1 and 2156 OTUs for EQ2. Diverse taxa from 12 phyla were identified, including Actinobacteria (3.17%), Bacteroidetes (25.11%), Chloroflexi (0.04%), Firmicutes (27.57%), Fusobacteria (5.15%), Proteobacteria (37.67%), Spirochaetes (0.15%), Synergistetes (0.22%), Tenericutes (0.16%), GN02 (0.19%), SR1 (0.01%) and TM7 (0.37%). Many OTUs were not closely related to known phylotypes, and may represent ‘equine-specific’ taxa. Phylotypes corresponding to Gammaproteobacteria were abundant, including Actinobacillus spp. (8.75%), unclassified Pasteurellaceae (9.90%) and Moraxella spp. (9.58%). PCR targeting the Synergistetes and Spirochaetes phyla was performed, and resultant plasmid libraries of 16S rRNA gene amplicons (ca. 1480 bp) were Sanger sequenced. Twenty-six Spirochaetes OTUs, and 16 Synergistetes OTUs were identified (99% identity cut-off). These ‘species-level’ OTUs were assigned Equine Oral Taxon (EOT) numbers, whose phylogenies and taxonomy were comprehensively investigated, in conjunction with corresponding Synergistetes and Spirochaetes OTUs identified by pyrosequencing. The vast majority of Spirochaetes taxa belonged to the genus Treponema, which corresponded to 7 of the 10 human oral treponeme phylogroups. Other Spirochaetes taxa belonging to the Leptospiraceae family were observed; but many treponemes commonly implicated in animal hoof/foot and non-oral soft tissue infections; e.g. Treponema phagedenis, Treponema pedis, Treponema refringens, Treponema calligyrum; were not identified here. Diverse Synergistetes taxa corresponding to oral clusters A and B were identified, which included Fretibacterium fastidiosum and Pyramidobacter piscolens. Taken together, our data reveals that equine subgingival plaque microbiota shares many similarities with the human, canine and feline oral microbiomes.

Detection of Treponemes in Canker Lesions of Horses by 16S rRNA Clonal Sequencing Analysis

Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponemal phylotypes originating from papillomatous digital dermatitis lesions of dairy cattle and as-yet uncultured human oral treponemes, indicating the presence of multiple treponemes in the lesions.

Chemotaxis of oral treponemes toward sera and albumin of rabbit.

The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin-binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin-binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin-binding polypeptide is responsible for chemotaxis toward rabbit serum.

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis.

Genetic characterization of an oral treponeme isolated from human subgingival plaque

We examined the genetic characteristics of a human oral treponeme isolated from subgingival plaque of a patient with periodontal disease that could not be assigned to any of the previously described oral Treponema species. The guanine-plus-cytosine content of its DNA was 51 mole%. The levels of DNA-DNA relatedness of the organism to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum and Treponema phagedenis, were less than 30%. This study clearly demonstrated that the isolated treponeme could be completely distinct from the established oral Treponema species genetically. Also, the study indicated remarkable genetic heterogeneity among human oral Treponema species.

Immunological studies on venereal spirochetosis of rabbits (rabbit syphilis).

White rabbits in a family, which were clinically diagnosed as moderately or severely diseased with spirochetosis, were bacteriologically and immunologically examined. The specimens from the diseased rabbits, including affected prepuces, scrotum, or skins with an occasional presence of the spirochetes, did not, however, result in growth in six conventional culture media. Serological tests, including quantitative complement fixation test, rapid plasma reagin card test, Treponema pallidum hemagglutination test, and microscopic agglutination test for leptospires using sera from diseased rabbits showed no differences when compared with those of pooled normal rabbit sera. Immunoblot analysis of the polypeptides from three human oral treponemes and three non-oral spirochetes demonstrated that antibodies against several treponemal polypeptides were detected.

Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp

A total of 22 strains of Treponema spp. including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae. None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria. All treponemes, eight Treponema denticola strains, eight T. socranskii strains, four oral pectinolytic treponemes, one T. pectinovorum strain, and one T. vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners. The partners consisted of one strain of Fusobacterium periodonticum, five F. nucleatum strains including all four subspecies of F. nucleatum, and a strain of F. simiae obtained from the dental plaque of a monkey. In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment. Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected. Only the T. denticola coaggregations were inhibited by lactose and D-galactosamine. None were inhibited by any of 23 other different sugars or L-arginine. Intragenic coaggregations were seen among the subspecies of F. nucleatum and with F. periodonticum, and none were inhibited by any of the sugars tested or by L-arginine. No intrageneric coaggregations were observed among the treponemes. These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners. Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.

Hydrolysis of the Leu-Gly bond of phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-D-Arg (a substrate of microbial collagenases) by treponemes isolated from the subgingival plaque of periodontitis patients

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl-l-prolyl-l-leucylglycyl-l-prolyl-d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US),Treponema denticola ATCC 35405, and 29 different clinical isolates ofT. denticola. TheTreponema US enzyme also hydrolyzed furylacryloyl-l-leucylglycyl-l-prolyl-l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators andp-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by “true” microbial collagenases.

Antisera to human oral treponemes and their use for detection of spirochetes in subgingival dental plaques.

Antisera to human oral treponemes and their use for detection of spirochetes in subgingival dental plaques.

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 micrometer to 0.2 micrometer in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.

Electron microscopy of the spherical body of oral spirochetes in vitro. Further studies.

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and th cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils. These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.