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To evaluate the association of oral Treponema (T.) spp. with severity of canine periodontitis, subgingival plaque samples of dogs of various breeds undergoing surgery were investigated. A wide range of oral Treponema spp. was analysed by a molecular and culture-independent approach applying DNA-DNA dot blot hybridization analysis and fluorescence in situ hybridization using Treponema specific oligonucleotide probes specific for phylogenetic groups I-VII of oral treponemes as well as probes specific for T. socranskii and T. denticola. To assess the periodontal status of affected dogs clinical parameters were measured and the periodontal status was classified from grade 0 (physiological periodont) to 3 (severe periodontitis). The periodontal status correlated significantly with an increasing concentration of volatile sulfur compounds (VSC, r=0.854) determined with a Halimeter, indicating a positive correlation between the presence of VSC-producing bacteria and periodontitis. In this study Treponema spp. of phylogenetic groups III, V-VII were not detected in any sample, whereas T. denticola-like treponemes were found only in 2 of 51 animals. However, treponemes belonging to phylogenetic groups I, II and IV of oral treponemes or T. socranskii were found in up to 64.84% of the dogs. The detection rate of Treponema spp. was significantly associated with an increased periodontal status. Treponemes present in periodontal lesions were also visualized by fluorescence in situ hybridization of gingival biopsies showing Treponema spp. not only in the microbial biofilm but also within the gingival tissue. The data presented here indicate that oral Treponema spp. are associated with canine periodontitis. Similar to human periodontitis, treponemes of groups I, II and IV and T. socranskii were found more frequently the higher the degree of periodontitis was.

Limit-dilution procedures were used to isolate seven, helically coiled bacterial strains from faeces of swine that constituted two unidentified taxa. Comparative 16S rRNA gene sequence analysis showed highest similarity values with species of the genus Treponema indicating that the isolates are members of this genus. Strain 7CPL208(T), as well as five further isolates, and 14V28(T) displayed the highest 16S rRNA gene sequence similarities with Treponema pectinovorum ATCC 33768(T) (92.3%) and Treponema parvum OMZ 833(T) (89.9%), respectively. Polar lipid profiles distinguished 7CPL208(T) and 14V28(T) from each other as well as from related species. Based on their phenotypic and genotypic distinctiveness, strains 7CPL208(T) and 14V28(T) are suggested to represent two novel species of the genus Treponema, for which the names Treponema berlinense sp. nov. and Treponema porcinum sp. nov. are proposed. The type strain for Treponema berlinense is 7CPL208(T) (=ATCC BAA-909(T)=CIP 108244(T)=JCM 12341(T)) and for Treponema porcinum 14V28(T) (=ATCC BAA-908(T)=CIP 108245(T)=JCM 12342(T)).

The genus Treponema consists of various species. Currently most of them are not cultivable because respective cultivation conditions are unknown. Therefore the biodiversity of treponemes was only appreciated recently by applying comparative 16S rRNA sequence analysis. Treponemes are mainly representatives of the gastrointestinal autochthonal flora, especially in termites, but they have also been described in swine and cattle. On the other hand treponemes are involved in different infectious diseases, the most well known being syphilis in humans or venereal spirochetosis in rabbits. Furthermore, treponemes are associated with several infectious periodontal diseases, e.g. gingivitis or periodontitis, where they can be detected regularly. Culture has not been successful for most of the oral treponemes, so the major part can only be identified by their 16S rRNA sequence. Similar to these oral disorders treponemes are also associated with digital dermatitis (DD), a chronic inflammatory disease of the bovine skin, where different treponemal phylotypes were found in large numbers. Treponema brennaborense was first identified and isolated in DD biopsies. Unravelling the pathogenic potential and aetiological significance of treponemes in chronic infectious diseases like peridontitis or DD remains a costly task. Although treponemes can be frequently detected in such lesions, it is often unclear to what extent treponemes are involved in pathogenesis of these diseases. The possession of various virulence features like high motility, the ability to adhere and invade as well as to cause cytopathic effects in eukaryotic cells are highly indicative of the aetiological relevance of treponemes.

The aim of this study was to establish an in vitro model that permits in vitro infection of bovine skin with Treponema spp. and enables to study the role of treponemes in the pathogenesis of digital dermatitis (DD). In all experiments, incubation with T. denticola or T. brennaborense was carried out simultaneously. Keratinocytes obtained from the claw were cultivated on cover glasses without antibiotics until they reached sub-confluence. Then they were incubated with OMIZ Pat medium containing treponemes for up to 96 h. Every 24 h two cover glasses were fixed and stained with the DAPI method. Skin explants were obtained from typical sites of DD lesions. First the explants were maintained in medium with antibiotics to eliminate bacterial contamination. Subsequently, they were rinsed thoroughly with medium without antibiotics and incubated with Treponema suspensions for 48 h. The treponemes stayed vital under culture conditions for even up to 96 h. They were still showing their typical spiral shaped morphology and adhered to the cultured keratinocytes at all time points. With prolonged incubation time cultured cells began to show morphologic damage and some cells detached from the cover glasses. Light and electron microscopical investigations of the explants revealed that treponemes were adhering to the surface of the epidermis. They were visible in often-enlarged intercellular spaces. In addition treponemes could be detected in deep epidermal layers and in very high concentrations in the dermis. In periodontal disease, spirochetes were observed in enlarged intercellular spaces. Our results support these findings suggesting that treponemes invade the deeper claw tissue via the intercellular spaces of the epidermis. We suggest that the enlargement of the intercellular spaces can improve an increased infection of deeper tissue layers and facilitates the way for infection with other anaerobic bacteria. [ABSTRACT FROM AUTHOR]