Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows.

Digital dermatitis (DD) of cattle leads to lameness and a decrease of milk production and is responsible for major economic losses worldwide. Although a bacterial aetiology is generally accepted, it still is unclear which microorganisms cause and/or maintain the disease. Recently, a previously undiscovered bacterial species, Guggenheimella bovis, has been isolated from the front of two DD lesions in Swiss cattle and suggested as a potential pathogen. The aims of the present study were to determine the prevalence of G. bovis in 58 German cows suffering from DD via dot blot hybridization, and to analyse the spatial distribution of G. bovis within the affected tissue by fluorescence in situ hybridization (FISH). A species-specific probe, GUBO1, was designed and evaluated. In none of the 58 samples Guggenheimella could be detected, while cultured G. bovis was reliably identified by GUBO1. Further FISH experiments were carried out on two additional biopsies of Swiss cattle tested positive for G. bovis by quantitative PCR and permitted visualization of the newly discovered bacteria in situ. In these biopsies G. bovis proved to be tissue invasive forming characteristic spherical microcolonies not only within the bacterial biofilm but also in seemingly unaffected parts of the tissue not yet reached by the advancing bacterial front. Although the presence of G. bovis does not constitute an essential premise for DD, it seems likely that the bacterial species involved in DD vary, and that in some cases G. bovis is crucial for the development of DD lesions.

Quantitation of Guggenheimella bovis and treponemes in bovine tissues related to digital dermatitis.

Digital dermatitis is an inflammation of uncertain aetiology in the skin of the foot of cattle. In 2005, a novel microorganism, Guggenheimella bovis, was isolated from the advancing front of digital dermatitis lesions, suggesting a possible role in pathogenesis. In the present study, tissue samples of 20 affected cows were examined by quantitative PCR for G. bovis, treponemes and the total eubacterial load. High numbers of eubacteria and treponemes were found in most lesions, whereas only a few lesions contained Guggenheimella, and only at low concentrations. The results argue against the relevance of G. bovis in the aetiology of digital dermatitis in cattle, but are consistent with a role for treponemes.

Molecular epidemiology of oral treponemes in patients with periodontitis and in periodontitis-resistant subjects.

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.

Gingival crevice microbiota from Chinese patients with gingivitis or necrotizing ulcerative gingivitis.

The objective of this study was to quantitatively compare the bacterial population structure in plaque from the gingival margin of two groups of 21 Chinese patients with gingivitis or necrotizing ulcerative gingivitis (NUG). Subjects were recruited in four dental clinics in Eastern China. Samples were quantitatively assessed by immunofluorescence and fluorescent in situ hybridization for taxa known to be associated with periodontal diseases. The analyses showed that the fusiform taxa (Fusobacterium nucleatum/Fusobacterium periodonticum, Leptotrichia buccalis, Tannerella forsythensis, and Capnocytophaga sp.), Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, Selenomonas sputigena, and treponemes were present in both groups with high prevalence. Porphyromonas gingivalis and Actinomyces gerencseriae were much more prevalent in the NUG group. Quantitatively, most taxa, including P. gingivalis, F. nucleatum and the treponemes, accounted, on average, for < 3% of the total bacterial cell number. Only P. intermedia/P. nigrescens, P. gingivalis, S. sputigena, A. gerencseriae, and the sum of all monitored suspected periodontal pathogens were significantly increased in the NUG group. The present study demonstrates for both groups a highly diverse plaque composition and suggests that, etiologically, the overall concentration and the concerted effects of the entire group of opportunistic pathogens thriving in NUG-associated plaque are of prime importance.

Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes.

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, "Treponema vincentii," and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5' hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes.