So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a ‘pathogen-related oral spirochaete’ due to the presence of a ∼37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1 % human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98·5 % similarity to its closest cultured relative, T. denticola. The name Treponema putidum sp. nov. is proposed (type strain OMZ 758T=ATCC 700334T=CIP 108088T).

Strong phospholipase A (PLA) and phospholipase C (PLC) activities as potential virulence factors are the outstanding characteristics of eight strains of small oral spirochaetes isolated from deep periodontal lesions. By qualitative dotblot DNA-DNA hybridization and 165 rDNA sequence comparison, these spirochaetes form a distinct phylogenetic group, with Treponema maltophilum as its closest cultivable relative. Growth of these treponemes, cells of which contain two endoflagella, one a t each pole, was autoinhibited by the PLAmediated production of lysolecithin unless medium OMIZ-Pat was prepared without lecithin. N-Acetylglucosamine was essential and D-ribose was stimulatory for growth. All isolates were growth-inhibited when 1 O/ O foetal calf serum was added to the medium. Growth on agar plates supplemented with human erythrocytes produced haemolysis. In addition to PLA and PLC, the new isolates displayed strong activities of alkaline and acid phosphatases, /?-galactosidase, /?-glucuronidase, N-acetyl-/?-glucosaminidase and sialidase, intermediate activities of C4-and C8-esterases, naphthol phosphohydrolase and a-fucosidase and a distinctive 30 kDa antigen detectable on Western blots. This phenotypically and genotypically homogeneous group is proposed as a novel species, Treponema lecithinolyticum sp. nov., with isolate OM2 684T designated as the type strain. A molecular epidemiological analysis using a T. lecithinolyticum-specif ic probe showed this organism to be associated with affected sites when compared with unaffected sites of periodontitis patients. This association was more pronounced in patients with rapidly progressive periodontitis than in those with adult periodontitis.

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.

No abstract

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.

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.

No abstract

Tierarztpraxis, CH-3186 Dü dingen, SwitzerlandDermatitis digitalis is an economically important ulcerative disease of undetermined aetiology affecting the hooves of cattle. Material was examined from two independent cases of this disease in Switzerland. Cultures from the advancing front of both lesions yielded large numbers of closely related short, mesophilic, non-motile, non-spore-forming, anaerobic, proteolytic, Gram-positive rods. The 16S rRNA gene sequences of strains OMZ 913 T and OMZ 915 were identical and indicate Tindallia magadiensis and Eubacterium saphenum as their closest relatives. Phenotypically, the novel isolates are clearly distinguished from related bacteria by protein and antigen patterns, by cellular fatty acids and by API ZYM activities. The diamino acid of the Gram-positive cell wall is ornithine and the G+C content of OMZ 913 T DNA is 44?4 mol%. The phylogenetic distance from recognized taxa in the phylum Firmicutes is sufficient to place these bovine isolates into a novel genus and species, for which the name Guggenheimella bovis gen. nov., sp. nov. is proposed, with OMZ 913 T (=CIP 108087 T =DSM 15657 T ) as the type strain.Dermatitis digitalis (DD), also known as Mortellaro disease or strawberry foot, is an economically important ulcerative disease affecting the bovine foot in an increasing number of countries including Germany, Italy, Japan, the Netherlands, Switzerland, the UK and the USA and may be the dominant cause of lameness in dairy cows (Blowey & Sharp, 1988;Choi et al., 1997;Collighan & Woodward, 1997; Collighan et al., 2000; Demirkan et al., 1998; Luginbühl & Kollbrunner, 2000; Moter et al., 1998;Schrank et al., 1999;Shibahara et al., 2002;Walker et al., 1995). The reported spread between herds by veterinarians, foot trimmers and purchased animals as well as curing of the condition by antibiotic therapy are characteristics of an infectious disease (Laven, 2001). However, despite extensive research, a specific infectious agent has not been identified, and it has been suggested that the disease may be a polymicrobial infection (Döpfer et al., 1997). The range of bacteria cultured from DD lesions includes Campylobacter sputorum (Shibahara et al., 2002), Porphyromonas levii, Fusobacterium necrophorum, Fusobacterium nucleatum, Prevotella oralis, Prevotella denticola, Prevotella bivia, Treponema brennaborense (Schrank et al., 1999) and as-yet unnamed treponemes (Demirkan et al., 1999;Stamm et al., 2002;Walker et al., 1995). By using culture-independent methods, such as immunocytochemistry and 16S rRNA gene sequence analysis, additional organisms were detected in lesions and were related to Bacteroides levii, Borrelia burgdorferi, Mycoplasma hyopharyngis and several Treponema species (Collighan & Woodward, 1997; Demirkan et al., 1998; Moter et al., 1998). Treponemes have been detected deep in the affected tissue by in situ hybridization using fluorescent 16S rRNAtargeted oligonucleotide probes; however, the eubacterial organisms seen at the front of the lesion appeared not to b...