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Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.

Fifty-one fluorescence-positive black-pigmented Bacteroides strains obtained from 51 patients with deep periodontal pockets (greater than 6 mm) were identified and characterized. Fifty of these strains were presumptively identified as Bacteroides intermedius according to the indole reaction. This was confirmed by further biochemical characterization. The 50 strains from diseased sites were then compared with 16 B. intermedius strains isolated from periodontally healthy individuals with no signs of destructive periodontal disease. Tests for antimicrobial susceptibility showed similar patterns for all 50 pocket-derived strains, except for one beta-lactamase-positive strain that was resistant to penicillin G and ampicillin. Forty-seven strains were tested for binding of three monoclonal antibodies defining three distinct serogroups of B. intermedius. Thirty-one strains belonged to serogroup I, three to serogroup II and thirteen to serogroup III. In comparison to the strains from the shallow periodontal pockets, serogroup I was significantly overrepresented in the patient group with periodontal disease. We conclude that saccharolytic black-pigmented Bacteroides species from deep periodontal pockets constituted, with very rare exceptions, a biochemically homogeneous but antigenically heterogeneous group of B. intermedius and that serogroup I is predominantly found in deep periodontal lesions.

The development of indirect immunofluorescence assays using MAbs as specific probes has made it possible to detect a variety of suspected periodontal pathogens in subgingival plaque, with exquisite sensitivity. The studies demonstrate high reproducibility of the results if different MAbs are used to assess the same bacterial species. Bacteroides forsythus, Bact. gingivalis, Bact. intermedius and Wolinella recta, but not Actinobacillus actinomycetemcomitans were found in sites of adult periodontitis with high prevalence but quite distinct degrees of colonization. The colonization levels of Bact. forsythus and Bact. gingivalis were significantly associated with the probing depth of the lesions. The investigations demonstrate the usefulness of serological analysis of plaque and indicate that several of the organisms implicated in the aetiology of periodontal diseases may be tolerated in high numbers in subgingival lesions, without periodontal destruction.

Several methods are currently being used to identify specific bacteria in dental plaque, namely direct culture, serological techniques and DNA probes. Culture methods are labour-intensive, dependent on the viability of the cells, and require fastidious growth conditions. Serological and DNA probes allow rapid strain-specific identification of periodontal pathogens with limited effort. The purpose of the present study was to compare results from serological and DNA probes in assessing and quantitating the presence of three suspected periodontal pathogens in subgingival plaque: Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bact. intermedius. Subgingival bacterial samples were obtained from 4 periodontal sites of each of 13 patients with untreated moderate to advanced adult periodontitis. Samples were taken using 2 paper points that were simultaneously inserted for 10 s to the bottom of the periodontal pockets. The plaque from one paper point was analysed by immunofluorescence using monoclonal antibodies. The other sample was analysed by species-specific cloned DNA probes. The number of bacteria per millilitre was calculated for both methods, and used for comparisons of results obtained with the two techniques. Results from indirect immunofluorescence and DNA hybridization analyses were negative for A. actinomycetemcomitans across all samples. Fluorescence did not detect bacteria at levels lower than 10(4), while the DNA probes identified organisms at levels of 10(3). Similar numbers of samples positive for Bact. gingivalis were obtained with either method (p = 0.227), and the results were not independent. A significantly greater proportion of Bact. intermedius-positive samples was detected by immunofluorescence (p = 0.0039), and the results of immunofluorescence and DNA hybridization were independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Four hybrid cell lines secreting monoclonal antibodies against antigens of Bacteroides intermedius were generated by fusing murine NSI cells with splenocytes from a rat immunized with B. intermedius strain OMZ248. An enzyme-linked immunosorbent assay was used to analyze the distribution of the recognized antigens on 39 strains from various Bacteroides species and on 5 strains from other genera. Only Bacteroides species B. intermedius, B. loescheii, B. melaninogenicus, and B. corporis were found to express at least one of the recognized antigens. Strains of the two asaccharolytic black-pigmenting Bacteroides species were negative. Among the strains capable of binding to one or more of the monoclonal antibodies, five groups with different reactivity patterns could be distinguished. Two of the monoclonal antibodies were specific for B. intermedius. The B. intermedius strains were metabolically almost identical, expressed at least three of the recognized antigens, and fell into three distinct antibody reactivity groups, suggesting a tentative separation of this species into three new serogroups. Oral and nonoral isolates of B. intermedius were, however, not distinguished by the monoclonal antibodies. One monoclonal antibody was directed against an antigen strongly expressed on all saccharolytic black-pigmenting Bacteroides strains tested so far, thus confirming the previously noted antigenic relationship between the species which had emerged from the former B. melaninogenicus subsp. intermedius and B. melaninogenicus subsp. melaninogenicus groups.

By using a sensitive enzyme-linked immunosorbent assay, 200 randomly selected sera from Red Cross blood donors were screened for immunoglobulin G (IgG), IgA, and IgM levels against Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius. A subgroup of 79 blood donors was clinically examined for type and extent of periodontal destruction, and serological and clinical data were subjected in all possible dual combinations to correlation analyses. The results revealed that the majority of the blood donors suffered from moderate to severe adult periodontitis, often coupled with severe gingival inflammation. No cases of localized juvenile periodontitis or rapidly progressive periodontitis were observed. The extent of periodontal destruction proved to be significantly correlated only to the IgG response levels against B. gingivalis. Corresponding correlation tests assessing the relationships of loss of attachment, bone loss, pocket depth, and papillary bleeding index with the IgG responses to A. actinomycetemcomitans were of marginal significance, while the IgG responses to B. intermedius revealed no relationship to the periodontal health status. The specific IgM responses proved to be unrelated to the clinical parameters, but interestingly, they were found to be highly correlated with each other. Specific IgA levels were frequently too low for enzyme-linked immunosorbent assay testing and, therefore, had to be exempted from statistical analyses. Assessments of the serotype specificity of strongly elevated IgG responses to A. actinomycetemcomitans disclosed no evidence for an association of a particular serotype-specific IgG response with the occurrence of adult periodontal destruction. In contrast to results of earlier studies, a number of sera were found to contain strongly elevated IgG levels against two or even all three serotypes. Although derived by an alternative approach, the reported results largely corroborate earlier observations linking only the occurrence of elevated anti-B. gingivalis IgG responses to the presence of marked periodontal lesions in adults.

The surface ultrastructure of 21 strains of Bacteroides intermedius was investigated by electron microscopy. Rat monoclonal antibodies (mAbs) were used to define serogroups and to detect the location of surface antigens. All 21 isolates had capsules as demonstrated by the use of wet and dry Indian ink stains. Negative staining of whole cells with 1% (w/v) methylamine tungstate showed that all 21 isolates carried clumped peritrichous fibrils with strain dependent morphology, density and length (less than or equal to 0.75 micron). Fibrils on 11 of 13 fresh clinical isolates were more conspicuously clumped and easily visible, whereas those on 6 of 8 laboratory strains were indistinct and were at the limits of the resolution of the negative staining technique. Staining with ruthenium red (RR), followed by thin sectioning, revealed a dense, amorphous RR staining layer (RRL), up to 24.8 +/- 3.0 nm thick, adjacent to the outer membrane on all of 15 strains examined. All isolates had a less dense RR staining matrix (RRM) extending away from the RRL. The structure of the RRM varied between strains. Four rat mAbs (37BI6.1, 38BI1, 39BI1.1 and 40BI3.2) were used to serogroup the 21 strains of B. intermedius. Immunonegative staining revealed that the mAbs were not directed against fibrilis. Antigens recognized by mAb 37BI6.1 and mAb 39BI1.1 were located on the surfaces of cells, beneath fibrils, and on extracellular vesicles. mAb 38BI1 recognized an antigen which was most accessible on lysed cells, and non-specific binding of mAb 40BI3.2 to grids prevented its localization on the cell surface.

The prevalence of Bacteroides forsythus and Bacteroides gingivalis in subgingival plaque of patients on short recall was analyzed in relation to the probing depth of the test sites. The subjects had excellent oral hygiene and therefore were unlikely to suffer from active periodontal destruction. Sixty-four subgingival plaque samples, taken from gingival or periodontal pockets with probing depths ranging from 1 to 8 mm, were quantitatively assessed for the presence of the two species using species-specific monoclonal antibodies in conjunction with a very sensitive indirect immunofluorescence technique. Both organisms were encountered in probes from sites as shallow as 2 mm, but the percentage of positive samples clearly rose in relation to the probing depth of the test sites. Overall, B. forsythus was found to colonize lesions earlier, that is at smaller probing depths, than B. gingivalis. Interestingly, whenever a sample was found to be positive for B. gingivalis it was also positive for B. forsythus. The numbers of B. forsythus and B. gingivalis and the total bacterial cell number found in the pockets were significantly correlated to the probing depth. However, with advancing probing depth the increase of the total cell numbers of the two Bacteroides species was considerably more pronounced than the increase of the total subgingival plaque cell number. The recall interval neither affected the frequency of sites positive for B. forsythus or B. gingivalis nor influenced significantly the proportions of the two species in subgingival plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrid cell lines producing monoclonal antibodies against Bacteroides forsythus or Wolinella recta were generated by fusion of SP2/0 or FO myeloma cells with splenocytes from Balb/c mice immunized with formalinized cells of B. forsythus FDC 331 or W. recta D13a-g, respectively. Enzyme-linked immunosorbent assays and indirect immunofluorescence tests were used to analyze the distribution of the recognized antigens on a panel of 70 strains representing 35 taxa, most of which are members of the oral flora. All monoclonal antibodies--eight against B. forsythus and six against W. recta--proved specific for the immunizing species. Four of the monoclonal antibodies against W. recta recognized antigens expressed by only some of the tested W. recta strains, thus confirming the earlier noted antigenic heterogeneity of this species. Antibody binding patterns consistent with those previously described or distinct for new serogroups could not, however, be observed. Four of the eight anti-B. forsythus monoclonal antibodies bound to only two of the three tested B. forsythus strains. All the remaining monoclonal antibodies detected every strain tested of the respective species against which they were raised. Preliminary results indicated that several of these antibodies should be very useful for the direct identification and quantification of these organisms in subgingival plaque.