A total of six active and six nonactive sites from six untreated periodontitis patients were examined for intragingival presence of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis. The active destructive periodontal disease was determined by the "tolerance method." The method of immunoperoxidase was used in the identification of intragingival microorganisms in active and nonactive periodontal sites. Light microscopic sections of gingival tissues consecutive to those with gram stain, revealing presence of bacteria (substantiated by electron microscopy), were stained with peroxidase-labeled antibodies against A. actinomycetemcomitans and B. gingivalis. B. gingivalis was found to be significantly elevated in the connective tissue of active sites when compared to nonactive sites. A statistically significant border-line difference was found between active and nonactive sites in the connective tissue invaded by A. actinomycetemcomitans. Our findings plus the well established periodontopathic potential of A. actinomycetemcomitans and B. gingivalis support the concept that these bacteria are important invasive pathogenic agents in periodontitis.

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The purpose of this study was to identify and quantify mononuclear cells and invasive bacteria in consecutive histological sections in diseased gingiva. Gingival biopsies were obtained from sites displaying evidences of severe Periodontitis (pocket depth >5 mm, bleeding on probing) in six patients. Specimens were frozen and serially sectioned at 8 fim in a cryostat. Monoclonal antibodies (mAb) directed against membrane markers of mononuclear infiltrate cells and rabbit polyclonal sera against specific bacteria shown to be invasive in association with an immunoperoxidase technique and specific point quantification were used. The mAb panel included Leu 1 (Pan T), Leu 2a (T suppressor/cytotoxic), Leu 3a (T helper/inducer), Leu 6 (Langerhans/dendritic), Leu 7 (NK/K), Leu M3 (monocyte/macrophage), and B7 (B cell). This methodology allows for in situ per cent quantification of mononuclear cell subsets along with identification and quantification of invasive bacteria in the same gingival tissue site. This may be a practical approach to establish the relationship between bacterial invasion and cellular immune response by the host. This technique enabled the characterization of the mononuclear infiltrate in relationship to specifically identified invasive bacteria in diseased gingiva.

The presence of bacteria within the gingival oral epithelium and adjacent connective tissue in cases of periodontitis and localized juvenile periodontitis have been described using scanning and transmission electron microscopy. The following bacterial morphotypes were identified: cocci, short rods, filaments and few spirochetes in periodontitis and mainly coccobacillary-shaped bacteria in localized juvenile periodontitis. Also Mycoplasma-like structures were identified in the localized juvenile periodontitis cases. Tunnel-like formations at different depths of the oral epithelium contained higher numbers of bacteria than those seen on the adjacent oral surface. Identification of specific bacteria in the oral epithelium may have important pathogenic and therapeutic implications.