Localization of Periodontopathic Bacteria in Gingival Tissues Using Novel mAb

Objectives: Recent publications have reported an association between periodontopathic bacteria and peripheral and central vascular diseases. However none of these studies show clear histological evidence with detection of bacteria in the diseased gingiva or in the vascular sites. Therefore our objectives were to develop monoclonal antibodies (mAbs) specific for Porphyromonas gingivalis and Tannerella forsythia and to locate these bacteria in the gingival tissues. Methods: mAbs were prepared in the conventional method and specificity and cross reactivity were confirmed with ELISA and immunohistochemistry (IHC) using rat liver infected with pathogenic and commensal bacteria commonly present in human body. IHC was done to locate these bacteria in gingival & subgingival granulation tissues collected from 102 patients who underwent periodontal surgery. The results were confirmed with real-time PCR. For selected cases, immuno-fluorescence double staining was done to detect intracellular positivity. Results: Both mAbs gave positive reactions only with respective bacteria infected rat liver sections and did not cross react with other bacteria. Out of 102 cases P. gingivalis was located in 53(65%) and T. forsythia in 66(80%) extracellularly in IHC. P. gingivalis was detected in 59(72%) and T. forsythia in 76(93%) in real time PCR. The correlation between the density of bacterial genomes and their respective IHC grading gave values < 0.001 for both the species. Conclusions: The produced mAbs could be used as specific tools to locate P. gingivalis and T. forsythia in human tissue sections that are formalin fixed and paraffin embedded. IHC is a reliable method of examination for these bacteria in gingival and subgingival granulation tissues because of the close correlation between real-time PCR data and IHC data. Also these bacteria may exist intracellularly in the squamous epithelial cells of diseased gingival tissues in patients with both chronic periodontitis and aggressive periodontitis.