Rat model of polymicrobial infection, immunity, and alveolar bone resorption in periodontal disease

Abstract

One of the predominant polymicrobial infections of humans is expressed clinically as periodontal disease. Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly implicated as members of a pathogenic consortium in the etiology of adult periodontitis. In this study we hypothesized that P. gingivalis, T. denticola, and T. forsythia are synergistic in terms of virulence potential and induce chronic periodontal inflammation that leads to alveolar bone resorption in a polymicrobial infection in rats. Groups of rats were infected with either P. gingivalis, T. denticola, or T. forsythia in monomicrobial infections or with all three species in polymicrobial oral infections with or without Fusobacterium nucleatum. PCR analyses of oral microbial samples demonstrated that rats infected with one bacterium were orally colonized by each of the bacteria during the study interval, and increased serum immunoglobulin G (IgG) antibody levels substantiated the interaction of the host with the infecting bacteria. PCR analyses of the rats with polymicrobial infections demonstrated that most rats were infected with P. gingivalis, T. denticola, and T. forsythia as a consortium. Furthermore, all rats exhibited a significant increase in the level of IgG antibody to the polymicrobial consortium. Radiographic measurement of alveolar bone resorption showed that rats infected with the polymicrobial consortium with or without F. nucleatum exhibited significantly increased alveolar bone resorption compared to the resorption in uninfected control rats, as well as the resorption in rats infected with one of the microbes. These results documented that P. gingivalis, T. denticola, and T. forsythia not only exist as a consortium that is associated with chronic periodontitis but also exhibit synergistic virulence resulting in the immunoinflammatory bone resorption characteristic of periodontitis.

FIG. 1.

Schematic diagram illustrating the experimental design, including rat acclimation, antibiotic treatment, PerioGard oral swabbing, preinfection oral microbial sample collection, monomicrobial infection (four to six infections) and polymicrobial infection (four infections), oral microbial sample collection (four to six times), PCR analysis, euthanasia, and gingival tissue and alveolar bone collection. For detailed information see Materials and Methods. Pg, P. gingivalis; Td, T. denticola; Tf, T. forsythia.

FIG. 2.

PCR analysis of oral microbial samples from rats infected with a polymicrobial inoculum (P. gingivalis, T. denticola, and T. forsythia or P. gingivalis, T. denticola, T. forsythia, and F. nucleatum). Agarose (1.5%) gels contained PCR products from reactions with P. gingivalis, T. denticola, T. forsythia, and F. nucleatum primers. Lane M, 1 Kb Plus DNA ladder; lane −, negative control containing the appropriate bacterial primer but no target DNA; lane +, positive control; lanes 1 to 11, oral microbial samples from individual infected rats. For P. gingivalis 381, a 600-bp amplicon was obtained and 9 of 11 infected rats were positive for P. gingivalis after the third infection. For T. denticola ATCC 35404, a 860-bp amplicon was obtained and all 11 infected rats were positive for T. denticola after the fourth infection (a few bands were faint). For T. forsythia ATCC 43037, a 426-bp amplicon was obtained and 9 of 11 infected rats were positive for T. forsythia after the fourth infection (a few bands were faint). For F. nucleatum ATCC 49256, a 360-bp amplicon was obtained and 7 of 11 infected rats were positive for F. nucleatum after the third infection (a few bands were faint).

FIG. 3.

Serum IgG antibody levels in serum from rats (collected at end of a 12- to 16-week infection) following monomicrobial infection (n = 4 to 9) or polymicrobial infection (n = 11). The graphs show the results for IgG antibody reactive with each of the three species of bacteria. The bars indicate the mean antibody levels in serum from rats orally infected with the individual bacteria or with polymicrobial consortia or from uninfected controls. The error bars indicate one standard deviation from the mean. An asterisk indicates that a value is significantly different (P < 0.001) than the value for uninfected controls or for antibody in serum from rats infected with a different microorganism. A number sign indicates that values for the monomicrobial and polymicrobial infection groups are significantly different (P < 0.001 to P < 0.02). An at symbol indicates that the antibody levels are significantly greater (P < 0.001) than the responses to T. forsythia in the rats infected with one species. An ampersand indicates that the antibody level is significantly greater (P < 0.01) than the level of antibody to T. denticola in rats infected with T. forsythia. Pg, P. gingivalis; Td, T. denticola; Tf, T. forsythia; Fn, F. nucleatum; Uninf, uninfected.

FIG. 4.

Maxillary and mandibular alveolar bone resorption in rats following monomicrobial infection with P. gingivalis, T. denticola, or T. forsythia or infection with the polymicrobial consortium containing P. gingivalis, T. denticola, and T. forsythia with or without F. nucleatum. Each bar indicates the mean alveolar bone resorption for two sites per tooth and three teeth in each quadrant for 4 to 11 rats per group. The error bars indicate one standard deviation from the mean. An asterisk indicates that a value is significantly different from the value for uninfected rats (P < 0.01), and a number sign indicates that there is a significant difference between a monomicrobial infection and a polymicrobial infection (P < 0.01). (A) Maxillary alveolar bone resorption in rats. (B) Mandibular alveolar bone resorption in rats. Pg, P. gingivalis; Td, T. denticola; Tf, T. forsythia; Fn, F. nucleatum; Uninf., uninfected.