Prevotella intermedia on Progression of Ligature-Induced

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INFECTION AND IMMUNITY, OCt. 1991, p. 3351-3359

Vol. 59, No. 10

0019-9567/91/103351-09$02.00/0 Copyright © 1991, American Society for Microbiology

Effects of Immunization with Porphyromonas gingivalis and Prevotella intermedia on Progression of Ligature-Induced Periodontitis in the Nonhuman Primate Macaca fascicularis J. L. EBERSOLE, M. BRUNSVOLD, B. STEFFENSEN, R. WOOD, AND S. C. HOLT*


of Periodontics,


The University of Texas Health Science Center San Antonio, San Antonio, Texas 78284

Received 12 February 1991/Accepted 3 July 1991

The nonhuman primate (Nhp) has proven to be a useful model of human periodontitis. This study describes the immunological characteristics of this model and the ability of active immunization to interfere with ecological changes in the microbiota and its associated disease symptoms. Nhps were parenterally immunized with whole-cell antigens of Porphyromonas gingivalis and Prevotella intermedia. The immunization elicited an approximate 2-log increase in serum immunoglobulin G (IgG), IgM, and IgA isotype antibody that was highly specific for these immunogens. Postimmunization and postligation, there was minimal change in the levels of specific antibody. P. gingivalis immunization significantly inhibited the emergence of this species during disease progression. In contrast, induction of anti-P. intermedia antibody had a minimal effect on this species within the subgingival plaque. Plaque indices showed few changes that could be attributed to active immunization. Both bleeding on probing and loss of attachment were higher in ligated sites of immunized animals than in the placebo-treated group. A signfficant increase in bone density loss was observed in the ligated teeth from immunized versus control animals. These findings indicate that active immunization of Nhps can elicit a substantial systemic immune response; however, while this response may effect the emergence of an individual microorganism, it appears that other ecological considerations are critical in disease progression. It is also possible that the induction of a broad-based immune response to multiple bacterial antigens can result in increased disease, potentially associated with hypersensitivity reactions to the bacteria in the subgingival plaque.

A considerable amount of research has determined that the various human periodontal diseases are microbiologically mediated. Of the vast numbers of bacterial species which occupy the gingival crevice and the developing periodontal pocket, only a small group are considered putative pathogens (3, 13, 38, 39, 42, 45, 46, 50, 54). Ethical restrictions on human experimentation do not permit studies which might conclusively implicate these pathogens as an etiologic agent(s) or elucidate the host-parasite interactions which occur in the progression of humnan periodontal disease. Therefore, we and others have documented both nonhuman primate (Nhp) (5, 6, 28, 31) and murine (mouse, rat, and hamster) (1, 7, 24, 35, 48, 51) models of periodontal disease. Importantly, cross-sectional microbiological studies have established a relationship between human periodontitis and ligature-induced periodontitis in the Nhp model (2, 23, 26, 27, 40). Although there is an extensive literature describing humoral immune responses to several of the putative periodontopathic bacteria in humans (14), it is difficult to characterize the exact nature of the host-parasite interactions which occur in humans, especially in the early stages of disease. With the exception of several investigations employing the rodent periodontal-disease model (35, 49, 51-53), current studies provide little information concerning longitudinal host immunological responses during periodontitis development. However, an inherent weakness in the rodent model is the difficulty in relating periodontal-disease progression in the rodent to that in humans. *

The Nhp model has been used to study the progression of periodontitis (20, 21, 23, 31) and provides insight into the possible participation of other members of the subgingival microbiota in disease progression as it might occur in a longitudinal fashion in humans (13, 19). The results presented here demonstrate an increase in select bacterial species during ligature-induced periodontitis in the Nhp, including Bacteroides melaninogenicus (Prevotella melaninogenica)Prevotella denticola Prevotella, loescheii, Bacteroides macacae, Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Wolinella recta. Therefore, all or several of these species might be major contributors to the inflammatory events of ligature-induced periodontitis in the Nhp. Thus, even in Nhps which appear to be free of P. gingivalis, ligature-induced periodontitis occurs, indicating the role of other members of this complex microbiota. While we have an excellent understanding of the clinical, microbiological, and cytological events in the progression of ligature-induced periodontitis in the Nhp, we still have little information relevant to the immunological responses of these animals to the microbiota of progressing disease or the function of this immune response (11, 23, 28). Further, classical microbiological and immunological approaches have revealed that control of a virulent microorganism or elimination of it from an environment would have a positive outcome on the protection of the host from infection with that microorganism. Therefore, in periodontal disease, it should be possible to control disease progression by either completely eliminating crucial members of the periodontopathic microbiota (e.g., P. gingivalis) or by maintaining these bacteria at very low levels. Since both the microbial

Corresponding author. 3351




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bIolog ----; Immunoogy



FIG. 1. Experimental protocol for examination of the effects of selected whole-cell antigens on immunological interference with ligature-induced periodontitis. After baseline samples for microbiological, immunological, clinical, and radiographic measurements, the Nhps were divided into four to six animals per group and were parenterally (actively) immunized (three times biweekly) with formalin-killed bacterial antigens or with IFA as a placebo. Two weeks after the last immunization, ligatures were placed, and the Nhps were sampled during the subsequent 35 weeks. (See text for detailed protocol.)

origin and the temporal progression of the disease are well understood, any interference with this ordered progression should arrest or even eliminate the disease itself (41). The present study extends our observations on ligatureinduced periodontitis in the Nhp Macaca fascicularis to determine whether there was a correlation between serum immunoglobulin G (IgG) levels elicited in response to the putative periodontopathic species P. gingivalis and P. intermedia and to investigate whether the levels of antibody to these species in serum were capable of protecting Nhps from the progression of ligature-induced periodontal disease. MATERIALS ANIl METHODS

Animals. Twenty adult female cynomolgus monkeys of the species M. fascicularis (Primate Imports, Port Washington, N.Y.) were used (27). The protocol as outlined in Fig. 1 was followed during the course of this experiment. Baseline microbiological and serum antibody levels to P. gingivalis, P. intermedia, P. loescheii, B. melaninogenicus, B. melaninogenicus-P. denticola, E. corrodens, A. actinomycetemcomitans, W. recta, and Capnocytophaga spp. were determined (27). Since the levels of these marker bacteria and their serum antibodies were found to vary among the animals, the monkeys were randomly divided into four experimental groups consisting of five animals per group immunized with P. intermedia, P. gingivalis, Bacteroidesfragilis, or placebo. The last two groups served as controls. All animals were maintained in accordance with the guidelines of the University of Texas Health Science Center at San Antonio, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Immunization. Whole-cell antigens were prepared by standard procedures (16). P. gingivalis 3079.03 (monkey isolate), P. intermedia 6235.2 (monkey isolate), and B. fragilis ATCC 25285 were grown as previously described (17), centrifuged into a pellet, and killed with 0.5% buffered formal saline. The fixed bacteria in phosphate-buffered saline containing 1 mM EDTA were emulsified with an equal volume of incomplete Freund's adjuvant (IFA), stored at 4°C, and used as the antigen in the immunization protocol. Each group was immunized intramuscularly with 109 total cells or with pla-

cebo (phosphate-buffered saline in IFA). Each animal received three separate injections separated by 2-week intervals. Serum samples were obtained by venipuncture from the femoral vein at the time points indicated in Fig. 1. All serum was stored frozen at -20°C. Specific-antibody determination. Levels of antibodies to the test bacteria were determined by quantitative enzymelinked immunosorbent assay using formalin-killed bacterial strains as antigens (15, 23). Previous studies (unpublished) using this procedure demonstrated that anti-human IgG reacted with Nhp IgG and was specific for purified human polyclonal IgG (Calbiochem), anti-human IgM reacted with Nhp IgM and was specific for human IgM (Calbiochem), and anti-human IgA reacted with Nhp IgA and was specific for purified human IgA (Calbiochem). A reference standard antiserum was prepared by hyperimmunization of three Nhps with the bacteria. The Nhp reference standard contained 100 enzyme-linked immunosorbent assay units (EU) of IgG, 6.3 EU of IgM, and S EU of IgA for P. gingivalis; 100 EU of IgG, 50 EU of IgM, and 50 EU of IgA for P. intermedia; and 50 EU of IgG, 200 EU of IgM, and 25 EU of IgA for B. fragilis. The reference standard was prepared such that for all microorganisms, 1 EU of an isotype was approximately the same. Microbiology. Microbiological sampling of the gingival crevice area and culturing procedures employed endodontic paper points (27). All isolated colonies were characterized biochemically and morphologically (27). Clinical observations. Clinical changes were assessed by employing standard plaque and bleeding indices, probing depth, and attachment level. All measurements were made by the same clinical examiner who remained blinded to all of the recorded observations. Plaque accumulation and gingival inflatnmation were recorded for two teeth per side per animal (teeth 3-6, 3-7, 4-6, and 4-7). The index of Silness and Loe (37) was selected for plaque assessment, while gingival inflammation was evaluated by the degree of bleeding on probing. As for the microbial sampling, clinical scoring was performed at the mesiobuccal corner close to the interdental contact point of each examined tooth. Attachment level measurements were also made by inserting the probe vertically, with the occlusal level of the marginal ridge proving to be a reliable reference point. Radiographic measurements were obtained by standardized methods for computer-assisted densitometric image analysis (CADIA) as described previously (4, 43, 44), and the interproximal areas corresponding to the sampling sites were analyzed for densitometric changes. For statistical analysis, the changes from baseline were expressed for each radiographic area by the parameter change in density. Ligation. After baseline sampling 2 (Fig. 1) and following active immunization, three adjacent teeth (3-5, 3-6, and 3-7) in the mandibular left quadrant (quadrant 3) were ligated with 3-0 silk sutures (Fig. 1) (27). The contralateral mandibular teeth (4-5, 4-6, 4-7) served as the nonligated controls. Statistical methods. Clinical changes were analyzed to evaluate the effects of ligation and immunization and the duration of these effects during the test period. Site measurements were used for correlating relationships between microbiological and clinical changes. Group effects were tested by the Kruskal-Wallis test, and the Wilcoxon matched-pair test (SAS statistical program) was applied in order to compare changes in the ligated sites compared with the nonligated sites as well as changes between baseline recordings and all subsequent observations.


VOL. 59, 1991


TABLE 1. Descriptive statistics of naturally existing serum antibody levels in Nhps to Porphyromonas, Prevotella, and Bacteroides speciesa Bacterium and antibody

P. intermedia IgG

IgM IgA P. gingivalis IgG IgM IgA B. fragilis IgG IgM IgA

ELISAb units Minimum SEM




89.6 21.0 17.9

81.5 13.5 14.0

9.8 4.2 2.0

20 5 7

184 70 39

24.7 5.6 1.9

16.0 4.0 1.5

4.9 1.0 0.3

2 2 1

95 16 6

4.7 353.8 10.2

4.5 255.0 8.0

0.3 69.0 1.8

3 75 5

10 1,253 43


aIn every case, the sample size was 20 animals. b ELISA, enzyme-linked immunosorbent assay.

For microbiological comparisons, the average CFU for the species and total counts for all sampled ligated and nonligated teeth were averaged for baseline determinations and for each immunization group at the eight sampling times. The levels of selected cultivable microbiota both pre- and postligation were assessed by determination of the maximum postligation value for the teeth under study and comparing it with the preligation value at week 16. Mean changes in percent microbiological values and their standard errors for each group were calculated for both ligated and nonligated teeth. RESULTS

Response to systemic immunization. All of the Nhps in these studies exhibited baseline levels of antibody to P. gingivalis, P. intermedia, and B. fragilis (Table 1). The predominant isotype against P. gingivalis and P. intermedia was IgG, with levels of antibody to P. intermedia (89.6 + 9.8 EU) being approximately fourfold greater than those to P. gingivalis (24.7 + 4.9 EU). In contrast, the principal response to the gut microorganism B. fragilis was the IgM isotype, with levels 30- to 100-fold greater than that of IgG or IgA antibody. Significant increases in serum antibody of all isotypes was detected following active immunization (Fig. 2). The level of serum IgA antibody was increased to a greater degree than the other isotypes with each of the microorganisms. Additionally, the antibody responses were specific for each of the immunizing antigens, since the levels of antibody to the homologous microorganism were 100- to 1,000-fold greater than the level of the antibody to the heterologous antigens. The kinetics of serum IgG responses to the three bacterial antigens are seen in Fig. 3. While there was a substantial increase in the antibody response following active imtnunization against the bacteria, the dynamics of the antibody response postimmunization and postligation differed with each species. Antibody to P. gingivalis increased through week 27 (11 weeks postligation) and remained elevated through approximately 39 weeks of the study, while IgG antibody to P. intermedia peaked at 2 weeks following the third immunization and decreased postligation to near-baseline levels by week 33. Anti-B. fragilis antibody increased to peak levels by 6 weeks postligation, after which it decreased

IgA 1gM IgG P. gingivalis

1gM IgA P. intemredia





B. fragi/is

IMMUNIZATION GROUP (Isotpe) FIG. 2. Serum antibody responses in Nhps following active immunization. The bars denote the mean levels of antibody against the homologous antigen. All postimmunization levels were significantly elevated at least at P < 0.001 compared with baseline. Antibody levels in placebo-immunized Nhps (IFA only) and levels of all isotypes to heterologous antigens were always

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