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1988 2: 260ADRK. Okuda and I. Takazoe

The Role of Bacteroides Gingivalis in Periodontal Disease  

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THE ROLE OF BACTEROIDES GINGIVALISIN PERIODONTAL DISEASE

K. OKUDA AND I. TAKAZOE

Department of Microbiology, Tokyo Dental College, 2-2-2 Masago, Chiba City, Japan

Adv Dent Res 2(2):260-268, November, 1988

ABSTRACT

The microbial flora in adult advanced periodontitis lesions is comprised of Gram-negative rods, withBacteroides gingivalis as one of the major representatives. This review deals with biological properties

of surface antigens, hemagglutinin (attachment factor), and capsular structure of B. gingivalis. Sera containinghigh IgG antibody levels to B. gingivalis enhanced the complement-mediated bactericidal activity in vitro, al-though the susceptibility to complement-mediated lysis differed among B. gingivalis strains. The protectiveeffect of immunization against B. gingivalis infection was examined in hamsters in which cotton threads hadbeen tied to the gingival margins of the mandibular first molar. Repeated oral topical application of hyper-immune sera against B. gingivalis resulted in effective elimination of the organisms from the periodontal lesionsin the experimental animals.

INTRODUCTION TAXONOMY AND SEROLOGY

In 1921, Oliver and Wherry named strains of obli-gately anaerobic, non-motile, gram-negative, sac-charolytic rods that produced black colonies on bloodagar as Bacterium melaninogenicum. Burdon (1928) re-ported that black-pigmented Bacteroides were presentin the oral cavity of man and that their number in-creases markedly in purulent periodontitis lesions.Recent studies have shown that the proportion ofGram-negative anaerobic organisms increases mark-edly in the subgingival microflora with increasing se-verity of periodontal disease (Slots, 1976,1979; Tanneret aL, 1979). It has also been shown that IgG antibodylevels in sera obtained from patients with adult peri-odontitis against B. gingivalis are significantly higherthan those in controls (Mouton et al, 1981; Ebersoleet aL, 1982, 1985; Naito et al, 1984, 1985a). Black-pigmented Bacteroides strains were found to be essen-tial in inducing transmissible infections by humangingival crevice organisms in animal models (Mac-Donald et al.f 1963; Socransky and Gibbons, 1965;Takazoe and Nakamura, 1971; Sundqvist et al., 1979).These findings indicated that black-pigmented Bac-teroides was associated with adult periodontitis.

The purpose of this paper is to review the role ofB. gingivalis in human periodontal disease.

Presented at the Sunstar Portside Symposium, November 14-15,1986, Kobe, Japan

260

Sawyer et al (1962) and Courant and Gibbons (1967)reported that the black-pigmented Bacteroides couldbe divided into non-fermenters, weak fermenters, andstrong fermenters. On this basis, Finegold and Barnes(1977) categorized three subspecies, B. melaninogeni-cus subspecies asaccharolyticus, B. melaninogenicus sub-species intermedius, and B. melaninogenicus subspeciesmelaninogenicus. The further classification of black-pigmented Bacteroides has been made mainly on thebasis of deoxyribonucleic acid (DNA) homology stud-ies (Coykendall et al, 1980; Kaczmarek and Coyken-dall, 1980; Holdeman and Johnson, 1982; Johnson andHoldeman, 1983; Van Steenbergen et al, 1984). Ninespecies are currently listed in Bergey's Manual ofSystematic Microbiology (Vol. 1) (Holdeman et al,1984).

Studies have shown that some black-pigmentedBacteroides species strains are serologically distinct(Reed et al, 1980) and can be identified by fluores-cent-antibody staining (Lambe, 1974; Lambe and Jer-ris, 1976; Mouton et al, 1980) or ELISA methods(Ebersole et al, 1984). In our studies, a total of 63black- or brown-pigmented Bacteroides strains was se-rologically characterized by adsorbed and unad-sorbed rabbit antisera (Okuda et al, 1986b).Immunodiffusion tests using sonicated antigen andan indirect immunofluorescent antibody methoddemonstrated that B. gingivalis, B. asaccharolyticus, B.macacae, and B. levii are antigenically distinct. All B.

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Vol. 2 No. 2 ROLE OF B. gingivalis IN PER10D0NTITIS 261

gingivalis strains formed precipitin lines, suggestingthat the B. gingivalis strains possessed an antigencommon in the species. However, the pattern of reac-tivity differed among the strains of B. gingivalis; eithersome antigenic differences exist within the species orcertain antigens were not fully expressed in sometested strains. Monoclonal antibodies against B. gin-givalis can also be utilized for rapid detection or iden-tification of B. gingivalis by immunological methods(Hanazawa et al., 1985b; Naito et al, 1985b; Simonsonet al, 1986; Chen et al., 1986). Further work is neededto characterize the specific antigens relevant to thepathogenesis of B. gingivalis.

PATHOGENIC FACTORS

Presumptive pathogenic factors of black-pig-mented Bacteroides relevant to periodontal disease havebeen reviewed by Slots (1982) and Slots and Genco(1984). This paper deals with the biological propertiesof surface antigens, including attachment factors andenzymatic activities of B. gingivalis.

ATTACHMENT FACTOR

The abilities of periodontopathic bacteria to attachto surfaces in the oral cavity are prerequisites for pro-duction of disease (Gibbons and Van Houte, 1975;Van Houte, 1982). Surface structures, including fim-briae of B. gingivalis, may play a significant role in B.gingivalis colonization in the subgingival area (Okudaand Takazoe, 1974; Slots and Gibbons, 1979; Okudaet al, 1981). Peros et al. (1985) have suggested thatthe hydrophobicity of B. gingivalis cells may be cor-related with the presence of fimbriae. Yoshimura etal. (1984b, 1986) purified B. gingivalis fimbriae anddemonstrated that they exhibited no hemagglutina-ting activity. Furthermore, Boyd and McBride (1984)demonstrated that removing fimbriae from B. gingi-valis cells had no effect on the hemagglutinating ac-tivity of whole cells. McKee et al. (1986) found thatthe susceptibility of mice to infection with a patho-genic strain of B. gingivalis W50 depended on cultureconditions. Cell grown under hemin excess condi-tions were heavily fimbriated and caused high mor-tality. In contrast, cells grown under hemin limitationpossessed few fimbriae and showed low mortality.To establish the true function of B. gingivalis fimbriae,one must develop a fimbriae-negative mutant strainor produce fimbriae manufactured by recombinantDNA techniques.

Boyd and McBride (1984) suggested that the outermembrane of B. gingivalis, which possesses bacterialaggregating activity, consists mainly of protein andcarbohydrate. The part which causes hemagglutina-tion, on the other hand, contains low-molecular-weightlipopolysaccharide (LPS), protein, and loosely boundlipid, but is not part of the pili. Inoshita et al. (1986)

extracted the exohemagglutinin of B. gingivalis anddemonstrated that its activity was inhibited by L-ar-ginine. Okuda et al. (1986c) characterized the hem-agglutinating factor from B. gingivalis strain 381. Thishemagglutinin was prepared from culture superna-tant by ammonium sulfate precipitation, hydropho-bic column chromatography on Phenyl-Sepharose CL-4B, ion-exchange chromatography on DE-SephadexA-50, and Sephadex G-100 gel filtration. The resultinghemagglutinating activity was 53.3 times higher thanthat of the initial ammonium sulfate precipitate. Thehemagglutinating activity of intact cells and crudehemagglutinin were completely destroyed by beingheated at 100°C for 10 min, but the activity of thepurified hemagglutinin was heat-stable. The activitywas inhibited by L-arginine and L-lysine, but was notaffected by proteolytic enzymes, neuraminidase, hy-aluronidase, lipase, or sugars. The purified hemag-glutinin appeared to be composed mainly of a 40-kDamaterial, as determined by SDS-PAGE.

Recently, Sako et al. (1988) examined the plasmidcontent in 134 strains of Bacteroides isolated from thehuman oral cavity. Although plasmids were isolatedfrom seven strains of asaccharolytic Bacteroides, no B.gingivalis strain contained plasmids. These findingsreveal that the strong hemagglutinating activity of B.gingivalis strain is encoded by chromosomal DNA.

It has been shown that lipopolysaccharides (LPS)possess the ability to adhere to erythrocytes (Stevenset al, 1980; Kirikae et al, 1986). Slots and^Genco (1984)also suggested that vesicles and LPS of B. gingivalisand Bacteroides intermedius may be among their at-tachment factors. LPS preparations from B. interme-dius ATCC strains directly agglutinated erythrocytes.However, the LPS extracted from B. gingivalis strain381 by the hot phenol-water method did not agglu-tinate any erythrocytes tested. The hemagglutinatingactivity of B. intermedius strains was completely elim-inated by treatment with lipase and phospholipase-C, suggesting that a lipid constituent is critical forhemagglutinating activity. However, phospholipidsfrom B. gingivalis and B. intermedius which were ex-tracted by chloroform-methanol did not agglutinatesheep erythrocytes (Okuda and Kato, 1987). It maybe that this method removes some other active com-ponent loosely bound to the lipid component. Therole of the lipid components of B. gingivalis in attach-ment is still debatable.

ANTIPHAGOCYTIC FACTOR

Sundqvist et al. (1979) found that black-pigmentedBacteroides species can produce transmissible soft tis-sue infections despite massive immigration of poly-morphonuclear leukocytes (PMNs) into the infectionsite. This suggests that bacterial factors protect theBacteroides from phagocytosis and intracellular killingby these PMNs. Encapsulated strains of black-pig-mented Bacteroides have produced experimental ab-

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262 OKUDA & TAKAZOE Adv Dent Res November 1988

scesses and remained viable during abscess formation(Takazoe et al., 1971). An encapsulated strain has alsobeen shown to resist phagocytosis by PMNs in vitro(Okuda and Takazoe, 1973). The capsular antigen ofblack-pigmented Baderoides is capable of masking LPSand preventing it from activating the complementsystem (Okuda et al, 1978). Furthermore, Ingham etal. (1977) and Codd and Selkon (1981) found that non-oral black-pigmented Baderoides significantly reducedphagocytosis and killing of intestinal facultative bac-teria. These authors, however, did not determinewhether a capsular structure was present on the strainsexamined. Okuda et al. (1987) found that strains ofblack-pigmented Baderoides species examined by In-dia-ink-staining or electron microscopy exhibited acapsular structure. Electron micrographs showed thatthe capsular structure of B. gingivalis strains was closelyassociated with the outer surface membrane. No sig-nificant differences in thickness of the B. gingivaliscapsule were found between strains sensitive and re-sistant to complement-mediated bactericidal activity.

CDCO

.C:

O106-]

E

oioH

XI(0

D 60 120 180m inIncubation time

-»:ST(11.7) •—•o:ABST o—

• -«:MI(<5)

o:ABPA•—-o:YN(<5)

Fig. —Complement-mediated bactericidal test of Baderoides gingi-valis 381 with various human sera diluted 1:5. Each point rep-resents the mean number ± standard deviation obtained fromduplicate plates. The antibody titer (log2) of the serum to ultrason-icated soluble antigens of B. gingivalis 381 strain is given in paren-theses. Sera labeled ST and PA possess high IgG levels to B. gingivalis381 from adults with periodontitis; sera ABST and ABPA are ad-sorbed with intact cells of B. gingivalis 381; sera MI and YN possessno reactive antibody to B. gingivalis 381.

Chen et al. (1987) showed the effect of immunizationon experimental B. gingivalis infection in a murinemodel using invasive (A7A1-28) and non-invasive (381)B. gingivalis strains. Mice immunized with the inva-sive B. gingivalis strain localized the infection to thechallenge site. On the other hand, mice immunizedwith the non-invasive strain developed spreading in-fections. They suggested that the protective anti-gen(s) of the invasive strain are heat-stable, and thatthe differences are due to the degree of encapsulationof the two B. gingivalis strains. The pathogenicity ofB. gingivalis has been attributed to capsule formationin vivo, although other factors may also have an in-fluence. It is likely that the availability of a highlypurified capsular antigen will facilitate studies of itsprecise role in pathogenesis.

LlPOPOLYSACCHARIDE (LPS)OF B. GINGIVALIS

It has been repeatedly demonstrated that LPS fromblack-pigmented Baderoides strongly stimulate boneresorption (Hausmann et al., 1970, 1972; Nair et al.,1983; Millar et al., 1986). Koga et al (1984) and Han-azawa et al. (1985a) demonstrated that the inductionof a number of biological activities — such as mitogen-icity, interleukin production, and activation of mac-rophages by LPS from B. gingivalis strain 381 — wascomparable with those provoked by LPS from £s-cherichia coli. More details of the relationship betweenextraction procedures, chemical structures, and thesebiological activities of subgingival Gram-negativebacteria (including B. gingivalis LPS) are discussed byHamada et al. (1988).

ENZYMATIC ACTIVITIES OF B. GINGIVALIS

It has been found that strains of black-pigmentedBaderoides are capable of producing many enzymaticactivities, including collagenase (Gibbons andMacDonald, 1961; Hausmann et al, 1967; Hausmannand Kaufman, 1969; Yamamoto and Takazoe, 1976;Toda et al., 1984) and other proteases (Fujimura andNakamura, 1981; Laughon et al., 1982; Slots, 1981;Kato et al., 1984; Suido et al., 1986). Strains of B. gin-givalis possess more proteolytic enzymatic activity(Slots, 1981), fibrinolytic activity (Wikstrom et al., 1983;Lantz et al., 1986), collagenase (Toda et al., 1984), ar-ylaminopeptidase activity (Suido et al, 1986), andtrypsin-like protease, which hydrolyzes Na-benzoyl-DL-arginine-p-nitroanilide (Yoshimura et al., 1984a;Ono et al, 1987; Fujimura and Nakamura, 1987) thando other black-pigmented Baderoides species. Kilian(1981), Carlsson et al (1984), and Nilsson et al (1985)demonstrated that B. gingivalis strains possessed en-zymes directed against serum factors such as im-munoglobulins. A pathogenic strain of B. gingivalis,W83, was resistant to phagocytic killing by leukocytes

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Vol. 2 No. 2 ROLE OF B. gingivalis IN PER10D0NTIT1S 263

TABLE 1OCCURRENCE OF B. gingivalis AND B. intcrmedius IN SAMPLES FROM HUMAN PERIODONTAL AREA

Group (number of patients)

Periodontally healthy (5)

Gingivitis (5)

Adult periodontitis (15)

-'Positive number in samples.

0/5a

1/5

11/15

B. gingivalis

0.61(0 -12.38(0 -

Mean

0

± 1.423.08)± 19.15

54.3)

percent ±(Range)

S.D.

5/5

5/5

15/15

B. intermedius

3.01 ± 2.36(0.62 - 6.32)8.56 ± 9.53(0.25 - 23.43)4.41 ± 4.42(0.15 - 17.18)

and possessed strong enzymatic activity able to de-grade immunoglobulins and complement compo-nents (Sundqvist and Johansson, 1982; Sundqvist etal, 1982, 1984). Although direct proof is still lacking,these enzymes may play a role in periodontal tissuedegradation and in evading host defense mecha-nisms in vivo.

EFFECTS OF B. GINGIVALIS ELIMINATION

Our studies (Okuda et al, 1988) on cultivable enu-meration of B. gingivalis and B. intermedius in plaquesamples from adult periodontitis, gingivitis, and peri-odontally healthy sites from each group are sum-marized in Table 1. The prevalence of B. gingivalis insubgingival dental plaque from periodontitis lesionswas significantly higher than in those from the peri-odontally healthy sites of another group (Mann-Whitney test, p < 0.01). We noted that the proportionof black-pigmented Bacteroides was significantly higherin samples obtained with paper points than in thoseobtained with a sealer (unpublished data). We em-phasize that comparisons between sampling methodsare needed, as suggested by Tanner and Goodson(1986). B. gingivalis has been shown to be a causativeagent of periodontitis in experimental animals (Heijlet al, 1980; Slots and Hausmann, 1979; Nagahata etal, 1982; Katahira et al, 1985); this depends upon itsability to colonize the periodontal region.

Many research groups (Osterberg et al, 1979;Loesche et al, 1981,1984; Slots et al, 1979) have shownthe effectiveness of antimicrobial agents in elimina-ting periodontopathogens such as B. gingivalis. Clin-damycin was administered to periodontal patientssystemically for five days at a total of 450 mg per day(Ohta et al, 1986). No B. gingivalis was detected inthe lesions, even three months after the treatment.There was a reduction in periodontal inflammationboth three and six months after the clindamycin treat-ment. These studies indicate that elimination of B.gingivalis is a rational treatment for such patients. Re-colonization of saccharolytic black-pigmented Bacter-

oides was found in periodontal pockets at three monthsafter clindamycin treatment, but no B. gingivalis wasdetected. These findings suggested the possibility ofbacterial interactions among black-pigmented Bacter-oides (Nakamura et al, 1981; Takazoe et al, 1984; Okudaet al, 1984). Yamada et al (1987) demonstrated thatBacteroides oris possessed strong bacteriocinogenic ac-tivity against B. gingivalis. We are planning a studyin vivo in order to clarify whether or not this kind ofbacteriocin would be useful in eliminating B. gingi-valis from peridontal lesions.

IGG RESPONSES TO B. GINGIVALIS

The serum IgG titers to bacterial sonicates deter-mined by micro-ELISA (Naito et al, 1984, 1985a) areshown in Table 2. The antibody levels to B. gingivalisin serum were significantly higher in samples frompatients with adult periodontitis than in sera fromhealthy individuals. Although there were individualdifferences within patient groups, positive correla-tions were found between serum IgG levels to B. gin-givalis and pocket depth (Mann-Whitney, p<0.01) andalveolar bone loss (Mann-Whitney, p<0.01). Theseresults agreed with findings from several other re-search groups (Mouton et al, 1981; Mansheim et al,1980; Taubman et al, 1982; Ebersole et al, 1982,1985).In contrast, Doty et al (1982) have reported decreasedlevels of serum immunoglobulins to B. gingivalis inperiodontitis patients. Martin et al (1986) demon-strated that gingival tissue supernatant fluid from adultperiodontitis patients exhibited increased antibodyreactivity to B. gingivalis. When autologous sera fromthe patients were assayed, no significant differencesin immunoglobulin levels to B. gingivalis were ob-served. These data indicate that differences exist inlocalized immune responses to B. gingivalis which arenot necessarily reflected in the systemic serum anti-body responses of these patients. Along this line, Ka-gan (1980) has shown that plasma cells in periodontallesions contain specific immunoglobulins to B. gin-givalis. Naito et al (1984) found that gingival crevi-

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264 OKUDA & TAKAZOE Adv Dent Res November 1988

TABLE 2IgG ANTIBODY LEVELS AGAINST ULTRASONICATED

SOLUBLE ANTIGENS OF Bacteroides gingivalis

Plaque Index0 ^ p l I < 0 . 50.5 ^ pi I < 1.51.5 £ pi I

Gingival IndexO ^ G I < 0.50.5 ^ G I < 1.51.5 ^ G I

Pocket Depth0 ^ PD < 22 ^ PD < 44 ^ P D

Alveolar Bone Loss0 ^ BL < 1515 ^ BL < 3030 ^BL

( n = 9)(n = 17)(n = 12)

(n = 11)(n = 15)(n = 12)

(n= 9)(n = 17)(n = 12)

(n = 14)(n= 8)(n = 16)

Mean Titers and Standard Error(Expressed as Log2)

5.53 ± 0.499.22 ± 0.479.58 ± 0.56

6.26 ± 0.698.90 ± 0.59

10.07 ± 0.29

5.53 ± 0.498.89 ± 0.40

10.20 ± 0.67

6.21 ± 0.549.16 ± 0.35

10.19 ± 0.48

cular fluid (GCF) from periodontitis patients had morespecific IgG to B. gingivalis than did samples fromhealthy individuals. B. gingivalis was not detected inhuman fecal samples from 20 adults, including 10periodontitis patients (Okuda et al., 1988). The find-ings may provide a basis for explaining that the an-tibody response to B. gingivalis is mainly induced byorganisms harbored in the periodontal pockets.

Recently, we found that most sera from patientswith adult periodontitis possessed high antibody lev-els to the hemagglutinin of B. gingivalis (Naito et al.,1987). Farida et al. (1986) also detected serum IgGantibodies to B. gingivalis LPS in various forms ofperiodontal disease in man. Further longitudinal de-terminations are required to clarify the role of theseantibodies to various surface antigens in the processof B. gingivalis infection.

ROLE OF SERUM IGG TO B. GINGIVALIS

Sundqvist and Johansson (1982) reported that thesusceptibility to complement-mediated lysis variedamong strains of black-pigmented Bacteroides species.They also found no correlation between the serumsensitivity of each strain and the specific antibodylevel in the serum. Okuda et al. (1986a) also investi-gated the relationship between human serum bacte-ricidal activity against B. gingivalis in human serumand IgG antibody levels to the organisms. The bac-tericidal activities of 20% human sera and comple-ment systems against B. gingivalis strain 381 are shownin the Fig. Serum containing high concentrations ofIgG antibody to B. gingivalis enhanced the bactericidalactivity. No enhancement was found in serum which

did not contain B. gingivalis-speciiic antibody or inserum adsorbed with intact B. gingivalis cells. Sensi-tivity to pooled human serum differed among B. gin-givalis strains. B. gingivalis activated the pooled humanserum complement through the classical pathway,indicating that the susceptibility of B. gingivalis to thebactericidal activity is classical-pathway-dependent.

EFFECTS OF VACCINATIONS

The effects of active and passive immunizationsagainst B. gingivalis infection were studied by use ofspecific-pathogen-free golden hamsters (Okuda et al.,1988). No black-pigmented Bacteroides were observedfrom swabbing of samples in the periodontal area ofthe experimental hamsters prior to the study. Blackcotton threads with a diameter of 0.2 mm were tiedcoronally to the gingival margins of the mandibularfirst molars in all hamsters by a method described byNagahata et al. (1982). The streptomycin-resistant B.gingivalis 381R' strain was inoculated into the oralcavity. The recovery of B. gingivalis 381R' was deter-mined by the number of cultivable cells on the liga-ture thread. The ligature thread from each hamsterwas removed and placed in a vial containing Tryp-ticase soy broth (BBL Microbiology Systems) with glassbeads, which was then sealed and vigorously shakenfor a minute. Then, 10-fold serial dilutions were made.A 100-mL aliquot of each dilution was placed on du-plicate blood plates, with or without 200 fxL/mLstreptomycin, which were cultured anaerobically for14 days. The proportion of B. gingivalis 381R' wasexpressed as the percentage of the total colony-form-ing units (CFU) on blood agar plates without strep-tomycin. Comparisons of independent recoveries wereanalyzed by non-parametric statistical analysis (Mann-Whitney test; Siegel, 1956).

SUBCUTANEOUS IMMUNIZATION

Five-week-old hamsters were divided into the fol-lowing groups: (1) 15 hamsters sham-immunized andinfected with B. gingivalis 381R'; (2) 15 hamsters im-munized with crude hemagglutinin and infected; and(3) 15 hamsters immunized with whole cells and in-fected. Grown cells of B. gingivalis strain 381 werewashed thoroughly with PBS and suspended at 20mg wet weight per mL. The extracted hemagglutininwas suspended in PBS at a concentration of 200 p,gdry weight per mL. The antigen suspensions wereemulsified in the same volume of incomplete Freund'sadjuvant (Difco Laboratories). Subcutaneous injec-tions of 100 jiL antigen were carried out at two-weekintervals into the inguinal region. Two weeks afterthe booster injection, the cotton threads were tied.

The total CFU from ligature threads ranged from1.2 x 108 to 1.3 x 1010, and the recovery number ofB. gingivalis 381R' was from 0 to 1.7 x 109. The mean

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Vol. 2 No. ROLE OF B. gingivalis IN PERIODONTITIS

recovery percentages of B. gingivalis 381R' in totalCFU at three weeks after infection are summarizedin Table 3. B. gingivalis 381R' was not recovered fromtwo samples out of 8 hamsters immunized with hem-agglutinin. Statistical analysis showed significantlylower recovery of B. gingivalis 381R' in the group im-munized with hemagglutinin and whole cells, ascompared with the sham-immunized group at threeweeks after infection. It has been reported that anti-bodies to pili (attachment factors) contribute to inhib-iting activity for a number of the bacterial colonizations(Nagy et al., 1978; Lee et al., 1983; Virji and Heckels,1985). In our study, complete colonization inhibitionwas not induced by the subcutaneous immuniza-tions. It is possible that the antibodies to hemagglu-tinin are not crucial, and that other antibodies toepitopes on other molecules present in the vaccineare active to a certain extent.

REPEATED PASSIVE LOCAL IMMUNIZATION

Forty hamsters were divided into four groups inthe experiment. All hamsters were ligated and in-fected with B. gingivalis 381R' for three days. MaleNew Zealand white rabbits, each weighing from 2.5to 3.1 kg, were immunized with either whole cells orextracted hemagglutinin. Grown cells of B. gingivalis381 were formalin-killed and washed with phos-phate-buffered saline (PBS). A 1.0-mL aliquot con-taining 20 mg wet weight cells per mL of PBS wasinjected intravenously on successive days. Sevenbooster immunizations with 2.0 mL of the immuno-

TABLE 3EFFECT OF SUBCUTANEOUS IMMUNIZATION WITHWHOLE CELLS OR EXTRACTED HEMAGGLUTININAGAINST B. gingivalis 381R' INFECTION IN LIGATED

HAMSTERS

Mean recovery % of B. gingivalis 381R'in total CFU

at 3 weeks after infection ± standardGroup deviation

Sham-immunized

Immunized withwhole cells

Immunized withhemagglutinin

l l / l l a

7.88 ± 1.5(2.6 - 19.85)b

7/71.80 ± 0.54

(0.26 - 4.88)6/8

1.14 ± 0.41c

(0 - 3.20)

TABLE 4EFFECT OF LOCAL PASSIVE IMMUNIZATION FOR

THREE WEEKS BY RABBIT ANTISERUM AGAINST B.gingivalis 381R' INFECTION IN LIGATED HAMSTERS

Mean recovery % of B. gingivalis 381R'Group in total CFU ± standard deviation

PBS 8/8a

Normal serum

Anti whole cellserum

Anti hemagglutininserum

7.17 ± 6.49(0.32 - 16.49)b

8/812.37 ± 17.20(0.58 - 48.46)

3/80.001 ± 0.001c-d

(0 - 0.0004)2/8

0.0004 ± 0.0009c-d

(0 - 0.0028)

-'Number of recoveries of B. gingivalis 381R' in hamsters.bRange of recovery percentage.^Significant difference from the sham-immunized group

(p < 0.01) by nonparametric statistical analysis (Mann-Whitney test).

c1Number of recoveries of B. gingivalis 381R' in hamsters.bRange of recovery percentage.^Significant difference from normal serum group (p <

0.001) by nonparametric statistical analysis (Mann-W7hitneytest).

dfrom the PBS group (p < 0.001).

gen were carried out at two- to four-day intervals.Two mL of the extracted hemagglutinin antigen (200fig dry weight per mL), emulsified with incompleteFreund's adjuvant (Difco), were injected into the rab-bits. Two booster immunizations of the antigens werecarried out. Blood samples were collected 10 daysafter the final immunization. The sera were inacti-vated at 56°C for 30 min. Forty-eight hamsters weredivided among four groups for this experiment. Afterbeing inoculated three times with B. gingivalis 381R',the ligated hamsters were orally inoculated with 50%rabbit serum sample. To avoid early dispersion of theserum from the periodontal regions, we added glyc-erol to each rabbit serum sample. A 100-fxL aliquotof each serum or PBS was inoculated into the oralcavity over three weeks. Immediately after the inoc-ulation, the ligated tooth regions of all hamsters werebrushed with a human inter-tooth brush for 30 sec toaid the serum in reaching the periodontal regions.

The repeated passive immunizations with rabbitantisera resulted in smaller numbers of B. gingivalis381R' colonization as compared with control groups(Table 4). Recoveries of B. gingivalis 381R' from ham-sters inoculated repeatedly with 50% antiserum againstwhole cells or hemagglutinin were significantly lowerthan those in control groups. No B. gingivalis 381R'cells were detected on 11 out of 16 ligature threadsfrom hamsters which were immunized with anti-serum for three weeks. The numbers of B. gingivalis381R' in samples immunized with antisera were neg-ligible as compared with numbers from hamsters

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266 OKUDA & TAKAZOE Adv Dent Res November 1988

treated with PBS or normal serum. Local passive im-munization with rabbit antiserum in the periodontalarea resulted in reduction of B. gingivalis 381R'. TheB. gingivalis colonization mechanisms may involve twosteps: attachment to the mucosal surface of plaquebacteria and then proliferation throughout the peri-odontal area. In this experiment, repeated inocula-tions with rabbit antisera might have played a role inthe second step. We are planning to examine whetherlocal passive immunization with IgG from rabbit anti-sera in the human periodontal region may help toeliminate B. gingivalis.

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