Bacteriology of Experimental Gingivitis in Children

Transcription

1 INFECTION AND IMMUNITY, Oct. 1984, p /84/ $02.00/0 Copyright 1984, Americn Society for Microbiology Vol. 46, No. 1 Bcteriology of Experimentl Gingivitis in Children W. E. C. MOORE,'* L. V. HOLDEMAN,' R. M. SMIBERT,' E. P. CATO,1 J. A. B3URMEISTER,2 K. G. PALCANIS,2 AND R. R. RANNEY2 Deprtment of Anerobic Microbiology, Virgini Polytechnic Institute nd Stte University, Blcksburg, Virgini 24061, nd Deprtment of Periodontics, School of Dentistry, Virgini Commonwelth University, Richmond, Virgini Received 16 April 1984/Accepted 6 July 1984 Children re more resistnt to gingivitis thn re dults. To determine possible differences in their periodontl flors, n experimentl gingivitis study, identicl in design to one reported erlier with young dults, ws conducted with four 4- to 6-yer-old children. The incidence of sites tht developed gingivl index scores of 2 in children ws less thn one-third of the incidence observed in dults. The composition of the flor of ech child ws sttisticlly significntly different from tht of ny other child or dult. The flors of the children s group were sttisticlly significntly different from those of the dults. Children hd 3-fold greter proportions of Leptotrichi species, 2.5-fold greter proportions of Cpnocytophg species, 2.3-fold greter proportions of Selenomons species, 2-fold greter proportions of bcteril species tht require formte nd fumrte, nd 1.5-fold greter proportions of Bcteroides species. Adults hd greter proportions of Fusobcterium, Eubcterium, nd Lctobcillus species. Fusobcterium nucletum, Actinomyces WV 963, Selenomons D04, nd Treponem socrnskii were predominnt species tht correlted with incresing gingivl index scores in both children nd dults. Experimentl gingivitis in humns (4) provides model for ssessing vribles tht my be relted to the development of gingivitis. The bcteriology of this model with young dult subjects, including the identifiction of ll rndomly selected isoltes nd the most likely custive bcteril species, ws previously described (12). Others (7, 9) hve shown tht prepubertl children do not develop gingivitis in this model to the sme extent or severity s do dults. Together with cross-sectionl dt indicting lesser severity of nturlly occurring gingivitis in children thn in dults (18), this indictes greter resistnce of children to the development of gingivitis. There re suggestions in the literture tht the host responses expressed in the gingiv of children my differ from those of dults. Histologiclly, gingivitis in children is dominted by lymphocytes nd includes fewer plsm cells thn does gingivitis in dults (5, 6). The child gingivitis lesion hs been chrcterized s T-cell dominted (15), wheres the predominnt cells in the infiltrte of dults re of the B-cell series (14). If the flors of developing gingivitis in children re identicl to those in dults, the greter resistnce of children my be ttributble to host response differences, quite possibly the immunologicl response differences suggested by the distinct chrcteristics of the inflmmtory infiltrtes. To determine whether the flors re the sme, the present study duplicted in children the previous design nd procedures in dults (12). MATERIALS AND METHODS Subjects. Four Cucsin mles, ges 4 to 6 yers nd hving primry dentition, prticipted s subjects. They were mediclly helthy by history nd received no medictions during the course of the study nd no ntibiotics for 2 months preceding the study. There were no instnces of probeble gingivl sulcus depth greter thn 3 mm nd no cliniclly detectble crious lesions of the teeth. Experimentl gingivitis. The experimentl gingivitis model s developed by Loe et l. (4), nd s utilized in the previous study of young dults (12), ws followed. This included the clening of ll teeth nd estblishing bse-line helth, which * Corresponding uthor. 1 ws verified by exmintion of the preselected sites on dy 0 of the experimentl phse (no brushing or other tooth clening). The experimentl phse continued for 25 dys. A second, identicl experiment ws conducted with ech child fter reestblishing gingivl helth nd llowing minimum of 3 months between the two trils. The gingivl index (GI) (3) nd plque index (16) scores were determined for four surfces of ll teeth present on ech smple dy. All exmintions were performed by the sme exminer, who lso hd done ll clinicl scoring in the previous experiment with dults. Smple sites. As in the dult experiment, smples were tken from two sites (one fcil nd one proximl) on ech of two teeth (one mndibulr nd one mxillry) on ech of dys 4, 11, nd 25 of the experimentl phse (Tble 1). In generl, the smple sites occupied the sme reltive loction in the dentl rches s the sites smpled previously in dults (12). However, smples were tken only from primry teeth. Additionlly, sites were chosen to be nondjcent to ny sites of lost primry incisors. As in dults, different surfce ws smpled ech time within ech experimentl replicte tril, nd no proximl smple site ws immeditely djcent to ny other smple site. Left cnines were used to void disturbing mesil smple sites on right primry molrs. Thus, ech smple cme f'rom site in which the plque ws completely undisturbed for the designted time. There were 24 fcil nd 24 proximl (mesil or distl) sites smpled in ech tril. Smpling. The teeth were isolted with cotton rolls, nd the smple sites were gently dried with sterile cotton swbs. The ccumulted suprgingivl plque t the sites to be smpled ws removed s completely s possible with sterile toothpicks. The smples were then tken with sterile nickelplted Morse 00 replceble scler tips by mking single pss from the depth of the sulcus to just coronl to the gingivl mrgin s ws done previously for the dult experiment. Microbiology. All smple dispersion, dilution, medi, cultures on pltes nd roll tubes, spirochete, nd other bcteril identifiction were s described previously for the dult experiment (12). In brief, rndomly selected isoltes from D-

2 2 MOORE ET AL. Dy TABLE 1. Smple site schedule for both replictes of experimentl gingivitis in children Smple site schedule for child: H (F,D) A (F,M) H (F,D) B (F,M) M (F,D) T (F,M) S (F,M) M (F,D) 11 A (F,M) H (F,D) B (F,M) A (F,M) T (F,M) S (F,M) T (F,M) T (F,M) 25 B (F,M) B (F,M) A (F,M,Lb) H (F,D) S (F,M) M (F,D) M (F,D) S (F,M) J (F bmb) T (Lb) L (F bdb) "Universl identifiction system for deciduous teeth (A to T), followed by smpled surfces within prentheses: F, fcil; D, distl; M, mesil; L, lingul. bextr sites smpled in tril I in n ttempt to increse the number of sites with GI scores of 2 for sttisticl nlyses. 4 medium (brin hert infusion bse) were subcultured in D-5 broth nd identified by comprison with type nd reference strins fter chrcteriztion by electrophoresis of soluble cellulr proteins (10), Grm stin, chromtogrphic nlysis of fermenttion cids nd hydrogen, growth nd ph in 30 medi, nd tests for indole, esculin hydrolysis, ctlse, protein digestion, gs production, motility, erotolernce, mmoni production, nd nitrte reduction (2), supplemented by other tests s required for differentition of species in severl gener. Strins of the genus Actinomyces were serotyped with monovlent fluorescent ntiser. Spirochetes were isolted from OTI medium or RIP broth on peptonefresh yest extrct-serum gr nd identified by morphology, serology, fermenttion ptterns, nd biochemicl rections (12). RESULTS AND DISCUSSION Of the 96 predesignted smple sites in both trils, 46 were found to hve GI scores of 0, 39 hd GI scores of 1, nd 11 hd GI scores of 2 (Tble 2); this compres with GI scores of 18, 42, nd 36, respectively, in dults (12) under the sme experimentl conditions (Tble 3). The men GI scores of smple sites were significntly lower for the children thn for the previously studied dults on corresponding smple dys (P < for dys 4 nd 11 nd P < for dy 25 by t tests). At the sme time the plque index scores for smple sites in children were higher thn for smple sites in dults t dy 4 (1.75 ± 0.08 versus 1.28 ± 0.08) nd essentilly the TABLE 3. INFECT. IMMUN. Child versus dult experimentl gingivitis Subjects (yr) GI No. of No. of No. of % score smples isoltes tx Coverge Children (4-6) , , Adults (22-31)b , , Includes four extr sites with GI scores of 1 nd two extr sites with GI scores of 2. b Dt re from reference 12. sme t dys 11 nd 25 (1.56 ± 0.09 versus 1.56 ± 0.12 nd 1.94 ± 0.04 versus 2.09 ± 0.07, respectively). These findings re consistent with previous reports tht children do not develop gingivitis to the sme extent or severity s do dults in the experimentl gingivitis model, even though estimtes of plque mss re comprble in the two popultions (7, 9). Trils. Neither the GI scores (P > 0.3) nor the plque index scores (P > 0.6) t smple sites differed significntly between trils in the children (two-wy nlysis of vrince). However, L nlysis (12) showed tht the composition of ll smples in tril I ws different from tht of ll smples in tril II (P = 0.01). In the experiment with dults, there lso ws sttisticlly significnt difference between trils I nd II (P = 0.004). In the dults it ppered tht there my hve been some crry-over between trils (12), but there ws no evidence for tht in the present experiment. In the children, the flors of dy 4 in trils I nd II were not significntly different (P = 0.29), nor were they significntly different on dy 11 (P = 0.44). However, the composition of the flors ws significntly different on dy 26 (P = 0.002). There were six predesignted sites with GI scores of 2 on dy 26 of the first tril nd only two such sites on dy 26 of the second tril. The proportions of most bcteril species in the flors were resonbly similr in both trils on dy 26. However, Actinomyces isrelii II, Actinomyces meyeri (-) (phenotypiclly like A. meyeri but not rective with monovlent A. meyeri ntiserum), Fusobcterium nucletum, Streptococcus Dll, Streptococcus mitis I, Streptococcus snguis I, Selenomons D04, nd Wolinell curv (19) were ech three times or more s numerous in tril I s in tril II on dy 26. Fctors tht my hve cused the different responses in the two trils re not obvious. Fcil sites versus proximl sites. The flors of fcil sites were not significntly different from those of proximl (distl or mesil) sites in children (P = 0.19); neither were GI scores TABLE 2. Distribution of GI scores mong sites' smpled in children Distribution of GI scores in: Tril Dy Child 1 site: Child 2 site: Child 3 site: Child 4 site: Men GI score ± SE I ± II ±0.11 The criteri of Loe nd Silness (3) were used to determine GI scores; essentilly, 0 is helthy, 1 is mild inflmmtion, nd 2 is moderte inflmmtion nd bleeding when gently probed. b Sites smpled re given in Tble 1 nd in the text. Sites 1 nd 2 were fcil nd proximl, respectively, of the first tooth listed; sites 3 nd 4 were fcil nd proximl, respectively, of the second tooth listed.

3 VOL. 46, 1984 TABLE 4. Percent similrity of flors of people' (720+ isoltes ech) % Similrity of flors of people: Person Cl C2 C3 C4 Al A2 A3 A4 Cl C C C Al A A A4 100 C, Child; A, dult. significntly different ( for fcil nd 0.58 ± 0.10 for proximl smple sites). These findings re in contrst to those in dults, in which both the flor composition (P = 0.004) nd GI scores differed between fcil nd proximl sites (12). These differences between children nd dults my relte to differences in tooth shpe or proximl tooth contct res which could produce different locl environment, flor, nd consequent pttern of gingivitis development. The flors of fcil sites in children were significntly different from those of fcil sites in dults (P < 0.001), nd the flors of proximl sites lso differed between children nd dults (P < 0.001). Comprisons of child proximl to dult fcil sites, nd child fcil to dult proximl sites, indicted eqully lrge differences (P < 0.001). Thus the differences in flor compositions pprently reflect difference in the totl flor of children versus tht of dults. Flor composition. There were 160 tx mong 3,217 isoltes from ll 102 smples from the children. The flors of the children were slightly more complex thn those of the dults. For exmple, in children 122 tx mong 1,363 isoltes from sites with GI scores of 1 ccounted for 97% of the cultivble flors, wheres in dults only 96 tx mong 1,332 isoltes ccounted for 98% of the flors, s estimted by formul Hg for coverge of Good (1) (Tble 3). The flors of children nd dults were compred by the L nlysis of Good (12). To compre the flors of children s group with the flors of dults, ll 24 or more smples from ech person were merged into single smple of c. 720 isoltes to represent the flor of tht person. L nlysis demonstrted tht the four children nd the four dults formed the two most distinct groups possible with probbility of (Tble 4). Becuse L nlysis is Monte Crlo test, is the minimum theoreticl probbility tht cn be TABLE 5. Men % similrity of 24 (30-colony) smples within nd between people Men % similrity of flors of people Person Cl C2 C3 C4 Al A2 A3 A4 Cl b C C C Al A A A4 20 C, Child; A, dult. b By L nlysis, P between Cl nd C4 = 0.003, nd P between ll others < EXPERIMENTAL GINGIVITIS IN CHILDREN 3 ttined with two groups of only four items. (Rndom ssignment of smples will produce the identicl two groups of four with 2.8% frequency.) The men similrity of the flors of different children ws 60%, of different dults ws 54%, nd of children compred with dults ws 51%. The compositions of ll 24 to 28 smples from ech child or dult were significntly different from those of the smples from ech other child or dult when one pir of people t time ws tested (Tble 5). In both children nd dults the men similrity of 24 to 28 smples ws 26%. The verge similrity of smples ws 22% mong different children nd 20% mong different dults. The verge similrity of smples from children s compred with smples from dults ws 20%. Differences in composition of the child nd dult flors re shown in Tbles 6 nd 7. These tbles list ll tx tht comprised t lest 0.5% of the flors of either dults or children. The children, who hd greter resistnce to gingivitis, hd 2.5-fold higher proportions of ll Cpnocytophg species combined, nd 2.2-fold higher proportions of ll nerobic vibrios (species of the combined gener Cmpylobcter, Selenomons, nd Wolinell). These dt suggest tht this flor composition is norml for children nd tht findings of significnt numbers of Cpnocytophg species in young persons, s reported recently in juvenile dibetics (8), should be compred with findings in helthy control subjects of the sme ge to ssess etiologicl significnce. Reltionship of flors to GI scores. The composition of the flors of sites with GI scores of 0 ws significntly different from those with GI scores of 1 (P < 0.001) or GI scores of 2 (P = 0.002), but the flors of sites with GI scores of 1 were not sttisticlly significntly different from those of sites with GI scores of 2 (P = 0.11). Flor compositions of sites with GI scores of 0 in children were significntly different from those in dults (P < 0.001), s were sites with GI scores of 1 (P < 0.001) nd sites with GI scores of 2 (P = 0.012). The lesser degree of significnce between child nd dult sites with GI scores of 2 my reflect the fct tht there were only 13 sites with GI scores of 2 in children nd 36 sites with GI scores of 2 in dults, wheres for GI scores of 0 there were 46 in children nd 18 in dults, nd for GI scores of 1 there were 43 in children nd 42 in dults. Bcteril species ssocited with gingivitis. The flors of dults, who were more susceptible to gingivitis, contined greter numbers of predominting grm-positive species (Tble 7). The notble exception is F. nucletum, which is grm negtive. Among the predominnt species in dult experimentl gingivitis, only five increse further in sulci of sites ffected with moderte or severe periodontitis (Tble 8). Of these five species, both A. isrelii I nd Propionibcterium cnes re most frequent in the gingivl crevices of people with helthy gingiv, wheres the incidence of Eubcterium timidum, F. nucletum nd Peptostreptococcus micros suggests tht they might mke the gretest contributions to the incresed sensitivity of dults to experimentl gingivitis nd to periodontitis. Mycoplsm. The incidence of mycoplsm mong smples from different popultions is shown in Tble 9. Their distribution indictes tht they do not contribute to gingivitis; rther, it ppers tht they occur s result of gingivitis nd might be importnt in periodontitis tissue destruction. Treponemes. Treponem socrnskii ws frequent in the gingivl crevice of people with helthy gingiv nd in children nd dults with experimentl gingivitis. In both children nd dults its frequency correlted with incresing GI

4 4 MOORE ET AL. INFECT. IMMUN. TABLE 6. Incidence of predominnt species more common in children, s percent of totl flor nd s percent of (30-isolte) smples tht re positive % of flor % of smples Species Child AdUltb Child/dult Child Adultb Child/dult Grm positive Actinomyces WV Actinomyces isrelii (-) Actinomyces isrelii II Actinomyces neslundii I Fc. Pos. Coccus D2SC Streptococcus nginosus Streptococcus D Streptococcus D Grm negtive Bcteroides D1C Bcteroides grcilis Bcteroides intermedius Cpnocytophg gingivlis Cpnocytophg ochrce Cpnocytophg sputigen Selenomons sputigen Selenomons DOI Selenomons D Selenomons D Veillonell dispr Wolinell curv Wolinell rect Children: 3,217 isoltes from 102 smples. b Adults: 3,035 isoltes from 96 smples (12). c Unnmed species re designted by letters nd numbers. TABLE 7. Incidence of predominnt species more common in dults, s percent of totl flor nd s percent of (30-isolte) smples tht re positive % of flor % of smples Species Child Adultb Child/dult Child Adultb Child/dult Grm positive Actinomyces isrelii I Actinomyces neslundii (-) Actinomyces neslundii III Actinomyces NVC Actinomyces odontolyticus (-) Actinomyces odontolyticus I Actinomyces viscosus II Coccus D30d Coccus SM Eubcterium sburreum Eubcterium timidum Lctobcillus D Propionibcterium cnes Peptostreptococcus micros Streptococcus D Streptococcus mitis Streptococcus SA Streptococcus snguis I Streptococcus snguis II Streptococcus SM Grm negtive Bcteroides oris Fusobcterium nucletum Veillonell prvul Children: 3,217 isoltes from 102 smples. b Adults: 3,035 isoltes from 96 smples (12). c Serologiclly relted to both A. neslundii nd A. viscosus II. d Unnmed species re designted by letters nd numbers.

5 VOL. 46, 1984 EXPERIMENTAL GINGIVITIS IN CHILDREN 5 TABLE 8. Concentrtions of some suspect etiologicl gents in subgingivl periodontl flors s % of flor Concn s % of flor in: Species Adult Child Adult Adult Adult (severe (helthy)' (experimentl (experimentl (moderte Aeidultti(sever gingivitis) gingivitis)b periodontitis) periodontitis)d Actinomyces isrelii I Eubcterium timidum Fusobcterium nucletum Propionibcterium cnes Peptostreptococcus micros Dt from periodontlly helthy dults, studied s control subjects (11-13). bdt from reference 12. c Dt from reference 11. d Dt from reference 13. scores, nd its higher incidence in moderte nd severe detect the incidence of treponemes, ech observtion results periodontitis suggests tht this species my be importnt in from treponemes mong 104 or more cells, wheres in the both gingivitis nd periodontitis. Severl other treponeml other bcteriologicl dt (Tbles 6 nd 7), the frequency of species pper to occur s result of gingivitis nd re detection is bsed upon smple size of only 30 colonies. ssocited primrily with moderte or severe periodontitis. The different flors mong nd between children, dults, The dt (Tble 9) indicte tht drk-field microscopic nd disese sttes my help to pinpoint the few bcteril observtions of smples dispersed in 1.5 ml of broth tend to species in common tht could be custive gents. Of 160 underestimte the incidence of treponemes tht cn be tx detected in this study, only T. socrnskii nd 11 cultured from helthy or gingivitis sites. Treponeml cul- nontreponeml species (Tble 10) incresed in both children tures often were obtined from low dilutions of smples even nd dults s the GI scores incresed. In children, the though no spirochetes were seen by drk-field exmintion. frequently detected nonspirochetl species (>0.05% of the Conversely, culturl results my underestimte the number flor) which incresed in proportion in the flor s GI of treponemes in moderte or severe periodontitis becuse score incresed were Actinomyces WVA 963, A. isrelii II, we hve not cultured the "lrge treponeme" tht is frequent Actinomyces neslundii (-), Cpnocytophg gingivlis, in these sites. Cpnocytophg ochrce, Cpnocytophg sputigen, The treponeme dt re not directly comprble to the Eubcterium sburreum, F. nucletum, Selenomons sputinontreponeme dt. Since selective medi were used to gen, Selenomons D01, Selenomons D04, nd Seleno- TABLE 9. Incidence of mycoplsm nd treponemes s percent of subgingivl smples tht were positive Incidences s % of subgingivl smples tht were positive in: Txon Children Adults GI = 0 GI = 1 GI = 2 GI=o GI = l GI = 2 Hlb ModC Sevd Culturl Mycoplsm Treponem denticol Treponem pectinovorume Treponem socrnskiif Treponem vincentii Treponeme C Treponeme D Treponeme E Treponeme F Treponeme G Treponeme L Treponeme M Treponeme N Treponeme P Treponeme R Treponeme S Microscopic Lrge treponeme Treponemes observed No. of smples Dt from reference 12. bdt from periodontlly helthy (Hl) dults studied s control subjects in references 11 to 13. C Dt from reference 11. Mod, Moderte periodontitis. d Dt from reference 13. Sev, Severe periodontitis. ' Dt from reference 17. f R. M. Smibert, J. L. Johnson, nd R. R. Rnney, submitted for publiction.

6 6 MOORE ET AL. TABLE 10. Tx tht incresed with incresing GI scores in both children nd dults GI Txon Fusobcterium nucletum Actinomyces WV Selenomons D Bcteroides oris Eubcterium timidum Leptotrichi D Selenomons D Bcteroides D FMNRD FPCD Bcteroides pneumosintes As percent of 2,011, 2,695, nd 1,546 isoltes from sites with GI scores of 0, 1, 2, respectively, from the present study nd reference 12. mons D06. Of the frequently detected nonspirochetl species most closely correlted with developing gingivitis in dults (12), only Actinomyces WVA 963, F. nucletum, nd Selenomons D04 pper in the bove list. The three mjor species of the genus Cpnocytophg nd severl of the most numerous nerobic vibrios incresed in frequency with incresing GI scores in children, which my indicte tht flors with lrger proportions of these grmnegtive species re somewht less potent gents of gingivitis production thn re the more grm-positive flors ssocited with the more rpidly developing gingivitis in dults. Cpnocytophg species nd nerobic vibrios do not increse further in frequency in proportion in periodontitis (Tble 8), which tends to diminish their potentil significnce s periodontl pthogens nd is consistent with the very low propensity of norml children to develop periodontitis. If the flors in children hd not been significntly different from those in dults, we could hve determined tht the greter resistnce of children to experimentl gingivitis reflects only host resistnce. Since the incidence of some predominnt species ws found to correlte with incresing GI scores in both children nd dults, host resistnce still could ply the greter role, or the custive gents could be different in the two popultions. ACKNOWLEDGMENTS We thnk Pulett C. Atkins, Sndr S. Brown, Polly H. Cooper, Lub S. Fbrycky, Lees R. Miller, nd Lind D. Stover for bcteriologicl nlyses; Lind L. Long for chromtogrphic nlyses; Kren S. Loferski for electrophoretic nlyses; Ruth Z. Beyer for dt mngement; Lind B. Cook for glsswre preprtion; nd Kimberly Hrdison nd Felici Cine for dentl prophylxis nd subject mngement. This work ws supported by Public Helth Service grnts DE from the Ntionl Institute of Dentl Reserch nd AI from the Ntionl Institute of Allergy nd Infectious Diseses nd by project from the Commonwelth of Virgini. INFECT. IMMUN. LITERATURE CITED 1. Good, I. J The popultion frequencies of species nd the estimtion of popultion prmeters. Biometrik 40: Holdemn, L. V., E. P. Cto, nd W. E. C. Moore (ed.) Anerobe lbortory mnul, 4th ed. Virgini Polytechnic Institute nd Stte University, Blcksburg. 3. Loe, H., nd J. Silness Periodontl disese in pregnncy. I. Prevlence nd severity. Act Odontol. Scnd. 21: Loe, H., E. Theilde, nd S. B. Jensen Experimentl gingivitis in mn. J. Periodontol. 36: Longhurst, P., R. Gillett, nd N. W. Johnson Electron microscope quntittion of inflmmtory infiltrtes in childhood gingivitis. J. Periodontl. Res. 15: Longhurst, P., N. W. Johnson, nd R. M. Hopps Differences in lymphocyte nd plsm cell densities in inflmmed gingiv from dults nd young children. J. Periodontol. 48: Mckler, S. B., nd J. J. Crwford Plque development nd gingivitis in the primry dentition. J. Periodontol. 44: Mshimo, P. A., Y. Ymmoto, J. Slots, B. H. Prk, nd R. J. Genco The periodontl microflor of juvenile dibetics. Culture, immunofluorescence, nd serum ntibody studies. J. Periodontol. 54: Mttson, L Development of gingivitis in pre-school children nd young dults. J. Clin. Periodontol. 5: Moore, W. E. C., D. E. Hsh, L. V. Holdemn, nd E. P. Cto Polycrylmide slb gel electrophoresis of soluble proteins for studies of bcteril flors. Appl. Environ. Microbiol. 39: Moore, W. E. C., L. V. Holdemn, E. P. Cto, R. M. Smibert, J. A. Burmeister, nd R. R. Rnney Bcteriology of moderte (chronic) periodontitis in mture dult humns. Infect. Immun. 42: Moore, W. E. C., L. V. Holdemn, R. M. Smibert, I. J. Good, J. A. Burmeister, K. G. Plcnis, nd R. R. Rnney Bcteriology of experimentl gingivitis in young dult humns. Infect. Immun. 38: Moore, W. E. C., L. V. Holdemn, R. M. Smibert, D. E. Hsh, J. A. Burmeister, nd R. R. Rnney Bcteriology of severe periodontitis in young dult humns. Infect. Immun. 38: Pge, R. C., nd H. E. Schroeder Pthogenesis of inflmmtory periodontl disese. Lb. Invest. 33: Seymour, G. J., M. S. Crouch, R. N. Powell, D. Brooks, I. Beckmn, H. Zol, J. Brdley, nd G. F. Burns The identifiction of lymphoid cell subpopultions in sections of humn lymphoid tissue nd gingivitis in children using monoclonl ntibodies. J. Periodontl Res. 17: Silness, J., nd H. Loe Periodontl disese in pregnncy. II. Correltion between orl hygiene nd periodontl condition. Act Odontol. Scnd. 22: Smibert, R. M., nd J. A. Burmeister Treponem pectinovorum sp. nov. isolted from humns with periodontitis. Int. J. Syst. Bcteriol. 33: Smith, L. V., L. B. Golub, nd D. F. Duperon An evlution of creviculr fluid nd gingivl tissue in children. J. Dent. Child. 41: Tnner, A. C. R., M. A. Listgrten, nd J. L. Ebersole Wolinell curv sp. nov.: "Vibrio succinogenes" of humn origin. Int. J. Syst. Bcteriol. 34: