Journal of Periodontal Research 17: 131-144, 1982

Periodontal disease associated withShigella flexneri in rhesus monkeysClinical, microbiologic and histopathologic findings

G. C. ARMITAGE, E . NEWBRUN, C. 1. HOOVER AND J. H. ANDERSON

Department of Pedodontology and Department of Oral Medicine/Hospital Dentistry,University of California, San Francisco and California Primate Research Center,

University of California, Davis, California, U.S.A.

The clinical, microbiologic, and histopatbologic features of Shigella flexneri-associatedperiodontal disease of rhestis monkeys were examined. Eighteen subgingival piaque samplesand 17 gingival biopsy specimens were taken from 8 rhesus monkeys harboring 5. flexneriorally. All plaque samples and three of tbe biopsy specimens were processed aerobicailyand anaerobically on seiective and nonselective media. The remaining biopsy specimenswere prepared for light or electron microscopic observation. An additional U subgingivalplaque samples from 6 monkeys not stispected of orally harboring S. flexneri were alsoprocessed microbiologically.

In the monkeys harboring Shigella orally, S. flexneri serotype 4 was isolated from 16 ofthe 18 subgingivai plaque samples. It constituted a minor plaque component in all 16 posi-tive sites, usually less than 0.1 % rf the recoverable flora. S. flexneri was not detected inany of the subgingival plaques from ccmtrol monkeys. Gingival sites harboring S. flexneriwere characterized by extremely intense clinical inflammation. Histopathologic observationsincluded ulceration of the pocket wall and heavy lymphocytic and plasma cell infUtrationof the connective tissue. At the ultrastructural level, unidentified bacteria were occasionallyobserved between, and possibly within, intact epithelial cells adjacent to areas of ulceration.

The results suggest that S. flexneri, though a numerically minor component of theplaque flora, may be important in the pathogenesis of the periodontal disease observed inthis study.

(Accepted for publication September 25, 1981)

nisms may be etiologically linked to indi-Inlroductlon vidual forans of gingivitis and periodontitis

It is well established that microorganisms (Newman & Socransky 1977, Slots 1976,are of major etiologic importance in the 1977, Darwish, Hyppa & Socransky 1978,pathogenesis of inflammatory periodontal Syed & Loesche 1978, Loesche & Syeddiseases of both humans and animals (Loe, 1978, Tanner et al. 1979). Most of the dataTheilade&Jensen 1965,Lindhe, Hamp&Loe implicating a specific group of bacteria in1975, Socransky 1977, Tanzer 1978, New- the etiology of a given periodontal diseaseman 1979, Newbrun 1979). Furthermore, come from studies of the predominant cul-data frcwn recent human studies suggest that tivable subgingival fiora. It is logically as-distinctly different groups of microorga- sumed that the numerically dominant orga-

132 ARMITAGE, NEWBRUN, HOOVER AND ANDERSON

nisms, particularly in the apical portion ofa periodontal lesion, have a probable etio-logic relationship to the disease.

Although periodontal disease occurs nat-urally in most nonhuman primates (for re-view see: Dreizen & Levy 1977), data sug-gesting an association between specificgroups of bacteria and periodontal disease inthese animals have only recently emerged.Monkeys (Macaea arctoides) fitted withsupragingival ligatures and maintained on asoft diet develop a form of periodontitis inwhich major segments of the subgingivalflora consist of various Haemophilus isolatesand Bacteroides melaninogenicus (Slots &Hausmann 1979). Concomitant with thedevelopment of alveolar bone loss in thesemonkeys, the proportion of Bacteroidesmelaninogenicus increased from a few per-cent to 66 % of the total cultivable flora.Earlier studies of other macaques failed toshow a relationship between specific bac-teria and periodontal disease, probably be-cause pooled plaque samples were used(Cock & Bowen 1967, Krygier et al. 1973).

McClure, Alford and Swenson (1976) atthe Yerkes Primate Center, Atlanta, werethe first to report an association betweenShigella flexneri and a destructive form ofperiodontal disease. They isolated this orga-nism from the mouths of 14 rhesus monkeyswhich had severely inflamed gingivae. Someof the affected monkeys also had extensivegingivai recession and alveolar bone loss.There was no apparent age or sex predilec-tion, and the infection occurred in outdoorgroups as well as in individually cagedmonkeys.

S. flexneri - associated periodontal dis-ease of rhesus monkeys has also been ob-served by Anderson et al. (1979) in 24 ani-mals (22 Macaea mulatta, 2 Macaea arc-toides) at the California Primate ResearchCenter, Davis. The monkeys ranged in agefrom 4 months to aged adults. In 12 ani-mals, fecal cultures positive for S. flexneri

were found after recognition of the perio-dontal disease. Diarrhea was a problem inonly two of these animals at the time theS. //e.inerj-associated periodontaJ diseasewas recognized. Gingival biopsy specimensfrom normal and severely inflamed sites in19 of the 24 atiimals were cultured for thepresence of 5. flexneri. All specimens fromnormal sites were negative for S. fiexneri;all those from severely inflamed sites werepositive.

These results suggest that S. flexneri, anintestinal pathogen, can colonize the gingivaand play an etioiogic role in a destructiveform of periodontaJ disease of rhesus mon-keys. However, many questions remain un-answered concerning the role of S. flexneriin the pathogenesis of this periodontal dis-ease: Is S. flexneri a dominant or a minorcomponent of the gingival flora? Is thisorganism responsible for the initiation and/or progression of the periodontal destruc-tion? How can S. //ocneri-associated perio-dontal disease be clinically distinguishedfrom other periodontal infections? As a firststep toward answering these questions, thepurpose of this report is to describe moreftilly some of the clinical, histopathologic,and microbiologic characteristics of S. flex-nm-associated periodontal disease of rhesusmonkeys.

Materials and Methoiit

Fourteen monkeys (13 Macaea mulatta, 1Macaea radiata) in the California PrimateResearch Center colony at the University ofCalifornia (Davis) were examined, severalof them on successive occasions over aperiod of up to 11 months. Eight animals(7 M. mulatta, 1 M. radiata) had severe gin-gival inflammation and were known toharbor S. flexneri orally (Anderson et al.1979). Four animals (controls) had eitherhealthy or moderately inflamed gingivaeand were not stispected of oraUy harboritigS. flexneri. Two animals had moderate gin-

P E R I O D O N T A L D I S E A S E A N D SHIGELLA FLEXNERI 133

gival inflamnsation but had previously beentreated for an 5. flexneri-associated perio-dontal infection by extraction, electro-cautery, and/or chemotherapy. The animalswere either caged individually indoors orin groups in larger outdoor facilities; allwere housed according to Institute of Lab-oratory Animal Resources guidelines. Thediet consisted of hard biscuits formulatedfor primates (Purina Monkey Lab Chow).The animals ranged in age from approxi-mately 1}^ years (with only primary teeth)to over 13 years old.

Clinical examinationTo facilitate intraoral examinations, col-lections of dental plaquS, and gingival biop-sies, the monkeys were immobilized withketamine hydrochloride (2.5-10 mg per kilo-gram). For each site from which plaquesamples or biopsy specimens were taken,Gingival Index (Loe & Silness 1963) andPlaque Index (Silness & Loe 1964) scoreswere recorded. Pocket depths were mea-sured to the nearest millimeter with aMichigan No. 0 periodontal probe (MarquisDental Mfg. Co.). In addition, loss of con-nective tissue attachment was estimated byrecording the distance from the cemento-enamel junction to the base of the probe-able crevice (Ramfjord 1974).

Mierobial samplingEighteen subgingival plaque samples wereobtained from different sites in the 8 mon-keys known to harbor S. flexneri orally.Severely inflamed and markedly erythema-tous sites were selected for sampling. Fromthe 4 control animals, a total of 5 subgin-gival plaque samples were taken. Of these 5sites, 3 were clinically healthy (GI = 0, noattachment loss), 1 had moderate gingivitis(GI = 2, no attachment loss), and 1 hadslight periodontitis (GI = 2, 1 mm attach-ment loss). Three subgingival samples werec^itained from different sites in each of the

2 animals with a past history (treated) of S.flexneri-a&socaXed periodontal disease.These sites had slight to moderate perio-dontitis (GI = 2,1 - 4 mm attachment loss).

Before the subgingival samples weretaken, supragingival plaque was removedwith a curet and the site wiped with a sterilecotton pellet and then irrigated with sterilephysiologic saline. Subgingival plaque wasobtained with a sterile thin plastic "scoop"prepared by cutting in half the tip of a dis-posable Eppendorf micropipette. The scoop,held in a pair of sterile hemostats, wasintroduced into the pocket at the site to besampled, and plaque was accumulated inthe hollow of the scoop.

Each scoop was dropped into a vial con-taining 1 ml of reduced transport fluid(RTF, Syed & Loesche 1972). The vialswere loosely capped and placed vertically inBio-Bag Anaerobic Culture Sets (MarionScientific Corp.). The bags were sealed andan anaerobic environment was generated;the resazurin indicator was usually colorlesswithin 30 min.

Culture methodsThe Bio-Bags containing the vials werebrought back from Davis to the laboratoryin San Francisco within 2 to 3 h after col-lection. The Bio-Bags were taken into ananaerobic glove box (Clinical Analysis Prod-ucts Co.) containing a mixttire of 85 % N^,10 % Ha, and 5 % COg (Cox & Mangels1976). Samples were dispersed, seriallydiluted, and plated within the box (Hoover& Newbrun 1977). Plaque samples weredispersed by tiltrasonic oscillation at 20 Wfor 10 s with a Lab-Line Ultratip LabsonicSystem equipped with a 1/8 in (ca. 3.17mm) titanium microtip (Lab-Line Instru-ments).

An undiluted 0.1 mJ portion of the dis-persed plaque sample and serial 10-folddilutions (lO-i to lO-S) were each plated onBrucella agar base (Difco) supplemented

134 ARMITAGE, NEWBRUN, HOOVER AND ANDERSON

with 5 % laked sheep blood, 10 jig/mlmenadione and 0.02 % dithiothreitol fornonselective bacterial growth and differen-tiation of Bacteroides melaninogenieus(black-pigmetited colonies). In addition, 0.1ml portions of the undiluted plaque sampleand seria! 10-fold dilutions (lO-i to 10-3)were plated on MacConkey agar (Difco) forenteric organisms, particularly S. flexneri. Aselective medium was needed since S. flex-neri was present only in low numbers inplaque and gingival biopsies. The Bnicelia-laked blood plates were incubated anaerobi-cally for 6 to 7 days at 37°C in GasPak jars(Baltimore Biological Laboratories, BBL).The MacConkey plates were incubatedaerobicaily for 48 h at 37°C.

In addition, some samples of plaque wereexamined directly in suspension at a mag-nification of 1000 X by Hoffman modula-tion contrast microscopy.

Gingival biopsiesFrom 7 of the 8 animals harboring S. flex-neri, gingival biopsy specimens were takenfrom severely inflamed sites and placed in50 % neutral phosphate-buffered formalin.A total of 10 specimens was obtained forhistopathological examination. After fixa-tion, each specimen was dehydrated, em-bedded in paraffin, and sectioned by routinehistoSogical procedures (Luna 1968). Sec-tions were stained with hematoxylin andeosin, methyl green-pyrontn, periodic acid -Schiff, and the Brown-Brenn method forbacteria (Pearse 1968).

An additional 4 biopsy specimens fromthese animals were prepared for electronmicroscopic examination. The tissues wereprefixed for 2-3 h in 5 % glutaraldehydeand 4 % paraformaldehyde buffered with0.02 M cacodylate (Karnovsky 1965). Theywere then post-fixed for 2 h at 4°C in 2 %s-collidine4)uffered osmic acid and sub-sequently dehydrated and embedded in Epon(Luft 1961). Thin sections were cut with a

diamond knife and stained on Formvar-coated grids with uranyl acetate and leadcitrate. They were examined in a SiemensElmiskop IA operated at 80 kv.

Three other biopsy specimens were usedto compare the percentage of S. flexneriassociated with the gingiva with the percent-age in the immediately adjacent subgingivalpiaque. For microbiological cultures eachspecimen was placed in RTF, subsequentlyminced with sterile scissors, and processedfor microbiological culture in the same wayas the plaque samples.

Identification and quantification of cul-tivable floraSince precise taxonomic identification ofmany ora! isolates is technically difficult,time-consuming, and beyond our resources,an exhaustive biochemical characterizationwas not attempted. 5. flexneri was quanti-fied on MacConkey agar; in most cases onlyone morphological type of colony (whichproved to be S. flexneri) could be observed.Representative isolates were biochemicallycharacterized to determine if they were S.fiexneri. Isolates of S. flexneri were testedfor serotype using commercial antisera(Difco). Isolates of Bacteroides melanino-genicus were differentiated by formation ofblack pigment on Brucella-laked bloodplates and subspeciated by acid formationfrom glucose, indole prodtiction, starchhydrolysis, and lipase activity as well asother biochemical tests (Anaerobe System20A, Analytab Products Inc., Plainvievi',N.y.).

Oral isolates of black-pigmented Bacte-roides that were asaccharolytic, indole-pos-itive, catalase-negative, lipase-negative, anddid not hydrolyze starch were classified asB. gingivalis (Coykendall, Kaczmarek &Slots 1980). B. melaninogenicus ss. inter-medius organisms fermented glucose, wereindole-positive, catalase-negative, lipase-pos-itive, and hydrolyzed starch. B. melanino-

P E R I O D O N T A L D I S E A S E A N D S i - I I G E L L A F L E X N E B I 135

Fig. t . Clinica! appearance of the gingival tissues ofan adult female il. mulatta (MMU 16591). Note ihemarked gingivaf inffamftiation. Shi;ieUa flcrncri wasisolated from several subgingivai plaque sampiestaken from this animal.

genicus ss. melaninogenicus organisms fer-mented glucose, were indole-negative, cata-lase-negative, iipase-negative, and hydro-lyzed starch.

Results

Clinical observationsGingival sites harboring S. flexneri werecharacterized by extremely intense clinical

•n- •

Pig. £. Deep (-̂ .7 mm) interproximai crater (arrow)between tiie maxillary molars of an adult male M.miihiftii jMMU 830). Shigella ilextwri was isolatedfrom subgingival piaque sampies and a gingivaibiopsy speoimen taken from tiiis site.

inflamraation. Eleven of the 16 sites positivefor Shigella had Gingival Index (GI) scoresof 3 (Table 1). Marked erythema, sponta-neous bleeding, and inflammatory hyper-plasia of the marginal and portions of theattached gingiva were particularly promi-nent (Fig. 1). In old monkeys O 12 years),gingival recession, extensive attachment loss,and deep interproximai cratering were com-mon (Fig. 2).

In the animals harboring S. flexneri, thedistribution and percentage of sites withintense gingival inflammation were variable.In some animals, severe gingival inflamma-tion was present around most remainingteeth; in others only one or two sites wereaffected. The percentage of sites with severeinflammation ranged from 10 to 82.3 % ofthe remaining dento-gingival units. Althoughthe whole-mouth distribution of the severeinflammation was variable, the gingivaaroond some or all of the anterior teeth wasaffected in every ShigeUa-^o%\tNz animal.In the 3 oldest monkeys O 12 years) manyteeth, including most or all of the anteriors,had been previously extracted because of5/n'ge//a-associated periodontal infections(Anderson et a!. 1979). Of the 5 youngerS/;/gc/i(7-positive monkeys « 9 years), thegingiva around some or all of the anteriorteeth was markedly inflamed. Indeed, in 4 ofthese animals only the anterior gingiva ex-hibited the marked inflammation. Of the 10plaque samples taken from the anterior teethof these younger animals, 8 were positivefor S. flexneri.

Over a period of several months, changesin the clinical appearance of the giogivaltissues were noted, especially in younganimals. One of these monkeys (MMU16492) initially presented with severe perio-dontal inflammation localized to the upperincisors, resembling necrotizing ulcerativegingivitis (Fig. 3A). Seven months laterthere was still gingival ulceration, but someinflammatory hyperplasia was also present

136 A R M i T A G E , N E W B R U N , H O O V E R A N D A N D E R S O N

Fig. 3. Progression of SA(f/''//'7.assocsated perjodontaidisease in a young .V. miiUiiln [MMU 16492) over anti-month period. At 15 months of age {A), liiefe wereinterproximai necrosis and uiceration resemblingnecrotizing ulcefative gingivitis. The gingivai lesionswere locatized to the upper anterior primary teeth.Seven months later (B), gingival recession and infiam-matory hyporplasia were noted. After 11 months {C).ulceration. marked infiammatory hyperplasia. and spon-taneous gingival bieeding were observed. At this time,the primary central incisors had been exfoliated andthe permanent incisors were erupting.

(Fig. 3B). After 11 months, in addition toulceration and inflammatory hyperplasia,spontaneous gingival bleeding was observed(Fig. 3C). Only on this last examination wasS. flexneri isolated from plaque adjacent tothe inflamed site.

Microbiologic findingsIn the 8 monkeys known to harbor Shigellaorally, S. flexneri serotype 4 was isolated

from 16 of the 18 subgingival plaque sam-ples (Table 1). S. flexneri constituted aminor piaque component in all 16 positivesites, usually less than 0.1 % of the recover-able flora, or about 10* to 10'̂ colony-form-ing units (CFU). The percentages of S.flexneri colonizing the gingival biopsy tissuewere higher than those in immediately ad-jacent subgingival plaque (Table 2). How-ever, S. flexneri still constituted only aminor component of the total cultivableflora.

S. flexneri was not detected in 6 sub-gingival samples from 2 monkeys which hadpreviously been treated for Shigella-asso-ciated periodontal disease, nor in 5 subgin-gival samples from controi monkeys (Table3).

Black-pigmented Bacteroides were pre-sent in ali of 12 S/iige//a-positive subgingivaiplaque samples cultured on Brucella-lakedblood agar (Table 1). They represented from1.6 to 57.7 % of the recoverable flora, orabout 10̂ to 10' CFU. In 3 of these 12samples, they constituted greater than 40 %of the total cultivable flora. Black-pig-mented Bacteroides were also cultured from3 of 7 control sites (Table 3).

Subspeciation of representative isolates in-dicated that B. melaninogenicus ss. inter-medius was the predominant type. B. mela-ninogenicus ss. melaninogenicus and B. gin-givalis were also present. We did not ob-serve catalase-positive, black-pigmentedBacteroides (B. macacae) as described bySlots and Genco (1980). The name B. asac-charolyticus was reserved for asaccharolyticblack-pigmented Bacteroides from sourcesother than the oral cavity (Coykendall et al.1980).

Very preliminary characterization of theplaque flora at M/geHa-positive sites indi-cated some variation from site to site, butin general. Gram-negative rods were themajor cultivable component. Examination ofdirect suspensions of the subgingival plaques

P E R I O D O N T A L D I S E A S E A N D SHIGELLA FLEXNERI 137

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138 A R M I T A G E , N E W B R U N , H O O V E R A N D A N D E R S O N

Table 2

Comparison of proportions of S, tiexneri andB. melaninogenicus isolated from subgingival

plaque and adjacenf gingivai biopsies

Monkey% CFU

B. rnclafu'no'f/enicus•/. CFU

TotalCFU

MMU 8021PlaqueBiopsy

MMU 630PlaqueBiopsy

MRA 303PlaqueBiopsy

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0.15

0.003

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17.1

39.5

11.8

20.7

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1.3 X 10'

1.7 X 10'

3.8 X 10«

3.8 X W

1.4 X 10'

revealed large numbers of motile bacteria,especially vibrios and spirochetes.

Histopaikological and ultrastructuralobservationsExamination of gingival biopsy tissue withthe light microscope revealed extensive ul-ceration of the pocket wall and the marginalgingiva. Heavy lymphocytic and plasmaceli infiltration of the connective tissue wasobserved. The gingival connective tissue wasalmost completely replaced by these in-flammatory cells. The pocket epithelium andportions of the oral epithelium were infil-trated with polymorphonuclear leukocytes.Microorganisms could not be detectedwithin the tissues by the Brown and BrennGram stain. At the ultrastructural level,numerous spirochetes were located nearulcerated surfaces of the pocket wall (Fig.4). The spirochetes were superficially posi-

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P E R I O D O N T A L D I S E A S E A N D SHIGELLA FLEXNERI 139

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P E R I O D O N T A L D I S E A S E A N D SHIGELLA FLEXNERI 141

tioned and in no instance were they ob-served deeper than 5 ^m into the connectivetissue. Of more interest was the occasionalobservation of unidentified microorganismsbetween, and possibly within, intact epi-thelial cells adjacent to areas of ulceration(Fig. 5).

Discussion

The results of this study confirm the ob-servations of McClure et al. (1976) andAnderson et al. (1979) that Shigella flexneri,an intestinal pathogen, can colonize dento-gingival sites in rhesus monkeys. Further-more, sites harboring Shigella eiihibit a formof periodontal disease characterized bymarked erythema, spontaneous bleeding,and inflammatory hyperplasia. The sheerintensity of the inflammation is clinicallystriking. Indeed, half of the Shigella-pos-itive monkeys studied by Anderson et al.(1979) were first suspected of harboring thismicroorganism on the basis of the clinicalappearance of the periodontai tissues. Inaddition, in infected animals, sites withoutintense inflammation were invariably neg-ative for Shigella.

In the present investigation, the pres-ence of clinically intense gingival inflam-mation was the main basis upon which siteswere suspected of harboring Shigella. Sub-sequent microbiologic examination ofplaques from these sites confirmed the pres-ence of Shigella in 16 out of 18 samples.The two sites negative for Shigella were inooe young animal (Fig. 3A—C) believed tohave early manifestations of the infection.An adjacent site, .sampled several monthslater, was positive for S. flexneri.

The finding that 5. flexneri was only a

minor component « 0 . 1 % CFU) of therecoverable dento-gingival flora does notnecessarily mean that this organism is un-important in the pathogenesis of the ob-served periodontal disease. It is not neces-sary for an organism to be numericallydominant in order to cause disease, espec-ially if it is highly virulent. Specific exam-ples of intestinal shigellosis illustrate thispoint. Gnotobiotic mice, which develop in-testinal shigeliosis when diassociated with S.flexneri and E. coli, have been found to havelO-'' to 10" S. flexneri per gram of cecai con-tents, whereas the numbers of E. coli werelO'c cells per gram. In similar experiments,involving diassociation of S. flexneri withBacteroides, Bacteroides constituted 10"cells per gram cecal content. Thus, in micewith intestinal shigellosis, S. fiexneri con-stituted only 0.0001 to 1.0 % of the intes-tinal flora (Maier & Hentges 1972). Whencynomolgus monkeys were experimentallyinfected with 5. flexneri, about 10' cells pergram were isolated from the dysentericstool. Since the feces normally contain atotal of about 10" cells per gram, it ap-pears that in monkeys suffering from bacil-lary dysentery, Shigella constitutes less than0.01 % of the total enteric flora (Honjo etal. 1964). These observations on experi-mental animals agree with clinical findingson human stools from individuals sufferingfrom bacillary dysentery, in which Shigellais also a minor component of the total fecalflora (W. K. Hadley, unpublished findingson patients at San Francisco General Hos-pital).

It is well established that Shigella infec-tions are not limited to the intestinal tractbut may cause copjunctiyitis, acute cystitis,respiratory disease, vulvpvaginitis (Davis

Fig. 5. Eieotron micrograph fA) of a microorganism (arrow) whicii appears to be within a gingivai epitheiiaicell at a S/rii/e^fa-positlve site. X 9.200. Higher magnilioation (B) showing desmosomes (d. arrows) and micro-organism (m. arrow), x 54,000.

142 A R M I T A G E , N E W B R U N , H O O V E R A N D A N D E R S O N

1975), and wound itifection (Gregory, Starr& Omdal 1974). Virulent strains are able toinvade epithelial cells and produce a toxinwhich inhibits the synthesis of protein byribosomes, ultimately leading to death ofthe epithelial cells and an inflammatoryreaction (Okatnura &. Nakaya 1977, Keusch& Jacewicz 1977, Keusch 1977). Viablebacilli, capable of undergoing ceil division,have been reported inside intestinal epi-thelial cells of monkeys experimentally in-fected with S. flexneri (Takeuchi, Formal &Spring 1968). In the present study, bacteriawere occasionally observed between, andpossibly within, intact gingival epithelialcells. Although the identity of these bac-teria remains unknown, their associationwith epithelial cells is consistent with theknown relationship of Shigella to epithelia!cells in. intestinal shigellosis.

Our results suggest that S. flexneri may beimportant in the pathogenesis of the severeform of periodontal disease observed inthis study. To our knowledge, this is thefirst reported example of a numericallyminor component of the plaque flora beingetiologically linked to a periodontal disease.From the present study, it is not possible todetermine if 5. flexneri initiates the perio-dontal destruction or if it plays a substan-tial role in the progression of the disease.Indeed, the data do not unequivocally showthat S. flexneri is a causative agent; experi-ments which satisfy Koch's postulates areneeded for this purpose.

Since 5. flexneri is a numerically minorcomponent of the plaque flora, it is un-likely that this microorganism alone is re-sponsible for most of the severe periodontaidestruction observed in the monkeys. It ismore likely that numerically dominantperiodontopathogens, such as Bacteroidessp. and spirochetes, are more important inthis regard. However, it is possible that S.flexneri, by virtue of its toxic effect on epi-thelial cells, may trigger an environmental

change favorable to the proliferation otperiodontopathogens.

It has reeently been established that avariety of factors can lead to shifts in thesubgingival flora. For example, the place-ment of ligatures around teeth encouragesthe proliferation of several Bacteroidesspecies (Slots & Hausmann 1979, Korn-man, Holt & Robertson 1980, Komman etal. 1981). Hormonal alterations associatedwith pregnancy have also been implicated inpromoting the growth of Bacteroides in sub-gingival plaques (Komman & Loesche1980). In these situations, ligatures andhormonal alterations are agents of environ-mental change. We believe that in additionto its direct toxicity, S. flexneri may alsobe an agent of environmental change.

Acknowledgements

The authors wish to thank Ms. EvangelineLeash for her editorial assistance. Apprecia-tion is also extended to Dr. Mark I. Ryderfor his aid in preparing the photographicprints.

References

Anderson, J., Armitage, G., Banks, T., Hoover,C , Newbrun, E. & Henrickson, R. 1979.Periodontal disease in macaques associatedwith Shigella flexneri. Abstract, Symposiumon Diseases of the Gastrointestinal Tract andPancreas of Non-human Primates, San Fran-cisco.

Cock, D. J. & Bowen, W. G. J967. Occurrenceof Bacterionema matruchotii and Bacteroidesmelaninogenicus in gingival plaque frommonkeys. Journal of Periodontal Research 2:36-39.

Cox, M. E. & Mangels, J. I. 1976. Improvedchamber for the isolation of anaerobic micro-organisms. Journal of Clinical Microbiology4: 40-45.

Coykendall, A. L., Kaczmarek, F. S. & Slots, J.1980. Genetic heterogeneity in Bacteroidesasaccharolytieus {Hddeman and Moore 1970).Finegold and Barnes 1977 (Aj^roved Lists,1980) and proposal of two new species: Bac-

P E R I O D O N T A L D I S E A S E A N D SHIGELLA FLEXNERI 143

teroides gingivalis sp. nov. and Bacteroidesmacacae com. nov. International Journal ofSystematic Bacteriology 36: 559-569.

Darwish, S., Hyppa, T. & Socransky, S. S. 1978.Studies of the predominant cuhivable micro-biota of early periodontitis. Journal of Perio-dontal Research 13: 1-16.

Davis, T. C. 1975. Chronic vulvovaginitis inchildren due to Shigeila flexneri. Pediatrics56: 41-44.

Dreizen, S. & Levy, B. M. 1977. Monkeymodels in dental research. Journal of MedicalPrimatology 6: 133-144.

Gregory, J. E., Starr, S. P. & Omdal, C. 1974.Letter: Wound infection with Shigella flex-neri. Journal of Infectious Diseases 129." 602—604.

Honjo, S., Takasaka, M., Fujiwara, T., Naka-gawa, M., Andoo, K., Ogawa, H., Takahashi,R. & Imaizumi, K. 1964. Shigellosis in cyno-molgus monkeys (Macaea irus). II. Experi-mental infection with Shigella flexneri withspecial references to clinical and bacteriolog-ical findings. Japanese Journal of MedicalSciences and Biology 17: 307-319.

Hoover, C. I. & Newbrun, E. 1977. Survival ofbacteria from human dental plaque undervarious transport conditions. Journal of Clin-ical Microbiology 6: 212-218.

Karnovsky, M. J. 1965. A formaldehyde-glu-taraldehyde fixative of high osmolarity foruse in electron microscopy. Journal of CellBiology 27: 137A-138A.

Keusch, G. T. 1977. Shigellosis control - a rosyfuture? Journal of Infectious Diseases 136:456-457.

Keusch, G. T. & Jacewicz, M. 1977. The patho-genesis of shigeila diarrhea. VI. Toxin andantitoxin in Shigella flexneri and Shigellasonnei infections in humans. Journal of In-fectious Diseases 135: 552-556.

Kornman, K. S., Holt, S. C. & Robertson, P. B.1980. Subgingival microflora associated withChe conversion of gingivitis to periodontitis inmonkeys. Journal of Dental Research 59:A388.

Komman, K. S. & Loesche, W. J. 1980. Thesubgingival microbial flora during pr^nancy.Journal of Periodontal Research 15: 111-122.

Komman, K. S., Siegrist, B., Soskolne, W. A.& Nuki, K. 1981. The predominant cultivablesubgingival flora of beagle dogs followingligature jdacement and metronidazole ther-apy. Journal of Periodontal Research 16:251-258.

Krygier, O., Genco, R. J., Mashimo, P. A. &

Hausmann, E. 1973. Experimental gingivitisin Macaea speciosa monkeys: Clinical, bac-teriological and histologic similarities tohuman gingivitis. Journal of Periodontology44: 454-463.

Lindhe, J., Hamp, S. E. & Loe, H. 1975.Plaque-induced periodontal disease in beagledogs. A 4-year clinical, roentgenographical,and histometrical study. Journal of Perio-dontal Research 10: 243-255.

Loe, H. & Siiness, J. 1963. Periodontal diseasein pregnancy. 1. Prevalence and severity.Acta Odontologica Scandinavica 21: 533-555.

Loe, H., Theiiade, E. & Jensen, S. B. 1965.Experimental gingivitis in man. Journal ofPeriodontology 36: 177-187.

Loesche, W. J. & Syed, S. A. 1978. Bacteri-ology of human experimental gingivitis: Ef-fect of plaque and gingivitis score. Infectionand Immunity 21: 830-839.

Luft, J. H. 1961. Improvements in epoxy-resinembedding methods. Journal of Biophysicaland Biochemical Cytology 9: 409-414.

Luna, L. G. 1968. Manual of Histologic Stain-ing Methods of the Armed Forces Institute ofPathology, 3rd ed.. New York: The BlakistonDiv., McGraw-Hill Book Company.

Maier, B. R. & Hraitges, D. J. 1972. Experi-mental Shigella infections in laboratory ani-mals. I. Antagonism by human normal floracomponents in gnotobiotic mice. Infectionand Immunity 6: 168-173.

McClure, H. N., Alford, T. & Swenson, D.1976. Non-enteric Shigella infections in non-human primates. Journal of the AmericanVeterinary Medical Association 169; 938-939.

Newbrun, E. 1979. Dentai plaque ecology re-lated to caries and periodontal diseases.Australian Dental Journal 24: 231-234.

Newman, M. G. 1979. The role of Bacteroidesmelaninogenicus and other anaerobes inperiodontal infections. Reviews of InfectiousDiseases 1: 313-323.

Newman, M. & Socransky, S. 1977. Predom-inant cultivable microbiota in periodontosis.Journal of Periodontal Research 12: 120-128.

Okamura, N. & Nakaya, R. 1977. Rough mu-tant of Shigella flexneri 2a that penetratestissue culture cells but does not evoke kerato-conjunctivitis in guinea pigs. Infection andImmunity 17: 4-8.

Pearse, A. G. 1968. Histochemistry. Theoreticaland Applied, 3rd ed., Boston: little. Brown &Co.

Ramfjord, S. P. 1974. Design of studies or clin-ical trials to evaluate the effectiveness of

144 A R M I T A G E , N E W B R U N , H O O V E R A N D A N D E R S O N

agents or procedures for the prevention, ortreatmrait, of loss of the periodontium. Jour-nal of Periodontal Research 9: (Suppl. 14)78-93.

Silness, J. & Loe, H. 1964. Periodontal diseasein pregnancy. IL Correlation between oralhygiene and periodontal condition. ActaOdontologica Scandinavica 22: 121-135.

Slots, J. 1976. The predominant cultivableorganisms in juvenile periodontitis. Scan-dinavian Journal of Dental Research 84: 1-10.

Slots, J. 1977. The predominant cultivablemicroflora of advanced periodontitis. Scan-dinavian Journal of Dental Research 85: 114-121.

Slots, J. & Hausmann, H. 1979. Longitudinalstudy of experimentally induced periodontaldisease in Macaea arctoides: relationship be-tween microflora and alveolar bone loss. In-fection and Immunity 23: 260-269.

Slots, J. & Genco, R. J. 1980. Bacteroidesmelaninogenicus subsp. macacae, a new sub-species from monkey periodontopathic indi-genous microflora. International Journal ofSystematic Bacteriology 30: 82-85.

Socransky, S. S. 1977. Microbiology of perio-dontal disease - present status and futureconsiderations. Journal of Periodontology 48:497-504.

Syed, S. A. & Loesche, W. J. 1972. Survival of

human dental plaque flora in various trans-port media. Applied Microbiology 24: 638-644.

Syed, S. A. & Loesche, W. J. 1978. Bacteriologyof human experimental gingivitis: Effect ofplaque age. Infection and Immunity 21: 821-829.

Takeuchi, A., Formal, S. F. & Spring, H. 1968.Experimental acute colitis in the rhesus mon-key following peroral infection with Shigellaflexneri. American Journal of Pathology 52:503-529.

Tanner, A. C. R., Haffer, C , Bratthall, G. T.,Visconti, R. A. & Socransky, S. S. 1979. Astudy of the bacteria associated with advanc-ing periodontitis in man. Journal of ClinicaiPeriodontology 6: 278-307.

Tanzer, J. M. 1978. Current status of themicrobiology of periodontal disease. In: Inter-national Conference on Research in Biologyof Periodontal Disease, University of Illinois.Chicago, June 1977 pp. 153-182.

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Department of Periodontology, 681 HSWSchool of DentistryUniversity of California, San FranciscoSan Francisco, CA 94143U.S.A.