Differences between gingivitis and periodontitis ...

Differences between gingivitisperiodontitis associated microbial flora

in the beagle dogRelationship of plaque parameters to histological

parameters of periodontal disease

G. K. SVANBERG, S. A. SYED AND B. W. SCOTT, JR.

Department of Oral Biology, Dental Research Institute, School of Dentistry,The University of Michigan, Ann Arbor, Michigan, U.S.A.

The predominant dental plaque flora was compared between two grouf)s of adult beagledogs. (1> ten with gingivitis and (2) ten with advanced periodontitis. Plaque from a max-illary third premolar was cultured under strict anaerobic conditions. Specimens comprisingthe marginal periodontal tissues were taken at the plaque sampling site and analyzed forsome histological parameters of periodontal disease. The periodontitis dogs scored signifi-cantly higher for crevice depth, length of ulcerated crevice epithelium, area of inflamedconnective tissue, and loss of attachment. Supragingival periodontitis plaque had signifi-cantly higher anaerobic to aerobic ratio, proportions and CFU of esculin negative strepto-cocci, but lower proportions of Actinomyces vhcosus, Subgingival periodontitis plaque hadsignificantly higher anaerobic to aerobic ratio, microscopic counts of spirochetes, totalviable CFU, proportions and CFU of esculin negative streptococci and Fusobacteriumnucleaium, as well as CFU of Bacieroides asaccharolyticus. Gram negative bacilli andcoccobacilli, but significantly lower proportions of A, viscaus and unspeciated actino-mycetes. The total viable CFU, proportions and CFU of esculin negative streptococcicorrelated with all four histological parameters of periodontal disease. The CFU of B,asaccharolyticus, bacilli, and coccobacilli correlated with the length of ulcerated creviceepithelium and loss of attachment, but F. nucleatum only with the area of inflamedconnective tissue and loss of attachment.

(Accepted for publication July 22, 19gJ)

& Going 1964, Soames & Davies 1975, List-Introduction garten, Lindhe & Parodi 1979, Syed, Svan-

In most studies on the etiology and patho- berg & Svanberg 1980) and with periodoD-genesis of periodontal disease in the beagle titis (Wunder, Briner & Calkins 1976, New-dog, only the quantity of dental plaque has man et al. 1977, Williams et al. 1979, Syed,been correlated with the severity of the Svanberg & Svanberg 1981). but only Cou-periodontal lesion. A few investigations have rant et al. (1968) made an attempt to com-been aimed at determining the cotnposition pare the bacterial flora in periodontal healthof the canine microbial flora associated with and disease. However, considering the vari-gingivitis (Krasse & Brill I960, De Castro ous levels of anaerobiosis described hy

S V A N B E R G , S Y E D A N D S C O T T , J R .

Aranki et al. (1969), Courant's study wasconducted using less than strict anaerobicconditions, and only a few selected orga-nisms were isolated for identification.

The aim of the present study was two-fold: to compare the cultivable microbialflora associated with gingivitis and perio-dontitis using strict anaerobic techniquesand to determine the relationship, if any,between certain microorganisms and somehistological parameters of periodontal dis-

ease.

Material and Methods

Twenty female beagle dogs were obtainedfrom the Laboratory Research EnterprisesInc., Kalamazoo, Michigan. The animalsare genotypically and phenotypicallyhighly standardized. After six to twelveweeks in the nursery, where the pups arekept in groups of about 100 animals, two orthree dogs are placed in woven steel rodcages where they remain for the rest oftheir stay. They are fed a standard pelletdog food (Bench and Field 26. MartinFeeds, New Paris, Indiatia) and water adlibitum.

Two age-matched sets of dogs, rangingin age from three to six years, were selectedby oral examination from a group of 800animals, 10 dogs for having minimal gin-gival inflammation and no detectable loss ofconnective tissue attachment (Gingivitisgroup) and 10 dogs with advanced perio-dontitis.

Table 1

Distribution of Piaque index (PII) scores andGingival Index (Gl) scores of upper left third

premoiar (p'')

Dogs

GingivitisPeriodontitisSignif.

PU

1 2

91

p < 0.002

3

19

1

1

Gl

2

910NS

3

Plaque Collection ProcedureThe animals were anesthetized with an intra-venous injection of Pentothal (Abbott,North Chicago, Illinois). The Plaque Indexscore (Silness & Loe 1964) and the GingivalIndex score (Loe & Silness 1963) were thenassessed for the upper left third premoiar(Ps). Supragingival plaque was collectedfrom a two millimeter wide zone adjacent tothe gingival margin of the distal root of P-*using a stainless steel plaque spoon (1.7 X15 mm) held in a hemostat. The plaquespoon was immediately placed in a 1 dratnscrew cap vial filled to the rim with pre-reduced transport fluid (RTF, Syed &Loesche 1972) and taken into an anaerobicglove box (Aranki et al. 1969) containing agaseous mixture of 85 % nitrogen, 10 %hydrogen, and 5 % carbon dioxide.

The remaining supragingival depositswere then removed with a curette operatedin a disto-mesial and slightly coronal direc-tion. Subgingival plaque was collected witha stainless steel wire loop (wire diameter0.3 mm, outer diameter of loop 1.5 mm)held in a hemostat. The loop was introducedinto the crevice at a slight angle to the toothsurface, moved apically until slight resis-tance was encountered, and then movedback and forth in a mesio-distal directionwith light pressure toward the tooth sur-face to free any adherent plaque. The loopholding the plaque sample was immediatelyplaced io RTF and taken into the anaerobicglove box. This method of sampling pro-vided a standard volume sample as plaquewas retained only in the interior of theloop.

Plaque Culturing and Characterization ofOrganismsPlaque culturing was done inside the an-aerobic glove box, and the procedures haveheen described in detail in a previous paper(Syed et al. 1980). Each sample was dis-persed for 20 seconds with an ultrasonic dis-

M I C R O B I O T A A N D P E R I O D O N T A L D I S E A S E I N B E A G L E S

rupter (Kontes, Vineland, New Jersey; fre-quency 23,500 cydes/s, power output 1,200watt/sq. in.) followed by Vortex mixing for15 seconds. Serial fivefold dilutions in RTFwere prepared, and 50 yd aliquots of thelast three dilutions were dispersed with anEppendorff pipette and spread with sterileL-shaped glass rods on duplicate plates ofthe following prereduced media: enrichedtrypticase soy agar (ETSA, Syed et al.1980), ETSA with 5 % sucrose, and Schaed-ler agar (Baltimore Biological Laboratories,Cockeysville, Maryland) with menadioneand 3 % defibrinated sheep blood. Theprocedure was completed 30 to 60 minutesafter collection of the plaque. The inocu-lated plates were incubated at 37°C in theglove box for six to eight days. One platefrom each sample showing 75 to 150 well-isolated colony forming units (CFU) waskept in the glove box and used for isolationand subculturing of the organistns. Theremaining plates were used for determina-tion of total and differential CFU and thendiscarded. A duplicate set of ETSA platesfrom each specimen was incubated aero-bically to determine the aerobic CFU.

Subculturing of the organisms on theprimary ETSA plates was done inside theglove box. Prior to transfer into broth mediathe colonies were examined for purity usinga stereomicroscope equipped with a zoomlens and illuminator. The microscope wasalso used when picking the colonies. Eachcolony was transferred into basal esculinbroth (Syed & Loesche 1978) and thegrowth used for esculin hydrolysis, nitratereduction and indole production tests. Gramstain, and inoculation of an ETSA plate foraerobic incubation. The purity of the sub-cultures was confirmed by microscopicexamination of Gram stained preparationsof the broth cultures and stereomicroscopicexamination of the growth on agar mediaused for various tests. Samples of the brothwere also passed into suitable media for

catalase production, glucose fermentation,and geiatinase tests (Syed 1976). Gas-liquidchromatographic analyses of volatile andnon-volatile fatty acid end products (Hol-deman & Moore 1975) from representativeisolates were done either to confirm theidentification or facilitate the characteriza-tion of the organisms. The organisms wereclassified according to the eighth edition ofBergey's Manual of Determinative Bacteri-ology (Buchanan & Gibbons 1974), the An-aerobic Laboratory Manual of Determi-native Bacteriology (Holdeman & Moore1975), and a taxonomic scheme (Syed &Loesche 1978).

Spirochete counts and anaerobic to aero-bic ratio: Following sonication and appro-priate dilution of the samples the micro-scopic counts of spirochetes were deter-tnined in a Petroff Hausser chamber withbuffered formalin-crystal violet added to thesuspending medium (Aranki et al. 1969).Since the number of spirochetes was low inthe gingivitis samples, the whole chamber(400 squares) was used.

The anaerobic to aerobic ratio was deter-mined as the ratio of tota! colonies grownanaerobically to total colonies grown aero-bically. Anaerobic counts included strictlyanaerobic and facultative organisms. Aero-bic counts included aerobic and facultativeorganisms.

Biopsy ProcedureSpecimens comprising parts of the toothand the adjacent marginal periodontal tis-sues (Schroeder et al. 1973) were taken ateach plaque sampling site, fixed in 5 %glutaraldehyde and 4 % paraformaldehydebuffered at pH 7.3 (Karnovsky 1965), decal-cified in formic acid-citrate (Luna 1968), andembedded in Epon (Luft 1961). Two micronthick bucco-lingual sections were taken andstained with methylene blue and basicfuchsin. Tracings of the sections were ob-tained by microscopic projection (linear

S V A N B E R G , S Y E D A N D S C O T T , J R .

Table 2

Histological parameters of periodontal disease in gingivitis and periodontitis dogs

GingivitisX ± SE

PeriodontitisX ± SE

Signif.

Loss of AttaciimBnt*Crevice Depth*Length of Uicerated Crevice Epitheiiutn'Area of Inflamed Connective Tissue"

0.03 ± 0.011.13 ± Q.110.26 ± 0.070.20 + 0.03

2.4B ±2 28 ±1.63 +1.61 ±

0.440.370.300.3S

p = 0.0001p = 0.0O4p = 0.0001p = 0 0003

* Measurements given in mm." Measurements given in mm^.

mag. 55 X) and analyzed for the followingfour parameters: (1) crevice depth, niea-sured from the free gingival margin to thefree surface of the junctional epithelium, (2)length of ulcerated crevice epithelium, iden-tified as crevice areas without any lightmicroscopically distinguishable epithelium,(3) area of inflamed connective tissue, com-prising the cell rich/collagen poor tissueportions, and (4) loss of attachment, mea-sured from the cemento-enamel junction tothe apical termination of the junctional epi-thelium.

Statistical AnalysisWhen comparing gingivitis and periodontitisdogs, the Median test was used for ordinallevel measurements (Plaque Index, Gingival

Index) and Student's t-test for ratio levelmeasurements (histological measurements,plaque parameters). The relationship be-tween subgingival microorganisms and thehistological parameters of periodontal dis-ease was analyzed by the Pearson Product-Moment Correlation test.

ResuHs

Clinical ParametersThe upper left third premoiar of the perio-dontitis dogs had significantly more supra-gingival piaque than those of the gingivitisdogs with a predominance of Plaque Indexscores of 3. The clinical signs of gingivalinflammation were similar in both groupswith a Gingival Index score of 2 (Table 1).

Table 3

Plaque parameters of supragingival and subgingival plaque'

Supragingival PiaqueGingivitis Psriodontilis

X + SE X ± SE Signif.

Subgingivai PiaqueGingivitis Periodontitis

X ± SE X ± SESignif.

Viable CFU per sampleX 10* 18.0 ± 4.6 32.9 + SB NS 0.09 ± 0.05 3.2 ± 0 . 8 p = 0.0003

Anaerotjic/Aerobic Rat io" 5.4 ± 1.4 46.9 + 18.7 p = 0.03 8.1 ± 2.7 76.6 + 29,1 p = 0.02Microscopic Counts of

Spirochetes x 10" 3.9 + 0.6 6.4 + 1 . 3 NS ND 15.0 ± 4 . 7 p = 0.0O3

* One sample was taken from 10 dogs in each group.Ail CFU recovered anaerobicatiy

• ' Anaerobic/aerobic ratio =All CFU recovered aerobicaiiy

ND = Not detectable

M I C R O B I O T A A N D P E R I O D O N T A L D I S E A S E I N B E A G L E S

Table 4

Differences in the proportions of the predominant cuitivable plaque fiora

Supragingrval PIsqueGingivitfs Periodontilis

X ± SE X ± SE Signif.

Subgingival PlaqueGingivitis Periodontitis

X ± SE X ± SESignif.

A viscosus 2.9 ± 1 . 1 "Unspeciated actinomycetesStreptococci (esculin neg.) 4.4 ± t.5F. nucfeatum

12.3 ± 2.2

p

p

= 0,05

= 0.008

1,63,52,2

20.7

± 0,5+ 1.0± 0,B± 6,5

0,3O.B

10,943,0

± 0,2± 0,4± 3,9± 8,4

p = 0,05p = 0.03p = 0,02p = 0.05

* Percentage of total cultivable bacteria on anaerobicaliy incubated ETSA plates,** Anaerobic and facultative streptococci which were not speciated.

Histological ParametersSpecimens taken at the plaque samplingsite in the gingivitis dogs showed insignifi-cant loss of connective tissue attachment(x = 0.03 mm), whereas the periodontitisspecimens revealed considerable breakdownof the supporting tissues (x = 2.48 mm.Table 2). The periodontitis specimens alsodisplayed significantly deeper crevices, moreextensive ulceration of the crevicular epi-thelium, and larger areas of inflamed con-nective tissue.

Plaque ParametersSupragingival plaques of both groups weresimilar in total viable CFU and microscopiccounts of spirochetes, whereas the anaerobicto aerobic ratio was significantly higher inthe periodontitis samples (Table 3). Sub-

gingival samples from the periodontitis dogshad significantly higher total viable CFU,anaerobic to aerobic ratio, and microscopiccounts of spirochetes (Table 3).

Bacteriological FindingsThe predominant cultivable flora was ana-lyzed for differences in the proportions ofmicroorganisms associated with gingivitisand periodontitis. Only those results whichshowed a significant difference between thetwo groups are given in Table 4. The gin-givitis dogs had significantly higher pro-portions of A. viscosus in supragingival andsubgingival plaque and unspeciated actino-mycetes in subgingival plaque. The perio-dontitis dogs had significantly higher pro-portions of esculin negative streptococci insupragingival and subgingival plaque and

Table 5Differences in viable counts x 1CH of predominant cultivable flora

Supragingival Plaque

Gingivitis PeriodontitisX ± SE x ± SE

Signif.

Subgingival PlaqueGingivitis Periodontitis

X ± SE X ± SESignif.

Gram pos, rods(escutin neg,)

Streptococci (esculin neF, nuc/eatum6, BsaccharolyticusGram neg, bacilliGram neg, coocobaciiii

9,; 48,0 ± 17,5 447,2 ± 186,9 p = 0.02

1,0 ± 0.7

0,22,3 :0,7 :0.1 :1,5 :

t 0,18t 1,8t 0.3b 0.03t 0,7

9,4 :

42.7122,1 .81.9 :21.8 :12,4 :

t 2,9

t 17,8t 41,3t 32.2t 7.2b 4.5

p = 0.005

p = 0,01p = 0,003p = 0.008p = 0.002p = 0,01

S V A N B E R G , S Y E D A N D S C O T T , J R .

Table 6Relationship between microorganisms of subgingival plaque and histological parameters

of periodontal disease

Total viable CFU

Viable counts of:

Gram positive organismsEsculin nagative rodsUnspeciated actinomycetesEsculin negative streptococci

Gram negative organismsF. nucleslumB. asBCcharolyticusBaciliiCoccobacilli

Proportions {"in) of:Escutin negative streptococci

Crevice

Corr.coeff.

0.63

0.55

0.64

0.60

0.65

depth

Signif.p value

0.G04

0.01

0.003

0.007

0.003

Lengtfi of

CreviceEpithelium

Corr.coeff.

0.82

0.550.620.79

0.B50.700.81

0.67

Signif.p value

0.0001

0.020.DO5

0.0001

0.0001

O.O0O9

0.0001

0.002

Area of Inflamed

Tissue

Corr.coeff.

0.62

0.66

0.52

0.60

0.57

Signif.p value

0.004

0.002

0.02

0.0O6

0.01

1 r\r. ^ 1

LOSS Ul

Attacf^ment

Corr.coeff.

0.B2

0.63

0.75

0.600.750.560.56

0.67

Signif.p value

0.0001

0.004

0.OOO2

0.007

0.0002

0.01

0.O1

0.002

significantly higher proportions of F. nu-cleatum in subgingival plaque.

The two groups were compared for differ-ences in the absolute levels of viable CFUof the various organisms (Table 5). Esculinnegative streptococci were the only orga-nisms that were found in significantlyhigher numbers in both supragingival andsubgingival plaque in the periodontitis dogs.This is a heterogeneous group of organismsand includes both anaerobic and faculta-tively anaerobic streptococci. They were notStreptococcus mutans or Streptococcus san-guis. We were not able to speciate theseorganisms by the criteria that were used inthis study. Therefore, these organisms weretreated as a group rather than a singlespecies. Subgingival samples from the perio-dontitis dogs also had significantly highercounts of esculin negative Gram positiverods, F. nucleatum, B. asaccharolyticus, andGram negative bacilli and coccobacilli.

The bacteriological parameters of thesubgingival samples were analyzed for cor-

relations, if any, with the histological para-meters of periodontal disease (Table 6). Lossof attachment and the length of ulceratedcrevice epithelium showed a high correlationwith the total viable CFU as well as theCFU of esculin negative streptococci and B.asaccharolyticus, and a moderate correla-tion with the CFU of esculin negative Grampositive rods and Gram negative bacilli.Also, loss of attachment displayed a mod-erate relation with the CFU of F. nucleatumand Gram negative coccobacilli. The lengthof ulcerated crevice epithelium revealed amoderate relation with the CFU of unspe-ciated actinomycetes and a high relation withthe CFU of Gram negative coccobacilli.Crevice depth and the area of inflamed con-nective tissue were correlated moderatelywith the total viable CFU and the CFU ofesculin negative Gram positive rods andstreptococci. In addition, a moderate rela-tion was seen between crevice depth and theCFU of B. asaccharolyticus as well as be-tween the area of inflamed connective tissue

M I C R O B I O T A A N D P E R I O D O N T A L D I S E A S E I N B E A G L E S

and the CFU of F. nucleatum. In terms ofproportions only the esculin negative Grampositive streptococci correlated signifi-cantly with the severity of the iesion andshowed a moderate relation with all fourhistologica! parameters of periodontal dis-

ease.

Discussion

In the present study on the dental micro-bial flora, beagle dogs with advanced perio-dontitis were compared with those that hadgingivitis but negligible loss of connectivetissue attachment. The two groups of dogshad similar GI scores, but the periodontitisdogs scored significantly higher for histo-logica! parameters of periodontal diseasesuch as crevice depth, length of ulceratedcrevice epithelium, area of inflamed con-nective tissue, and loss of attachment. Theresults showed that periodontal pocketsharbored significantly more Gram negativeanaerobes and spirochetes than did gingivitissites. F. nucleatum accounted for a majorportion of the periodontal pocket florawhich also contained significantly higherproportions of esculin negative streptococci.

It has been reported that Gram negativeanaerobic organisms account for a majorportion of the subgingival flora of thebeagle dog (Newman et al. 1977, Williamset al. 1979, Syed et al. 1980, 1981) and thatthe mean proportions of B. melaninogenicus(B. asaccharolyticus) are higher in advancedthan incipient periodontal lesions (Newmanet al. 1977). Our results indicate that theproportions of B. asaccharolyticus insamples from periodontal pockets are high(20.3 + 5.4 %; x ± SE), but not signifi-cantly different from the gingivitis samples(19.0 ± 8.4 %; X ± SE). However, thelevels of subgingival B. asaccharolyticus ex-hibited strong correlations with the lengthof ulcerated crevice epithelium and loss ofattachment, and the subgitigival periodont-itis samples had significantly higher CFU

of this organism than did the subgingivalgingivitis samples. These findings imply arelationship between B. melaninogenicus (B.a.^accharolyticus) and the severity of perio-dontal disease in the beagle dog. B. asac-charolyticus and closely related strains alsohave been described as important pathogensin actively destructive periodontal disease inthe Macaca arctoides monkey (Slots &Hausmann 1979). This is in agreement withhuman studies which implicate this organismas a periodontal pathogen (Slots 1977,Loesche et al. 1981) that is isolated fromsites with the most inflammation and sup-puration (Tanner et al. 1979). However, B.asaccharolyticus isolated from the oral floraof beagles (Syed 1980) and Macaca arc-toides (Slots & Hausmann 1979) show cata-lase activity whereas the human oral strainsare catalase negative and have higher con-tents of guanine - plus - cytosine of thedeoxyribonucleic acids (Coykendall, Kacz-marek & Slots 1980).

Newman et al. (1977) found similar andfairly low proportions of F. nucleatum insubgingival samples from incipient ('^ 2 %)and advanced (^^A%) periodonta! lesionsin the beagle dog. In comparison with thesefigures, the mean proportions of F. nucle-atum in our study were about ten timeshigher in both the gingivitis and periodon-titis samples. Our findings are compatiblewith what has been described for the humansubgingivai flora in that F, nucleatum mayaccount for a considerable portion of thecultivable flora recovered from relativelyhealthy sites (Tanner et al. 1979). How-ever, the general pattern seems to be thatsubgingival F. nucleatum are low in clin-ically healthy sites (Slots 1977, Newman etal. 1978), increase with increasing GI scores(van Palenstein Helderman 1975) and, to-gether with B. melaninogenicus (B. asac-charolyticus), account for the major portionof the viable CFU in periodontal pockets(Slots 1977). This is analogous to our find-

S V A N B E R G , S Y E D A N D S C O T T , J R ,

ings in that, although high in the subgin-gival gingivitis samples, the proportions ofF. nucleatum were significantly higher inthe subgingival periodontitis samples andtogether with B. asaccharo}yncus accountedfor an average of about 60 % of the cul-tivable subgingival periodontitis flora.

Our gingivitis samples harbored strepto-cocci at levels similar to what has beenreported for the beagle dog (Wunder et al,1976, Williams et al, 1979), The proportionsof esculin negative streptococci were signifi-cantly higher in the periodontal pockets butlower than what has been described forperiodontal pockets in humans (Gibbons etal, 1963, 1964, Dwyer & Socransky 1968),Their less than strict anaerobic techniquesmost likely discriminated against the fastid-ious Gram negative anaerobes resulting inan overestimation of the Gram positiveorganisms e,g, streptococci. When the hu-man subgingival flora of periodontal pocketswas cultured under strict anaerobiosis.Gram positive cocci (Williams, Pantalone &Sherds 1976) and facultatively anaerobiccocci (Slots 1977) accounted for about 15 %and 6 % respectively, which is similar to ourfindings in periodontal pockets in the beagledog,

Actinomyces species were among the pre-dominant cultivable organisms of the supra-gingival and subgingival gingivitis flora inthe beagle dog (Syed et al, 1980) but werepresent in low proportions in periodontitisdogs, particularly in the subgingival samples(Syed et al, 1981). High proportions ofactinomycetes have been reported for thehuman gingivitis flora (Loesche & Syed1978, Slots et al, 1978), but these organismswere found to be less numerous in de-structive disease sites (Tanner et al. 1979),This is in keeping with the results of thepresent study in that Actinomyces specieswere very low in the periodontal pockets.

Although Courant et al, (1968) could notdetect any spirochetes in dogs with perio-

dontitis, these organisms were seen in mostsites with gingivitis (Swensen & Muhter1947, Krasse & Brill 1960) and found topredominate in the apical part of the gin-gival crevice (Soames &, Davies 1975). Inour study, the spirochete counts were sim-ilar in supragingival gingivitis and perio-dontitis plaque, but although found in sig-nificant numbers in the subgingival perio-dontitis flora, spirochetes were not detectedin the subgingival gingivitis samples. Thesefindings suggest a relationship betweenspirochetes and the severity of periodontaldisease in the beagle dog, which also hasbeen reported for humans (Socransky et al,1963, Lindhe, Liljenberg & Listgarten 1980),However, our data reveal no correlation ofspirochetes to crevice depth, length ofulcerated crevice epithelium, area of in-flamed connective tissue or loss of attach-ment. Spirochetes require some essentialnutrients that can be provided by other or-ganisms of the oral flora and might be con-tained in the crevicular fluid (Loesche1976), Thus, it is conceivable that theestablishment of a complex flora associatedwith periodontal disease will favor thegrowth of spirochetes. The seepage of ex-udate from the periodonta! lesion might helpexplain why the surface portion of subgin-gival plaque has a layer of spirochetes {List-garten 1976) and why the apical part of sub-gingival plaque may consist entirely ofspirochetes (Soames & Davies 1975),

A fall in the oxidation-reduction potentialoccurs with increasing age of the plaque(Kenney & Ash 1969) which will favor thegrowth of facultative and strict anaerobicorganisms, and bleeding will provide forgrowth factors that are essential for someorganisms (Loesche 1968), Such considera-tions shouid call for caution when drawingany conclusions regarding the cause andeffect relationship between the bacteriolog-ical and histological parameters of perio-dontal disease. It is true that in the present

M I C R O B I O T A A N D P E R I O D O N T A L D I S E A S E I N B E A G L E S

study the total viable CFU were signifi-cantly higher in the periodontal pockets andshowed a strong correlation with the lengthof ulcerated crevice epithelium and loss ofattachment. However, not only the propor-tions of F. nucleatum and esculin negativestreptococci but also the CFU of these twoorganisms as well as the CFU of otherGram negative organisms such as B. asac-charolyticus, bacilli and coccobacilli weresignificantly higher in the subgingival perio-dontitis samples and also showed a signifi-cant correlation with the length of ulceratedcrevice epithelium and loss of attachment.These results indicate that specific orga-nisms are correlated with some histologicalparameters of periodontal disease in thebeagle dog and suggest that, in this analysis,not only the proportions but also the CFUof the organisms should be taken into con-sideration. It is tempting to hypothesize thatwhen the numbers of certain microorga-nisms reach a critical level their pathogenicpotential is increased beyond the capacity ofthe host defense system resulting in break-down of the tissues. If this is true it mighthelp explain why, although in significantlylower numbers, periodontitis associatedbacteria also are found in gingivitis sites.

Acknowledgments

Dr. Walter Loesche provided encourage-ment in the initiation of this study andvaluable criticism during the preparationof the manuscript. Janice Stoll and Dr.Mona Svanberg assisted with the bacterio-logical aspects of this study.

This investigation was supported by GrantDE 02731 from the National Institute ofDental Research.

References

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by means of simplified anaerobic glcr\'e boxprocedure. Applied Microbiology 17: 568-576.

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Courant, P. R., Saxe, S. R., Nash, L. & Roddy,S. 1968. Sulcular bacteria in the beagle dog.Periodontics 6: 250-252.

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De Castro, C & Going, D. H 1964. A bac-teriologic and histologic investigation of thehealthy gingival sulcus of young dogs andchildren. Journal of Periodontotogy 35: 216-221.

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Gibbons, R. J.. Socransky, S. S., Sawyer, S.,Kapsimalis, B. & Macdonald, J. B. 1963. Themicrobiota of the gingi '̂ai crevice area ofman - II. The predominant cultivable or-ganisms. Archives of Oral Biology 8: 281-289.

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

Kenney, E. B. & Ash, M. M. 1969. Oxidationreduction potential of developing plaque,periodontal pockets, and gingival suki. Jour-nal of Periodontology 40: 630-633.

Krasse, B. & Brill, N. 1960. Effect of con-sistency of diet on bacteria in gingival pocketin dogs. Odontologisk Revy 11: 152-165.

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10 S V A N B E R G , S Y E D A N D S C O T T , JR.

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Address:

The University of MichiganSchool of DentistryDepartment of PeriodonticsAnn Arbor, Michigan 48109U.S.A.