Vol. 43, No. 1INFECTION AND IMMUNITY, Jan. 1984, p. 308-3130019-9567/84/010308-06$02.00/0Copyright © 1984, American Society for Microbiology

Amount and Avidity of Salivary and Serum Antibodies AgainstStreptococcus mutans in Two Groups of Human Subjects with

Different Dental Caries SusceptibilityOLLI-PEKKA J. LEHTONEN,l* ERIK M. GRAHN,2 TOM H. STAHLBERG,3 AND LAURI A. LAITINEN3

Department of Medical Microbiology, Turku University, SF-20520 Turku 52'; Turku Naval Base, Turkii2; and NavalResearch Unit, Central Military Hospital, Helsinki,3 Finland

Received 21 March 1983/Accepted 26 September 1983

Immunoglobulin A (IgA) and IgG antibodies against Streptococcus mutans KlR and 10449 weremeasured in serum and in stimulated whole saliva from two groups of naval recruits, representing high orlow caries susceptibility. The antibody assays were performed by using the enzyme-linked immunosorbentassay, and the results were expressed by a method able to estimate the amount of high-avidity and totalspecific antibodies. As a control, concentrations of salivary total immunoglobulins were related to theamounts of specific antibodies. Further, antibodies were assayed against three antigens, unrelated to thestreptococci. No clear differences were observed in serum antibodies between the subjects with high or lowcaries susceptibility. However, in saliva, low caries susceptibility was associated with a high amount of totalantigen-specific IgA, and possibly IgG, against S. mutans. This difference between the groups still existedwhen the amounts of specific antibodies were related to the amounts of salivary immunoglobulins. Therewere no differences in the amounts of total specific antibodies against the unrelated antigens. No differenceswere observed in the estimates of high-avidity anti-S. mutans antibodies between the groups, either inserum or saliva. Thus, within the limitations of the assays and crude antigen, lack of high-avidity antibodiesis not responsible for caries susceptibility. Instead, the amount of anti-S. mutans antibodies seems to belinked with caries protection. The results of the present study indicate that salivary antibodies are linkedwith the control of human dental caries.

Streptococcus mutans is a major etiological agent inhuman dental caries. Specific antibodies against this bacteri-um are found in serum and saliva (6). Salivary antibodiesoriginate either from salivary glands (3) or from serumantibodies via crevicular fluid (4). Local gingival immuno-globulin synthesis also occurs, at least when gingival inflam-mation is involved (2). The exact role of these antibodies inthe control of dental caries is still unclear despite beingintensively studied.

Low-caries experience in humans is associated with highlevels of serum antibodies against S. mutans (5-7, 16). Inimmunization experiments with primates, a rise in serumimmunoglobulin G (IgG) antibodies against S. mutans pro-tein antigen is associated with caries protection (22, 23).

Salivary antibodies, in contrast to serum antibodies, arenot so clearly shown to be protective. In rodents, salivaryantibodies against S. mutans antigens protect against caries(27, 29, 33). In immunization experiments with primates, IgAantibodies against S. mutans were associated with a reduc-tion of the amount of the bacterium in the oral cavity (10),but this protective response has invariably not been record-ed (8). A negative correlation between salivary IgA antibod-ies against S. mutans and caries index has been found inchildren (12), but in several studies with adults, no caries-reducing effect of salivary antibodies has been detected (4, 5,15). Further, there is even some evidence that salivary IgAagainst S. mutans is caries enhancing (7). In humans,indirect evidence of the protective role of salivary IgAantibodies has been obtained in patients with IgA deficiency(18). The differing results of the previous studies may be dueto the fact that the measurement of salivary antibodies isdifficult to standardize because of differing flow rates (14, 30)

* Corresponding author.

and considerable day-to-day variation of individual salivaryimmunoglobulin concentrations (13).

In this study, anti-S. mutans antibodies of IgA and IgGclasses in serum and stimulated whole saliva were measuredin subjects with high- or low-caries susceptibility. Antibodyavidity may have a role in protection against caries (19, 20,31). Therefore, in this study, a modified enzyme-linkedimmunosorbent assay (ELISA) was employed, enabling usto perform independent estimations of high-avidity and totalantigen-specific antibodies (25, 26). In addition, salivary andserum total IgA and IgG were assayed. To evaluate thevalidity of anti-S. mutans antibodies, three unrelated anti-gens were also used in the antibody assays.

MATERIALS AND METHODSSubjects. The dental status of 290 recruits serving at Turku

Naval Base was examined to find two different groupsrepresenting high or low caries susceptibility. The high-caries-susceptibility group (HCS group), consisted of 12subjects with a high decayed, missing, or filled tooth index(mean + standard deviation, 21.4 ± 5.8) and numerousdentine carious lesions (7.1 + 5.7). Furthermore, paraffin-stimulated whole saliva samples from all HCS subjectsyielded 2105 CFU of aciduric bacteria per ml as determinedwith the Dentocult dip-slide test (Orion Diagnostica, Espoo,Finland) (17). The 12 subjects of the low-caries-susceptibilitygroup (LCS group) had a low decayed, missing, or filledtooth index (6.8 ± 2.4), no present dentine caries, and i104CFU of aciduric bacteria in their saliva. Visible plaque (1)was encountered in 7.2 ± 6.4 and 2.6 ± 3.2 teeth in the HCSgroup and the LCS group, respectively. Gingival bleedingindices (1) were 7.3 ± 5.5 and 3.1 + 3.0, correspondingly.The flow rate of paraffin-stimulated saliva was 1.3 + 0.4ml/min in the HCS group and 1.8 ± 0.5 ml/min in the LCS

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S. MUTANS ANTIBODIES AND DENTAL CARIES 309

group. All of the subjects were 18 to 22 years of age and hadbeen recently vaccinated against tetanus and parotitis. All ofthe subjects had received pertussis vaccination in theirchildhood.

Saliva and blood samples. Whole saliva samples werecollected between 9 and 11 a.m., at least 2 h after the lastmeal, in a quiet room. The subjects chewed a 5-g piece ofparaffin wax. The saliva secreted within the first 30 s wasswallowed and saliva was collected for the next 5 min. Thesaliva was immediately frozen and transported to the labora-tory. The saliva was inactivated by heating at 56°C for 30min, centrifuged at 1,000 x g for 30 min, and stored at-700C.Venous blood was drawn for serum samples after the

collection of the saliva. The sera were stored at -200C.Assay of total IgA and IgG in whole saliva. The total

amount of IgA and IgG in saliva was assayed by using a"trapping antibody"-type enzyme immunoassay. For IgAmeasurement, polystyrene microtiter plates (Linbro; FlowLaboratories, Inc., McLean, Va.) were coated by adding 75,ul of rabbit anti-human IgA (Dako-Immunoglobulins a/s,Copenhagen, Denmark) diluted 1:500 in bicarbonate buffer(pH 9.6) and incubated overnight at room temperature. Theplates were washed twice by adding 0.9% NaCI containing0.05% Tween 20 (NaCI-Tween) and once with phosphate-buffered saline (PBS), pH 7.4. The plates were incubated for1 h with PBS containing 1% normal sheep serum (NSS-PBS)and washed three times as described above. Thereafter, 75,ul of the saliva samples, diluted 1:10,000 in NSS-PBS wereincubated in duplicate on the anti-human IgA-coated platesfor 2 to 3 h at 37°C. After being washed, horseradishperoxidase-conjugated anti-human IgA (Dako-Immunoglob-ulins a/s) diluted 1:10,000 in NSS-PBS containing 5% poly-ethylene glycol 4000 was added, and the plates were in-cubated for 1.5 h at 37°C. After a threefold wash, 75 ,ul of1,2-phenylenediamine (3 mg/ml) in 0.1 M citrate buffer (pH5.6) containing 0.02% H202 was added and incubated for 15to 30 min. The incubation was terminated by adding 75 ,ul of1.0 M HCI. The absorbances were read by an automaticphotometer (Titertek Multiskan; Eflab Oy, Helsinki, Fin-land) at 492 nm. The human serum IgA standard (Behring-werke AG, Marburg, Federal Republic of Germany), 0.86mg/ml, was diluted from 1:103 to 1:107.The measurement of total IgG in saliva was accomplished

in a similar way; rabbit anti-human IgG and horseradishperoxidase-conjugated anti-human IgG (Dako-Immunoglob-ulins a/s) were used. Samples were incubated in PBS con-taining 0.5% bovine serum albumin (Miles Ltd., StokePoges, Slough, United Kingdom) and 0.5% Tween 20, in-stead of NSS-PBS. The human serum IgG standard (Beh-ringwerke AG), 0.34 mg/ml, was diluted from 1:103 to 1:107.

Assay of total IgA and IgG in serum. In serum samples thetotal concentrations of IgA and IgG were measured by singleradial immunodiffusion. This was performed by Tri-PartigenIgA and Tri-Partigen IgG (Behringwerke AG). Three stan-dards were used for each determination.

Antigens for ELISA. S. mutans KlR (ATCC 27351, sero-type g) and 10449 (ATCC 25175, serotype c) were grownanaerobically on brucella blood agar (Difco Laboratories,Detroit, Mich.) for 48 h. The colonies were scraped into PBScontaining 0.5% formaldehyde. The suspension was allowedto stand overnight at 4°C and was washed three times withPBS. The suspension was sonicated at 50 W for 2 min(Branson Sonifier B15; Branson Instruments Co., Stamford,Conn.).Tetanus toxoid and killed Bordetella pertussis were ob-

tained from the Central Public Health Laboratory, Helsinki,Finland. Parotitis virus was grown in African green monkeykidney cells, and the cell lysate was used as the antigen (28).ELISA. Specific antibodies were assayed by using ELISA.

The antigens were diluted in PBS for coupling onto polysty-rene microtiter plates (Linbro). In the coupling solution, theoptical density of the sonicated streptococcal suspensionswas 1.0 at 500 nm (Beckman Acta CII spectrophotometer).The concentrations of tetanus toxoid, B. pertlussis suspen-sion, and parotitis cell lysate protein were 9 ,ug/ml, 5 x 108bacteria per ml, and 5 ,ug/ml, respectively. The couplingsolution, 100 pA/well, was incubated overnight at 37°C. Theplates were washed twice with NaCl-Tween and once withPBS. Thereafter, 120 RI of NSS-PBS was added into eachwell, incubated for 1 h at 37°C, and washed as previouslydescribed.

Six dilutions of each sample in NSS-PBS were pipetted induplicate into the wells at 100 pA/well. Saliva samples werediluted from 1:20 on and serum samples from 1:60 on atthreefold intervals. The dilutions were incubated for 4 h at37°C. After being washed, 100 pA of alkaline phosphatase-conjugated anti-human IgG or IgA (Orion Diagnostica) dilut-ed 1:200 or 1:150, respectively, in NSS-PBS was added toeach well. After 12 to 16 h at room temperature and afterbeing washed, 100 pL of 1,2-nitrophenyl phosphate (OrionDiagnostica) in diethanolamine buffer (Orion Diagnostica)(pH 10.5) was added, and the plates were incubated at 37°Cfor 15 to 40 min. After termination of the reaction with 100 plAof 1.0 M NaOH, the absorbances of each well were mea-sured at 405 nm with the Titertek Multiskan.

All the assays of the samples for a certain antibody classand specificity were performed at the same time. A blankabsorbance obtained by substituting antibody sample withplain buffer was subtracted from all absorbances.

Interpretation of ELISA results. The dose-response curvesof each sample in ELISA were subjected to a curve-fittingprocedure (26). For each sample this gives two parameterswhich indicate in arbitrary units how much of the sampleantibodies can bind either in antibody or antigen excess.These parameters have been shown to correlate separatelywith the amounts of high-avidity and total antigen-specificantibodies (25, 26) and are thus estimates of high-avidityantibodies and total specific antibodies.

Statistics. The means were compared by the Student t test.In addition, the results from the assays of specific salivaryantibodies were subjected to an analysis of covariance inwhich the total salivary immunoglobulin concentrations ofeach subject were taken as covariates. The correlationbetween antibodies and the number of carious lesions wasstudied by Spearman's rank correlation analysis.

RESULTSThe total IgA and IgG concentrations in saliva and serum

of the two groups of subjects are presented in Table 1. Inserum immunoglobulins, there were no differences betweenthe groups. However, the saliva of subjects in the HCSgroup contained more IgA than that of subjects in the LCSgroup, P = 0.05. With IgG, there was a similar tendency, butno statistical significance.

Figure 1 depicts the mean ELISA dose-response curves ofthe two groups in the assay of salivary IgA antibodies againstS. mutans KMR. Saliva from the HCS group gave slightlyhigher mean absorbances than saliva from the LCS groupwhen the assay was performed in a low sample dilution (1:20or 1:60). However, the mean dose-response curve of theHCS group was steep, so that in higher dilutions (1:540,

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310 LEHTONEN ET AL.

TABLE 1. Total immunoglobulin concentration expressed asmean ± standard deviation

Concn (g/liter) in:Test Serum Salivagroup

IgG IgA IgG IgAbHCS 10.8 ± 3.4 1.64 ± 0.74 0.027 ± 0.038 0.180 ± 0.250LCS 10.6 ± 3.5 1.81 ± 1.22 0.004 ± 0.004 0.076 ± 0.030a n = 12 for both groups.b Significant difference by Student's t test, P = 0.05.

1:1,620, 1:4,860) the difference was reversed. The dose-response curves differed in shape, and this difference wastaken into account by computing the estimates for high-avidity antibodies and total specific antibodies.The results from assays of serum high-avidity antibodies

are shown in Table 2. The only significant difference be-tween the antibodies against the five antigens was that theHCS group had more high-avidity IgG antibodies againsttetanus toxoid than the LCS group.

Table 3 shows the amounts of serum total antigen-specificantibodies. There is only one significant difference; the LCSgroup had more high-avidity IgA antibodies against parotitisvirus antigen than the HCS group.

In saliva (Table 4), the HCS group had more high-avidityIgG antibodies against S. mutans 10449, S. mutans KMR, andtetanus toxoid, as well as high-avidity IgA antibodies againstS. mutans KlR and B. pertussis. Because of the differencebetween total salivary immunoglobulins (Table 1), totalsalivary IgA and IgG concentrations of each subject weretaken as covariates in the comparison of specific IgA andIgG antibodies. The results of this analysis of covariance arerepresented in addition to the Student t test in Tables 4 and5. After the analysis of covariance, no difference in high-avidity antibodies against the S. mutans strains was detect-ed. There were significant differences between the groups inhigh-avidity IgG antibodies against B. pertussis and high-avidity IgA antibodies against tetanus toxoid (Table 4).The LCS group had more total specific salivary IgA

antibodies against S. mutans KlR and 10449 and IgG anti-bodies against S. mutans KlR than the HCS group, andthese differences also remained after the analysis of covari-ance (Table 5). These antibodies did not correlate signifi-cantly to the number of carious lesions in the whole material(Spearman's correlation coefficients, -0.333, 0.210, and-0.225, respectively). No significant differences in antibod-ies against the unrelated antigens were noticeable after theanalysis of covariance.

DISCUSSIONMost of the studies have revealed a negative correlation

between decayed, missing, or filled score and serum anti-S.mutans antibodies, thus suggesting a protective role forthese antibodies (5-7, 16, 21). Serum antibody titers againstS. mutans are higher in caries-active individuals than inindividuals with low caries activity (15, 24). Serum antibod-ies against S. mutans decrease after treatment of caries (5).In the present study, the HCS group contained subjects withmany dentine lesions, and these subjects might have moreantibodies than they would have without these lesions.The HCS group had more IgA in whole saliva than the

LCS group. Several investigators have found more IgA inthe saliva of caries-resistant subjects than in that of caries-prone subjects (11, 19, 21, 30, 34), but in some studies therewere no significant differences (32, 35, 37). These results, in

contrast to the results of the present study, were obtainedwith radial immunodiffusion. This method underestimatessecretory IgA as compared with monomeric IgA (3). Theenzyme immunoassay used in the present work does notnecessarily detect secretory IgA with a poorer efficacy thanmonomeric IgA (9). The discrepancy between the presentresults and the literature could be explained by differentratios of monomeric to polymeric IgA in subjects with highor low caries susceptibility.The differences in the shapes of the ELISA dose-response

curves of salivary antibodies show that antibody samplesshould be titrated for a proper comparison. Otherwise,information about the qualitative differences of the antibodysamples might be lost. Contrasting results may be obtained ifonly one arbitrary dilution is used in the assay.On the basis of the different shapes of the dose-response

curves, the results are interpreted so that the HCS group hadmore high-avidity and less total specific antibodies against S.mutans than the LCS group. When the total immunoglobulinconcentrations were taken as covariates, it was revealed thatthe differences in high-avidity antibodies against S. mutanswere due to differences in total salivary immunoglobulinconcentrations. The analysis of covariance gave significantdifferences in high-avidity antibodies against tetanus toxoidand B. pertussis, but on the other hand, these antibodyestimates did not clearly differ without the analysis ofcovariance.There was both an absolute and a relative difference in the

amount of total specific salivary IgA antibodies against S.mutans KlR and 10449 and the amount of IgG antibodiesagainst strain KlR between the LCS and HCS groups. Thisindicates a protective role for IgA and possibly IgG antibod-ies in whole saliva against S. mutans. The gingival bleedingindices of the HCS and LCS groups, however, differed fromeach other, and this differing degree of local inflammationmight affect salivary antibodies (2). The large amount ofbacteria in the saliva of the individuals in the HCS groupmight also absorb antibodies to a larger extent than the salivaof those in the LCS group. Further, the current caries statusof the groups differed from each other. However, no signifi-

OD405nm

0*5

0.4

0.3

0.2

0.1

1 1 1 1 1 120 60 180 540 1620 4860

FIG. 1. Mean dose-response curves of the subjects with high (0)or low (0) caries susceptibility in ELISA of salivary IgA antibodiesagainst S. mutans K1R. Each point represents the mean of absor-bances obtained in samples from twelve subjects. Bars, standarddeviations. Abscissa, sample dilution in the assay. Ordinate, opticaldensity.

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VOL. 43, 1984 S. MUTANS ANTIBODIES AND DENTAL CARIES 311

TABLE 2. High-avidity antibodies in serum expressed as mean t standard deviation'

AntigenAntibody Test group"

nie

S. mutans KlR S. mutans 10449 Tetanus toxoid B. pertussis Parotitis virus

IgA HCS 0.55 ± 0.53 0.40 t 0.24 1.13 ± 0.77 1.36 t 0.99 0.29 t 0.13LCS 0.44 t 0.38 0.41 t 0.17 0.96 t 0.50 0.94 t 0.56 0.35 t 0.25

IgG HCS 1.87 t 0.71 1.35 t 0.34 0.77 t 0.15 0.88 t 0.32 0.42 ± 0.17LCS 1.55 ± 0.54 1.29 t 0.25 0.65 t 0.20' 0.79 ± 0.39 0.54 ± 0.22

"Antibodies expressed as the estimates of high-avidity antibodies (arbitrary units). Note that the antibody estimates against differentantigens are not comparable to each other.

b n = 12 for both groups.Significant difference by Student's t test between the HCS and the LCS groups for IgG antibodies to tetanus toxoid, P = 0.025.

TABLE 3. Total specific antibodies in serum expressed as mean t standard deviation'

AntigenAntibody Test grouph S. muitans KlR S. muitatis 10449 Tetanus toxoid B. pertiussis Parotitis virusIgA HCS 2.72 t 0.37 2.59 t 0.27 2.17 t 0.22 2.89 ± 0.23 2.78 t 0.57

LCS 2.74 t 0.26 2.58 t 0.42 2.17 t 0.32 2.04 ± 0.22' 3.21 t 0.58

IgG HCS 2.51 t 0.16 3.28 t 0.31 3.58 t 0.44 2.76 t 0.37 2.76 t 0.37LCS 2.31 t 0.36 3.31 t 0.33 3.65 t 0.30 2.85 t 0.28 2.59 t 0.44

a Antibodies expressed as the log 10 of the estimates of total specific antibodies (arbitrary units). Note that the antibody estimates againstdifferent antigens are not comparable to each other.

b n = 12 for both groups.'*Significant difference by Student's t test between the HCS and the LCS groups for IgA antibodies to B. pertussis, P = 0.02.

TABLE 4. High-avidity antibodies in whole saliva expressed as mean ± standard deviation"Antigen

Antibody Test group"nie

S. mutans KlR S. mutans 10449 Tetanus toxoid B. pertussis Parotitis virus

IgA HCS 0.48 ± 0.13 0.39 ± 0.42 0.12 ± 0.13 0.12 ± 0.11 0.16 ± 0.12LCS 0.37 ± 0.10' 0.22 ± 0.23 0.09 ± 0.04" 0.08 ± 0.03 0.10 ± 0.15

IgG HCS 0.28 ± 0.14 0.19 ± 0.09 0.20 ± 0.13 0.08 ± 0.03 0.07 t 0.04LCS 0.17 ± 0.05e 0.07 ± 0.04f 0.11 ± 0.07k 0.07 ± 0.04" 0.07 t 0.02

aAntibodies expressed as the estimates of high-avidity antibodies (arbitrary units). Note that the antibody estimates against differentantigens are not comparable to each other.

n = 12 for both groups.Significant difference by Student's t test between the HCS and the LCS groups for IgA antibodies to S. mutans KMR, P = 0.01.

d The total immunoglobulin A or G concentrations in saliva were taken as covariates in the analysis of covariance. P = 0.05.e As in footnote c except for IgG antibody, P = 0.005.f As in footnote c except for IgG antibody to S. mutans 10449, P = 0.02.g As in footnote c except for IgG antibody to tetanus toxoid, P = 0.01.h As in footnote d except for IgG antibody to B. pertussis, P = 0.05.

TABLE 5. Total specific antibodies in whole saliva expressed as mean ± standard deviation"Antigen

Antibody Test grouph S. mnutans KlR S. mutans 10449 Tetanus toxoid B. pertiussis Parotitis virusIgA HCS 3.10 t 0.20 2.04 ± 0.10 3.40 ± 0.58 3.10 t 0.58 1.82 ± 0.28

LCS 3.45 ± 0.32' 2.18 ± 0.55 3.37 ± 0.97 3.24 ± 0.74 1.95 ± 0.73

IgG HCS 2.90 ± 0.51 4.38 ± 0.63 3.51 ± 1.18 4.12 ± 0.79 3.08 ± 1.25LCS 3.42 ± 0.42' 3.93 ± 0.53f 3.96 ± 0.27 4.02 ± 1.86 3.40 ± 0.68

a Antibodies expressed as the log 10 of the estimates of total specific antibodies (arbitrary units). Note that the antibody estimates againstdifferent antigens are not comparable to each other.bn = 12 for both groups.' Significant difference by Student's t test, P = 0.001. Significant difference by analysis of covariance as described in Table 4, footnote d, P

= 0.05.d Significant difference by analysis of covariance as described in Table 4, footnote d, P = 0.05.

Significant difference by Student's t test and analysis of covariance as described in Table 4, footnote d. P = 0.001 and P = 0.05,respectively.f Significant difference by Student's t test, P = 0.02.

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312 LEHTONEN ET AL.

cant correlation was observed between the above-mentionedsalivary antibodies and the individual number of cariouslesions.The HCS group had a lower level of total specific antibod-

ies against S. miutans than the LCS group, but on the otherhand, the HCS group had more high-avidity antibodies whenno comparison to the total salivary immunoglobulins wasperformed. This means that the HCS group had a greaterproportion of high-avidity antibodies than the LCS group.This difference in the avidity distributions of the antibodiesmight reflect a more established state of immunization in theHCS than in the LCS group (36), perhaps due to differencesin the ecology of S. mutans or in the disease history of thegroups.We could not demonstrate significant differences in the

estimates of serum high-avidity anti-S. mutans antibodiesbetween the HCS and LCS groups. This suggests thatdifferences between individual immunoreactivities against S.mutans are not due to differing B-cell repertoires. Thedifferences could merely be due to the regulation of themagnitude of antibody production. Recently, T helper cellshave been shown to have dose dependency in the responseagainst S. mutans antigen, and this dependency is linked toHLA-DR antigen (20). However, it should be rememberedthat the antigen in the present study was crude, containingan undefined number of antigenic determinants. Furtherstudies should be performed with purified antigens, enablingan exact avidity (affinity) determination.The present study suggests that antibodies in whole saliva

have a role in the control of caries. In previous studies, thismay have been unnoticed because of methodological diffi-culties in the measurement of salivary antibodies. Thus,measurement of secretory immunity deserves attention inthe development of vaccination against dental caries.

ACKNOWLEDGMENTS

We thank Erkki Nieminen for excellent technical assistance.Parotitis virus antigen was a generous gift from Olli Meurman,Department of Virology, Turku University. Constructive criticismby Jorma Tenovuo, Institute of Dentistry, Turku University, isgratefully acknowledged.

This study is one of the research projects of The Naval ResearchUnit aimed at developing the health care of recruits and was alsosupported by The Finnish Dental Society.

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