Vol. 55, No. 10INFECTION AND IMMUNITY, OCt. 1987, p. 2341-23470019-9567/87/102341-07$02.00/0Copyright © 1987, American Society for Microbiology

Protection of Gnotobiotic Rats against Dental Caries by PassiveImmunization with Bovine Milk Antibodies to Streptococcus mutans

SUZANNE M. MICHALEK,* RICHARD L. GREGORY,t CECILY C. HARMON, JENNY KATZ,GLORIA J. RICHARDSON, TRACY HILTON, STEVEN J. FILLER, AND JERRY R. McGHEE

Department of Microbiology, Institute ofDental Research, and School ofDentistry, University ofAlabama atBirmingham, University Station, Birmingham, Alabama 35294

Received 24 February 1987/Accepted 19 June 1987

A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, dand g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animalscontained immunoglobulin Gl (IgGl) antibody activity to seven serotypes (a to g) of mutans streptococci. Wheyobtained from the animal with the highest serum antibody activity, which also contained high levels of IgGlantibody, was used in passive caries immunity studies. Gnotobiotic rats monoinfected with Streptococcusmutans MT8148 serotype c or Streptococcus sobrinus OMZ176 (d) or 6715 (g) and provided a caries-promotingdiet containing immune whey had lower plaque scores, numbers of streptococci in plaque, and degree of cariesactivity than similarly infected animals given a diet containing control whey obtained from nonimmunizedcows. To establish the nature of the protective component(s) present in the immune whey, an ultrafiltratefraction of the whey was prepared. This preparation contained higher levels of IgGl anti-S. mutans antibodyactivity than the immune whey. Rats monoinfected with S. mutans MT8148 and provided with a dietsupplemented with 0.1% of this fraction exhibited a degree of caries protection similar to that seen in animalsprovided a diet containing 100% immune whey. In fact, a diet containing as little as 0.01% of the ultrafiltratefraction gave some degree of protection against oral S. mutans infection. The active component in the immunewhey was the IgGl anti-S. mutans antibody, since rats monoinfected with S. mutans MT8148 and provided adiet supplemented with purified immune whey IgGl had significantly reduced plaque scores, numbers of S.mutans in plaque, and caries activity compared with control animals. Prior adsorption of the IgG fraction withkilled S. mutans MT8148 whole ceUs removed antibody activity and abrogated caries protection.

The demonstration that Streptococcus mutans serotype cis a major etiologic agent of human dental caries (4, 16, 17)has led to investigations aimed at developing a means forprotecting the host against this infectious disease. Extensivestudies in experimental rodent models have shown thatactive immunization with S. mutans antigen, either by localinjection in the region of the salivary glands (22, 23, 35, 38)or by oral administration (25, 26, 28, 36), results in theinduction of high levels of specific salivary antibodies whichcorrelate with protection against caries lesion formation (seereference 21 for a review). Other studies (13-15) have shownthat systemic immunization of primates with S. mutansantigens results in significant antibody activity, especially ofthe immunoglobulin G (IgG) isotype, in serum and crevicularfluid and protection against dental caries. All of these studiesdemonstrate that active immunization with S. mutans anti-gens is an effective method for inducing antibody responseswhich protect against experimental dental caries. Otherstudies in rats (22, 24), primates (11, 12), and humans (18)have shown that passively derived serum (12, 22) or murinemonoclonal IgG (11, 18) or colostrum and milk IgA (22, 24)antibodies to S. mutans can also confer protection against S.mutans colonization and caries lesion formation.The concept of protecting a host with passively derived

antibodies is not new (reviewed in references 2 and 32), andperhaps the best source of passive antibodies other thanthose derived from the mother would be from bovine milk, anutrient available to most of the human population. In cattle,

* Corresponding author.t Present address: Department of Oral Biology, Emory Univer-

sity School of Dentistry, Atlanta, GA 30322.

serum IgG consists of two subclasses, IgGl and IgG2, inapproximately equal amounts, with lesser amounts of IgAand IgM (reviewed in reference 6). Bovine colostrum con-tains predominantly IgGl subclass antibodies which areserum derived and secretory IgA and IgM. Milk containsmuch lower immunoglobulin levels than colostrum; how-ever, the proportion of isotypes is similar, with the majorfraction being of the IgGl subclass. Thus, hyperimmuniza-tion of pregnant heifers results in the development of highserum antibody levels of the IgGl subclass which subse-quently transudate into milk (6).

Several studies have reported the protection of newborncalves by vaccination of the pregnant mother with organismsthat cause severe gastrointestinal disease. When pregnantcows were immunized with a K99 extract derived fromEscherichia coli K-12 (30) or with purified K99 pili (1),significant colostral antibody titers were induced and suck-ling calves were protected against challenge with K99+enteropathogenic E. coli. Furthermore, colostrum fromcows immunized with rotavirus which contained significantantiviral activity protected newborn calves from challenge,whereas calves fed control colostrum developed severediarrheal disease (34). Others have shown that bovinecolostrum with significant antibody titers to rotavirus alsoprotected piglets from this disease (5). These studies andothers (20, 40) clearly indicate that bovine colostral antibod-ies survive proteolytic digestion during passage through theintestine and function in the milieu to protect the sucklingfrom disease.

In the present study, we provide evidence that systemicimmunization of cows with a mutans streptococcal wholecell vaccine results in the appearance of IgGl antibody

2341

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2342 MICHALEK ET AL.

activity in both serum and whey. This immune whey, whenadded to a caries-promoting diet and provided to gnotobioticrats moninfected with virulent S. mutans, conferred protec-tion against dental caries formation. Systematic analysis offractionated immune whey demonstrated that the IgGl anti-S. mutans antibody was the protective component.

MATERIALS AND METHODS

Bovine milk products. The bovine whey and serum sam-ples used in this study were kindly provided by the StolleResearch and Development Corp. (Cincinnati, Ohio). Briefdescriptions of the vaccine preparation, immunization pro-tocol, and methods for processing the bovine milk are givenbelow.

Vaccine preparation. Seven strains of mutans streptococ-ci, which were obtained from the American Type CultureCollection (Rockville, Md.) (31377 [serotype b], 10449 [c],31341 [c], 27607 [d], 27947 [d], 27351 [g], and 27352 [g]), wereused to prepare a multivalent vaccine (file IND BB-IND 857;Stolle Research and Development Corp., Cincinnati, Ohio).Each bacterium was cultured separately in Todd-Hewittbroth (Difco Laboratories, Detroit, Mich.) for 24 h at 37°C ina 5% C02-in-air atmosphere. The bacteria were killed byheating for 30 min at 60°C, harvested (10,000 x g, 15 min),and washed five times with sterile saline and three times withsterile distilled water. The bacteria were lyophilized, and anequal weight of each of the seven bacterial strains wascombined and suspended in sterile saline to a final concen-tration of 108 equivalent CFU/ml. Inoculation of the suspen-sion on mitis salivarius agar (Difco) did not reveal thepresence of viable streptococci.

Immunization procedure. Pregnant cows were injectedwith 5 ml of the multivalent mutans streptococcus vaccine inthe gluteus maximus muscle of the hind leg. This procedurewas repeated at 2-week intervals beginning 3 to 4 weeksbefore the predicted day of parturition and continuedthroughout the period that the cow gave milk. For thestudies reported here, milk was collected and processedaccording to routine dairy procedures. Control milk wascollected from cows in the same herd that had not beenimmunized with the streptococcal vaccine. Pre- and postim-munization serum samples were also obtained from the samecows and stored frozen for analysis of antibody activity asdescribed below.

Bovine whey and concentrated whey fractions. Fresh wholemilk was centrifuged (14,000 x g, 30 min) to remove the fatlayer. The skim milk was then warmed to 40°C, adjusted topH 4.6 with 5 N HCl, and passed through cheesecloth toremove the casein. The whey was then adjusted to pH 7.0with 5 N NaOH and either spray dried and stored at 4°C untilreconstituted for use or further purified. The ultrafiltratefraction was prepared by passing the whey through anAmicon membrane (Amicon Corp., Danvers, Mass.) with a100,000-molecular-weight cutoff, which separated the immu-noglobulins and other high-molecular-weight proteins fromlactalbumin. This fraction was lyophilized and stored at 4°Cuntil used.

Purification of bovine whey IgGI. Purified IgGl from eitherimmune or control whey was obtained by ion-exchangechromatography by a modification of a previously describedmethod (6). The ultrafiltrate whey fraction was reconstitutedin 0.01 M phosphate buffer (pH 7.4) and applied to aDEAE-cellulose column (5 by 50 cm, DE-52; Whatman Ltd.,Maidstone, Kent, England) equilibrated with the samebuffer. The column was eluted stepwise, and fractions were

monitored for protein by spectrophotometric analysis at 280nm and for IgGl by Ouchterlony and immunoelectrophoreticanalysis with rabbit antibodies specific for bovine IgG heavychain and for bovine IgGl (Nordic Immunology, Tilburg,The Netherlands). The IgGl-rich fractions (eluting with 0.1M NaCI) were pooled, concentrated, dialyzed against 0.01 Mphosphate buffer (pH 5.5), and applied to a CM-50 Sephadexcolumn (5 by 50 cm) previously equilibrated with the samebuffer. The fall-through peak consisted of pure IgGl asdetermined by Ouchterlony and immunoelectrophoreticanalysis. Similar amounts of IgGl were recovered from theimmune and control whey.

In one series of experiments, a portion of the immuneIgGl was adsorbed with Formalin-killed S. mutans MT8148(serotype c) whole cells. Briefly, packed S. mutans wholecells (2 ml) were mixed with purified IgGl (20-ml volumes)and incubated with rotation for 2 h at 25°C and thenovernight at 4°C. The cells were removed by centrifugation(12,000 x g, 30 min), and the supernatant was collected andfilter sterilized. Both the pre- and postadsorbed IgGl frac-tions were tested for anti-S. mutans antibody activity byenzyme-linked immunosorbent assay (ELISA) (see below).ELISA. Bovine whey, purified whey fractions, and serum

samples were assayed for IgGl antibody activity againstrepresentative strains of the seven serotypes (a through g) ofmutans streptococci (3, 7, 33) by an indirect ELISA. Wholecell antigen of S. mutans MT8148 (serotype c), LM7 (e), andOMZ175 (f); Streptococcus rattus BHT (b); Streptococcuscricetus AHT (a); and Streptococcus sobrinus OMZ176 (d)and 6715 (g) were prepared as previously described (9, 28)and adjusted to an absorbance of 0.660 at 660 nm in 0.1 Mcarbonate buffer (pH 9.6). Individual wells of Linbro flat-bottom enzyme immunoassay plates (Flow Laboratories,McLean, Va.) were coated with whole cell antigens byadding 100 ,ul of the antigen per well and incubating platesfor 3 h at 37°C and then overnight at 4°C. Unbound antigenwas removed by washing wells three times with salinecontaining 0.05% Tween 20 (Sigma Chemical Co., St. Louis,Mo.; Tween-saline), and 200 pul of a solution containing 10,ug of globulin-free human serum albumin (Sigma) per ml inTween-saline was added to each well. Plates were thenincubated at room temperature for 1.5 h to block unreactedsites. After wells were washed, various dilutions of whey,purified whey fractions, or serum in Tween-saline wereadded to the wells, and plates were incubated for 1 h at 37°Cand then washed three times. Rabbit IgG anti-bovine IgGlheavy-chain antibody was added (100 plI), and the plateswere incubated at 37°C for 1 h. The wells were washed andalkaline phosphatase (Sigma)-labeled goat IgG anti-rabbitIgG chain reagent (Behring Diagnostics, American HoechstCorp., Somerville, N.J.) was added (100 ,ul) to each well andincubated at 37°C for 3 h and then overnight at 4°C. Afterwells were washed three times, p-nitrophenyl-phosphate(Sigma) in 10% diethanolamine (Sigma) buffer (1 mg/ml, pH9.6) was added (100 ,ul/well), and the amount of color whichdeveloped after 1.5 h was read at 405 nm with a TitertekMultiskan photometer (Flow).

Rats. Germfree Fischer [CDF(F-344)/Crl BR] rats (originalbreeding colony was obtained from Charles River BreedingLaboratories, Wilmington, Mass.) were bred and maintainedin Trexler plastic isolators (27, 39) at The University ofAlabama at Birmingham Gnotobiotic Rat Facility. Adultbreeding rats were provided autoclaved Wayne Lab-Bloxsterilizable, fluoride-free rat chow (Allied Mills, Inc., Chi-cago, Ill.) and sterile, distilled drinking water ad libitum. Onthe day of parturition, all litters were reduced to eight pups

INFECT. IMMUN.

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

BOVINE IgG ANTIBODIES PROTECT AGAINST DENTAL CARIES 2343

TABLE 1. IgGl antibody activities to seven serotypes of mutans streptococci in sera from five cows immunized with themultivalent vaccine

Bacterial Serum A405 of serum from animal no.:strain erlumio

(serotype) dilution 1831 2078 2199 2217 2286

AHT (a) 1/2,000 <0.150 0.214 ± 0.003 0.276 ± 0.010 0.171 ± 0.063 0.277 ± 0.031BHT (b) 1/3,000 0.856 ± 0.045 0.964 ± 0.065 0.571 ± 0.015 0.356 ± 0.017 0.742 ± 0.052MT8148 (c) 1/2,000 0.216 ± 0.011 0.314 ± 0.012 0.191 ± 0.009 <0.100 0.274 ± 0.014OMZ176 (d) 1/2,000 0.837 ± 0.071 0.947 ± 0.063 0.646 ± 0.024 0.682 ± 0.037 0.747 ± 0.053LM7 (e) 1/1,000 0.215 ± 0.015 0.335 ± 0.021 0.243 ± 0.019 0.295 ± 0.021 0.289 ± 0.017OMZ175 (f) 1/2,000 0.371 ± 0.016 0.834 ± 0.110 0.427 ± 0.011 0.323 ± 0.060 0.643 ± 0.0276715 (g) 1/1,000 0.345 ± 0.021 0.565 ± 0.032 0.201 ± 0.008 <0.100 0.245 ± 0.016

a Values are the means ± standard errors of the absorbance at 405 nm on quadruplicate determinations of diluted serum samples from individual cows. Serumsamples (1/50 dilution) from control cows had absorbance values of less than 0.1 to the seven bacterial strains.

per dam. At weaning (19 days of age), all pups to beemployed in the experiments were divided at random, trans-ferred to experimental isolators, and provided caries-promoting diet 305 (27) containing whey or purified wheyfractions from immunized or control cows (see below).

Experimental design. In our initial studies, weanlinggermfree rats (age 19 days) were divided into groups andprovided diet 305 containing bovine whey. The lyophilizedbovine whey was added to the diet to substitute for thedietary protein lactalbumin (20% by weight of diet). Sinceapproximately one-third of the whey product consists ofprotein, the remaining whey constituent (40% by weight ofdiet) was substituted for cornstarch. All other ingredientswere exactly as previously described (27, 31). In the studieswith purified whey fractions, these products were substi-tuted for lactalbumin on a basis of percentage by weight ofthe total dietary ingredients (27).

In all experiments, rats were challenged orally at 19 daysof age with an 18-h brain heart infusion broth (Difco) cultureof S. mutans MT8148 or S. sobrinus OMZ176 or 6715(approximately 5 x 106 CFU) with the aid of an automaticpipetter and were provided the appropriate diet 305 adlibitum. At 45 days of age, rats were removed from theisolators, weighed individually, and sacrificed by injection ofsodium nembutal. Both mandibles from each rat were asep-

tically removed and defleshed. One mandible was used forenumerating the number of viable S. mutans or S. sobrinuspresent, and the other was stained with safranin and scoredfor plaque as described previously (23, 27). Both mandibleswere then stained with murexide and hemisectioned with theaid of a dental drill, and buccal, sulcal, and proximal carieslesions were scored by the method of Keyes (10) as reportedpreviously (23, 27).

Statistics. The caries scores, numbers of S. mutans inmolar plaque, and levels of IgGl antibody in bovine whey,purified whey fractions, and bovine serum were statisticallyreduced by computing means and standard errors of themean. Differences among means were evaluated by an

analysis of variance and by multiple-mean comparisons withthe Duncan test (8). Values of the individual and pooledbovine whey, purified whey fractions, and serum samplesare expressed as the mean + standard error of the mean ofthe absorbance at 405 nm after control background absorb-ance was subtracted. The values represent the mean ofquadruplicate determinations per sample.

RESULTS

Analysis of bovine serum and whey IgGl antibody levels.All five cows injected with the multivalent vaccine devel-

oped good serum IgGl antibody responses to whole cells ofrepresentative strains of seven different serotypes of mutansstreptococci (serotypes a through g), as determined byELISA (Table 1). In general, serum from cow 2078 exhibitedsomewhat higher levels of antibody activity to the seven

serotypes of mutans streptococci than was observed inserum samples from the other four cows, although allanimals responded well to the vaccine. A similar pattern ofIgGl antibody activity was observed in whey samples fromthese animals and the antibody levels in immune whey fromcow 2078 (Table 2). Control whey had essentially no anti-body activity to the seven serotypes tested, although a lowamount of activity to a serotype d strain of S. sobrinus was

observed when a 1/50 dilution of whey sample was assayed(Table 2). All subsequent in vivo rat studies employed theseimmune and control wheys.

Effect of bovine immune whey on protection of gnotobioticrats against S. mutans- and S. sobrinus-induced dental caries.Gnotobiotic rats provided diets containing various amountsof immune whey and challenged with S. mutans MT8148(serotype c) had lower plaque scores, numbers of S. mutansin plaque, and caries scores than animals challenged with S.mutans and provided a diet containing only control whey(group D, Table 3). A diet containing as little as 10% immunewhey provided significant protection against buccal andproximal lesion (P s 0.01) and sulcal lesion (P < 0.05)formation in rats monoassociated with S. mutans (group C,Table 3). These results were not due to a nutritional effect,since no significant difference was observed in the meanbody weights of rats in the four different groups (Table 3).The bovine immune whey was also effective in protecting

rats from infection with S. sobrinus OMZ176 (serotype d)and 6715 (serotype g) (Tables 4 and 5). Rats provided diet

TABLE 2. IgGl antibody activities to seven serotypes of mutansstreptococci in immune and control wheya

Bacterial strain A45(serotype) Immune whey Control whey

AHT (a) 0.063 + 0.001 0.001 ± 0.001BHT (b) 0.412 ± 0.037 0.016 ± 0.006MT8148 (c) 0.137 ± 0.012 0.004 ± 0.001OMZ176 (d) 0.323±+ 0.075 0.088 ± 0.003LM-7 (e) 0.083 ± 0.013 0.024 ± 0.006OMZ175 (f) 0.371 ± 0.046 0.022 ± 0.0176715 (g) 0.175 ± 0.023 0.025 ± 0.005a Immune whey (derived from animal 2078) and control whey samples (100

mg/ml of 0.01 M phosphate buffer [pH 7.4]) were diluted (immune 1/1000,control 1/50) for use in the ELISA. Values are the means + standard errors ofthe absorbance at 405 nm on quadruplicate determinations.

VOL. SS, 1987

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2344 MICHALEK ET AL.

TABLE 3. Effect of immune whey on protection of gnotobiotic rats monoassociated with S. mutans MT8148 (serotype c)

No. of Caries scoredgroup (immune! Plaque CFU/ Buccal Sulcal Proximal boywaruimue

scoreb mandible'coywcontrol whey)' (106) Enamel Dentinal Enamel slighta Enamel setnlghtslightslgtsihA (100/0) 2.1 (76) 0.7 ± 0.2 0.8 ± 0.4 0.1 ± 0.1 10.8 ± 0.6 4.3 ± 0.6 0.0 (100) 0.0 (100) 111 ± 7

(92) (94) (99) (35) (66)B (33/67) 2.9 (67) 1.0 ± 0.2 3.2 ± 1.0 0.6 ± 0.3 14.2 ± 1.2 8.7 ± 1.3 1.0 ± 0.4 0.0 (100) 124 ± 12

(90) (77) (95) (14) (32) (56)C (10/90) 3.9 (56) 2.8 ± 0.3 5.4 ± 0.9 3.2 ± 0.4 14.9 ± 0.3 10.3 ± 0.2 1.4 ± 0.5 0.0 (100) 118 ± 8

(70) (61) (71) (10) (20) (39)D (0/100) 8.9 9.2 ± 0.6 13.9 ± 0.4 11.1 ± 0.3 16.6 ± 0.7 12.8 ± 0.6 2.3 ± 0.7 0.9 ± 0.4 117 ± 7

a Values within parentheses are the percent immune versus control whey in the diet. Each group contained seven rats.bValues are the mean plaque scores derived from one mandible per rat. Numbers within parentheses are the percent decrease in the mean plaque score of the

experimental groups (A through C) compared with the control group (D).c Values are the means ± standard errors of the number of mutans streptococci CFU per mandible of duplicate determinations per sample as determined on

mitis salivarius agar. Numbers within parentheses are the percent decrease in the mean number of viable S. mutans of the experimental groups (A through C)compared with the control group (D).

d Values are the mean caries scores ± standard error. Numbers within parentheses are the percent decrease in the level of lesions in experimental animals(groups A through C) compared with control animals (group D).

containing 10% immune whey (group C) and challenged withS. sobrinus OMZ176 (Table 4) or 6715 (Table 5) exhibitedsignificantly lower (P c 0.05) plaque scores, numbers of S.mutans in plaque, and buccal caries scores than the valuesobtained in infected animals which received diet containingonly control whey (group D, Tables 4 and 5). These resultsclearly indicate that the bovine immune whey, when pro-vided in diet to gnotobiotic rats, was effective in protectingthese animals from infection with virulent serotype c, d, or gstrains of mutans streptococci.

Effect of a bovine immune whey fraction on protection ofgnotobiotic rats against S. mutans-induced dental caries. Toestablish the nature of the component(s) present in immunewhey which conferred protection against S. mutans infec-tion, whey protein and IgGl were purified and tested in ourgnotobiotic rat model. A diet containing 1% (group A) or0.1% (group B) of the immune whey ultrafiltrate (Table 6)was effective in significantly (P c 0.01) reducing plaquescores, numbers of S. mutans in plaque, and caries scores ofgnotobiotic rats infected with S. mutans MT8148 comparedwith the values obtained with infected animals providedunsupplemented diet 305 (group D, Table 6). Animals pro-vided diet containing only 0.01% of the immune wheyultrafiltrate (group C) showed reduced plaque and S. mutanslevels; however, the degree of caries protection was lessthan that seen in animals given diet containing 10- or 100-foldhigher levels of this ultrafiltrate fraction (Table 6).

Evidence that IgGl anti-S. mutans antibodies effect cariesimmunity. The major component in immune whey responsi-ble for caries protection appears to be IgGl anti-S. mutansantibody. This conclusion is based upon evidence providedin Table 7, which shows that purified IgGl from immunewhey, with anti-S. mutans antibody activity, conferredcaries immunity. Rats provided normal purified whey IgGl(group C) exhibited caries scores which were slightly lower,but not significantly different from those obtained in rats thatreceived normal caries-promoting diet (group D). Moredefinitive evidence for IgGl antibody-induced immunity wasprovided by the demonstration that removal of specificantibodies from the immune whey IgGl by adsorption withS. mutans MT8148 abrogated the caries protection (groupB), which was noted with unadsorbed IgGl antibodies(group A, Table 7).

DISCUSSIONPreviously we showed that whey from cows immunized

with a vaccine consisting of whole cells of serotype a, b, andg strains of mutans streptococci, when provided to ratschallenged with a mixture of virulent homologous microor-ganisms, protected these animals from dental caries forma-tion (25). The present study was designed to establish theeffectiveness of immune bovine whey from cows immunizedwith a mutans streptococcal vaccine in protecting the hostagainst infection with virulent mutans streptococci, espe-

TABLE 4. Effect of immune whey on protection of gnotobiotic rats monoassociated with S. sobrinus OMZ176 (serotype d)

Experimental No. of CrescedMeangroup (immune! Plaque CFU/ Buccal Sulcal Proximal boywcontrol whey)' scoentandbae Dentinal Dentinal (g)(105) Enamel Dentia Enamel Enamel

slight Easlight slightA (100/0) 2.0 (76) 1.6 + 0.3 6.3 ± 0.7 4.8 ± 0.7 15.1 ± 0.5 11.0 ± 0.4 0.0 (100) 0.0 (100) 113 ± 5

(80) (57) (34) (15) (12)B (33/67) 4.2 (49) 2.4 ± 0.6 8.5 ± 0.6 5.3 ± 0.4 16.9 ± 0.4 11.6 ± 0.5 1.5 ± 0.5 0.0 (100) 115 ± 7

(70) (42) (27) (5) (7) (50)C (10/90) 5.1 (39) 5.4 ± 0.7 9.6 ± 0.4 6.0 ± 0.2 18.0 ± 0.4 12.4 ± 0.2 2.6 ± 0.7 0.0 (100) 121 ± 5

(32) (35) (18) (- 1) (0.8) (13)D (0/100) 8.3 7.9 ± 0.8 14.7 t 0.2 7.3 ± 0.6 17.8 ± 0.7 12.5 ± 0.2 3.0 ± 0.7 1.9 ± 0.5 122 ± 6

a See Table 3, footnote a. Each group contained seven rats.b See Table 3, footnote b.c See Table 3, footnote c.d See Table 3, footnote d.

INFECT. IMMUN.

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

BOVINE IgG ANTIBODIES PROTECT AGAINST DENTAL CARIES 2345

TABLE 5. Effect of immune whey on protection of gnotobiotic rats monoassociated with S. sobrinus 6715 (serotype g)Experimental No. of Caries scored

(immune! Plaque CFU/ Buccal Sulcal Proximal bean(immune/ scoreb mandible' oywcontrol sdle6 Dentinal Dentinal Dentinal (g)whey)' (16 Enamel slight Enamel slight Enamel slight

A (100/0) 4.2 (70) 1.3 ± 0.3 9.6 ± 0.6 6.4 + 0.8 15.6 t 0.6 11.8 ± 0.4 0.0 (100) 0.0 (100) 125 ± 7(89) (44) (56) (24) (25)

B (33/67) 6.5 (54) 3.1 + 0.7 12.6 ± 0.6 9.1 ± 0.6 18.4 ± 0.5 12.6 ± 0.2 0.3 + 0.3 0.0 (100) 122 ± 7(74) (26) (37) (11) (20) (92)

C (10/90) 8.7 (39) 7.8 ± 0.9 14.0 ± 0.4 10.6 ± 0.6 19.3 ± 0.6 13.1 ± 0.4 2.7 ± 0.4 0.6 ± 0.4 116 ± 5(35) (18) (26) (6) (17) (32) (70)

D (0/100) 14.2 1.2 ± 0.1 17.1 ± 0.5 14.4 ± 0.4 20.6 ± 0.7 15.7 ± 0.4 4.0 t 0.3 2.0 ± 0.3 128 ± 8

a See Table 3, footnote a. Each group contained seven rats.b See Table 3, footnote b.See Table 3, footnote c.

d See Table 3, footnote d.

cially S. mutans serotype c, and to determine the nature ofthe component(s) present in the immune bovine whey im-portant in caries immunity.

Systemic immunization of cows with a vaccine composedof whole cells of seven strains of mutans streptococci,representing serotypes b, c, d, and g, resulted in the induc-tion of serum IgG antibody activity to the seven serotypes (athrough g) of mutans streptococci (data not shown), whichwas primarily of the IgGl subclass (Table 1). When wheyfrom these cows was assessed for antibody levels to theseven serotypes of mutans streptococci, the pattern of IgGlantibody activity was similar to that seen in serum (Table 2).These results are consistent with previous findings of others(6), which have shown that bovine IgGl is transported fromserum into colostrum and milk and represents the majorimmunoglobulin isotype found in this external secretion.To determine the effectiveness of the immune bovine

whey in protection against dental caries formation, variousamounts of the whey were substituted into a caries-promoting diet and provided to gnotobiotic rats monoinfec-ted with a virulent serotype c, d, or g strain of mutansstreptococci. In all cases, rats provided diet containingimmune whey were protected against infection and subse-quent caries formation (Tables 3 through 5). In fact, a dietcontaining only 6% immune whey conferred caries immunityto rats monoinfected with a virulent serotype c strain of S.

mutans (Table 3). These results extend our previous findings(25) and provide additional evidence that whey from cowsimmunized with S. mutans antigen would be an effectivemeans of passively protecting against dental caries.

Previous investigations by others have reported the poten-tial of bovine whey for passive protection against gastroin-testinal infections. It has been shown that calves providedwhey from cows immunized with a crude K99 extract (30) or

with purified K99 pili or whole cell bacterins (1) wereprotected from challenge with an enterotoxigenic E. coli. Inthe latter study (1), the extent of protection was correlatedwith the level of anti-K99 colostral antibody. Other studieshave shown that piglets provided bovine whey containingrotavirus-neutralizing activity were protected against intra-nasal inoculation with a porcine rotavirus, whereas controlpiglets developed diarrhea, excreted virus, and died within 6days after infection (5).To determine the nature of the whey component(s) which

provided caries immunity, an ultrafiltrate fraction was pre-pared and tested in the gnotobiotic rat model. This prepara-tion contained IgGl anti-S. mutans serotype c antibodyactivity and provided caries immunity to rats when as littleas 0.01% was added to the caries-promoting diet (Table 6).These results suggest that the IgGl antibodies were impor-tant in protection, which would be in agreement with thestudies of others (34, 37) demonstrating that the ability of

TABLE 6. Effect of an ultrafiltrate fraction of immune whey on protection of gnotobiotic rats monoassociated with S. mutans MT8148(serotype C)a

Caries scoreeNo. of MeanExperimental Plaque CFU/ Buccal Sulcal Proximal body wtgroupb (%) scorec mandibled

(106) Enamel Dentinal Enamel Dentinal Enamel Dentinal (g)slight slight sih

A (1) 3.3 (74) 1.8 ± 0.5 8.3 ± 0.3 6.8 ± 1.0 10.9 ± 0.8 9.5 ± 0.6 0.0 (100) 0.0 (100) 119 ± 7(89) (48) (49) (39) (34)

B (0.1) 3.5 (72) 2.2 + 0.7 9.8 ± 0.5 7.8 ± 0.5 12.7 ± 1.0 10.7 + 0.7 0.0 (100) 0.0 (100) 118 ± 5(87) (38) (41) (29) (26)

C (0.01) 6.8 (46) 4.7 ± 0.8 13.0 ± 0.8 9.3 ± 1.0 14.1 ± 1.0 11.8 ± 0.3 2.7 + 0.4 0.3 ± 0.3 110 ± 7(72) (18) (30) (22) (19) (37) (80)

D (0.0) 12.5 16.8 ± 5.7 15.8 ± 0.7 13.3 ± 0.8 18.0 ± 0.6 14.5 ± 0.3 4.3 ± 0.4 1.5 ± 0.4 110 ± 7a IgGl antibody activity to S. mutans MT8148 in the ultrafiltrate fraction of immune whey (100 mg/ml of 0.01 M phosphate buffer, pH 7.4) was determined by

ELISA: absorbance at 405 nm of a 1/1000 dilution was 0.479 + 0.015.b Values in parentheses are the percent composition of the immune whey fraction added to diet 305. Each group contained 7 rats.See Table 3, footnote b.

d See Table 3, footnote c.e See Table 3, footnote d.

VOL. 55, 1987

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2346 MICHALEK ET AL.

TABLE 7. Effect of purified whey IgGl on protection of gnotobiotic rats monoassociated with S. mutans MT8148 (serotype C)a

No. of Caries scoreeExperimental groupb Plaque CFU/ Buccal Sulcal Proximal body wt

score' mandible'(106) Enamel Dentinal Enamel Dentinal Enamel Dentinal (g)

slight slight slightA (0.1% immune IgGl) 3.1 (74) 2.2 ± 0.4 8.8 ± 0.7 6.5 ± 0.8 10.4 ± 0.7 9.0 ± 0.5 0.0 (100) 0.0 (100) 106 ± 4

(88) (45) (48) (40) (36)B (0.1% immune IgGl 10.4 (13) 17.3 ± 5.2 15.5 ± 0.7 11.7 ± 1.0 17.8 ± 0.7 14.6 ± 0.5 2.7 ± 0.3 1.1 ± 0.2 114 ± 6absorbed with (6) (3) (7) (-3) (-3) (29) (15)MT8148)

C (0.1% control IgGl) 9.7 (19) 19.1 ± 2.0 14.1 ± 1.1 10.8 ± 0.9 15.5 ± 0.4 12.8 ± 0.2 2.6 ± 0.4 0.9 ± 0.1 117 ± 3(-5) (12) (14) (10) (9) (32) (31)

D (0.0%) 12.0 17.8 ± 3.2 16.0 ± 0.7 12.6 ± 0.8 17.2 ± 0.8 14.1 ± 0.2 3.8 ± 0.6 1.3 ± 0.3 128 ± 8

a Antibody activity to S. mutans MT8148 in the purified IgGl fraction of control and immune whey (100 mg/ml in 0.01 M phosphate buffer, pH 7.4) wasdetermined by ELISA: A405 was 0.014 ± 0.002 and 0.842 ± 0.087 for control and immune whey IgGl (1/50 and 1/1000 dilution, respectively). The absorbed IgGfraction (group B) had an absorbance of 0.017 ± 0.001 at a 1/50 dilution.

b See Table 6, footnote b. Each group contained 7 rats.See Table 3, footnote b.

d See Table 3, footnote c.See Table 3, footnote d.

bovine whey to protect calves from rotavirus infectioncorrelated with the level of IgGl anti-rotavirus antibodyactivity.When the caries-promoting diet was supplemented with

0.1% purified immune whey IgGl and provided to gnotobi-otic rats moninfected with S. mutans MT8148, protectionagainst infection and caries formation was noted, an effectwhich was abrogated by prior adsorption of the purified IgGlwith whole cell antigen of S. mutans MT8148. These resultsdemonstrate that the protective component in the immunewhey is the IgGl anti-S. mutans antibodies.

Bovine milk is a dietary nutrient which is available to mostof the human population, and the results presented hereprovide evidence that bovine milk containing antibodies to amucosally associated infectious organism could provide pas-sive immune protection against the disease. This conceptgains support from the finding that bovine colostral IgGlantibodies remain functionally active in the gastrointestinaltracts of infant and adult rabbits and after in vitro exposureto pepsin and trypsin (20). In one clinical study (29), it wasshown that provision of a bovine milk immunoglobulinconcentrate containing antibodies to enteropathogenic E.coli to infected patients resulted in the elimination of theseE. coli from stool samples of 84.3% of the subjects. Theseresults provide evidence that bovine IgGl antibodies remainfunctionally active even when exposed to proteolytic en-zymes which are present in external secretions.

In rats, functional immunoglobulins can be transportedacross the intestinal mucosa during neonatal life; however,this process ceases within the first 18 to 21 days of life (seereference 19 for review). Therefore, in the present study, theability of the bovine IgGl antibody to protect against S.mutans-induced dental caries would most likely be a localpassive protection in the oral cavity. This possibility gainssupport from the recent findings of Lehner et al. (11) and Maand co-workers (18), who showed passive protection againstS. mutans colonization and caries formation when murinemonoclonal IgG antibody to the streptococcal antigen I/IIwas applied to the tooth surfaces of monkeys and humans,respectively.

In a recent study, we also observed that volunteers usingthe bovine immune whey, which contained IgGl anti-S.mutans antibody activity, as a daily mouth rinse showed adecrease in the number of S. mutans cells in plaque ascompared with subjects using a control whey mouth rinse

(manuscript in preparation). In preliminary in vitro studies,we have observed that the bovine IgGl antibodies inhibit S.mutans growth, as well as GTF, lactate dehydrogenase, andglucose-phosphotransferase activities. Thus, the immunewhey IgGl used in this study contained antibodies withdifferent functional specificities, each of which could beimportant in protection. In this regard, the bovine antibodiescould adhere to tooth surfaces, and those specific for GTFcould interfere with the accumulation of growing S. mutansplaque, whereas those specific for lactate dehydrogenaseand for glucose-phosphotransferase could interfere withgrowth and acid production. The relative importance of eachof these antibody specificities and of antibodies of a singlespecificity versus multiple specificities in passive protectionagainst S. mutans colonization and caries formation willrequire further investigation.

ACKNOWLEDGMENTS

We thank Lee R. Beck for providing the bovine whey and theultrafiltrate fraction and for his advice and support of these studies.We also thank Dawn E. Colwell for her critical review of this workand Sandra Roberts for secretarial support.These studies were supported in part by a grant from the Stolle

Research and Development Corp., Cincinnati, Ohio, and by PublicHealth Service grants DE 04217, DE 05358, and DE 02670 andContract DE 42551 from the National Institutes of Health.

LITERATURE CITED1. Acres, S. D., R. E. Isaacson, L. A. Babiuk, and R. A. Kapitany.

1979. Immunization of calves against enterotoxigenic colibacil-losis by vaccinating dams with purified K99 antigen and wholecell bacterins. Infect. Immun. 25:121-126.

2. Brambell, F. W. R. 1970. The transmission of passive immunityfrom mother to young. North Holland Publishing Co., Amster-dam.

3. Bratthall, D. 1970. Demonstration of five serological groups ofstreptococcus strains resembling Streptococcus mutans. Odon-tol. Revy 21:143-152.

4. Bratthall, D., and B. Kohler. 1976. Streptococcus mutansserotypes: some aspects of their identification, distribution,antigenic shifts and relationship to caries. J. Dent. Res. 55:C15-C21.

5. Bridger, J. C., and J. F. Brown. 1981. Development of immunityto porcine rotavirus in piglets protected from disease by bovinecolostrum. Infect. Immun. 31:906-910.

6. Butler, J. E. 1983. Bovine immunoglobulin: an augmentedreview. Vet. Immunol. Immunopathol. 4:43-152.

INFECT. IMMUN.

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

BOVINE IgG ANTIBODIES PROTECT AGAINST DENTAL CARIES 2347

7. Coykendall, A. L., and K. B. Gustafson. 1986. Taxonomy ofStreptococcus mutans, p. 21-28. In S. Hamada, S. M.Michalek, H. Kiyono, L. Menaker, and J. R. McGhee (ed.),Molecular microbiology and immunobiology of Streptococcusmutans. Elsevier Science Publishers, Amsterdam.

8. Duncan, D. B. 1955. Range and multiple tests. Biometrics11:1-42.

9. Gregory, R. L., S. M. Michalek, I. L. Shechmeister, and J. R.McGhee. 1983. Effective immunity to dental caries: protectionof gnotobiotic rats by local immunization with a ribosomalpreparation from Streptococcus mutans. Microbiol. Immunol.27:787-800.

10. Keyes, P. H. 1958. Dental caries in the molar teeth of-rats. Il. Amethod for diagnosing and scoring several types of lesionssimultaneously. J. Dent. Res. 37:1088-1099.

11. Lehner, T., J. Caldwell, and R. Smith. 1985. Local passiveimmunization by monoclonal antibodies against streptococcalantigen I/II in the prevention of dental caries. Infect. Immun.50:796-799.

12. Lehner, T., S. J. Challacombe, M. W. Russell, C. M. Scully, andJ. E. Hawkes. 1978. Passive immunization with serum andimmunoglobulin against dental caries in rhesus monkeys. Lan-cet i:693-694.

13. Lehner, T., M. W. Russell, and J. Caldwell. 1980. Immunizationwith a purified protein from Streptococcus mutans againstdental caries in rhesus monkeys. Lancet i:995-996.

14. Lehner, T., M. W. Russell, J. Caldwell, and R. Smith. 1981.Immunization with purified protein antigens from Streptococcusmutans against dental caries in rhesus monkeys. Infect. Immun.34:407-415.

15. Lehner, T., M. W. Russell, C. M. Scully, S. J. Challacombe, andJ. Caldwell. 1979. The role of IgG, IgA and IgM classes ofantibodies in protection against caries in rhesus monkeys, p.216-217. In M. T. Parker (ed.), Pathogenic streptococci.Reedbook, Ltd., Chertsey, Surrey.

16. Loesche, W. J., J. Rowan, L. H. Straffon, and P. J. Loos. 1975.Association of Streptococcus mutans with human dental decay.Infect. Immun. 11:1252-1260.

17. Loesche, W. J., and L. H. Straffon. 1979. Longitudinal investi-gation of the role of Streptococcus mutans in human tissuedecay. Infect. Immun. 26:498-507.

18. Ma, J. K.-C., R. Smith, and T. Lehner. 1987. Use of monoclonalantibodies in local passive immunization to prevent colonizationof human teeth by Streptococcus mutans. Infect. Immun.55:1274-1278.

19. Mackenzie, N. 1984. Fc receptor-mediated transport of immu-noglobulin across the intestinal epithelium of the neonatalrodent. Immunol. Today 5:364-366.

20. McClead, R. E., and S. A. Gregory. 1984. Resistance of bovinecolostral anti-cholera toxin antibody to in vitro and in vivoproteolysis. Infect. Immun. 44:474-478.

21. McGhee, J. R., and S. M. Michalek. 1981. Immunobiology ofdental caries: microbial aspects and local immunity. Annu. Rev.Microbiol. 35:595-638.

22. McGhee, J. R., S. M. Michalek, J. M. Navia, and A. J. Narkates.1976. Effective immunity to dental caries: studies of active andpassive immunity to Streptococcus mutans in malnourishedrats. J. Dent. Res. 55:C206-C214.

23. McGhee, J. R., S. M. Michalek, J. Webb, J. M. Navia, A. F. R.Rahman, and D. W. Legler. 1975. Effective immunity to dentalcaries: protection of gnotobiotic rats by local immunization withStreptococcus mutans. J. Immunol. 114:300-305.

24. Michalek, S. M., and J. R. McGhee. 1977. Effective immunity todental caries: passive transfer to rats of antibodies to Strepto-coccus mutans elicits protection. Infect. Immun. 17:644-650.

25. Michalek, S. M., J. R. McGhee, R. R. Arnold, and J. Mestecky.1978. Effective immunity to dental caries: selective induction ofsecretory immunity by oral administration of Streptococcusmutans in rodents. Adv. Exp. Med. Biol. 107:261-269.

26. Michalek, S. M., J. R. McGhee, J. Mestecky, R. R. Arnold, andL. Bozzo. 1976. Ingestion of Streptococcus mutans inducessecretory immunoglobulin A and caries immunity. Science192:1238-1240.

27. Michalek, S. M., J. R. McGhee, and J. M. Navia. 1975. Viru-lence of Streptococcus mutans: a sensitive method for evaluat-ing cariogenicity in young, gnotobiotic rats. Infect. Immun.12:69-75.

28. Michalek, S. M., I. Morisaki, R. L. Gregory, H. Kiyono, S.Hamada, and J. R. McGhee. 1983. Oral adjuvants enhance IgAresponses to Streptococcus mutans. Mol. Immunol. 20:1009-1018.

29. Mietens, C., H. Keinhorts, H. Hilpert, H. Gerber, H. Amster,and J. J. Pahud. 1979. Treatment of infantile E. coli gastroen-teritis with specific bovine anti-E. coli milk immunoglobulins.Eur. J. Pediatr. 132:239-252.

30. Nagy, B. 1980. Vaccination of cows with a K99 extract toprotect newborn calves against experimental entertoxic coli-bacillosis. Infect. Immun. 27:21-24.

31. Navia, J. M., H. Lopez, and R. S. Harris. 1969. Purified diet fordental caries research with rats. J. Nutr. 97:133-140.

32. Ogra, P. L., and D. H. Dayton. 1979. Immunology of breastmilk. Raven Press, New York.

33. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemical andserological properties of Streptococcus mutans from varioushumans and animals sources. Acta Pathol. Microbiol. Scand.Sect. B 82:357-370.

34. Saif, L. J., D. R. Redman, K. L. Smith, and K. W. Theil. 1983.Passive immunity to bovine rotavirus in newborn calves fedcolostrum supplements from immunized or nonimmunizedcows. Infect. Immun. 41:1118-1131.

35. Smith, D. J., M. A. Taubman, and J. L. Ebersole. 1978. Effectsof local immunization with glucosyltransferase fractions fromStreptococcus mutans on dental caries in hamsters caused byhomologous and heterologous serotypes of Streptococcusmutans. Infect. Immun. 21:843-851.

36. Smith, D. J., M. A. Taubman, and J. L. Ebersole. 1979. Effect oforal administration of glucosyltransferase antigens on experi-mental dental caries. Infect. Immun. 26:82-89.

37. Snodgrass, D. R., K. J. Fahey, P. W. Wells, I. Campbell, and A.Whitelaw. 1980. Passive immunity in calf rotavirus infections:maternal vaccination increases and prolongs immunoglobulinGl antibody secretion in milk. Infect. Immun. 28:344-349.

38. Taubman, M. A., and D. J. Smith. 1976. Effects of localimmunization with glucosyltransferase fractions from Strepto-coccus mutans on dental caries in rat and hamsters. J. Immunol.118:710-716.

39. Trexler, P. C. 1959. The use of plastics in the design of isolatorsystem. Ann. N.Y. Acad. Sci. 78:29-36.

40. Zinkernagel, R. M., and A. Colombini. 1975. Passive oralimmunization with bovine immunoglobulins: enterpathogenicEscherichia coli from infants and bovine anti-E. coli lactoserumassayed in the rabbit ileal loop model. Med. Microbiol. Immu-nol. 162:1-7.

VOL. 55, 1987

on Septem

ber 6, 2020 by guesthttp://iai.asm

.org/D

ownloaded from