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Article

Bovine Milk Antibodies against Cell Surface Protein Antigen PAc-Glucosyltransferase Fusion Protein Suppress Cell Adhesion and Alter Glucan Synthesis of Streptococcus mutans¹

Department of Preventive Dentistry, Kyushu University Faculty of Dentistry, Fukuoka 812-8582 and ^* University Farm, Kyushu University Faculty of Agriculture, Fukuoka 811-2307, Japan

²To whom correspondence should be addressed.

	ABSTRACT

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Cell surface protein antigen (PAc) and glucosyltransferases (GTF) produced by Streptococcus mutans are considered major colonization factors of the organism, and the inhibition of these factors is thought to prevent dental caries. In this study, 8-mo-old pregnant Holstein cows were immunized with fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose. High titers of immunoglobulin antibodies specific for the fusion protein were found in normal milk after reimmunization, and they persisted for ~3 mo. The immunoglobulin G (IgG) antibodies against PAcA-GB were purified from immunized milk. The antibodies significantly inhibited the adhesion of S. mutans cells to saliva-coated hydroxyapatite beads. IgG antibodies purified from immunized milk also inhibited total glucan synthesis by cell-associated GTF preparation and GTF-I from S. mutans. The immunized milk may be useful as a means of passive immunization for the prevention of dental caries in humans.

KEY WORDS: • bovine milk • Streptococcus mutans • dental caries • passive immunization

	INTRODUCTION

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Streptococcus mutans has been implicated as the primary causative organism of dental caries, one of the most common diseases in humans. Colonization of tooth surfaces by S. mutans is thought to be an important step for the initiation of dental caries. This process is mediated by both sucrose-independent and sucrose-dependent mechanisms. The former mechanism involves interaction between cell surface components of S. mutans, such as a 190-kDa surface protein antigen (PAc),³ and acquired pellicles formed on tooth surfaces (Koga et al. 1990 ). The latter is due to the synthesis of water-insoluble glucan from sucrose catalyzed by glucosyltransferases (GTF) (Kuramitsu et al. 1995 ). In addition, the glucan firmly coats the tooth surface, forming a barrier that prevents the diffusion of acid produced by the organism. The important roles of PAc and water-insoluble glucan-synthesizing GTF (GTF-I) in the cariogenicity of S. mutans make them rational targets for the development of an anticaries vaccine and an adhesion inhibitor. Simultaneous inhibition of these colonization factors may result in the protection of teeth from dental caries. In a previous study, we constructed a fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose (Yu et al. 1997 ). We demonstrated the inhibitory effects of rabbit antibodies against the fusion protein on the adhesion of S. mutans to saliva-coated hydroxyapatite (S-HA) and on glucan synthesis by GTF from the organism.

Bovine milk has been used as a means of passive immunization for prevention measures targeting several pathogens (Ebina et al. 1983 , Ishida et al. 1992 , Murosaki et al. 1991 , Tacket et al. 1988 ). In trying to eradicate dental caries, several attempts have been made to develop a method for the passive oral administration of preformed antibodies to S. mutans (Hamada and Kodama 1996 ). Early experiments included attempted passive oral immunization using colostral antibodies against whole-cell antigens of mutans streptococci (Filler et al. 1991 , Michalek et al. 1987 ). Because colostrum is not suitable for drinking on a daily basis, a method is needed to produce normal milk containing antibodies that protect humans from dental caries. In this study, we immunized cows with a fusion protein PAcA-GB and obtained normal milk containing antibodies specific for the fusion protein. We also discuss the inhibitory effects of these antibodies, both on the in vitro adhesion of S. mutans to S-HA and on glucan synthesis by S. mutans GTF, when they are purified from immunized milk.

	MATERIALS AND METHODS

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Bacterial strains.

S. mutans MT8148 was used as a representative strain of S. mutans serotype c. S. mutans TK18 is a recombinant strain that produces a large amount of PAc (Koga et al. 1990 ). S. mutans strains UA130B⁺, UA130C⁺ and UA130D⁺ are transformants, which express a single type of GTF (GTF-I, GTF-SI and GTF-S, respectively) (Yu et al. 1997 ). Escherichia coli XL1-Blue was obtained from Stratagene (La Jolla, CA). The culture media used were 2 x TY broth (Laloi et al. 1996 ) for E. coli and brain heart infusion (BHI; Difco Laboratories, Detroit, MI) broth for S. mutans.

Immunization procedure.

The fusion protein PAcA-GB was purified according to the method described previously (Yu et al. 1997 ). Briefly, E. coli XL1-Blue harboring the plasmid pQEPG22 expressing the PAcA-GB was incubated in 2 x TY broth containing 200 mg/L ampicillin and 20 g/L glucose at 37°C until an A550 of 1.0 was attained. Isopropyl-ß-D-thiogalactopyranoside was added to the culture to a final concentration of 1 mmol/L, and the culture was grown for 3 h. The cells were harvested by centrifugation at 5,000 x g for 20 min, and one-step purification of the fusion protein with a Qiagen kit was performed using a Ni-nitrilotriacetic acid resin column according to the manufacture's instructions (Qiagen, Chatworth, CA). The purified fusion protein was analyzed by SDS-PAGE followed by immunoblotting. Two pregnant Holstein cows (1 and 2) were immunized by subcutaneous injections with 100 mg of fusion protein PAcA-GB emulsified in Freund's complete adjuvant (Difco Laboratories) 9 wk before the calculated date of delivery. The cows were immunized again subcutaneously 3 and 7 wk later with 50 mg of the protein emulsified in Freund's incomplete adjuvant (Difco Laboratories). After delivery, cow 1 was reimmunized twice by injections into the lymph nodes near the thymus with 50 mg of the protein emulsified in Freund's incomplete adjuvant (Ebina 1996 ). Two control pregnant cows (3 and 4) did not receive any injections of immunogen.

These experiments were conducted under the control of the guideline for Animal Experiment in Faculty of Agriculture and the Graduate Course, Kyushu University and the Law (No. 105) and Notification (No. 6) of the Japanese Government.

Preparation of milk immunoglobulins.

Colostrum and normal milk were collected from the cows for 5 mo after calving. The milk fat was removed by centrifugation at 12,000 x g for 15 min and the skimmed milk was pasteurized at 65°C for 30 min. The milk was adjusted to pH 4.6 with 12.8 mol/L HCl, and casein was separated from the whey by centrifugation at 12,000 x g for 15 min; subsequently, the pH of the whey was adjusted to 7.0 with 1 mol/L NaOH. Milk immunoglobulins were concentrated by 50% saturated ammonium sulfate precipitation and dialyzed against potassium PBS (pH 7.0).

Dialyzed and concentrated immunoglobulin was subjected to affinity chromatography on a HiTrap Protein G column (5 mL) (Pharmacia, Uppsala, Sweden) equilibrated with PBS. The column was washed extensively with PBS, and immunoglobulin G (IgG) antibody was eluted with 0.1 mol/L glycine-HCl buffer, pH 2.7. Fractions containing IgG antibody were identified by immunoblotting with alkaline phosphatase–conjugated sheep anti-bovine IgG antibody (heavy chain) (Bethyl Laboratories, Montgomery, TX), collected, and used as IgG antibody.

The concentrations of IgG, IgA and IgM in normal milk were determined by ELISA using bovine IgG, IgA and IgM (Intercell Technologies, Hopewell, NJ) as standards. Alkaline phosphatase–conjugated sheep anti-bovine IgG (heavy chain) (Bethyl Laboratories), alkaline phosphatase-conjugated rabbit anti-bovine IgA ( chain) (Bethyl Laboratories), and alkaline phosphatase–conjugated rabbit anti-bovine IgM (µ chain) (Bethyl Laboratories) were used as antibodies to detect the immunoglobulins.

rPAc.

Recombinant PAc (rPAc) was purified from the culture supernatants of transformant S. mutans TK18 by ammonium sulfate precipitation, chromatography on DEAE-cellulose and subsequent gel filtration on Sepharose CL-6B (Pharmacia) (Koga et al. 1990 ).

Preparation of GTF.

Cell-free and cell-associated GTF were prepared as described by Hamada et al. (1989) . In brief, S. mutans strains were grown in BHI broth at 37°C for 18 h. Cell-associated GTF was extracted from whole cells of S. mutans MT8148 by treatment with 8 mol/L urea at 25°C for 1 h. The extract was centrifuged at 5,000 x g for 20 min, and the supernatant was dialyzed against 0.1 mol/L potassium phosphate buffer (KPB, pH 6.0). The supernatant was used as the cell-associated GTF preparation. Cell-free GTF was prepared from culture supernatants of S. mutans MT8148. To examine the effects of antibodies on each GTF of S. mutans, transformants producing a single species of GTF were used (Yu et al. 1997 ). GTF-I and GTF-SI were extracted from whole cells of the transformants UA130B⁺ and UA130C⁺, respectively, by treatment with 8 mol/L urea at 25°C for 1 h. GTF-S was prepared from culture supernatants of the transformant UA130D⁺.

ELISA.

For the ELISA, 96-well microtiter plates were coated with 100 µL of rPAc or GTF-I (5 µg/mL) in 50 mmol/L carbonate-bicarbonate buffer (pH 9.6). After incubation at 37°C for 90 min, the plates were washed with PBS containing 0.05% Tween 20 (PBST) and blocked with PBST containing 10 g/L chicken egg albumin at 37°C for 90 min. After the plates were washed three times with PBST, twofold serial dilutions of bovine pasteurized milk were added (100 µL/well) and the plates were incubated at 37°C for 90 min. The bound antibodies were detected with alkaline phosphatase–conjugated rabbit anti-bovine IgG (heavy and light chains) (Zymed Laboratories, South San Francisco, CA) followed by the addition of p-nitrophenylphosphate substrate solution (1 g/L). After a 30 min incubation at 37°C, the A405 was measured with a microplate reader (Bio-Rad Laboratories, Richmond, CA). The ELISA antibody titer was expressed as the reciprocal of the highest dilution giving an A405 of 0.1 above the conjugated control (no sample added) after 30 min of incubation with the substrate.

Adhesion of S. mutans cells to S-HA.

Paraffin-stimulated whole saliva was collected from a healthy adult subject and clarified by centrifugation at 12,000 x g for 20 min. Spheroidal hydroxyapatite beads (5mg; BDH, Poole, England) were incubated with 200 µL of clarified whole saliva for 1 h at 37°C and washed three times with buffered KCl (Gibbons and Hay 1989 ). S. mutans MT8148 was grown at 37°C for 18 h in BHI broth; 2',7'-bis[2-carboxyethyl]-5[6]-carboxyfluorescein acetoxymethyl ester (BCECF-AM) (Sigma Chemical, St. Louis, MO) was added to the bacterial culture to a final concentration of 10 µmol/L and incubated for an additional 30 min in the dark (Martin and Bhakdi 1992 ). BCECF-labeled bacteria (4 x 10⁷) were allowed to react with S-HA beads (5 mg) in 200 µL buffered KCl. After incubation at 37°C for 3 h, the beads were washed three times with buffered KCl, and the fluorescence intensity associated with the S-HA beads was determined with a Spectramax Gemini microplate reader (Molecular Devices, Sunnyvale, CA). The number of bacteria adsorbed was determined from the calculated specific fluorescence intensity of the bacteria. To evaluate the inhibitory effects of antibodies on the adherence of S. mutans cells to S-HA beads, BCECF-labeled bacteria (4 x 10⁷) were allowed to react with S-HA beads (5 mg) in 200 µL buffered KCl containing various amounts of IgG antibodies at 37°C for 3 h.

GTF assay.

To evaluate total glucan synthesis activity, a reaction mixture containing 10 g/L sucrose and GTF (2.5 µg of protein) in a total volume of 50 µL of KPB (pH 6.0) was incubated at 37°C for 3 h. After incubation, 75 µL of 100% ethanol was added to the mixture, and synthesized glucan was precipitated at -30°C for 30 min. The precipitated glucan was centrifuged at 16,000 x g for 15 min and washed three times with 60% ethanol. The precipitates were suspended in 50 µL of KPB (pH 6.0), and the total amount of glucan was determined by the phenol-sulfuric acid method with glucose as a standard (Dubois et al. 1956 ). To determine the inhibitory effects of antibodies on glucan synthesis, various amounts of purified IgG antibodies were added to the reaction mixture, and the amount of glucan synthesized by GTF was measured as described above.

SDS-PAGE and immunoblotting.

SDS-PAGE was performed using 7.5% polyacrylamide gels according to the method of Laemmli (1970) . rPAc and GTF-I were subjected to SDS-PAGE and transferred electrophoretically to nitrocellulose membranes according to the method of Burnette (1981) . After blocking with 10 g/L chicken egg albumin in Tris-buffered saline (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.5) plus 1% Triton X-100 (TBS-Triton), the membranes were treated with bovine IgG (10 mg/L) purified from milk. After washing with TBS-Triton, the antibodies bound to proteins immobilized on the membrane were detected with alkaline phosphatase–conjugated rabbit anti-bovine IgG (heavy and light chains) (Zymed Laboratories).

Statistical analysis.

Quantitative differences in the adhesion of S. mutans to S-HA and glucan synthesis were analyzed by Student's t test.

	RESULTS

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Induction of antibodies in milk.

Immunization of cows with the fusion protein PAcA-GB raised immunoglobulin antibody titers in colostrum and normal milk. The time course of antibody titers is shown in Figure 1 A and B. Very high antibody titers against rPAc and GTF-I (>10,000) were found in the colostrum from the first four or five milkings of immunized cows. The antibody titer against rPAc was higher than that against GTF-I in immunized cows. The antibody titer against GTF-I decreased to ~100 3 wk after calving; the titer against rPAc decreased to the same level 6 wk after calving. To raise antibody titers in normal milk, cow 1 was reimmunized twice into the lymph nodes. The antibody titers subsequently increased. Maximal antibody titers against rPAc and GTF-I in normal milk were detected 8 d after the first reimmunization (d 55), and the high titers persisted for ~3 mo. On the other hand, antibody titers against rPAc and GTF-I were very low even in the colostrum from the first milking of nonimmunized cows.

View larger version (21K): [in this window] [in a new window]   Figure 1. ELISA titers of bovine milk immunoglobulins (A) to recombinant protein antigen serotype c (rPAc)and (B) glucosyltransferase-I (GTF-I). Titers are expressed as the reciprocal of the highest dilution giving an A405 of 0.1 above the conjugated control (no sample added) after 30 min of incubation with the substrate. At d 47, the first reimmunization was performed, and the highest titer values for antibodies to both rPAc and to GTF-I were found at d 55. Symbols: •, milk immunoglobulins from cow 1, which received immunization and reimmunization; , milk immunoglobulins from cow 2, which received immunization only before delivery; and , milk immunoglobulins from nonimmunized control cows 3 and 4, respectively.

The concentration of each immunoglobulin class in normal milk was determined by ELISA. The mean concentrations of IgG, IgA and IgM in the milk of immunized cow 1 (d 55) were 0.440, 0.053 and 0.032 g/L milk, respectively. On the other hand, the mean concentrations of IgG, IgA and IgM in the milk of nonimmunized cow 3 (d 55) were 0.420, 0.057 and 0.022 g/L milk, respectively. These results indicate that IgG is the major immunoglobulin class in bovine milk and that there are few differences in the amount of each immunoglobulin class from immunized and nonimmunized milk. The IgG antibodies were purified from the immunized milk (cow 1) and nonimmunized milk (cow 3) on d 55 by affinity chromatography and were used as IgG antibodies against PAcA-GB and control IgG, respectively, in the following inhibition experiments. The specificities of the IgG antibodies were examined by immunoblotting. IgG antibodies against PAcA-GB reacted with rPAc and GTF-I (Fig. 2 , lanes 2 and 4). On the other hand, IgG antibodies purified from nonimmunized milk did not react with these protein antigens (Fig. 2 , lanes 1 and 3).

View larger version (48K): [in this window] [in a new window]   Figure 2. Specificity of bovine milk immunoglobulin G (IgG) antibodies against a fusion of the saliva-binding alanine-rich region (PAcA) of protein antigen serotype c (PAc) with the glucan binding (GB) domain of glucosyltransferase-I (GTF-I) (PAcA-GB). Purified antigen samples (1 µg each) were suspended in SDS-PAGE reducing buffer (1% SDS, 1% 2-mercaptoethanol) and heated at 100°C for 3 min. Samples were subjected to SDS-PAGE (7.5% polyacrylamide) and then electrophoretically transferred to nitrocellulose membranes. The membranes were reacted with bovine IgG antibody purified from the milk of nonimmunized cow 3 (d 55) (lanes 1 and 3) and IgG antibody purified from the milk of immunized cow 1 (d 55) (lanes 2 and 4). Lanes: 1 and 2, recombinant PAc (rPAc); 3 and 4, GTF-I.

The effect of IgG antibodies of normal milk on the adhesion of S. mutans MT8148 cells to S-HA beads was examined. The IgG antibodies against PAcA-GB significantly inhibited the adhesion of S. mutans cells in a dose-dependent manner (Fig. 3 ). On the other hand, the IgG purified from nonimmunized milk had no influence on the adhesion.

View larger version (20K): [in this window] [in a new window]   Figure 3. Inhibition of adhesion of Streptococcus mutans to saliva-coated hydroxyapatite (S-HA) beads by bovine immunoglobulin G (IgG) antibodies. 2',7'-bis[2-Carboxyethyl]-5[6]-carboxyfluorescein (BCECF)-labeled S. mutans MT8148 cells (4 x 107) were allowed to react with 5 mg of S-HA beads with various amounts of IgG antibodies purified from the milk of immunized cow 1 (d 55) (•) and the milk of nonimmunized cow 3 (d 55) (). Values are reported as the means ± SD of triplicate independent assays. The experiments were separately performed three times, and similar results were obtained in each experiment. *P < 0.05; **P < 0.01 compared with control.

Total glucan synthesis by cell-associated GTF preparation from S. mutans MT8148 was significantly inhibited by the addition of IgG antibodies against PAcA-GB in a dose-dependent manner (Fig. 4 A). In contrast, IgG from nonimmunized milk enhanced glucan synthesis by cell-associated GTF. Glucan synthesis by cell-free GTF preparation was not influenced by the addition of IgG against PAcA-GB (Fig. 4 B). Further experiments were performed with three GTF preparations obtained from transformants UA130B⁺, UA130C⁺ and UA130D⁺, each expressing a single type of GTF (GTF-I, GTF-SI and GTF-S, respectively). Anti-PAcA-GB IgG antibodies markedly inhibited glucan synthesis by GTF-I but moderately inhibited that by GTF-SI (Table 1 ). Anti-PAcA-GB IgG antibodies weakly suppressed glucan synthesis by GTF-S. Nonimmunized IgG antibodies enhanced glucan synthesis by all three GTF.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of bovine immunoglobulin G (IgG) antibodies on glucan synthesis by crude glucosyltransferase (GTF) preparations from Streptococcus mutans MT8148. (A) Cell-associated GTF; (B) cell-free GTF. The reaction mixture consisted of 10 g/L sucrose, crude GTF preparation (2.5 µg of protein), and bovine IgG antibodies purified from the milk of immunized cow 1 (d 55) (•) or the milk of nonimmunized cow 3 (d 55) () in a total volume of 50 µL of 0.1 mol/L potassium phosphate buffer (KPB) (pH 6.0). After incubation for 3 h at 37°C, the total amount of glucan synthesized was measured. Values are reported as the means ± SD of triplicate independent assays. The experiments were separately performed three times, and similar results were obtained in each experiment. *P < 0.05; **P < 0.01 compared with control.

	DISCUSSION

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Bovine milk has several advantages for use in humans as a means of passive immunization. It is a common daily food that is easily delivered to the oral cavity, and it is produced on a large scale at low cost. It also contains various natural defense factors to prevent microbial activity, such as lysozyme and lactoperoxidase (Takahashi et al. 1992 , Tenovuo et al. 1982 ). Because bovine milk contains a large amount of immunoglobulins in the colostrum (Butler 1983 ), colostral antibodies have been used for passive immunization for the prevention of infectious diseases. However, providing the colostrum as a daily food for humans is prohibited, making it necessary to produce normal milk containing high titers of antibodies. In this study, reimmunization into the lymph nodes resulted in the induction of high titers of immunoglobulins against a fusion protein PAcA-GB, and the induction persisted for ~3 mo.

In this study, we pasteurized normal milk at 65°C for 30 min after the removal of fat and casein. ELISA was used to examine the influence of heating on the ability of milk immunoglobulins to react with rPAc and GTF-I. The binding capacity of immunoglobulins to coated antigens remained stable even after treatment at 65°C for 30 min, but it was markedly reduced by treatment at 70°C for 30 min and was lost completely when heated at 75°C for 30 min (data not shown). This means that milk pasteurized at 65°C for 30 min contains effective antibodies that can be delivered easily to the human oral cavity.

This study showed that the major immunoglobulin class in bovine milk is IgG. Mach and Pahud (1971) reported that the major immunoglobulin class in bovine milk is IgG, although secretory IgA is most abundant in most other external secretions, including saliva, lacrimal, nasal and gastrointestinal secretions. Butler (1983) also reported the same results concerning the major immunoglobulin class in bovine milk. Immunoglobulins in bovine milk (mostly IgG) are either derived from serum or synthesized locally at a lower rate than milk-specific proteins, and serum is the chief source of IgG (Newby and Bourne, 1977 ). IgG antibodies were therefore purified from bovine milk and were used for the inhibition experiments.

The IgG antibodies purified from immunized milk suppressed the adhesion of S. mutans cells to S-HA. The first step in the eventual formation of dental caries is the adsorption of S. mutans to tooth surfaces, which is caused by an interaction between the organism and salivary proteins on tooth surfaces. This interaction is essential for the initiation of dental caries, even though the binding affinity may be low. The interaction between S. mutans cells and salivary proteins is mediated by PAc of the organism. Nakai et al. (1993) used several truncated PAc fragments to show that the functional domain for the binding of PAc is the A region. IgG antibodies against PAcA-GB inhibited the adhesion of the organism, which suggests that the inactivation of the functional domain of PAc could lead to the prevention of dental caries.

Purified IgG against PAcA-GB inhibited glucan synthesis by cell-associated GTF, but not that by cell-free GTF. S. mutans produces both water-soluble and water-insoluble glucans from sucrose by the combined action of three forms of GTF (GTF-I, GTF-SI and GTF-S) (Kuramitsu et al. 1995 ). GTF-I and GTF-SI, which synthesize primarily water-insoluble glucan from sucrose, are associated mainly with the bacterial cell surface (Hamada et al., 1989 ). On the other hand, GTF-S is released from cell surfaces and synthesizes water-soluble glucan. These enzymes are homologous proteins of ~1500 amino acid residues organized into two relatively independent domains, an N-terminal sucrose-binding domain and a C-terminal GB domain (Kuramitsu et al. 1995 ). The fusion protein PAcA-GB used in this study as an immunogen was constructed with the A fragment of PAc and the GB domain of GTF-I.

The nonimmunized IgG significantly enhanced glucan synthesis by all forms of GTF (Fig. 4 A, B and Table 1 ). Similar results have been reported for control and/or immune sera from rabbits, rats, monkeys and humans (Schachtele et al. 1978 ). It is thought that phospholipids present in sera cause the observed stimulation of GTF activity (Schachtele et al. 1978 ). The same mechanism may be responsible for the results seen in this study because the majority of IgG in bovine milk is transferred from serum (Butler 1983 ).

In conclusion, we immunized pregnant cows with a fusion protein PAcA-GB and obtained normal milk containing a high titer of antibodies to the protein. The IgG antibodies against PAcA-GB inhibited both the adhesion of S. mutans to S-HA and glucan synthesis catalyzed by GTF-I from S. mutans. Immunized milk may have a high potential as a means of passive immunization for preventing dental caries in humans. Further experiments are required to examine the inhibitory effects of immunized milk on the colonization of human teeth by S. mutans.

	ACKNOWLEDGMENTS

 
We thank Takusaburo Ebina, of the Division of Immunology, Research Institute Miyagi Cancer Center, Miyagi, Japan for helpful suggestions concerning the immunization method of cows.

	FOOTNOTES

 
¹ Supported in part by Grants-in-Aids for Developmental Scientific Research (C)11672051 (T.O.) from the Ministry of Education, Science, Sport and Culture of Japan and by the Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (T.K.).

³ Abbreviations used: BCECF-AM, 2',7'-bis[2-carboxyethyl]-5[6]-carboxyfluorescein acetoxymethyl ester; BHI, brain heart infusion; GB, glucan-binding; GTF, glucosyltransferase; IgG, immunoglobulin G; KPB, potassium phosphate buffer; PAc, protein antigen serotype c; PBST, PBS containing 0.05% Tween 20; rPAc, recombinant PAc; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; S-HA, saliva-coated hydroxyapatite; TBS-Triton, Tris-buffered saline plus 1% Triton X-100.

Manuscript received June 18, 1999. Initial review completed July 6, 1999. Revision accepted July 27, 1999.

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