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In recent years, we have demonstrated a new immunization route, tonsillar application, for inducing mucosal immunity (2-8). This immunization induced antibodies more effectively than nasal application and oral administration. The tonsillar application of formalin-killed whole cells of Streptococcus sobrinus and S. mutans in rabbits induced salivary immunoglobulin A (IgA) and blood plasma IgG against these cells (7). It was also found that these antibodies in the blood plasma induced by tonsillar application did not contain antibodies cross-reacting with human cardiac muscles; such antibodies have been known to be induced in the blood plasma by intramuscular injection. Further, the antibodies induced by tonsillar application of S. sobrinus AHT-k (serotype g) selectively reacted with S. sobrinus AHT-k and serologically related mutans group streptococci (serotypes a, d and h), whereas those induced by intramuscular injection reacted with a variety of oral streptococci, including unrelated mutans group streptococci (serotypes b, c, e, and f) in addition to the aforementioned streptococci (unpublished observations). In our unpublished observation, the antibodies induced by tonsillar application of S. sobrinus inhibited S. sobrinus colonization onto enamel slices in test tubes.

In the present study, we inoculated living S. sobrinus cells into rabbits immunized with formalin-killed cells of S. sobrinus by tonsillar application and the anticaries potential was examined.

Immunization and antibody induction. S. sobrinus AHT-k was cultured in brain heart infusion broth for 18 h at 37°C and killed with 10% formalin for 24 h. Twenty-four 6-week-old rabbits were used. Three hundred microliters of the killed-S. sobrinus suspension (10¹⁰ cells/ml) was dropped by a micropipette onto the surface of the palatine tonsil of nine rabbits, instilled by a conductor into the stomach of three other rabbits, and intramuscularly injected without any adjuvants into the femoral region of three more rabbits every week for 6 weeks. In the three control rabbits, phosphate-buffered saline (PBS) alone was applied to the tonsil. There was a further group of six nonimmune rabbits. The saliva and blood plasma of these rabbits were collected once a week after immunization. Immunoglobulin concentrations in the saliva and blood plasma were examined once a week for 21 weeks by enzyme-linked immunosorbent assay using plates coated with the ultrasonic fragments of S. sobrinus as described before (7). In the case of IgA, IgA concentration in saliva was represented as that of a monomer calculated by the value based on the standard curve obtained by enzyme-linked immunosorbent assay using purified rabbit IgA from serum (Inter-cell Technologies Inc., Hopewell, N.J.).

Inoculation of living S. sobrinus cells. Three days after the final immunization, the living S. sobrinus cells (10⁵ cells) in 1 ml of saline were orally inoculated into the rabbits every 3 days for 15 weeks, 35 times in total, with 1 ml of 3 M sucrose solution. In three of the nine rabbits immunized by tonsillar application, 1 ml of 1 mM galactose solution was orally administered every 3 days in addition to the above-described inoculation. We confirmed that indigenous mutans group streptococci were negligible. All rabbits were maintained on a solid diet (Cler Japan, Inc., Tokyo, Japan).

Detection of living S. sobrinus and caries regions. The cells were separately collected three times from one of the five lower left mandibular tooth surfaces in each rabbit by swabbing with a cotton bud and were directly cultured for 18 h at 37°C on plates of mitis salivarius agar medium, and 100 random colonies were blotted onto nitrocellulose membranes. The blotted strips were reacted with rat antiserum against S. sobrinus (1:100 diluted) and horseradish peroxidase-labeled goat IgG antibody against rat IgG (1:200 diluted; ICN Pharmaceuticals Inc.). The anti-S. sobrinus antiserum used was prepared from rats immunized with S. sobrinus whole cells by intramuscular injection every week for 6 weeks. The serum was absorbed by whole cells of S. sanguis ATCC 10556, S. salivarius IFO13956, S. pyogenes ATCC 12344, and serologically related S. downei ATCC 33748 (MFe28). The absorbed serum was confirmed to react selectively against S. sobrinus and not to react with other oral streptococci. The positive spots on the blotted strips were counted as S. sobrinus cells. The proportion of S. sobrinus colonies in 100 random colonies was determined as the mean of nine specimens separately obtained from three rabbits in each group. We confirmed that S. sobrinus proportions obtained by this method were similar to the proportion of mutans group streptococci obtained with Dentcult-SM (Orion Diagnostica, Espoo, Finland) or mitis salivarius-bacitracin (0.2 U/ml) agar medium supplemented with 15% sucrose, both of which allow selective growth of mutans group streptococci from the oral cavity.

Statistical analysis. The twenty-four rabbits used were divided into eight groups of three rabbits each. Three of the groups were immunized by tonsillar application, one group was immunized by intramuscular injection, and another was immunized by intragastric instillation. Also, there were one control group (PBS applied) and two nonimmune groups. All experimental results were expressed as the means ± standard deviations (SD). Statistical analysis was carried out by using the Friedman test. When significances were observed, the differences were determined by using the post hoc test (Scheffe's F method).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Concentrations of anti-S. sobrinus IgA antibody in saliva (a) and anti-S. sobrinus IgG antibody in blood plasma (b) after tonsillar application (circle), intragastric instillation (triangle), and intramuscular injection (solid square) of formalin-killed S. sobrinus cells. The results of control rabbits (PBS applied) are also shown (open square). The results of antibody concentrations show the average for the three rabbits of each group. Error bars indicate SD. Symbols for statistical significance: *, P < 0.005; **, P < 0.01 (5 to 21 weeks versus control with PBS applied); , P < 0.01 (5 to 21 weeks versus intramuscular injection); , P < 0.01 (5 to 21 weeks versus intragastric instillation).

View larger version (80K): [in this window] [in a new window]   FIG. 2.   Teeth after inoculation with S. sobrinus for 15 weeks in nonimmune rabbits (A), rabbits immunized by intramuscular injection (B), rabbits immunized by intragastric instillation (C), rabbits immunized by tonsillar application (D), rabbits inoculated with S. sobrinus with galactose (E), and noninoculated rabbits (F). The dark parts of the tooth surface are the caries regions. In noninoculated rabbits, no caries regions were found.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Proportion of living S. sobrinus colonies in 100 random colonies (open column) and area of caries regions (solid column) on the tooth surface after immunization and subsequent S. sobrinus inoculation for 15 weeks. The proportion of living S. sobrinus colonies and the area of caries regions are shown as an average of three individual rabbits in each group. Error bars indicate SD. Abbreviations: non-immun., nonimmune rabbits; i.m. inject., rabbits immunized by intramuscular injection; i.g. instil., rabbits immunized by intragastric instillation; ton. appl., rabbits immunized by tonsillar application; gal., galactose. Symbols for statistical significance: *, P < 0.05; **, P < 0.01 (versus S. sobrinus-inoculated nonimmune rabbits); , P < 0.01 (versus S. sobrinus-inoculated rabbits receiving tonsillar application).