Effective immunity to dental caries: gastric intubation of Streptococcus mutans whole cells or cell walls induces protective immunity in gnotobiotic rats.

Gnotobiotic rats were given Streptococcus mutans 6715 whole cells (WC), purified cell walls (CW), or cell wall lysate by gastric intubation (GI), and assessments were made of humoral immune responses in serum and saliva and of caries protection. Levels of secretory immunoglobulin A (IgA) and IgG antibodies to S. mutans WC in saliva samples from experimental rats were determined by an enzyme-linked immunosorbent assay. Serum antibody levels of the IgM, IgG, and IgA isotypes were also determined. Similar levels of salivary antibodies were induced in rats given S. mutans WC or CW by GI, whereas lower salivary antibody titers were observed in rats given cell wall lysate by the oral route. The level of serum antibodies in the various groups of rats also reflected the oral antigen used. The specificity of salivary IgA and serum IgG antibodies in the various groups of rats was determined by enzyme-linked immunosorbent assay with lipoteichoic acid, serotype g carbohydrate, dextran, CW, and WC as coating antigens. Salivary IgA and serum IgG antibodies in rats given S. mutans WC or CW by GI were primarily directed to lipoteichoic acid and serotype g carbohydrate. The presence of salivary IgA antibodies to S. mutans in rats given either S. mutans WC or CW by GI correlated with a significant reduction in the levels of plaque, numbers of viable S. mutans in plaque, and caries scores when compared with the control animals (infected only). These results demonstrate that particulate antigens of S. mutans induce salivary immune responses when given by GI to gnotobiotic rats and that the presence of these antibodies correlates with caries protection.     

Effective immunity to dental caries: enhancement of salivary anti-Streptococcus mutans antibody responses with oral adjuvants.

In the present study, we compared the ability of the soluble adjuvants concanavalin A (ConA), muramyl dipeptide (MDP), and peptidoglycan (PG) to enhance immune responses to orally administered particulate antigens of Streptococcus mutans 6715 in gnotobiotic rats. The isotype and levels of antibody in saliva and in serum from experimental rats were determined by an enzyme-linked immunosorbent assay using S. mutans whole cells (WC) as the coating antigen. The specificities of salivary and serum immunoglobulin A (IgA) antibodies to particulate S. mutans antigens, lipoteichoic acid, S. mutans serotype g carbohydrate, and dextran were also determined. When 50 micrograms of ConA was used as the oral adjuvant with S. mutans 6715 WC immunogen, a slight enhancement of immune responses was obtained. A higher dose of ConA suppressed humoral responses to the immunogen. Enhanced immune responses, especially of the IgA isotype, in both serum and saliva were induced in gnotobiotic rats given MDP and either S. mutans 6715 WC or purified cell walls (CW) by gastric intubation. Elevated IgA antibody levels to CW, lipoteichoic acid, and carbohydrate were observed in rats given S. mutans WC and MDP by gastric intubation, whereas oral immunization with S. mutans CW and MDP resulted in higher antibody levels to CW and carbohydrate and lower levels to lipoteichoic acid when compared with the antibody levels in rats given antigen alone. Rats orally immunized with either S. mutans WC or CW and MDP and challenged with virulent S. mutans 6715 exhibited significantly (P less than or equal to 0.05) lower plaque scores, numbers of viable S. mutans in plaque, and caries scores than did rats immunized with antigen alone or in infected-only controls. In another series of experiments, a PG fraction derived from S. mutans 6715 CW was assessed for adjuvant properties. The oral administration of PG and either S. mutans WC or CW induced good salivary and serum IgA antibody responses. The specificity of the antibodies was similar to that obtained in rats given antigen and MDP. Rats receiving either S. mutans WC or CW and PG and challenged with virulent S. mutans 6715 had lower plaque scores, fewer numbers of viable S. mutans in plaque, and lower caries activity than did infected rats receiving S. mutans WC or CW immunogen alone. These results provide evidence that soluble adjuvants derived from the gram-positive bacterial CW, e.g., MDP and PG, are effective oral adjuvants and augment IgA immune responses to particulate S. mutans antigens which are protective against the mucosally associated disease, dental caries.     

Haptenated streptococcal antigens elicit either T cell-dependent type 1 or T cell-independent type 2 immune responses

Antigens of Streptococcus mutans 6715 (alternatively designated serotype g Streptococcus sobrinus), including whole cells (WC g), cell walls (CW g), peptidoglycan (PG g) and serotype carbohydrate (Ml g) were coupled with trinitrophenyl (TNP), and the nature of the immune response to each immunogen was determined in normal and X-linked immunodeficient (xid) murine spleen cell cultures. Responses to TNP-WC g, -CW g and -PG g and to the classical type 1 antigen TNP-Brucella abortus occurred in both xid and normal splenic cultures, while TNP-Ml g only triggered immune responses in normal spleen cell cultures, suggesting that the former three antigens are type 1 and the latter type 2. Further support for the type 2 nature of TNP-Ml g was the finding that Peyer's patch cell cultures from both xid (which contain mature B cells) and normal mice supported responses to TNP-Ml g and TNP-Ficoll, while xid splenic cultures failed to support responses to either type 2 antigen. The three type 1 TNP-S. mutans antigens induced responses in nude spleen cell and in purified splenic B cell cultures, but required T cells for in vitro responses to lower doses of immunogen. On the other hand, TNP-Ml g induced anti-TNP PFC responses at several antigen concentrations in purified B cell cultures, without requirement for added T cells. These studies show that the intact S. mutans cell, as well as CW g and PG g, acts as a T cell-dependent (TD) type 1 antigen, while the serotype carbohydrate (Ml g) induces a T cell-independent (TI) type 2 response. Thus, the intact bacterium is a TD type 1 antigen, whereas its purified components are either type 1 or type 2 antigens and differ significantly in terms of their T cell dependence.     

Purification and immunological characterization of a rhamnose-glucose antigen from Streptococcus mutans 6517-T2 (serotype g).

The serotype antigens of Streptococcus mutans have been described as cell wall-associated polysaccharides. In this study, an additional wall polysaccharide antigen was purified and characterized from S. mutans strain 6715-T2, a mutant of 6715 (serotype g). Strain 6715-T2 lost the serotype antigen during animal passage. Rhamnose-containing carbohydrate fractions were solubilized from bacterial cells by extraction with 5% trichloroacetic acid at 4 degrees C for 18 h and with 0.01 N HCl at 100 degrees C for 20 min. Extracts were combined and purified on columns of diethylaminoethyl-Sephadex A-25 and Sephadex G-100. The purified sample contained 59% rhamnose, 31% glucose, 2.2% protein, and 0.24% phosphorus. The purified rhamnose-glucose polysaccharide (RGP/6715-T2) reacted strongly with antisera to whole cells of 6715-T2. Agar gel diffusion and comparative immunoelectrophoresis studies revealed that RGP/6715-T2 was serologically distinct from the serotype g and d polysaccharide antigens. These techniques also indicated immunological identity between RGP/6715-T2 and RGP/B13, a rhamnose-glucose polymer previously isolated from S. mutans B13, a serotype d strain. Antigen immunologically identical to RGP/6715-T2 was detected both in Rantz-Randall extracts from whole cells of S. mutans strains 6715, OMZ-65, and 6715-PT and in extracts from cells of 6715-T2 and C307, two mutant serotype g strains that lacked the serotype g antigen.     

Natural transmission of Streptococcus sobrinus in rats: saliva and serum antibody responses to colonization.

One hundred and twenty weanling rats fed diet NIH 2000 that were free of Streptococcus sobrinus and other mutans streptococci were employed in this study. Sixty rats were inoculated orally with S. sobrinus 6715. Each infected rat (donor) was paired and housed with an uninfected recipient. Saliva and serum samples were collected from 24 (12 donor and 12 recipient) rats at the baseline (day 0) and from groups of 12 recipients sacrificed on days 10, 24, 38, and 52, and the level of infection with S. sobrinus was monitored. Salivary immunoglobulin A (IgA) and IgG and serum IgM and IgG antibodies reactive with whole cells (WC), glucosyltransferase (GTF), and the serotype carbohydrate (g) of S. sobrinus were measured by an indirect enzyme-linked immunosorbent assay. Although the rats were free of S. sobrinus and other mutans streptococci at baseline, they exhibited salivary IgA and serum IgM antibodies reactive with S. sobrinus WC, GTF, and g and serum IgG antibodies reactive with WC and GTF. Infection of recipients with S. sobrinus did not induce salivary antibodies reactive with WC, GTF, or g. In contrast, increases in serum IgM and IgG antibodies reactive with WC and serum IgM antibodies reactive with g were observed.     

Effect of neonatal thymectomy on immune responses of rats to Streptococcus mutans.

The effect of neonatal thymectomy on secretory and systemic antibody responses in rats was studied. Groups of normal or thymectomized (Tx) rats were infected or immunized and infected with Streptococcus mutans 6715. Tx rats exhibited a significantly lower level of salivary immunoglobulin A (IgA) antibody to S. mutans after a 45- to 65-day infection. Similarly, after multiple local injections of formalinized S. mutans, Tx rats showed a delay in the appearance and lower levels of salivary IgA antibody to S. mutans. Serum IgG antibody levels were also decreased in Tx rats with both experimental protocols. In contrast, salivary IgG and serum IgM anti-S. mutans activity in Tx and normal rats were similar during the experiments. These results demonstrated that thymus deprivation at birth produces profound effects on the ability of rats to manifest secretory IgA antibody responses to the pathogenic microorganism S. mutans.     

Enhancement of Murine Immune Responses to Orally Administered Haptenated Streptococcus mutans

The induction of immune responses to orally administered trinitrophenyl (TNP)-haptenated Streptococcus mutans and its enhancement with muramyldipeptide (MDP), peptidoglycan (PG), and concanavalin A (Con A) were investigated in lipopolysaccharide (LPS)-non-responsive C3H/HeJ mice and the syngeneic, LPS-responsive C3H/HeN strain. Both mouse strains manifested similar immune responses, primarily of the IgM isotype, after a single gastric intubation (GI) with TNP-S. mutans. However, when groups of animals were first carrier-primed by GI with S. mutans for 2 consecutive days, followed by a single GI with TNP-S. mutans 1 week later, C3H/HeJ mice gave a significantly higher (P less than or equal to 0.01) splenic IgA anti-TNP plaque-forming cell (PFC) response than identically treated C3H/HeN mice. Furthermore, saliva, urine and serum from these C3H/HeJ mice possessed high levels of IgA anti-TNP antibodies as determined by the enzyme-linked immunosorbent assay, whereas C3H/HeN mice exhibited low antibody levels. Oral administration of Con A (either 250 micrograms or 500 micrograms/mouse) or purified PG (1 mg/mouse) at the time of TNP-S. mutans immunization resulted in significantly (P less than or equal to 0.01) enhanced splenic IgA anti-TNP PFC responses, especially in C3H/HeJ mice. On the other hand, MDP promoted IgA anti-TNP PFC responses in LPS-responsive C3H/HeN mice but did not augment responses in C3H/HeJ animals. A similar immune response pattern was seen when antibody levels were measured in serum, saliva, and urine of both mouse strains. These results demonstrate that haptenated S. mutans is a good antigen for the induction of high IgA responses in orally immunized C3H/HeJ mice and that this high response can be enhanced with the adjuvants Con A and PG. However, MDP is ineffective in C3H/HeJ mice but enhances IgA responses in normal LPS-responsive C3H/HeN animals.     

Virulence of Streptococcus mutans: immunochemical characterization of a serotype g-defective mutant (C307).

A mutant of Streptococcus mutans 6715 wild type designated C307 has been shown to possess a small amount of either Lancefield- or Rantz-Randall-extractable serotype antigen. Quantitative analysis employing combined immunoabsorption and radial immunodiffusion of anti-S. mutans serotype-specific serum demonstrated that C307 exhibited less than 1% of the amount of serotype g antigen normally expressed in S. mutans 6715 wild type.     

Oral adjuvants enhance IgA responses to Streptococcus mutans

The induction of immune responses to orally-administered trinitrophenyl (TNP)-haptenated Streptococcus mutans or its cell wall components and enhancement of immune responses with oral adjuvants has been studied in high IgA responsive C3H/HeJ mice and in gnotobiotic rats. Gastric intubation of TNP-S. mutans to LPS non-responsive C3H/HeJ or syngeneic, LPS responsive C3H/HeN mice induced IgA responses as determined by measuring splenic plaque-forming cell (PFC) responses and IgA anti-TNP antibodies in serum, saliva, and urine. Higher IgA responses always occurred in C3H/HeJ mice given oral S. mutans antigen than similarly treated C3H/HeN animals. Oral administration of the adjuvants concanavalin A or S. mutans cell wall peptidoglycan (PG) with antigen resulted in augmented IgA responses, especially in C3H/HeJ mice. On the other hand, oral administration of muramyl dipeptide (MDP) with antigen boosted anti-TNP responses in C3H/HeN, but not in C3H/HeJ, mice. Gnotobiotic rats given S. mutans whole cells (WC) or purified cell walls (CW) by the oral route exhibited a salivary IgA immune response which was potentiated greater than twofold when antigen was given with PG or MDP. In other studies, S. mutans WC or CW antigen in water-oil-water (W/O/W) emulsion or liposomes was administered by gastric intubation to rats. Significant salivary IgA responses were induced with these antigen-adjuvant preparations. Although rats given S. mutans WC or CW were protected from S. mutans challenge, the greatest degree of caries immunity was obtained in animals which received antigen and adjuvant and which exhibited significant salivary IgA antibody levels. In preliminary studies, it was observed that local injection of rats in the salivary gland region with a ribosomal preparation from S. mutans resulted in a significant salivary IgA response and caries immunity. The potential for soluble and lipid carrier adjuvants in oral vaccines for induction of protective antibodies to S. mutans is discussed.     

Virulence of Streptococcus mutans: cariogenicity of S. mutans in adult gnotobiotic rats.

Gnotobiotic rats infected with Streptococcus mutant 6715, mutans C211 at 45 days of age on provided a purified diet containing 5% sucrose developed carious lesions on buccal, sulcal, and proximal molar surfaces within 15 days (60 days of age). The level of caries increased significantly (P less than or equal to 0.01) within the next 15 days (by day 75), an extensive decay was observed on all three molar surfaces of 90-day-old infected rats (45 days after challenge). Mutant C211 was previously shown to exhibit increased glucosyltransferase activity and greater adherence and virulence than S. mutans 6715 wild type (wt). Gnotobiotic rats (90 days of age) infected with either S. mutans AHT or S. mutans 6715 (wt) at 45 days of age developed significantly (P less than or equal to 0.01) fewer caries on all molar surfaces than rats of the same age that were infected with S. mutans 6715, mutant C211. The level of plaque increased 2-fold, and the number of viable S. mutans in plaque increased 10-fold between days 60 and 90 in rats infected with S. mutans 6715, mutant C211. Ninety-day-old rats infected with either S. mutans AHT or S. mutans 6715 (wt) had similar levels of plaque and numbers of S. mutans in plaque; however, these values were two- to fourfold lower than those observed in rats of the same age that were infected with S. mutans 6715, mutant C211.     

Humoral and cell-mediated responses to a ribosomal preparation from Streptococcus mutans.

Streptococcus mutans 6715 ribosomes disrupted in a Braun homogenizer were isolated in sodium dodecyl sulfate by differential centrifugation. This preparation contained 80% RNA and 20% protein, and carbohydrate was not detected by phenol-sulfuric acid and methyl pentose assays. The sedimentation coefficient of the ribosomes was 70S. After dialysis in 0.01 M phosphate buffer containing 10(-4) M MgCl2, the ribosomes dissociated into 54S and 32S particles. Leukocytes from rabbits immunized intramuscularly with the ribosomal preparation showed transformation and migration indices of 13.0 and 0.71, which were significantly different (P less than 0.05) from the respective indices of 0.9 and 0.98 in nonimmunized animals. Hyperimmune serum from these rabbits agglutinated representative Formalin-killed strains of all seven serotypes of S. mutans, inhibited adherence of live S. mutans 6715 to glass, and agglutinated S. mutans 6715 ribosomes adsorbed upon erythrocytes. These findings suggested that animals immunized with S. mutans ribosomes may be protected from caries caused by any of the seven serotypes of this organism.     

Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions.

Quantitative analyses of cell walls from Streptococcus mutans Ingbritt grown under carbohydrate limitation in the chemostat showed that growth conditions had no statistically significant effect on the composition of polysaccharide, peptidoglycan, or the proportion of polysaccharide in the cell wall. Lysis of cell wall preparations with a muramidase supported this conclusion and further indicated that there was little difference in their overall structure. In contrast, there was a consistent difference between the rates of lysis by this enzyme of organisms grown in 0.2% glucose and 0.5% glucose. Extremes of pH or dilution rate essentially did not influence the immunogenicity of type c antigen in whole organisms irrespective of whether the carbohydrate source was glucose or sucrose. However, differences were found in the immunogenicity of lipoteichoic acid under similar circumstances. The results indicated there was an inherent phenotypic stability in the cell walls of S. mutans Ingbritt despite changes in pH, generation time, and carbohydrate source, and that any changes that did occur were probably due to associated cell-surface components.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of antibodies to Streptococcus mutans surface antigens

An ELISA was developed to quantitate the level of antibodies to various cell surface antigens of the Gram positive bacterium. Streptococcus mutans. Whole cells and purified cell wall components of S. mutans, lipoteichoic acid (LTA) from Streptococcus pyogenes, and dextran T 2000 were employed as coating antigens in this study. Cell walls of S. mutans were purified by mechanical disruption of whole cells followed by differential centrifugation and proteolytic enzyme treatment. Serotype-specific carbohydrate was purified from an autoclaved, lyophilized S. mutans whole cell preparation by column chromatography. LTA was prepared by Sepharose 4B chromatography of a phenol-water extract of S. pyogenes and used for detection of anti-polyglycerophosphate (PGP) antibodies. A rabbit antiserum to S. mutans 6715 (serotype g), which precipitated with purified carbohydrate antigen (RR g), gave good reactions with purified cell walls and whole cells of S. mutans 6715, less activity with RR g and low activity to LTA and dextran when tested by ELISA. Adsorption of this antiserum with whole cells of S. pyogenes resulted in antibody activity with specificity only to the serotype carbohydrate. The specificity of the antibody for homologous coating antigen was RR g > cell wall > whole cells. An antiserum to S. mutans MT573 (serotype e) contained antibody predominantly to LTA, whereas, anti- S. mutans MT703 (serotype e) reacted with both dextran and LTA; however, the activity to LTA was removed by prior adsorption of the antiserum with S. pyogenes cells. This treatment did not alter the antibody activity to dextran. To establish the sensitivity of ELISA, a purified IgG anti-serotype carbohydrate antibody was prepared by adsorption of anti-S, mutans 6715 antiserum with a mutant of S. mutans which lacks serotype carbohydrate followed by adsorption and elution of specific antibodies from S. mutans 6715 whole cells. The minimum level of sensitivity of ELISA was 12.5 ng of IgG anti-serotype carbohydrate.     

Surface glycoproteins as markers of the cellular status of B chronic lymphocytic leukaemia lymphocytes.

In order to indirectly assess T and B cell function in vivo in spontaneously autoimmune MRL mice, IgM plaque forming cell (PFC) responses to the thymus-independent antigens type III pneumococcal polysaccharide (S3) and polyvinylpyrrolidone (PVP) were determined. Both MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp+/+ (MRL/n) mice responded well to S3 and, in fact, low doses of S3 which are not immunogenic in normal strains of mice elicited good responses in MRL mice. PVP was less immunogenic than S3, however, doses of PVP which are considered sub-immunogenic in normal mice did elicit responses in MRL mice. The effect of ageing on S3 and PVP responsiveness in MRL mice was also determined. Responses to S3 and PVP declined minimally in MRL/l mice and were unchanged in MRL/n mice. Amplifier T cell (TA) activity in MRL mice was indirectly assessed by determining the effect on concanavalin A or anti-lymphocyte serum on PFC responses to S3 and PVP. Whereas significant enhancement of the S3 and PVP IgM PFC responses occurred in MRL/n mice, neither method elicited remarkable enhancement in MRL/l mice. The lack of IgM enhancement was not due to altered kinetics of activation nor to a switch to IgG PFC responses. Possible reasons for the apparent dysfunction of TA in MRL/l mice are discussed.     

Function of anti-Streptococcus mutans antibodies: anti-ribosomal antibodies inhibit acid production, growth, and glucose phosphotransferase activity.

Antibodies induced in sera and saliva of rats and rabbits immunized with ribosomal preparations from Streptococcus mutans 6715 inhibited transport of glucose by the phosphotransferase system by greater than 60%, acid production from sucrose by greater than 95%, and growth of the homologous S. mutans by greater than 59%. Inhibition of growth and acid production by immune sera and saliva were abrogated by prior adsorption with S. mutans 6715 whole cells, glucosyltransferase, lipoteichoic acid, or alpha 1-6 or alpha 1-3 dextran. These results indicate that antibodies induced to an S. mutans ribosomal preparation react with cell surface determinants and suggest that the antibodies inhibit sucrose-induced acid formation and growth of virulent S. mutans by neutralizing the glucose-phosphotransferase system.     

Effective immunity to dental caries: dose-dependent studies of secretory immunity by oral administration of Streptococcus mutans to rats.

Rats (COBS/CD) provided Formalin-killed Streptococcus mutans 6715, C211 in their drinking water (10(8) to 10(9) equivalent colony-forming units [CFU] per ml) had high levels of specific antibodies in saliva, colostrum, and milk. Rats provided a lower concentration of S. mutans antigen (10(7) CFU per ml) in water had agglutinin titers in secretions that were similar to those in controls. Gnotobiotic rats provided S. mutans antigen in food (10(7) to 10(8) equivalent CFU per g of diet) manifested a secretory immune response as evidenced by the presence of specific immunoglobulin A antibodies in saliva, colostrum, and milk. Gnotobiotic rats provided a higher concentration of antigen (10(9) CFU per g) in food had levels of specific antibodies in their secretions that were similar to those in controls. No significant antibody activity to S. mutans was observed in sera of any group of animals. Furthermore, the presence of specific salivary immunoglobulin A antibodies in gnotobiotic rats correlated with a reduction in the level of plaque, numbers of viable S. mutans in plaque, and levels of S. mutans-induced dental caries. This paper discusses the importance of antigen dosage for induction of a secretory immune response that is protective against S. mutans-induced dental caries.     

Virulence of Streptococcus mutans: a sensitive method for evaluating cariogenicity in young gnotobiotic rats.

Gnotobiotic rats infected with Streptococcus mutans 6715 at 19 days of age and fed a purified diet (305) containing 5% sucrose developed extensive caries lesions on all molar surfaces within 16 days (35 days of age). Approximately twice as many lesions developed when infected rats were maintained until 45 days of age, whereas noninfected rats did not develop caries when fed diet 305. Gnotobiotic rats infected with S. mutans 6715 and fed a purified diet containing no sucrose (300) until day 25 and subsequently fed diet 305 for 10 days developed lesions similar to rats fed diet 305 for 16 days. Furthermore, rats infected with S. mutans 6715 and fed diet 300 until 45 days of age developed approximately one-half the smooth surface lesions as infected rats fed diet 305 for the same length of time. The level of caries on buccal and proximal molar surfaces in 45-day-old gnotobiotic rats varied when animals were infected with S. mutans AHT, BHT, NCTC 10449, 6715, or LM-7. Animals infected with S. mutans AHT showed more severe lesions on the buccal surfaces than those observed in animals infected with the other strains of S. mutans tested, whereas S. mutans 6715 caused significantly more caries on proximal surfaces. On the other hand, rats infected with S. mutans LM-7 exhibited the lowest level of caries on all molar surfaces of the five strains of S. mutans tested.     

Oral ecology and virulence of Lactobacillus casei and Streptococcus mutans in gnotobiotic rats.

Lactobacilli comprise a small percentage of the normal oral microbial flora of humans and are isolated commonly from saliva and frequently from an active caries lesion. We have compared the pathogenesis and colonization pattern of Lactobacillus casei with that of Streptococcus mutans strain 6715 in gnotobiotic rats. Of the two L. casei strains tested, L. casei strain ATCC 4646 caused slightly more caries than L. casei strain ATCC 11578. However, the level of caries induced by either L. casei strain was significantly lower (P less than 0.01) than that observed in similar-aged rats monoassociated with S. mutans strain 6715. When groups of rats were infected with mixtures of L. casei strain ATCC 4646 and S. mutans strain 6715, or with L. casei followed by S. mutans, higher numbers of L. casei than S. mutans were found associated with the tongue and in saliva; S. mutans always predominated in plaque. The level of caries observed in these groups of rats was similar to that seen with rats monoassociated with S. mutans except when L. casei comprised greater than 1% of the plaque microflora. In this latter situation, the level of caries was significantly lower (P less than or equal to 0.05) than that obtained in S. mutans-monoassociated rats. The results of this study suggest that L. casei colonizes sites in the oral cavity (including the tongue and saliva) other than the tooth surface in rats. The effect of L. casei in plaque toward reduction of S. mutans-induced dental caries in rats is discussed.     

Extractability of Cell Wall Polysaccharide from Lactobacilli and Streptococci by Autoclaving and by Dilute Acid

Autoclaving cell wall of Streptococcus mutans Ingbritt for 15 min under the Rantz and Randall conditions released one-tenth of the total cell wall carbohydrate, whereas two-thirds was extracted after autoclaving for 180 min. The extract contained the serotype c-specific antigen but lacked the lipoteichoic acid component extracted when whole cells were autoclaved. Autoclaving cell wall preparations from other strains of S. mutans and also Streptococcus salivarius and Streptococcus mitis in 0.85% NaCl for 180 min released the major proportion of the wall polysaccharide fraction. Approximately 50 to 90% of wall carbohydrate of Lactobacillus fermentum and Lactobacillus casei was released when cell wall preparations were autoclaved in 0.85% NaCl for 180 min. For wall preparations from several strains of S. mutans, autoclaving for 60 min at pH 3.75 released only 39 to 62% of wall carbohydrate, whereas almost total release could be achieved with the lactobacilli. Heating S. mutans Ingbritt cell wall for 24 h at 60 degrees C in 0.1 N H(2)SO(4) released only two-thirds of the wall carbohydrate; by comparison nearly all of the wall carbohydrate was released in 3 h from L. casei and L. fermentum. Autoclaving L. casei cell wall and purified soluble wall fractions hydrolyzed the phosphodiester bond between the polysaccharide and peptidoglycan. This was shown by the release of reactive N-acetylhexosamine in both cases and the presence of a phosphomonoester in the autoclaved soluble wall fractions. The results indicate that autoclaving can hydrolyze covalent linkages, and this must be considered when the Rantz and Randall procedure is used to obtain antigen preparations.     

Isolation and immunochemical characterization of the group-specific antigen of Streptococcus mutants 6715.

The group d antigen of Streptococcus mutans 6515 was isolated from a buffer (pH 7.3)-boiled extract of whole cells and analyzed immunochemically. Rabbits immunized in three different fashions with whole S. mutans 6715 each responded to the same antigenic cell surface component. This presumptive major antigen was found in culture supernatant, sonically treated supernatant, acid and buffer extracts of whole cells, and trichloroacetic acid extract of cell membranes. A crude preparation of this antigen could completely inhibit antibody-mediated cell (S. mutans 6715) agglutination in a spectrophotometric analysis. The antigen was purified from buffer-boiled extracts by gel filtration on columns of Sepharose 4B. The antigen did not migrate to the anode on electrophoresis nor did it contain appreciable quantities of phosphorus, glycerol, or ribitol. This suggested that the d antigenicity did not reside in a teichoic acid. The d antigen contained galactose and glucose as the sole saccharides, in a ratio of 5.9:1.0. Protein (9.5%) appeared to be a portion of the antigen, although Pronase-digested antigen retained the same electrophoretic mobility and could precipitate virtually all (98.6%) purified antibody directed to the intact antigen. The data obtained from hapten innvolved. Glucose also contributed to the immunodominant region. Antibody directed to the d antigen may be of importance in the inhibition of adherence phenomena manifested by S. mutans organisms of the d group.