Conventional Sprague-Dawley Rats Research Articles

Changes in Bowel Microbiota Induced by Feeding Weanlings Resistant Starch Stimulate Transcriptomic and Physiological Responses

The ability to predictably engineer the composition of bowel microbial communities (microbiota) using dietary components is important because of the reported associations of altered microbiota composition with medical conditions. In a synecological study, weanling conventional Sprague-Dawley rats (21 days old) were fed a basal diet (BD) or a diet supplemented with resistant starch (RS) at 5%, 2.5%, or 1.25% for 28 days. Pyrosequencing of 16S rRNA genes and temporal temperature gradient electrophoresis (TTGE) profiles in the colonic digesta showed that rats fed RS had altered microbiota compositions due to blooms of Bacteroidetes and Actinobacteria. The altered microbiota was associated with changes in colonic short-chain fatty acid (SCFA) concentrations, colonic-tissue gene expression (Gsta2 and Ela1), and host physiology (serum metabolite profiles and colonic goblet cell numbers). Comparisons between germ-free and conventional rats showed that transcriptional and serum metabolite differences were mediated by the microbiota and were not the direct result of diet composition. Altered transcriptomic and physiological responses may reflect the young host's attempts to maintain homeostasis as a consequence of exposure to a new collection of bacteria and their associated biochemistry.

Intranasal immunization against dental caries with a Streptococcus mutans-enriched fimbrial preparation.

Streptococcus mutans has been identified as the major etiological agent of human dental caries. The first step in the initiation of infection by this pathogenic bacterium is its attachment (i.e., through bacterial surface proteins such as glucosyltransferases, P1, glucan-binding proteins, and fimbriae) to a suitable receptor. It is hypothesized that a mucosal vaccine against a combination of S. mutans surface proteins would protect against dental caries by inducing specific salivary immunoglobulin A (IgA) antibodies which may reduce bacterial pathogenesis and adhesion to the tooth surface by affecting several adhesins simultaneously. Conventional Sprague-Dawley rats, infected with S. mutans at 18 to 20 days of age, were intranasally immunized with a mixture of S. mutans surface proteins, enriched for fimbriae and conjugated with cholera toxin B subunit (CTB) plus free cholera toxin (CT) at 13, 15, 22, 29, and 36 days of age (group A). Control rats were either not immunized (group B) or immunized with adjuvant alone (CTB and CT [group C]). At the termination of the study (when rats were 46 days of age), immunized animals (group A) had significantly (P < 0.05) higher salivary IgA and serum IgG antibody responses to the mixture of surface proteins and to whole bacterial cells than did the other two groups (B and C). No significant differences were found in the average numbers of recovered S. mutans cells among groups. However, statistically fewer smooth-surface enamel lesions (buccal and lingual) were detected in the immunized group than in the two other groups. Therefore, a mixture of S. mutans surface proteins, enriched with fimbria components, appears to be a promising immunogen candidate for a mucosal vaccine against dental caries.

Clindamycin-induced alterations in intestinal microflora-associated characteristics in rats.

Conventional Sprague-Dawley rats were treated with clindamycin, 40 mg/kg/day and 0.04 mg/kg/day, for 5 days. At a dose of 40 mg/kg/day, microflora-associated characteristics (MACs), such as shape, color, and consistency of feces, proteolytic activity, electrophoretic pattern, and cholesterol and bilirubin metabolism were transformed into values like those found in germfree rats: germfree animal characteristics (GACs). The effect on the proteolytic activity lasted longest. It did not disappear until one or two enemas with cecal contents from intact conventional rats were administered. At a dose of 0.04 mg/kg/day, effects on the proteolytic activity and cholesterol metabolism were seen. With the exception of one rat, the effect on proteolytic activity did not disappear until one or two enemas were given. The results indicate that clindamycin, even in very small daily doses, has a profound and long-lasting influence on many intestinal MACs in rats.

The influence of certain added water-borne trace elements on the percentage bacterial composition of tooth fissure plaque from conventional sprague-Dawley rats

The effects of 12 water-borne trace elements (Hg, Sn, V, Mo, Mn, Fe, F, AI, Sr, Pb, Se and Li) on the percentage bacterial composition of tooth fissure plaque from Sprague-Dawley rats were investigated. The diet contained 65 per cent sucrose and distilled water supplemented with one or other of the trace elements for 14 days. The major plaque bacteria isolated from the rats were Streptococcus mutans, actinomycetes, neisseria, veillonellae, lactobacilli and actinobacilli. Rats receiving Hg, V, Mo, Fe and F had significantly lower levels of Strep. mutans in their plaque. Those receiving Hg and V had significantly raised levels of actinomycetes; those receiving Mo and F had raised levels of Gram-negative bacteria (neisseriae and Gram-negative rods). In-vitro experiments suggested that the changes in the plaque flora may be mediated by differential effects on the growth of plaque bacteria. Rats receiving drinking water supplemented with the other trace elements harboured a plaque flora not significantly different from the control animals.

Relationship Between the Populations of Streptococcus mutans in the Mouth and Feces of Conventional Sprague-Dawley Rats

The relationship between oral and fecal S mutans populations has been studied in rats fed high sucrose or glucose diet. All molar teeth were extracted either prior to or following oral challenge with strain 6715. In certain instances this procedure was supplemented with amputation of the incisors and disinfection of the remaining incisor surface area with iodine solution. The results suggest that in rats the colonization of S mutans on the teeth is required for its presence in the intestinal canal as well as the mouth. The significance of the observations has been discussed.

Impaired enteric degradation of pancreatic endopeptidases in antibiotic-treated rats.

Conventional Sprague-Dawley rats were fed by gastric tube for 5 days with either benzylpenicillin, ampicillin, doxycycline, or clindamycin. In contrast to the pretreatment period fibrinolytic activity and active and immunoreactive trypsin and immunoreactive elastase were present in fecal extracts after 4 days of antibiotic administration. This is consistent with findings in germfree rats and represents an alteration in the intestinal microflora. The germfree characteristics persisted until a suspension of cecal contents from normal rats was administered by enema on the 26th day. In fecal extracts from the clindamycin-treated rats considerable amounts of active and immunoreactive enzymes were, however, still found 10 days after the enema. A possible explanation is that clindamycin or a metabolite remains in the intestinal tract for a long time. The pathophysiological significance of this finding is unknown.

Environmental origin of natural antibodies to teichoic acid

In an effort to determine the origin of natural antibodies to teichoic acid, rats were fed a sterile liquid diet free of detectable teichoic acid and virtually free of gram-positive bacteria. Both germ-free and conventional Sprague-Dawley rats raised on this diet failed to produce antibodies to polyglycerophosphate, whereas 100% of their counterparts fed the usual teichoic acid-containing diet did produce these antibodies. The intestinal flora was similar in both groups of animals. When the test animals were immunized intraperitoneally or orally with gram-positive bacteria, 100% displayed immunocompetency by producing significant levels of antibody. These results demonstrate the environmental nature of the antigenic stimulus for these antibodies and suggest the importance of food as the major source of stimulation. The experimental model described here furnishes a valuable tool for studies of immunologic responses where a single known specificity and a controlled system would be advantageous.

Effect of age, sex, and intestinal flora on the induction of colon tumors in rats.

Germfree and conventional Sprague-Dawley rats were assessed for their susceptibility to intrarectally injected N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU). Adenocarcinoma of the colon was induced in germfree and conventional rats by both MNNG and MNU. The colons of germfree rats were more susceptible to the direct-acting carcinogens, as manifested by earlier morbidity and development of colon tumors (50% tumors within 30-35 wk), than were those of conventional rats (50% colon tumors within 48-50 wk). Germfree and conventional male rats were more susceptible to the carconogens than were their female germfree and conventional counterparts. Young (30 days old at the start of the experiment) germfree rats developed colon tumors more quickly (15-20 wk) than did older (60 days) germfree rats after intrarectal injections of MNNG. No colon tumors were observed in germfree and conventional rats after 75 weekly intrarectal injections with a buffer. Transplantation of an adenocarcinoma induced with MNU in a female rat to germfree and conventional rats showed that it was easily transplantable, required no immunosuppression, and had essentially the same morphologic characteristics as did the primary tumor.

Inhibition of rat dental caries by dextranase from a strain of Spicaria violacea.

Dextranase AD17 obtained from a culture liquor of a strain of Spicaria violacea was assessed for its ability to inhibit the development of dental caries in conventional Sprague-Dawley rats which had been infected with one of the Streptococcus mutans strains. MT6R (serotype c), OMZ 176R (d), or MT-703R (e). These experiments showed that caries was significantly inhibited when rats were given cariogenic diets No. 2000 and drinking water containing AD17 at a concentration of 10 units/g, as compared to control rats not given dextranase. The inhibitory effects of AD17 were more prominent in smooth surface caries than in total caries. AD17 had a tendency to retard both the establishment of inoculated S. mutans and plaque deposition on tooth surfaces. However, S. mutans could be implanted in the rat oral cavity after repeated inoculation of the bacteria, even in the presence of AD17. These results suggest that the anticaries activity of AD17 is due to not only inhibition of adherence of S. mutans cells on tooth surfaces but also to physiochemical changes of dental plaque formed under the enzymatic action of AD17. Preliminary histophatological examination showed that AD17 had no significant toxicity in rats.

Role of sucrose in colonization of Streptococcus mutans in conventional Sprague-Dawley rats.

The role of sucrose in the colonization of S mutans strain 6715 in conventional Sprague-Dawley rats was studied. A diet with 56% sucrose favored the oral colonization of the test strain compared to diets with 56% glucose or fructose or to laboratory chow as determined by recoveries from extracted teeth ground in tissue grinders. S mutans strain 6715 cells became well established in all rats fed a high sucrose diet with cell inoculums ranging from 10(8) to the lowest effective dose of 10(5) CFU once orally administered; in rats on nonsucrose diets, inoculation with even the highest dose only infrequently resulted in the establishment of S mutans strain 6715. Sucrose- and glucose- grown cells appeared to behave similarly. Colonization of S mutans strain 6715 occurred in all rats fed diets with a sucrose content ranging from 56 to as low as 1%. The establishment of S mutans strain 6715 on the teeth of rats fed diets with a sucrose concentration of 0.1 or 0.01% was impaired and comparable to the diet containing 56% glucose. In rats fed a high glucose diet, uniform establishment and persistence of the test strain occurred after frequent inoculations with about 5 X 10(8) CFU. The colonization under these conditions appeared to be independent of the intestinal canal as a bacterial cell source. These data suggest the possibility that S mutans can establish itself in the human mouth in the absence of dietary sucrose. In rats fed a high glucose diet and inoculated with 10(7) CFU or less, the cells gradually disappeared from the teeth; in contrast, the test strain implanted well in rats fed the sucrose favors firmer attachment of initially weakly attached cells via in situ new glucan synthesis. S mutans strain 6715 also appeared to have some affinity for teeth in the absence of dietary sucrose that may be of ecological significance. Once firmly established in rats fed a high sucrose diet, S mutans strain 6715 maintained itself in high numbers on the teeth after a switch to a high glucose diet during a 14-week period.

Studies of the mechanism of sucrose-associated colonization of Streptococcus mutans on teeth of conventional rats.

The mechanism of the colonization of S mutans strain 6715 on teeth in the presence of sucrose has been investigated using conventional Sprague-Dawley rats. Experiments were performed using rats fed diets with 56% sucrose or 56% glucose; oral inoculations with sucrose-grown, extracellular glucan-coated cells and glucose-grown, uncoated cells; and teeth treated with dextran 2000 via its incorportation in the diet, or teeth left untreated. In all instances, sucrose was required for satisfactory colonization of the test strain. Preformed extracellular glucan, whether associated with the bacterial cell or with the tooth surface, did not facilitate bacterial colonization when compared with cells or teeth devoid of extracellular glucan under the conditions used. The evidence suggests that glucan-mediated attachment of S mutans strain 6715 in the presence of sucrose involves an initial phase in which the cells are relatively weakly associated with the tooth surface and a subsequent phase during which firmer attachment occurs via new glucan synthesis.

The effects of a dextranase from Spicaria violacea IFO 6120 on experimental dental caries in rat

A partially purified dextranase preparation (AD 17) from culture supernatant of a strain of Spicaria violacea was tested for the ability to inhibit the developmentof dental caries in conventional Sprague-Dawley rats which had been inoculated with one of the Streptococcus mutans strains MT6R (Bratthall's serotype c), OMZ176R (d), or MT703R (e). AD17 including in the cariogenic diet #2000 and drinking water inhibited the development of dental caries with statistical significance. Serotypes of S. mutans used in this experiment have been detected among Japanesechildren in various proportions. The results suggest a possibiity of clinical application of AD17 to human children for the purpose of caries prevention.

An environmental chamber for the study of the physiology of conventional and germ-free laboratory animals.

The design and performance of a fully-climatized environmental chamber for the study of the physiology of conventional or germ-free research animals are described. The chamber temperature can be regulated between 14°–36°C ± 1°C, the humidity between 45–95 ± 2% at 14°C and between 20–95 ± 2% at 36°C. Bilaterally symmetrical lighting is variable over 15 stages between 50–2,400 lux. The performance of the chamber was evaluated in a trial study in which the effect of different chamber temperatures on the O2-consumption of 24 conventional Sprague-Dawley rats held in groups of six to a cage with sexes separate. This showed a mean O2-consumption at 14° to 16°C of 743 ml/(kg·hr), at 18° to 20°C of 653 ml/(kg·hr) at 26° to 28°C of 629 ml/(kg·hr) and at 30° to 32°C of 627 ml/(kg·hr). The differences between O2-consumption at 14° to 16°C and 26° to 28°C and higher were significant (0.05 > p > 0.01).

AUTOANTIBODIES TO COLON IN GERMFREE RATS MONOCONTAMINATED WITH CLOSTRIDIUM DIFFICILE

Germfree rats monocontaminated with the anaerobic microorganisms Clostridium difficile or another Clostridium species (strain G 62) produce auto-antibodies to colon antigen. The antigen can be extracted with phenol water from the feces of germfree rats. Antibodies, demonstrable by means of passive hemagglutination of antigen sensitized sheep erythrocytes appear after monocontamination for 35 days or longer. The indirect immunofluorescence techniques, applied to sections of germfree rat colon, gave positive mucosal staining. The staining was similar to that obtained with sera from patients with ulcerative colitis or from rats immunized with rabbit colon. No antibodies were found in the sera of germfree rats, germfree rats monocontaminated with various other bacteria, conventional rats of germfree origin, or conventional Sprague-Dawley rats. Although the anti-colon antibodies of the Clostridium infected rats reacted with the same feces extract as the antibodies of ulcerative colitis patients or of rabbit colon immunized rats, their specificity was different. While the latter cross-react with polysaccharide from E. coli O14, those from the Clostridium-infected exgermfree rats did not. Rats monocontaminated with Cl. difficile also developed antibodies to this organism, but no cross-reaction between Cl. difficile antigen and colon antigen could be demonstrated. This speaks against breakage of tolerance by cross-reacting bacterial antigen as the cause of autoimmunity in these rats. Other possible mechanisms for autoantibody production in this model are immunogenic alteration of gastrointestinal mucins by bacterial degradation, adjuvant effects of bacterial products, or both.

The exposed germ-free pulp: Effects of topical corticosteroid medication and restoration

The dental pulps of germ-free and conventional Sprague-Dawley rats were surgically exposed and treated with corticosteroid and obtundent drugs and were either sealed by means of a temporary cement or left exposed to the oral environment. The results showed that, regardless of medication or closure, the pulps of conventional animals degenerated, whereas the pulps of germ-free animals healed uneventfully as substantiated by the formation of a reparative dentine bridge. The application of the steroid formula immediately following pulpal exposure was neither helpful nor harmful.

Properties of the rat submaxillary gland virus hemagglutinin and antihaemagglutinin and their incidence in apparently healthy gnotobiotic and conventional rats.

SUMMARY The properties of the rat submaxillary gland (RSMG) virus haemagglutinin and its serum antihaemagglutinin, and their incidence and relative titres in gnotobiotic and conventional Sprague-Dawley rats of various ages were investigated. The haemagglutinin was stable at -55° for as long as 2 1 2 years and retained its activity after 2 hr at 60°. Various carbohydrases and cobra venom had no effect upon its titre; however, 2 × crystallized trypsin and pronase reduced the titre substantially. The haemagglutinin was not recovered from the submaxillary glands of rats less than 8 weeks of age, though 3-, 5-, and 6-week-old animals had low serum antihaemagglutinin titres. A general correlation was observed between the haemagglutinin and antihaemagglutinin titres of the animals; higher serum antihaemagglutinin titres occurred in those rats with higher haemagglutination titres. No significant difference was usually seen between the haemagglutination titres of gnotobiotic and conventional rats of the same age. The antihaemagglutinin titres of some conventional rats, however, tended to be two- to fourfold greater than those of gnotobiotic animals of comparable age with identical haemagglutination titres. Sera which were heated at 56° for 30 min. absorbed with kaolin, incubated with trypsin, potassium or sodium periodate, or receptor-destroying enzyme showed no reduction in their antihaemagglutinin titres. Sucrose density-gradient centrifugation studies indicated that the antihaemagglutinin was associated with the 7 S fraction; however, some sera showed antihaemagglutinin activity in the 19 S fraction. The haemagglutinin of rat submaxillary gland virus was not inhibited by antisera prepared in mice against reovirus 3, Kilham’s rat virus, Toolan’s H-1 virus or Crawford’s minute virus of mice.

Effect of fluoride on hypomineralized areas in the molars of rats fed a cariogenic diet

Hypomineralized areas (HMA) were shown to exist in the molars of different types of young (22–24-day-old) rats, including germfree Sprague-Dawley rats. Under normal conditions these HMA undergo mineralization or maturation in conventional Sprague-Dawley rats fed a diet of laboratory chow. Concomitant with this mineralization an increase in resistance to decalcification occurred. Conversely, when young 22-day-old conventional rats were placed on a cariogenic diet, a great increase in HMA score during the first week on the diet was observed, but little or no increase during the following 2 weeks. When rats 21 days old were fed a cariogenic diet and treated topically with a solution containing 4500 μg/ml fluoride a decrease in HMA score was revealed (by silver nitrate staining) suggesting that mineralization had taken place under these conditions. Furthermore, this decrease in HMA score in the fluoride-treated group fed a cariogenic diet was even greater than that of littermate animals fed a chow diet. These data support the hypothesis that in rats fed a cariogenic diet an effective, topically applied agent might not only be cariostatic, but might even favour mineralization.

Cycasin excretion in conventional and germfree rats.

Urinary and fecal excretion of the naturally occurring toxic and carcinogenic glycoside, cycasin, β-D-glycosyloxyazoxymethane, was determined in germfree and conventional Sprague-Dawley male rats. Excretion in germfree rats was nearly quantitative while only 18 to 35% was recovered in conventional rats. Since cycasin is inert until acted upon by a β-glucosidase, the lack of enzymatic cleavage in germfree rats suggests the bacterial flora as the most likely source for the enzyme in conventional rats. Considerable variation was encountered in the amount of cycasin which was not recovered and was presumably metabolized in conventional rats. The possible significance of this variability is discussed in relation to toxicity and carcinogenicity.