Antibiotic-treated Rats Research Articles

Stimulation of Mucosal Inflammatory Activity by the Normal Fecal Flora in a Rat Model of Colitis

Bacteria and their products stimulate inflammatory responses and may play a role in the pathogenesis of inflammatory bowel diseases. We investigated interactions between fecal flora and injured colonic mucosa using the trinitrobenzenesulfonic acid model of colitis in the rat. First, we tested the effects of broad-spectrum antibiotics administered at different times after induction of colitis. Second, in rats pretreated with antibiotics, we studied the effects of reintroduction of fecal flora on the release of inflammatory mediators and on the morphology of colonic lesions. Antibiotics reduced colonic lesion scores when started on day 1 after induction of colitis, but had no significant effect when started on day 7. In antibiotic-treated rats, intracolonic administration of a suspension of fecal contents 6 h after induction of colitis enhanced mucosal inflammatory activity and aggravated lesion scores. This effect was not observed when the suspension was administered 3 days after induction. Sonication of the suspension inhibited bacterial growth in aerobic and anaerobic cultures, and abolished its effect on colonic inflammation. In this model, bacteria of the common flora stimulate inflammatory activity and play a role in the induction of colonic mucosal lesions.

Gut bacteria provide precursors of benzodiazepine receptor ligands in a rat model of hepatic encephalopathy

Benzodiazepine receptor (BZR) ligands are elevated in animals and humans with fulminant hepatic failure (FHF) and contribute to the pathogenesis of the associated hepatic encephalopathy (HE). As gut factors are proposed to play a role in the pathogenesis of HE, we investigated gut flora as a source of BZR ligands. Rats received daily oral neomycin, vancomycin and metronidazole (AB +) or saline (AB −) before and concurrent with the induction of FHF with thioacetamide. BZR ligands were extracted from brain and plasma and quantified using radiometric techniques. Plasma BZR ligand concentrations in AB(+) and AB(−) rats with HE were higher than AB(+) and AB(−) control rats. Brain BZR ligand concentrations increased in AB(+) and AB(−) rats with HE. Stool cultures from antibiotic treated rats with HE indicated the presence of multidrug resistant Acinetobacter lwoffii. Although no significant concentrations of BZR ligands were detected in culture media inoculated with A. lwoffii, administering A. lwoffii to normal rats significantly elevated BZR ligand levels in brain, but not plasma. Thus, antibiotic treatment of rats is associated with the growth of a resistant strain of bacterium which produces an inactive BZR ligand precursor. BZR ligands may be synthesized from these precursors in the brain and efficiently cleared by a normal liver following brain-to-plasma transfer. Impairment of this clearance process in FHF facilitates their accumulation, enabling agonist BZR ligands to contribute to the pathogenesis of HE by further enhancing GABAergic neurotransmission.

Role of intestinal microflora in chronic inflammation and ulceration of the rat colon.

Bacteria and their products stimulate inflammatory responses. The effects of different antimicrobial regimens (amoxicillin/clavulanic acid, tobramycin, imipenem, vancomycin, metronidazole) were investigated on the course of experimental colitis induced by trinitrobenzenesulphonic acid (TNB) in the rat. On day 7 and 21 after the induction of colitis, matched groups of control and antibiotic treated rats were subjected to colonic dialysis to measure eicosanoid release, and killed for morphological assessment of the colonic lesions (macro and microscopic scores). Stool samples were cultured. Selective antibiotic treatment against Gram positive, Gram negative or anaerobic bacteria had no effect on colonic lesion scores. By contrast, certain broad spectrum antibiotics (amoxicillin/clavulanic acid or the association of imipenem plus vancomycin) significantly reduced macro and microscopic scores. Rats receiving these antibiotics did not develop chronic colitis as shown by the virtual absence of colonic strictures, adhesions, fibrosis, and granulomas. On day 21 after TNB, the intracolonic release of prostaglandin E2, thromboxane B2, and leukotriene B4 was significantly higher in control than in antibiotic treated rats. Control stool cultures showed abundant colony forming units of both aerobic and anaerobic bacteria. Amoxicillin/clavulanic acid and imipenem plus vancomycin induced appreciable reductions in luminal bacteria. In conclusion, certain broad spectrum antibiotics prevent chronic colitis. The normal colonic flora seems to play an important pathogenetic part in the progression of inflammatory colonic lesions to chronicity.

The effect of fibrin glue on intraperitoneal contamination in rats treated with systemic antibiotics

Intraperitoneal fibrin sealant lowers septic mortality in a rat model of peritoneal contamination (2 × 10 6 organism inoculum) at the cost of increased late intraabdominal abscesses. This study utilized parenteral antimicrobials to determine if the protective effect of intraperitoneal fibrin could be achieved without increasing the late abscess formation rate. One hundred and fiftyfive rats were divided into four groups. Gelatin capsules containing various dilutions of feces (10 10 CFU/ml) and barium sulfate were placed into the abdomen in all groups. Group I controls had no antibiotics or fibrin. In group II, the capsule was surrounded by a solution of cryoprecipitate, thrombin, and calcium (fibrin “glue”). Groups III (no fibrin, antibiotics) and IV (fibrin, antibiotics) received a broad-spectrum cephalosporin intramuscularly postoperatively and then daily. Surviving rats were sacrificed on the tenth postoperative day. At a moderate volume of fecal inoculum (0.3 ml), fibrin reduced mortality from 100% in the control group to 0% in treated animals ( P < 0.001) that did not receive antibiotics. Abscesses formed in 10% of the surviving fibrin-treated rats which were implanted with 0.1 ml of inoculum. In the 0.2 and 0.3 ml inoculum groups substantially more abscesses occurred (75 and 70%, respectively). The protective effect of fibrin was not manifested in the antibiotic-treated rats since no deaths occurred in either group. At higher and lower inoculum doses, no significant differences between fibrin and control groups were observed in mortality or abscess formation, whether or not antibiotics were given. We conclude that intraabdominal abscess formation among survivors is not intrinsic to fibrin but rather, a consequence of survival in rats that otherwise would have succumbed to early sepsis. Fibrin glue can be used topically in the peritoneum without increased abscess formation, providing antibiotics are given.

Utilization and Excretion of a New Sweetener, Fructooligosaccharide (Neosugar), in Rats

In order to study the digestibility of the fructooligosaccharide "Neosugar," [U-14C]Neosugar or [U-14C]sucrose was orally administered to germfree, conventional and antibiotic-treated rats and the radioactivities of expired 14CO2, urine and feces were determined 24 h later. More than 50% of the Neosugar was expired as CO2 in conventional rats. This was the same as for sucrose, but the time course was delayed by about 2 h. In germfree rats, no 14CO2 was released for the first 8 h, and 14CO2 released after 8 h probably reflected bacterial colonization of the gut. The radioactivity of the urine was about 3-4% in all groups, but that of the feces from germfree rats was about eight times higher than the level in conventional rats. When [U-14C]Neosugar was anaerobically incubated with the cecal contents of conventional rats, more than 10% of the added Neosugar was metabolized to CO2, about 66% to volatile fatty acids and about 7% to microbes. More than 58% of 1-14C-volatile fatty acids such as acetic acid, propionic acid or butyric acid injected directly into the cecum of conventional rats was excreted as CO2 within 24 h. These results indicate that Neosugar given orally to rats is metabolized mainly to volatile fatty acids and CO2 by intestinal microorganisms, and the volatile fatty acids produced are absorbed and further converted to CO2 in the body. Thus, the data indicate that Neosugar is partially utilized as an energy source.

Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations

We examined the effects of subinhibitory concentrations of ciprofloxacin, tobramycin, and ceftazidime on Pseudomonas aeruginosa exoenzyme expression in vitro and in vivo. Exotoxin A, exoenzyme S, phospholipase C, elastase, and total protease activities were suppressed by antibiotics at concentrations as low as 1/20 of the MIC over a 24-h period in broth. Continuous 10-day exposure of P. aeruginosa DG1 broth cultures to antibiotic levels equal to 1/10 of the MIC reduced exoenzyme S activity in all treatment groups. Elastase activity was reduced only by ciprofloxacin and tobramycin treatment. This suppressive effect of the antibiotics persisted throughout the 10 days and was not influenced by the increase in MIC of ciprofloxacin detected during the course of the experiment. Rats chronically infected with P. aeruginosa were treated with subinhibitory doses of antibiotics and compared with untreated controls. Bacterial numbers in lung homogenates from each of the four study groups were identical. However, the lungs from antibiotic-treated rats had significantly less histological damage than those from control rats (P less than 0.001). The protective effect was greatest for ciprofloxacin and tobramycin. Further, P. aeruginosa isolates from ciprofloxacin- and tobramycin-treated rats demonstrated significantly less exoenzyme S and elastase activity than isolates from untreated rats (P less than 0.001). Isolates from ceftazidime-treated lungs expressed less exoenzyme S activity (P less than 0.001) but an equivalent amount of elastase activity as isolates from controls. The suppression of P. aeruginosa exoenzymes may arrest progressive lung injury during chronic P. aeruginosa lung infections.

Subinhibitory antibiotics reduce Pseudomonas aeruginosa tissue injury in the rat lung model

Using the agar-bead rat lung model, we evaluated the effects of subinhibitory antibiotic treatment upon Pseudomonas aeruginosa exoenzyme expression and lung injury in vivo. One hundred and twenty-eight animals were separated into two groups of 64 animals. One group was inoculated with P. aeruginosa DG1, and the other with P. aeruginosa 3740. Each of these two groups was divided into four subgroups of 16 animals on the basis of ten-day antibiotic treatment with ciprofloxacin, tobramycin and ceftazidime or untreated controls. P. aeruginosa DG1 is non-mucoid and expresses significant yields of exoenzyme S and elastase. P. aeruginosa 3740 is a mucoid organism isolated from the sputum of a cystic fibrosis patient, and demonstrates modest elastase activity only (10% of DG1 levels). Lung bacterial counts were similar in treatment and control groups. Lungs from antibiotic-treated rats demonstrated fewer histological changes than those from untreated animals (P less than 0.001). DG1 lung isolates from antibiotic-treated animals yielded less elastase and exoenzyme S compared with isolates from untreated animals (P less than 0.001). No detectable decrease in elastase or mucoid phenotype was observed in 3740 lung isolates from antibiotic treated animals. Thus, antibiotic protection against lung injury by P. aeruginosa may involve modulation of virulence factors.

In vivo effects of β-lactam antibiotics and heterocyclic thiol compounds on vitamin K-dependent carboxylation activity and blood coagulation factors in vitamin K-deficient rats

The vivo effects of heterocyclic thiol compounds, corresponding to the 3′-position substituents of several β-lactam antibiotics, on blood coagulation factors and on liver microsomal γ-glutamylcarboxylation (γ-carboxylation) activity were evaluated in rats maintained on a vitamin K-deficient diet. These rats, when compared to normal control animals, exhibited hypoprothrombinemic changes: prolongation of both prothrombin time and activated partial thromboplastin time, decreases in factor VII and plasma prothrombin, and increases in PIVKA II (descarboxyprothrombin) both in plasma and liver. They also displayed a marked increase in liver microsomal γ-carboxylation activity. These blood coagulation variables could be altered markedly by administering various heterocyclic thiol compounds to the vitamin K-deficient rats, although these compounds did not inhibit γ-carboxylation activity in an assay system using phylloquinone. A similar pattern of alteration was observed when some β-lactam antibiotics were administered. Increased microsomal γ-carboxylation activity in antibiotic-treated vitamin K-deficient rats was normalized by the administration of vitamin K, concomitant with the recovery of blood coagulation variables to the normal range. The results indicate that antibiotic-induced hypoprothrombinemia in vivo is not caused by inhibition of enzymes of the γ-carboxylation system, such as vitamin K reductase and γ-glutamylcarboxylase, but is related to the endogenous vitamin K level.

Antibiotic efficacy in intraabdominal sepsis: a clinically relevant model.

We present preliminary data on the role of antibiotics in intraabdominal sepsis using a new, clinically relevant animal model. Peritoneal cavity infection was induced by ligation and perforation of the cecum in adult rats. Surviving rats were randomized to receive either saline or cefoxitin at the time of cecal excision and peritoneal lavage, 18 h after the onset of infection. This is different from previous models of abdominal sepsis (in which antibiotics are given within 4 h of peritoneal contamination) and mimics the clinical setting in which antibiotics are initiated much later, at the time of operation. Antibiotic-treated rats received 20 mg cefoxitin i.m. every 8 h for 7 days; controls received saline at similar times. Thirty-nine of 67 control rats died (58%) versus 20 of 64 (31%) that received cefoxitin (p less than 0.005). We conclude that even with delayed administration, antibiotics appear to improve the outcome of intraabdominal sepsis. With further characterization of this model we plan to use it as an in vivo assay to compare the efficacy of different antimicrobial agents in intraabdominal sepsis.

Ability of intestinal Escherichia coli to survive within mesenteric lymph nodes.

Identification of mesenteric lymph node (MLN) bacteria showed that indigenous streptomycin-sensitive Escherichia coli could be recovered from MLN at least 48 h after this organism had been essentially eliminated from the cecal flora by antibiotics and replaced with exogenous streptomycin-sensitive E. coli JK. Additional experiments with antibiotic-treated rats also showed that indigenous streptomycin-sensitive E. coli could be recovered from the MLN 4 days after elimination of this organism from the cecal flora. These findings suggest that the time of bacterial translocation to MLN may be kinetically different from the time of recovery of bacteria from MLN and that the MLN may be a focus of infection with intestinal bacteria.

Suppression of carbon monoxide excretion rate by tin protoporphyrin.

The effect of a single prophylactic dose of tin protoporphyrin on the carbon monoxide (CO) excretion rate of antibiotic-treated neonatal rats before and after hematoma formation was evaluated. The CO excretion rate, reflecting the rate of bilirubin production, of tin protoporphyrin-treated (TP-H) rats 24 hours after injection of 65 mole of tin protoporphyrin per kilogram (time [t] = 0 hours) was approximately 18% lower than those of the saline-control (S-C) and saline-hematoma (S-H) rats, but this difference was no longer evident at t = 43 hours. After hematoma formation at t = 44 hours, the CO excretion rate of the S-H rats increased rapidly; this increase was delayed and lessened in the TP-H rats. At eight hours posthematoma (t = 52 hours), the CO excretion rate of the TP-H rats was significantly lower than that of the S-H rats, 53 +/- 2 vs 73 +/- 3 microL/kg/hr, respectively. A maximal rate of 89 +/- 5 microL/kg/hr was reached 25 hours posthematoma in the S-H rats (t = 69 hours), as compared with 80 +/- 3 microL/kg/hr at 44 hours posthematoma in the TP-H rats (t = 88 hours). The recovery of injected blood as CO over a 68-hour study period was approximately 90% for the S-H rats and approximately 65% for the TP-H rats. At t = 112 hours, hepatic heme oxygenase activity of the TP-H rats was still significantly lower than that of the S-H and S-C rats; however, plasma bilirubin concentrations of all three groups were similar. These studies demonstrate that tin protoporphyrin is an effective in vivo inhibitor of endogenous heme catabolism as measured by the CO excretion rate in antibiotic-treated neonatal rats with and without artificially created hematomas.

Contribution of intestinal microfloral metabolism to the total macromolecular covalent binding of 1-nitropyrene in the lung and liver of the rat

1-Nitropyrene (NP) is a direct acting mutagen found in diesel exhaust and coal-combustion fly ash. The purpose of this study was to investigate the contribution of gut microfloral metabolism to the macromolecular covalent binding (MCB) of NP and/or its metabolites in lungs and liver of the rat. Normal and antibiotic treated rats were administered [ 14C]NP and MCB was quantitated at various times in lungs and livers. Abolition of gut microfloral metabolism by antibiotic treatment significantly altered total MCB in lungs. MCB in lungs of antibiotic-treated animals 4 h after oral administration of NP was 0.15 nmol NP equivalents/g and was significantly ( P < 0.05) decreased to less than one-half of control values (0.42 nmol NP equivalents/g). MCB in lungs of antibiotic-treated rats was no different from the controls 1 week after NP administration (0.1 nmol NP equivalents/g). Comparison of livers from control and antibiotic-treated rats demonstrated the same pattern of MCB as lungs but differences were not significant. These results reveal that metabolism by gut microflora may play a role in the activation and covalent binding of NP to macromolecules. However, the alteration of covalent binding observed after antibiotic treatment was a change in the time course of formation and breakdown of covalently bound forms and not an effect on the quantity of bound material remaining at 1 week indicating that gut microfloral metabolism is not an exclusive pathway for bioactivation of NP in the rat.

1486 TIN PROTOPORPHYRIN (TP) AND THE EXCRETION RATE OF CO (VeCO) IN ANTIBIOTIC-TREATED NEONATAL RATS WITH ARTIFICIAL HEMATOMAS

Previous studies showed that TP inhibits tissue heme oxygenase activity (HO) and lowers serum total bilirubin levels [B], but does not effect the VeCO of neonatal rats with and without artificially created hematomas (H). It is possible that intestinal bacteria play a role in this model by metabolizing excreted heme to CO. Rat litters, with intestines sterilized by antibiotics, were divided into 3 groups of 3 rats each. At 12 h postpartum (t=0), the TP/H rats were injected with TP (65 μmoles/kg). Hematomas were given to H and TP/H rats (t=45 h). Rats were sacrificed at t=113 h. The [B] of the TP/H rats was not reduced, but hepatic HO activity was suppressed (p < .005) when compared to the H control group. The overall average VeCO of H rats was increased by ∼ 45% over S rats and TP was effective in delaying and suppressing this increase; the inhibition of average VeCO was ∼ 25%. The results suggest that in antibiotic-treated rats with artificial hematomas: 1) single dose TP was effective in lowering VeCO and thus total bilirubin formation and 2) intestinal bacteria may contribute significantly to VeCO, if endogenous heme degradation is inhibited.

Effects of acute and short-time antibiotic treatment on renal lithium elimination and serum lithium levels in the rat.

Male Wistar rats received a single stomach load (10 ml/kg) of a 150 mmol LiCl solution, either alone or together with tetracycline (33.5 mg/10 ml), ampicillin (33.5 mg/10 ml), or metronidazole (15 mg/10 ml). Urine was collected 1-5 hours after administration and blood samples were drawn after 1, 6, and 24 hours. All antibiotics caused a reduction in urinary lithium excretion but did not affect renal lithium clearance. Serum lithium levels were reduced by tetracycline and metronidazole 6 hours after administration but increased after 24 hours. Additional experiments including frequent mapping of serum lithium levels confirmed these findings. Tetracycline, also reduced renal sodium clearance and increased distal sodium reabsorption. Short-term daily treatment during one week with tetracycline or metronidazole showed that these initial changes were only transient, since after treatment for one week no differences could be observed between antibiotic-treated rats and control rats. The results indicate that antibiotics may cause a delay but no decrease of the gastrointestinal absorption of lithium and that they do not affect renal lithium clearance. Signs of lithium intoxication during combined use of lithium and antibiotics are therefore probably not caused by a renal interaction mechanism affecting the renal lithium clearance.

Nitrosoproline formation in control and antibiotic-treated rats given nitrate and proline

The role of the gastrointestinal microflora in the nitrate-dependent formation of nitrosoproline was assessed in control and antibiotic-treated rats. Urinary nitrosoproline excretion as an index of in vivo nitrosamine formation was shown to be unaffected by bacterial decontamination of the alimentary tract, and proceeded in the absence of detectable nitrate reductase activity in the intestinal contents. These observations suggest that the gut microflora are not required for the formation of nitrosamino acid from nitrate and proline.

Enterohepatic circulation and biliary secretion of 5,6-dihydro-5,6-dihydroxy[ 14C]carbaryl glucuronide in the rat

Intestinal absorption (enterohepatic circulation) and biliary secretion of 14C from a metabolite of carbaryl isolated from rat bile, 5,6-dihydro-5,6-dihydroxy[ 14C]carbaryl glucuronide, and its aglycone were observed: Lincomycin and kanamycin sulfate were also given to rats to determine the effect of an altered intestinal microflora on the above processes. Net absorption of 14C from the glucuronide occurred in the small intestine and cecum of control rats (68.5%); 10% of the infused 14C was secreted in the bile. Antibiotic treatment affected the site of absorption and the biliary secretion of 14C from the glucuronide since net 14C absorption occurred only in the small intestine of antibiotic-treated rats (32.5%) and biliary secretion accounted for less than 1% of the infused 14C. The site of absorption of 14C from the aglycone and biliary secretion of 14C (17%, control rats; 14%, antibiotic-treated rats) were not affected by antibiotic treatment. Carbon-14 from the aglycone was absorbed primarily in the small intestine (89.3%, control rats; 84.2%, antibiotic-treated rats). The results indicate that the intestinal microflora influence the enterohepatic circulation and biliary secretion of the glucuronic acid conjugate of 5,6-dihydro-5,6-dihydroxycarbaryl.

Studies on the re-establishment of the intestinal microflora in germ-free rats with special reference to the metabolism of N-isopropyl-alpha-chloroacetanilide (propachlor).

Conventional rats metabolize 2-chloro-N-isopropylacetanilide (propachlor) mainly to various 2-methylsulphonylacetanilides and to residues in the faeces that are unextractable. Mercapturic acid pathway (MAP) metabolites of propachlor are the source of the methylsulphonyl sulphur and the insoluble faecal residues. Germ-free and antibiotic-treated rats quantitatively metabolized propachlor only to MAP metabolites, which were excreted in the urine and faeces (all water-soluble). Germ-free rats given enemas of caecal contents from conventional rats were qualitatively similar to conventional rats with respect to the metabolism of propachlor within 56 days of the inoculations i.e. they excreted 2-methylsulphonyl acetanilides in the urine and insoluble residues in the faeces. They approached quantitative similarity within 180 days. Antibiotic-treated rats spontaneously recovered the ability to metabolize propachlor as conventional rats, qualitatively within 6 days and quantitatively within 21 days. While differences occurred in the faecal metabolites of propachlor, biliary secretion of metabolites by germ-free rats was not different from that of conventional or antibiotic-treated rats.

Formation route of sulfur-containing metabolites of afloqualone, a new centrally acting muscle relaxant, in rat.

The formation route of the sulfur-containing metabolites of afloqualone [6-amino-2-fluoromethyl-3-(o-tolyl)-4(3H)-quinazolinone, AFQ], a centrally acting muscle relaxant, was studied in rats. When 14C-N-acetyl AFQ 2-mercapturate (AFQM) methyl ester was administered orally to normal rats, it was excreted in the bile as AFQM, one of the major biliary metabolites of AFQ, and in the urine as sulfur-containing metabolites of AFQ such as methylsulfinyl and methylsulfonyl metabolites of AFQ. This indicated that AFQM was a precursor of these methylsulfinyl and -sulfonyl metabolites. Pretreatment of rats with antibiotics significantly reduced the urinary excretion of 14C-AFQM and its metabolites as compared with normal rats. Oral administration of 14C-AFQ to antibiotics-treated rats also significantly reduced the amounts of the sulfur-containing metabolites excreted into the urine, but did not affect the amounts of other non-sulfur-containing metabolites. In addition, oral administration of 14C-AFQ to bile-duct-ligated rats gave results similar to those in the antibiotics-treated rats. These results indicate that in normal rats microfloral metabolism is necessary for the formation of the methylsulfinyl and -sulfonyl metabolites from AFQM and other unidentified mercapturate pathway conjugates excreted into the bile. The metabolites of AFQ produced by the microflora are reabsorbed into the systemic circulation, processed by further metabolism in the liver, and in turn excreted in the urine.

Influence of diet and microbial activity in the digestive tract on digestibility, and nitrogen and energy metabolism in rats and pigs

1. Balance trials with respiration measurements were performed with twelve rats and twelve pigs given either low- or high-crude-fibre diets. There were six collection periods with the rats over a live-weight range of 86-264 g and three collection periods with the pigs over a live-weight range of 30-55 kg. Measurements were made on the influence of microbial activity in the digestive tract on digestibility and nitrogen and energy metabolism. Dietary inclusion of the antibiotic Nebacitin was the method used to reduce the microbial population. 2. The microbial activity in the hind-gut (mumol ATP/g air-dry contents) of antibiotic-treated rats was reduced to approximately one-tenth of that of untreated rats. 3. Live-weight gain was not significantly affected in either species by a reduction in the microbial activity, in spite of a reduction in dry matter digestibility in animals with reduced microflora. 4. For rats on low-crude-fibre diets, a reduction in microflora reduced digestibility of all nutrients and energy and metabolizability of digestible energy by approximately 5.4%. All differences were highly significant. On high-crude-fibre diets the decrease was approximately 5.9%. In pigs on both crude fibre levels, the digestibility was also influenced by the level of microflora, but the pattern was somewhat different from that obtained with rats, with the Nebacitin treatment increasing the digestibility of N slightly, and the digestibility of fat markedly. 5. Retained N in rats reached a maximum when the rats were approximately 60 d old and thereafter decreased with increasing age. However, for pigs daily N retention increased with age. The retained N: digested N value decreased linearly with age in the rats, but varied little with age over the range (104-146 d) studied in the pigs. 6. The metabolizability of gross energy (metabolizable energy (ME): gross energy) was significantly reduced with an increase in crude fibre level and by the addition of Nebacitin. 7. Retained energy (RE) in relation to ME (RE:ME), was not significantly affected either by level of microbial activity or by crude fibre. 8. The ratio, RE as fat (RF): RE as protein (RP) increased as the animals grew. In the rat experiment there was a tendency for RP to be higher for animals with normal microflora than for animals with reduced microflora for both crude fibre levels. 9. With rats, the regression analyses indicated that the energy requirement for maintenance could be influenced by both the level of microbial activity in the digestive tract and by the level of fibre in the diet. The net availability of ME for maintenance and growth by rats averaged 0.72 for all treatments. 10. The net availability of ME for growth in the pigs averaged 0.65 for all treatments.

Influence of eating frequency upon plaque formation and periodontal bone loss.

The normal oral flora of weanling Osborne-Mendel rats was suppressed with penicillin, then bone resorption was induced in the test group by inoculation with Actinomyces viscosus Ny 1. In the control group the oral flora was suppressed with erythromycin. A programmable feeding machine forced the rats into extremely varied eating frequencies. Rats which ate less frequently than normal exhibited significantly less bone loss. Frequent eating was correlated with elevated plaque accumulation. AVIS-inoculated animals exhibited significantly (P less than 0.001) more plaque than antibiotic-treated rats. Both inoculated as well as non-inoculated animals exhibited significantly more plaque (P less than 0.001) when fed 28 times per day, in comparison to all other feeding schedules. AVIS-inoculated animals exhibited significantly (P less than 0.001) more bone loss than the antibiotic-treated rats. Inoculated animals which were fed only seven times per day exhibited significantly (P less than 0.05) less bone loss than similarly inoculated animals fed more frequently. Bone loss in the antibiotic-treated animals was independent of feeding frequency. Among the other feeding frequencies (14 and 28 x/day, and ad libitum), there were no significant differences either in AVIS-inoculated or in antibiotic-treated animals.