The 'normalization' of germ-free guineapigs with host-specific caecal microflora

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guineapigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), beta-aspartylglycine (faeces), volatile fatty acids (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guineapigs, showed an improvement in CR and RCW, yielding values found in control animals.     

beta-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic-treated mice.

The caecal supernatants from germ-free, antibiotic-treated and control mice were compared with respect to their content of low-molecular-weight substances (less than 3500 mol. wt.). The supernatants contained about the same amount of free amino acids. After acid hydrolysis, the caecal supernatants of germ-free and antibiotic-treated mice showed a 2.9-fold increase in free amino acids, whereas a similar treatment of the supernatant from control mice resulted in a 2.6-fold increase. By gel filtration on Sephadex G-25, and high-voltage paper electrophoresis at pH 3.5 of the fractions eluted after the void volume, it was found that the caecal supernatants of germ-free and antibiotic-treated mice contained a substance more acidic than aspartic acid. Preparative high-voltage electrophoresis, dansylation, amino acid analysis and a specific colour reaction showed the substance to be beta-aspartylglycine. After a minimal 36 h of treatment with neomycin and bacitracin, a high concentration of beta-aspartylglycine was found, and no enterococci and aerobic Gram-negative rods could be cultured from the caecal contents. The possibility that in one mouse the appearance of beta-aspartylglycine was related to a decrease in Gram-negative rods was ruled out by selective elimination of aerobic Gram-negative rods by using polymyxin B. This suggests that other bacteria concomitantly eliminated with the enterococci and aerobic Gram-negative rods, directly or indirectly, could play a role in the accumulation of beta-aspartylglycine.     

Intestinal accumulation of urea in germ-free animals--a factor in caecal enlargement

To examine the connection between caecal size and urea concentration in the caecal contents urease inhibition was tested in conventional animals and urea and urease were administered to germ-free rats and mice. Administration of alloxan and barbituric acid and immunization with urease led to slightly larger caeca in conventional animals. Neomycin treatment caused a clear enlargement of the caecum. In germ-free animals urease administration led to a reduction in the caecal size, whereas urea in the drinking water caused enlargement. The urea concentration and the urease activity in the caecal contents correlated well with the caecal weights.     

Fate of salmonellas in the alimentary tract of chicks pre-treated with a mature caecal microflora to increase colonization resistance

Oral challenge with a strain of Salmonella kedougou , resistant to nalidixic acid, gave a time-course for salmonella survival in the the alimentary tract of chicks pre-treated with a caecal culture from an eight-week-old bird to increase colonization resistance. In untreated, control chicks, salmonella colonization of the caeca resulted in counts of > 10⁶/g within 48 h and a mean generation time of 1·6 h. With treated birds, however, the salmonellas failed to multiply in the caeca and decreased to a low level over a 48-h period, thus suggesting a mainly bacteriostatic effect. Pretreatment of chicks with the caecal culture also reduced the proportion of salmonella-positive crop samples. Growth of the salmonella in a feed-slurry system resembling moistened crop contents occurred rapidly at 37°C but was prevented by addition of the treatment culture, a decline in numbers coinciding with Lactobacillus populations of ca 10⁹/g and a pH value of 5·5. Incorporation of a feed decontaminant, 1% formic acid (pH 4·0), rapidly eliminated both salmonellas and organisms added from the treatment culture.     

Fate of salmonellas in the alimentary tract of chicks pre-treated with a mature caecal microflora to increase colonization resistance

Oral challenge with a strain of Salmonella kedougou, resistant to nalidixic acid, gave a time-course for salmonella survival in the alimentary tract of chicks pre-treated with a caecal culture from an eight-week-old bird to increase colonization resistance. In untreated, control chicks, salmonella colonization of the caeca resulted in counts of greater than 10(6)/g within 48 h and a mean generation time of 1.6 h. With treated birds, however, the salmonellas failed to multiply in the caeca and decreased to a low level over a 48-h period, thus suggesting a mainly bacteriostatic effect. Pre-treatment of chicks with the caecal culture also reduced the proportion of salmonella-positive crop samples. Growth of the salmonella in a feed-slurry system resembling moistened crop contents occurred rapidly at 37 degrees C but was prevented by addition of the treatment culture, a decline in numbers coinciding with Lactobacillus populations of ca 10(9)/g and a pH value of 5.5. Incorporation of a feed decontaminant, 1% formic acid (pH 4.0), rapidly eliminated both salmonellas and organisms added from the treatment culture.     

Effect of mannanoligosaccharides supplementation on caecal microbial activity of rabbits

A total of 200 weaned (35 days) hybrid Hyla rabbits were randomly divided among five groups housed in bicellular cages (20 cages per group). Between 35 and 60 days of age, the groups were submitted to the following treatments: group ANT (positive control) fed a basal diet supplemented with antibiotics (colistin sulphate, 144 mg/kg; tylosin, 100 mg/kg; and oxytetracyclin, 1000 mg/kg); groups MOS_0.5, MOS_1.0 and MOS_1.5 fed the basal diet supplemented with 0.5, 1.0 and 1.5 g/kg mannanoligosaccharides (MOS), respectively; another group fed the basal diet without antibiotics or mannanoligosaccarides supplementation (negative control). Along the trial, an episode of epizootyc rabbit enteropathy occurs so that in the control group mortality rate was very high (78%) and survivor rabbits showed severe symptoms of disease (diarrhoea). Thus, the control group was discarded from the trial. At 60 days of age, samples of caecal content were collected from 10 rabbits per group and used as inocula for an in vitro gas production trial. At the end of fermentation (120 h of incubation), organic matter digestibility (OMd), cumulative gas production, fermentation kinetics, pH, volatile fatty acid (VFA) and NH3 productions were measured. Inoculum from MOS_1.0 rabbits showed the significant higher values of OMd (64.21%, P < 0.05), gas production (262.32 ml/g, P < 0.05), acetate (96.99 mmol/g OM, P < 0.05) and butyrate (26.21 mmol/g OM, P < 0.05) than the other groups. Slight differences were recorded among the groups ANT, MOS_0.5 and MOS_1.5. In addition, branched chain acids, in proportion to total VFAs, were significantly higher in MOS_1.0 inoculum (0.04, P < 0.05). MOS are able to affect fermentation activity of caecal micro-organism, but their activities seem not proportional to their level in the diet.     

Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice

115 strains of clostridia were accumulated from 3 separate isolations from the faeces of 1 limited flora (LF) mouse produced by inoculation of germ-free mice with chloroform-treated faeces of conventional mice. These strains were divided into 36 types on the basis of conventional biochemical characteristics. There was some variation in types isolated on the 3 occasions. Although 3 groups of polyassociated (PA) mice were produced from mixing 46 monoassociated mice, each inoculated with 1 of 46 strains of 36 bacterial types, the caecal size of PA mice was still greater than that of the control mice prepared by inoculation of chloroform-treated faeces of an LF mouse. After PA mice of each group were housed together, the caecal size became smaller and was only slightly larger than the control.     

The effect of dietary mannitol on the caecal microflora and short-chain fatty acids in rats

Wistar rats fed 5%-mannitol diet showed significant decreases of viridans streptococci and bifidobacteria, and a remarkable increase of butyric acid concentration and a decrease of acetic acid in the caecal contents compared with the sucrose and basal diet groups. Stocky fusiform bacteria were increased in concentration in the rats on mannitol compared with the other groups.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Gut immune maturation depends on colonization with a host-specific microbiota

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.     

A comparative study of raw garlic extract and tetracycline on caecal microflora and serum proteins of albino rats

Studies conducted on the comparative action of raw garlic extract and tetracycline hydrochloride on equal concentrations showed the raw garlic extract to be a more potent antimicrobial agent than tetracycline. The reversion to normalcy of caecal microflora and serum proteins after the withdrawal of test materials indicated an inverse relation between intestinal microflora and serum globulins.     

Biodiversity and fermentative activity of caecal microbial communities in wild and farm rabbits from Spain

In order to study the microbial caecal ecosystem of wild and domestic rabbits through the fermentation characteristics and concentration and diversity of bacterial and archaeal communities, caecal samples from sixteen wild rabbits (WR) were contrasted with two groups (n = 4) of farm rabbits receiving low (LSF) or high (HSF) soluble fibre diets from 28 (weaning) to 51 days of age. DNA was extracted for quantifying bacteria and Archaea by qPCR and for biodiversity analysis of microbial communities by DGGE. Samples from WR had lower caecal pH and ammonia and higher volatile fatty acids concentration than farm animals. Lower acetate and higher butyrate proportions were detected in WR. Bacterial and archaeal DGGE profiles were clearly different between wild and farm rabbits, and diet-affected population of farm rabbits. Similarity index of bacteria was lower than 0.40 among WR, and 0.52 among farm rabbits. In conclusion, caecal fermentation characteristics differ between wild and farm rabbits, which harbour clearly different bacterial and archaeal communities. In farm rabbits, diversity is influenced by the dietary level of soluble fibre.     

Stress-induced inhibition of the ileal, caecal, and colonic contractile activity in rabbits]

In alert rabbits, immobilisation stress decreased the spike burst rate and amplitude in ileum, caecum, and colon for 20 min. Following beta-adrenoceptor blockade, the contractile activity suppression was aggravated. The stress seems to lead to suppression of the ileal, caecal, and colonic contractile activity for up to 40 min which is unrelated to adrenergic inhibition of the smooth muscle activity mediated by beta-adrenoceptors.     

Caecal microflora and fermentation pattern in exclusively milk-fed young rabbits.

Thirty-two SPF New Zealand White rabbits of both sexes were used in this experiment. They were sequentially slaughtered at 22, 29, 35 and 42 days of age. The rabbits were only milked by their mother and by foster mothers (no access to solid food) throughout the experiment. They exhibited a caecal fermentation pattern that turned towards a proteolytic metabolic activity (high levels in NH3 and in branched-chain fatty acids and valeric acid: 17 mmol.L-1 and 1 mmol.L-1, on average, respectively). The absence of caecal cellulolytic microflora and the low concentration of the total volatile fatty acids (tVFA) could be explained by the lack of substrate that would have been brought by a solid feed. From day 29 onwards, the low tVFA (12.5 mmol.L-1) and high NH3 concentrations (16.5 mmol.L-1) explained the high pH value (6.8 on average). This could be considered as a pathological value in weaned rabbits, but in our case no clinical signs of diarrhoea were observed. The evolution of the colibacilli flora according to age was similar to that usually described and thus was not correlated with pH, tVFA or cellulolytic flora. The evolution of the colibacilli population (from 10(7) bact/g on day 22 to 10(3) on day 42) seemed dependent on ontogenic factors rather than on the composition of the caecal media (pH, etc.).     

酸马奶提取Kluyveromyces marxianus代谢抗菌复合物对感染致病性Escherichia coli O8小鼠的免疫机能及其

【目的】酸马奶可防治心血管、消化系统、肺结核、糖尿病和腹泻等疾病,尤其马克斯克鲁维酵母Kluyveromyces marxianus对单核细胞增生李斯特菌Listeria monocytogenes有抑菌作用,但对酸马奶提取K. marxianus代谢抗菌复合物抑菌作用的研究报道较少。为此,研究酸马奶提取K. marxianus代谢抗菌复合物对感染致病性大肠杆菌Escherichia coli O8小鼠免疫机能及其盲肠菌群的影响。【方法】将128只小鼠随机分为4组,空白组、致病对照组、K2组和K8组,致病对照组小鼠连续7 d灌胃无菌PBS,并于第4天注射E. coli O8;K2组灌胃抗菌复合物K. marxianus pH 2.0,并注射E. coli O8;K8组灌胃抗菌复合物K. marxianus pH 8.0,并注射E. coli O8。采用常规HE染色法观察0、4和7 d小鼠小肠病理切片,称重法测定小鼠免疫器官指数,ELISA法测定血清中免疫球蛋白,流式细胞术测定T细胞亚群,平板涂布法测定盲肠菌群。【结果】致病对照组小鼠在实验第4天注菌后出现一系列患病临床症状和小肠组织病理变化。K2组和K8组小鼠整体精神状态好于致病对照组,死亡小鼠数量较少,可一定程度上缓解和改善感染致病性E. coli O8小鼠的小肠组织病理变化。致病对照组在第7天胸腺指数显著低于空白组(P<0.05)。K2组和K8组在第4天和第7天脾脏指数显著高于空白组(P<0.05)。致病对照组在第7天IgA显著低于空白组(P<0.05)。K2组在第4天IgA、IgG显著高于空白组(P<0.05)。K2组在第7天IgG和IgM显著高于空白组(P<0.05)。K8组在第7天IgM显著高于空白组(P<0.05)。K2组在第4天和第7天CD8+显著低于空白组,CD4+/CD8+显著高于空白组(P<0.05)。K8组在第4天CD8+显著低于空白组(P<0.05)。K8组在第7天CD8+显著低于空白组,CD4+/CD8+显著高于空白组(P<0.05)。致病对照组在第7天盲肠E. coli数量显著高于空白组,双歧杆菌数量显著低于空白组(P<0.05)。K2组在第7天盲肠E. coli数量显著低于空白组,肠球菌数量显著低于空白组,乳酸杆菌数量显著高于空白组(P<0.05)。K8组在第7天盲肠肠球菌数量显著低于空白组,乳酸杆菌数量显著高于空白组(P<0.05)。【结论】酸马奶提取K. marxianus代谢抗菌复合物K2和K8能缓解感染致病性E. coli O8小鼠临床症状,提高其免疫机能,并影响其盲肠菌群,提高双歧杆菌和乳酸杆菌,降低E. coli和肠球菌的数量。     

The influence of mannan oligosaccharides, acidifiers and their combination on caecal microflora of Japanese quail (Coturnix japonica)

The aim of this study was to investigate the effects of the dietary supplementation of mannan oligosaccharides (MOS) extracted from yeast Saccharomyces cerevisiae, acidifiers -calcium formate (CF), calcium propionate (CP)- and their combination on the caecal microflora of Japanese quail (Coturnix japonica). Four hundred and fifty 1-day old quail where divided in six groups with three replicates each. One group that served as control received the basal diet. The five experimental diets consisted of the basal diet to which either 1 g MOS/kg, or 6 g CF/kg, or 6 g CP/kg, or 1 g MOS plus 6 g CF/kg or 1 g MOS plus 6 g CP/kg were added. The body weight was examined at weekly intervals and mortality was recorded daily. At days 21 and 42 of age, the total count of aerobic bacteria, lactic acid bacteria, enterobacteriaceae and coliforms in the caecal content of one bird of each replicate was determined. Also, at day 42 of age, two birds of each replicate were slaughtered and their carcass weight was determined. The results showed that MOS significantly (P ≤ 0.050) increased the total aerobic plate and lactic acid bacteria counts on day 21. Furthermore, CP significantly (P ≤ 0.050) decreased the total aerobic plate and lactic acid bacteria counts compared to controls on day 21. Significant interaction between MOS and acidifiers was noticed on total aerobic plate count on day 21. No significant (P > 0.050) difference was found in the caecal microflora on day 42. Finally, no significant (P > 0.050) difference was noticed on mortality, body and carcass weight.