Electron microscopy study of the bacteria adherent to the rumen wall in young conventional lambs

The lamb rumen walls were rapidly colonized by an abundant bacterial population after birth. This colonization was examined by electron microscopy in neonatal conventional lambs. The sequence of establishment of the epimural species during the 3 weeks following birth, and the distribution of bacteria on the different sacs of the rumen, were examined by scanning electron microscopy. The population was very dense and consisted of a limited number of morphological types by 2 days after birth. Three types of rods were dominant at that time. The microflora was more complex 2 weeks later. Observations by transmission electron microscopy of desquamated epithelial cells revealed the presence of adherent bacteria that are surrounded by fibrous carbohydrate coats and sometimes partially enclosed by invaginations of the epithelial cell.     

Isolation and identification of adherent epimural bacteria during succession in young lambs.

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Isolation and identification of adherent epimural bacteria during succession in young lambs

Successive changes in aerobic and anaerobic bacterial counts and changes in the generic composition of the epimural community in lambs from 1 to 10 weeks were determined. Bacterial culture counts revealed a predominantly anaerobic community, with the mean anaerobic count being 1.4 X 10(7) CFU/cm2 of tissue surface. The aerobic count was highest at 1 week of age and declined significantly thereafter to a mean of 1.8 X 10(4) CFU/cm2, thus representing only 0.13% of the mean anaerobic count after week 1. Of the 345 strains isolated anaerobically at 1, 2, 4, 6, 8, and 10 weeks of age, 47, 32, 12, 32, 2, and 5% were capable of growth in a partially reduced medium, indicating a reduction in the number of facultative anaerobes with time. The majority of isolated strains were identified as belonging to genera commonly isolated from rumen contents. In some instances, however, strains did not correspond to previously described species, and some genera were present in proportions different from those expected in rumen fluid. At three of the sampling times, one genus was dominant, constituting 45 to 55% of the isolates. These dominant isolates were Streptococcus bovis, Bacteroides sp., and an anaerobic Streptococcus sp. for weeks 1, 2, and 10, respectively. During the transition period (weeks 4 to 8), two or more groups were codominant.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Establishment of Bacteroides succinogenes and measurement of the main digestive parameters in the rumen of gnotoxenic lambs

We attempted to determine the degree of diversification of the microflora that allow the establishment of Bacteroides succinogenes S85 in the rumen of gnotoxenic lambs. Four lambs (group I) received an inoculum orally, composed of 182 noncellulolytic bacterial strains (inoculum 1) previously isolated from the rumen of conventional young lambs. Two lambs (group II) were inoculated with 32 strains (inoculum 2) selected among the 182 strains of inoculum 1. Two lambs (group III) received an inoculum (inoculum 3) composed of 106 noncellulolytic bacterial strains previously isolated from the rumen of meroxenic lambs. Two lambs (group IV) were inoculated with 16 strains (inoculum 4) chosen among the 106 strains of inoculum 3. All lambs were inoculated from birth except two lambs of group I, which were inoculated from 1 month of age. Each lamb then received orally a pure culture of B. succinogenes. This strain became established more easily in the rumen of lambs that had received complex inocula (group I). Its population reached a level close to that generally observed in conventional lambs (10(7)-10(8) bacteria.mL-1). In contrast, B. succinogenes became established in only one lamb of group II, but bacterial numbers varied considerably. In group III, repeated inoculations were necessary to obtain its definitive establishment (10(7)-10(8) bacteria.mL-1 after weaning). In spite of several inoculations, this cellulolytic species failed to establish in the rumen of lambs of group IV, which had received the less complex inoculum. The volatile fatty acid levels were very different from one lamb group to another. The more complex the inoculum administered to the animals, the higher the concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Successive changes in the epimural bacterial community of young lambs as revealed by scanning electron microscopy.

Scanning electron microscopy was used to determine the time of initial colonization of the rumen epithelium of young lambs and successive changes with time in the morphological composition of the epimural community. Tissue samples were obtained from two groups of lambs at 1, 2, 4, 6, 8, and 10 weeks of age. Comparisons were made with the epimural communities observed at 12 well-distributed sites in the rumen of a mature wether. Epimural bacteria were already present on the epithelium at 1 week of age. The morphological composition of the epimural community changed with age, with the pattern of succession being similar in both groups of lambs. A total of 24 morphotypes were distinguished by scanning electron microscopy; 17 were rod shaped, 4 were cocci, 2 were spiral, and 1 was filamentous. These morphotypes were further subdivided into: (i) those persisting after their initial colonization in young lambs and present in the adult (7 morphotypes), (ii) those seen only in the adult (2 morphotypes), and (iii) those present only in young lambs (15 morphotypes). The seven morphotypes present in both the lamb and the adult could be considered indigenous members of the epimural community. Several morphotypes appeared restricted in their colonization to certain regions of the papillae, suggesting the presence of microhabitats within the epithelial habitat. Two rod-shaped bacteria were repeatedly seen specifically attached to one another, suggesting an interspecific association.     

Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation.

Eight strains of rumen bacteria capable of degrading phloroglucinol (1,3,5-trihydroxybenzene) under anaerobic conditions were isolated from enrichment cultures of the bovine rumen microflora established in a prereduced medium containing 0.02 M phloroglucinol. Five of the strains were facultatively anaerobic Gram-positive streptococci which were identified as Streptococcus bovis. Three strains of obligately anaerobic Gram-positive cocci were assigned to the genus Coprococcus. Anaerobic cultures of the Streptococcus bovis strains in a 40% rumen fluid medium initially containing 0.02 M phloroglucinol degraded 50-80% of the substrate within 2 days, whereas cultures of the Coprococcus strains degraded more than 80% of the substrate under the same conditions. The Streptococcus bovis strains were incapable of degrading phloroglucinol in brain heart infusion or in the medium of de Man, Rogosa, and Sharpe (MRS broth) incubated aerobically.     

Gram-Negative Anaerobes in the Intestinal Flora of Pigs

A differential count of the gram-negative, anaerobic, non sporeforming bacteria in different segments of the pig intestinal tract was performed in 2 groups of pigs. Seventy-seven strains were identified, belonging to 6 groups: Sphaerophorus necrophorus, anhemolytic Sphaerophorus spp., Bacteroides (Eggerthella) spp., Fusobactenum spp., Veillonella/Acidaminococcus spp., and Peptostreptococcus elsdenii. The characters of these groups are described, and quantitative data on their occurrence in a group of normal porkers and a group of pigs with experimental swine dysentery are given.     

The immune response to Lactococcus lactis: Implications for its use as a vaccine delivery vehicle

Abstract The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. ¹⁴CO₂ and ¹³CO₂ incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows

The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined. The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus. Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet. Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture. For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep. bovis. The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.     

Transformation of mercuric chloride and methylmercury by the rumen microflora

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Studies on the Cecal Microflora of Commercial Broiler Chickens

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.     

The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen

Tetracycline resistant (Tc^R) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens , were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline-resistant strains of M. elsdenii , and in some strains of S. ruminantium.     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

Changes in bacterial diversity associated with epithelial tissue in the beef cow rumen during the transition to a high-grain diet

Our understanding of the ruminal epithelial tissue-associated bacterial (defined as epimural bacteria in this study) community is limited. In this study, we aimed to determine whether diet influences the diversity of the epimural bacterial community in the bovine rumen. Twenty-four beef heifers were randomly assigned to either a rapid grain adaptation (RGA) treatment (n = 18) in which the heifers were allowed to adapt from a diet containing 97% hay to a diet containing 8% hay over 29 days or to the control group (n = 6), which was fed 97% hay. Rumen papillae were collected when the heifers were fed 97%, 25%, and 8% hay diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis were used to characterize rumen epimural bacterial diversity and to estimate the total epimural bacterial population (copy numbers of the 16S rRNA gene). The epimural bacterial diversity from RGA heifers changed (P = 0.01) in response to the rapid dietary transition, whereas it was not affected in control heifers. A total of 88 PCR-DGGE bands were detected, and 44 were identified from phyla including Firmicutes, Bacteroidetes, and Proteobacteria. The bacteria Treponema sp., Ruminobacter sp., and Lachnospiraceae sp. were detected only when heifers were fed 25% and 8% hay diets, suggesting the presence of these bacteria is the result of adaptation to the high-grain diets. In addition, the total estimated population of rumen epimural bacteria was positively correlated with molar proportions of acetate, isobutyrate, and isovalerate, suggesting that they may play a role in volatile fatty acid metabolism in the rumen.     

The Isolation and Characterization of Strains of Lipolytic Bacteria from the Ovine Rumen

The first anaerobic lipolytic bacterium isolated from the rumen was Anaerovibrio lipolytica . In this study strains of anaerobic lipolytic bacteria were isolated from a sheep rumen. All the new isolates were Gram negative curved rods with flagella. The bacteria, which produced propionic acid as a major fermentation product, could ferment only a small range of substrates. The new isolates are thought to belong to the same genus as Anaerovibrio lipolytica .     

Microbial Ecology of the Gut in Laboratory Stocks of the Migratory Grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae)

Mean pH values in pooled samples of foregut, midgut, and hindgut from adult Melanoplus sanguinipes, which had been raised in the laboratory on barley shoots and wheat bran, were 5.15, 6.39, and 5.98, respectively. Homogenates of midgut/hindgut sections and frass (feces) yielded colony counts of bacteria by the spread plate method of 5.7 to 5.9 and 5.3 to 5.5 log₁₀ colonies per mg, respectively; there were no significant differences (P > 0.05) between counts obtained on several media or on media incubated aerobically or anaerobically. There was no evidence of significant populations of protozoa, fungi, or obligately anaerobic bacteria associated with the gut. A total of 168 pure strains of bacteria isolated from the gut sections were characterized and assigned to 11 taxonomic groups, including Enterococcus spp., Serratia liquefaciens, Pseudomonas spp., and Enterobacter spp. Numbers of Enterococcus spp. in the gut were 2 to 3 orders of magnitude higher than those of the other genera. Strains representing only four of the groups were recovered from bran fed to the grasshoppers; the barley shoots, which were raised in sterile soil, appeared virtually sterile. Examination of the gut wall by scanning electron microscopy revealed the presence of epimural bacteria in the foregut and hindgut but not in the midgut. The distribution of epimural cocci and bacilli differed with the gut section examined. Numerous spherical to ovoid structures up to 10 μm in diameter, which were not identified, were associated with the microvillous surface of the midgut epithelium. Acetate was present in gut, hemolymph, and frass, and it was shown that representative isolates of Enterococcus spp. and Enterobacter agglomerans produced acetate when incubated in an aqueous suspension of bran. The egestion time of solid digesta, as measured with methylene blue-stained barley shoots, was 3.0 to 5.7 h. The results show that M. sanguinipes supported extensive indigenous populations of luminal and epimural bacteria in the gut which were composed predominantly of facultatively anaerobic species; the relatively short egestion time, indicating rapid passage of digesta through the gut, was consistent with the microscopic appearance of digesta residues in frass and could account, at least in part, for the absence of a significant population of obligately anaerobic bacteria from the gut.     

Isolation and characterization of large treponemes from the bovine rumen.

Ten strains of strictly anaerobic spiral organisms were isolated in pure culture from a 10(-7) dilution of bovine rumen contents. Three strains were studied in detail. These strains morphologically resembled previously isolated and described rumen treponemes except the new isolates were larger, 0.7 micrometer wide and 12 to 25 micrometer long. They rapidly fermented pectin and, less readily, L-arabinose, inulin, and sucrose. Acetic and formic acid were the main fermentation products from pectin; small amounts of succinic acid were also formed.