The rumen microbiology of seaweed digestion in Orkney sheep

The microbial populations of the rumens of seaweed-fed and pasture-fed Orkney sheep were examined. The populations in the pasture-fed sheep were similar to those of other domestic ruminants fed on land plants, but those of the seaweed-fed animals showed major differences in the dominant species. Total ciliate populations were quantitatively similar, but in the seaweed-fed animals Dasytricha ruminantium was one of the most dominant species. No phycomycete fungi or cellulolytic bacteria were found in the seaweed-fed animals, and the bacterial population was dominated by Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens and lactate-utilizing species. Electron microscopy revealed that spirochaetes and an unidentified filamentous bacterium were probably of major significance in seaweed digestion. The ability of bacterial strains from both groups of animals to metabolize plant and algal constituents was examined.     

The effects of organic selenium supplementation on the rumen ciliate population in sheep

The effect of selenium supplementation on the rumen protozoan population of sheep was demonstrated. Both the total and generic counts of rumen ciliates in sheep fed a diet with basal Se content (70 microg/kg dry matter) were compared to those of animals given feed supplemented with inorganic (disodium selenite) or organic Se (selenized yeast) (310 microg/kg dry matter). The genera of Entodinium, Isotricha, Dasytricha, Ophryoscolex, Diploplastron and Polyplastron occurred in all sheep except for the control, in which Ophryoscolex was not observed. The population of Ophryoscolex caudatus f. tricoronatus was significantly higher in sheep supplemented with organic Se than in animals given inorganic Se (by 160 %). Supplementation of feed with selenized yeast induced significant growth in the Diploplastron population (by 63 %) while no change occurred in sheep given selenite. The populations of Dasytricha ruminantium and Polyplastron multivesiculatum were higher than control in both Se-supplemented groups. The ciliate population of Entodinium spp. was not influenced by Se supplements. Our results suggest a protective effect of Se feed supplementation on the development of some rumen ciliate species in young ruminants.     

Competence of the African tortoise tick, Amblyomma marmoreum (Acari: Ixodidae), as a vector of the agent of heartwater (Cowdria ruminantium)

The ability of the African tortoise tick, Amblyomma marmoreum, to acquire and transmit Cowdria ruminantium infection was investigated experimentally with transmission trials and with a C. ruminantium-specific polymerase chain reaction (PCR) detection assay. Laboratory-reared A. marmoreum larvae and nymphs were fed on small ruminants with clinical heartwater. After molting, the resultant nymphs were fed on Cowdria ruminantium-naive sheep (n = 3), and the adults were ground and inoculated intravenously into sheep (n = 5). Fatal heartwater developed in the 5 recipient animals, demonstrating larvae-nymph transmission and nymph-adult acquisition of infection. Cowdria ruminantium infection was also detected in adult A. marmoreum by PCR analysis, although at lower frequency (10%) than in Amblyomma hebraeum ticks (43%), the major vector of C. ruminantium in southern Africa, which had been fed simultaneously on the infected animals (P<0.0001). Amblyomma marmoreum, therefore, can be an effective vector of C. ruminantium. The potential role of this species in heartwater epidemiology and in the spread of the disease to new areas is highlighted by these results and by the fact that immature stages of this tick feed readily on domestic and wild animals susceptible to C. ruminantium.     

Natural variability and diurnal fluctuations within the bacteriophage population of the rumen.

To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time. Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid. It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals. Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen. In sheep fed once daily, a distinct diurnal variation in the phage population was observed. Two hours postfeeding, total phage DNA dropped to its lowest level. The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle. The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily.     

Evidence to show that an agent that cross-reacts serologically with Cowdria ruminantium in Zimbabwe is transmitted by ticks

The serological diagnosis of heartwater based on reactions to the immunodominant Cowdria ruminantium major antigen protein-1 (MAP-1) is impaired by the detection of false-positive reactions. In this study, the prevalence of false-positive reactions on seven heartwater-free farms in Zimbabwe was determined to be 8-94% by immunoblotting against C. ruminantium antigens. The highest prevalence of false-positives on Spring Valley Farm correlated with the presence of Rhipicephalus evertsi evertsi ticks. The other tick species found on these seven farms were Hyalomma truncatum and Hyalomma marginatum rufipes. Rhipicephalus evertsi evertsi ticks collected from Spring Valley Farm and fed on seronegative sheep caused seroconversion in one of two sheep. This sheep developed a mild febrile reaction and C. ruminantium MAP-1 antigen reactive antibodies 3 weeks after the ticks started feeding. Polymerase chain reactions (PCRs), conducted using C. ruminantium-specific primers on ticks collected from the seven farms and on some of the R. e. evertsi ticks that had caused seroconversion in one sheep, were negative. However, some of these ticks gave positive PCRs with DNA primers which amplify a 350 bp DNA fragment of the 16s rRNA gene from all ehrlichial agents indicating the presence of infection with one or more Ehrlichia species. Although attempts to isolate the cross-reacting agent from the sheep were unsuccessful, this study demonstrates that false-positive reactions with the MAP-1 C. ruminantium antigen are associated with agents transmitted by ticks.     

Comparison of efficacy of American and African Amblyomma ticks as vectors of heartwater (Cowdria ruminantium) infection by molecular analyses and transmission trials

The ability of Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, and Amblyomma variegatum to acquire and transmit Cowdria ruminantium infection was investigated. Uninfected nymphs were fed on clinically reacting C. ruminantium-infected sheep and then analyzed for infection by specific DNA detection assays and by tick transmission trials. By polymerase chain reaction (PCR), the mean infection prevalence of A. maculatum ticks (50.7%) was similar to that of A. variegatum, Elevage strain (43.5%; P = 0.83) and Petit Bourg strain (45.9%; P = 0.26) ticks. Though Amblyomma hebraeum were not tested by PCR, by DNA probe their infection prevalence was 94%. In contrast, A. americanum and A. cajennense ticks demonstrated very low susceptibility to C. ruminantium, and the prevalence of infection by PCR was approximately 1%. The higher susceptibility of A. maculatum and A. variegatum to C. ruminantium correlated with superior vector efficiency, depicted by similar prepatent periods and severity of disease transmissions to sheep. Amblyomma americanum and A. cajennense failed to transmit infection, confirming that low susceptibility to C. ruminantium correlates with the poor vector status of these species. These results highlight the importance of A. maculatum as a potential vector that is likely to play a major role in the establishment and maintenance of heartwater, if the disease were to be introduced to the U.S.A., Central, and South America.     

Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens.

Cross-reactivity among four species of ruminal bacteria was examined by using egg yolk antibodies from immunized Leghorn laying hens and an enzyme-linked-immunosorbent assay. The effects of the four species on the hens were compared on various days postimmunization. Hens injected with the same bacterial species had similar apparent antibody levels over the entire postimmunization period, but only Bacteroides ruminicola B1(4) and Selenomonas ruminantium D antigens elicited early increases in apparent antibody levels during weeks 2 and 3. Antibody cross-reactivity was greatly reduced by week 2, except for antibodies against Streptococcus bovis JB1.     

Differentiation of ruminal bacterial species by enzyme-linked immunosorbent assay using egg yolk antibodies from immunized chicken hens

Cross-reactivity among four species of ruminal bacteria was examined by using egg yolk antibodies from immunized Leghorn laying hens and an enzyme-linked-immunosorbent assay. The effects of the four species on the hens were compared on various days postimmunization. Hens injected with the same bacterial species had similar apparent antibody levels over the entire postimmunization period, but only Bacteroides ruminicola B1(4) and Selenomonas ruminantium D antigens elicited early increases in apparent antibody levels during weeks 2 and 3. Antibody cross-reactivity was greatly reduced by week 2, except for antibodies against Streptococcus bovis JB1.     

Methanogen community structure in the rumens of farmed sheep, cattle and red deer fed different diets

Development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on knowledge of the methanogens present. We investigated the composition of archaeal communities in the rumens of farmed sheep (Ovis aries), cattle (Bos taurus) and red deer (Cervus elaphus) using denaturing gradient gel electrophoresis (DGGE) to generate fingerprints of archaeal 16S rRNA genes. The total archaeal communities were relatively constant across species and diets, and were less variable and less diverse than bacterial communities. There were diet- and ruminant-species-based differences in archaeal community structure, but the same dominant archaea were present in all rumens. These were members of three coherent clades: species related to Methanobrevibacter ruminantium and Methanobrevibacter olleyae; species related to Methanobrevibacter gottschalkii, Methanobrevibacter thaueri and Methanobrevibacter millerae; and species of the genus Methanosphaera. Members of an archaeal group of unknown physiology, designated rumen cluster C (RCC), were also present. RCC-specific DGGE, clone library analysis and quantitative real-time PCR showed that their 16S rRNA gene sequences were very diverse and made up an average of 26.5% of the total archaea. RCC sequences were not readily detected in the DGGE patterns of total archaeal 16S rRNA genes because no single sequence type was abundant enough to form dominant bands.     

Similarity of the ruminal bacteria across individual lactating cows

Dairy cattle hold enormous significance for man as a source of milk and meat. Their remarkable ability to convert indigestible plant mass into these digestible food products resides in the rumen - an anaerobic chambered compartment - in the bovine digestive system. The rumen houses a complex microbiota which is responsible for the degradation of plant material, consequently enabling the conversion of plant fibers into milk and meat and determining their quality and quantity. Hence, an understanding of this complex ecosystem has major economic implications. One important question that is yet to be addressed is the degree of conservation of rumen microbial composition across individual animals. Here we quantified the degree of similarity between rumen bacterial populations of 16 individual cows. We used real-time PCR to determine the variance of specific ruminal bacterial species with different metabolic functions, revealing that while some bacterial strains vary greatly across animals, others show only very low variability. This variance could not be linked to the metabolic traits of these bacteria. We examined the degree of similarity in the dominant bacterial populations across all animals using automated ribosomal intergenic spacer analysis (ARISA), and identified a bacterial community consisting of 32% operational taxonomic units (OTUs) shared by at least 90% of the animals and 19% OTUs shared by 100% of the animals. Looking only at the presence or absence of each OTU gave an average similarity of 75% between each cow pair. When abundance of each OTU was added to the analysis, this similarity decreased to an average of less than 60%. Thus, as suggested in similar recent studies of the human gut, a bovine rumen core microbiome does exist, but taxa abundance may vary greatly across animals.     

Evidence for the possible involvement of Selenomonas ruminantium in rumen fiber digestion

Selenomonas ruminantium strains were isolated from sheep rumen, and their significance for fiber digestion was evaluated. Based on the phylogenetic classification, two clades of S. ruminantium (clades I and II) were proposed. Clade II is newly found, as it comprised only new isolates that were phylogenetically distant from the type strain, while all of the known isolates were grouped in the major clade I. More than half of clade I isolates displayed CMCase activity with no relation to the degree of bacterial adherence to fibers. Although none of the isolates digested fiber in monoculture, they stimulated fiber digestion when co-cultured with Fibrobacter succinogenes, and there was an enhancement of propionate production. The extent of such synergy depended on the clade, with higher digestion observed by co-culture of clade I isolates with F. succinogenes than by co-culture with clade II isolates. Quantitative PCR analysis showed that bacterial abundance in the rumen was higher for clade I than for clade II. These results suggest that S. ruminantium, in particular the major clade I, is involved in rumen fiber digestion by cooperating with F. succinogenes.     

<Emphasis Type="Italic">Methanobrevibacter</Emphasis> Phylotypes are the Dominant Methanogens in Sheep from Venezuela

Rumen methanogens in sheep from Venezuela were examined using 16S rRNA gene libraries and denaturing gradient gel electrophoresis (DGGE) profiles prepared from pooled and individual PCR products from the rumen contents from 10 animals. A total of 104 clones were examined, revealing 14 different 16S rRNA gene sequences or phylotypes. Of the 14 phylotypes, 13 (99 of 104 clones) belonged to the genus Methanobrevibacter, indicating that the genus Methanobrevibacter is the most dominant component of methanogen populations in sheep in Venezuela. The largest group of clones (41 clones) was 97.9-98.5% similar to Methanobrevibacter gottschalkii. Two sequences were identified as possible new species, one belonging to the genus Methanobrevibacter and the other belonging to the genus Methanobacterium. DGGE analysis of the rumen contents from individual animals also revealed 14 different bands with a range of 4-9 bands per animal.     

Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Utilization of xylooligosaccharides by selected ruminal bacteria.

The ability of ruminal bacteria to utilize xylooligosaccharides was examined. Xylooligosaccharides were prepared by partially hydrolyzing oat spelt xylan in phosphoric acid. This substrate solution was added (0.2%, wt/vol) to a complex medium containing yeast extract and Trypticase that was inoculated with individual species of ruminal bacteria, and growth and utilization were monitored over time. All of the xylanolytic bacteria examined were able to utilize this oligosaccharide mixture as a growth substrate. Butyrivibrio fibrisolvens, Eubacterium ruminantium, and Ruminococcus albus used xylooligosaccharides and whole, unhydrolyzed xylan to similar extents, while Prevotella ruminicola used twice as much xylooligosaccharides as xylan (76 versus 34%). Strains of Selenomonas ruminantium were the only nonxylanolytic species that were able to grow on xylooligosaccharides. The ability of individual S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidases activities.     

Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan^® probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups ( Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium ) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.     

Vaccine strategies for Cowdria ruminantium infections and their application to other ehrlichial infections.

Suman Mahan and co-authors review the strategies applied to develop improved vaccines for Cowdria ruminantium infections (heartwater). Inactivated vaccines using cell-cultured C. ruminantium organisms combined with an adjuvant are capable of protecting goats, sheep and cattle against lethal C. ruminantium challenge. Immune responses induced with this vaccine, or after recovery from infection, target outer membrane proteins of C. ruminantium, in particular the major antigenic protein 1 (MAP-1). Genetic immunizations with the gene encoding MAP-1 induce protective T helper cell type 1 responses against lethal challenge in a mouse model. Similarly, homologues of MAP-1 in other phylogenetically and antigenically related ehrlichial agents such as Anaplasma marginale and Ehrlichia chaffeensis are also targets of protective responses. Given the antigenic similarities between the related ehrlichial agents, common strategies of vaccine development could be applied against these agents that cause infections of importance in animals and humans.     

Molecular diversity of methanogens in feedlot cattle from Ontario and Prince Edward Island, Canada

The molecular diversity of rumen methanogens in feedlot cattle and the composition of the methanogen populations in these animals from two geographic locations were investigated using 16S rRNA gene libraries prepared from pooled PCR products from 10 animals in Ontario (127 clones) and 10 animals from Prince Edward Island (114 clones). A total of 241 clones were examined, with Methanobrevibacter ruminantium accounting for more than one-third (85 clones) of the clones identified. From these 241 clones, 23 different 16S rRNA phylotypes were identified. Feedlot cattle from Ontario, which were fed a corn-based diet, revealed 11 phylotypes (38 clones) not found in feedlot cattle from Prince Edward Island, whereas the Prince Edward Island cattle, which were fed potato by-products as a finishing diet, had 7 phylotypes (42 clones) not found in cattle from Ontario. Five sequences, representing the remaining 161 clones (67% of the clones), were common in both herds. Of the 23 different sequences, 10 sequences (136 clones) were 89.8 to 100% similar to those from cultivated methanogens belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales, and the remaining 13 sequences (105 clones) were 74.1 to 75.8% similar to those from Thermoplasma volcanium and Thermoplasma acidophilum. Overall, nine possible new species were identified from the two clone libraries, including two new species belonging to the order Methanobacteriales and a new genus/species within the order Methanosarcinales. From the present survey, it is difficult to conclude whether the geographical isolation between these two herds or differences between the two finishing diets directly influenced community structure in the rumen. Further studies are warranted to properly assess the differences between these two finishing diets.     

Alkaline phosphatase activity of rumen bacteria.

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.     

Interaction of ruminal bacteria in the production and utilization of maltooligosaccharides from starch.

The degradation and utilization of starch by three amylolytic and one nonamylolytic species of ruminal bacteria were studied. Pure cultures of Streptococcus bovis JB1, Butyrivibrio fibrisolvens 49, and Bacteroides ruminicola D31d rapidly hydrolyzed starch and maltooligosaccharides accumulated. The major starch hydrolytic products detected in S. bovis cultures were glucose, maltose, maltotriose, and maltotetraose. In addition to these oligosaccharides, B. fibrisolvens cultures produced maltopentaose. The products of starch hydrolysis by B. ruminicola were even more complex, yielding glucose through maltotetraose, maltohexaose, and maltoheptaose but little maltopentaose. Selenomonas ruminantium HD4 grew poorly on starch, digested only a small portion of the available substrate, and generated no detectable oligosaccharides as a result of cultivation in starch containing medium. S. ruminantium was able to grow on a mixture of maltooligosaccharides and utilize those of lower degree (less than 10) of polymerization. A coculture system containing S. ruminantium as a dextrin-utilizing species and each of the three amylolytic bacteria was developed to test whether the products of starch hydrolysis were available for crossfeeding to another ruminal bacterium. Cocultures of S. ruminantium and S. bovis contained large numbers of S. bovis but relatively few S. ruminantium and exhibited little change in the pattern of maltooligosaccharides observed for pure cultures of S. bovis. In contrast, S. ruminantium was able to compete with B. fibrisolvens and B. ruminicola for these growth substrates. When grown with B. fibrisolvens, S. ruminantium grew to high numbers and maltooligosaccharides accumulated to a much lesser degree than in cultures of B. fibrisolvens alone. S. ruminantium-B. ruminicola cultures contained large numbers of both species, and maltooligosaccharides never accumulated in these cocultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkaline phosphatase activity of rumen bacteria

Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.