Adaptation to herbivory by the Tammar wallaby includes bacterial and glycoside hydrolase profiles different from other herbivores.

Metagenomic and bioinformatic approaches were used to characterize plant biomass conversion within the foregut microbiome of Australia's "model" marsupial, the Tammar wallaby (Macropus eugenii). Like the termite hindgut and bovine rumen, key enzymes and modular structures characteristic of the "free enzyme" and "cellulosome" paradigms of cellulose solubilization remain either poorly represented or elusive to capture by shotgun sequencing methods. Instead, multigene polysaccharide utilization loci-like systems coupled with genes encoding beta-1,4-endoglucanases and beta-1,4-endoxylanases--which have not been previously encountered in metagenomic datasets--were identified, as were a diverse set of glycoside hydrolases targeting noncellulosic polysaccharides. Furthermore, both rrs gene and other phylogenetic analyses confirmed that unique clades of the Lachnospiraceae, Bacteroidales, and Gammaproteobacteria are predominant in the Tammar foregut microbiome. Nucleotide composition-based sequence binning facilitated the assemblage of more than two megabase pairs of genomic sequence for one of the novel Lachnospiraceae clades (WG-2). These analyses show that WG-2 possesses numerous glycoside hydrolases targeting noncellulosic polysaccharides. These collective data demonstrate that Australian macropods not only harbor unique bacterial lineages underpinning plant biomass conversion, but their repertoire of glycoside hydrolases is distinct from those of the microbiomes of higher termites and the bovine rumen.

Development of a colorimetric colony-screening assay for detection of defluorination by micro-organisms.

AIMS: To develop a colorimetric colony-screening assay to facilitate the isolation of micro-organisms capable of defluorination. METHODS AND RESULTS: A metal-dye chelate, zirconium-xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1:2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l(-1) respectively), the assay could detect a fluoride application spot (5 mmol l(-1)) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0.1 g l(-1) and some proteins digest to between 1 and 5 g l(-1). A microbial enrichment culture growing on solidified medium containing 20 mmol l(-1) fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. CONCLUSIONS: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro-organisms growing on solidified medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can be used to facilitate the isolation of micro-organisms capable of defluorination.

An improved method for RNA extraction from carcass samples for detection of viable Escherichia coli O157:H7 by reverse-transcriptase polymerase chain reaction.

AIMS: To develop a rapid RNA extraction procedure for maximizing bacterial RNA yield from carcass samples with low abundance of Escherichia coli O157:H7 without pre-enrichment. METHODS AND RESULTS: Nontarget bacterial cells were added to the sample prior to RNA extraction, facilitating the co-precipitation of target RNA along with nontarget RNA and thus enhancing the recovery. This method was developed using a serial dilution of log phase target cells (E. coli O157:H7), combined with a high number of nontarget cells (E. coli K12). Cells were lysed by a bead beating method followed by RNA purification using a commercial kit. A reverse-transcriptase PCR assay for the detection of rfbE gene in E. coli O157:H7 was used to demonstrate that the procedure increased the recovery of amplifiable RNA target with a detection limit of approximately 63 CFU ml(-1) in cultures and 27.5 CFU ml(-1) in carcass liquor. CONCLUSIONS: An RNA extraction procedure was developed to detect low numbers (<30 viable cells ml(-1)) of E. coli O157:H7 in carcass liquor without pre-enrichment. SIGNIFICANCE AND IMPACT OF THE STUDY: This method could be applied for the detection of E. coli O157:H7 in low abundance on carcasses where rapid detection and early intervention is essential for safety in the livestock industry.

Effect of tea saponin on methanogenesis, microbial community structure and expression of mcrA gene, in cultures of rumen micro-organisms.

AIMS: To determine the in-vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. METHODS AND RESULTS: Saponin extracted from tea seeds was added to (1) an in-vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme-M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real-time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. CONCLUSIONS: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal-associated methanogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.

Detection and monitoring of anaerobic rumen fungi using an ARISA method.

AIM: To develop an automated ribosomal intergenic spacer region analysis (ARISA) method for the detection of anaerobic rumen fungi and also to demonstrate utility of the technique to monitor colonization and persistence of fungi, and diet-induced changes in community structure. METHODS AND RESULTS: The method could discriminate between three genera of anaerobic rumen fungal isolates, representing Orpinomyces, Piromyces and Neocallimastix species. Changes in anaerobic fungal composition were observed between animals fed a high-fibre diet compared with a grain-based diet. ARISA analysis of rumen samples from animals on grain showed a decrease in fungal diversity with a dominance of Orpinomyces and Piromyces spp. Clustering analysis of ARISA profile patterns grouped animals based on diet. A single strain of Orpinomyces was dosed into a cow and was detectable within the rumen fungal population for several weeks afterwards. CONCLUSIONS: The ARISA technique was capable of discriminating between pure cultures at the genus level. Diet composition has a significant influence on the diversity of anaerobic fungi in the rumen and the method can be used to monitor introduced strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of ARISA analysis, a better understanding of the effect of diets on rumen anaerobic fungi populations is provided.

Review: The application of omics to rumen microbiota function.

Rumen microbiome profiling uses 16S rRNA (18S rRNA, internal transcribed spacer) gene sequencing, a method that usually sequences a small portion of a single gene and is often biased and varies between different laboratories. Functional information can be inferred from this data, but only for those that are closely related to known annotated species, and even then may not truly reflect the function performed within the environment being studied. Genome sequencing of isolates and metagenome-assembled genomes has now reached a stage where representation of the majority of rumen bacterial genera are covered, but this still only represents a portion of rumen microbial species. The creation of a microbial genome (bins) database with associated functional annotations will provide a consistent reference to allow mapping of RNA-Seq reads for functional gene analysis from within the rumen microbiome. The integration of multiple omic analytics is linking functional gene activity, metabolic pathways and rumen metabolites with the responsible microbiota, supporting our biological understanding of the rumen system. The application of these techniques has advanced our understanding of the major microbial populations and functional pathways that are used in relation to lower methane emissions, higher feed efficiencies and responses to different feeding regimes. Continued and more precise use of these tools will lead to a detailed and comprehensive understanding of compositional and functional capacity and design of techniques for the directed intervention and manipulation of the rumen microbiota towards a desired state.

Shifts in Host Mucosal Innate Immune Function Are Associated with Ruminal Microbial Succession in Supplemental Feeding and Grazing Goats at Different Ages.

Gastrointestinal microbiota may play an important role in regulating host mucosal innate immune function. This study was conducted to test the hypothesis that age (non-rumination, transition and rumination) and feeding type [Supplemental feeding (S) vs. Grazing (G)] could alter ruminal microbial diversity and maturation of host mucosal innate immune system in goat kids. MiSeq sequencing was applied to investigate ruminal microbial composition and diversity, and RT-PCR was used to test expression of immune-related genes in ruminal mucosa. Results showed that higher (P < 0.05) relative abundances of Prevotella, Butyrivibrio, Pseudobutyrivibrio, Methanobrevibacter.gottschalkii, Neocallimastix, Anoplodinium-Diplodinium, and Polyplastron, and lower relative abundance of Methanosphaera (P = 0.042) were detected in the rumen of S kids when compared to those in G kids. The expression of genes encoding TLRs, IL1α, IL1ß and TICAM2 was down-regulated (P < 0.01), while expression of genes encoding tight junction proteins was up-regulated (P < 0.05) in the ruminal mucosa of S kids when compared to that in G kids. Moreover, irrespective of feeding type, relative abundances of ruminal Prevotella, Fibrobacter, Ruminococcus, Butyrivibrio, Methanobrevibacter, Neocallimastix, and Entodinium increased with age. The expression of most genes encoding TLRs and cytokines increased (P < 0.05) from day 0 to 7, while expression of genes encoding tight junction proteins declined with age (P < 0.05). This study revealed that the composition of each microbial domain changed as animals grew, and these changes might be associated with variations in host mucosal innate immune function. Moreover, supplementing goat kids with concentrate could modulate ruminal microbial composition, enhance barrier function and decrease local inflammation. The findings provide useful information in interpreting microbiota and host interactions, and developing nutritional strategies to improve the productivity and health of rumen during early life.

Screening of bacteria from the cattle gastrointestinal tract for inhibitory activity against enterohemorrhagic Escherichia coli O157:H7, O111:H-, and O26:H11.

A quick and reproducible microgel plate assay was adapted to screen bacteria from cattle gastrointestinal tracts for production of compounds inhibitory to the growth of three enterohemorrhagic Escherichia coli (EHEC) serotypes: O157:H7, O111:H-, and O26:H11. The inhibitory activity of 309 bacteria, isolated on several agar media, was assessed by a microgel assay performed in 96-well microtiter plates. Fifty-three isolates secreted inhibitory compounds with a molecular weight of less than 1,000. In 12 isolates, the inhibitory activity was attributable to compounds other than lactic or acetic acid. These compounds were highly heat tolerant, with varying sensitivity to digestion by proteolytic enzymes. The inhibitory isolates were identified as lactic acid-producing bacteria on the basis of a combination of analyses, including 16S-rDNA restriction fragment length polymorphisms, 16S-rDNA gene sequences, and fermentation end products. The lactic acid bacteria of ruminants may contain antibacterial compounds not yet described. Naturally occurring populations of lactic acid bacteria may have potential as probiotics, to reduce the carriage of EHEC in the gastrointestinal tract of ruminants.

An omaso-abomasal cannula used to assess efflux from the omasum of sheep.

An omaso-abomasal cannulation technique was developed for sheep to assess efflux from the omasum. The procedure enabled the collection of the total omasal effluent and avoided transection of the innervation and circulation of this region of the gut. Eating was associated with an increase in flow of the fluid and dry matter fractions of digesta passing from the omasum but efflux was unaffected by rumination. There were few protozoa and flagellated organisms in the omasal effluent relative to the numbers in ruminal fluid. The preparation was used over 3 months of study.

A comparative study of the anatomy of the omasum in domesticated ruminants.

The structure of the omasum was compared in 10 water buffalo (Bubalus bubalis), 10 Shorthorn cattle (Bos indicus), 9 Merino sheep (Ovis aries) and 10 cross-bred goats (Capra hircus), and the functional significance of the differences is discussed. The weight of the omasum and area of the internal laminae appeared to be smaller in Brahman cattle than the buffalo and British cattle. When compared in relation to body weight, the omasums of caprid animals were smaller than those of bovids, by a factor of 0.33-0.67 but the omasums of goats were appreciably larger than sheep. The number of laminae ranged from 33 to 35 for the sheep and goats compared with 122 to 169 for the buffalo and cattle. It is suggested that the marked difference in structure of the omasum between the small and large ruminants may influence the food intake of these animals through the effects of this gut compartment on absorption of nutrients and passage of digesta.

Residues of endosulfan in the tissues of lactating goats.

The sites of tissue accumulation in lactating goats of the organochlorine insecticide endosulfan were studied. Twelve lactating goats were dosed orally with endosulfan (1 mg/kg body weight per day) for 28 days. Groups of 3 animals were killed on days 1, 8, 15, and 21 after endosulfan treatment ended and their tissues examined for the presence of endosulfan. Total residues of alpha and beta endosulfan and endosulfan sulphate (mg/kg) were detected in kidney (0.29), gastro-intestinal tract (0.20), liver (0.12), brain (0.06), muscle and spleen (0.04), lung and heart (0.01) and milk (0.02) on the first sampling day but within 15 days, concentrations had fallen to < 0.01 mg/kg in all tissues except kidney (0.20). Endosulfan could not be detected in animals 21 days after dosing had ceased. The residue in milk could only be detected on day 1 of sampling. This study indicates that kidney rather than fatty tissue should be used to monitor the presence of endosulfan in animals intended for human consumption.

Effects of disodium fumarate on ruminal fermentation and microbial communities in sheep fed on high-forage diets.

This study was conducted to investigate effects of disodium fumarate (DF) on fermentation characteristics and microbial populations in the rumen of Hu sheep fed on high-forage diets. Two complementary feeding trials were conducted. In Trial 1, six Hu sheep fitted with ruminal cannulae were randomly allocated to a 2 × 2 cross-over design involving dietary treatments of either 0 or 20 g DF daily. Total DNA was extracted from the fluid- and solid-associated rumen microbes, respectively. Numbers of 16S rDNA gene copies associated with rumen methanogens and bacteria, and 18S rDNA gene copies associated with rumen protozoa and fungi were measured using real-time PCR, and expressed as proportion of total rumen bacteria 16S rDNA. Ruminal pH decreased in the DF group compared with the control (P < 0.05). Total volatile fatty acids increased (P < 0.001), but butyrate decreased (P < 0.01). Addition of DF inhibited the growth of methanogens, protozoa, fungi and Ruminococcus flavefaciens in fluid samples. Both Ruminococcus albus and Butyrivibrio fibrisolvens populations increased (P < 0.001) in particle-associated samples. Trial 2 was conducted to investigate the adaptive response of rumen microbes to DF. Three cannulated sheep were fed on basal diet for 2 weeks and continuously for 4 weeks with supplementation of DF at a level of 20 g/day. Ruminal samples were collected every week to analyze fermentation parameters and microbial populations. No effects of DF were observed on pH, acetate and butyrate (P > 0.05). Populations of methanogens and R. flavefaciens decreased in the fluid samples (P < 0.001), whereas addition of DF stimulated the population of solid-associated Fibrobacter succinogenes. Population of R. albus increased in the 2nd to 4th week in fluid-associated samples and was threefold higher in the 4th week than control week in solid samples. Analysis of denaturing gradient gel electrophoresis fingerprints revealed that there were significant changes in rumen microbiota after adding DF. Ten of 15 clone sequences from cut-out bands appearing in both the 2nd and the 4th week were 94% to 100% similar to Prevotella-like bacteria, and four sequences showed 95% to 98% similarity to Selenomonas dianae. Another 15 sequences were obtained from bands, which appeared in the 4th week only. Thirteen of these 15 sequences showed 95% to 99% similarity to Clostridium sp., and the other two showed 95% and 100% similarity to Ruminococcus sp. In summary, the microorganisms positively responding to DF addition were the cellulolytic bacteria, R. albus, F. succinogenes and B. fibrisolvens as well as proteolytic bacteria, B. fibrisolvens, P. ruminicola and Clostridium sp.

Isolation of Succinivibrionaceae implicated in low methane emissions from Tammar wallabies.

The Tammar wallaby (Macropus eugenii) harbors unique gut bacteria and produces only one-fifth the amount of methane produced by ruminants per unit of digestible energy intake. We have isolated a dominant bacterial species (WG-1) from the wallaby microbiota affiliated with the family Succinivibrionaceae and implicated in lower methane emissions from starch-containing diets. This was achieved by using a partial reconstruction of the bacterium's metabolism from binned metagenomic data (nitrogen and carbohydrate utilization pathways and antibiotic resistance) to devise cultivation-based strategies that produced axenic WG-1 cultures. Pure-culture studies confirm that the bacterium is capnophilic and produces succinate, further explaining a microbiological basis for lower methane emissions from macropodids. This knowledge also provides new strategic targets for redirecting fermentation and reducing methane production in livestock.

The stimulatory effect of the organic sulfur supplement, mercaptopropane sulfonic acid on cellulolytic rumen microorganisms and microbial protein synthesis in cattle fed low sulfur roughages.

Two metabolism trials (experiments 1 and 2) were conducted to examine the effect of the organic S compound, sodium 3-mercapto-1-propane sulfonic acid (MPS) on feed intake, fiber digestibility, rumen fermentation and abundance of cellulolytic rumen microorganisms in cattle fed low S (<0.11%) roughages. Urea was provided in all treatments to compensate for the N deficiency (<0.6%) in the roughages. In experiment 1, steers (333 ± 9.5 kg liveweight) were fed Angleton grass (Dicanthium aristatum) supplemented with S in equivalent amounts as either MPS (6.0 g/day) or sodium sulfate (9.56 g/day). Supplementation of Angelton grass with either sulfate or MPS resulted in an apparent increase in flow of rumen microbial protein from the rumen. Sulfur supplementation did not significantly change whole tract dry matter digestibility or intake, even though sulfate and MPS supplementation was associated with an increase in the relative abundance of the fibrolytic bacteria Fibrobacter succinogenes and anaerobic rumen fungi. Ruminal sulfide levels were significantly higher in the sulfate treatment, which indicated that the bioavailability of the two S atoms in the MPS molecule may be low in the rumen. Based on this observation, experiment 2 was conducted in which twice the amount of S was provided in the form of MPS (8.0 g/day) compared with sodium sulfate (6.6 g/day) to heifers (275 ± 9 kg liveweight) fed rice straw. Supplementation with MPS compared with sulfate in experiment 2 resulted in an increase in concentration of total volatile fatty acids, and ammonia utilization without a change in feed intake or whole tract fiber digestibility even though S and N were above requirement for growing cattle in both these treatment groups. In conclusion, supplementation of an S deficient low-quality roughage diet with either MPS or sodium sulfate, in conjunction with urea N, improved rumen fermentation, which was reflected in an increase in urinary purine excretion. However, MPS appeared to have a greater effect on stimulating short-chain fatty acid production and ammonia utilization when provided at higher concentrations than sulfate. Thus, the metabolism of MPS in the rumen needs to be investigated further in comparison with inorganic forms of S as it may prove to be more effective in stimulating fermentation of roughage diets.

Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet.

AIM: To examine the effect of sulfur-containing compounds on the growth of anaerobic rumen fungi and the fibrolytic rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in pure culture and within the cattle rumen. METHODS AND RESULTS: The effect of two reduced sulfur compounds, 3-mercaptopropionic acid (MPA) or 3-mercapto-1-propanesulfonic acid as the sole S source on growth of pure fibroyltic fungal and bacterial cultures showed that these compounds were capable of sustaining growth. An in vivo trial was then conducted to determine the effect of sulfur supplements (MPA and sodium sulfate) on microbial population dynamics in cattle fed the roughage Dichanthium aristatum. Real-time PCR showed significant increases in fibrolytic bacterial and fungal populations when cattle were supplemented with these compounds. Sulfate supplementation leads to an increase in dry matter intake without a change in whole tract dry matter digestibility. CONCLUSIONS: Supplementation of low S-containing diets with either sodium sulfate or MPA stimulates microbial growth with an increase in rumen microbial protein supply to the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of real-time PCR monitoring, a better understanding of the effect of S supplementation on discrete microbial populations within the rumen is provided.

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples.

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.