Changes in rumen microbial community composition in yak in response to seasonal variations.

AIMS: Yak is a dominant ruminant, well adapted to grazing on pasture year around in the harsh climate of the 3000-meter-high Qinghai-Tibetan Plateau. The complex microbial community that resides within the yak rumen is responsible for fermentation and contributes to its climatic adaptation. This study aimed to characterize the rumen microbiota responses to wide seasonal variations, especially those necessary for survival in the cold seasons. METHODS AND RESULTS: In the present study, we performed 16s rRNA gene sequencing to investigate the seasonal variations in microbiota composition, diversity and associated volatile fatty acids (VFAs) in yak rumen. The results showed that rumen microbiota were dominated by Bacteroides (72.13%-78.54%) and Firmicutes; the relative abundance of Firmicutes was higher in summer (17.44%) than in winter (10.67%; p < 0.05). The distribution of taxa differed among spring, summer and winter rumen communities (PERMANOVA, p = 0.001), whereas other taxa (e.g., Fibrobacter, Verrucomicrobia, Anaerostipes and Paludibacter), which could potentially help overcome harsh climate conditions were observed in higher abundance during the cold spring and winter seasons. The highest total VFA concentration in the yak rumen was obtained in summer (p < 0.05), followed by spring and winter, and both positive and negative correlations between VFAs and specific genera were revealed. CONCLUSIONS: Microbiota in yak rumen appear to be highly responsive to seasonal variations. Considering environmental factors, we suggest that seasonal adaptation by microbial communities in rumen enables their hosts to survive seasonal scarcity and cold stress in the spring and winter. SIGNIFICANCE AND IMPACT OF STUDY: The present study furthers our understanding of how microbial adaptation to seasonal variations in nutrient availability and climate may function in high plateau ruminants, providing insights into the tripartite relationship between the environment, host and microbiota.

Addition of arabinoxylan and mixed linkage glucans in porcine diets affects the large intestinal bacterial populations.

PURPOSE: To investigate the effects of two cereal soluble dietary fibres (SDF), wheat arabinoxylan (AX) and oat-mixed linkage glucans (MLG), on fermentative end-products and bacterial community profiles of the porcine caecum (Cae) and distal colon (DC). We hypothesised that feeding pigs these SDF would stimulate Cae and DC carbohydrate fermentation, resulting in a modification of the resident bacterial communities. METHODS: Five groups of six pigs were each fed one diet based on wheat starch (WS) only, or treatment diets in which some WS was replaced by 10 % AX, or 10 % MLG, a combination of 5 % AX:5 % MLG (AXMLG), or completely replaced with ground whole wheat. Post-euthanasia, Cae and DC digesta were collected for analysis of fermentative end-products, and bacterial community profiles were determined by 16S rRNA gene amplicon 454 pyrosequencing. RESULTS: Across all the SDF-containing diets, predominantly in the proximal region of the large intestine, Prevotella, Lactobacillus, Mitsuokella and Streptococcus were most significantly influenced (P < 0.05), while notable changes were observed for the Ruminococcaceae and Lachnospiraceae families in the Cae and DC. The addition of MLG or AXMLG had the greatest effect of influencing bacterial profiles, reducing sequence proportions assigned to the genus Clostridium, considered detrimental to gut health, with associated increases in short-chain fatty acid and reduced ammonia concentrations. CONCLUSIONS: This study demonstrated how the cereal SDF AX and MLG altered the large intestinal bacterial community composition, particularly proximally, further giving insights into how diets rich in specific complex carbohydrates shift the bacterial population, by increasing abundance and promoting greater diversity of those bacteria considered beneficial to gut health.

Methanogen Diversity in Indigenous and Introduced Ruminant Species on the Tibetan Plateau.

Host factors are regarded as important in shaping the archaeal community in the rumen but few controlled studies have been performed to demonstrate this across host species under the same environmental conditions. A study was designed to investigate the structure of the methanogen community in the rumen of two indigenous (yak and Tibetan sheep) and two introduced domestic ruminant (cattle and crossbred sheep) species raised and fed under similar conditions on the high altitude Tibetan Plateau. The methylotrophic Methanomassiliicoccaceae was the predominant archaeal group in all animals even though Methanobrevibacter are usually present in greater abundance in ruminants globally. Furthermore, within the Methanomassiliicoccaceae family members from Mmc. group 10 and Mmc. group 4 were dominant in Tibetan Plateau ruminants compared to Mmc. group 12 found to be highest in other ruminants studied. Small ruminants presented the highest number of sequences that belonged to Methanomassiliicoccaceae compared to the larger ruminants. Although the methanogen community structure was different among the ruminant species, there were striking similarities between the animals in this environment. This indicates that factors such as the extreme environmental conditions and diet on the Tibetan Plateau might have a greater impact on rumen methanogen community compared to host differences.

Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Investigation of a new acetogen isolated from an enrichment of the tammar wallaby forestomach.

BACKGROUND: Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative acetogens in the tammar wallaby forestomach. RESULTS: Methanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative acetogens in the cultures. A new acetogen (growth on H2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated >97% rrs identity to previously isolated kangaroo acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive acetogenesis under high hydrogen concentration (>5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively. CONCLUSIONS: The unique properties of tammar wallaby acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or acetogen stimulation, for example by harnessing mixotrophic growth capabilities.

Effects of feeding plant-derived agents on the colonization of Campylobacter jejuni in broiler chickens.

The aim of this work was to test the potential use of plant-derived extracts and compounds to control Campylobacter jejuni in broiler chickens. Over a 7-wk feeding period, birds were fed a commercial diet with or without plant extracts (Acacia decurrens, Eremophila glabra), essential oil [lemon myrtle oil (LMO)], plant secondary compounds [terpinene-4-ol and α-tops (including α-terpineol, cineole, and terpinene-4-ol)], and the antibiotic virginiamycin. Traditional culture and real-time quantitative PCR techniques were used to enumerate the numbers of C. jejuni in chicken fecal and cecal samples. In addition, BW and feed intake were recorded weekly for the calculation of BW gain and feed conversion ratio. The mean log10 counts of C. jejuni were similar (P > 0.05) across treatments. However, significantly lower levels of fecal Campylobacter counts (P < 0.05) were recorded at d 41 for the α-tops treatment by culture methods. No differences (P > 0.05) in BW gain were obtained for dietary supplementation, except for the E. glabra extract, which had a negative impact (P < 0.001) on BW, resulting in sporadic death. Results from this study suggest that supplemental natural compounds used in the current study did not reduce the shedding of C. jejuni to desired levels.

The extent of nitrogen isotopic fractionation in rumen bacteria is associated with changes in rumen nitrogen metabolism.

Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet (Δ15Nanimal-diet). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural 15N enrichment of the bacterial biomass over their substrates (Δ15Nbacteria-substrate) and thereby further impacting Δ15Nanimal-diet. To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains (Fibrobacter succinogenes, Eubacterium limosum and Xylanibacter ruminicola) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ15Nbacteria-substrate was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ15Nbacteria-substrate was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ15Nbacteria-substrate and Rikenellaceae_RC9_gut_group, a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ15Nbacteria-substrate may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ15Nanimal-diet.

Resistant starches protect against colonic DNA damage and alter microbiota and gene expression in rats fed a Western diet.

Resistant starch (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), opposes dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed Western-type diets moderate in fat (19%) and protein (20%) containing digestible starches [low amylose maize starch (LAMS) or low amylose whole wheat (LAW)] or RS [HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference] for 11 wk (n = 10/group). A control diet included 7% fat, 13% protein, and LAMS. Colonocyte DNA single-strand breaks (SSB) were significantly higher (by 70%) in rats fed the Western diet containing LAMS relative to controls. Dietary HAW, HAMS, and HAMSB opposed this effect while raising digesta levels of SCFA and lowering ammonia and phenol levels. SSB correlated inversely with total large bowel SCFA, including colonic butyrate concentration (R(2) = 0.40; P = 0.009), and positively with colonic ammonia concentration (R(2) = 0.40; P = 0.014). Analysis of gut microbiota populations using a phylogenetic microarray revealed profiles that fell into 3 distinct groups: control and LAMS; HAMS and HAMSB; and LAW and HAW. The expression of colonic genes associated with the maintenance of genomic integrity (notably Mdm2, Top1, Msh3, Ung, Rere, Cebpa, Gmnn, and Parg) was altered and varied with RS source. HAW is as effective as HAMS and HAMSB in opposing diet-induced colonic DNA damage in rats, but their effects on the large bowel microbiota and colonocyte gene expression differ, possibly due to the presence of other fiber components in HAW.

Responses in digestion, rumen fermentation and microbial populations to inhibition of methane formation by a halogenated methane analogue.

The effects of the anti-methanogenic compound, bromochloromethane (BCM), on rumen microbial fermentation and ecology were examined in vivo. Japanese goats were fed a diet of 50 % Timothy grass and 50 % concentrate and then sequentially adapted to low, mid and high doses of BCM. The goats were placed into the respiration chambers for analysis of rumen microbial function and methane and H2 production. The levels of methane production were reduced by 5, 71 and 91 %, and H2 production was estimated at 545, 2941 and 3496 mmol/head per d, in response to low, mid and high doses of BCM, respectively, with no effect on maintenance feed intake and digestibility. Real-time PCR quantification of microbial groups showed a significant decrease relative to controls in abundance of methanogens and rumen fungi, whereas there were increases in Prevotella spp. and Fibrobacter succinogenes, a decrease in Ruminococcus albus and R. flavefaciens was unchanged. The numbers of protozoa were also unaffected. Denaturing gradient gel electrophoresis and quantitative PCR analysis revealed that several Prevotella spp. were the bacteria that increased most in response to BCM treatment. It is concluded that the methane-inhibited rumen adapts to high hydrogen levels by shifting fermentation to propionate via Prevotella spp., but the majority of metabolic hydrogen is expelled as H2 gas.

An arabinoxylan-rich fraction from wheat enhances caecal fermentation and protects colonocyte DNA against diet-induced damage in pigs.

Population studies show that greater red and processed meat consumption increases colorectal cancer risk, whereas dietary fibre is protective. In rats, resistant starches (a dietary fibre component) oppose colonocyte DNA strand breaks induced by high red meat diets, consistent with epidemiological data. Protection appears to be through SCFA, particularly butyrate, produced by large bowel carbohydrate fermentation. Arabinoxylans are important wheat fibre components and stimulate large bowel carbohydrate SCFA production. The present study aimed to determine whether an arabinoxylan-rich fraction (AXRF) from wheat protected colonocytes from DNA damage and changed colonic microbial composition in pigs fed with a diet high (30 %) in cooked red meat for 4 weeks. AXRF was primarily fermented in the caecum, as indicated by higher tissue and digesta weights and higher caecal (but not colonic) acetate, propionate and total SCFA concentrations. Protein fermentation product concentrations (caecal p-cresol and mid- and distal colonic phenol) were lower in pigs fed with AXRF. Colonocyte DNA damage was lower in pigs fed with AXRF. The microbial profiles of mid-colonic mucosa and adjacent digesta showed that bacteria affiliating with Prevotella spp. and Clostridial cluster IV were more abundant in both the mucosa and digesta fractions of pigs fed with AXRF. These data suggest that, although AXRF was primarily fermented in the caecum, DNA damage was reduced in the large bowel, occurring in conjunction with lower phenol concentrations and altered microbial populations. Further studies to determine the relationships between these changes and the lowering of colonocyte DNA damage are warranted.

Methanogen colonisation does not significantly alter acetogen diversity in lambs isolated 17 h after birth and raised aseptically.

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17 h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.

Screening of Australian plants for antimicrobial activity against Campylobacter jejuni.

Campylobacter jejuni is the most common cause of acute enteritis in humans, with symptoms such as diarrhoea, fever and abdominal cramps. In this study, 115 extracts from 109 Australian plant species were investigated for their antimicrobial activities against two C. jejuni strains using an in vitro broth microdilution assay. Among the plants tested, 107 (93%) extracts showed activity at a concentration between 32 and 1024 µg/mL against at least one C. jejuni strain. Seventeen plant extracts were selected for further testing against another six C. jejuni strains, as well as Campylobacter coli, Escherichia coli, Salmonella typhimurium, Bacillus cereus, Proteus mirabilis and Enterococcus faecalis. The extract from Eucalyptus occidentalis demonstrated the highest antimicrobial activity, with an inhibitory concentration of 32 µg/mL against C. jejuni and B. cereus. This study has shown that extracts of selected Australian plants possess antimicrobial activity against C. jejuni and thus may have application in the control of this organism in live poultry and retail poultry products.

High-yield and phylogenetically robust methods of DNA recovery for analysis of microbial biofilms adherent to plant biomass in the herbivore gut.

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the "repeated bead beating and column (RBB+C) purification" method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.

The effects from DNA extraction methods on the evaluation of microbial diversity associated with human colonic tissue.

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host-microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account.

Correction: Martinez-Fernandez et al. Seasonal and Nutrient Supplement Responses in Rumen Microbiota Structure and Metabolites of Tropical Rangeland Cattle. Microorganisms 2020, 8, 1550.

We have found one inadvertent error in our paper published in Microorganisms [...].

Response to Climate Change: Evaluation of Methane Emissions in Northern Australian Beef Cattle on a High Quality Diet Supplemented with Desmanthus Using Open-Circuit Respiration Chambers and GreenFeed Emission Monitoring Systems.

The main objective of this study was to compare the effect of supplementing beef cattle with Desmanthus virgatus cv. JCU2, D. bicornutus cv. JCU4, D. leptophyllus cv. JCU7 and lucerne on in vivo methane (CH4) emissions measured by open-circuit respiration chambers (OC) or the GreenFeed emission monitoring (GEM) system. Experiment 1 employed OC and utilized sixteen yearling Brangus steers fed a basal diet of Rhodes grass (Chloris gayana) hay in four treatments-the three Desmanthus cultivars and lucerne (Medicago sativa) at 30% dry matter intake (DMI). Polyethylene glycol (PEG) was added to the diets to neutralize tannin binding and explore the effect on CH4 emissions. Experiment 2 employed GEM and utilized forty-eight animals allocated to four treatments including a basal diet of Rhodes grass hay plus the three Desmanthus cultivars in equal proportions at 0%, 15%, 30% and 45% DMI. Lucerne was added to equilibrate crude protein content in all treatments. Experiment 1 showed no difference in CH4 emissions between the Desmanthus cultivars, between Desmanthus and lucerne or between Desmanthus and the basal diet. Experiment 2 showed an increase in CH4 emissions in the three levels containing Desmanthus. It is concluded that on high-quality diets, Desmanthus does not reduce CH4 emissions.

Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach.

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.

Diversity of anaerobic gut fungal populations analysed using ribosomal ITS1 sequences in faeces of wild and domesticated herbivores.

Gut fungal-specific PCR primers have been used to selectively amplify the ITS1 region of gut fungal rDNA recovered from faeces of domestic and wild animals to investigate population diversity. Two different gel-based methods are described for separating populations of gut fungal rDNA amplicons, namely (1) denaturing gradient gel electrophoresis (DGGE) and (2) separation according to small size differences using Spreadex, a proprietary matrix for electrophoresis. Gut fungal populations were characterised by analysis of rDNA in faeces of seventeen domesticated and ten wild herbivores. Sequences derived from these gel-based characterisations were analysed and classified using a hidden Markov model-based fingerprint matching algorithm. Faecal samples contained a broad spectrum of fungi and sequences from five of the six recognised genera were identified, including Cyllamyces, the most recently described gut fungal genus, which was found to be widely distributed in the samples. Furthermore, four other novel groupings of gut fungal sequences were identified that did not cluster with sequences from any of the previously described genera. Both gel- and sequence- based profiles for gut fungal populations suggested a lack of geographical restriction on occurrence of any individual fungal type.

Seasonal and Nutrient Supplement Responses in Rumen Microbiota Structure and Metabolites of Tropical Rangeland Cattle.

This study aimed to characterize the rumen microbiota structure of cattle grazing in tropical rangelands throughout seasons and their responses in rumen ecology and productivity to a N-based supplement during the dry season. Twenty pregnant heifers grazing during the dry season of northern Australia were allocated to either N-supplemented or un-supplemented diets and monitored through the seasons. Rumen fluid, blood, and feces were analyzed before supplementation (mid-dry season), after two months supplementation (late-dry season), and post supplementation (wet season). Supplementation increased average daily weight gain (ADWG), rumen NH3-N, branched fatty acids, butyrate and acetic:propionic ratio, and decreased plasma δ15N. The supplement promoted bacterial populations involved in hemicellulose and pectin degradation and ammonia assimilation: Bacteroidales BS11, Cyanobacteria, and Prevotella spp. During the dry season, fibrolytic populations were promoted: the bacteria Fibrobacter, Cyanobacteria and Kiritimatiellaeota groups; the fungi Cyllamyces; and the protozoa Ostracodinium. The wet season increased the abundances of rumen protozoa and fungi populations, with increases of bacterial families Lachnospiraceae, Ruminococcaceae, and Muribaculaceae; the protozoa Entodinium and Eudiplodinium; the fungi Pecoramyces; and the archaea Methanosphera. In conclusion, the rumen microbiota of cattle grazing in a tropical grassland is distinctive from published studies that mainly describe ruminants consuming better quality diets.