Polyomic tools for an emerging livestock parasite, the rumen fluke Calicophoron daubneyi; identifying shifts in rumen functionality.

BACKGROUND: Diseases caused by parasitic flatworms of rumen tissues (paramphistomosis) are a significant threat to global food security as a cause of morbidity and mortality in ruminant livestock in subtropical and tropical climates. Calicophoron daubneyi is currently the only paramphistome species commonly infecting ruminant livestock in temperate European climates. However, recorded incidences of C. daubneyi infection in European livestock have been increasing over the last decade. Whilst clinical paramphistomosis caused by adult worms has not been confirmed in Europe, fatalities have been attributed to severe haemorrhagic enteritis of the small intestine resulting from the migration of immature paramphistomes. Large numbers of mature adults can reside in the rumen, yet to date, the impact on rumen fermentation, and consequently on productivity and economic management of infected livestock, have not been resolved. Limited publicly available nucleotide and protein sequences for C. daubneyi underpin this lack of biological and economic understanding. Here we present for the first time a de novo assembled transcriptome, with functional annotations, for adult C. daubneyi, which provides a reference database for protein and nucleotide sequence identification to facilitate fundamental biology, anthelmintic, vaccine and diagnostics discoveries. RESULTS: This dataset identifies a number of genes potentially unique to C. daubneyi and, by comparison to an existing transcriptome for the related Paramphistomum cervi, identifies novel genes which may be unique to the paramphistome group of platyhelminthes. Additionally, we present the first coverage of the excretory/secretory and soluble somatic proteome profiles for adult C. daubneyi and identify the release of extracellular vesicles from adult C. daubneyi parasites during in vitro, ex-host culture. Finally, we have performed the first analysis of rumen fluke impacting upon rumen fermentation parameters using an in vitro gas production study resulting in a significant increase in propionate production. CONCLUSIONS: The resulting data provide a discovery platform (transcriptome, proteomes, EV isolation pipeline and in vitro fermentation system) to further study C. daubneyi-host interaction. In addition, the acetate: propionate ratio has been demonstrated to decrease with rumen fluke infection suggesting that acidotic conditions in the rumen may occur.

The rumen microbiome: an underexplored resource for novel antimicrobial discovery.

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.

Changes in the Total Fecal Bacterial Population in Individual Horses Maintained on a Restricted Diet Over 6 Weeks.

Twelve mature (aged 5-16 years) horses and ponies of mixed breed and type were fed restricted (1.25% BM Dry matter) quantities of one of two fiber based diets formulated to be iso-caloric. Diet 1 comprised of 0.8% body mass (BM) of chaff based complete feed plus 0.45% BM low energy grass hay (the same hay used for both diets). Diet 2 comprised 0.1% BM of a nutrient balancer plus 1.15% BM grass hay. Fecal samples were collected at week 10 and week 16. DNA was extracted and the V1-V2 regions of 16SrDNA were 454-pyrosequenced to investigate the bacterial microbiome of the horse. The two most abundant phyla found in both diets and sampling periods were the Firmicutes and Bacteroidetes. There was a clear reduction in Bacteroidetes with a concordant increase in Firmicutes over time. There was a limited degree of stability within the bacterial community of the hindgut of horses, with 65% of bacteria retained, over a 6 week period whilst on a uniform diet. The presence of a core community defined by being present in all samples (each animal/diet combination) included in the study and being present at 0.1% relative abundance (or greater) was identified. In total 65 operational taxonomic units (OTUs) were identified that fit the definition of core making up 21-28% of the total sequences recovered. As with total population the most abundant phyla were the Bacteroidetes followed by the Firmicutes, however there was no obvious shift in phyla due to period. Indeed, when the relative abundance of OTUs was examined across diets and periods there was no significant effect of diet or period alone or in combination on the relative abundance of the core OTUs.

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis.

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Can the Bacterial Community of a High Arctic Glacier Surface Escape Viral Control?

Glacial ice surfaces represent a seasonally evolving three-dimensional photic zone which accumulates microbial biomass and potentiates positive feedbacks in ice melt. Since viruses are abundant in glacial systems and may exert controls on supraglacial bacterial production, we examined whether changes in resource availability would promote changes in the bacterial community and the dynamics between viruses and bacteria of meltwater from the photic zone of a Svalbard glacier. Our results indicated that, under ambient nutrient conditions, low estimated viral decay rates account for a strong viral control of bacterial productivity, incurring a potent viral shunt of a third of bacterial carbon in the supraglacial microbial loop. Moreover, it appears that virus particles are very stable in supraglacial meltwater, raising the prospect that viruses liberated in melt are viable downstream. However, manipulating resource availability as dissolved organic carbon, nitrogen, and phosphorous in experimental microcosms demonstrates that the photic zone bacterial communities can escape viral control. This is evidenced by a marked decline in virus-to-bacterium ratio (VBR) concomitant with increased bacterial productivity and number. Pyrosequencing shows a few bacterial taxa, principally Janthinobacterium sp., dominate both the source meltwater and microcosm communities. Combined, our results suggest that viruses maintain high VBR to promote contact with low-density hosts, by the manufacture of robust particles, but that this necessitates a trade-off which limits viral production. Consequently, dominant bacterial taxa appear to access resources to evade viral control. We propose that a delicate interplay of bacterial and viral strategies affects biogeochemical cycling upon glaciers and, ultimately, downstream ecosystems.

Temporal dynamics of the metabolically active rumen bacteria colonizing fresh perennial ryegrass.

This study investigated successional colonization of fresh perennial ryegrass (PRG) by the rumen microbiota over time. Fresh PRG was incubated in sacco in the rumens of three Holstein × Friesian cows over a period of 8 h, with samples recovered at various times. The diversity of attached bacteria was assessed using 454 pyrosequencing of 16S rRNA (cDNA). Results showed that plant epiphytic communities either decreased to low relative abundances or disappeared following rumen incubation, and that temporal colonization of the PRG by the rumen bacteria was biphasic with primary (1 and 2 h) and secondary (4-8 h) events evident with the transition period being with 2-4 h. A decrease in sequence reads pertaining to Succinivibrio spp. and increases in Pseudobutyrivibrio, Roseburia and Ruminococcus spp. (the latter all order Clostridiales) were evident during secondary colonization. Irrespective of temporal changes, the continually high abundances of Butyrivibrio, Fibrobacter, Olsenella and Prevotella suggest that they play a major role in the degradation of the plant. It is clear that a temporal understanding of the functional roles of these microbiota within the rumen is now required to unravel the role of these bacteria in the ruminal degradation of fresh PRG.

Characterization of the rumen lipidome and microbiome of steers fed a diet supplemented with flax and echium oil.

Developing novel strategies for improving the fatty acid composition of ruminant products relies upon increasing our understanding of rumen bacterial lipid metabolism. This study investigated whether flax or echium oil supplementation of steer diets could alter the rumen fatty acids and change the microbiome. Six Hereford × Friesian steers were offered grass silage/sugar beet pulp only (GS), or GS supplemented either with flax oil (GSF) or echium oil (GSE) at 3% kg(-1) silage dry matter in a 3 × 3 replicated Latin square design with 21-day periods with rumen samples taken on day 21 for the analyses of the fatty acids and microbiome. Flax oil supplementation of steer diets increased the intake of polyunsaturated fatty acids, but a substantial degree of rumen biohydrogenation was seen. Likewise, echium oil supplementation of steer diets resulted in increased intake of 18:4n-3, but this was substantially biohydrogenated within the rumen. Microbiome pyrosequences showed that 50% of the bacterial genera were core to all diets (found at least once under each dietary intervention), with 19.10%, 5.460% and 12.02% being unique to the rumen microbiota of steers fed GS, GSF and GSE respectively. Higher 16S rDNA sequence abundance of the genera Butyrivibrio, Howardella, Oribacterium, Pseudobutyrivibrio and Roseburia was seen post flax feeding. Higher 16S rDNA abundance of the genus Succinovibrio and Roseburia was seen post echium feeding. The role of these bacteria in biohydrogenation now requires further study.

Characterisation of the faecal bacterial community in adult and elderly horses fed a high fibre, high oil or high starch diet using 454 pyrosequencing.

Faecal samples were collected from seventeen animals, each fed three different diets (high fibre, high fibre with a starch rich supplement and high fibre with an oil rich supplement). DNA was extracted and the V1-V2 regions of 16SrDNA were 454-pyrosequenced to investigate the faecal microbiome of the horse. The effect of age was also considered by comparing mature (8 horses aged 5-12) versus elderly horses (9 horses aged 19-28). A reduction in diversity was found in the elderly horse group. Significant differences between diets were found at an OTU level (52 OTUs at corrected Q<0.1). The majority of differences found were related to the Firmucutes phylum (37) with some changes in Bacteroidetes (6), Proteobacteria (3), Actinobacteria (2) and Spirochaetes (1). For the forage only diet,with no added starch or oil, we found 30/2934 OTUs (accounting for 15.9% of sequences) present in all horses. However the core (i.e. present in all horses) associated with the oil rich supplemented diet was somewhat smaller (25/3029 OTUs, 10.3% ) and the core associated with the starch rich supplemented diet was even smaller (15/2884 OTUs, 5.4% ). The core associated with samples across all three diets was extremely small (6/5689 OTUs accounting for only 2.3% of sequences) and dominated by the order Clostridiales, with the most abundant family being Lachnospiraceae. In conclusion, forage based diets plus starch or oil rich complementary feeds were associated with differences in the faecal bacterial community compared with the forage alone. Further, as observed in people, ageing is associated with a reduction in bacterial diversity. However there was no change in the bacterial community structure in these healthy animals associated with age.

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems.

Identification of a core bacterial community within the large intestine of the horse.

The horse has a rich and complex microbial community within its gastrointestinal tract that plays a central role in both health and disease. The horse receives much of its dietary energy through microbial hydrolysis and fermentation of fiber predominantly in the large intestine/hindgut. The presence of a possible core bacterial community in the equine large intestine was investigated in this study. Samples were taken from the terminal ileum and 7 regions of the large intestine from ten animals, DNA extracted and the V1-V2 regions of 16SrDNA 454-pyrosequenced. A specific group of OTUs clustered in all ileal samples and a distinct and different signature existed for the proximal regions of the large intestine and the distal regions. A core group of bacterial families were identified in all gut regions with clear differences shown between the ileum and the various large intestine regions. The core in the ileum accounted for 32% of all sequences and comprised of only seven OTUs of varying abundance; the core in the large intestine was much smaller (5-15% of all sequences) with a much larger number of OTUs present but in low abundance. The most abundant member of the core community in the ileum was Lactobacillaceae, in the proximal large intestine the Lachnospiraceae and in the distal large intestine the Prevotellaceae. In conclusion, the presence of a core bacterial community in the large intestine of the horse that is made up of many low abundance OTUs may explain in part the susceptibility of horses to digestive upset.

Strong stability and host specific bacterial community in faeces of ponies.

The horse, as a hindgut fermenter, is reliant on its intestinal bacterial population for efficient diet utilisation. However, sudden disturbance of this population can result in severe colic or laminitis, both of which may require euthanasia. This study therefore aimed to determine the temporal stability of the bacterial population of faecal samples from six ponies maintained on a formulated high fibre diet. Bacterial 16S rRNA terminal restriction fragment length polymorphism (TRFLP) analyses of 10 faecal samples collected from 6 ponies at regular intervals over 72 hour trial periods identified a significant pony-specific profile (P<0.001) with strong stability. Within each pony, a significantly different population was found after 11 weeks on the same diet (P<0.001) and with greater intra-individual similarity. Total short chain fatty acid (SCFA) concentration increased in all ponies, but other changes (such as bacterial population diversity measures, individual major SCFA concentration) were significant and dependent on the individual. This study is the first to report the extent of stability of microbes resident in the intestinal tract as represented with such depth and frequency of faecal sampling. In doing so, this provides a baseline from which future trials can be planned and the extent to which results may be interpreted.