The microbial population in the hindgut plays a key role in the health and welfare of the horse: an active and functional fibrolytic bacterial population in the hindgut will convert fibrous feeds into volatile fatty acids which make a significant contribution to the energy requirements of the host, whilst an unbalanced fermentation leading to the proliferation of lactic acid producing bacteria will decrease fibre breakdown and lead to the accumulation of toxic by-products which can have wide ranging effects on the animal's metabolism including but not limited to the onset of laminitis. In the wider area of gut microbiology there is an active debate concerning the existence of a core stable microbial microbiota. It is estimated that there are perhaps 5000 unique bacterial phylotypes in the human gut when considered over a range of individuals under different spatial and temporal conditions. However, it is speculated that there are perhaps 300 phylotypes that make up a core stable microbial population in a healthy individual. This population is unique to individuals and changes only slightly as people age being resistant to modification and resilient to antibiotics. In addition, to the core group, there also are 'passengers' or transients, sometimes in great numbers, sometimes undetectable. The size of the transitory population may depend on external or coincidental influences such as diet, travelling or infections. Our recent observations have suggested that equine hindgut might not fit this model, in grazing horses the faecal microbiota (as relieved by TFRLP) was as different between individual horses on consecutive days as it was between individual horses on the same day. Whilst in horses being fed a commercial compounded feed there appear to be some stability in the faecal microbiota of individual horses during an intensive sampling regime over 72 hours, but when the same animals were sampled some 11 weeks later (having been on the same diet throughout) although again a stable population was determined over an intensive 72 hour sampling period the new population had little or no relationship to the orginal population, suggesting that the population was not stable over this 11 week period (Newbold et al unpublished observations). What is not clear from these studies is whether this represents a true instability in the equine microbiota and thus a striking departure from the human gut or merely that the equine gut contains a large volatile transient population. Unfortunately there is only very limited characterisation of the equine hindgut bacterial population either at the level of genomic or phenotypic diversity and we are unable to speculate on either the core microbiota nor its associated microbiome. High throughput pyrosequencing approaches such as 454 sequencing have allowed high density sequencing of environmental 16s rDNA transcriptomes providing previously unobtainable insights into bacterial diversity and population dynamics. Here we propose to combine highly controlled equine feeding trials using practically applicable diets and 454 based 16S rDNA transcriptomics to define the core microbiota in the equine hindgut and to provide information on it stability in comparison to other gut ecosystems. We wish to establish not only the stability of the equine hindgut microbiota but also the extent to which it is affected by diet and animal characteristics.

The oral bacterial flora is a large and complex community. It has long since been known that shifts in the members and proportions of bacterial species occurs with the onset of various diseases that either result in specific oral manifestations linked with systemic disease or that actually occur in the oral environment. Periodontal disease is the most significant oral disease in dogs with signs of this disease observed in over 70% of dogs over the age of 3 years. Furthermore multiple breeds characteristically suffer from an aggressive form of early-onset periodontitis which may progress to severe disease by the age of 2-3 years. Human periodontal disease is associated with the loss of so called 'beneficial' species and the appearance or overgrowth of 'disease-associated' species. Studies of the canine oral flora have also resulted in the identification of various organisms associated with the periodontally diseased state. In fact the development of oral disease has been described as involving three independent factors: 1) a change in the immunocompetency of the host; 2) a change in the numbers of beneficial bacteria in the mouth and 3) the succession of specific bacteria associated with disease from the normal 'health-associated' flora. It is unclear which or what interplay of these factors are responsible for the progression of breed-associated early onset canine periodontitis. Screening of amplified 16S rDNA libraries has identified that this same scenario exists in the canine oral flora with respect to periodontal disease. In this project we propose the use of 454 pyrosequencing using a metagenomic approach based upon analysis of 16S rRNA sequences from pooled plaque samples obtained from clinically healthy animals and from five breed-specific pooled plaque samples acquired from dogs with severe periodontal disease, where those breeds are associated with the early onset of disease (~2 years of age). This study will differ from those already done in two ways. First there are reports that DNA from some groups of microorganisms in complex communities is not amplified well by DNA polymerase, but can be analysed in a quantitative manner if amplified from a cDNA template, so rRNA species will be directly converted to cDNA, and this cDNA will be subjected to 454 pyrosequencing. Second, the sequencing will be done at a level to facilitate the analysis of over 333,000 sequences per pooled sample which allows a much greater depth of analysis of species and their represented numbers than can be easily done through a standard 16S rDNA plasmid-based library. The aims of this study will be to identify bacterial species that are associated with health and to determine whether any bacterial species are associated with predisposition of breeds to periodontitis. Following the identification of such species molecular tools will be developed that will allow the presence and numbers of these beneficial bacteria to be assessed from individual dogs in a quantitave and repeatable manner using qPCR. These protocols will support the industrial partner in studying the relative metabolic activity of the beneficial or disease associated species in response to nutritional formulations. Through comparison of such qPCR analyses on rRNA sequences and rDNA sequences the longitudinal monitoring of the effects of modifications in canine dietary formulation will be possible.

Articular cartilage degradation is the major cause of joint dysfunction and disability in osteoarthritis (OA) in humans and companion animals and leads to chronic morbidity, pain and impaired quality of life. OA is a complex, multifactorial disease and we are investigating the early stages of disease and the possible role of diet/functional actives to support joint health. As part of this work, we have recently developed and exploited an explant culture model of equine articular cartilage to identify secreted biomarkers using proteomics and cross-species peptide matching techniques. We have also used human Affymetrix GeneChip Microarrays to study gene expression in cartilage by cross-hybridizing messenger RNA from equine articular chondrocytes onto this human microarray platform. This work is the result of an ongoing BBSRC CASE studentship in collaboration with WALTHAM and the School of Veterinary Medicine and Science at the University of Nottingham which started in October 2006 and has just entered its third year (BBSRC grant BS/S/M/2006/13141). This approach has been fruitful and is generating large amounts of proteomic and gene expression datasets that require detailed and comparative analysis by bioinformatics and data-mining techniques. This CASE project will apply data mining and network biology approaches to identify potential biomarkers of early OA development, with an expectation that this work will also provide insight into the response of chondrocyte to inflammatory perturbation. We aim to achieve this by comparing differences between control cartilage samples and experimental samples exposed to a variety of disease relevant catabolic stimuli. The catabolic stimuli we propose to use include pro-inflammatory cytokines (i.e. interleukin 1 beta (IL-1beta), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha)), reactive oxygen species, static mechanical load and reduced extracellular pH. We will also study the effects of anabolic growth factors such as insulin-like growth factor I (IGF-I), transforming growth factor beta (TGF-beta), connective tissue growth factor (CTGF) and a panel of carefully selected nutrient derived actives known for their putative anti-inflammatory properties in the cartilage explant system. The proteomic and gene expression data extracted using this approach will help identify biomarkers with the capacity to discriminate between control and stimulated explants. We will develop the most appropriate model to discriminate between samples and determine at which stage of the protocol the data mining process is better able to identify individual biomarkers for the domain at hand, using data from proteomic and mass spectroscopy experiments and differentially expressed genes from microarray studies. Additionally a bioinformatics-intensive stage will focus on the characterization of sequence fragments to identify which proteins the peptide fragments originate from, identify potential role(s) for the secreted proteins present in the samples and, finally, infer the biological functions and interaction networks of the proteins.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.