The relationship between age of sheep and the microbial community structure in their udders will be investigated to test the hypotheses: 1. With increasing lactation number, the species and strains of bacteria colonising the udder increase 2. The bacterial load of pathogens and the ratio of pathogenic to benign strains of microbes affect weight gain in lambs, a marker for milk production 3. Pathogen load and presence of specific virulence markers in sheep with either clinical or sub-clinical mastitis is related to sheep age (number of lactations). This will contextualise the processes by which udders of ruminants become infected, the importance of the balance between benign and pathogenic strains and begin to understand the dynamics of the interactions between the host and its microbes, whether good or bad. To address Hypothesis 1, 10 - 30 sheep (200 in total) from one farm of each year group will be sampled 48 hrs after lambing. We will record the identity, age (from teeth), breed, body condition, number and birth weight of lambs and physical appearance and texture of the udder and teat. Ewes will be body condition scored and lambs weighed at 1, 3, 5, and 7 weeks. The udder bacterial community will be investigated using culture and molecular-based whole community approaches. Milk samples will be split into two aliquots, one frozen for molecular analysis and the other chilled for culturing. Chilled samples will be cultured according to standard practice. Isolates from these plates will be characterised using biochemical tests and selected isolates will be characterised more fully using molecular diagnostic tests. Isolates will be stored at -80 in 10% glycerol. The whole bacterial community in the milk will be analysed using a general bacterial PCR approach. DNA isolated from the milk samples will be amplified using general bacterial PCR primers and the community diversity determined using a fingerprinting technique such as DGGE. Using the fingerprinting analysis selected samples will be deep-sequenced using long-read amplicon 454 pyrosequencing. Such an analysis will provide complete coverage of the bacterial community in all of the sampled udders of sheep at different ages and so allow an analysis of how the microbial community changes as sheep age and whether specific populations are linked to reduced milk production and the healthy development of lambs. The impact of the community diversity of bacteria and presence of specific genotypes on lamb growth having adjusted for birth weight, litter size, ewe body condition and any other circumstances that might have affected growth e.g. an episode of diarrhoea in the lamb will be used to assess the association between infected udders and milk production. This type of in-depth analysis has not been done before. The presence and number of virulence determinants, such as the coagulase genes in a detected staphylococcal community, will be analysed using qPCR. The ratio of pathogenic to benign strains will be determined with reference to the bacterial community structure and statistical analyses used to determine relationships and patterns in the data. Hypothesis 3 will be tackled primarily using isolated strains grown from the sampled milk. The presence of virulence factors in isolates will be analysed with a particular focus on determining whether there are other physiological or genetic factors that can be determined to gain a deeper understanding of the process from infection to disease in ruminant mastitis. An overall aim will be to determine whether microbial colonisation of the udder is inevitable, always detrimental or potentially beneficial if pathogen strain load is limited. We will test results for hypothesis 3 by manipulating the udder of sheep in vivo by introducing intramammary treatment five sheep of different ages and assessing the impact of this compared with controls not given such treatment.

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.

Contagious ovine digital dermatitis (CODD) is an extremely severe and relatively recent cause of infectious lameness in sheep in the UK, affecting approximately 35-53% of UK sheep farms and 300,000 sheep annually. The economic impact of CODD is unknown but likely similar to that of footrot, another common cause of lameness which is estimated at £24million per year. The Farm Animal Welfare Council, who report to the UK government on farm animal welfare issues, identified that research into this new disease should be a priority. Research into footrot has led to substantial improvements for the evidenced based advice available to farmers and vets on this aspect of lameness, and has been widely taken up by the sheep industry. However many farmers are struggling to control CODD infection in their flocks and substantial research support is required here. The applicants are currently the only research group studying CODD and through funding from the British Veterinary Association, HCC and EBLEX, this research group is making advances in our understanding of the epidemiology, pathology and microbiology of the disease, including the development of control strategies using antimicrobial drugs. The outputs of this work will produce practical solutions for the farming industry. Given the severe pressure on antimicrobial usage world-wide, sustainable, non-antibiotic solutions to CODD must focus on preventative approaches such as stopping the introduction of disease into naïve flocks through biosecurity and improvement in the sheep's ability to resist infection through vaccination. Both these solutions require a greater knowledge of the agent(s) causing the disease than we currently possess. We have already demonstrated evidence that specific culturable treponemes identical to those from bovine digital dermatitis have an association with CODD lesions and data from other studies has suggested a role for Dichelobacter nodosus and Fusobacterium necrophorum. This study proposes to i) carry out substantial metagenomic (entire microbial community) investigations of CODD lesions to identify the key microbes involved and ii)to characterise the host immune response to the identified microbes and determine whether specific antigens may allow a protective immune response to underpin future vaccine studies and iii) to use additional metagenomic surveys of the host farm environment and faeces to detect the relevant microbes which should allow for relevant farmer / veterinary guidelines to improve disease control. This proposal would ensure we obtain a greater understanding of the role of different bacteria in CODD development. This information will be key to future vaccine studies so that the correct microorganisms are targeted for vaccine design. Monitoring specific immunity in the naive sheep flock to be used in this study after exposure to CODD will allow assessment of the different stages of the infective process. This will not only describe stages of microbial infections but may also identify whether some animals are able to produce an effective immune response allowing for subsequent antigen discovery for effective vaccines. Identification of the considered causal microorganisms will also allow for more appropriate antimicrobial treatments thus reducing the risk of antibiotic resistance developing. Monitoring bacterial populations of CODD lesions post treatment should inform biosecurity practices i.e. whether treated animals result in bacteriological cure or become asymptomatic CODD "carriers". Monitoring faeces and environmental samples prior and post antibiotic treatment will also allow for development of best farm practices to minimise reinfection of sheep with CODD thus preventing failure / overuse of antibiotic treatment at the flock level. Investigating CODD using the various methods described above, should improve understanding of the disease and contribute towards the eradication of this painful and expensive disease.

This project is focussed on the health, productivity and welfare of sheep. We will concentrate on aspects of metabolism that affect lifelong health and wellbeing. Specifically, we will study a key aspect of metabolism referred to as one-carbon (1C) metabolism. This is important because it affects many key processes in the cell, including DNA synthesis, DNA methylation and cell proliferation. It does this by delivering methyl groups, which are central to these biochemical reactions. Deficiencies in metabolites involved in these pathways, such as choline, methionine, folate and vitamin B12, have adverse effects on animal development and wellbeing. For example, deficiencies in vitamin B12 and/or folate can affect fertility and fetal development, and lead to poor growth, vascular disease and metabolic syndrome in adult animals and humans. 1C metabolism pathways are complex and are affected by many genes. We hypothesise that mutations in these genes will affect 1C metabolism and the vulnerability of animals to micronutrient deficiencies. In this study we will identify such mutations, determine their functional significance (i.e. what they do and how important they are), test their impact in animals fed different diets, and find ways to use this information to improve the welfare of farm animals. Firstly, we will identify mutations (i.e. single nucleotide polymorphisms or SNPs) in around 40 genes directly affecting 1C metabolism, and in very closely related pathways involved in energy metabolism, cell proliferation, DNA synthesis and DNA methylation. To achieve this in sheep we will create our own sequence data. We estimate that we will identify 2000-4000 SNPs by these methods. We will then conduct a large-scale study with liver samples collected from around 300 sheep slaughtered at local abattoirs. We will genotype each sample (to determine which SNPs are present), measure gene expression and conduct a comprehensive analysis of all metabolites involved in 1C metabolism. Interpretation of these data will inform us on SNP function and how these SNPs affect pathways involved in 1C metabolism. To achieve this we will collate the required information and, using our knowledge of these genomes combined with complex bioinformatic analyses, determine the subset of SNPs that have the greatest impact on 1C metabolism. We expect to identify an estimated 100 or so functionally significant SNPs in these genes. We will then construct a 'SNP chip'; i.e. a tool to genotype sheep simultaneously and cheaply for many SNPs. This chip will be used to screen several flocks of sheep to identify 24 'Low-risk' and 24 'High-risk' weaned lambs, and 24 'Low-risk' and 24 'High-risk' breeding ewes. We will then monitor these animals in separate studies involving Control and Methyl-Deficient diets (i.e. two genotypes by two diets). For weaned lambs we will focus on effects on growth, animal health and liver metabolism, and carcass yields and composition. For breeding ewes we will also focus on animal health and liver metabolism, but extend studies to consider effects on chemical modifications to DNA (i.e. DNA methylation) in early (Day 16) male and female embryos. This research will provide novel insights into nutrient x gene interactions for many components of 1C metabolism and how they affect (a) lamb production efficiency, health and welfare, and (b) early development of mammalian embryos influencing fertility and the long-term health and wellbeing of offspring. We will be able to use this information to help breed animals with better functioning 1C metabolism (leading to permanent improvements in welfare and productivity) and/or to improve animal diets. As 1C metabolism also influences human development and health our results will also have biomedical research benefits, and increase the utility of sheep as a model species for this type of research.

Liver fluke is a common parasite that affects sheep and cattle in the UK. It is found throughout the world and in some countries it affects humans too, causing serious and sometimes fatal disease. Fluke infected cattle lose weight, become anaemic, lethargic and stop being productive. This has a serious effect on the welfare of the animal and serious economic consequences for the farmer. It is thought that fluke costs UK agriculture at least £300million pounds a year through direct losses, but real costs are probably much higher. Fluke has become much more common over the past 10 years, due in part to our changing weather patterns, wet summers and mild winters favour the development of the parasite and its vector - a mud snail, found commonly throughout Britain. In a recent study we found 75% of dairy herds had evidence of fluke infection. Future climate change is predicted to have a significant impact on prevalence of infection, changing the epidemiology and increasing incidence of disease. Increased cattle movements and changes to both farm management and environmental schemes are exacerbating the problem. A limited range of drugs is available to control fasciolosis. Only one drug - triclabendazole (TCBZ), is effective against early and late juvenile and adult stages of the parasite and is used extensively for prophylaxis and treatment of disease. There is growing evidence of resistance to TCBZ in fluke populations, moreover the European Medicines Agency has recently revised its advice on drugs used to treat fluke such that they are now contra-indicated in dairy animals. Targeted use of drugs, at specific times of year will slow the development of drug resistance and reduce the overall quantity of drug used, but a better understanding of the epidemiology and transmission of disease is vital if we are to develop control programmes that rely on improved on farm management practises rather than depending solely on drugs. This ultimately will be a sustainable and cost-effective way to control both clinical and sub-clinical disease in cattle and is the express desire of the livestock industry. Specifically requested by the farming industry, the purpose of this project is to produce new, sustainable, bespoke control programmes for beef and dairy farms, to reduce losses associated with fluke infection. In order to achieve this we must first develop diagnostic tests to identify infected herds. We already have good tools that we can use on milk samples to detect infected dairy herds but we need similar tests that are appropriate for beef herds. In addition we are aware of a newly emerging parasite problem, the rumen fluke. It is not clear if this parasite causes disease but it has the potential to interfere with the diagnostic tests we are developing for fluke. Therefore we will also develop a molecular test for rumen fluke. Secondly we will develop a system to categorise snail habitats that can be used to analyse satellite maps on a regional geographic scale to obviate the need to visit every farm to investigate snail habitat. We will also investigate how cow behaviour affects how the parasite gets to a snail host and from the snail host back to the cow. These are risk factors for fluke infection on a farm. Other risk factors, particularly husbandry practices, physical and environmental factors will be obtained from a study of 250 farms and these data fed into statistical and mathematical models to determine theoretically which of these factors are the most important in determining whether a farm has fluke or not. Concurrently we will assess the cost-benefit of changing these practices. Finally we will conduct a trial to evaluate if changing farm practice is effective in reducing levels of infection. We are working in partnership with the Agricultural Levy boards of the UK to implement improved control of fluke infection to benefit animal health, welfare and profitability of livestock farming in the UK.