The intestinal tracts of animals are populated by hundreds, perhaps thousands of bacterial species in vast numbers - tens of billions of bacterial cells per gram of intestinal contents. Collectively these bacteria make up the microbiota, and in its overall composition and genetic makeup, the population is called the microbiome. The metagenome can be defined as the totality of DNA sequences of all the component organisms. Many of these species have not been cultured in the laboratory and most are poorly characterised. Yet they are crucial partners to their animal hosts in nutrition and health - and include the so-called 'good bacteria' of the digestive tract. For the first time, a new approach, high-throughput DNA sequencing (HTS), makes it possible to identify and count on a large scale each bacterial species in a microbiome using specific sequence 'signatures'. These are obtained from a gene, present in all bacteria, that codes for an RNA molecule that is part of the protein synthesis machinery. This gene (16S rDNA) includes both near-constant regions, useful for its specific enrichment from the metagenome, and highly variable regions where the sequence is characteristic of the bacterial species concerned. It is these variable sequence 'signatures' that will identify component bacteria. It is also feasible to infer the biochemical activities of the microbiome by using HTS to identify genes in the metagenome that encode enzymes, capable of digesting dietary substances that could enhance the nutrition of the host organism. For HTS analysis, DNA is extracted from intestinal (caecal) contents or faeces. The16SrDNA sequences are enriched by amplification, using a method called PCR, to create a pool of fragments representing all the bacteria present. These fragments are then individually analysed and their sequences, amounting to one million or more per analysis run, matched computationally to those of all known bacterial species. In this way the bacteria from which they were derived can be identified. If there is no exact match, the closest known relative can be identified. 'Proof of principle' experiments have established the practicality of this approach to unravel the complexities of intestinal microbiology. However few published studies have rigorously defined the variability inherent in the technology, or between individuals, or from day to day and as dietary intake changes. Such data are essential if the enormous power of the technology is to be exploited in rational, hypothesis-based scientific studies. We propose to obtain these data using broiler chickens, so that groups of birds of defined and matched age, breed and diet can be accessed at relatively low cost. Having established robust baselines for analysis, we will tackle some key questions about the role of the microbiota. How does the microbiome change as birds age and change diet? What is the effect of colonisation of the intestinal tract by food borne pathogens such as Campylobacter, and can this information be used to enhance levels of bacteria that may suppress the invading pathogen? We will also assess the potential of sequencing the entire metagenome, the gene pool representing the microbiota as a whole. We will seek evidence for bacterial enzymes that may add to the digestive capacity of the host and thus enhance growth and productivity of the birds. We believe this proposal will firmly establish the scientific credentials of intestinal microbiome research on food animals, and prepare the way for future research into the role of the microbiome in animal health and welfare, efficient utilisation of feed, emergence of antibiotic resistance, and the establishment of intestinal pathogens.

The intestinal tracts of animals are populated by hundreds, perhaps thousands of bacterial species in vast numbers - tens of billions of bacterial cells per gram of intestinal contents. Collectively these bacteria make up the microbiota, and in its overall composition and genetic makeup, the population is called the microbiome. The metagenome can be defined as the totality of DNA sequences of all the component organisms. Many of these species have not been cultured in the laboratory and most are poorly characterised. Yet they are crucial partners to their animal hosts in nutrition and health - and include the so-called 'good bacteria' of the digestive tract. For the first time, a new approach, high-throughput DNA sequencing (HTS), makes it possible to identify and count on a large scale each bacterial species in a microbiome using specific sequence 'signatures'. These are obtained from a gene, present in all bacteria, that codes for an RNA molecule that is part of the protein synthesis machinery. This gene (16S rDNA) includes both near-constant regions, useful for its specific enrichment from the metagenome, and highly variable regions where the sequence is characteristic of the bacterial species concerned. It is these variable sequence 'signatures' that will identify component bacteria. It is also feasible to infer the biochemical activities of the microbiome by using HTS to identify genes in the metagenome that encode enzymes, capable of digesting dietary substances that could enhance the nutrition of the host organism. For HTS analysis, DNA is extracted from intestinal (caecal) contents or faeces. The16SrDNA sequences are enriched by amplification, using a method called PCR, to create a pool of fragments representing all the bacteria present. These fragments are then individually analysed and their sequences, amounting to one million or more per analysis run, matched computationally to those of all known bacterial species. In this way the bacteria from which they were derived can be identified. If there is no exact match, the closest known relative can be identified. 'Proof of principle' experiments have established the practicality of this approach to unravel the complexities of intestinal microbiology. However few published studies have rigorously defined the variability inherent in the technology, or between individuals, or from day to day and as dietary intake changes. Such data are essential if the enormous power of the technology is to be exploited in rational, hypothesis-based scientific studies. We propose to obtain these data using broiler chickens, so that groups of birds of defined and matched age, breed and diet can be accessed at relatively low cost. Having established robust baselines for analysis, we will tackle some key questions about the role of the microbiota. How does the microbiome change as birds age and change diet? What is the effect of colonisation of the intestinal tract by food borne pathogens such as Campylobacter, and can this information be used to enhance levels of bacteria that may suppress the invading pathogen? We will also assess the potential of sequencing the entire metagenome, the gene pool representing the microbiota as a whole. We will seek evidence for bacterial enzymes that may add to the digestive capacity of the host and thus enhance growth and productivity of the birds. We believe this proposal will firmly establish the scientific credentials of intestinal microbiome research on food animals, and prepare the way for future research into the role of the microbiome in animal health and welfare, efficient utilisation of feed, emergence of antibiotic resistance, and the establishment of intestinal pathogens.

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.

Piglet weaning through separation from the sow is a critical and stressful period in the life of a pig. At this stage piglets are particularly vulnerable to gut problems and have a tendency to develop a condition called post-weaning diarrhoea. This disease is primarily caused by bacteria and viruses which challenge the piglet's immature gut, and is a welfare and economic concern to farmers, because piglets suffer weight loss, discomfort and sometimes death. Zinc oxide is currently added to pig feed as a dietary supplement to reduce post-weaning diarrhoea and boost growth. However, whilst Zinc oxide improves piglet gut health, there are environmental concerns due to the potential contamination of land with zinc through pig manure and waste. High zinc levels are also thought to promote the ability of bacteria to evade antibiotics, resulting in antimicrobial resistance. For these reasons, from June 2022 the supplementation of pig feed with high levels of Zinc oxide will be banned in the UK and Europe. Within the pig farming sector there are serious concerns that this ban will have a damaging impact on the health and welfare of piglets during weaning. Farmers and veterinarians anticipate post-weaning diarrhoea will become harder to manage and will require more medical treatment. As a consequence, antibiotic use to treat piglet gut infections may increase, exacerbating already high usage within the pig industry. This has implications for limiting levels of antimicrobial resistance in livestock and the food-chain. Working directly with farmers during the transitional period to "zero zinc", in this study we aim to investigate the impact of the ban on piglet health and growth. Our goal is to identify practical measures that will make an on-farm difference to reducing disease, whilst improving animal welfare and productivity. We will examine a range of farm management, husbandry and biological measures, to see which factors influence post-weaning diarrhoea in piglets. We will do this by collecting pig faecal samples from farms both before and after the introduction of the June 2022 ban, and identifying any changes in the type and numbers of microbes found in piglet faeces. We will look at the composition of the "friendly" gut bacteria, as well as disease-causing bugs, and examine levels of resistance to antibiotics. At the same time we will analyse data from participating farms to measure piglet health status, and track any management changes farmers may have introduced to lessen the impact of the Zinc oxide ban. This, together with a questionnaire-based survey of pig farmers across the UK, will provide a picture of both the perceptions and reality of the Zinc oxide ban on piglet weaning. Crucially, by working directly with farmers, we hope to find acceptable solutions that may reduce disease in piglets, whilst providing information to help offset the uncertainty accompanying the journey to "zero zinc".