The chicken caecal microbiome: from baselines to biological impact

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.

We propose to exploit the enormous opportunity offered by high throughput DNA sequencing (HTS) to make good the current deficit in knowledge of gut bacteria and their biological significance, in the chicken: the most important food animal exploited by humans. The intestinal microbiota play a central role in every aspect of alimentary tract function. Yet full scientific investigation of their composition and function has been thwarted by technical and logistic difficulties, both in culture-based conventional microbiology and in clone library approaches to phylogenetic analysis. HTS technology, while very cost effective in yielding very large amounts of information, is nevertheless not accessible without significant expenditure. We therefore propose first to undertake the crucial, but non-trivial, scoping experiments required to assess technical reproducibility and biological variability in the chicken caecal microbiome. We will use the classical approaches of experimental replication and statistical analysis to enable robust experimental design for subsequent studies. These will be the first such extensive baseline experiments to our knowledge in the emerging field of microbiomics based on HTS. We will then define the effects of fundamental variables in the life of a broiler chicken: through developmental change and growth, with dietary change to maturity, and in different host genetic backgrounds. In a key experiment relating to food safety, we will explore the effect of colonisation of the caeca with the most prevalent bacterial foodborne diarrhoeal pathogen, Campylobacter jejuni. Lastly we will probe the chicken caecal metagenome, to seek knowledge of the role of the microbiota in processing feed components such as complex plant-derived glycans. We believe this work will lay the foundation for a wide range of new research in the chicken, and on a wider front establish standards for future HTS-based microbiomics research.

Total income from farming in the UK was estimated to be £2.54 billion in 2007 and sustainable agriculture remains a cornerstone of BBSRC and Defra research requirements. In particular poultry health is vital to the supply and safety of food. According to DEFRA, in the UK alone, ~29 million laying hens and > 850 million chickens are killed each year. Securing the supply of food will be vital to meet the needs of the growing and increasingly affluent population and was recently identified by BBSRC as a priority for future research. Defining the basis of beneficial and detrimental effects of diets and husbandry practices at the level of microbial communities is therefore timely and highly relevant to industry. There is considerable potential for translation of the research to practice, for example through the identification of novel constituents of the microbiota or microbial factors that could be given in early life to improve gut health. as is already commonplace in poultry farming with ill-defined probiotics such as Aviguard and Briolact. Functional metagenomics may also identify metabolic activities upon which food-producing animals are reliant for extraction of nutrients and energy, providing key data to improve the health and yield of animals. And crucially on this point, embedded within the project is a collaborator from the chicken feed industry: Mike Bedford from AB Vista. It is also important to consider the impact of changing husbandry practices on gut health and the potential for spread and emergence of pathogens, in particular owing to consumer demand for free-range poultry and EC directives to change the way poultry are reared. Thus the research will benefit the UK poultry industry and ultimately its customers and consumers. The proposed work will contribute new and fundamental knowledge on numerous fronts relevant to BBSRC strategy and priorities, including aspects of Integrative Biology, Sustainable Agriculture, The Healthy Organism and Tools and Technology. Thus there will be impacts in UK Industry especially in relation to animal husbandry and efficient use of feeds, in Society in terms of inputting into Food Security policy and Diet and Health research, and a major impact in new knowledge and techniques for the exploitation of high throughput sequencing methods to underpin scientific advances. The methods developed will underpin new research into diet and health in both humans and food animals, where there is enormous potential to adopt and develop the technology to better understand the intimate relationship between higher animals and their microbiome in health and disease. With regard to the human microbiome, the impacts are potentially very large, for example in relation to changes in the microbiome during the aging process and the contribution this makes to health in an aging population. On a broader front, the investigation of complex microbial communities has implications in a wide range of spheres of activity. The technology is largely generic and major opportunities exist to exploit it in fields such as soil and environmental water and associated microbiomes in relation to agriculture, pollution and environmental sustainability; in waste treatment by composting or sewage treatment; in the food industry where complex fermented foodstuffs may involve the action of microbial communities; and in medicine where it has already been exploited in, for example, direct characterisation of the complex microbiota of mixed infections and abscesses. In all of these areas, there are major implications for society and impacts in relation to key policy areas such as Food Security, Climate Change, Control of Infectious Diseases and Healthier Aging. Finally the composition and stability of the microbiome are intrinsically complex in nature so the proposed work has considerable relevance to 'systems' approaches to bioscience.