POULTRY CRC LTD FINAL REPORT Sub-Project No: Poultry CRC 2.1.5 PROJECT LEADER: R.J. Hughes Sub-Project Title: Identification of microbial and gut-related factors driving bird performance DATE OF COMPLETION: 31 July 2016 © 2016 Poultry CRC Ltd All rights reserved. ISBN 1 921010 67 3 Identification of microbial and gut-related factors driving bird performance Sub-Project No. 2.1.5 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Poultry CRC, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, Poultry CRC encourages wide dissemination of its research, providing the Centre is clearly acknowledged. For any other enquiries concerning reproduction, contact the Communications Officer on phone 02 6773 3767. Researcher Contact Details Dr Robert J. Hughes Dr Mark S. Geier South Australian Research and Development Research and Innovations Services, Institute, The University of Adelaide, University of South Australia, Mawson Lakes Roseworthy Campus, Roseworthy, South Campus, Mawson Lakes, South Australia, Australia, 5371 5095 Phone: +61 8 8303 7788 Phone: +61 8 8302 3256 Email: [email protected] Email: [email protected] Prof Robert J. Moore Dr Dana Stanley Host-Microbe Interactions Laboratory, School of Institute for Future Farming Systems, Central Science, Royal Melbourne Institute of Queensland University, Rockhampton, Technology University, Bundoora, Victoria, 3083 Queensland ,4702 Phone: +61 3 9925 7580 Phone: +61 8 8302 3256 Email: [email protected] Email: [email protected] Dr Shubiao Wu Prof Zhongtang Yu Environmental and Rural Science, University of Animal Sciences, The Ohio State University, New England, Armidale, New South Wales, Columbus, Ohio, 43210. United States of 2351 America Phone: +61 2 6773 2238 Phone: +1 614 292 3057 Email: [email protected] Email: [email protected] In submitting this report, the researchers have agreed to the Poultry CRC publishing this material in its edited form. Poultry CRC Ltd Contact Details PO Box U242, University of New England, Armidale, New South Wales, 2351 Phone: 02 6773 3767 Fax: 02 6773 3050 Email: [email protected] Website: http://www.poultrycrc.com.au Published in 2016 ii Executive Summary Improving growth performance in chickens has long been one of the most important goals in poultry research. Previous Poultry CRC projects demonstrated that there was considerable bird-to- bird variation in performance and that particular gut microbes and intestinal gene expression patterns were linked to birds with high or low apparent metabolisable energy (AME). Further CRC projects identified a number of specific gut microbes that were indicative of high or low-performing birds, and demonstrated the important role of gene expression in the small intestine in affecting performance of meat chickens. Despite all the improvements in genetics, nutrition, disease control and husbandry over many decades, performance within and between flocks varies considerably and this variation causes significant losses to the industry both during production and processing. This sub-project brought together the collective knowledge and skills of many researchers in six institutions (five in Australia and one in the USA) to undertake excellent quality research to deliver significant benefits to the Australian chicken meat industry. The research team utilised recent technological advances in this area of science to comprehensively characterise the gut environment which is a critical factor in determining productivity of meat chickens, and reducing reliance on in-feed antibiotics. The main aims of this sub-projects were (1) customise an existing gut bacterial microarray chip for usage in Australia, (2) advance our understanding of gut microbes and host gene expression patterns underpinning bird performance, (3) identify feeding regimes (including probiotics) that can consistently facilitate the establishment of a healthy gut environment, (4) identify organisms with the potential to become new probiotics, and (5) advance our understanding of microbial changes during necrotic enteritis infection and identify dietary additives to reduce the severity of necrotic enteritis. The first generation gut microarray, referred to as Poultry Intestinal Tract Chip 1 or PITChip 1 originating from The Ohio State University was customised for usage in Australia by addition of DNA probes for caecal bacteria taken from low and high performing chickens in a series of experiments conducted at the University of Adelaide. PITChip2 was then used to evaluate bacterial DNA from further experiments conducted in Australia. PITChip3 was an advancement on earlier versions by inclusion of a wider region of highly conserved DNA to enable more accurate classification of gut bacteria. Differences in gut microbiota revealed by PITChip3 point to interactions between the gut microbiota and diet, and subsequent effects on growth performance and NE resistance seen in chickens fed with either corn- and wheat-based diets. iii The most technologically advanced methods available for microbiota analysis have been implemented and applied within this project, putting this poultry capability at the cutting edge within this area of research. During the life of this sub-project, DNA sequencing technologies advanced at such a rapid rate that it now possible to analyse hundreds of gut samples by 16S mRNA sequencing for less than AUD$8 per sample. Furthermore, the current technologies identify all the significant bacterial populations present in the samples, which make these technologies the preferred choice of most if not all researchers conducting the latest studies on gut microbial ecology. A key starting point for this project was to determine the reproducibility of previous results that had indicated correlations between microbiota composition and growth performance. Therefore, a series of three experiments were conducted on different batches of chickens all fed the same commercial diet and grown in the same facility under identical physical conditions. Growth rate and feed conversion of the chickens were comparable to the breeder’s expectations, however, within each trial there were differences in the performance parameters of individual birds and so relatively high and low performance birds could be identified. Analyses of the same bacterial DNA samples by PITChip2 and Roche pyrosequencing demonstrated (1) that there was wide variation in background gut microbiota between different batches of birds, (2) there were no specific gut microbes that were consistently associated with good or poor performance in three separate experiments, and (3) numerous consortia of gut microbes can support high performance in meat chickens. A further series of three experiments utilising different antibiotics and cereal grains further demonstrated that numerous consortia of gut microbes can support high performance in meat chickens. This finding of great variability in the microbiota of different batches of high performance birds was new and somewhat unexpected. It has very important implications for the direction of future research to understand the basis of gut health and is of fundamental importance in helping to understand the way in which products aimed at manipulating gut health (e.g., phytogenics, prebiotics, and probiotics) can be identified, developed and implemented. It is clear that they must be effective on and within a wide variety of different gut environments or must be effectively tailored for use under defined circumstances. Gene expression differences observed in the duodenum and other intestinal sections were not directly linked to differences in performance and hence did not provide useful tools to monitor the effects of dietary manipulation for productivity gain or identify host genes for marker assisted breeding for improved performance. We concluded that differences in gene expression seen in and across batches of birds may be due to influences other than performance, for example, slight variations in environmental conditions, differences in colonising gut microflora, breeder flock age and health status, and cleanliness of breeder farms and hatchery. Relatively early in the life of the iv project the decision was made to discontinue work on gene expression in the gut as it was not clear that there would be any downstream implementation of the knowledge that would be of direct benefit to the industry. The in vitro methods to identify potentially probiotic strains of bacteria that have been used by others and reported in the scientific literature were recognised as being largely unproven and having limitations. Therefore we initiated work to develop new in vivo based methods to identify probiotic strains. The resulting gut bacterial isolates from healthy chickens have potential for development into commercial probiotic products, not only for application in Australia, but also as exportable commodities. In ovo application of these pure cultures should be explored, along with administration of probiotics via other routes and at other times during the