Alma Mater Studiorum – Università di Bologna DOTTORATO DI RICERCA IN Scienze e Tecnologie Agrarie, Ambientali e Alimentari Ciclo XXIX° Settore Concorsuale di afferenza: 07/H2 Settore Scientifico disciplinare: VET04 Assessment of the impact of different feeding strategies on chicken gastrointestinal tract by shotgun metagenomic sequencing to fight colonization by potential foodborne pathogens Tesi presentata da: Paola Moniaci CoordinatoreDottorato Relatore Prof. Giovanni Dinelli Prof. Gerardo Manfreda Correlatore Dott.ssa Alessandra De Cesare Esame finale anno 2017 To my parents, for believing in me and for always supporting me in my journey even when it did not seem right, for always telling me that everything happens for a reason, and that I must face every situation with courage and without fear. Thank to them I learnt how to befriend strangers, to be always honest and kind with everyone. Thank you for sharing your love of life and sense of humour with me and for showing me how hard work looks like. Thank you for being such caring and dedicated parents always willing to help and protect me and my beloved sister. Without you, I would not be nowhere near the person I would like to became and this thesis would certainly have not existed. Thank you… I owe you everything. CONTENTS 1 1. INTRODUCTION 4 1.1 The chicken gut microbiota: composition and development 4 1.2 Interactions between populations belonging to the chicken gut microbiota 7 1.3 Microbiota’s role in host physiology 9 1.3.1 Immunostimulation, immunomediation and mucosal development 10 1.3.2 Synthesis of dietary compounds 11 1.4 Factors affecting and modulating the chicken GI microbiota 14 1.4.1 Environment, age and diet 14 1.4.2 Diet and feed additives 18 1.5 Analysis of chicken gut microbiota: from traditional techniques to metagenomic analysis 22 1.5.1 16S rRNA gene based metataxonomic analysis 25 1.5.2 Shotgun whole genome metagenomic sequencing 26 1.5.3 Sequences’ data analysis and MG-RAST platform 27 2. OBJECTIVES 30 3. TRIALS PERFORMED 32 3.1 Trial on metagenomic investigation of caeca of chickens fed with Lactobacillus acidophilus D2/CSL 32 3.1.1 Background 32 3.1.2 Methodology 34 3.1.2.1 Animals and diet groups 34 3.1.2.2 Sample collection 37 3.1.2.3 DNA extraction from chicken caecum contents 37 3.1.2.4 Library preparation and metagenomic sequencing 37 3.1.2.5 Sequences analysis 37 3.1.2.6 Statistical analysis 38 3.1.3 Results 39 3.1.3.1 Sequences obtained 39 3.1.3.2 Caeca microbiota composition 41 3.1.3.3 Caeca metabolic genes composition 47 3.2 Trial on metagenomic investigation of caeca of chickens fed with serine protease 53 3.2.1 Background 53 1 3.2.2 Methodology 55 3.2.2.1 Animals and diet groups 55 3.2.2.2 Sample collection 57 3.2.2.3 DNA extraction from chicken caecum contents 57 3.2.2.4 Library preparation and metagenomic sequencing 57 3.2.2.5 Sequences analysis 58 3.2.2.6 Statistical analysis 59 3.2.3 Results 60 3.2.3.1 Sequences obtained 60 3.2.3.2 Caeca microbiota composition 62 3.2.3.3 Impact of diet, age and their interaction on microbiota composition 69 3.2.3.4 Identification of signature species 75 3.2.3.5 Caeca metabolic genes composition 77 3.3 Trial on metagenomic investigation of caeca of chickens fed with phytase and corresponding carcasses 87 3.3.1 Background 87 3.3.2 Methodology 89 3.3.2.1 Animals and diet groups 89 3.3.2.2 Sample collection 91 3.3.2.3 DNA extraction from chicken caecum contents 91 3.3.2.4 DNA extraction from chicken carcass skin and washing water 92 3.3.2.5 Library preparation and metagenomic sequencing 92 3.3.2.6 Sequences analysis 93 3.3.2.7 Statistical analysis 94 3.3.3 Results 94 3.3.3.1 Sequences obtained 94 3.3.3.2 Caeca microbiota composition 97 3.3.3.3 Caeca metabolic genes composition 106 3.3.3.4 Chicken carcass microbiota composition 108 4. DISCUSSION AND CONCLUSIONS 116 4.1 Impact of feed supplemented with Lactobacillus acidophilus D2/CSL on chicken gastrointestinal tract 116 2 4.2 Impact of feed supplemented with serine protease on chicken gastrointestinal tract 118 4.3 Impact of feed supplemented with phytase on chicken gastrointestinal tract 121 4.4 Impact of feed supplemented with phytase on chicken carcass microbiota 122 4.5 Future prospectives 123 5. REFERENCES 126 6. LIST OF PUBBLICATIONS AND ATTENDANCE TO CONFERENCES 149 3 1. INTRODUCTION 1.1 The chicken gut microbiota: composition and development Domestic chickens are the most common avian species and valuable sources of proteins for humans. The chicken gastrointestinal tract is densely populated by microorganisms, which closely and intensively interact with the host, the diet and the ingested feed. A better understanding of these interactions, impacting on animal nutrition and health, is required to further enhance poultry productions, safety of poultry products and host growth performance (Rinttilä and Apajalahti, 2013; Pan and Yu, 2014). The total DNA that can be extracted from the chicken gut is the metagenome and is the aggregate of the DNA of the host and the microbiota. Microbiota is the collective microbial community inhabiting the chicken gut. Metagenome and microbiome are often used interchangeably but the microbiome is the collective genomic content of a microbiota and indicates the total genetic capacity of the community (Tremaroli and Bäckhed, 2012). Between all body sites the gastrointestinal tract (GIT) is the most densely colonized organ by different microbial cells representing the GIT microbiota (Scott et al., 2013; Doré and Blottière, 2015; Jandhyala et al., 2015; Yoon et al. 2015). The microbiota consists of trillions of symbiotic microbial cells harbored by each host, primarily bacteria in the gut; furthermore, the microbiome consists of the genes these cells harbor. In fact, the microbiome is defined as the combined genetic material of the microorganisms in a particular environment (Ursell et al., 2013). All animals coexist with their microbiota establishing a symbiotic equilibrium that confers them a variety of physiologic benefits. The composition of the microbiota is host specific, evolve during the animal's lifetime and is susceptible to both exogenous and endogenous modifications (Neish, 2009; Sekirov et al., 2010). Rawls et al. (2006) showed that the transplantation of microbial communities between different host species results in the transplanted community transforming to resemble the native microbiota of the recipient host. The symbiotic equilibrium between host and microbiota is established soon after the animal birth. Later on, changes in its composition are influenced by the exposure to the microorganisms present in the surrounding environment by diet (Pan and Yu, 2014; Blottière Doré, 2015; Wang et al., 2016). The loss of balance settled between the various populations that compose each microbiota may lead to serious repercussions on the host, such as the onset of a large number of metabolic, immune-mediated, allergic and inflammatory pathologies (Cogen et al., 2008; Yoon et al. 2015). Compared with mammals, chicken and other poultry, as turkey and duck, have a shorter gastrointestinal tract causing a faster digesta transit, a relatively short retention time for ingested food and consequently selecting a deeply different intestinal microbiota compared to other food animals. 4 The average transit time through the whole chicken gastrointestinal tract is less than 3.5 h (Hughes, 2008; Pan and Yu, 2014). The chicken gastrointestinal apparatus is divided into histologically and anatomically distinct structure, named esophagus, stomach, small intestine (duodenum, jejunum and ileum) and large intestine (caecum, colon and rectum). The ingested food is initially stored in the crop, an extension of the esophagus, which inner surface is covered with non-secretory stratified squamous epithelium, then it passes to the stomach (Van de Graaff, 1986; Grist, 2004). The food begins the digestion process in the crop where it is fermented by bacteria, especially Lactobacillus genus, since crop is the election site for bacteria colonization. From the crop, food passes into the proventriculus and successively into the gizzard, that constitute the glandular and muscular parts of the avian stomach. From the gizzard, the digesta passes into the small intestine which is comprised of the duodenum, jejunum and ileum, where it is mixed with bile salts and proteinases, amylases and lipases secreted in enzyme secretions from the pancreas and others digestive enzyme produced by the secretory mucosa of the small intestine. The small intestine is the major site of chemical digestion and nutrient absorption due, other than presence of pancreatic and small intestine-produced enzymes, to villi and microvilli. The digesta then passes into the large intestine where two caeca branch out forming two separate blind ended pouches (Fuller and Brooker, 1974; Barnes et al., 1980; McLelland, 1989; Mead, 1997) that are filled trough retrograde peristalsis from the colon. The caeca are thought to be involved in the breakdown of plant material indigestible for the host and the absorption of water, glucose and volatile fatty acids. From the ileo-caecal junction the digesta enters the colon where there is very little absorption or digestion processes and then finally the feces pass into the cloaca and expelled mixed with uric acid (McLelland, 1989). The short digesta retention time, during chicken digestion process, selects bacteria that can adhere to the mucosal layer and/or grow fast. The regions which have less tolerable conditions and faster passage of contents have lower numbers of bacteria. However, abundance and diversity of microbiota are different along the gastrointestinal tract and the ceca.