An Integrated Study on Chicken Gut Microbiome Associated with Diets and Feed Utilization Using Microarray and Illumina Sequencing

The gastrointestinal (GI) tract of chicken harbors a complex and dynamic microbiome that is almost exclusively composed of bacteria. This bacterial community is considered as an essential component which contributes to the wellbeing of animal host in a wide range of aspects, especially nutrition and disease resistance. The overall objective of the studies presented here was to better understand the role gut microbiome plays in host growth performance and disease resistance by investigating the diversity of gut microbiome and identifying bacteria with different relative abundance in chickens with different feed utilization efficiencies and fed with different diets.;In the first study (Chapter 3), the relationship between gut microbiome and host feed utilization efficiency was investigated. Poultry Intestinal Tract Chip version 2 (PITChip2), a poultry specific phylogenetic microarray was used to characterize gut microbiome structures in 24 male Cobb 500 broiler chickens with low feed conversion ratio (FCR) and 24 chickens with high FCR. No clear separation in the gut microbiome structure was observed between good- and bad-performance chickens as indicated by hierarchical clustering (HCL) and principal component analysis (PCA). As indicated by significant difference (p≤0.05) in relative abundance between high FCR and low FCR chickens, 100 phylotypes were found to correlate with bird performance, with 48 of them showing greater than 3-fold differences. Five phylotypes were more abundant in good-performance low FCR chickens. Three of them represent species of Bacteroides, Peptococcus, and Ruminococcaceae . The other two can only be classified as Bacteria. These five phylotypes may serve as indicators of feed utilization efficiency in chicken and also as potential probiotic candidates or targets of enhancement by prebiotics to improve growth performance of broilers.;In the second study (Chapter 4), PITChip3, the latest version of PITChip, was developed. It has 1,204 customized oligonucleotide probes designed based on the V1-V3 region of bacterial 16S rRNA and are able to detect 62 genus-level phylotypes, 662 species-level phylotypes, and 34 pathogens. Each PITChip3 slide has six microarrays and every probe has 4 replicates randomly located on each microarray. The optimal hybridization temperature of PITChip3 was determined to be 48°C. At the optimal hybridization temperature, PITChip3 has a linear detection range from 3x107 to 3x10 10 copies of a target per hybridization reaction. The utility of PITChip3 was tested using metagenomic DNA samples extracted from the cecal content samples of broilers fed either corn-based or wheat-based diet. Clear separations between chickens fed with different diets and between chickens at two different ages were observed on PCA plots. Eighteen species-level phylotypes, including Akkermansia muciniphila, Clostridium perfringens, and Corynebacterium variabile, along with 2 genus-level phylotypes Bacteroides and Escherichia/Shigella/ Salmonella, were further identified with different relative abundance in chickens fed different diets. Such difference in gut microbiome revealed by PITChip3 may help to better understand the interactions between gut microbiome and diet and the benefits and risk associated with the two common diets.;In the last study (Chapter 5), the difference in gut microbiome between chickens fed with corn- or wheat-based diet was further investigated using Illumina sequencing. Two hundred 1-day-old straight run Cobb 500 broiler chicks were randomly assigned to one of two dietary treatments. Each dietary treatment had 20 replicate pens with 5 birds per pen. At 14 and 35 days of age, ileal mucosa and cecal content were collected from one bird per pen. Metagenomic DNA was extracted from those samples and Illumina MiSeq platform was used to sequence the V1-V3 hypervariable region (2x300bp paired-end) of bacterial 16S rRNA gene. A phylotype-based analysis was performed and no clear difference in microbial diversity between the two dietary treatments was observed. Yet we were able to identify phylotypes that have different relative abundance in 14-day-old chickens fed with the two different diets. One phylotype representing unclassified members in the family Ruminococcaceae and one representing unclassified Bacteria had higher relative abundance in the cecal content of chickens fed with corn-based diet, whereas a phylotype classified as Escherichia/Shigella had higher relative abundance in chickens fed with wheat-based diet. It is known that chickens fed with corn-based diet perform better and are less likely to have Clostridium perfringens induced necrotic enteritis (NE). Further studies on the phylotypes identified may help to find non-antibiotic alternatives to enhance growth and protect chickens from disease such as NE.;Taken together, the series of studies provided a comprehensive characterization of gut microbiome in chickens fed with corn- and wheat-based diets and also in chickens with different feed utilization efficiencies. (Abstract shortened by UMI.).