The intestinal microbiome in poultry tracts functions as an interface between the host and the feed ingested and has important roles in nutrition and health of the host. This intestinal microbiome consisting of bacteria, archaea, fungi and few protozoa, with bacteria being the most abundant domain and greatly affecting the performance of the host. Modulation of the intestinal microbiome of poultry through dietary and managerial interventions has been used to improve poultry growth and health. However, the diversity and complexity of the intestinal microbiome hinders understanding of the mechanism of these interventions and achieving the intended modulations. Cultivation-based methods have been applied to intestinal microbiome studies, providing useful information. However, the fact that the majority of poultry intestinal microbiome are uncultivable limits the knowledge that can be learned about this microbiome. PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and cloning and sequencing are primary cultivation-independent 16S rRNA gene-targeted molecular techniques commonly used in characterizing diversity and composition of poultry intestinal microbiome. However, they can only provide limited information because neither can provide detailed information on most of the bacteria present in the microbiome in a cost-effective manner. Recently, microarray and next-generation sequencing (NGS) technologies have been widely applied to investigation of microbiomes in various habitats, including guts of humans and animals, greatly advancing our knowledge on microbiomes. However, few metagenomics or microarray studies have been conducted to investigate the poultry intestinal microbiome. The objectives of my study were to i) achieve a comprehensive understanding of the intestinal microbiomes of broiler chickens and turkeys, ii) establish a global phylogenetic framework of bacterial diversity of this microbiome, iii) examine how litter conditions and bacitracin (as a model antimicrobial growth promoter) affect colonization and abundance of common enteric pathogens in broiler chickens, and iv) develop a habitat-specific phylochip that can support comprehensive studies on poultry intestinal microbiome.
In the first study (chapter 3), a naїve-analysis of all the available 16S rRNA gene sequences of poultry gut origin archived in the public databases was performed. By analyzing high quality sequences of chicken and turkey gastrointestinal origin (3,184 and 1,345, respectively) collected from the GenBank, Ribosomal Database Project (RDP), and Silva database, I identified 915 and 464 species-equivalent operational taxonomic units (OTUs, defined at 0.03 phylogenetic distance) in the chicken and the turkey intestinal microbiomes, respectively. Firmicutes, Bacteroidetes, and Proteobacteria were the largest phyla in both chicken and turkey, accounting for > 90% of all the sequences. The estimated coverage of bacterial diversity of chicken and turkey reached 89% and 68% at species-equivalent level, respectively. More than 7,000 bacterial sequences from each bird species would be needed to reach 99% diversity coverage for either bird species.
In the second study (chapter 4), we used 454 pyrosequencing in investigating the bacterial diversity of cecal content and ileal mucosa samples of chickens and turkeys with two technical replicates. The objectives of this study were to uncover the major bacterial composition of poultry intestinal microbiome and to investigate the variations of bacteria diversity as affected by different sequencing runs and data processing pipelines. The 338,177 sequences analyzed represented on average 3,401 and 125 OTUs in chicken cecal content and ileal mucosa as well as 1,687 and 16 OTUs in turkey cecal content and ileal mucosa, respectively. The sequences from each bird species reached > 95% bacterial diversity in the intestinal microbiomes, except for the turkey mucosa microbiome. When compared to the public databases, this study identified 39 and 50 new genera in the chicken and turkey cecal microbiome, respectively. Noticeable variations were observed from the number of OTUs revealed by different sequencing runs and data processing pipelines. This study provided comprehensive perspectives on the chicken and turkey intestinal microbiota.
In the third study (chapter 5), a habitat-specific phylochip microarray (referred to as PITChip) was developed based on the sequences collected in the first and the second studies. The PITChip enables simultaneous detection and semi-quantification of 1,848 different bacterial OTUs and 105 genera of bacteria. After careful validation, the PITChip was used to analyze microbiomes of ileal mucosa and cecal content of broiler chicken reared on fresh and reused litters to assess how litter conditions affected the intestinal microbiome. More than 85 groups of bacteria were found to be influenced by litter status in each sampling location. This study indicated that litter management can modulate the intestinal microbiome of broiler chickens and may have a profound effect on bird health and performance. This is the first phylochip developed for comprehensive studies of the intestinal microbiome of poultry. The PITChip may be used in future integrated studies to investigate interrelations between intestinal microbiomes, diets, feed additives, and managerial factors to promote flock health and performance.
In the last study (chapter 6), we further investigated the effects of supplemental bacitracin and litter management (fresh vs. reused) on the abundance of Campylobacter, Clostridium perfringens, and Salmonella in broiler chickens using specific quantitative PCR (qPCR) assays. This is the first study that has examined the effect of dietary bacitracin and litter conditions on the prevalence of these three common enteric pathogens. Bacitracin was shown to be effective to reduce abundance of C. perfringens, but it had no effect on Campylobacter or Salmonella. A somewhat negative correlation was noted between the abundance of C. perfringens and Salmonella. Unless contaminated by pathogens from a previous flock, litter reuse did not appear to increase the risk of necrotic enteritis caused by C. perfringens or Salmonella infection in subsequent flocks.
Taken together, the findings of the series of studies on poultry intestinal microbiome advanced our knowledge on the bacterial communities of poultry, provided new insight into the effects of litter management and bacitracin on the intestinal microbiome of broiler chickens, and established a new microarray that can be used to support comprehensive analysis of the intestinal microbiome of chickens and turkeys that are often needed in studies of poultry nutrition and host health.