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      Age-Related Differences in the Luminal and Mucosa-Associated Gut Microbiome of Broiler Chickens and Shifts Associated with Campylobacter jejuni Infection

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          Abstract

          Despite the importance of gut microbiota for broiler performance and health little is known about the composition of this ecosystem, its development and response towards bacterial infections. Therefore, the current study was conducted to address the composition and structure of the microbial community in broiler chickens in a longitudinal study from day 1 to day 28 of age in the gut content and on the mucosa. Additionally, the consequences of a Campylobacter (C.) jejuni infection on the microbial community were assessed. The composition of the gut microbiota was analyzed with 16S rRNA gene targeted Illumina MiSeq sequencing. Sequencing of 130 samples yielded 51,825,306 quality-controlled sequences, which clustered into 8285 operational taxonomic units (OTUs; 0.03 distance level) representing 24 phyla. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Tenericutes were the main components of the gut microbiota, with Proteobacteria and Firmicutes being the most abundant phyla (between 95.0 and 99.7% of all sequences) at all gut sites. Microbial communities changed in an age-dependent manner. Whereas, young birds had more Proteobacteria, Firmicutes, and Tenericutes dominated in older birds (>14 days old). In addition, 28 day old birds had more diverse bacterial communities than young birds. Furthermore, numerous significant differences in microbial profiles between the mucosa and luminal content of the small and large intestine were detected, with some species being strongly associated with the mucosa whereas others remained within the luminal content of the gut. Following oral infection of 14 day old broiler chickens with 1 × 10 8 CFU of C. jejuni NCTC 12744, it was found that C. jejuni heavily colonized throughout the small and large intestine. Moreover, C. jejuni colonization was associated with an alteration of the gut microbiota with infected birds having a significantly lower abundance of Escherichia (E.) coli at different gut sites. On the contrary, the level of Clostridium spp. was higher in infected birds compared with birds from the negative controls. In conclusion, the obtained results demonstrate how the bacterial microbiome composition changed within the early life of broiler chickens in the gut lumen and on the mucosal surface. Furthermore, our findings confirmed that the Campylobacter carrier state in chicken is characterized by multiple changes in the intestinal ecology within the host.

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          Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

          Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.
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            Diversity and succession of the intestinal bacterial community of the maturing broiler chicken.

            The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of Lactobacillus, with the majority of the rest being related to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus (6.5%). In contrast, Clostridiaceae-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.
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              Bacterial census of poultry intestinal microbiome.

              The objective of this study was to generate a phylogenetic diversity census of bacteria identified in the intestinal tract of chickens and turkeys using a naïve analysis of all the curated 16S rRNA gene sequences archived in public databases. High-quality sequences of chicken and turkey gastrointestinal origin (3,184 and 1,345, respectively) were collected from the GenBank, Ribosomal Database Project, and Silva comprehensive ribosomal RNA database. Through phylogenetic and statistical analysis, 915 and 464 species-equivalent operational taxonomic units (defined at 0.03 phylogenetic distance) were found in the chicken and the turkey sequence collections, respectively. Of the 13 bacterial phyla identified in both bird species, Firmicutes, Bacteroidetes, and Proteobacteria were the largest phyla, accounting for >90% of all the sequences. The chicken sequences represent 117 established bacterial genera, and the turkey sequences represent 69 genera. The most predominant genera found in both the chicken and the turkey sequence data sets were Clostridium, Ruminococcus, Lactobacillus, and Bacteroides, but with different distribution between the 2 bird species. The estimated coverage of bacterial diversity of chicken and turkey reached 89 and 68% at species-equivalent and 93 and 73% at genus-equivalent levels, respectively. Less than 7,000 bacterial sequences from each bird species from various locations would be needed to reach 99% coverage for either bird species. Based on annotation of the sequence records, cecum was the most sampled gut segment. Chickens and turkeys were shown to have distinct intestinal microbiomes, sharing only 16% similarity at the species-equivalent level. Besides identifying gaps in knowledge on bacterial diversity in poultry gastrointestinal tract, the bacterial census generated in this study may serve as a framework for future studies and development of analytic tools.
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                Author and article information

                Contributors
                Journal
                Front Cell Infect Microbiol
                Front Cell Infect Microbiol
                Front. Cell. Infect. Microbiol.
                Frontiers in Cellular and Infection Microbiology
                Frontiers Media S.A.
                2235-2988
                22 November 2016
                2016
                : 6
                : 154
                Affiliations
                [1] 1Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine Vienna, Austria
                [2] 2Department of Animal Hygiene, Poultry and Environment, Faculty of Veterinary Medicine, South Valley University Qena, Egypt
                [3] 3Department for Farm Animals and Veterinary Public Health, Institute of Milk Hygiene, University of Veterinary Medicine Vienna, Austria
                Author notes

                Edited by: Avelino Alvarez-Ordóñez, Teagasc Food Research Centre, Ireland

                Reviewed by: Héctor Arguello-Rodriguez, University of Córdoba, Spain; Marc Maresca, Aix Marseille Université, France

                *Correspondence: Wageha A. Awad wageha.awad@ 123456vetmeduni.ac.at
                Article
                10.3389/fcimb.2016.00154
                5118433
                27921008
                4ff4a790-f790-49ad-8c4e-463269f10d5e
                Copyright © 2016 Awad, Mann, Dzieciol, Hess, Schmitz-Esser, Wagner and Hess.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 July 2016
                : 01 November 2016
                Page count
                Figures: 7, Tables: 3, Equations: 0, References: 49, Pages: 17, Words: 10537
                Funding
                Funded by: European Regional Development Fund 10.13039/501100008530
                Award ID: L00112
                Categories
                Microbiology
                Original Research

                Infectious disease & Microbiology
                broiler chickens,microbiota,16s rrna gene,age,luminal content,mucosa,campylobacter jejuni,miseq sequencing

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