Age-related differences in the luminal and mucosa-associated gut microbiome of broiler chickens and shifts associated with Campylobacter jejuni infection

ORIGINAL RESEARCH published: 22 November 2016 doi: 10.3389/fcimb.2016.00154 Age-Related Differences in the Luminal and Mucosa-Associated Gut Microbiome of Broiler Chickens and Shifts Associated with Campylobacter jejuni Infection Wageha A. Awad 1, 2*, Evelyne Mann 3 , Monika Dzieciol 3 , Claudia Hess 1 , Stephan Schmitz-Esser 3 , Martin Wagner 3 and Michael Hess 1 1 Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine, Vienna, Austria, 2 Department of Animal Hygiene, Poultry and Environment, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt, 3 Department for Farm Animals and Veterinary Public Health, Institute of Milk Hygiene, University of Veterinary Medicine, Vienna, Austria 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 Received: 06 July 2016 Accepted: 01 November 2016 Published: 22 November 2016 Citation: Awad WA, Mann E, Dzieciol M, Hess C, Schmitz-Esser S, Wagner M and Hess M (2016) Age-Related Differences in the Luminal and Mucosa-Associated Gut Microbiome of Broiler Chickens and Shifts Associated with Campylobacter jejuni Infection. Front. Cell. Infect. Microbiol. 6:154. doi: 10.3389/fcimb.2016.00154 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 × 108 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 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 November 2016 | Volume 6 | Article 154 Awad et al. Campylobacter and Gut Microbiota 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. Keywords: broiler chickens, microbiota, 16S rRNA gene, age, luminal content, mucosa, Campylobacter jejuni, MiSeq sequencing INTRODUCTION et al., 2014). In addition, Awad et al. (2016) showed that Campylobacter had the ability to reduce butyrate, isobutyrate, valerate, and isovalerate which might be due to the utilization of short-chain fatty acids (SCFAs) as a carbon source (Masanta et al., 2013) or due to the reduction of butyric acid producing bacteria amongst the microbiota. In general, there is a complex interplay between microbiota composition and SCFAs concentration and it was found that the type and level of SCFAs in the gut can affect different members of the microbial community in various ways (Mon et al., 2015). It is still unknown how C. jejuni affects the ecology of the chicken gut, a feature of high importance considering a possible detrimental effect on the health of birds associated with C. jejuni colonization. Haag et al. (2012) demonstrated that C. jejuni colonization in mice depends on the microbiota of the host and vice versa and Campylobacter colonization induces a shift of the intestinal microbiota. Thus, it can be hypothesized that Campylobacter colonization is associated with an alteration in the intestinal microbiota of chickens as well. Therefore, the second aim of the actual study was to investigate the dynamics of an experimental Campylobacter jejuni NCTC 12744 infection in 14– 28 days old chickens and the consequences on the alteration of the gut microbiome. A diverse microbiota is found throughout the gastrointestinal tract (GIT) of chickens, most predominant in the cecum (Mead, 1997; Videnska et al., 2014). The gut microbiota plays an essential role in nutrition, detoxification of certain compounds, growth performance and protection against pathogenic bacteria. The microbiota is crucial to strengthen the immune system, thereby affecting growth, health, and wellbeing of chicken. Generally, the gut microbiota modulates host responses to limit the colonization of pathogens (Rehman et al., 2007). There is little information about the diversity and function of the gut microbiota in chickens, its impact on the host and the impact of certain pathogens. Development of the gut microbiota in chickens occurs immediately after hatching and is influenced by both genetic and external factors like diet and environment (Apajalahti et al., 2004). It was reported that disturbances in the intestinal microbiota leads to a delay in growth, weakens the host resistance and increases the susceptibility to various infectious diseases (Lan et al., 2004). Gong et al. (2002) demonstrated that the cecal microbiota protects chickens against bacterial infections, while microbiota in the small intestine contributes significantly to its function, including digestion and nutrient absorption, which significantly determines the growth rate of the bird. Studies on gut microbiota have mostly been performed with chickens older than (...truncated)