Conserved developmental trajectories of the cecal microbiota of broiler chickens in a field study

There is great interest in identifying gut microbiota development patterns and underlying assembly rules that can inform strategies to improve broiler health and performance. Microbiota stratification using community types helps to simplify complex and dynamic ecosystem principles of the intestinal microbiota. This study aimed to identify community types to increase insight in intestinal microbiota variation between broilers and to identify factors that explain this variation. A total of 10 well-performing poultry flocks on four farms were followed. From each flock, the cecal content of nine broilers was collected at 7, 14, and 35 days posthatch. A total of two robust community types were observed using different clustering methods, one of which was dominated by 7-day-old broilers, and one by 35-day-old broilers. Broilers, 14-day-old, were divided across both community types. This is the first study that showed conserved cecal microbiota development trajectories in commercial broiler flocks. In addition to the temporal development with age, the cecal microbiota variation between broilers was explained by the flock, body weight, and the different feed components. Our data support a conserved development of cecal microbiota, despite strong influence of environmental factors. Further investigation of mechanisms underlying microbiota development and function is required to facilitate intestinal health promoting management, diagnostics, and nutritional interventions

Host and environmental factors affecting the intestinal microbiota in chickens

The initial development of intestinal microbiota in poultry plays an important role in production performance, overall health and resistance against microbial infections. Multiplexed sequencing of 16S ribosomal RNA gene amplicons is often used in studies, such as feed intervention or antimicrobial drug trials, to determine corresponding effects on the composition of intestinal microbiota. However, considerable variation of intestinal microbiota composition has been observed both within and across studies. Such variation may in part be attributed to technical factors, such as sampling procedures, sample storage, DNA extraction, the choice of PCR primers and corresponding region to be sequenced, and the sequencing platforms used. Furthermore, part of this variation in microbiota composition may also be explained by different host characteristics and environmental factors. To facilitate the improvement of design, reproducibility and interpretation of poultry microbiota studies, we have reviewed the literature on confounding factors influencing the observed intestinal microbiota in chickens. First, it has been identified that host-related factors, such as age, sex, and breed, have a large effect on intestinal microbiota. The diversity of chicken intestinal microbiota tends to increase most during the first weeks of life, and corresponding colonization patterns seem to differ between layer- and meat-type chickens. Second, it has been found that environmental factors, such as biosecurity level, housing, litter, feed access and climate also have an effect on the composition of the intestinal microbiota. As microbiota studies have to deal with many of these unknown or hidden host and environmental variables, the choice of study designs can have a great impact on study outcomes and interpretation of the data. Providing details on a broad range of host and environmental factors in articles and sequence data repositories is highly recommended. This creates opportunities to combine data from different studies for meta-analysis, which will facilitate scientific breakthroughs toward nutritional and husbandry associated strategies to improve animal health and performance

Odds ratios needed to produce a receiver operating characteristic (ROC) curve that did not have a rounded shape.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152359#sec006" target="_blank">methods</a> for definition of rounded shape. AUC = area under receiver operating characteristic curve, OR = odds ratio, Freq = frequency. The odds ratio and frequency refer to the single binary variable in the risk model.</p

Examples of rounded and non-rounded shapes of receiver operating characteristic (ROC) curves.

<p>Examples of rounded and non-rounded shapes of receiver operating characteristic (ROC) curves.</p

Histograms of predicted risks of patients and nonpatients for scenarios with varying odds ratios.

<p>The area under the receiver operating characteristic curve was 0.70 and risk allele frequencies were 30%. OR = odds ratio.</p

Comparison of different invasive and non-invasive methods to characterize intestinal microbiota throughout a production cycle of broiler chickens

In the short life of broiler chickens, their intestinal microbiota undergoes many changes. To study underlying biological mechanisms and factors that influence the intestinal microbiota development, longitudinal data from flocks and individual birds is needed. However, post-mortem collection of samples hampers longitudinal data collection. In this study, invasively collected cecal and ileal content, cloacal swabs collected from the same bird, and boot sock samples and cecal droppings from the litter of the broilers’ poultry house, were collected on days 0, 2, 7, 14 and 35 post-hatch. The different sample types were evaluated on their applicability and reliability to characterize the broiler intestinal microbiota. The microbiota of 247 samples was assessed by 16S ribosomal RNA gene amplicon sequencing. Analyses of α and β measures showed a similar development of microbiota composition of cecal droppings compared to cecal content. Furthermore, the composition of cecal content samples was comparable to that of the boot socks until day 14 post-hatch. This study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. Specifically, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally.</p

Comparison of Different Invasive and Non-Invasive Methods to Characterize Intestinal Microbiota throughout a Production Cycle of Broiler Chickens

In the short life of broiler chickens, their intestinal microbiota undergoes many changes. To study underlying biological mechanisms and factors that influence the intestinal microbiota development, longitudinal data from flocks and individual birds is needed. However, post-mortem collection of samples hampers longitudinal data collection. In this study, invasively collected cecal and ileal content, cloacal swabs collected from the same bird, and boot sock samples and cecal droppings from the litter of the broilers' poultry house, were collected on days 0, 2, 7, 14 and 35 post-hatch. The different sample types were evaluated on their applicability and reliability to characterize the broiler intestinal microbiota. The microbiota of 247 samples was assessed by 16S ribosomal RNA gene amplicon sequencing. Analyses of α and β measures showed a similar development of microbiota composition of cecal droppings compared to cecal content. Furthermore, the composition of cecal content samples was comparable to that of the boot socks until day 14 post-hatch. This study shows that the value of non-invasive sample types varies at different ages and depends on the goal of the microbiota characterization. Specifically, cecal droppings and boot socks may be useful alternatives for cecal samples to determine intestinal microbiota composition longitudinally

Cereal type and combined xylanase/glucanase supplementation influence the cecal microbiota composition in broilers

Dietary fiber-degrading enzyme supplementation in broilers aims at off-setting the anti-nutritive effect of non-starch polysaccharides and at promoting broiler health. Recently, we demonstrated that xylanase/glucanase addition in wheat-based diet improved nutrient digestibility, arabinoxylan fermentability and broiler growth. Conversely, maize arabinoxylan was found to be recalcitrant to xylanase action. These findings suggested that enzyme-mediated improvement of nutrient digestion and carbohydrate fermentation depended on the cereal type present in the diet, and may have contributed to broiler growth. Hence, we aimed at further investigating the link between dietary enzymes and carbohydrate fermentation in broilers, by studying the impact of enzyme supplementation in cereal-based diets, to the microbial communities in the ileum and ceca of broilers. For that purpose, 96 one-day-old male broilers were randomly reared in two pens and received either wheat-based or maize-based starter and grower diets. At d 20, the broilers were randomly assigned to one out of four dietary treatments. The broilers received for 8 d the wheat-based or maize-based finisher diet as such (Control treatments; WC, MC) or supplemented with a xylanase/glucanase combination (Enzyme treatments; WE, ME). At d 28, samples from the digestive tract were collected, and the ileal and cecal microbiota composition was determined by 16S ribosomal RNA gene amplicon sequencing. A similar phylogenetic (alpha) diversity was observed among the four treatments, both in the ileal and the cecal samples. Furthermore, a similar microbial composition in the ileum (beta diversity) was observed, with lactobacilli being the predominant community for all treatments. In contrast, both cereal type and enzyme supplementation were found to influence cecal communities. The type of cereal (i.e., wheat or maize) explained 47% of the total variation in microbial composition in the ceca. Further stratifying the analysis per cereal type revealed differences in microbiota composition between WC and WE, but not between MC and ME. Furthermore, the prevalence of beneficial genera, such as Faecalibacterium and Blautia, in the ceca of broilers fed wheat-based diets coincided with arabinoxylan accumulation. These findings indicated that fermentable arabinoxylan and arabinoxylo-oligosaccharides released by dietary xylanase may play an important role in bacterial metabolism