Similarity and stability of the gut microbiota composition over time
The microbial community similarity among all samples over time was assessed by calculating a Bray-Curtis similarity matrix. Community similarity analysis based on the Bray-Curtis index showed clear differences between gut sites and age, indicating strong shifts in microbial community structures (Figure 5). In addition, the Bray-Curtis index suggested that the birds at the first day of age displayed a high degree of dissimilarity compared with the other ages. It was also apparent that microbiota compositions of older birds were more similar compared with young birds.
Figure 5  Microbial community similarity between all samples calculated with Bray-Curtis similarities, which displays the similarity results between the control and infected groups according to age and gut sites. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content. The Bray-Curtis index revealed clear differences between jejunum and cecum from infected birds at the two sampling time points post infection. Furthermore, the comparison of the microbiota between control and infected birds showed that community structures were more dissimilar at the OTUs level, demonstrating that the gut microbial communities changed as a result of infection.
To measure the similarity between microbial communities in all birds at different ages, principal component analysis (PCA) was performed (Figure 6). PCA analysis showed that there was a clear clustering of the birds at days 1 and 7 of age in the jejunum and cecum compared with the other days. In addition, the microbial community of the older chickens clustered with less variation compared to young birds. PCA plots also demonstrate that, the microbial community was more separated in the ceca than in the jejunum.
Figure 6  Principal component analysis (PCA) was analyzed for the control (A) and infected birds (B). Orange (day 1), Red (day 7), Green (day 14), Blue (day 21), and Black (day 28). Each symbol indicates an individual bird. All PCA plots include the data of all samples; symbols not belonging to the sample group indicated in the header are displayed faded. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content. To delineate the shared species among the groups, a Venn diagram displaying the overlaps between gut sites at different ages and groups was performed (Figure S3). The proportions of shared OTUs appear to be low at each gut site from day 1 to day 28 of age. These shared species, however, varied from one site to another.
Furthermore, the analysis showed that only 399 OTUs (n = 1847 OTUs) were shared among the jejunal mucosa in the control and infected birds, while 745 OTUs (n = 2401 OTUs) were shared between the jejunal content in the control and infected birds at the two time points post infection (Figure 7A). In the cecal mucosa and the cecal content, the comparison revealed that only 2218 OTUs (n = 6736 OTUs) and 2617 OTUs (n = 6860 OTUs) were shared, among control and infected birds combining the two time points post infection (Figure 7B). These data demonstrated that 25–36% of the observed OTUs in the jejunum and cecum were shared between the control and infected birds, respectively.
Figure 7  Venn diagrams showing the shared OTUs between the control and infected birds at different gut sites at the two sampling points post infection (A) jejunum; (B) cecum. JM, jejunal mucosa; JC, jejunal content; CM, cecum mucosa; CC, cecum content; control (c); infected (i); d, day.