Phyla composition exhibited significant microbial differences between theALV-J infected chickens and the control
Each sample was rareﬁed to 17,595 sequences; with a threshold of 97% sequence identity, 16,740 unique OTUs were identiﬁed in the samples. Total sequences were assigned to 38 phyla (3 archaeal phyla and 35 bacterial phyla). Bacterial phyla isolated from the samples included Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Distribution of the four phyla, in group A (viral control), showed that the gut microflora was primarily dominated by Firmicutes phyla, the other phyla appeared in smaller quantities including Actinobacteria, Bacteroidetes, and Proteobacteria. Meanwhile, in group B (ALV-J infected chickens), gut microbiota was dominated by both Firmicutes and Proteobacteria, with other phyla noted in smaller quantities including Bacteroidetes,Actinobacteria, and a few other unknown phyla (Fig 1). These results matched a recent study related to microbial diversity in chickens which also found that Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes were the top four phyla in the intestinal tract of chickens .
Interestingly, two of the phyla including Firmicutes and Proteobacteria were found to be proportionally signiﬁcantly different between the A and B groups (P < 0.05). Among the two phyla, Proteobacteria concentration was much higher in group B than in group A (Fig 1). These results indicated that ALV-J infection had a signiﬁcant impact on the proportion of Firmicutes and Proteobacteria at the phylum level.
Bacterial taxonomic clades showed signiﬁcant differences between between the ALV-J infected chickens and the control
Principal coordinate analysis was conducted based on weighted UniFrac distances to assess the microbial distribution between the two groups. Results of weighted UniFrac analysis depicted a notable distribution difference for PC2; However, no difference in distribution for PC1 was observed. The gut microbial community of group A was substantially separated from that of group B (Fig. 2A). In group A(the control), all samples clustered together, while in group B(ALV-J infected chickens), all samples except B2 clustered together. This result indicates that ALV-J infection significantly altered the gut microbiota distribution in chickens.
To investigate which OTUs could serve as biomarkers in an unbiased manner, we used the LEfSe classification tool. The analysis detected 15 bacterial taxonomic clades showing signiﬁcant differences among the two groups. In group B, key phylotypes were Proteobacteria, Helicobacter,Helicobacteraceae,Comamonas,Betaproteobacteria, Burkholderiales, Gammaproteobacteria, Comamonadaceae, Actinomycetales,Stenotrophomonas, Methylobacterium,Xanthomonadaceae, Rickettsiales, Corynebacteriaceae, and Propionibacterium, while Lactobacillales and Lactobacillaceae were present in group A (Fig. 2B). The defined taxa are potential biomarkers for group A and group B. For example, Proteobacteria can serve as biomarkers for ALV-J infection in chickens at the phylum, order, and class levels, while a few taxa were markers for the healthy chickens, the most prominent were members of the family Lactobacillaceae. The heatmap displayed a similar pattern in Fig. 2C. These results suggested that the composition of chicken gut microflora was significantly altered due to ALV-J infection.
Difference for composition of probiotics in chicken gut microbial flora found in he ALV-J infected chickens and the control
We further identified dominant species found in the gut flora between the two groups. Results showed significant differences (P<0.05) between eight species including Propionibacteriumacnes, Lactobacilluscoleohominis, Lactobacillushelveticus, Lactobacillusreuteri, and rarely identified species such as Mycoplana spp.,Comamonas spp., Delftia spp., and Helicobacter spp. (Figure 3). In the group B(ALV-J infected chickens) three species exhibited a significant reduction in abundance including Lactobacilluscoleohominis, Lactobacillushelveticus, and Lactobacillus reuteri compared with the group A (the control). Two of these species, Lactobacillushelveticus, and Lactobacillus reuteri are probiotics. These results suggested that at the species level ALV-J infection significantly altered the composition of probiotics in chicken gut microbes.