Phyla composition exhibited significant microbial differences between ALV-J-infected chickens and controls
Each sample was rareﬁed to 17,595 sequences; using a threshold of 97% sequence identity, 16,740 unique operational taxonomic units (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. The distribution of the four phyla, in group A (viral control), showed that the gut microbiota was dominated by Firmicutes, whereas other phyla appeared in smaller quantities including Actinobacteria, Bacteroidetes, and Proteobacteria. In group B (ALV-J-infected chickens), the gut microbiota was dominated by both Firmicutes and Proteobacteria, with other phyla found in smaller quantities including Bacteroidetes, Actinobacteria, and a few other unknown phyla (Fig 1).
Interestingly, two phyla, Firmicutes and Proteobacteria, were found to be proportionally signiﬁcantly different between groups A and B (P < 0.05). Among the two phyla, the Proteobacteria concentration was much higher in group B than in group A (Fig 1). These results indicate that ALV-J infection signiﬁcantly impacted the proportion of Firmicutes and Proteobacteria at the phylum level.
Bacterial taxonomic clades showed signiﬁcant differences between ALV-J-infected chickens and controls
Principal coordinate analysis was conducted based on weighted UniFrac distances to assess the microbial distribution between the two groups. The results of weighted UniFrac analysis revealed a notable distribution difference for PC2; However, no difference in the distribution of PC1 was observed. The gut microbial community of group A was substantially separated from that of group B (Fig. 2A). In group A (control), all samples clustered together, whereas in group B (ALV-J-infected chickens), all samples except B2 clustered together. This indicates that ALV-J infection significantly altered the gut microbiota distribution in chickens.
To investigate which OTUs can serve as biomarkers in an unbiased manner, we used the linear discriminant analysis effect size (LEfSe) classification tool. The analysis detected 15 bacterial taxonomic clades showing signiﬁcant differences among the two groups (P < 0.05). In group B, the key phylotypes were Proteobacteria, Helicobacter, Helicobacteraceae, Comamonas, Betaproteobacteria, Burkholderiales, Gammaproteobacteria, Comamonadaceae, Actinomycetales, Stenotrophomonas, Methylobacterium, Xanthomonadaceae, Rickettsiales, Corynebacteriaceae, and Propionibacterium, whereas Lactobacillales and Lactobacillaceae were present in group A (Fig. 2B). The heat map displayed a similar pattern, as shown in Fig. 2C. These results suggest that the composition of the chicken gut microbiota was significantly altered by ALV-J infection.
Difference in composition of probiotics in chicken gut microbiota in ALV-J-infected chickens and controls
We further identified the dominant species in the gut microbiota between the two groups. The results revealed significant differences (P < 0.05) between eight species including Propionibacterium acnes, Lactobacillus coleohominis, Lactobacillus helveticus, Lactobacillus reuteri, and rarely identified species such as Mycoplana spp.,Comamonas spp., Delftia spp., and Helicobacter spp. (Figure 3). In group B (ALV-J-infected chickens), three species exhibited a significant reduction in abundance including L. coleohominis, L. helveticus, and L. reuteri compared with group A (control). Two of these species, L. helveticus and L. reuteri are probiotics. These results suggest that at the species level, ALV-J infection significantly altered the composition of probiotics in chicken gut microbes.