Comparison of the Luminal and Mucosa-Associated Microbiota in the Colon of Pigs with and without Swine Dysentery

Abstract

Colonic contents and mucosal scrapings from pigs inoculated with Brachyspira hyodysenteriae or Brachyspira hampsonii were collected at necropsy and classified as either positive (n = 29) or negative (n = 7) for swine dysentery (SD) based upon lesions and positive culture from the source pig. The microbiota in each sample was analyzed by bacterial census taking (16S rRNA gene sequencing). Procrustes analysis revealed similar clustering by disease classification with a relatively high M2 value (0.44) suggesting differences in the microbiota between mucosal and luminal samples from the same pig. In both sample types, differences in richness and beta diversity were observed between disease statuses (P ≤ 0.014). The relative abundance of Brachyspirales, Campylobacterales, Desulfovibrionales, and Enterobacteriales was higher in pigs with dysentery for both mucosal scrapings and luminal samples while Clostridiales, Erysipelotrichales, and Fusobacteriales were significantly more abundant in the luminal contents only. For inoculated pigs that did not develop dysentery, Burkholderiales were more abundant in both sample types, Bacteroidales and Synergistales were more abundant in mucosal scrapings, and Lactobacillales and Bifidobacteriales were more abundant in luminal contents when compared with diseased pigs. Linear discriminant analysis of effect size revealed Brachyspira, Campylobacter, Mogibacterium, and multiple Desulfovibrio spp. as differential features in mucosal scrapings from pigs with dysentery while Lactobacillus and a Bifidobacterium spp. were differential in pigs without disease. These differential features were not observed in luminal samples. In summary, microbial profiles in both sample types differ significantly between disease states; however, evaluation of the mucosal microbiome specifically may be of higher value in elucidating bacterial mechanisms underlying development of SD.

Keywords: Brachyspira hampsonii; Brachyspira hyodysenteriae; metagenomics; microbial profiling; swine; swine dysentery.

Figure 1

Rarefaction curves comparing alpha diversity (chao1) of microbiota samples from pigs with swine dysentery (blue lines) and without dysentery (red lines) after experimental inoculation. Samples of both colonic luminal contents (A) and colonic mucosal scrapings (B) reveal significant differences in richness between disease states (P = 0.014 and P = 0.001, respectively).

Figure 2

Stacked bar charts representing proportional abundance of major phyla in microbiota from 9-week-old pigs with and without swine dysentery (SD). The bars on the left reflect comparison of colonic luminal content samples and bars at the right refelct comparison of colonic mucosal scrapings. The Firmicutes:Bacteroidetes ratios in luminal content samples were significantly higher in pigs with SD relative to inoculated pigs that did not develop disease (P = 0.001).

Figure 3

Stacked bar charts representing proportional abundance of major bacterial orders in the microbiota of colonic mucosal scrapings from 9-week-old pigs with or without swine dysentery following experiemntal inoculation with Brachyspira hyodysenteriae or Brachyspira hampsonii. Bars represent results of 16S rRNA gene sequencing followed by assignment of operational taxonomic units. Pigs that developed dysentery had an increased relative abundance of Brachyspirales (total percentage too small to be visible in this graph), Campylobacterales, and Desulfovibrionales, whereas those pigs that did not develop disease had increases in abundance of Bacteroidales and Synergistales (not visible in this graph). A false discovery rate of 5% was used to determine significance.

Figure 4

Comparative weighted Venn tree (CoVennTree) based on partial 16S rRNA gene sequences in the microbiota of colonic mucosal scrapings from pigs that did (blue circles) and did not (red circles) develop swine dysentery after experimental inoculation. The numbers in parentheses refer to Venn decomposition similarity (VDS value) and reflect the degree of similarity of all child nodes after the parent node (1 = identical). The overlap of weighted Venn circles of parental nodes reflects sequence reads originating from the same organism (group). Libraries were normalized to 150,000 reads and singletons were excluded.

Figure 5

Histogram of linear discriminant analysis (LDA) scores computed by LEfSe revealing differentially abundant taxa in the microbiota of mucosal scrapings from pigs with or without swine dysentery (SD) following experimental inoculation with Brachyspira hyodysenteriae or Brachyspira hampsonii. Brachyspira, Campylobacter, Mogibacterium, Anaerotruncus, Oscillospira, and multiple Desulfovibrio spp. were differential features in mucosal scrapings from pigs with SD while Lactobacillus, Roseburia, Synergistales, a Bifidobacterium spp., and a specific Desulfovibrio spp. were characteristic of samples from those pigs that were resistant to infection and disease development.

Figure 6

Bar graphs representing relative abundance in individual pig mucosal scraping samples of specific genera identified as differential features in LEfSE for pigs with (class: Pos) and without (class: Neg) swine dysentery (SD) after experimental inoculation. Solid black horizontal lines represent the mean abundance within each class. Panels (A–D) represent genera that were differential features of the microbiota from pigs with SD, whereas panels (E,F) represent two genera that were differential for the mucosal microbiota from pigs without SD.