Modulation of the porcine intestinal microbiota in the course of Ascaris suum infection

Abstract

Background: The porcine roundworm Ascaris suum impairs feed conversion and weight gain, but its effects on intestinal microbiota remain largely unexplored.

Methods: Modulation of the intestinal microbiota was assessed in pigs that were infected once with 10,000 A. suum eggs and pigs that received a trickle infection (1000 eggs/day over 10 days), compared with a non-infected control group. Six pigs each were sacrificed per group at days 21, 35 and 49 post-infection (p.i.). Faecal samples taken weekly until slaughter and ingesta samples from different intestinal compartments were subjected to next-generation sequencing of the bacterial 16S rRNA gene.

Results: The results revealed marked differences between the single- and the trickle-infected group. Single infection caused a remarkable but transient decrease in microbial diversity in the caecum, which was not observed in the trickle-infected group. However, an increase in short-chain fatty acid-producing genera in the caecum on day 21 p.i., which shifted to a decrease on day 35 p.i., was common to both groups, possibly related to changes in excretory-secretory products following the parasite's final moult. Faecal microbial interaction networks were more similar between the single-infected and control group than the trickle-infected group. In addition, a lower degree of similarity over time indicated that A. suum trickle infection prevented microbiota stabilization.

Conclusions: These different patterns may have important implications regarding the comparability of experimental infections with natural scenarios characterized by continuous exposure, and should be confirmed by further studies.

Keywords: Ascarids; Intestinal microbiota; Microbiome; Network analysis; Roundworms; Short-chain fatty acids; Trickle infection.

Fig. 1

Sampling strategy to assess the effect of A. suum infection on the gastrointestinal microbiota of pigs

Fig. 2

Microbial diversity (A observed OTUs, B inverse Simpson index) in faecal samples of A. suum-infected and non-infected control pigs. Shown are boxplots of original values (left) with boxes extending from the 25th to the 75th percentile, a line at the median, whiskers extending to 1.5 the interquartile range and dots indicating outliers; and GLMM-derived estimates with shaded areas indicating 95% confidence intervals (right). Note the different y-axis limits. The GLMMs indicated significantly higher inverse Simpson indices in the trickle-infection than the control group (P = 0.024) as well as a significant reduction in inverse Simpson indices over time in the single-infection group (P < 0.001)

Fig. 3

Heatmap of differentially abundant species in the faecal microbiota of A. suum-infected pigs compared with a non-infected control group, as determined by DESeq2 analysis. Only differences with Benjamini–Hochberg adjusted P-values ≤ 0.01 are shown as coloured tiles

Fig. 4

Association networks of the faecal microbiota of A. suum single-infected and trickle-infected pigs on day −3 (prior to infection) and on days 14, 21 and 35 p.i., compared with a non-infected control group. Only the top 100 nodes (i.e. OTUs) with the largest eigenvector centrality and only positive associations are shown. Node colour indicates taxonomy at the family level, while node size is proportional to eigenvector centrality. Hub nodes (based on eigenvector centrality) are highlighted by a bold rim

Fig. 5

Microbial diversity (observed OTUs, Shannon index and inverse Simpson index) in the small intestine (A) and large intestine (B) of A. suum-infected and non-infected control pigs. Asterisks indicate statistically significant differences (Kruskal–Wallis test, FDR-corrected P < 0.05)

Fig. 6

Non-metric multidimensional scaling (NMDS) plot of microbial community composition in the ileum (A), caecum (B) and colon (C) of A. suum-infected and non-infected pigs. Results of PERMANOVAs are shown in blue boxes. S single-infection group, T trickle-infection group, C non-infected control group

Fig. 7

Genus-level taxonomic composition of the microbiota in the ileum, caecum and colon of A. suum-infected vs non-infected control pigs on days 21 and 35 p.i.