A Comparison of the Colonic Microbiome and Volatile Organic Compound Metabolome of Anoplocephala perfoliata Infected and Non-Infected Horses: A Pilot Study

Abstract

Anoplocephala perfoliata is a common equine tapeworm associated with an increased risk of colic (abdominal pain) in horses. Identification of parasite and intestinal microbiota interactions have consequences for understanding the mechanisms behind parasite-associated colic and potential new methods for parasite control. A. perfoliata was diagnosed by counting of worms in the caecum post-mortem. Bacterial DNA was extracted from colonic contents and sequenced targeting of the 16S rRNA gene (V4 region). The volatile organic compound (VOC) metabolome of colonic contents was characterised using gas chromatography mass spectrometry. Bacterial diversity (alpha and beta) was similar between tapeworm infected and non-infected controls. Some compositional differences were apparent with down-regulation of operational taxonomic units (OTUs) belonging to the symbiotic families of Ruminococcaceae and Lachnospiraceae in the tapeworm-infected group. Overall tapeworm burden accounted for 7-8% of variation in the VOC profile (permutational multivariate analysis of variance). Integration of bacterial OTUs and VOCs demonstrated moderate to strong correlations indicating the potential of VOCs as markers for bacterial OTUs in equine colonic contents. This study has shown potential differences in the intestinal microbiome and metabolome of A. perfoliata infected and non-infected horses. This pilot study did not control for extrinsic factors including diet, disease history and stage of infection.

Keywords: anoplocephala perfoliata; equine; gut microbiome; omics integration; volatile organic compounds (VOCs).

Figure 1

Taxonomic summary at phylum level for all samples. Key: TU = tapeworm positive with burden unrecorded, TL = low tapeworm (1–20 worms), TM = medium tapeworm (21–49 worms), TH = high tapeworm (≥50 worms).

Figure 2

Alpha (A,B) and beta diversity (Bray-Curtis distance is shown) (C,D) indices (OTU level) of the colonic contents of horses infected with Anoplocephala perfoliata and non-infected controls. Alpha diversity was not significantly different between groups, pair-wise ANOVA (p > 0.05). The beta diversity between groups was not significant (PERMANOVA, p > 0.05). In (A,C) CO (n = 28) vs. TP (n = 20), (B,D) CO vs. TP_21 (n = 9). Key: TP = tapeworm positive (n = 20), TP_21 = tapeworm samples with >21 worms (n = 9), CO = control (tapeworm negative), NMDS = non-metric multidimensional scaling.

Figure 3

Alpha (A) and beta (B) (Bray-Curtis distance is shown) diversity indices for horses with low (<10 e.p.g) and high (>100 e.p.g) strongyle FECs. Alpha diversity was not significantly different between groups, pair-wise ANOVA (p > 0.05). The beta diversity between groups was not significant (permutational multivariate analysis of variance, p > 0.05) Key: High_FEC = high strongyles (>100 e.p.g) n = 24, Low_FEC = low strongyles (<10 e.p.g) n = 10, FEC = faecal egg count, e.p.g = eggs per gram, NMDS = non-metric multidimensional scaling.

Figure 4

Box and whisker plots of VOC abundance of the colonic contents of horses infected with Anoplocephala perfoliata and non-infected controls. The three volatile organic compounds (VOCs) with the smallest p-values, identified by t-test for both TP vs. CO (A–C) and TP_21 vs. CO (A,D,E) are shown. Plots were constructed to show VOC abundance change in individual groups: control, low (1–20 worms), medium (21–49 worms) and high (≥50 worms) to show gradient change of compounds with level of tapeworm burden.

Figure 5

A Pearson’s correlation plot of bacteria and volatile organic compound (VOC) data of the colonic contents of horses infected with Anoplocephala perfoliata and non-infected controls. Component 1 is shown in (A) and component 2 is shown in (B). The ability of the model to separate CO and TP by bacteria alone is demonstrated by plot (C), and VOCs alone by plot (D). Key: TP = tapeworm positive (n = 20), CO = control (tapeworm negative, n = 28).

Figure 6

A heatmap of correlations (sorted by correlation values) between bacterial operational taxonomic units (OTUs) and volatile organic compounds (VOCs) found in the colonic contents of horses infected with Anoplocephala perfoliata and non-infected controls. Details of OTU taxonomic identification are in Table S4.