Untargeted metabolomics of the intestinal tract of DEV-infected ducks
Authors: Cai Haiqing , Yang Xia , Yang Yunyun , Feng Yi , Wen Anlin , Yang Ying , Wen Ming , Ou Deyuan 
Published: December 2023

Abstract
Introduction
Duck enteritis virus (DEV) mainly causes infectious diseases characterized by intestinal haemorrhage, inflammation and parenchymal organ degeneration in ducks and other poultry. However, the mechanism by which it causes intestinal damage in ducks is not well understood. Metabolomics can provide an in-depth understanding of the full complexity of the disease.

Methods
In this study, 24 clinically healthy green-shell ducks (weight 1.5 kg ± 20 g) were randomly divided into 2 groups (experimental group, 18; control group, 6). The experimental group was intramuscularly injected with 0.2 mL of DEV virus in solution (TCID50 3.16 × 108 PFU/mL), and the control group was injected with 0.2 mL of sterile normal saline. Duck duodenum and ileum tissue samples were collected at 66 h, 90 h and 114 h post-injection (12 h of fasting before killing), and metabolomics analysis of duck duodenum and ileum tissues at the three time points (66, 90, 114 h) was performed by liquid chromatography–mass spectrometry (LC–MS) to screen for and analyse the potential differentiated metabolites and related signalling pathways.

Results
Screening was performed in the positive/negative mode (Pos: Positive ion mode; the ionization of substances at the ion source with positive ions such as H+, NH4+, Na+ and K+; Neg: Negative ion mode; the ionization of substances at the ion source with negative ions such as Cl−, OAc−), and compound abundance was compared to that in the control group. The total number of differentially abundant compounds in the duodenum at 66 h, 90 h and 114 h of DEV infection gradually increased, and metabolites such as cytidine, 2′-deoxyriboside and 4-guanidinobutyric acid were differentially abundant metabolites common to all three time periods. The metabolic pathways related to inflammatory response and immune response were tryptophan acid metabolism, cysteine-methionine metabolism, histidine metabolism and other amino acid metabolism and fat metabolism. Among them, the metabolic pathways with more differentially abundant metabolites were amino acid biosynthesis, cysteine and methionine metabolism, tryptophan metabolism, unsaturated fatty acid biosynthesis and purine metabolism, and the metabolic pathways with more enrichment factors were the IgA-related intestinal immune network pathway and lysosome pathway. Compared with the control group, there were 16 differentially abundant metabolites in the ileum tissue of DEV-infected ducks at 66 h of infection, 52 at 90 h of infection, and 40 at 14 h of infection with TD114. The metabolic pathways with more enriched differentially abundant metabolites were pyrimidine metabolism, tyrosine metabolism, phenylalanine metabolism and tryptophan biosynthesis. The metabolic pathways with the most enrichment factors were the mTOR signalling pathway, ferroptosis pathway, tryptophan metabolism pathway and caffeine metabolism pathway.

Conclusion
Comparative analysis showed that the number of differentially abundant metabolites in the duodenum and ileum differed to some extent after DEV infection, with significantly more differentially abundant metabolites in duodenal tissues and fewer in ileal tissues; after DEV infection, the highest number of differentially abundant metabolites was obtained at 114 h of DEV infection, followed by the second highest at 90 h of infection and the lowest at 66 h of infection. The common differentially abundant metabolites in duodenal and ileal tissues were prostaglandins, arachidonic acid, and arachidonic ethanolamine. The main metabolic pathways in the duodenum were the IgA-associated intestinal immune network pathway and the lysosomal pathway, and the metabolic pathways with more enriched factors in the ileum were the mTOR signalling pathway, the ferroptosis pathway, and the tryptophan metabolism pathway.