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As one of the most prevalent contaminants in animal and human food, the deleterious effects of trichothecene mycotoxin deoxynivalenol (DON) warrant extensive investigation. Here, to assess the effects of DON exposure to the populations of gut microbiota, four-weeks-old mice were exposed to different doses (1.0 and 5.0 mg/kg) of DON every two days for 14 days. The contents of the cecum were then collected for DNA extraction and metagenomic shotgun sequencing, in order to detect alterations of the gut microbiota. We found that the average body weight and daily gain in the high dose DON treated group decreased. Metagenomic analysis demonstrated that the relative abundance of Firmicutes in the low and Bacteroidetes in the high dose groups increased compared to that in the untreated control group. Moreover, using gene calling and functional annotation, we found that large numbers of biosynthesis and degradation dependent populations were altered. As a result, metabolism pathways including sphingolipid, protein digestion/absorption, and lipoic acid pathways in the high dose DON exposed group dramatically fluctuated in comparison to the control and low dose groups. In addition, metagenomic binning identified ten microbiota genome drafts, with high levels of completeness, that further explain the DON-induced intestinal toxicity. Our findings suggested that DON exposure significantly impacted the microbiota community in the mouse, causing biosynthesis and degradation damage and metabolism pathway disorders.Deoxynivalenol exposure decreased the body weight in mice.Deoxynivalenol exposure altered the composition of gut microbiota in mice.Deoxynivalenol exposure affected microbial biosynthesis and degradation, and changed the normal metabolism in intestine.Ten microbiota genome drafts with high quality were assembled based on the sequence data in the study.