Composition and metabolic potential of the intestinal microbiome of Gallus gallus L. Broilers under experimental T-2 toxicosis as influenced by feed additives

Mycotoxins can adversely affect the composition and function of the poultry gut microbiota, with implications for host health. The introduction of feed additives into contaminated feed is a strategy for restoring the intestinal microbiome under mycotoxicoses. This paper shows for the first time that the feed additive Zaslon 2+ effectively improves the structure and metabolic potential of the intestinal microbiome in broiler chickens with experimental T-2 mycotoxicosis. Our goal was to identify changes in the chyme microbiota and its functional annotation after 14-day exposure to T-2 toxin, artificially introduced with feed, and under the influence of the feed additive Zaslon 2+, fed alone and in combination with the proteolytic drug Axtra Pro. The experiments were carried out in the vivarium of the Federal Scientific Center ARRTPI RAS in 2021. Broiler chickens of the Smena 8 cross aged 33 days were assigned into four groups of 5 birds each. Control group I received a basal diet (BD) without T-2 toxin, group II was fed with BD added with T-2 toxin (200 µg/kg) (BD + T-2), group III - BD + T-2 + additive Zaslon 2+ (1 g/kg feed) (BIOTROF Ltd, Russia), group IV - BD + T-2 + additive Zaslon 2+ added with proteolytic preparation Axtra Pro (DuPont de Nemours, Inc., USA) (100 mg/kg feed). Zaslon 2+ contains diatomite, two Bacillus strains, and a mixture of natural essential oils (eucalyptus, thyme, garlic, and lemon). Feed intake averaged 150 g/day, i.e. the birds of the experimental groups received 30 µg T-2 toxin daily. At the end of the experiment, the caecum content was sampled from three broilers of each group. Total DNA was isolated from the samples using the Genomic DNA Purification Kit (Fermentas, Inc., Lithuania). The caecal bacterial community was assessed by NGS sequencing on the MiSeq platform (Illumina, Inc., USA) using primers for the V3-V4 region of the 16S rRNA gene. Bioinformatic data analysis was performed using QIIME2 ver. 2020.8 (https://docs.qiime2.org/2020.8/). The reconstruction and prediction of the functional content of the metagenome, gene families, and enzymes was carried out using the PICRUSt2 (v.2.3.0) software package (https://github.com/picrust/picrust2). MetaCyc base data (https://metacyc.org/) was used to analyze metabolic pathways and enzymes. NGS-sequencing revealed changes in biodiversity and composition of the gut microbiota at the level of phyla. I.e., in group II, the population of superphylum Actinobacteriota and phylum Proteobacteria increased 1.8 and 3.5 times, respectively (p ≤ 0.05) while the superphylum Desulfobacterota , on the contrary, decreased 2.2 times (p ≤ 0.05). In group IV (BD + T-2 supplemented with Zaslon 2+ and Axtra Pro), the abundance of superphylum Actinobacteriota and phylum Proteobacteria also increased compared to group I (p ≤ 0.05), while in group III (BD + T-2 supplemented with Zaslon 2+) no change occured. The members of superphylum Verrucomicrobiota completely disappeared in groups II and IV, while in group I they accounted for 14.1±0.8 %. In group III compared to group I, bacteria of the genus Lactobacillus increased (p ≤ 0.01) from 15.9±1.32 to 30.7±1.84 %. The genus Akkermansia represented by the only species A. muciniphila sharply decreased in all groups fed T-2 toxin (groups II, III, and IV) as compared to group I (p ≤ 0.001), up to a complete absence in groups II and IV. Pathogenic microorganisms which were absent in group I ( Enterococcus cecorum , Campylobacter concisus , Campylobacter gracilis , Streptococcus gordonii , Flavonifractor spp.) appeared in group II. In groups III and IV, these pathogens were either absent or were present in a significantly smaller amount than in group II (p ≤ 0.05). Gut microbial community showed differences between groups (p ≤ 0.05) in 163 predicted metabolic pathways. When exposed to T-2 toxin (group II compared to group I, p ≤ 0.05), there was an increase in the predicted metabolic pathways for the degradation of aromatic compounds, including xenobiotics, and amino acids and for the synthesis of coenzymes, cofactors and formation of biofilms, cell walls, spores and protective substances in cells. The feed additive Zaslon 2+ contributed to the adjustment of metabolic pathways to the level of group I. The combined use of the feed additive Zaslon 2+ and protease (group IV) had no positive effect on the potential of metabolic pathways. Thus, feed contamination with T-2 toxin has a negative impact on the composition and predicted metabolic potential of the gut microbiome of Smena 8 cross broiler chickens. In general, the effect of the feed additive Zaslon 2+ and its complex with protease was positive though the additive without the enzyme showed greater efficiency.