Effect of T-2 toxin on expression of genes associated with immunity in tissues of the blind processes of the intestinal and pancreas of broilers (Gallus gallus L.)

A significant proportion of poultry feed is contaminated with T-2 toxin. The bird’s immune system is one of the targets of this xenobiotic. However, the results of studying the effect of T-2 toxin on the expression of immunity genes in birds are extremely limited. In the present study, we have shown that contamination of broiler feed with T-2 toxin affects the level of expression of genes associated with the functioning of the immune system in the cecum and pancreas. The aim of the work was to assess the effect of T-2 toxin on the level of expression of genes involved in the immune system responses in the tissues of the intestine and pancreas cecum in broilers. The feeding trials with T-2 toxin added to the feed were carried out on broilers of the Smena 8 cross from 30 to 50 days of age (the vivarium of the Federal Research Center VNITIP RAS, 2020). Broilers were assigned to four treatments. The control group I received a diet with no T-2 toxin added, group II received a diet added with 100 μg/kg T-2 toxin, group III with 200 μg/kg, and group IV with 400 μg/kg. Gene expression was analyzed by quantitative PCR with reverse transcription (RT-qPCR). A reverse transcription reaction was performed to generate cDNA on an RNA template using the iScript™ Reverse Transcription Supermix (Bio-Rad, USA). The following primer pairs were used: for Interleukin 6 ( IL6 ) F - 5'-AGGACGAGATGTGCAAGAAGTTC-3', R - 5'-TTG-GGCAGGTTGAGGTTGTT-3'; for Interleukin 8 ( IL8 ) F - 5'-GGAAGAGAGGTGTGCTTGGA-3', R - 5'-TAACATGAGGCACCGA-TGTG-3'; for Interferon 7 ( IRF7 ) F - 5'-ATCCCTTGGAAGCACAACGCC-3', R - 5'-CTGAGGCAACCGCGTAGACCTT-3'; for Prostaglandin-endoperoxide synthase 2 ( PTGS2) F - 5'-TC-GAGATCACACTTGATTGACA-3', R - 5'-TTTGTGCCTTGTGGGTCAG-3'; for avian beta-defensin 9 ( AvBD9 ( Gal9 )) F - 5'-AACACCGTCAGGCATCTTCACA-3', R - 5'-CGTCTTCTTGGCTGTAAGCTGGA-3', for avian beta-defensin 10 ( AvBD10 ( Gal10 )) F - 5'-GCTCTTCGCT-GTTCTCC-TCT-3', R - 5'-CCAGAGATGGTGAAGGTG-3'; for Caspase 6 ( Casp6 ) F - 5'-CAGAGGAGACAAGTGCCAGA-3', R - 5'-CCAGGAGCCGTTTACAGTTT-3'. The beta-actin protein gene was a reference control. Amplification reactions were performed using a SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, USA). The amplification mode and conditions corresponded to those proposed by the primer developers. The relative expression level was estimated by the 2-ΔΔCT method. Biochemical blood profiles of broilers were analyzed (a Sinnowa BS3000P semi-automatic biochemical analyzer, SINNOWA Medical Science & Technology Co., Ltd, China) with a set of veterinary diagnostic reagents (DIAKON-VET, Russia). Principal component analysis (PCA) was used to compare gene expression levels and blood biochemical parameters. The expression of genes associated with the inflammatory response, apoptosis, antimicrobial and antiviral protection was evaluated. Activation (p ≤ 0.05) of the expression of the pro-inflammatory genes IL6 and PTGS2 occurred in broilers fed T-2 toxin. This can adversely affect the health and productivity of the birds, since the overproduction of proinflammatory cytokines is involved in the pathogenesis of several diseases. An increase (up to 41.7-fold, p = 0.0005) in the PTGS2 gene expression in the pancreas was characteristic of all groups fed T-2 toxin compared to the control. In the tissues of the intestinal cecum, there was a decrease (up to 12.5-fold, p = 0.02) in the expression level of the Casp6 gene of the apoptosis factor regardless of the T-2 toxin dosage. In the pancreas, there was a reverse tendency of a sharp increase in the Casp6 gene expression as the T-2 toxin concentration increased (p ≤ 0.0008). In group II, the expression increased 22.4 times (p = 0.0008), in group III 715.8 times (p = 0.0003), in group IV 31288.3 times (p = 0.0003) compared to the control. The expression of AvBD9 and AvBD10 genes of avian β-defensins which are associated with a higher bacteriostatic activity against many pathogens decreased 2.1 to 5.3 times (p ≤ 0.05) in the caecum of broilers fed 200 and 400 200 μg/kg T-2 toxin. In the pancreas, regardless of the T-2 toxin dosage, on the contrary, the expression of these genes significantly increased (p ≤ 0.04). In the caecum, 100 μg/kg T-2 toxin exposure inhibited the IRF7 gene expression 3-fold (p = 0.03) compared to the control. This can negative affect birds’ health, since the IRF7 gene of the interferon regulatory factor 7 participates in counteraction against many viruses. In general, the pancreas was found to be more sensitive to the effects of the T-2 toxin because the expression of almost all studied genes was significantly increased as compared to that in the cecum tissue. This difference in the immune response may be due to the functional divergence between the intestine and the pancreas.