Hepatotoxic and Immunomodulatory Transcriptome Responses to Aflatoxin B1 in the Turkey (Meleagris gallopavo)

Abstract

Hepatoxicity and immunotoxicity from dietary exposure to aflatoxin B1 (AFB1) adversely affect poultry health and production. Domestic turkeys (Meleagris gallopavo) are especially sensitive to AFB1 since they have a deficiency in glutathione-mediated detoxification of the reactive AFB1 intermediate. Changes in gene expression can be used to characterize the molecular mechanisms of toxicity; transcriptome analysis allows investigation of differential expression at the genome-wide level. In this research, Illumina RNA-sequencing (RNA-seq) was used to examine transcriptome responses to AFB1 exposure in the turkey. As the liver is the primary site of AFB1 activation and toxicity, the effects of dietary AFB1 on the domestic turkey liver transcriptome was first investigated by sequencing 4 pooled libraries representing 3 individuals for each of 4 treatment groups. As detailed in Chapter 2, predicted transcripts were de novo assembled and differential expression analysis identified significant effects on transcripts from genes involved in apoptosis, cell cycle regulation and lipid metabolism (like E3 ubiquitin-protein ligase Mdm2 and lipoprotein lipase). In Chapter 3, RNA-seq and de novo transcriptome assembly were performed on 3 individual spleen samples per treatment group (n = 12) collected from the same AFB1 challenge trial. Significant down-regulation of antimicrobial genes (like beta-defensin 1) and up-regulation of cytotoxic and antigen presentation genes (such as granzyme A) were observed after AFB1 treatment. Another aspect of these studies was to evaluate the ability of a Lactobacillus-based dietary probiotic to reduce AFB1-effects in the liver and spleen. Addition of probiotics during AFB1 exposure modulated expression in both tissues. Many AFB1-induced expression changes were not mitigated in liver, and although probiotics had some amelioratory effects in the spleen, they were also broadly suppressive of immune genes. Multiple genes impacted in the spleen transcriptome belonged to the Major Histocompatibility Complex (MHC), a region of the genome with genes essential to immune functions. The functions and expression patterns of many of the genes located in the turkey MHC have not been characterized. A single-gene investigation (Chapter 4) characterized expression patterns of 29 MHC genes in the domestic turkey and provided first evidence for expression of B-butyrophilin 2 in muscle tissue. Understanding these expression profiles will help determine MHC gene functions and provide background for expression changes from immunological challenges like AFB1. Unlike the domestic turkey, Eastern wild turkeys (M. g. silvestris) are more resistant to aflatoxicosis due at least in part to their ability to detoxify AFB1. In Chapter 5, an in ovo exposure model was utilized to directly compare the effects of AFB1 exposure in domestic and wild turkey embryos. Embryonic exposure has applicability to poultry production since AFB1 can be maternally transferred into eggs. RNA-seq datasets from embryonic liver tissue in domestic (n = 24) and wild (n =15) turkeys were mapped to a MAKER turkey gene set. Differential expression and pathway analysis identified conserved effects on cell cycle regulators (like E3 ubiquitin-protein ligase Mdm2) and variable effects in genes encoding detoxifying and anti-oxidant enzymes (like glutathione S-transferases) in domestic and wild turkeys. Overall, transcriptome analysis identified hepatic and splenic responses to AFB1, evaluated the use of probiotics, directly compared domestic and wild turkeys, and provided gene targets for future investigation of the molecular mechanisms of aflatoxicosis.