Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B₁

Abstract

The food-borne mycotoxin aflatoxin B₁ (AFB₁) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. AFB₁ toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB₁-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB₁, and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB₁ were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB₁-treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB₁-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis.

Keywords: Key Contribution; RNA-seq; Results of this study support the hypothesis that the greater ability of wild turkeys to detoxify AFB1 is related to higher constitutive expression of GSTA3 coupled with an inherited (genetic) difference in functional expression in domesticated birds. Key differences in GSTA3 expression between the Eastern wild and domesticated turkeys is not unique to these genetic lines but is a broader phenomenon indicating lower fitness in domesticated birds. Results of RNA-seq analysis emphasize the differential response of these genetically distinct birds, demonstrating significant differences in expression of Phase I and Phase II genes and in genes important in toxic response.; aflatoxin B1; differential expression; domesticated turkey; liver; wild turkey.

Figure 1

Principal component analysis (PCA) of normalized RNA-seq read counts. For each treatment group, sample to sample distances (within- and between-treatments) are illustrated on the first two principal components comprising approximately 95% of the variation.

Figure 2

Distribution of differentially expressed genes in turkey. For each comparison, the number of genes with FDR p-value < 0.05 and |log₂FC| > 2.0 shared or unique to each treatment are indicated in the Venn diagram. Circle size is proportional to the number of genes and direction of expression change (↑ or ↓) is given for the genes in each group.

Figure 3

Distribution of differentially expressed of liver genes between turkey types (Wild and Domesticated). For each comparison, the number of genes with FDR p-value < 0.05 and |log₂FC| > 2.0 shared or unique to each treatment group are indicated. Circle size is proportional to the number of genes and direction of expression change (↑ or ↓) is given for the genes in each group.

Figure 4

Effect of AFB₁ on expression of GSTA3 in the livers of turkeys. (A) Mean normalized RNA-seq read counts. For each treatment group, individual read counts are indicated by closed circles. Error bars denote standard deviation of the mean. (B) Relative expression as measured by qRT-PCR. For each group the fold change (ΔΔCt) between AFB₁-treated and control birds is given. Asterisks denote significant comparisons (p-value < 0.01).