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Title: High-throughput sequencing reveals differing immune responses in the intestinal mucosa of two inbred lines afflicted with Necrotic enteritis

item TRUONG, AND DUC - Chung-Ang University
item HONG, YEONG HO - Chung-Ang University
item Lillehoj, Hyun

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/20/2015
Publication Date: 6/25/2015
Publication URL:
Citation: Truong, A., Hong, Y., Lillehoj, H.S. 2015. High-throughput sequencing reveals differing immune responses in the intestinal mucosa of two inbred lines afflicted with Necrotic enteritis. Veterinary Immunology and Immunopathology. 166(3-4):116-124.

Interpretive Summary: Necrotic enteritis (NE) is a gut infection caused by the bacterium, Clostridium perfringens (C. perfringens) that costs US industry more than $ 2 billion. With an increasing push for a regulatory ban of antibiotics in animal agriculture, there is a critical need to understand process of pathogen infection and to develop logical solutions to decrease economic losses due to NE. However, the molecular mechanisms underlying the pathology of NE remain to be determined first. ARS scientists collaborated with scientists at Korean universities to find out what proteins in the chicken are changed by NE infection using state of the art technology for gene analysis. The results showed that NE infection process induced significant gene changes in the intestinal genes that are involved in defending host against Clostridium bacteria. The characterization of genes identified in this study will lead to a better understanding of molecular mechanisms involved in the NE disease and an understanding of the function of the genes will lead to the novel strategies to effectively control gut infection and decrease economic losses caused by NE. Furthermore, the results we reported in this paper will provide a foundation for future studies that can be more targeted towards a for benefit agricultural science and animal immunology.

Technical Abstract: We investigated the necrotic enteritis (NE)-induced transcripts of immune-related genes in the intestinal mucosa of two highly inbred White Leghorn chicken lines, line 6.3 and line 7.2, which share the same MHC haplotype and show different levels of NE susceptibility using high-throughput RNA sequencing (RNA-Seq) technology. NE was induced by the previously described co-infection model using Eimeria maxima and Clostridium perfringens. The RNA-Seq generated over 38 million sequence reads for Marek's disease (MD)-resistant line 6.3 and over 40 million reads for the MD-susceptible line 7.2. Alignment of these sequences with the Gallus gallus genome database revealed the expression of over 29,900 gene transcripts induced by NE in these two lines, among which 7,841 genes were significantly upregulated and 2,919 genes were downregulated in line 6.3 chickens and 6,043 genes were significantly upregulated and 2,764 genes were downregulated in NE-induced line 7.2 compared with their uninfected controls. Analysis of 560 differentially expressed genes (DEGs) using the gene ontology database revealed annotations for 246 biological processes, 215 molecular functions, and 81 cellular components. Among the 53 cytokines and 96 cytokine receptors, 15 cytokines and 29 cytokine receptors were highly expressed in line 6.3, whereas the expression of 15 cytokines and 15 cytokine receptors was higher in line 7.2 than in line 6.3 (fold change=2, p<0.01). In a hierarchical cluster analysis of novel mRNAs, the novel mRNA transcriptome showed higher expression in line 6.3 than in line 7.2, which is consistent with the expression profile of immune-related target genes. In qRT-PCR and RNA-Seq analysis, all the genes examined showed similar responses to NE (correlation coefficient R=0.85-0.89, p<0.01) in both lines 6.3 and 7.2. This study is the first report describing NE-induced DEGs and novel transcriptomes using RNA-seq data from two inbred chicken lines showing different levels of NE susceptibility. These findings provide important insights into our current knowledge of host-pathogen interaction and the nature of host genes that can serve as NE resistance markers for molecular breeding.