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

Truong, Anh Duc, Hong, Yeong Ho, Lillehoj, Hyun S.
Veterinary immunology and immunopathology 2015 v.166 no.3-4 pp. 116
Clostridium perfringens, Eimeria maxima, Gallus gallus, Marek disease, White Leghorn, breeding, chickens, cluster analysis, coccidiosis, cytokine receptors, cytokines, databases, gene expression, gene expression regulation, genes, genetic markers, haplotypes, high-throughput nucleotide sequencing, host-pathogen relationships, immune response, inbred lines, intestinal mucosa, messenger RNA, mixed infection, necrotic enteritis, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, transcriptome
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.