Printer Friendly

RNA-seq profiles of immune related genes in the spleen of necrotic enteritis-afflicted chicken lines.

INTRODUCTION

Necrotic enteritis (NE) is an economically important disease of the poultry industry worldwide, and is mediated by Clostridium perfringens strains that produce necrotic enteritis toxin B-like (NetB), a [beta]-pore-forming toxin (Yan et al., 2013). NE was described in chicken for the first time by Parish in England in 1961 (Parish, 1961). The disease has been estimated to cost the world poultry industry roughly $2 billion annually (McReynolds et al., 2004). Over the past several years, several studies have profiled the gene expression of two genetically disparate inbred lines (resistant and susceptible line) with regard to their resistance or susceptibility against bacteria, protozoa and virus, including expression of Toll-like receptors (TLRs) and interleukin (IL) genes in response to Salmonella (Pan et al., 2011), beta-defensins in response to Eimeria maxima (Hong et al., 2012), and Th1 cytokines genes in response to Newcastle disease virus (Liu et al., 2012).

Recently, next-generation sequencing technology (RNA-Seq) has become available as a powerful tool to investigate transcriptional profiles for gene expression analysis of many organisms, such as Homo sapiens (Mortazavi et al., 2008), Chlamydomonas reinhardtii (Simon et al., 2013), Saccharomyces cerevisiae (Nagalakshmi et al., 2008), Fusarium graminearum (Zhao et al., 2013), Streptococcus mutans (Zeng et al., 2013), Catharanthus roseus (Van Moerkercke et al., 2013), mice (Cloonan et al., 2008), Arabidopsis (Lister et al., 2008) and Candida parapsilosis (Guida et al., 2011). RNA-Seq data has been shown to be efficiently used to improve gene model prediction, identify novel transcripts, and measure transcript expression in a single assay (Mortazavi et al., 2008; Trapnell et al., 2012). Furthermore, RNA-Seq technology is much more sensitive and efficient than the dedicated microarrays previously used to compare gene expression profiles (Mortazavi et al., 2008). RNA-Seq data also has been successfully used to identify alternative splicing in the genes of different species (Huang et al., 2013).

In addition, the different expression of miRNA were also examined in spleen and intestine of two chicken lines, namely, line 6.3 and line 7.2 which have been used in our previous small RNA next generation sequencing (NGS) studies (Hong et al., 2014). Herein, we used an RNA-Seq approach to perform transcriptome analysis in relation to the immune response in NE-afflicted chicken, and identified statistically significant gene expression differences of individual genes or transcripts between the spleens of two genetically disparate chicken lines. RNA-seq was carried out herein to identify mRNAs which are differentially expressed in the spleens of the two chicken lines experimentally afflicted with NE using co-infection with E. maxima and C. perfringens, and to gain an understanding of how immune related genes are regulated during NE for disease control in chicken.

MATERIALS AND METHODS

Experimental animal and necrotic enteritis disease model

White Leghorn chickens were used for the experiments, including the Marek's disease (MD) resistant line 6.3, and the susceptible line 7.2. These chicken lines have been selected and maintained for decades after exposure to avian leukosis virus and MD virus; line 6.3 being resistant, and line 7.2 being sensitive to these pathogens at the Avian Disease and Oncology Laboratory (ADOL), of the Agriculture Research Service, United States Department of Agriculture (USDA, East Lansing, MI, USA). Twenty chickens per group were randomly selected and infected with E. maxima strain 41A (1.0 x [10.sup.4] oocysts/chicken) by oral gavage on day 14, followed by oral gavage with C. perfringens strain Del-1 (1.0 x [10.sup.9] colony forming units [CFU]/chicken) 4 days later, as previous reported (Jang et al., 2012). The protocol for the development of NE was approved by the Beltsville Area Institutional Animal Care and Use Committee, USDA.

Total RNA preparation and quality control

On day 20 post-hatch, spleens were collected from five chickens per group. The spleens were carefully homogenized with a mortar and pestle after freezing with liquid nitrogen, and the total RNA from both lines was isolated using TRizol reagent (Invitrogen, Carlsbad, CA, USA) as described (Hong et al., 2012). RNA concentrations were quantified using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) at the wavelength of 260/280 nm ratio between 1.7 and 2.0. Integrity of the total RNA samples was evaluated using an Agilent 2100 (Agilent Technologies Inc., Santa Clara, CA, USA) and Tecan F2000 (Tecan Group Ltd., Mannedorf, Switzerland), and only the samples with RNA integrity number above 7.0 which were of high-quality (28S/18S > 1) were used for the following experiments.

RNA-seq library construction, RNA sequencing and data analysis

RNA-seq library construction and RNA high-throughput sequencing were performed by Theragen Bio Institute (Suwon, Korea) using an Illumina HiSeq 2000 high throughput sequencer, according to the manufacturer's specifications. The RNA-Seq data were analyzed according to the method described (Trapnell et al., 2012). Briefly, reads were mapped to the Gallus gallus reference genome (v.4.0) obtained from the University of California, Santa Cruz (UCSC) database (UCSC: http://genome.ucsc.edu/) using TopHat v.2.0.3 (http://tophat.cbcb.umd.edu/), and Bowtie v.0.12.8 (http://bowtie-bio.sourceforge.net/index.shtml) from Illumina iGenomes (http://support.illumina.com/). Transcript abundance and differential expression were calculated with the Cufflinks program v.2.0.1 (http://cufflinks.cbcb.umd.edu/) as described (Trapnell et al., 2012). Levels of gene expression were normalized using values of the fragments per kilobase of exon per million mapped reads (FPKM). The following equation was used: RPKM = [10.sup.9](C)/(N x L), where C is the uniquely mapped counts determined from the high quality category, L is the length of the cDNA for the longest splice variant for a particular gene model, and N is the total mappable reads, which was determined as the sum of the high quality reads and the highly repetitive reads. Log2-transformations of the normalization were performed as specified in the methods described (Mortazavi et al., 2008), and the p-values calculated using the right-tailed Fisher exact test were < 0.01. Subsequently, differential expression pattern analysis of the known mRNA and predicted novel mRNA was performed using unannotated sRNAs. Functional annotation in the form of GO was subsequently extracted from the database using Blast2GO v2.7.1 (http://www.geneontology.org/). The "TreeMap" view of differences in the expression of GO terms was generated using REVIGO software (http://revigo.irb.hr/), as described (Supek et al., 2011). Further, the genes that significantly differed from the corresponding library were searched against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to determine the pathways using DAVID Bioinformatics Resources version 6.7, NIAID/NIH (http://david.abcc. ncifcrf.gov/tools.jsp) with p < 0.01.

Hierarchical cluster analysis for mRNA

Hierarchical cluster analysis of expressed genes was performed using Cluster software version 4.49 (http://www.bram.org/serf/Clusters.php) and Java Treeview software (http://jtreeview.sourceforge.net/). Cluster map analysis of the splenic genes detected between the two chicken lines was carried out using Euclidean distance. The p-values were calculated using the right-tailed Fisher's exact test at the significance level of p < 0.01.

Quantitative real-time PCR

For analysis of mRNA gene expression, 5 [micro]g of total RNA were treated with 1.0 unit of DNase I and reverse transcribed using the Maxima first strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's recommendations. After cDNA synthesis, gene expression profiles were detected and quantified using an equivalent amount of cDNA. Briefly, cDNA (100 ng) was added to a reaction mix including 10 [micro]L of 2 x Power SYBR Green Master Mix (Roche, IN, USA), 0.5 [micro]L of each primer, and RNase-free water to a total volume of 20 [micro]L. Real-time PCR (RT-PCR) was performed on a LightCycler 96 system (Roche) following the standard cycling program. Each analysis was performed in triplicate. Standard curves were generated using log10 diluted cDNA from individual and pooled total RNA, as described by Hong et al. Data from quantitative real-time PCR (qRT-PCR) were normalized relative to the expression of GAPDH using the [2.sup.-[DELTA][DELTA]Ct] method, as previously described (Livak and Schmittgen, 2001). Oligonucleotide primers for analysis and the GAPDH control were designed based on sequences available in public databases using Lasergene software (DNASTAR Inc., Madison, WI, USA) as indicated in Table 1.

Statistical analysis

Statistical analyses were performed with IBM SPSS ver. 20 (IBM Co., Armonk, NY, USA). The data were expressed as the mean [+ or -] SD for each group (N = 5), and comparisons between groups were carried out using Student t-test. Statistical significance was defined as p < 0.05 and p = 0.01.

RESULTS

RNA sequence alignment

A total of 42.7 and 42.6 million sequence reads for chicken lines 6.3 and 7.2 were obtained by RNA-seq as indicated in Table 2. Of the total reads, 79.15% and 78.50% were successfully mapped to the chicken genome in lines 6.3 and 7.2, respectively. In each group, 57.11% and 56.57% of the sequences were properly paired, respectively. The percentage of properly paired sequences was not higher because only the reads aligned entirely inside exonic regions were included, and 54.77% and 52.49% of the reads were mapped to intergenic/exon-intron regions in the spleens of lines 6.3 and 7.2, respectively. Overall, line 6.3 had a higher number of successfully mapped reads than line 7.2 for the spleen.

Based on the number of sequence reads, the expression levels of the genes in each sample can be estimated. These fragments or reads were used to measure the levels of gene expression, and to identify novel splice variants of genes. As a result, the mRNA of 29,997 genes was detected, which was composed of 14,479 (49%) known genes and 15,518 (51%) novel genes.

To evaluate the quality of the RNA-Seq data, several quality control analyses were performed. In this study, gene coverage was used for evaluation of RNA-seq. Gene coverage is defined as the percentage of a gene covered by reads, and is equal to the ratio of the base number in a gene covered by unique mapping reads to the total base numbers of that gene. As shown in Supplementary Figure S1, the distribution of distinct reads over different read abundance categories showed similar patterns in the RNA-Seq libraries of the two chicken lines. In both NE-afflicted chicken lines, the read coverage was more than 90% of the chicken genome for 67% of the read sequences in both NE-afflicted chicken lines and 70% of the chicken genome for 12% of the read sequences.

Gene ontology analysis

Gene ontology (GO) plays an important role in the annotation and categorization of sequences, including unidentified and unannotated sequences. GO enrichment analysis provides all GO terms enriched significantly in the differentially expressed genes (DEGs) compared to the genome background, and filters the DEGs which correspond to biological functions. This method maps all DEGs to GO terms in the database, calculating gene numbers for every term, then uses a hypergeometric test to find the GO terms which are significantly enriched in the DEGs compared to the genome background with p < 0.01, fold change [greater than or equal to] 2, and then the "TreeMap" view can be used to visualize the differentially expressed GO terms using REVIGO software.

In chicken line 6.3, 88 GO molecular functions were revealed to be annotated for 313 genes, in which 244 of the genes were upregulated and 69 genes were downregulated. In addition, 135 GO biological processes had annotations for 249 genes, of which 153 were upregulated and 96 were downregulated. For 22 GO cellular components which contained 176 genes, 134 of the genes were upregulated while 42 were downregulated (Table 3 and Supplementary Table S1a). On the other hand, chicken line 7.2 had 54 GO molecular functions for which there were 108 genes annotated genes: 67 upregulated and 41 downregulated. Of 70 GO biological processes annotated with 127 genes, 68 genes were upregulated while 59 were downregulated. 10 GO cellular components containing 29 genes, of which 12 were upregulated and 7 were downregulated (Table 3 and Supplementary Table S1b).

When NE-afflicted MD-resistant chicken line 6.3 was compared to the MD-susceptible line 7.2, the following GO annotations were revealed: 76 GO molecular functions were annotated with 239 genes, of which 149 were upregulated and 90 were downregulated; 141 GO biological processes were annotated with 234 genes, of which 83 were upregulated and 151 were downregulated; and 34 GO cellular components were annotated with 221 genes, of which 134 were upregulated and 87 were downregulated (Table 3 and Supplementary Table S1c). Overall, the number of genes up and downregulated in the spleen of line 6.3 was higher than in line 7.2. The NE-afflicted line 6.3 contained more upregulated genes in the molecular function and cellular component categories than line 7.2, whereas the inverse was observed in biological processes, as summarized in Table 3.

The four major categories clustered in the biological process ontology of chicken line 6.3 were signal cellular histone metabolism, positive regulation of polarized epithelial cell differentiation, nitrogen compound transport and cellular response to cell-matrix adhesion. In the spleen of line 7.2, most of the DEG categories in the biological process were related with arginine metabolism and ion transport (Figure 1). However, two major clusters of the cellular components in chicken line 6.3 were focused intrinsic to the membrane and extracellular space, and the major components of four clusters fell into lipid particles, outer mucus layer, anchored to the membrane, and membrane in line 7.2 (Figure 1). On the other hand, molecular functions for metallopeptidase activity, transmembrane transport activity and calcium ion binding were also clustered in line 6.3, while four major clusters indicated lytic transglycosylase activity, bile acid activity, short chain carboxylesterase activity and phosphoendopyruvate carbonxykinase activity in line 7.2 (Figure 1.)

Identification of differentially expressed genes in two chicken lines

Generally, the number of mRNA-seq reads generated from a transcript is directly proportional to that transcript's relative abundance in the samples (Trapnell et al., 2012). In this study, DEGs of chicken lines 6.3 and 7.2 were compared with p < 0.01 and fold change [greater than or equal to] 2.

First, the gene expression profiles of the two NE-afflicted chicken lines were examined. The results indicated that 2,163 genes were significantly upregulated while 1,092 genes were downregulated in chicken line 6.3, with p < 0.01 and fold change [greater than or equal to] 2, whereas 1,388 genes were significantly upregulated and 1,080 genes were downregulated in line 7.2 (Table 2). In addition, comparison of the spleen expression files of the two NE-afflicted chickens revealed 2,234 genes to be significantly expressed between the two NE-afflicted chicken lines. Of the 2,234 genes, 1,239 were abundantly expressed in chicken line 6.3 compared to line 7.2, and 995 were highly expressed in line 7.2 with p<0.01 and fold change [greater than or equal to] 2(Table 2 and Figure 2).

To understand the functions of the DEGs, we mapped them using the Kyoto Encyclopedia of Genes and Genomes databases (KEGG) (http://www.genome.jp/kegg/) for signalling pathways analysis based on using DAVID Bioinformatics Resources version 6.7 with p < 0.01. In KEGG pathway mapping, 1,311 genes could be assigned gene identification number with the National Center for Biotechnology Information (NCBI Gene ID). We identified 1,129 and 1,165 DEGs in chicken line 6.3 and line 7.2, respectively that could be mapped into various pathways at the KEGG (data not shown). The top 14 most differentially expressed signalling pathways from each comparison group are listed in Table 4. Specifically, the number of up and downregulated genes involved in mitogen activated protein kinase (MAPK) signalling pathway, endocytosis signalling pathway, cytokine-cytokine receptor interaction, JAK-STAT signalling pathway, Toll like receptor pathway, focal adhesion, erythroblastic leukemia viral oncogene homolog (ErbB) signalling pathway and transforming growth factor beta (TGF-[beta]) signalling pathway were significantly represented the largest group, which may have an important role in response of spleen in two chicken lines to the E. maxima/C. perfringens (EM/CP) infections.

Hierarchical clustering was then performed for the analysis of 107 novel genes (Supplementary Table S2) found to be differentially expressed in the spleens between the two NE-afflicted chicken lines using Euclidean distance. In Figure 3, red indicates the upregulated and green the downregulated mRNAs between the two lines. Over half of the genes showed higher expression in resistant chicken line 6.3 than in susceptible line 7.2. Among the novel genes of interest, many were associated with response to stress, cellular, binding, membrane and adenosine triphosphate (ATP) activity. A number of the transcripts have specific involvement in the immune response.

Immune-related genes

Immune-related genes are biologically important for the host response to pathogens. Due to differences in the genetic background between chicken line 6.3 and line 7.2, it was expected that some immune related genes would be differentially expressed between the two lines of chicken. The data revealed a total of 139 immune related genes among the samples, with 130 genes in chicken line 6.3 and 117 genes in line 7.2 that were upregulated at p < 0.01, and fold change [greater than or equal to] 2. Comparison of chicken line 6.3 to line 7.2 indicated 12 immune related genes to be expressed at higher level in line 7.2 than line 6.3, whereas 18 immune related genes were more abundantly expressed in line 6.3 than line 7.2. Among the large number of DEGs, most of the up- and downregulated genes were related to protein transportation, modification and degradation in immunization. These genes are likely related to the degradation and processing of antigens for major histocompatibility complex (MHC) class I and II molecules. Most of the DEGs in the spleen were related to MHC-II antigen processing pathways, such as immunoglobulin, interferon, interleukin, heat shock protein 70 (Hsp70), and hemoglobulin (Supplementary Table S3). Moreover, we mapped all of DEGs immune-related genes in two chicken lines to the KEGG database for signalling pathways analysis and particularly, those involved in cell adhesion molecules pathway, Integrin signalling pathway and JAK/STAT signalling pathway.

Innate immune responses in necrotic enteritis-afflicted chicken lines

Transcription analysis of 53 cytokines and 97 cytokine receptors of the two NE-afflicted chicken lines are shown in Supplementary Table S4. The expression of 15 cytokines genes (ADIPOQ, BMP2K, CCL3, CXCL13, IL-16, IL-28B, IL-34, SPP1, TGFB1, TNFAIP2, TNFAIP3, TNFAIP8, TNFAIP8L1, TNFSF11, and TNFSF13B) was significantly upregulated by 2.1- to 9.25-fold (Supplementary Table S4, Figure 4), while 46 cytokine receptors (CSF3R, CXCR7, IL-17RA, IL-18R1, IL-1R1, IL-1R2, IL-20RA, IL-21R, IL-2RG, IL-4I1, IL-5RA, IL-7R, IL-9R, LILRB3, TGFBR1, TLR1, TLR15, TLR21, TLR5, TLR6, TLR7, TNFRSF13B, TNFRSF13C, TNFRSF1B, TRAT1, XCR1, etc.) were also significantly upregulated by 2.04- to 10.16-fold in chicken line 6.3 with p < 0.01 and fold [greater than or equal to] 2 (Supplementary Table S4, Figure 5). In contrast, 18 cytokines genes (BMPER, CCL19, CCL20, CCL4, CXCL14, IFNB, IL-12A, IL-17B, IL-17D, IL-17F, IL-19, IL-22, IL-26, IL-7, LIF, NLRC3, TGFA, and VEGFA) and 14 receptor genes (CXCR3, IFITM5, IL-13RA1, IL-17RC, IL-1RAPL2, IL-22RA1, IL-22RA2, ILDR1, LILRA5, LTBP2, PDGFD, PDGFRL, TNFRSF14, and TNFRSF6B) were markedly downregulated by 2.05- to 7.48-fold and 2.1- to 10.1-fold, respectively, in chicken line 6.3 with p < 0.01 and fold [greater than or equal to] 2 (Supplementary Table S4, Figures 4 and 5).

Similarly, the expression of 18 cytokine genes (IL-7, IL-10, TNFAIP8, SPP2, TNFAIP3, IFNB, CCL19, TNFAIP8L1, IL-16, CNTF, CXCL13, TNFAIP8L3, TNFSF13B, IL-34, TGFB1, TNFAIP2, CCL3, and ADIPOQ) and 39 cytokine receptor genes (CXCR7, CSF2RA, IL-1RL1, I-L1R2, IL-17RA, IFNAR1, IL-18RAP, IL-31RA, IL-9R, IL-18R1, IL-20RB, IL-21R, IL-2RG, IL-20RA, IL-4I1, LILRB1, TLR1, IL-17REL, LILRB5, TGFBR1, TLR6, TNFRSF13B, XCR1, TLR7, TNFRSF13C, TLR15 etc.) was also markedly upregulated by 2.03- to 8.41-fold and 2.08- to 9.12-fold, respectively, in chicken line 7.2 with p < 0.01 and fold [greater than or equal to] 2 (Supplementary Table S4, Figures 4 and 5). In addition, the expression of 7 cytokines genes (IL-22, VEGFA, IL26, CXCL14, BMPER, IL-17D, and CCL20) and 16 receptor genes (IL-22RA1, CXCR3, IL-17RC, IL-1RAPL2, IFITM5, NRG2, TNFRSF14, LEPREL2, IL-11RA, LTBP2, ILDR1, IL-17RD, MC1R, IL-22RA2, LEPREL1, and LILRA5) were significantly downregulated by 3.6- to 5.7-fold and 2.14- to 9.12-fold, respectively with p < 0.01 and fold [greater than or equal to] 2 (Figures 4 and 5).

When the cytokine genes were compared between the two lines, 6 genes (CXCL13, IL-28B, IFNA1, TNFSF11, SPP1, and TNFSF13B) showed higher expression in line 6.3, while the gene expression levels of 16 genes (IL-10, SPP2, CMC1, IL-19, LIF, TAP1, IFNG, IL-6, FASLG, IL-7, CCL19, TNFAIP8L3, IL-34, CCL3, IL-10, and SPP2) were higher in line 7.2 (Supplementary Table S4, Figure 4). Of the cytokine receptors, 20 showed significantly higher expression in line 6.3, while 8 were more abundantly expressed in line 7.2 with p < 0.01 and fold [greater than or equal to] 2 (Supplementary Table S4, Figure 5).

Transcription analysis of 44 CD genes was also carried out, indicating 24 CD genes (CD3E, CD244, CD69, CD247, CD40L, CD38, CD5L, CD44, CD5, CD83, CD163L1, CD36, CD14, CD28, CD200R1A, CD40, CD59, CD200, CD4, CD2, CD72, CD180, CD79B, and CD93) to be markedly increased by 2.1- to 8.4-fold, and 4 CD genes (CD163, CD8A, CD34, and CD7) to be significantly decreased by 2.1- to 8.2-fold in chicken line 6.3 with p < 0.01 and fold [greater than or equal to] 2. In contrast, 23 CD genes (CD3E, CD69, CD48, CD274, CD40L, CD59, CD40, CD93, CD247, CD28, CD5L, CD38, CD44, CD200R1A, CD14, CD5, CD83, CD2, CD200, CD4, CD79B, CD72, and CD180) were significantly increased by 2.1- to 8.7-fold while 2 CD genes (CD163 and CD36) were significantly decreased by 5.0- and 6.8-fold in chicken line, 7.2 with p < 0.01 and fold [greater than or equal to] 2 (Supplemtary Table S5. Figure 6).

Discovery of novel genes in two chicken lines

For exploration of novel mRNA transcripts, the RNA-seq data were used to identify novel spliced transcripts consisting of novel splicing events of known annotated exons. Several programs are available for such analysis, including Qpalma, Splicemap, Mapsplice, TopHat, GSNAP and PALMapper, in which the TopHat algorithm pairs candidate exons and evaluates the alignment of reads to such candidates (Trapnell et al., 2009). In this study, mRNA for 29,997 genes was detected including 15,518 novel genes. Among the total novel genes, 3,700 and 3,407 were found to be upregulated in chicken lines 6.3 and 7.2, respectively with p < 0.01 and fold [greater than or equal to] 2. Moreover, comparison of the individual genes between the lines showed that the number of individual genes differentially expressed was generally equal in the NE-afflicted chicken line 6.3 (810 novel genes) and chicken line 7.2 (813 novel genes), with p < 0.01 and fold [greater than or equal to] 2. In addition, comparison of the expression of candidate novel genes between line 6.3 and line 7.2 revealed 1,503 and 1,047 novel genes to be significantly upregulated in chicken lines 6.3 and 7.2, respectively, with p < 0.01 and fold [greater than or equal to] 2 (data not shown).

Quantitative real-time PCR for DEG

In order to validate the DEGs identified by RNA-Seq, 14 genes were selected for analysis via qRT-PCR. The genes were selected based on functional enrichment and pathway results from those with differing expression patterns. Primers were designed based on available sequences to amplify the specific altered genes. Primer sequences and the sizes of expected products are shown in Table 1. Fold changes from qRT-PCR were compared between the two chicken lines in Figure 7 and Supplementary Figure S2.

The qPCR results revealed consistent expression patterns with the differential expression observed by RNA-Seq analysis for all 14 genes. The expression trends were consistent for all transcripts in both analyses, with a correlation coefficient of [R.sup.2] = 0.81 for chicken line 6.3 and [R.sup.2] = 0.87 for chicken line 7.2 line 7.2 as shown in Figure 8. The expression of seven genes (IL-16, LILRB3, TLR21, LILRB5, IL-7R, TNFAIP2, and CXCL13) was significantly higher in chicken line 6.3 compared to line 7.2, while the other seven genes (CCL3, CCL4, CSF3R, IL-34, LILRB1, LILRB4, and IL-1R2) showed significantly higher expression in chicken line 7.2 than line 6.3 (Figures 7 and Supplementary Figure S2).

DISCUSSION

High throughput RNA-Sequencing technology is a recently developed approach to transcriptome profiling using deep-sequencing technologies and generates concomitant gene expression and polymorphism data over the whole transcriptome through a single experiment (Ozsolak and Milos, 2011). NE disease mainly targets in the intestine and several publications demonstrated that the level of mRNA expression were significantly regulated in intestine of two chicken lines coinfected with E. maxima and C. perfringenes, in which the expression of mRNA was highly increased in chicken line 6.3 than chicken line 7.2 (Kim et al., 2014) but the global mRNA expression in spleen of two chicken lines coinfected by E. maxima and C. perfringenes is not investigated yet. On the other hand, the spleen is involved in both the humoral and cellular immune responses through its role in the generation, maturation and storage of lymphocytes (Redmond et al., 2010). Gene expression study of the spleen of chicken is commonly used as an indicator of immune response (Redmond et al., 2010). In the present study, RNA-seq technology was utilized in the analysis of DEGs and novel transcriptome in the spleen of two NE-afflicted chicken lines, lines 6.3 and 7.2 from ADOL that carry the same MHC haplotype ([B.sup.2]) but differ in their response to MD (Briles et al., 1977) or NE infection (Kim et al., 2014).

The draft sequences of spleen were generated from the two genetically disparate chicken lines with approximately 41.7 and 41.6 million sequences reads for NE-afflicted chicken lines 6.3 and 7.2, respectively, in which at least 78% of the reads aligned to the chicken genome (Table 2). In total, the mRNA of 29,997 genes was built with alternatively spliced transcripts. These genes profiles are very attractive, because difference in the expression levels of genes between the two chicken lines may provide clues for underlying protective immune response mechanisms in NE. Recently, several groups were used RNA-Seq to analysis of gene profiles and they show that the minimum 70% of reads were aligned to reference genome and 40% reads were mapped uniquely to the reference genome (Park et al., 2014).

The DEGs profiles of the spleen of two NE-afflicted chicken lines were generated herein, and significantly higher expression was observed in the NE-afflicted line 6.3 than line 7.2, as shown in Table 2. To better understand the gene expression patterns of two NE-afflicted chicken lines, a rigorous algorithm was developed to identify the DEGs between the two lines. By comparing NE-afflicted chicken lines 6.3 and 7.2, more significant DEGs were identified. A total of 2,234 genes were found: 1,239 upregulated and 995 downregulated. DEGs analysis by RNA-seq carried out on hematopoietic stem cells detected a higher number of DEGs than reported previously, which may have been related to the more comprehensive transcriptome discovery method (Park et al., 2014). Herein, GO functional enrichment of the gene expression was performed. Blast2GO software (v.2.7.1) with the GO database was used for the analysis of DEGs in both chicken lines, revealing 135 associated GO biological processes, 88 molecular functions and 22 cellular components in the NE-afflicted chicken line 6.3, and 70 associated biological processes, 10 molecular functions and 54 cellular components in the NE-afflicted chicken line 7.2. When comparing the DEGs between the two NE-afflicted chicken lines, the GO database indicated the annotations to be associated with 141 GO biological processes, 76 molecular functions and 34 cellular components. Most of the DEGs were involved in various immunological responses of the KEGG pathways, such as the JAK-STAT signalling pathway, TGF-[beta] signaling pathway, toll like receptor signaling pathway, MAPK signaling pathway, and the cytokine-cytokine receptor interaction pathway, particularly in NE-afflicted chicken line 6.3 (data not shown). Changes in the expression of genes in these pathways have also been discovered in the spleen after Clostridium infection, including signal transducer and activator of transcription, growth factor receptor-bound protein, cytokines and cytokine receptor genes (Zhou et al., 2009). Analyzed DEGs in the spleen of broiler chickens after vaccination and challenge by Escherichia coli, finding that most of the DEGs influencing the KEGG pathways were involved in the JAK-STAT signalling pathway, toll like receptor signalling pathway and the cytokine-cytokine receptor interaction pathway. The GO terms in which DEGs of the two NE-afflicted chicken lines were found uniquely helped us to provide insight into identifying the expression of which genes are different, as well as the overall processes defined by those genes. Changes in the genes in these pathways highlight the importance of proper signalling cascades to fight NE infection. The results from DEGs add greater depth to the knowledge base about host response to NE disease.

One of the most important findings from the RNA sequences is the finding of a large number of novel genes in the two NE-afflicted chicken lines. TopHat algorithm mapping to the chicken reference genome was used to find candidates novel genes, and 51% of the consensus sequences generated from the de novo assembly were annotated. This analysis included a large number of transcripts (15,518 contigs) not present in public databases. Overall, chicken line 6.3 showed a substantially larger number of novel genes than line 7.2. The false positive rate needs further investigation. The functional classification of the novel transcripts identified many transcripts specifically involved in the immune response, such as in the MAPK signaling pathway, JAK-STAT signaling pathway, TGF-[beta] signaling pathway, and toll like receptor signaling pathway. Therefore, a large number of the candidate novel genes were highly diverse, with a large proportion involved in transport, developmental processes, stress response, and cell adhesion. Based on the large number of new exons observed, analyses of the total RNA extracted from the spleen of the two NE-afflicted chicken lines may be a useful approach for the mining of candidate novel genes (data not shown).

In this study, 150 chicken cytokine genes were found to be related to the NE-afflicted chicken lines. Overall, this number is similar to the number of genes identified in duck (150 genes) and zebra finches (150 genes) and is substantially lower than number of mammalian cytokine genes, such as the 230 genes identified in humans and 218 genes in mice (Huang et al., 2013). It i s suggested that the RNA-seq analysis performed in this study will provide useful information on the altered expression of innate immune genes, or the discovery of novel genes in the spleen, after NE infection in the two chicken lines. Cytokines, including the IL-2, IL-4, IL-5, TGF-[beta], and IL-10 family, play a major role in the adaptive immune system (Wright et al., 2013). First, TGF-[beta] is produced by T cells and many other cell types. It is primarily an inhibitory cytokine, and inhibits the proliferation of T cells as well as the activation of macrophages. It also acts on polymorphonuclear neutrophil granulocytes and endothelial cells to block the effects of pro-inflammatory cytokines (Gandrillon et al., 1999). Among the TGF-[beta] family, TGF-[beta]1 was significantly increased by 7.34- and 7.46-fold in chicken lines 6.3 and 7.2 after co-infection with EM/CP. In addition, TGF-[beta] receptors (TGF-[beta]R2, TGF-[beta]RAP1, and TGF-[beta]R1) were also upregulated, and shown to have higher expression in chicken line 6.3 (by 2.18-, 1.22, 3.91-fold, respectively) than in line 7.2 (0.96-, 0.4-, and 3.41-fold, respectively) with p < 0.05 (Table S4, Figure 5). Second, interleukin 2 (IL-2) and interleukin 4 (IL-4) are produced by T helper cells, although they can also be produced by cytotoxic T cells to a lesser extent. They are major growth factors for T cells, and also promote the growth of B cells while having the ability to activate NK cells and monocytes (Hemmerle and Neri, 2013). The data obtained herein indicated marked upregulation of IL-2 receptors (IL-2RB and IL-2RG) in the two NE-afflicted chicken lines: by 2.20- and 3.56-fold in chicken line 6.3 and 1.54- and 3.58-fold in line 7.2 for the two genes, respectively. Interleukin 4 receptors were also significantly upregulated in both chicken lines (Supplementary Table S4, Figures 4 and 5).

Interleukin 5 (IL-5) is produced by Th2 cells and functions to promote the growth and differentiation of B cells and eosinophils, as well as activate mature eosinophiles (Johansson et al., 2013). In this study, IL-5 was markedly downregulated in line 7.2 by 0.04-fold, but significantly upregulated by 0.4-fold in line 6.3. Moreover, the expression of IL-5R was significantly increased by 1.22 and 7.26-fold in both lines, respectively. Third, the IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29, in mammals. Evolutionary, IL-10 family cytokines emerged before the adaptive immune response (Wolk et al., 2002). These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers (Wolk et al., 2002). Herein, 5 genes and 5 receptors of the IL-10 family were observed: IL-10, IL-19, IL-22, IL-26, and IL-28B genes, and IL-20RA, IL-20RB, IL-20RA1, IL-20RA2, and IL-28RA receptors. Among the IL-10 family transcripts, three interleukins (IL-19, IL-22 and IL-26) were downregulated by 2.87- to- 7.84 fold in the two chicken lines with p<0.01 and fold change [less than or equal to] 2 (Supplementary Table S4, Figures 4 and 5). The IL-28B gene (IFN-[lambda]3) was upregulated in chicken line 6.3 by 2.98-fold but downregulated in line 7.2 by 0.3-fold, while IL-10 was significantly upregulated in both chicken lines by 0.5- and 2.32-fold in lines 6.3 and 7.2, respectively. In addition, the interleukin 10 family receptors, IL-22RA1 and RA2, were downregulated in both chicken lines by 2.11- to 10.08fold. The IL-20 receptor genes, IL-20RA and IL-20RB, were also upregulated in the two NE-afflicted chicken lines by 1.53- to 3.59-fold (Supplementary Table S4, Figures 4 and 5). Previous studies reported that IL-10 and its family members share common receptors (Commins et al., 2008). Often, however, cytokines have distinct, if not antagonistic, functions. As an example, the signals of both IL-10 and IL-22 go through IL-10R2 to activate JAK1 and TYK2, respectively, thus resulting in STAT3 activation. IL-22, however, also induces serine phosphorylation of STAT3 (while IL-10 does not), an event that is associated with MAP kinase pathway activation (Sabat, 2010).

Of this family of pattern recognition receptors, the TLRs appear to be the most important, and have been the subject of intensive research over the past decade. TLRs are preferentially expressed on immune cells, including macrophages, DCs, monocytes, neutrophils, eosinophils, natural killer cells, platelets, and T and B lymphocytes (Shi et al., 2007). More recently, increasing evidence has indicated that the engagement of TLRs can promote cancer cell growth, induce evasion of immune surveillance, and enhance tumor metastasis and chemoresistance, or rather, induction of tumor cell apoptosis depending on ligands (Gonzalez-Reyes et al., 2010). The avian genome encodes 10 functional TLRs that are located either on the cell surface or within endosomes (Ramasamy et al., 2014). In a previous study, the TLR genes were found to be differentially expressed during the early stages of infection by Salmonella Pullorum in chicks, with significant upregulation of the expression of TLR2, TLR4, and TLR6 in the gastrointestinal tissues, but significant downregulation of TLR3 and TLR15 (Ramasamy et al., 2014). In addition, the expression of TLR3 and TLR7 was significantly upregulated in both susceptible and resistant chicken lines treated with polyionosinic-polycytidylic acid, being significantly higher in the resistant chickens compared with susceptible chickens (Haunshi and Cheng, 2014). In this study, the expression of 6 TLR genes (TLR1, TLR5, TLR6, TLR7, TLR15, and TLR21) was detected. Expression of the 6 TLR genes was significantly upregulated by 2.28- to 8.04-fold in chicken line 6.3, while that of 5 of the TLR genes (excluding TLR5) was significantly upregulated by 2.23- to 8.26-fold in chicken line 7.2 at p < 0.01 (Supplementary Table S4, Figures 4 and 5). These results revealed that genes of the TLR pathway play an important role in the pathogenicity of EM/CP coinfection model of NE. The findings are helpful for understanding the molecular basis of pathogenesis and the underlying mechanism of NE response, and also provide strong evidence of TLR involvement in the innate immune response to NE disease in poultry.

We also investigated 139 immune related genes for which higher expression was observed among the chicken lines (18 genes upregulated in line 6.3 and 12 genes in line 7.2, comparatively with p < 0.01 and fold [greater than or equal to] 2). In a previous report, search of the 450,000 sequences in the chicken expressed sequence tag collection enabled identification of 185 immune-related sequences which are also members of the cytokines, chemokines, antigens, cell surface proteins, receptors and MHC-associated genes (Smith et al., 2004). In the present study, most of the immune-related genes were also members of the antigens, cell surface proteins, receptors and MHC-associated genes, STAT family, TRAF family, interleukins and differentiation antigens. An interesting finding is that most of the immunoglobulin (Ig) genes were upregulated in the two NE-afflicted chicken lines. However, some of the Ig genes were more highly expressed in the MD-resistant chicken line 6.3 than in the spleen of the susceptible chicken line 7.2, including IgJ, IgA, Ig rearranged H-chain, Immunoglobulin heavy chain variable region, and Ig germline heavy chain VD region (Supplementary Table S3). The investigation of these genes will advance basic avian immunology of immunoglobulins and will pave the way for large-scale immune-related microarray experiments, providing new insight into functional and evolutionary studies.

Moreover, the CD molecular genes were also markedly upregulated: 24 CD genes in chicken line 6.3 and 23 CD genes in line 7.2 with p < 0.01 and fold [greater than or equal to] 2. The role of CD presented on the cell membrane is to allow molecular response to pathogens. CD molecular genes remain specific to a development period or as characteristic markers until destruction of the cell membrane. The majority of CD antigens are involved in immune functions of organisms and include the receptors for antigens, MHC glycoproteins, adhesive molecules, receptors for immunoglobulins, receptor for complement, receptors for lymphocytes and other growth and differentiation factors, membrane enzymes or transport molecules and other molecules (Fabryova and Simon, 2009).

Additional validation of the RNA-seq analysis was performed through qRT-PCR analysis of selected genes in the two NE-afflicted chicken lines. Gene expression changes for the 14 genes as observed by qRT-PCR were compared for the two chicken lines, as shown in Figures 7 and S1. Of those analyzed, the gene expression levels of 7 genes (IL-16, LILRB3, CXCL13, IL-7R, LILRB5, TLR21, and TNFAIP2) were higher in chicken line 6.3 than in line 7.2, while the other 7 genes (CCL3, IL-34, LILRB1, LILRB4, CCL4, CSF3R, and IL-1R2) showed significantly higher expression in chicken line 7.2 (Figures 7 and Supplementary Figure S1). All altered genes examined herein showed similar responses to co-infected EM/CP exposure in the qRT-PCR and RNA-Seq analyses. A high correlation between RNA-seq and qRT-PCR was observed at the range of 0.81 to 0.87 (p < 0.01) as shown in Figure 8. Quantitative RT-PCR validation was also an important indication that the pooled samples (2 pools of each 10 chickens) used for RNA-seq analysis reflected the expression levels in the individual pools. While pooled samples obviously could have masked individual variation, the goal in the present study was to gain a broader understanding of gene responses to NE infection in the spleen and to provide insights into important pathways and processes.

We draw four noteworthy conclusions from our results. First, using next-generation sequencing technology, we generated the first draft sequence for two chicken lines, one of which is a natural host of NE disease. Second, we identified 139 immune-related genes that were differentially expressed in the two NE-induced chicken lines. Further efforts identified 150 cytokines with differential expression in the two chicken lines. Third, we performed a deep transcriptome analysis to characterize gene expression profiles and to identify genes that are responsive to NE disease. Fourth, we found 15,518 candidate novel genes that may be involved in the host immune response to NE disease whose infection was examined in the two chicken lines. Overall, the genes of chicken line 6.3 were more highly expressed than those of chicken line 7.2 in response to NE induction. This dataset will be helpful for gene discovery, function, mapping, and genomic evaluation in chickens. Moreover, the significant DEGs will useful for future studies to understand the regulation and function of signalling pathways in the two genetic chicken lines of the present study. Collectively, the results generated in this study have provided information that our current knowledge of how chicken genes control NE disease and further study to develop disease resistance markers for molecular breeding.

http://dx.doi.org/10.5713/ajas.15.0143

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

This work was supported by the Next-Generation BioGreen 21 Program (PJ00808401) and Golden Seed Project (PJ009925022015), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS), Korea. The chicken lines provided by the Avian Disease and Oncology Laboratory (ADOL), USDA Agriculture Research Service, in East Lansing, MI.

REFERENCES

Briles, W. E., H. A. Stone, and R. K. Cole. 1977. Marek's disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195:193-195.

Cloonan, N., A. R. Forrest, G. Kolle, B. B. Gardiner, G. J. Faulkner, M. K. Brown, D. F. Taylor, A. L. Steptoe, S. Wani, G. Bethel, A. J. Robertson, A. C. Perkins, S. J. Bruce, C. C. Lee, S. S. Ranade, H. E. Peckham, J. M. Manning, K. J. McKernan, and S. M. Grimmond. 2008. Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5:613-619.

Commins, S., J. W. Steinke, and L. Borish. 2008. The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J. Allergy Clin. Immunol. 121:1108-1111.

Fabryova, K. and M. Simon. 2009. Function of the cell surface molecules (CD molecules) in the reproduction processes. Gen. Physiol. Biophys. 28:1-7.

Gandrillon, O., U. Schmidt, H. Beug, and J. Samarut. 1999. TGFbeta cooperates with TGF-alpha to induce the self-renewal of normal erythrocytic progenitors: evidence for an autocrine mechanism. EMBO J. 18:2764-2781.

Gonzalez-Reyes, S., L. Marin, L. Gonzalez, L. O. Gonzalez, J. M. del Casar, M. L. Lamelas, J. M. Gonzalez-Quintana, and F. J. Vizoso. 2010. Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis. BMC Cancer 10:665.

Guida, A., C. Lindstadt, S. L. Maguire, C. Ding, D. G. Higgins, N. J. Corton, M. Berriman, and G. Butler. 2011. Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis. BMC Genomics 12:628.

Haunshi, S. and H. H. Cheng. 2014. Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek's disease. Poult. Sci. 93:550-555.

Hemmerle, T. and D. Neri. 2014. The antibody-based targeted delivery of interleukin-4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer. Int. J. Cancer 134:467-477.

Hong, Y H., H. Dinh, H. S. Lillehoj, K. D. Song, and J. D. Oh. 2014. Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease. Poult. Sci. 93:1383-1395.

Hong, Y H., W. Song, S. H. Lee, and H. S. Lillehoj. 2012. Differential gene expression profiles of [beta]-defensins in the crop, intestine, and spleen using a necrotic enteritis model in 2 commercial broiler chicken lines. Poult. Sci. 91:1081-1088.

Huang, Y, Y Li, D. W. Burt, H. Chen, Y Zhang, W. Qian, H. Kim, S. Gan, Y Zhao, J. Li, K. Yi, H. Feng, P. Zhu, B. Li, Q. Liu, S. Fairley, K. E. Magor, Z. Du, X. Hu, L. Goodman, H. Tafer, A. Vignal, T. Lee, K. W. Kim, Z. Sheng, Y An, S. Searle, J. Herrero, M. A. Groenen, R. P. Crooijmans, T. Faraut, Q. Cai, R. G. Webster, J. R. Aldridge, W. C. Warren, S. Bartschat, S. Kehr, M. Marz, P. F. Stadler, J. Smith, R. H. Kraus, Y Zhao, L. Ren, J. Fei, M. Morisson, P. Kaiser, D. K. Griffin, M. Rao, F. Pitel, J. Wang, and N. Li. 2013. The duck genome and transcriptome provide insight into an avian influenza virus reservoir species. Nat. Genet. 45:776-783.

Jang, S. I., H. S. Lillehoj, S. H. Lee, K. W. Lee, E. P. Lillehoj, Y. H. Hong, D. J. An, W. Jeong, J. E. Chun, F. Bertrand, L. Dupuis, S. Deville, and J. B. Arous. 2012. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens. Vaccine 30:5401-5406.

Johansson, M. W., D. S. Annis, and D. F. Mosher. 2013. [alpha](M)P(2) integrin-mediated adhesion and motility of IL-5-stimulated eosinophils on periostin. Am. J. Respir. Cell Mol. Biol. 48:503-510.

Kim, D. K., H. S. Lillehoj, S. I. Jang, S. H. Lee, Y H. Hong, and H. H. Cheng. 2014. Transcriptional profiles of host-pathogen responses to necrotic enteritis and differential regulation of immune genes in two inbreed chicken lines showing disparate disease susceptibility. PLoS One 9:e114960.

Lister, R., R. C. O'Malley, J. Tonti-Filippini, B. D. Gregory, C. C. Berry, A. H. Millar, and J. R. Ecker. 2008. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133:523-536.

Liu, W. Q., M. X. Tian, Y P. Wang, Y Zhao, N. L. Zou, F. F. Zhao, S. J. Cao, X. T. Wen, P. Liu, and Y. Huang. 2012. The different expression of immune-related cytokine genes in response to velogenic and lentogenic Newcastle disease viruses infection in chicken peripheral blood. Mol. Biol. Rep. 39:3611-3618.

Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(T)) Method. Methods 25:402-408.

McReynolds, J. L., J. A. Byrd, R. C. Anderson, R. W. Moore, T. S. Edrington, K. J. Genovese, T. L. Poole, L. F. Kubena, and D. J. Nisbet. 2004. Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poult. Sci. 83:1948-1952.

Mortazavi, A., B. A. Williams, K. McCue, L. Schaeffer, and B. Wold. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5:621-628.

Nagalakshmi, U., Z. Wang, K. Waern, C. Shou, D. Raha, M. Gerstein, and M. Snyder. 2008. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344-1349.

Ozsolak, F. and P. M. Milos. 2011. RNA sequencing: advances, challenges and opportunities. Nat. Rev. Genet. 12:87-98.

Pan, Z., Q. Fang, S. Geng, X. Kang, Q. Cong, and X. Jiao. 2012. Analysis of immune-related gene expression in chicken peripheral blood mononuclear cells following Salmonella enterica serovar Enteritidis infection in vitro. Res. Vet. Sci. 93:716-720.

Parish, W. E. 1961. Necrotic enteritis in the fowl (Gallus gallus domesticus). I. Histopathology of the disease and isolation of a strain of Clostridium welchii. J. Comp. Pathol. 71:377-393.

Park, S. J., T. Umemoto, M. Saito-Adachi, Y Shiratsuchi, M. Yamato, and K. Nakai. 2014. Computational promoter modeling identifies the modes of transcriptional regulation in hematopoietic stem cells. PLoS One 9:e93853.

Ramasamy, K. T., P. Verma, and M. R. Reddy. 2014. Toll-like receptors gene expression in the gastrointestinal tract of Salmonella serovar Pullorum-infected broiler chicken. Appl. Biochem. Biotechnol. 173:356-364.

Redmond, S. B., R. M. Tell, D. Coble, C. Mueller, C. Palic, C. B. Andreasen, and S. J. Lamont. 2010. Differential splenic cytokine responses to dietary immune modulation by diverse chicken lines. Poult. Sci. 89:1635-1641.

Sabat, R. 2010. IL-10 family of cytokines. Cytokine Growth Factor Rev. 21:315-324.

Shi, Y, D. White, L. He, R. L. Miller, and D. E. Spaner. 2007. Toll-like receptor-7 tolerizes malignant B cells and enhances killing by cytotoxic agents. Cancer Res. 67:1823-1831.

Simon, D. F., R. F. Domingos, C. Hauser, C. M. Hutchins, W. Zerges, and K. J. Wilkinson. 2013. Transcriptome sequencing (RNA-seq) analysis of the effects of metal nanoparticle exposure on the transcriptome of Chlamydomonas reinhardtii. Appl. Environ. Microbiol. 79:4774-4785.

Smith, J., D. Speed, A. S. Law, E. J. Glass, and D. W. Burt. 2004. In-silico identification of chicken immune-related genes. Immunogenetics 56:122-133.

Supek, F., M. Bosnjak, N. Skunca, and T. Smuc. 2011. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:e21800.

Trapnell, C., L. Pachter, and S. L. Salzberg. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105-1111.

Trapnell, C., A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. Salzberg, J. L. Rinn, and L. Pachter. 2012. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7:562-578.

Van Moerkercke, A., M. Fabris, J. Pollier, G. J. Baart, S. Rombauts, G. Hasnain, H. Rischer, J. Memelink, K. M. Oksman-Caldentey, and A. Goossens. 2013. CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data. Plant Cell Physiol. 54:673-685.

Wolk, K., S. Kunz, K. Asadullah, and R. Sabat. 2002. Cutting edge: immune cells as sources and targets of the IL-10 family members? J. Immunol. 168:5397-5402.

Wright, H. L., H. B. Thomas, R. J. Moots, and S. W. Edwards. 2013. RNA-seq reveals activation of both common and cytokine-specific pathways following neutrophil priming. PLoS One 8:e58598.

Yan, X. X., C. J. Porter, S. P. Hardy, D. Steer, A. I. Smith, N. S. Quinsey, V Hughes, J. K. Cheung, A. L. Keyburn, M. Kaldhusdal, R. J. Moore, T. L. Bannam, J. C. Whisstock, and J. I. Rood. 2013. Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens. MBio 4:e00019-13.

Zeng, L., S. C. Choi, C. G. Danko, A. Siepel, M. J. Stanhope, and R. A. Burne. 2013. Gene regulation by CcpA and catabolite repression explored by RNA-Seq in Streptococcus mutans. PLoS One 8:e60465.

Zhao, C., C. Waalwijk, P. J. de Wit, D. Tang, and T. van der Lee. 2013. RNA-Seq analysis reveals new gene models and alternative splicing in the fungal pathogen Fusarium graminearum. BMC Genomics 14:21.

Zhou, H., J. Gong, J. Brisbin, H. Yu, A. J. Sarson, W. Si, S. Sharif, and Y. Han. 2009. Transcriptional profiling analysis of host response to Clostridium perfringens infection in broilers. Poult. Sci. 88:1023-1032.

Anh Duc Truong, Yeong Ho Hong *, and Hyun S. Lillehoj (1)

Department of Animal Science and Technology, Chung-Ang University, Anseong 456-756, Korea

* Corresponding Author: Yeong Ho Hong. Tel: +82-31-670-3025, Fax: +82-31-671-3025, E-mail: yhong@cau.ac.kr

(1) Animal Biosciences and Biotechnology Laboratory, Agricultural Research Services, U. S. Department of Agriculture, Beltsville, MD 20705, USA.

Submitted Feb. 17, 2015; Revised Apr. 10, 2015; Accepted Apr. 20, 2015

Table 1. Oligonucleotide primers used for quantitative RT-PCR

Target              Nucleotides                             Size
gene                                                        (bp)

GAPDH     Forward   5'- TGC TGC CCA GAA CAT CAT CC -3'      142
          Reverse   5'- ACG GCA GGT CAG GTC AAC AA -3'

CCL3      Forward   5'-CAT TGC CTC CGC CTA CAT -3'           98
          Reverse   5'-ACT CCT CGG GGT TTA CAC ATA -3'

CCL4      Forward   5'-CCC CTT GTC ATC GGT CAC -3'           94
          Reverse   5'-AGA GGC AGG AGC AGA GCA -3'

CSF3R     Forward   5'-CCT CTT CGG GAC GCT GTA -3'          110
          Reverse   5'-GGG CTT TCT CAT GGG TAG TG -3'

CXCL13    Forward   5'-GCC TGT GCC TGG TGC TC -3'           116
          Reverse   5'-TGC CCC CTT CCC CTA AC -3'

IL1R2     Forward   5'-AAC TGC CCA GAT CAC ACC AT -3'       112
          Reverse   5'-TCAAGG CAG AAA AAT CCA CAT -3'

IL7R      Forward   5'-CGG CAG GAA GAA AGC ACT -3'           97
          Reverse   5'-TCA CCT CAT ACA ACG CAT CTG-3'

IL16      Forward   5'-TGC CTC ACA AGA ATC AAC AAC T-3'     102
          Reverse   5'-ATA GAG CCC TTC CCA CCT TC-3'

IL34      Forward   5'-GCC CGC TCC CTT TTC TT -3'           117
          Reverse   5'-TGA TGG CTC TGC TTC TCT TTA TT-3'

LILRB1    Forward   5'-ACA CGA CCG AGT TCT TCT TTG-3'       112
          Reverse   5'-TCC ACG GGG TCA CTC TTC T-3'

LILRB3    Forward   5'-CCC CCA TCC AGC ATT TC -3'            93
          Reverse   5'-GGT GGC CCC ATA GTC CTT -3'

LILRB4    Forward   5'-GGC CGA GTT CTC CTT TGT TA -3'       107
          Reverse   5'-CCA CGG GGT CAC TCT TCT C -3'

LILRB5    Forward   5'-GGC CGA GTT CTC CTT TGT TA -3'       112
          Reverse   5'-CAG CTT CAT GGG GTC ACT CT -3'

TLR21     Forward   5'-GCT CAC AGG CAA AAT CAC G -3'        102
          Reverse   5'-AGC AGC AGG GTT CTC AGG T -3'

TNFAIP2   Forward   5'-CCA CGG CGT ACC CAG ATA -3'           95
          Reverse   5'-GCC CAG GAT GCT TTT GAG T -3'

Target              GenBank accession no.
gene

GAPDH     Forward   NM_204305
          Reverse

CCL3      Forward   EU999777
          Reverse

CCL4      Forward   NM_204720
          Reverse

CSF3R     Forward   NM_001030898
          Reverse

CXCL13    Forward   XM_420474
          Reverse

IL1R2     Forward   XM_416914
          Reverse

IL7R      Forward   NM_001080106
          Reverse

IL16      Forward   AB104417
          Reverse

IL34      Forward   XM_003641892
          Reverse

LILRB1    Forward   XM_004950708
          Reverse

LILRB3    Forward   XM_004951056
          Reverse

LILRB4    Forward   XM_003643693
          Reverse

LILRB5    Forward   XM_003643376
          Reverse

TLR21     Forward   XM_003641158
          Reverse

TNFAIP2   Forward   XM_421383
          Reverse

Statistical significance was defined as p < 0.05 and p = 0.01.

Table 2. Number of reads and number of genes detected using RNA
sequencing in the two NE-afflicted chicken lines

Sample         Category                   No. of read (%)

                                       Genome               Gene

Line 6.3 (R)   Total read         42,736,296 (100)    41,772,762 (100)
               Mapped read       33,827,317 (79.15)  22,877,379 (54.77)
               Properly paired   24,406,606 (57.11)  21,660,998 (51.85)

Line 7.2 (S)   Total read         42,617,720 (100)    41,686,454 (100)
               Mapped read       33,453,716 (78.50)  22,068,712 (52.94)
               Properly paired   24,107,042 (56.57)  20,848,772 (50.01)

Sample         Category          Expressed      DEGs (a)
                                 gene
                                             DEG (b)    DEGs
                                               set

Line 6.3 (R)   Total read         29,897      2,234     3,255
               Mapped read
               Properly paired

Line 7.2 (S)   Total read         29,833        --      2,468
               Mapped read
               Properly paired

Ne, necrotic enteritis; DEG, differentially expressed gene; R,
resistant; S, susceptible.

(a) DEGs are genes that showed significantly different expression
with p < 0.01 and fold change > 2.

(b) DEG set are genes that showed significantly different
expression in NE afflicted chicken line 6.3 and line 7.2 with
p < 0.01 and fold change > 2.

Table 3. Gene ontology (GO) and numbers were up and down
regulated in the spleen of both chicken lines with p < 0.05 and
Log2-transformation of this normalization [greater than or equal to] 2

                                      Spleen       GO term

                                    Up    Down

Line 6.3 (a)   Molecular function   244    69      88
               Biological process   153    96      135
               Cellular component   134    42      22

Line 7.2 (a)   Molecular function   67     41      40
               Biological process   68     59      70
               Cellular component   12     7       10

Line 6.3       Molecular function   149    90      76
vs 7.2 (b)     Biological process   83    151      141
               Cellular component   134    87      34

(a) Up and down regulated genes in necrotic enteritis
(NE)-afflicted chickens to control.

(b) Up and down regulated genes in the two chicken lines infected
with NE.

Table 4. Top 14 most abundant differentially expressed signalling
pathways

ID         Pathway                         Line 6.3        Line 7.2

                                         DEG   p-value   DEG   p-value

gga04010   MAPK signalling pathway       57    0.00028   48    3.9E-3
gga04144   Endocytosis pathway           43    8.6E-3    23    2.1E-3
gga04630   JAK-STAT signalling pathway   29    0.0014    19    0.0018
gga04510   Focal adhesion                24    0.0013    14    0.0034
gga04012   ErbB signalling pathway       22    2.9E-4    18    0.0027
gga04350   TGF-[beta] signalling
             pathway                     21    0.0014    13    2.0E-3
gga04622   RIG-I like receptor           19    3.7E-4    19    1.9E-4
             signalling pathway
gga04020   Calcium signalling pathway    19    4.6E-3    18    3.2E-06
gga04620   Toll like receptor pathway    15    0.0029     8    0.0012
gga04370   VEGF signalling pathway       15    0.0021    10    0.0003
gga04115   p53 signalling pathway        15    3.2E-4    10    0.0016
gga04672   Intestinal immune network     13    0.0012    11    0.0021
             for IgA production
gga04910   Insulin signalling pathway    11    3.4E-3     9    0.0034
gga04512   ECM-receptor interaction      11    0.0005     6    0.0056

DEG, differentially expressed gene; MAPK, mitogen activated
protein kinase; ErbB, erythroblastic leukemia viral oncogene
homolog; TGF, transforming growth factor; RIG-I, retinoic
acid-inducible gene 1; VEGF, vascular endothelial growth factor;
Ig, immunoglobulin; ECM, The extracellular matrix.


Supplementary Data

TABLE OF CONTENTS

Supplementary Tables

Supplementary Table S1
Supplementary Table S2
Supplementary Table S3
Supplementary Table S4
Supplementary Table S5

Supplementary Figure

Supplementary Figure S1
Supplementary Figure S2


Sequence data availability

All raw Illumina sequence data can be obtained freely by contacting the Department of Animal Science and Technology. The AMG_Gallus database has been uploaded in the National Agricultural Biotechnology Information Centrer (NABIC, http://nabic.rda.go.kr/) [ID: NN-0915-000001 and NN-0915-000002].

Supplementary Table S1a. Gene ontology enrichment test in the
spleen of resistant chicken line 6.3 with p < 0.05 and
Log2-transformations of the normalization [greater than or equal;
to] 2 (MF: molecular function, BP: biological process and CC:
cellular component). The data showed the number of genes which
were up-and downregulated in resistant chicken line 6.3.

ACC          Category   Definition

GO:0043169      MF      cation binding
GO:0043167      MF      ion binding
GO:0046872      MF      metal ion binding
GO:0008237      MF      metallopeptidase activity
GO:0004089      MF      carbonate dehydratase activity
GO:0004222      MF      metalloendopeptidase activity
GO:0004869      MF      cysteine-type endopeptidase inhibitor
                          activity
GO:0052582      MF      (+)-menthofuran synthase activity
GO:0043825      MF      succinylornithine transaminase activity
GO:0016705      MF      oxidoreductase activity, acting on
                          paired donors, with incorporation or
                          reduction of molecular oxygen
GO:0004026      MF      alcohol O-acetyltransferase activity
GO:0034318      MF      alcohol O-acyltransferase activity
GO:0034319      MF      alcohol O-butanoyltransferase activity
GO:0034323      MF      O-butanoyltransferase activity
GO:0034326      MF      butanoyltransferase activity
GO:0034338      MF      short-chain carboxylesterase activity
GO:0016836      MF      hydro-lyase activity
GO:0004611      MF      phosphoenolpyruvate carboxykinase
                          activity
GO:0004613      MF      phosphoenolpyruvate carboxykinase (GTP)
                          activity
GO:0005499      MF      vitamin D binding
GO:0016829      MF      lyase activity
GO:0004566      MF      beta-glucuronidase activity
GO:0045294      MF      alpha-catenin binding
GO:0046914      MF      transition metal ion binding
GO:0003824      MF      catalytic activity
GO:0070011      MF      peptidase activity, acting on L-amino
                          acid peptides
GO:0016835      MF      carbon-oxygen lyase activity
GO:0005509      MF      calcium ion binding
GO:0003674      MF      molecular function
GO:0008743      MF      L-threonine 3-dehydrogenase activity
GO:0008233      MF      peptidase activity
GO:0035529      MF      NADH pyrophosphatase activity
GO:0070573      MF      metallodipeptidase activity
GO:0004668      MF      protein-arginine deiminase activity
GO:0015204      MF      urea transmembrane transporter activity
GO:0042887      MF      amide transmembrane transporter activity
GO:0005506      MF      iron ion binding
GO:0046996      MF      oxidoreductase activity, acting on
                          paired donors
GO:0050046      MF      lathosterol oxidase activity
GO:0070704      MF      sterol desaturase activity
GO:0047756      MF      chondroitin 4-sulfotransferase activity
GO:0032217      MF      riboflavin transporter activity
GO:0004175      MF      endopeptidase activity
GO:0008239      MF      dipeptidyl-peptidase activity
GO:0004497      MF      monooxygenase activity
GO:0005215      MF      transporter activity
GO:0004528      MF      phosphodiesterase I activity
GO:0005184      MF      neuropeptide hormone activity
GO:0001968      MF      fibronectin binding
GO:0016813      MF      hydrolase activity, acting on
                          carbon-nitrogen

GO:0022857      MF      transmembrane transporter activity
GO:0004551      MF      nucleotide diphosphatase activity
GO:0016805      MF      dipeptidase activity
GO:0004866      MF      endopeptidase inhibitor activity
GO:0061135      MF      endopeptidase regulator activity
GO:0034481      MF      chondroitin sulfotransferase activity
GO:0009055      MF      electron carrier activity
GO:0015116      MF      sulfate transmembrane transporter
                          activity
GO:0051183      MF      vitamin transporter activity
GO:0047429      MF      nucleoside-triphosphate diphosphatase
                          activity
GO:0019842      MF      vitamin binding
GO:0043027      MF      cysteine-type endopeptidase inhibitor
                          activity involved in apoptotic process
GO:0017171      MF      serine hydrolase activity
GO:0020037      MF      heme binding
GO:0030414      MF      peptidase inhibitor activity
GO:0016787      MF      hydrolase activity
GO:0016491      MF      oxidoreductase activity
GO:0045296      MF      cadherin binding
GO:0046906      MF      tetrapyrrole binding
GO:0061134      MF      peptidase regulator activity
GO:0015291      MF      secondary active transmembrane
                          transporter activity
GO:0016413      MF      O-acetyltransferase activity
GO:0015301      MF      anion:anion antiporter activity
GO:0008009      MF      chemokine activity
GO:0043499      MF      eukaryotic cell surface binding
GO:0042379      MF      chemokine receptor binding
GO:0005488      MF      binding
GO:0005328      MF      neurotransmitter:sodium symporter
                          activity
GO:0022891      MF      substrate-specific transmembrane
                          transporter activity
GO:0043028      MF      cysteine-type endopeptidase regulator
                          activity involved in apoptotic process
GO:0008483      MF      transaminase activity
GO:0016709      MF      oxidoreductase activity, acting on
                          paired donors,
GO:0072341      MF      modified amino acid binding
GO:0005326      MF      neurotransmitter transporter activity
GO:0004091      MF      carboxylesterase activity
GO:0016769      MF      transferase activity, transferring
                          nitrogenous groups
GO:0004857      MF      enzyme inhibitor activity
GO:0016831      MF      carboxy-lyase activity
GO:0019752      BP      carboxylic acid metabolic process
GO:0043436      BP      oxoacid metabolic process
GO:0006082      BP      organic acid metabolic process
GO:0042180      BP      cellular ketone metabolic process
GO:0016114      BP      terpenoid biosynthetic process
GO:0016054      BP      organic acid catabolic process
GO:0046395      BP      carboxylic acid catabolic process
GO:0006520      BP      cellular amino acid metabolic process
GO:0006721      BP      terpenoid metabolic process
GO:0044282      BP      small molecule catabolic process
GO:0008299      BP      isoprenoid biosynthetic process
GO:0071705      BP      nitrogen compound transport
GO:0051792      BP      medium-chain fatty acid biosynthetic
                          process
GO:0051793      BP      medium-chain fatty acid catabolic
                          process
GO:0044106      BP      cellular amine metabolic process
GO:0021849      BP      neuroblast division in subventricular
                          zone
GO:0030860      BP      regulation of polarized epithelial
                          cell differentiation
GO:0030862      BP      positive regulation of polarized
                          epithelial cell differentiation
GO:0009063      BP      cellular amino acid catabolic process
GO:0006720      BP      isoprenoid metabolic process
GO:0019087      BP      transformation of host cell by virus
GO:0006105      BP      succinate metabolic process
GO:0019545      BP      arginine catabolic process to succinate
GO:0009308      BP      amine metabolic process
GO:0051791      BP      medium-chain fatty acid metabolic
                          process
GO:0021873      BP      forebrain neuroblast division
GO:0009310      BP      amine catabolic process
GO:0006567      BP      threonine catabolic process
GO:0019518      BP      L-threonine catabolic process to glycine
GO:0050667      BP      homocysteine metabolic process
GO:0000052      BP      citrulline metabolic process
GO:0018101      BP      peptidyl-citrulline biosynthetic process
                          from peptidyl-arginine
GO:0019240      BP      citrulline biosynthetic process
GO:0071918      BP      urea transmembrane transport
GO:0015816      BP      glycine transport
GO:0071460      BP      cellular response to cell-matrix adhesion
GO:0071404      BP      cellular response to low-density
                          lipoprotein particle stimulus
GO:0043627      BP      response to estrogen stimulus
GO:0032218      BP      riboflavin transport
GO:0009068      BP      aspartate family amino acid catabolic
                          process
GO:0016053      BP      organic acid biosynthetic process
GO:0046394      BP      carboxylic acid biosynthetic process
GO:0030859      BP      polarized epithelial cell
                          differentiation
GO:0032328      BP      alanine transport
GO:0008150      BP      biological process
GO:0071402      BP      cellular response to lipoprotein
                          particle stimulus
GO:0055098      BP      response to low-density lipoprotein
                          particle stimulus
GO:0015840      BP      urea transport
GO:0006527      BP      arginine catabolic process
GO:0006566      BP      threonine metabolic process
GO:0044281      BP      small molecule metabolic process
GO:0015824      BP      proline transport
GO:0006722      BP      triterpenoid metabolic process
GO:0016104      BP      triterpenoid biosynthetic process
GO:0019742      BP      pentacyclic triterpenoid metabolic
                          process
GO:0019745      BP      pentacyclic triterpenoid biosynthetic
                          process
GO:0021591      BP      ventricular system development
GO:0021670      BP      lateral ventricle development
GO:0055094      BP      response to lipoprotein stimulus
GO:0051547      BP      regulation of keratinocyte migration
GO:0051549      BP      positive regulation of keratinocyte
                          migration
GO:0044283      BP      small molecule biosynthetic process
GO:0021846      BP      cell proliferation in forebrain
GO:0006633      BP      fatty acid biosynthetic process
GO:0030574      BP      collagen catabolic process
GO:0015820      BP      leucine transport
GO:1900755      BP      branched-chain amino-acid anion
                          transport
GO:0051546      BP      keratinocyte migration
GO:0044003      BP      modification by symbiont of host
                          morphology or physiology
GO:0055057      BP      neuroblast division
GO:0015803      BP      branched-chain aliphatic amino acid
                          transport
GO:0035094      BP      response to nicotine
GO:0044243      BP      multicellular organismal catabolic
                          process
GO:0042886      BP      amide transport
GO:0031915      BP      positive regulation of synaptic
                          plasticity
GO:0055093      BP      response to hyperoxia
GO:0018195      BP      peptidyl-arginine modification
GO:0006508      BP      proteolysis
GO:0042493      BP      response to drug
GO:0009065      BP      glutamine family amino acid catabolic
                          process
GO:0030858      BP      positive regulation of epithelial cell
                          differentiation
GO:0060033      BP      anatomical structure regression
GO:0050775      BP      positive regulation of dendrite
                          morphogenesis
GO:0031668      BP      cellular response to extracellular
                          stimulus
GO:0021872      BP      forebrain generation of neurons
GO:0060479      BP      lung cell differentiation
GO:0060487      BP      lung epithelial cell differentiation
GO:0008272      BP      sulfate transport
GO:0008610      BP      lipid biosynthetic process
GO:0007566      BP      embryo implantation
GO:0006525      BP      arginine metabolic process
GO:0007405      BP      neuroblast proliferation
GO:0048103      BP      somatic stem cell division
GO:0072537      BP      fibroblast activation
GO:0071496      BP      cellular response to external stimulus
GO:0051180      BP      vitamin transport
GO:0016999      BP      antibiotic metabolic process
GO:0034332      BP      adherens junction organization
GO:0015804      BP      neutral amino acid transport
GO:0051817      BP      modification of morphology or
                          physiology of other organism involved
                          in symbiotic interaction
GO:0060428      BP      lung epithelium development
GO:0050819      BP      negative regulation of coagulation
GO:0035690      BP      cellular response to drug
GO:0017145      BP      stem cell division
GO:0016051      BP      carbohydrate biosynthetic process
GO:0048545      BP      response to steroid hormone stimulus
GO:0006094      BP      gluconeogenesis
GO:0055114      BP      oxidation-reduction process
GO:0019048      BP      virus-host interaction
GO:0017144      BP      drug metabolic process
GO:0045746      BP      negative regulation of Notch signaling
                          pathway
GO:0016126      BP      sterol biosynthetic process
GO:0006629      BP      lipid metabolic process
GO:0006631      BP      fatty acid metabolic process
GO:0044419      BP      interspecies interaction between
                          organisms
GO:0071347      BP      cellular response to interleukin-1
GO:0043648      BP      dicarboxylic acid metabolic process
GO:0032963      BP      collagen metabolic process
GO:0044259      BP      multicellular organismal macromolecule
                          metabolic process
GO:0035821      BP      modification of morphology or physiology
                          of other organism
GO:0043154      BP      negative regulation of cysteine-type
                          endopeptidase activity involved in
                          apoptotic process
GO:2000117      BP      negative regulation of cysteine-type
                          endopeptidase activity
GO:0042398      BP      cellular modified amino acid
                          biosynthetic process
GO:0006730      BP      one-carbon metabolic process
GO:0019319      BP      hexose biosynthetic process
GO:0030334      BP      regulation of cell migration
GO:0044236      BP      multicellular organismal metabolic
                          process
GO:0061351      BP      neural precursor cell proliferation
GO:2000145      BP      regulation of cell motility
GO:0007586      BP      digestion
GO:0048814      BP      regulation of dendrite morphogenesis
GO:0070555      BP      response to interleukin-1
GO:0034612      BP      response to tumor necrosis factor
GO:0019233      BP      sensory perception of pain
GO:0032355      BP      response to estradiol stimulus
GO:0016020      CC      membrane
GO:0005576      CC      extracellular region
GO:0005575      CC      cellular component
GO:0044425      CC      membrane part
GO:0031224      CC      intrinsic to membrane
GO:0016021      CC      integral to membrane
GO:0017090      CC      meprin A complex
GO:0005615      CC      extracellular space
GO:0072562      CC      blood microparticle
GO:0072563      CC      endothelial microparticle
GO:0005886      CC      plasma membrane
GO:0071944      CC      cell periphery
GO:0044421      CC      extracellular region part
GO:0005624      CC      membrane fraction
GO:0005626      CC      insoluble fraction
GO:0019031      CC      viral envelope
GO:0000267      CC      cell fraction
GO:0055036      CC      virion membrane
GO:0045177      CC      apical part of cell
GO:0030424      CC      axon
GO:0005811      CC      lipid particle
GO:0043034      CC      costamere

ACC          Regulated
             gene

             Up   Down     p-value

GO:0043169   13    4      6.14E-05
GO:0043167   13    4      6.27E-05
GO:0046872   12    4     0.000196789
GO:0008237   3     1     0.000268084
GO:0004089   2     0     0.000299687
GO:0004222   2     1     0.00082792
GO:0004869   2     0     0.000947924
GO:0052582   1     0     0.00174286
GO:0043825   1     0     0.00194545
GO:0016705   3     0     0.00196149
GO:0004026   0     1      0.0019911
GO:0034318   0     1      0.0019911
GO:0034319   0     1      0.0019911
GO:0034323   0     1      0.0019911
GO:0034326   0     1      0.0019911
GO:0034338   0     1      0.0019911
GO:0016836   2     0     0.00296521
GO:0004611   1     0     0.00343723
GO:0004613   1     0     0.00343723
GO:0005499   1     0      0.0034967
GO:0016829   1     1     0.00366269
GO:0004566   1     0     0.00397161
GO:0045294   1     0     0.00420115
GO:0046914   8     1     0.00445236
GO:0003824   18    10    0.00447815
GO:0070011   3     2     0.00454432
GO:0016835   2     0     0.00464331
GO:0005509   3     2     0.00475453
GO:0003674   28    7     0.00478706
GO:0008743   1     0     0.00482599
GO:0008233   3     2      0.0050348
GO:0035529   1     0     0.00510374
GO:0070573   1     0      0.0051373
GO:0004668   0     1     0.00537643
GO:0015204   1     0     0.00548746
GO:0042887   1     0     0.00548746
GO:0005506   3     0     0.00550626
GO:0046996   1     0      0.0058867
GO:0050046   1     0      0.0058867
GO:0070704   1     0      0.0058867
GO:0047756   1     0     0.00624023
GO:0032217   1     0     0.00640645
GO:0004175   2     2     0.00718106
GO:0008239   1     0     0.00731233
GO:0004497   2     0     0.00792078
GO:0005215   5     1     0.00913711
GO:0004528   1     0     0.00952365
GO:0005184   1     0      0.0108094
GO:0001968   0     1      0.0119692
GO:0016813   0     1      0.0141259
GO:0022857   5     0      0.014237
GO:0004551   1     0      0.0149732
GO:0016805   1     0      0.0152864
GO:0004866   2     0      0.0175173
GO:0061135   2     0      0.0177896
GO:0034481   1     0      0.018335
GO:0009055   2     0      0.0185937
GO:0015116   1     0      0.0187158
GO:0051183   1     0      0.0190009
GO:0047429   1     0      0.0194876
GO:0019842   2     0      0.0201883
GO:0043027   1     0      0.0210609
GO:0017171   0     2      0.021913
GO:0020037   2     0      0.0225592
GO:0030414   2     0      0.0231552
GO:0016787   5     4      0.0245724
GO:0016491   4     0      0.0252204
GO:0045296   1     0      0.0255924
GO:0046906   2     0      0.0259168
GO:0061134   2     0      0.029062
GO:0015291   2     0      0.0301934
GO:0016413   0     1      0.0309025
GO:0015301   1     0      0.0311367
GO:0008009   0     1      0.0324486
GO:0043499   0     1      0.0332507
GO:0042379   0     1      0.0339999
GO:0005488   20    5      0.0367398
GO:0005328   0     1      0.0373173
GO:0022891   5     0      0.0394981
GO:0043028   1     0      0.0409408
GO:0008483   1     0      0.0418697
GO:0016709   1     0      0.0433748
GO:0072341   1     0      0.0439676
GO:0005326   0     1      0.0451888
GO:0004091   0     1      0.0454664
GO:0016769   1     0      0.0469751
GO:0004857   2     0      0.0470614
GO:0016831   1     0      0.0491927
GO:0019752   4     2     0.000212763
GO:0043436   4     2     0.000212763
GO:0006082   4     2     0.000217525
GO:0042180   4     2     0.000253846
GO:0016114   2     0     0.000543184
GO:0016054   2     1     0.000978565
GO:0046395   2     1     0.000978565
GO:0006520   3     1     0.00120223
GO:0006721   2     0     0.00128429
GO:0044282   2     1      0.0015781
GO:0008299   2     0     0.00185047
GO:0071705   2     1     0.00190925
GO:0051792   0     1      0.0019911
GO:0051793   0     1      0.0019911
GO:0044106   2     2     0.00220763
GO:0021849   1     0     0.00221879
GO:0030860   1     0     0.00221879
GO:0030862   1     0     0.00221879
GO:0009063   2     0     0.00279384
GO:0006720   2     0     0.00354996
GO:0019087   0     1     0.00358255
GO:0006105   1     0      0.0036492
GO:0019545   1     0      0.0036492
GO:0009308   2     3     0.00385351
GO:0051791   0     1     0.00395518
GO:0021873   1     0     0.00417217
GO:0009310   2     0     0.00424347
GO:0006567   1     0     0.00482599
GO:0019518   1     0     0.00482599
GO:0050667   1     0     0.00527964
GO:0000052   0     1     0.00537643
GO:0018101   0     1     0.00537643
GO:0019240   0     1     0.00537643
GO:0071918   1     0     0.00548746
GO:0015816   0     1     0.00553173
GO:0071460   0     1     0.00555438
GO:0071404   0     1     0.00577902
GO:0043627   0     2     0.00613608
GO:0032218   0     1     0.00640645
GO:0009068   0     1     0.00650831
GO:0016053   1     2     0.00653598
GO:0046394   1     2     0.00653598
GO:0030859   1     0     0.00681453
GO:0032328   0     1      0.0070819
GO:0008150   26    8     0.00731891
GO:0071402   0     1     0.00777455
GO:0055098   0     1     0.00821599
GO:0015840   1     0     0.00867569
GO:0006527   1     0     0.00872043
GO:0006566   1     0     0.00882713
GO:0044281   6     2     0.00909546
GO:0015824   0     1     0.00951104
GO:0006722   1     0     0.00954903
GO:0016104   1     0     0.00954903
GO:0019742   1     0     0.00954903
GO:0019745   1     0     0.00954903
GO:0021591   1     0      0.009551
GO:0021670   1     0      0.009551
GO:0055094   0     1      0.0102067
GO:0051547   0     1      0.0103724
GO:0051549   0     1      0.0103724
GO:0044283   1     2      0.0111576
GO:0021846   1     0      0.0111652
GO:0006633   1     1      0.0115736
GO:0030574   0     1      0.0117449
GO:0015820   0     1      0.0117529
GO:1900755   0     1      0.0117529
GO:0051546   0     1      0.0119144
GO:0044003   0     1      0.013036
GO:0055057   1     0      0.013864
GO:0015803   0     1      0.0142855
GO:0035094   0     1      0.0143034
GO:0044243   0     1      0.0146319
GO:0042886   1     0      0.015095
GO:0031915   0     1      0.0165538
GO:0055093   0     1      0.0168077
GO:0018195   0     1      0.0169921
GO:0006508   3     2      0.0178713
GO:0042493   1     1      0.0179224
GO:0009065   1     0      0.0185135
GO:0030858   1     0      0.0194834
GO:0060033   0     1      0.0198409
GO:0050775   1     0      0.0198532
GO:0031668   1     1      0.0201539
GO:0021872   1     0      0.0217616
GO:0060479   1     0      0.0218822
GO:0060487   1     0      0.0218822
GO:0008272   1     0      0.0218896
GO:0008610   2     1      0.022386
GO:0007566   0     1      0.0232497
GO:0006525   1     1      0.023798
GO:0007405   1     0      0.026249
GO:0048103   1     0      0.0262981
GO:0072537   1     0      0.0271172
GO:0071496   1     1      0.027315
GO:0051180   1     0      0.0278874
GO:0016999   1     0      0.0281423
GO:0034332   1     0      0.0283483
GO:0015804   0     1      0.0284833
GO:0051817   0     1      0.0287122
GO:0060428   1     0      0.0297913
GO:0050819   0     1      0.0301457
GO:0035690   1     0      0.0304062
GO:0017145   1     0      0.031876
GO:0016051   2     0      0.0325671
GO:0048545   0     2      0.0326385
GO:0006094   1     0      0.0327722
GO:0055114   4     0      0.0349281
GO:0019048   0     1      0.0359779
GO:0017144   1     0      0.0366382
GO:0045746   1     0      0.037303
GO:0016126   1     0      0.038073
GO:0006629   3     1      0.038336
GO:0006631   1     1      0.0387578
GO:0044419   1     1      0.0398852
GO:0071347   0     1      0.0403745
GO:0043648   1     0      0.0405356
GO:0032963   0     1      0.0411421
GO:0044259   0     1      0.0411421
GO:0035821   0     1      0.0413052
GO:0043154   1     0      0.0420778
GO:2000117   1     0      0.0420778
GO:0042398   0     1      0.0423695
GO:0006730   1     0      0.0425298
GO:0019319   1     0      0.0434838
GO:0030334   1     1      0.0435087
GO:0044236   0     1      0.0439448
GO:0061351   1     0      0.0445523
GO:2000145   1     1      0.0461137
GO:0007586   1     0      0.0463652
GO:0048814   1     0      0.0471453
GO:0070555   0     1      0.048985
GO:0034612   0     1      0.0490052
GO:0019233   1     0      0.049195
GO:0032355   0     1      0.0499926
GO:0016020   15    9      3.55E-05
GO:0005576   6     4     0.000157129
GO:0005575   30    8     0.000246254
GO:0044425   17    2     0.000315492
GO:0031224   16    1      0.0004088
GO:0016021   15    1     0.000985323
GO:0017090   1     0     0.00188578
GO:0005615   1     3     0.00527238
GO:0072562   0     1     0.00579968
GO:0072563   0     1     0.00579968
GO:0005886   8     3     0.00621419
GO:0071944   8     3      0.0100907
GO:0044421   2     3      0.0113203
GO:0005624   3     0      0.0209092
GO:0005626   3     0      0.0237271
GO:0019031   1     0      0.0286916
GO:0000267   3     0      0.035624
GO:0055036   1     0      0.0422082
GO:0045177   2     0      0.0424893
GO:0030424   2     0      0.0442162
GO:0005811   0     2      0.0461664
GO:0043034   0     1      0.0479751

Supplementary Table S1b. Gene ontology enrichment test in the
spleen of susceptible chicken line 7.2 with p < 0.05 and
Log2-transformations of the normalization [greater than or equal to] 2
(MF: molecular function, BP: biological process and CC: cellular
component). The data showed the number of genes which were up- and
downregulated in susceptible chicken line 7.2.

ACC            Category   Definition

GO:0008933        MF      lytic transglycosylase activity
GO:0022892        MF      substrate-specific transporter activity
GO:0004055        MF      argininosuccinate synthase activity
GO:0043825        MF      succinylornithine transaminase activity
GO:0004026        MF      alcohol O-acetyltransferase activity
GO:0034318        MF      alcohol O-acyltransferase activity
GO:0034319        MF      alcohol O-butanoyltransferase activity
GO:0034323        MF      O-butanoyltransferase activity
GO:0034326        MF      butanoyltransferase activity
GO:0034338        MF      short-chain carboxylesterase activity
GO:0005215        MF      transporter activity
GO:0005499        MF      vitamin D binding
GO:0004611        MF      phosphoenolpyruvate carboxykinase
                            activity
GO:0004613        MF      phosphoenolpyruvate carboxykinase (GTP)
                            activity
GO:0008743        MF      L-threonine 3-dehydrogenase activity
GO:0008508        MF      bile acid:sodium symporter activity
GO:0008139        MF      nuclear localization sequence binding
GO:0015204        MF      urea transmembrane transporter activity
GO:0042887        MF      amide transmembrane transporter activity
GO:0005184        MF      neuropeptide hormone activity
GO:0015125        MF      bile acid transmembrane transporter
                          activity NAD(P)+protein-arginine ADP-
GO:0003956        MF      ribosyltransferase activity
GO:0015103        MF      inorganic anion transmembrane
                            transporter activity
GO:0005344        MF      oxygen transporter activity
GO:0022891        MF      substrate-specific transmembrane
                            transporter activity
GO:0016740        MF      transferase activity
GO:0003918        MF      DNA topoisomerase (ATP-hydrolyzing)
                            activity
GO:0008553        MF      hydrogen-exporting ATPase activity,
                            phosphorylative mechanism
GO:0019825        MF      oxygen binding
GO:0022857        MF      transmembrane transporter activity
GO:0008509        MF      anion transmembrane transporter activity
GO:0003916        MF      DNA topoisomerase activity
GO:0003810        MF      protein-glutamine gamma-
                          glutamyltransferase activity
GO:0022804        MF      active transmembrane transporter
                            activity
GO:0019842        MF      vitamin binding
GO:0015116        MF      sulfate transmembrane transporter
                            activity
GO:0005048        MF      signal sequence binding
GO:0004364        MF      glutathione transferase activity
GO:0008009        MF      chemokine activity
GO:0042379        MF      chemokine receptor binding
GO:0016853        MF      isomerase activity
GO:0016755        MF      transferase activity, transferring
                          amino-acyl groups
GO:0016413        MF      O-acetyltransferase activity
GO:0008028        MF      monocarboxylic acid transmembrane
                            transporter activity
GO:0015075        MF      ion transmembrane transporter activity
GO:0015301        MF      anion:anion antiporter activity
GO:0003824        MF      catalytic activity
GO:0015291        MF      secondary active transmembrane
                            transporter activity
GO:0005343        MF      organic acid:sodium symporter activity
GO:0016829        MF      lyase activity
GO:0004181        MF      metallocarboxypeptidase activity
GO:0008483        MF      transaminase activity
GO:0004091        MF      carboxylesterase activity
GO:0016831        MF      carboxy-lyase activity
GO:0006525        BP      arginine metabolic process
GO:0009308        BP      amine metabolic process
GO:0016054        BP      organic acid catabolic process
GO:0046395        BP      carboxylic acid catabolic process
GO:0044282        BP      small molecule catabolic process
GO:0044106        BP      cellular amine metabolic process
GO:0009063        BP      cellular amino acid catabolic process
GO:0009064        BP      glutamine family amino acid metabolic
                            process
GO:0051792        BP      medium-chain fatty acid biosynthetic
                          process
GO:0051793        BP      medium-chain fatty acid catabolic
                            process
GO:0006811        BP      ion transport
GO:0006567        BP      threonine catabolic process
GO:0019518        BP      L-threonine catabolic process to glycine
GO:0006105        BP      succinate metabolic process
GO:0019545        BP      arginine catabolic process to succinate
GO:0009310        BP      amine catabolic process
GO:0016360        BP      sensory organ precursor cell fate
                            determination
GO:0009068        BP      aspartate family amino acid catabolic
                            process
GO:0051791        BP      medium-chain fatty acid metabolic
                            process
GO:0048241        BP      epinephrine transport
GO:0048242        BP      epinephrine secretion
GO:0016338        BP      calcium-independent cell-cell adhesion
GO:0071918        BP      urea transmembrane transport
GO:0000270        BP      peptidoglycan metabolic process
GO:0006566        BP      threonine metabolic process
GO:0006527        BP      arginine catabolic process
GO:0015840        BP      urea transport
GO:0006607        BP      NLS-bearing substrate import into
                            nucleus
GO:0008052        BP      sensory organ boundary specification
GO:0006526        BP      arginine biosynthetic process
GO:0019752        BP      carboxylic acid metabolic process
GO:0043436        BP      oxoacid metabolic process
GO:0006082        BP      organic acid metabolic process
GO:0006520        BP      cellular amino acid metabolic process
GO:0042180        BP      cellular ketone metabolic process
GO:0015698        BP      inorganic anion transport
GO:0042886        BP      amide transport
GO:0042415        BP      norepinephrine metabolic process
GO:0015671        BP      oxygen transport
GO:0015669        BP      gas transport
GO:0009065        BP      glutamine family amino acid catabolic
                            process
GO:0030325        BP      adrenal gland development
GO:0010160        BP      formation of organ boundary
GO:0055010        BP      ventricular cardiac muscle tissue
                          morphogenesis
GO:0048859        BP      formation of anatomical boundary
GO:0003229        BP      ventricular cardiac muscle tissue
                            development
GO:0006471        BP      protein ADP-ribosylation
GO:0008272        BP      sulfate transport
GO:0006584        BP      catecholamine metabolic process
GO:0006094        BP      gluconeogenesis
GO:0006265        BP      DNA topological change
GO:0009712        BP      catechol-containing compound metabolic
                            process
GO:0006820        BP      anion transport
GO:0071705        BP      nitrogen compound transport
GO:0050432        BP      catecholamine secretion
GO:0072537        BP      fibroblast activation
GO:0034311        BP      diol metabolic process
GO:0003208        BP      cardiac ventricle morphogenesis
GO:0019319        BP      hexose biosynthetic process
GO:0009084        BP      glutamine family amino acid biosynthetic
                            process
GO:0018149        BP      peptide cross-linking
GO:0055008        BP      cardiac muscle tissue morphogenesis
GO:0051937        BP      catecholamine transport
GO:0006810        BP      transport
GO:0043648        BP      dicarboxylic acid metabolic process
GO:0035270        BP      endocrine system development
GO:0018958        BP      phenol-containing compound metabolic
                            process
GO:0051234        BP      establishment of localization
GO:0006313        BP      transposition, DNA-mediated
GO:0060415        BP      muscle tissue morphogenesis
GO:0005811        CC      lipid particle
GO:0031225        CC      anchored to membrane
GO:0070701        CC      mucus layer
GO:0070702        CC      inner mucus layer
GO:0070703        CC      outer mucus layer
GO:0005833        CC      hemoglobin complex
GO:0016471        CC      vacuolar proton-transporting V-type
                            ATPase complex
GO:0005865        CC      striated muscle thin filament
GO:0016020        CC      membrane
GO:0033176        CC      proton-transporting V-type ATPase
                            complex

ACC            Regulated   p-value
               gene

               Up   Down

GO:0008933     0     1     0.0010512
GO:0022892     6     1     0.00105122
GO:0004055     0     1     0.00213383
GO:0043825     1     0     0.00273775
GO:0004026     0     1     0.00283308
GO:0034318     0     1     0.00283308
GO:0034319     0     1     0.00283308
GO:0034323     0     1     0.00283308
GO:0034326     0     1     0.00283308
GO:0034338     0     1     0.00283308
GO:0005215     6     1     0.0032377
GO:0005499     1     0     0.00362091
GO:0004611     1     0     0.00370373
GO:0004613     1     0     0.00370373
GO:0008743     1     0     0.00371402
GO:0008508     1     0     0.00607798
GO:0008139     0     1     0.00648464
GO:0015204     1     0     0.00665044
GO:0042887     1     0     0.00665044
GO:0005184     1     0     0.00862614
GO:0015125     1     0     0.0102939
GO:0003956     0     1     0.0111821
GO:0015103     2     0     0.011549
GO:0005344     1     0     0.0124526
GO:0022891     5     0     0.012562
GO:0016740     2     7     0.01265
GO:0003918     0     1     0.0134776
GO:0008553     1     0     0.0148506
GO:0019825     1     0     0.0180823
GO:0022857     5     0     0.019283
GO:0008509     2     0     0.0201608
GO:0003916     0     1     0.0219479
GO:0003810     0     1     0.0221803
GO:0022804     3     0     0.0225543
GO:0019842     2     0     0.0236602
GO:0015116     1     0     0.0238317
GO:0005048     0     1     0.0242385
GO:0004364     0     1     0.0244004
GO:0008009     0     1     0.0266425
GO:0042379     0     1     0.0278065
GO:0016853     1     1     0.0293523
GO:0016755     0     1     0.0297397
GO:0016413     0     1     0.0297724
GO:0008028     1     0     0.030432
GO:0015075     4     0     0.0304919
GO:0015301     1     0     0.0381749
GO:0003824     6     10    0.0387819
GO:0015291     2     0     0.0397036
GO:0005343     1     0     0.0401934
GO:0016829     1     1     0.0415912
GO:0004181     1     0     0.0418653
GO:0008483     1     0     0.0451902
GO:0004091     0     1     0.0467262
GO:0016831     1     0     0.0488677
GO:0006525     1     1     0.00025555
GO:0009308     2     3     0.00056722
GO:0016054     2     1     0.0010466
GO:0046395     2     1     0.0010466
GO:0044282     2     1     0.00169233
GO:0044106     2     2     0.00268388
GO:0009063     2     0     0.00270262
GO:0009064     1     1     0.00271965
GO:0051792     0     1     0.00283308
GO:0051793     0     1     0.00283308
GO:0006811     5     1     0.00348546
GO:0006567     1     0     0.00371402
GO:0019518     1     0     0.00371402
GO:0006105     1     0     0.00431246
GO:0019545     1     0     0.00431246
GO:0009310     2     0     0.00439359
GO:0016360     0     1     0.00504016
GO:0009068     1     0     0.00521027
GO:0051791     0     1     0.00561711
GO:0048241     0     1     0.006018
GO:0048242     0     1     0.006018
GO:0016338     0     1     0.00626024
GO:0071918     1     0     0.00665044
GO:0000270     0     1     0.00773822
GO:0006566     1     0     0.00831129
GO:0006527     1     0     0.00895028
GO:0015840     1     0     0.00906918
GO:0006607     0     1     0.0106659
GO:0008052     0     1     0.0107772
GO:0006526     0     1     0.0124525
GO:0019752     2     2     0.0124589
GO:0043436     2     2     0.0124589
GO:0006082     2     2     0.0126423
GO:0006520     2     1     0.013138
GO:0042180     2     2     0.013814
GO:0015698     2     0     0.0140833
GO:0042886     1     0     0.0146989
GO:0042415     0     1     0.0152179
GO:0015671     1     0     0.0152386
GO:0015669     1     0     0.0168414
GO:0009065     1     0     0.0181944
GO:0030325     0     1     0.0182192
GO:0010160     0     1     0.0206901
GO:0055010     0     1     0.020926
GO:0048859     0     1     0.02221
GO:0003229     0     1     0.02375
GO:0006471     0     1     0.0253868
GO:0008272     1     0     0.0267008
GO:0006584     0     1     0.0274192
GO:0006094     1     0     0.0274712
GO:0006265     0     1     0.0284711
GO:0009712     0     1     0.0287056
GO:0006820     2     0     0.0288136
GO:0071705     1     1     0.0297978
GO:0050432     0     1     0.0303896
GO:0072537     1     0     0.0305875
GO:0034311     0     1     0.0315112
GO:0003208     0     1     0.0348399
GO:0019319     1     0     0.0363584
GO:0009084     0     1     0.0364427
GO:0018149     0     1     0.0376767
GO:0055008     0     1     0.0381418
GO:0051937     0     1     0.0383056
GO:0006810     7     3     0.0403819
GO:0043648     1     0     0.0408411
GO:0035270     0     1     0.0424684
GO:0018958     0     1     0.0442745
GO:0051234     7     3     0.0451024
GO:0006313     0     1     0.0489602
GO:0060415     0     1     0.0498833
GO:0005811     0     2     0.00111071
GO:0031225     0     2     0.0064666
GO:0070701     0     1     0.00906814
GO:0070702     0     1     0.00906814
GO:0070703     0     1     0.00906814
GO:0005833     1     0     0.0103916
GO:0016471     1     0     0.0159078
GO:0005865     0     1     0.0190633
GO:0016020     9     9     0.0205417
GO:0033176     1     0      0.025971

Supplementary Table S1c. Gene ontology enrichment test of
resistant line 6.3 versus susceptible line 7.2 in the spleen with
ACC p < 0.05 and Log2-transformations of the normalization of
resistant-susceptible [greater than or equal to] 2 (MF: molecular
function, BP: biological process and CC: cellular component). The
data showed the number of genes which were up-and downregulated
in resistant chicken line 6.3 compared to susceptible chicken
line 7.2.

ACC          Category   Definition

GO:0004175      MF      endopeptidase activity
GO:0047429      MF      nucleoside-triphosphate diphosphatase
                          activity
GO:0070011      MF      peptidase activity, acting on L-amino
                          acid peptides
GO:0004533      MF      exoribonuclease H activity
GO:0008233      MF      peptidase activity
GO:0004527      MF      exonuclease activity
GO:0051786      MF      all-trans-retinol 13,14-reductase
                          activity
GO:0008933      MF      lytic transglycosylase activity
GO:0016829      MF      lyase activity
GO:0015563      MF      uptake transmembrane transporter
                          activity
GO:0080138      MF      borate uptake transmembrane transporter
                          activity
GO:0052582      MF      (+)-menthofuran synthase activity
GO:0004532      MF      exoribonuclease activity
GO:0016896      MF      exoribonuclease activity, producing
                          5'-phosphomonoesters
GO:0016796      MF      exonuclease activity, active with either
                          ribo- or deoxyribonucleic acids and
                          producing 5'-phosphomonoesters
GO:0004518      MF      nuclease activity
GO:0004222      MF      metalloendopeptidase activity
GO:0031694      MF      alpha-2A adrenergic receptor binding
GO:0003964      MF      RNA-directed DNA polymerase activity
GO:0004611      MF      phosphoenolpyruvate carboxykinase
                          activity
GO:0004613      MF      phosphoenolpyruvate carboxykinase (GTP)
                          activity
GO:0015105      MF      arsenite transmembrane transporter
                          activity
GO:0004566      MF      beta-glucuronidase activity
GO:0004523      MF      ribonuclease H activity
GO:0047547      MF      2-methylcitrate dehydratase activity
GO:0034061      MF      DNA polymerase activity
GO:0001733      MF      galactosylceramide sulfotransferase
                          activity
GO:0050694      MF      galactose 3-O-sulfotransferase activity
GO:0015370      MF      solute:sodium symporter activity
GO:0016836      MF      hydro-lyase activity
GO:0004190      MF      aspartic-type endopeptidase activity
G0:0070001      MF      aspartic-type peptidase activity
G0:0035529      MF      NADH pyrophosphatase activity
G0:0046715      MF      borate transmembrane transporter activity
G0:0016891      MF      endoribonuclease activity, producing
                          5'-phosphomonoesters
G0:0015168      MF      glycerol transmembrane transporter
                          activity
G0:0004519      MF      endonuclease activity
G0:0015204      MF      urea transmembrane transporter activity
G0:0042887      MF      amide transmembrane transporter activity
G0:0004668      MF      protein-arginine deiminase activity
G0:0008508      MF      bile acid:sodium symporter activity
G0:0016893      MF      endonuclease activity, active with
                          either ribo- or deoxyribonucleic acids
                          and producing 5'-phosphomonoesters
G0:0005355      MF      glucose transmembrane transporter
                          activity
G0:0015145      MF      monosaccharide transmembrane transporter
                          activity
G0:0015149      MF      hexose transmembrane transporter activity
G0:0004521      MF      endoribonuclease activity
G0:0003858      MF      3-hydroxybutyrate dehydrogenase activity
G0:0016835      MF      carbon-oxygen lyase activity
G0:0016491      MF      oxidoreductase activity
G0:0004170      MF      dUTP diphosphatase activity
G0:0003824      MF      catalytic activity
G0:0004540      MF      ribonuclease activity
G0:0004528      MF      phosphodiesterase I activity
G0:0015125      MF      bile acid transmembrane transporter
                          activity
G0:0015166      MF      polyol transmembrane transporter
                          activity
G0:0008237      MF      metallopeptidase activity
G0:0015294      MF      solute:cation symporter activity
G0:0008242      MF      omega peptidase activity
G0:0001968      MF      fibronectin binding
G0:0043169      MF      cation binding
G0:0043167      MF      ion binding
G0:0015665      MF      alcohol transmembrane transporter
                        activity
G0:0016779      MF      nucleotidyltransferase activity
G0:0004497      MF      monooxygenase activity
G0:0016813      MF      hydrolase activity, acting on carbon-
                          nitrogen
G0:0004551      MF      nucleotide diphosphatase activity
G0:0031690      MF      adrenergic receptor binding
G0:0046872      MF      metal ion binding
G0:0015081      MF      sodium ion transmembrane transporter
                          activity
G0:0016787      MF      hydrolase activity
G0:0051119      MF      sugar transmembrane transporter activity
G0:0004089      MF      carbonate dehydratase activity
G0:0015293      MF      symporter activity
G0:0004866      MF      endopeptidase inhibitor activity
G0:0061135      MF      endopeptidase regulator activity
G0:0008028      MF      monocarboxylic acid transmembrane
                        transporter activity
G0:0044419      BP      interspecies interaction between
                          organisms
G0:0030574      BP      collagen catabolic process
G0:0044243      BP      multicellular organismal catabolic
                          process
G0:0006720      BP      isoprenoid metabolic process
G0:0007566      BP      embryo implantation
G0:0016032      BP      viral reproduction
G0:0043627      BP      response to estrogen stimulus
G0:0051704      BP      multi-organism process
G0:0032963      BP      collagen metabolic process
G0:0044259      BP      multicellular organismal macromolecule
                          metabolic process
G0:0044236      BP      multicellular organismal metabolic
                          process
GO:0046080      BP      dUTP metabolic process
GO:0007584      BP      response to nutrient
GO:0009211      BP      pyrimidine deoxyribonucleoside
                          triphosphate metabolic process
GO:0009219      BP      pyrimidine deoxyribonucleotide metabolic
                          process
G0:0009200      BP      deoxyribonucleoside triphosphate
                          metabolic process
GO:0032355      BP      response to estradiol stimulus
GO:0019629      BP      propionate catabolic process, 2-
                          methylcitrate cycle
GO:0009394      BP      2'-deoxyribonucleotide metabolic process
GO:0006721      BP      terpenoid metabolic process
GO:0015850      BP      organic alcohol transport
GO:0045471      BP      response to ethanol
GO:0097305      BP      response to alcohol
GO:0051701      BP      interaction with host
GO:0007565      BP      female pregnancy
GO:0009262      BP      deoxyribonucleotide metabolic process
GO:0008299      BP      isoprenoid biosynthetic process
GO:0006508      BP      proteolysis
GO:0031667      BP      response to nutrient levels
GO:0080029      BP      cellular response to boron-containing
                          substance levels
GO:0001955      BP      blood vessel maturation
GO:0006278      BP      RNA-dependent DNA replication
GO:0009147      BP      pyrimidine nucleoside triphosphate
                          metabolic process
GO:0019896      BP      axon transport of mitochondrion
GO:0015700      BP      arsenite transport
GO:0032526      BP      response to retinoic acid
GO:0035445      BP      borate transmembrane transport
GO:0019087      BP      transformation of host cell by virus
GO:0009991      BP      response to extracellular stimulus
GO:0019541      BP      propionate metabolic process
GO:0019543      BP      propionate catabolic process
GO:0033189      BP      response to vitamin A
GO:0009719      BP      response to endogenous stimulus
GO:0010036      BP      response to boron-containing substance
GO:0046713      BP      borate transport
GO:0071705      BP      nitrogen compound transport
GO:0048241      BP      epinephrine transport
GO:0048242      BP      epinephrine secretion
GO:0044403      BP      symbiosis, encompassing mutualism
                          through parasitism
GO:0015816      BP      glycine transport
GO:0051385      BP      response to mineralocorticoid stimulus
GO:0051412      BP      response to corticosterone stimulus
GO:0015793      BP      glycerol transport
GO:0071918      BP      urea transmembrane transport
GO:0000052      BP      citrulline metabolic process
GO:0018101      BP      peptidyl-citrulline biosynthetic process
                          from peptidyl-arginine
GO:0019240      BP      citrulline biosynthetic process
GO:0071460      BP      cellular response to cell-matrix adhesion
GO:0071404      BP      cellular response to low-density
                          lipoprotein particle stimulus
GO:0015074      BP      DNA integration
GO:0006220      BP      pyrimidine nucleotide metabolic process
GO:0042572      BP      retinol metabolic process
GO:0032328      BP      alanine transport
GO:0048545      BP      response to steroid hormone stimulus
GO:0033273      BP      response to vitamin
GO:0000270      BP      peptidoglycan metabolic process
GO:0071402      BP      cellular response to lipoprotein
                          particle stimulus
GO:0055098      BP      response to low-density lipoprotein
                          particle stimulus
GO:0072527      BP      pyrimidine-containing compound metabolic
                          process
GO:0015840      BP      urea transport
GO:0015824      BP      proline transport
GO:0019047      BP      provirus integration
GO:0019061      BP      uncoating of virus
GO:0030069      BP      lysogeny
GO:0060346      BP      bone trabecula formation
GO:0061430      BP      bone trabecula morphogenesis
GO:0009725      BP      response to hormone stimulus
GO:0090305      BP      nucleic acid phosphodiester bond
                          hydrolysis
GO:0055094      BP      response to lipoprotein stimulus
GO:0071702      BP      organic substance transport
GO:0051547      BP      regulation of keratinocyte migration
GO:0051549      BP      positive regulation of keratinocyte
                          migration
GO:0051546      BP      keratinocyte migration
GO:0022415      BP      viral reproductive process
GO:0055114      BP      oxidation-reduction process
GO:0008643      BP      carbohydrate transport
GO:0015820      BP      leucine transport
GO:1900755      BP      branched-chain amino-acid anion transport
GO:0001957      BP      intramembranous ossification
GO:0036072      BP      direct ossification
GO:0032868      BP      response to insulin stimulus
GO:0042415      BP      norepinephrine metabolic process
GO:0006776      BP      vitamin A metabolic process
GO:0044003      BP      modification by symbiont of host
                          morphology or physiology
GO:0007412      BP      axon target recognition
GO:0060612      BP      adipose tissue development
GO:0001666      BP      response to hypoxia
GO:0015803      BP      branched-chain aliphatic amino acid
                          transport
GO:0019059      BP      initiation of viral infection
GO:0030260      BP      entry into host cell
GO:0044409      BP      entry into host
GO:0051806      BP      entry into cell of other organism
                          involved in symbiotic interaction
GO:0051828      BP      entry into other organism involved in
                          symbiotic interaction
GO:0042886      BP      amide transport
GO:0055093      BP      response to hyperoxia
GO:0046685      BP      response to arsenic-containing substance
GO:0035094      BP      response to nicotine
GO:0015791      BP      polyol transport
GO:0031915      BP      positive regulation of synaptic
                          plasticity
GO:0001523      BP      retinoid metabolic process
GO:0070482      BP      response to oxygen levels
GO:0019069      BP      viral capsid assembly
GO:0046797      BP      viral procapsid maturation
GO:0060325      BP      face morphogenesis
GO:0060416      BP      response to growth hormone stimulus
GO:0016101      BP      diterpenoid metabolic process
GO:0030325      BP      adrenal gland development
GO:0018195      BP      peptidyl-arginine modification
GO:0060343      BP      trabecula formation
GO:0060033      BP      anatomical structure regression
GO:0060323      BP      head morphogenesis
GO:0046627      BP      negative regulation of insulin receptor
                          signaling pathway
GO:1900077      BP      negative regulation of cellular response
                          to insulin stimulus
GO:0055010      BP      ventricular cardiac muscle tissue
                          morphogenesis
GO:0043434      BP      response to peptide hormone stimulus
GO:0046626      BP      regulation of insulin receptor signaling
                          pathway
GO:1900076      BP      regulation of cellular response to
                          insulin stimulus
GO:0003229      BP      ventricular cardiac muscle tissue
                          development
GO:0060324      BP      face development
GO:0052126      BP      movement in host environment
GO:0052192      BP      movement in environment of other organism
GO:0021700      BP      developmental maturation
GO:0007628      BP      adult walking behavior
GO:0006584      BP      catecholamine metabolic process
GO:0006260      BP      DNA replication
GO:0061383      BP      trabecula morphogenesis
GO:0042493      BP      response to drug
GO:0042755      BP      eating behavior
GO:0015804      BP      neutral amino acid transport
GO:0009712      BP      catechol-containing compound metabolic
                          process
GO:0050432      BP      catecholamine secretion
GO:0044423      CC      virion part
GO:0020002      CC      host cell plasma membrane
GO:0055036      CC      virion membrane
GO:0033644      CC      host cell membrane
GO:0044218      CC      other organism cell membrane
GO:0044279      CC      other organism membrane
GO:0019012      CC      virion
GO:0018995      CC      host
GO:0033643      CC      host cell part
GO:0043657      CC      host cell
GO:0043245      CC      extraorganismal space
GO:0044215      CC      other organism
GO:0044216      CC      other organism cell
GO:0044217      CC      other organism part
GO:0005576      CC      extracellular region
GO:0031225      CC      anchored to membrane
GO:0044421      CC      extracellular region part
GO:0005811      CC      lipid particle
GO:0071575      CC      integral to external side of plasma
                          membrane
GO:0016020      CC      membrane
GO:0016590      CC      ACF complex
GO:0031224      CC      intrinsic to membrane
GO:0032588      CC      trans-Golgi network membrane
GO:0005678      CC      chromatin assembly complex
GO:0031233      CC      intrinsic to external side of plasma
                          membrane
GO:0005578      CC      proteinaceous extracellular matrix
GO:0019028      CC      viral capsid
GO:0071944      CC      cell periphery
GO:0044425      CC      membrane part
GO:0031012      CC      extracellular matrix
GO:0016021      CC      integral to membrane
GO:0005886      CC      plasma membrane
GO:0005887      CC      integral to plasma membrane
GO:0019031      CC      viral envelope

             Regulated
ACC          genes        p-value

             Up   Down

GO:0004175   2     2      7.41E-05
GO:0047429   1     0     0.000534732
GO:0070011   6     2     0.000766021
GO:0004533   1     1     0.000845852
GO:0008233   6     2     0.000892198
GO:0004527   2     1     0.00159076
GO:0051786   1     0     0.00182499
GO:0008933   0     1     0.00219589
GO:0016829   3     1      0.0023682
GO:0015563   1     0     0.00318461
GO:0080138   1     0     0.00318461
GO:0052582   1     0     0.00352433
GO:0004532   1     1     0.00356907
GO:0016896   1     1     0.00356907
GO:0016796   1     1     0.00388849
GO:0004518   2     3     0.00421236
GO:0004222   2     1     0.00452514
GO:0031694   0     1     0.00562692
GO:0003964   1     3     0.00598317
GO:0004611   1     0     0.00614515
GO:0004613   1     0     0.00614515
GO:0015105   1     0     0.00696569
GO:0004566   1     0      0.0073504
GO:0004523   1     2     0.00751456
GO:0047547   0     1     0.00789084
GO:0034061   1     3     0.00853451
GO:0001733   0     1     0.00873618
GO:0050694   0     1     0.00873618
GO:0015370   0     2     0.00904613
GO:0016836   2     0     0.00949916
GO:0004190   1     2     0.00975985
G0:0070001   1     2     0.00975985
G0:0035529   1     0      0.0100091
G0:0046715   1     0      0.0100095
G0:0016891   1     2      0.0101006
G0:0015168   1     0      0.0103365
G0:0004519   3     1      0.010481
G0:0015204   1     0      0.0109939
G0:0042887   1     0      0.0109939
G0:0004668   0     1      0.0110373
G0:0008508   1     0      0.0110595
G0:0016893   1     2      0.0113342
G0:0005355   0     1      0.0119593
G0:0015145   0     1      0.0119593
G0:0015149   0     1      0.0119593
G0:0004521   1     2      0.013506
G0:0003858   1     0      0.0143252
G0:0016835   2     0      0.0144079
G0:0016491   6     0      0.0145508
G0:0004170   0     1      0.0151129
G0:0003824   16    10     0.0155904
G0:0004540   1     2      0.0177235
G0:0004528   1     0      0.0179027
G0:0015125   1     0      0.0181056
G0:0015166   1     0      0.0182634
G0:0008237   3     1      0.0184625
G0:0015294   0     2      0.0189906
G0:0008242   0     1      0.0199636
G0:0001968   0     1      0.0201884
G0:0043169   14    4      0.0206679
G0:0043167   14    4      0.0209605
G0:0015665   1     0      0.0216736
G0:0016779   1     3      0.0231699
G0:0004497   2     0      0.0252525
G0:0016813   0     1      0.0269252
G0:0004551   1     0      0.0270368
G0:0031690   0     1      0.0288964
G0:0046872   13    4      0.0361807
G0:0015081   0     2      0.0377544
G0:0016787   9     4      0.0384575
G0:0051119   0     1      0.0436929
G0:0004089   2     0      0.0445549
G0:0015293   0     2      0.0478865
G0:0004866   2     0      0.0482536
G0:0061135   2     0      0.0490373
G0:0008028   1     0      0.0499747
G0:0044419   1     1      2.12E-05
G0:0030574   0     2     0.000172781
G0:0044243   0     2     0.000244847
G0:0006720   3     0     0.000529324
G0:0007566   1     1     0.000690813
G0:0016032   1     3     0.000938757
G0:0043627   0     2      0.0011929
G0:0051704   1     6     0.00171616
G0:0032963   1     1     0.00227394
G0:0044259   1     1     0.00227394
G0:0044236   0     2     0.00250464
GO:0046080   0     2     0.00262305
GO:0007584   1     2     0.00263182
GO:0009211   0     2      0.0028462
GO:0009219   0     2     0.00304728
G0:0009200   0     2     0.00352186
GO:0032355   0     2     0.00371478
GO:0019629   0     1     0.00400373
GO:0009394   0     2      0.0040798
GO:0006721   2     0     0.00411059
GO:0015850   1     1     0.00445688
GO:0045471   1     1     0.00455246
GO:0097305   1     1     0.00455246
GO:0051701   1     1     0.00491978
GO:0007565   0     2      0.0049753
GO:0009262   0     2     0.00503283
GO:0008299   2     0     0.00590401
GO:0006508   6     2     0.00592331
GO:0031667   2     2     0.00599592
GO:0080029   1     0     0.00603303
GO:0001955   0     1      0.0063067
GO:0006278   1     3     0.00643116
GO:0009147   0     2     0.00656845
GO:0019896   0     1     0.00687467
GO:0015700   1     0     0.00696569
GO:0032526   0     2     0.00714133
GO:0035445   1     0     0.00717238
GO:0019087   0     1     0.00723965
GO:0009991   2     2     0.00729875
GO:0019541   0     1     0.00789084
GO:0019543   0     1     0.00789084
GO:0033189   0     2     0.00804444
GO:0009719   1     4     0.00995205
GO:0010036   1     0      0.0100095
GO:0046713   1     0      0.0100095
GO:0071705   2     1      0.0101633
GO:0048241   0     1      0.0102815
GO:0048242   0     1      0.0102815
GO:0044403   1     1      0.0105244
GO:0015816   0     1      0.0108562
GO:0051385   1     0      0.0109053
GO:0051412   1     0      0.0109053
GO:0015793   1     0      0.0109934
GO:0071918   1     0      0.0109939
GO:0000052   0     1      0.0110373
GO:0018101   0     1      0.0110373
GO:0019240   0     1      0.0110373
GO:0071460   0     1      0.0111982
GO:0071404   0     1      0.0112234
GO:0015074   1     2      0.0119014
GO:0006220   0     2      0.0121271
GO:0042572   1     0      0.0125948
GO:0032328   0     1      0.0126352
GO:0048545   1     2      0.0138253
GO:0033273   0     2      0.0142373
GO:0000270   0     1      0.0144731
GO:0071402   0     1      0.0151801
GO:0055098   0     1      0.0151914
GO:0072527   0     2      0.0157354
GO:0015840   1     0      0.0165188
GO:0015824   0     1      0.0180736
GO:0019047   1     0      0.0181211
GO:0019061   1     0      0.0181211
GO:0030069   1     0      0.0181211
GO:0060346   0     1      0.0187984
GO:0061430   0     1      0.0187984
GO:0009725   2     2      0.0189292
GO:0090305   1     3      0.0189859
GO:0055094   0     1      0.0191323
GO:0071702   1     3      0.01926
GO:0051547   0     1      0.0193231
GO:0051549   0     1      0.0193231
GO:0051546   0     1      0.0217672
GO:0022415   1     1      0.0220443
GO:0055114   6     0      0.0224917
GO:0008643   1     1      0.0228045
GO:0015820   0     1      0.0231931
GO:1900755   0     1      0.0231931
GO:0001957   0     1      0.0234858
GO:0036072   0     1      0.0234858
GO:0032868   1     1      0.0246032
GO:0042415   0     1      0.0251005
GO:0006776   1     0      0.0255507
GO:0044003   0     1      0.0259203
GO:0007412   0     1      0.0259526
GO:0060612   1     0      0.0264902
GO:0001666   0     2      0.0267097
GO:0015803   0     1      0.0271136
GO:0019059   1     0      0.0271628
GO:0030260   1     0      0.0271628
GO:0044409   1     0      0.0271628
GO:0051806   1     0      0.0271628
GO:0051828   1     0      0.0271628
GO:0042886   1     0      0.0273651
GO:0055093   0     1      0.0277426
GO:0046685   1     0      0.027967
GO:0035094   0     1      0.0285816
GO:0015791   1     0      0.0298649
GO:0031915   0     1      0.0301821
GO:0001523   1     0      0.0305911
GO:0070482   0     2      0.0321618
GO:0019069   1     0      0.0325204
GO:0046797   1     0      0.0325204
GO:0060325   0     1      0.0325838
GO:0060416   1     0      0.0327757
GO:0016101   1     0      0.0328931
GO:0030325   0     1      0.0328938
GO:0018195   0     1      0.0333781
GO:0060343   0     1      0.0334187
GO:0060033   0     1      0.0346531
GO:0060323   0     1      0.0351934
GO:0046627   0     1      0.0353221
GO:1900077   0     1      0.0353221
GO:0055010   0     1      0.0364566
GO:0043434   1     1      0.0372623
GO:0046626   0     1      0.0388746
GO:1900076   0     1      0.0388746
GO:0003229   0     1      0.0403232
GO:0060324   0     1      0.0410943
GO:0052126   1     0      0.0413725
GO:0052192   1     0      0.0413725
GO:0021700   1     1      0.042601
GO:0007628   0     1      0.0432687
GO:0006584   0     1      0.0455282
GO:0006260   1     3      0.0473241
GO:0061383   0     1      0.047444
GO:0042493   1     1      0.0481178
GO:0042755   0     1      0.0481504
GO:0015804   0     1      0.0483029
GO:0009712   0     1      0.0484574
GO:0050432   0     1      0.049897
GO:0044423   2     3      2.65E-05
GO:0020002   1     3      5.48E-05
GO:0055036   1     0      5.77E-05
GO:0033644   1     3      7.41E-05
GO:0044218   1     3      7.92E-05
GO:0044279   1     3      7.92E-05
GO:0019012   1     4      8.63E-05
GO:0018995   1     3      0.000268057
GO:0033643   1     3      0.000268057
GO:0043657   1     3      0.000268057
GO:0043245   1     3      0.000280882
GO:0044215   1     3      0.000280882
GO:0044216   1     3      0.000280882
GO:0044217   1     3      0.000280882
GO:0005576   8     4      0.00111473
GO:0031225   1     2      0.00119462
GO:0044421   5     3      0.00261723
GO:0005811   0     2      0.00316573
GO:0071575   0     1      0.00385823
GO:0016020   21    9      0.00416239
GO:0016590   0     1      0.0104395
GO:0031224   21    0      0.0112493
GO:0032588   0     1      0.0118958
GO:0005678   0     1      0.0143956
GO:0031233   0     1      0.0206332
GO:0005578   0     4      0.02211
GO:0019028   1     1      0.0306338
GO:0071944   12    3      0.0307419
GO:0044425   20    2      0.0316867
GO:0031012   0     4      0.0324155
GO:0016021   18    1      0.0337337
GO:0005886   11    3      0.0374987
GO:0005887   0     4      0.0433339
GO:0019031   1     0      0.047653

Supplementary Table S2. Details of the 107 novel up-and
downregulated genes in the spleen of the two chicken lines. The
data showed significant changes in the gene expression of the 107
novel genes in the NE-afflicted chicken lines. The genes included
here showed significant differences in gene expression (p <
0.001, Log2-transformations of the normalization).

Align        ID         Line 6.3     Line 7.2     P-Value

1       MERGEG000551     2136.97     0.299379     0.00001
2       MERGEG000617     5.35256    0.0971332     0.00003
3       MERGEG000618     3.50802     0.188697    0.000026
4       MERGEG000944    0.131426      3.34162    0.000013
5       MERGEG001890     33.1856      2.09431    0.000024
6       MERGEG001892     1.94946    0.0565148    0.000002
7       MERGEG001986     1.62426    0.0174069    0.000223
8       MERGEG003380     2.36542     1.09E-08     0.00002
9       MERGEG004114     2542.29      418.361     0.00012
10      MERGEG004703     27.5891      8.14928    0.000052
11      MERGEG006359    0.347298      4.20567    0.000181
12      MERGEG006582   0.0887352      2.36354    0.000212
13      MERGEG006584   0.0960111      3.63323     0.00003
14      MERGEG006615     203.441       4.1005     0.00048
15      MERGEG007249     1807.33      131.641     0.00028
16      MERGEG007250     3656.99      370.669     0.00024
17      MERGEG008766    0.115329      645.198    0.000005
18      MERGEG009685      135.27      11.3463    0.000248
19      MERGEG009859   0.0158767      1.66938     0.00013
20      MERGEG009862   0.0147668      1.11988    0.000079
21      MERGEG011093    0.260391      9.21101    0.000079
22      MERGEG011720   0.0939934      2.42406    0.000163
23      MERGEG011796     1.17005      9.83704    0.000165
24      MERGEG011843     189.587      38.0563     0.00063
25      MERGEG012002     5.91058    0.0707595    0.000001
26      MERGEG012743     101.042      21.7745    0.000063
27      MERGEG012902     1.52806    0.0779027    0.000103
28      MERGEG013363     42.4419      11.5543    0.000001
29      MERGEG014107     14.2182    0.0221362    0.000022
30      MERGEG014878     94.7909      26.3151    0.000002
31      MERGEG016657    0.210053     0.010179    0.000263
32      MERGEG016754      1.9062     0.201944    0.000114
33      MERGEG017199      3.5207     0.160044    0.000011
34      MERGEG017201     3.94706     0.199663     0.00018
35      MERGEG017202     2.71074    0.0834113     0.00001
36      MERGEG017811     51.1047     0.968184    0.000002
37      MERGEG017813     127.141      6.51689    0.000004
38      MERGEG018593     7.47913     0.624983    0.000105
39      MERGEG019909     22.6466      3.91193    0.000001
40      MERGEG023028     17.0676     0.651791    0.000001
41      MERGEG023671     41.0883      6.09047    0.000039
42      MERGEG025414     2.48584    0.0616675    0.000194
43      MERGEG025515      4.8911     0.211326    0.000001
44      MERGEG025584     8.08979    0.0975418    0.000009
45      MERGEG025588     6.15473     0.169144    0.000098
46      MERGEG025594      6.4614    0.0935933    0.00004
47      MERGEG025619     5.55924    0.0589723    0.000007
48      MERGEG025745     1.19203      12.2097    0.000055
49      MERGEG025809     44.6485     0.794799    0.00001
50      MERGEG025958     3.13714      4.03167    0.000011
51      MERGEG025992     320.944    0.0198383    0.000031
52      MERGEG026053     9.61143     0.479531    0.000164
53      MERGEG026071      105.14      29.9747    0.000108
54      MERGEG026263     102.943      15.0452    0.000001
55      MERGEG026303       722.45    0.911791    0.000069
56      MERGEG026452     0.691953      23.303    0.000232
57      MERGEG026502      17.3414     2.29048    0.000194
58      MERGEG026554     0.498798     17.4815    0.00029
59      MERGEG026559       56.695     1.21542    0.000011
60      MERGEG026611   0.00466288    0.358285    0.000037
61      MERGEG026690            0      150808    0.000169
62      MERGEG026742      17.7466    0.111535    0.000002
63      MERGEG026743      12.0938    0.296218    0.000017
64      MERGEG026977    0.0841445     6.22033    0.000089
65      MERGEG027165      7.39997    0.373603    0.000195
66      MERGEG027183       516.88    0.427449    0.000209
67      MERGEG027402     0.166846     9.39216    0.000047
68      MERGEG027408     0.116773     9.01353    0.000007
69      MERGEG027425      31.3806     3.20114    0.000269
70      MERGEG027438      52.4236     1.53551    0.000097
71      MERGEG027503     0.506263     14.3587    0.000001
72      MERGEG027638      28.9093     4.60078    0.000003
73      MERGEG027776      3.48675     29.1998    0.000005
74      MERGEG027843     0.265967     3.62531    0.000127
75      MERGEG027851      9.92577     1.11266    0.000149
76      MERGEG027864      5.03866    0.0589511   0.000035
77      MERGEG027951     0.424828     14.1378    0.000013
78      MERGEG028014     0.140447      4.5427    0.000004
79      MERGEG028162       45.366     3.25828    0.000108
80      MERGEG028374      1.14716      21.675    0.000001
81      MERGEG028465      31.9067    0.121619    0.000022
82      MERGEG028470       34.982     3.54287    0.000052
83      MERGEG028475      30.2852    0.441379    0.000004
84      MERGEG028484    0.0788914     2.33215    0.000108
85      MERGEG028525     0.314738       5.959    0.000095
86      MERGEG028547    0.0387693     3.16784    0.000089
87      MERGEG028556     0.168418     12.3046    0.00008
88      MERGEG028770      11.7212    0.434151    0.000083
89      MERGEG028908      824.188     2388.62    0.000042
90      MERGEG028967      8.73409    0.203217    0.000003
91      MERGEG028983   0.00234716     3.17869    0.000269
92      MERGEG029032      0.49295     15.8866    0.000007
93      MERGEG029081     0.012317      4.0843    0.0001
94      MERGEG029100      13.0374    0.319954    0.00018
95      MERGEG029165      15.0278     436.474    0.00008
96      MERGEG029168      209.505     1467.47    0.00010
97      MERGEG029220      3.30095    0.0202832   0.000002
98      MERGEG029362       7.7485    0.033152    0.000121
99      MERGEG029392     0.208215     34.6643    0.000002
100     MERGEG029474      6.33528    0.0620386   0.000001
101     MERGEG029569       7.9446    0.216283    0.000048
102     MERGEG029570      6.57315    0.252599    0.000038
103     MERGEG029610      7.61695       69.81    0.000166
104     MERGEG029676      37.4481     1.32459    0.000046
105     MERGEG029773      7.31126    0.131129    0.000256
106     MERGEG029791      25.0124     3.28569    0.000117
107     MERGEG030130      31.3201     493.883    0.00026

Supplementary Table S3. Differentially expressed genes related to
antigen processing between the spleen of each of the samples and
the two chicken lines. This data showed significant changes in
the gene expression of the 139 immune-related genes in the two
NE-afflicted chicken lines. The genes included here showed
significant differences in gene expression with p < 0.01,
Log2-transformations of the normalization.

Gene name                        Accession no     Fold changes

                                                  Line 6.3   Line 7.2

Beta-defensin 1 (AvBDl) gene     NM 204993          6.27       3.09

Beta-defensin 3 ST1 (AvBD3ST1)   XM 004934447      -1.73      -0.82
gene

Beta-defensin 3 ST2 (AvBD3ST2)   NM 001277431      -1.19      -1.38
gene

Beta-defensin 9 (AvBD9) gene     AY621321           7.01       7.04

Beta-defensin 7 (AvBD7) gene     AY621322           5.68       0.00

Beta-defensin 10 (AvBD10) gene   AY621323            0          0

Bundling protein                 NM 204127          8.38       8.18

Rho GDP dissociation inhibitor   NM 001277364       7.73       7.71
(GDI) beta

Actin related protein 2/3        NM 001030632       8.82       8.75
complex, subunit 1B

Brain abundant, membrane         NM 204116          7.99       8.29
attached signal protein 1

Beaded filament structural       NM 205151          3.87       2.77
protein 1

Chromosome 6 open reading        XM 001231980       6.68       6.48
frame

Complement component 1, q        XM 417654          7.66       8.00
subcomponent, A chain

Complement component 1, q        XM 417653          6.57       6.86
subcomponent, C chain

Coiled-coil domain containing    XM 415635         -3.32       9.33
57

CD3e molecule, epsilon           NM 206904          8.48       8.80

Immunoglobulin-associated beta   NM 001006328       7.06       7.14

Carbohydrate (N-                 XM 003641759       6.27       6.96
acetylglucosamine-6-
O)sulfotransferase 2

Cold inducible RNA binding       NM 001031347       8.97       9.30
protein

CKLF-like MARVEL transmembrane   NM 001001778       7.52       7.99
domain containing 3

Complement component             XM 002196472       6.87       6.10
(3d/Epstein Barr virus)
receptor 2

Cellular repressor of            XM 003640464       8.70       8.19
E1A-stimulated genes 1

Colony stimulating factor 3      NM 001030898       7.43       6.99
receptor

C-src tyrosine kinase            NM 205425         10.16       9.96

Cytochrome b-245, beta           NM 001100286       6.73       7.17
polypeptide

Cytohesin 1                      NM 001031105       7.61       7.95

Dual adaptor of                  NM 001012933       6.04       6.08
phosphotyrosine and
3-phosphoinositides

Deleted in malignant brain       XM 003643135.1     6.23       6.22
tumors 1

Ets variant gene 6               NM 001199273       7.46       0.00

Extracellular fatty acid         AY545055           8.36       8.14
binding protein

Formin-like 1                    XM 001234504       8.03       8.05

Guanine nucleotide               NM 205402          8.18       8.09
binding protein

GRB2-related adaptor protein     NM 001277553       7.20       7.61

Alpha-D-globin                   NM 001004375      10.82       7.04

Hepatic and glial cell           XM 425793          7.92       6.70
adhesion molecule

HtrA serine peptidase 1          XM 003641501       6.00       5.92

Hydrogen voltage-gated channel   NM 001030663       7.08       7.40
1

Interferon, gamma-inducible      XM 418246          8.61       8.86
protein 30

Immunoglobulin J polypeptide     NM 204263          8.81      10.21

Immunoglobulin superfamily,      XM 003643874       6.02       3.85
member 1

Inverted formin, FH2 and WH2     XM 421396          4.86       3.46
domain

Interferon regulatory factor 5   NM 001031587       6.69       6.98
Janus kinase 3                   NM 204996          7.68       7.97

Lysosomal protein                XM 417701          8.86       9.12
transmembrane 5

Macrophage receptor with         NM 204736          8.43       8.27
collagenous structure

Muscleblind-like                 NM 001163327       8.02       7.94

Microtubule associated           NM 001031576       8.06       8.63
monoxygenase, calponin and LIM

Myosin IF                        NM 205254          7.01       7.30

NUAK family, SNFl-like kinase    XM 416310          5.76       5.43

OTU domain containing 1          XM 418604          7.58       6.41

Purinergic receptor P2Y          NM 001008679       7.21       7.17

Phosphatidylinositol transfer    NM 001039266       6.63       6.65
protein, beta

Pleckstrin                       NM 204863          7.78       7.33

Protein phosphatase 1,           XM 003644026.1     7.72       7.97
regulatory (inhibitor) subunit
9B

Protein tyrosine phosphatase,    NM 204417          8.57       8.71
receptor type, C

Quaking homolog, KH domain RNA   NM 204310          7.07       6.79
binding

Ras association (RalGDS/AF-6)    NM 001030884       7.29       7.33
domain family member 2

Ribosomal protein L29            NM 001171677      10.74      11.22

Leukocyte ribonuclease A-2       NM 001007942       8.33       8.47

Selectin E                       XM 422207.3        7.22       6.86

Small ArfGAP2                    NM 001030902       7.93       7.96

SPEG complex locus               XM 004176748        0         7.20

Spi-C transcription factor       XM 425488          6.69       6.46

Serine/threonine kinase 38       XM 003642721       6.62       6.57

Synaptonemal complex central     XM 001231763        0         7.61
element protein 3

Transcription factor 7 (T-cell   NM 204547          6.68       7.08
specific, HMG-box)

Transforming growth factor,      NM 205036          6.84       6.63
beta-induced

Tetratricopeptide repeat         NM 001012567       4.79       4.97
domain 27

Unc-119 homolog B                XM 415263          6.36       6.23

WDFY family member 4             XM 003641478       6.25       6.39

Zinc finger E-box binding        XM 422151          6.30       5.93
homeobox 2

Allograft inflammatory factor    XM 415461          8.34       8.20
1-like

Argininosuccinate synthase 1     NM 001013395       7.97       8.24
(ASS1)

ATPase, Na+/K+ transporting,     NM 205535          5.85       5.81
beta 3 polypeptide

Avidin-related protein           XM 001233209       4.97       8.95
4/5-like

Uncharacterized LOC419592        XM 417739          8.59       8.44

Carbonic anhydrase 13-like       XM 003640811       6.94       5.15

Chemokine (C-C Motif) Ligand     XM 415780          6.39       7.30

C-type lectin domain family 17   XM 423779          6.01       6.41

Complement receptor type         XM 001235095       5.79       5.85
2-like

Friend leukemia virus            M 001030908        6.94       6.72
integration 1 (FLI1)

DEAD (Asp-Glu-Ala-Asp) box       XM 416260          8.56       8.65
polypeptide 17

Major histocompatibility         S66480            10.15      10.49
complex class II beta chain

Viral interleukin-8 homolog      XM 420473          8.09       7.96

Chicken Ig rearranged            CHKIGLAVD         10.72      12.12
light-chain mRNA V-J-C region

ADP-ribosylation factor-like     XM 001237034       6.02       5.66
4C (ARL4C)

Actin related protein 2/3        NM 001031561      10.12      10.58
complex, subunit 5, 16kDa
ARPC5)

Bruton agamma globulinemia       NM 204233          6.60       6.64
tyrosine kinase (BTK)

Coiled-coil domain containing    XM 423729           0         7.13
165

Clusterin (CLU)                  NM 204900          9.30       9.40

Colony stimulating factor 1      XM 414597          7.46       6.93
receptor(CSF1R)

Tyrosine kinase protooncogene    CHKTCKL            6.86       6.90
(c-tkl)

Ganglioside GM2 activator-like   XM 003642059       7.68       6.43

Hemoglobin, gamma A (HBG1)       NM 001004390      11.31       8.71

Histone deacetylase 8 (HDAC8)    XM 420178          9.96      10.49

Hermansky-Pudlak syndrome 5      XM 421011          8.74       8.13
(HPS5)

Heat shock protein Hsp70         AY143692           9.46       8.55
(hsp70) gene

Immunoglobulin heavy chain       AM773251          10.03      -0.32
variable region (IGVH gene)

Immunoglobulin heavy chain       AM773267           9.56       5.96
variable region (IGVH gene)

GTPase IMAP family member        XM 418473          7.12       6.73
8-like

Integrin, alpha 4 (alpha 4       XM 421974          7.55       7.44
subunit of VLA-4 receptor)

Integrin, beta 2 (complement     NM 205251          8.27       8.54
component 3 receptor 3 and 4)

Integrin beta 3 (platelet        NM 204315          6.57       4.72
glycoprotein IIIa, antigen
CD61)

Janus kinase 1 (JAK1)            NM 204870          8.35       8.30

Lymphocyte cytosolic protein 1   NM 001008440       9.01       8.88
(L-plastin)

Leukocyte cell-derived           NM 205478          7.67       2.04
chemotaxin 2

Lymphocyte-specific protein 1    NM 204342          8.57       8.61

126 MRP mRNA                     X61200             9.37       9.52

Moesin (MSN)                     XR 140102          8.87       8.83

Nerve growth factor receptor     NM 001146133       6.46       6.62

Nucleophosmin (nucleolar         NM 205267         11.14      11.41
phosphoprotein B23, numatrin)

Protein kinase C, beta           XM 414868          7.43       7.59

Ribosomal protein S6             NM 205225          9.81      10.54

Septin 6 (SEPT6)                 NM 001031125       7.98       8.09

GB-2 T cell receptor alpha       GDU04611           7.57       7.95

AA2 T cell receptor beta         EF554758           7.91       8.54

Transmembrane protein 123        NM 001006279       7.82       8.17
(TMEM123)

Ig germline heavy chain VD       CHKIGVHAH          9.37       7.58
region (VH15-9)

Ig germline heavy chain VD       CHKIGVHAJ          9.19       7.26
region (VH57-1)pseudogene.

Ig germline heavy chain VD       CHKIGVHAR          7.38       7.53
region (VH57-13)

Zinc finger protein 598,         XM 414850          6.96       7.15
transcript variant 3 (ZNF598)

Gatinaise disrupted tyrosinase   DQ118701          11.06      -1.74
gene, intron 4

Hemoglobin subunit               XM 003643191      11.84       8.53
alpha-A-like

High mobility group 1 protein    Y17968             8.76       8.42

Hypothetical protein, clone      AJ719941          10.66      10.81
8j21

Ig heavy-chain gene V region     CHKIGHC            8.72       7.80

Ig rearranged H-chain gene       CHKIGHAZE         10.52       7.71
V-D-J-region

Ig rearranged H-chain gene       CHKIGHAZE         12.45      11.15
V-D-J-region

Ig rearranged heavy chain V      CHKIGVHAAK         8.49       7.94
region (VH1)

Ig rearranged lambda-chain       CHKIGLA12          8.25       6.48
pseudogene psi-V14 V1 region

Ig rearranged lambda-chain       CHKIGLA19          9.45       7.02
pseudogene psi-V7 V1 region

Immunoglobulin alpha heavy       S40610             7.74       5.00
chain

Mitochondrial genome             X52392            12.53      12.30

Ras-related protein              XM 419183          5.79       5.46
Rab-10-like

SH2 domain-containing adapter    XM 003643441      10.53       7.62
protein E-like

T-cell-interacting, activating   XM 003644001       6.60        0
receptor on myeloid cells
protein 1-like

T-lymphocyte surface antigen     XM 003642660       6.86       7.24
Ly-9-like

Transmembrane 6 superfamily      XM 423447           0         8.26
member 2-like

Uncharacterized LOC415472        XM 429332          5.85       6.42

Supplementary Table S4. Description of cytokine and receptor
genes in the spleens of chicken lines 6.3 and 7.2 responsive to
co-infection with EM-CP. The data showed significant changes in
gene expression of the 53 cytokine genes and 97 cytokine receptor
genes in NE-afflicted chicken lines. The genes included here
showed significant differences in gene expression (p < 0.01,
Log2-transformations of the normalization).

Gene        Full name                                 Fold changes

                                                  Line 6.3   Line 7.2

ADIPOQ      Adiponectin, C1Q And Collagen           8.15       8.41
BMP2K       BMP-2-Inducible Protein Kinase          2.04       1.75
BMPER       BMP Binding Endothelial Regulator      -3.10      -3.82
CCL1        Chemokine (C-C Motif) Ligand 1         -1.25       1.57
CCL17       Chemokine (C-C Motif) Ligand 17        -0.20      -0.19
CCL19       Chemokine (C-C Motif) Ligand 19        -2.30       2.70
CCL20       Chemokine (C-C Motif) Ligand 20        -3.77      -3.62
CCL3        Chemokine (C-C Motif) Ligand 3          3.42       4.68
CCL4        Chemokine (C-C Motif) Ligand 1          1.85       5.78
CMC1        COX Assembly Mitochondrial Protein     -0.60       0.32
              1 Homolog
CNTF        Ciliary Neurotrophic Factor             0.93       3.45
CXCL13      Chemokine (C-X-C Motif) Ligand 13       9.25       2.93
CXCL14      Chemokine (C-X-C Motif) Ligand 14      -2.66      -3.95
FASLG       Fas Ligand (TNF Superfamily,           -1.74       1.95
              Member 6)
IFITM10     Interferon Induced Transmembrane        0.00       0.00
              Protein 10
IFNA1       Interferon, Alpha 1                     1.92      -0.59
IFNB        Interferon, Beta                       -2.64       2.62
IFNG        Interferon, Gamma                      -1.80       1.48
IFNK        Interferon, Kappa                      -1.57      -1.81
IL-10       Interleukin 10                          0.50       2.32
IL-12A      Interleukin 12A                        -2.81       0.99
IL-16       Interleukin 16                          2.06       2.05
IL-17B      Interleukin 17B                        -2.57       0.00
IL-17D      Interleukin 17D                        -3.94      -3.62
IL-17F      Interleukin 17F                        -3.40      -0.61
IL-19       Interleukin 19                         -2.87       0.48
IL-22       Interleukin 22                         -7.48      -5.77
IL-26       Interleukin 26                         -7.08      -4.93
IL-28B      Interleukin 28B                         2.98      -0.30
IL-34       Interleukin 34                          2.94       2.94
IL-5        Interleukin 5                           0.44      -0.04
IL-6        Interleukin 6                           0.95       1.60
IL-7        Interleukin 7                          -2.73       2.03
IL-8        Interleukin 8                          -0.77      -0.12
ILF2        Interleukin Enhancer Binding Factor     0.66       0.76
              2
LIF         Leukemia Inhibitory Factor             -2.38       0.76
NLRC3       NLR Family, CARD Domain Containing     -2.64      -0.46
              3
NLRC5       NLR Family, CARD Domain Containing     -1.16      -0.63
              5
NRG4        Neuregulin 4                           -1.48      -1.89
SPP1        Secreted Phosphoprotein 1               2.50       0.35
SPP2        Secreted Phosphoprotein 2              -0.08       2.43
TAP1        Transporter 1, ATP-Binding              0.11       1.10
              Cassette, Sub-Family B
TAP2        Transporter 2, ATP-Binding              0.26       0.55
              Cassette, Sub-Family B
TGFA        Transforming Growth Factor, Alpha      -2.05      -0.80
TGFB1       Transforming Growth Factor, Beta 1      7.34       7.46
TNFAIP2     Tumor Necrosis Factor,                  4.97       4.64
              Alpha-Induced Protein 2
TNFAIP3     Tumor Necrosis Factor,                  2.11       2.53
              Alpha-Induced Protein 3
TNFAIP8     Tumor Necrosis Factor,                  2.45       2.42
              Alpha-Induced Protein 8
TNFAIP8L1   Tumor Necrosis Factor,                  2.80       2.94
              Alpha-Induced Protein 8 Like 1
TNFAIP8L3   Tumor Necrosis Factor,                  0.04       3.96
              Alpha-Induced Protein 8 Like 3
TNFSF11     Tumor Necrosis Factor (Ligand)          3.52       1.64
              Superfamily, Member 11
TNFSF13B    Tumor Necrosis Factor (Ligand)          6.70       4.69
              Superfamily, Member 13B
VEGFA       Vascular Endothelial Growth Factor     -4.24      -5.06
              A
CSF2RA      Colony Stimulating Factor 2             5.23       7.77
              Receptor, Alpha
CSF2RB      Colony Stimulating Factor 2             5.33       7.68
              Receptor, Beta
CSF3R       Colony Stimulating Factor 3            10.16       8.52
              Receptor
CXCR3       Chemokine (C-X-C Motif) Receptor 3     -8.22      -7.88
CXCR7       Chemokine (C-X-C Motif) Receptor 7      4.33       2.40
GDF8        Growth differentiation factor 8         0.00       1.87
IFIH1       Interferon Induced With Helicase C     -1.46      -0.16
              Domain 1
IFITM5      Interferon Induced Transmembrane       -8.38      -4.84
              Protein 5
IFNAR1      Interferon (Alpha, Beta And Omega)      0.98       2.09
              Receptor 1
IFNAR2      Interferon (Alpha, Beta And Omega)      0.78       1.03
              Receptor 2
IFNGR1      Interferon Gamma Receptor 1             1.75       0.95
IFNGR2      Interferon Gamma Receptor 2            -0.64       0.08
IGF1R       Insulin-Like Growth Factor 1            1.53       1.43
              Receptor
IL-11RA     Interleukin 11 Receptor, Alpha          0.92      -3.29
IL-12RB1    Interleukin 12 Receptor, Beta 1         1.91       0.84
IL-12RB2    Interleukin 12 Receptor, Beta 2         0.89       1.32
IL-13RA1    Interleukin 13 Receptor, Alpha 1       -2.86      -1.92
IL-17RA     Interleukin 17 Receptor, Alpha          2.39       2.08
IL-17RC     Interleukin 17 Receptor C              -4.96      -5.02
IL-17RD     Interleukin 17 Receptor D              -1.25      -2.70
IL-17REL    Interleukin 17 Receptor E-Like          1.58       4.28
IL-18R1     Interleukin 18 Receptor 1               5.88       5.01
IL-18RAP    Interleukin 18 Receptor Accessory       6.13       3.11
              Protein
IL-1R1      Interleukin 1 Receptor 1                6.49       1.97
IL-1R2      Interleukin 1 Receptor 2                3.55       7.91
IL-1RAP     Interleukin 1 Receptor Accessory        6.63       5.81
              Protein
IL-1RAPL2   Interleukin 1 Receptor Accessory       -5.00      -4.99
              Protein-Like 2
IL-1RL1     Interleukin 1 Receptor-Like 1           2.82       6.98
IL-1RL2     Interleukin 1 Receptor-Like 2           4.46      -0.07
IL-20RA     Interleukin 20 Receptor, Alpha          2.48       3.59
IL-20RB     Interleukin 20 Receptor, Beta           1.53       2.23
IL-21R      Interleukin 21 Receptor                 3.04       3.49
IL-22RA1    Interleukin 22 Receptor, Alpha 1       -10.08     -9.12
IL-22RA2    Interleukin 22 Receptor, Alpha 2       -2.11      -2.43
IL-23R      Interleukin 23 Receptor                 1.71      -0.34
IL-28RA     Interleukin 28 Receptor, Alpha         -1.43      -1.03
IL-2RB      Interleukin 2 Receptor, Beta            2.20       1.54
IL-2RG      Interleukin 2 Receptor, Gamma           3.56       3.58
IL-31RA     Interleukin 31 Receptor, Alpha          6.40       4.78

IL-4I1      Interleukin 4 Induced 1                 2.23       3.92
IL-4R       Interleukin 4 Receptor                  1.71       1.64
IL-5RA      Interleukin 5 Receptor, Alpha           7.26       1.22
IL-6ST      Interleukin 6 Signal Transducer         6.02       5.67
IL-7R       Interleukin 7 Receptor                 10.67       8.23
IL-9R       Interleukin 9 Receptor                  5.31       4.90
ILDR1       Immunoglobulin-Like Domain             -3.04      -2.78
              Containing Receptor 1
ILDR2       Immunoglobulin-Like Domain              1.54       2.60
              Containing Receptor 2
ILF3        Interleukin Enhancer Binding Factor     1.02       1.22
              3
LEPRE1      Leucine Proline-Enriched                1.31       1.73
              Proteoglycan (Leprecan) 1
LEPREL1     Leprecan-Like 1                         3.61      -2.15
LEPREL2     Leprecan-Like 2                        -0.86      -3.41
LEPREL4     Leprecan-Like 4                        -0.24       0.52
LEPROTL1    Leptin Receptor Overlapping             0.51       1.34
              Transcript-Like 1
LIFR        Leukemia Inhibitory Factor Receptor     2.72       3.66
              Alpha
LILRA2      Leukocyte Immunoglobulin-Like           5.09       1.75
              Receptor, Subfamily A, Member 2
LILRA4      Leukocyte Immunoglobulin-Like           0.82      -0.54
              Receptor, Subfamily A, Member 4
LILRA5      Leukocyte Immunoglobulin-Like          -2.17      -2.14
              Receptor, Subfamily A, Member 5
LILRA6      Leukocyte Immunoglobulin-Like           4.99       0.00
              Receptor, Subfamily A, Member 6
LILRB1      Leukocyte Immunoglobulin-Like           2.47       4.64
              Receptor, Subfamily B, Member 1
LILRB3      Leukocyte Immunoglobulin-Like           5.49       0.58
              Receptor, Subfamily B, Member 3
LILRB4      Leukocyte Immunoglobulin-Like           3.24       2.52
              Receptor, Subfamily B, Member 4
LILRB5      Leukocyte Immunoglobulin-Like           4.31       3.65
              Receptor, Subfamily B, Member 5
LTB4R       Leukotriene B4 Receptor                 7.16       7.33
LTBP1       Latent Transforming Growth Factor       1.65       1.75
              Beta Binding Protein 1
LTBP2       Latent Transforming Growth Factor      -3.63      -3.18
              Beta Binding Protein 2
MC1R        Melanocortin 1 Receptor                 0.00      -2.65
NRG2        Neuregulin 2                           -0.09      -4.76
NRG3        Neuregulin 3                            0.38       0.00
PDGFC       Platelet Derived Growth Factor C        1.79      -1.03
PDGFD       Platelet Derived Growth Factor D       -2.19       2.30
PDGFRL      Platelet-Derived Growth Factor         -4.61      -0.03
              Receptor, Alpha Polypeptide
TGFBR2      Transforming Growth Factor, Beta        2.18       0.96
              Receptor II
TGFBRAP1    Transforming Growth Factor, Beta        1.22       0.40
              Receptor Associated Protein 1
TGFBR1      Transforming Growth Factor, Beta        3.91       3.41
              Receptor I
TLR1        Toll-Like Receptor 1                    3.34       4.11
TLR15       Toll-Like Receptor 15                   5.30       8.26
TLR21       Toll-Like Receptor 21                   4.32       2.23
TLR5        Toll-Like Receptor 5                    2.28      -0.47
TLR6        Toll-Like Receptor 6                    3.82       5.46
TLR7        Toll-Like Receptor 7                    8.04       7.08
TNFRSF11A   Tumor Necrosis Factor Receptor         -0.88      -1.59
              Superfamily, Member 11a,
TNFRSF11B   Tumor Necrosis Factor Receptor         -0.19      -1.00
              Superfamily, Member 11b
TNFRSF13B   Tumor Necrosis Factor Receptor          4.04       5.59
              Superfamily, Member 13B
TNFRSF13C   Tumor Necrosis Factor Receptor          6.73       7.94
              Superfamily, Member 13C
TNFRSF14    Tumor Necrosis Factor Receptor         -4.47      -3.48
              Superfamily, Member 14
TNFRSF18    Tumor Necrosis Factor Receptor          1.28       1.84
              Superfamily, Member 18
TNFRSF1B    Tumor Necrosis Factor Receptor          4.61       5.83
              Superfamily, Member 1B
TNFRSF21    Tumor Necrosis Factor Receptor          0.90       1.75
              Superfamily, Member 21
TNFRSF25    Tumor Necrosis Factor Receptor          2.80       1.92
              Superfamily, Member 25
TNFRSF4     Tumor Necrosis Factor Receptor         -1.20      -1.20
              Superfamily, Member 4
TNFRSF6B    Tumor Necrosis Factor Receptor         -6.36      -0.46
              Superfamily, Member 6B
TNFRSF9     Tumor Necrosis Factor Receptor          1.63       1.98
              Superfamily, Member 9
TRAT1       T Cell Receptor Associated              4.37       5.10
              Transmembrane Adaptor 1
TRDC        T Cell Receptor Delta Constant          2.43       2.14
TRGC1       T Cell Receptor Gamma Constant 1        1.69       1.84
XCR1        Chemokine (C Motif) Receptor 1          6.65       6.25

Supplementary Table S5. Description of CD molecular genes
responsive to NE-affliction in the spleen of the two chicken
lines. The data showed significant changes in the gene expression
of 44 CD molecular genes in the two NE-afflicted chicken lines.
The genes included here showed significant differences in gene
expression (p < 0.01, Log2-transformations of the normalization).

Symbols    Full gene name                    Fold changes

                                         Line 6.3   Line 7.2

CD2        CD2 Molecule                    6.03       5.02
CD200      CD200 Molecule                  5.62       6.03
CD200R1    CD200 Molecule                  1.14      -1.06
             receptor 1
CD200R1A   CD200 Molecule                  4.38       3.75
             receptor 1 Like
CD226      CD226 Molecule                  1.42       0.65
CD244      CD244 Molecule                  2.12       1.51
CD247      CD247 Molecule                  2.71       3.27
CD274      CD274 Molecule                 -0.13       2.54
CD276      CD276 Molecule                 -0.09       0.73
CD28       CD28 Molecule                   4.34       3.31
CD2AP      CD2 Molecule                   -1.51      -1.50
             Associated Protein
CD300A     CD300A Molecule                 1.38       1.69
CD300C     CD300C Molecule                 1.86       1.05
CD300LF    CD300 molecule-like             0.00       0.00
             family member f
CD34       CD34 Molecule                  -2.94      -1.18
CD36       CD36 Molecule                   4.28      -5.01
CD38       CD38 Molecule                   2.89       3.32
CD3D       T-Cell Surface                  1.32       1.61
CD3E       T-Cell Surface Glycoprotein     2.10       2.08
             CD3 Epsilon
CD4        CD4 Molecule                    5.86       6.15
CD40       CD40 Molecule                   4.43       3.23
CD40L      CD40 Molecule Ligand            2.88       2.57
CD44       CD44 Molecule                   3.49       3.69
CD5        CD5 Molecule                    3.74       4.56
CD55       CD55 Molecule                  -1.03      -1.15
CD59       CD59 Molecule                   4.78       2.70
CD5L       CD5 Molecule-Like               3.25       3.31
CD69       CD69 Molecule                   2.30       2.15
CD7        CD7 Molecule                   -2.18      -0.62
CD72       CD72 Molecule                   6.10       8.48
CD79B      CD79b molecule,                 6.62       8.14
             immunoglobulin-associated
             beta
CD80       CD80 Molecule                   1.37       1.70
CD8A       CD8a Molecule                  -4.90      -1.56
CD93       CD93 Molecule                   8.42       3.25
CD99       CD99 Molecule                  -0.39      -0.46
CD99L2     CD99 molecule-like 2            0.99      -1.03
CD101      CD101 Molecule                 -0.70      -1.60
CD109      CD109 Molecule                  1.48      -0.62
CD14       CD14 Molecule                   4.30       3.89
CD163      CD163 Molecule                 -8.26      -6.88
CD163L1    CD163 Molecule Like 1           4.20       1.28
CD180      CD180 Molecule                  6.53       8.70
CD48       CD48 Molecule                  -0.81       2.34
CD83       CD83 Molecule                   3.95       4.69

Supplementary Figure S1. Percent of coverage representing the
percentage of genes expressed in the spleen of the two chicken lines.
The distribution of distinct reads over different read abundance
categories showed similar patterns for both RNA-Seq libraries. The
similarity distribution displayed a comparable pattern with
approximately 73% of the sequences having a similarity of 80%, while
approximately 27% of the hits had a similar range in the spleen of
lines 6.3 and 7.2.

Distribution of Genes' Coverage in NE samples of line 6.3

0.90~1.00 (12691)        67%
0.80~0.90 (1456)          8%
0.70~0.80 (788)           4%
0.60~0.70 (623)           3%
0.50~0.60 (530)           3%
0.40~0.50 (490)           3%
0.30~0.40 (551)           3%
0.20~0.30 (613)           3%
0.10~0.20 (675)           4%
0.00~0.10 (583)           3%

Distribution of Genes' Coverage in NE samples of line 7.2

0.90~1.00 (12484)        67%
0.80~0.90 (1415)          8%
0.70~0.80 (767)           4%
0.60~0.70 (598)           3%
0.50~0.60 (513)           3%
0.40~0.50 (519)           3%
0.30~0.40 (527)           3%
0.20~0.30 (611)           3%
0.10~0.20 (710)           4%
0.00~0.10 (609)           3%

Note: Table made from pie chart.
COPYRIGHT 2015 Asian - Australasian Association of Animal Production Societies
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Truong, Anh Duc; Hong, Yeong Ho; Lillehoj, Hyun S.
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Author abstract
Date:Oct 1, 2015
Words:22281
Previous Article:Effects of post-harvest storage duration and variety on nutrient digestibility and energy content wheat in finishing pigs.
Next Article:The effects of gilts housed either in group with the electronic sow feeding system or conventional stall.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters