RNA-seq Profiles of Immune Related Genes in the Spleen of Necrotic Enteritis-afflicted Chicken Lines

  • Truong, Anh Duc (Department of Animal Science and Technology, Chung-Ang University) ;
  • Hong, Yeong Ho (Department of Animal Science and Technology, Chung-Ang University) ;
  • Lillehoj, Hyun S. (Animal Biosciences and Biotechnology Laboratory, Agricultural Research Services, U. S. Department of Agriculture)
  • 투고 : 2015.02.17
  • 심사 : 2015.04.20
  • 발행 : 2015.10.01


The study aimed to compare the necrotic enteritis (NE)-induced transcriptome differences between the spleens of Marek's disease resistant chicken line 6.3 and susceptible line 7.2 co-infected with Eimeria maxima/Clostridium perfringens using RNA-Seq. Total RNA from the spleens of two chicken lines were used to make libraries, generating 42,736,296 and 42,617,720 usable reads, which were assembled into groups of 29,897 and 29,833 mRNA genes, respectively. The transcriptome changes were investigated using the differentially expressed genes (DEGs) package, which indicated 3,255, 2,468 and 2,234 DEGs of line 6.3, line 7.2, and comparison between two lines, respectively (fold change ${\geq}2$, p<0.01). The transcription levels of 14 genes identified were further examined using qRT-PCR. The results of qRT-PCR were consistent with the RNA-seq data. All of the DEGs were analysed using gene ontology terms, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the DEGs in each term were found to be more highly expressed in line 6.3 than in line 7.2. RNA-seq analysis indicated 139 immune related genes, 44 CD molecular genes and 150 cytokines genes which were differentially expressed among chicken lines 6.3 and 7.2 (fold change ${\geq}2$, p<0.01). Novel mRNA analysis indicated 15,518 novel genes, for which the expression was shown to be higher in line 6.3 than in line 7.2 including some immune-related targets. These findings will help to understand host-pathogen interaction in the spleen and elucidate the mechanism of host genetic control of NE, and provide basis for future studies that can lead to the development of marker-based selection of highly disease-resistant chickens.


RNA-seq;Chicken;Necrotic Enteritis;Cytokine;Eimeria maxima;Clostridium perfringens


연구 과제 주관 기관 : Ministry of Agriculture Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA), Korea Forest Service (KFS)


  1. 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.
  2. Fabryova, K. and M. Simon. 2009. Function of the cell surface molecules (CD molecules) in the reproduction processes. Gen. Physiol. Biophys. 28:1-7.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 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.
  13. 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.
  14. Johansson, M. W., D. S. Annis, and D. F. Mosher. 2013. ${\alpha}(M){\beta}(2)$ integrin-mediated adhesion and motility of IL-5-stimulated eosinophils on periostin. Am. J. Respir. Cell Mol. Biol. 48:503-510.
  15. 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.
  16. 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.
  17. 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.
  18. 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.
  19. 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.
  20. 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.
  21. 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.
  22. Ozsolak, F. and P. M. Milos. 2011. RNA sequencing: advances, challenges and opportunities. Nat. Rev. Genet. 12:87-98.
  23. 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.
  24. 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.
  25. 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.
  26. 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.
  27. 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.
  28. Sabat, R. 2010. IL-10 family of cytokines. Cytokine Growth Factor Rev. 21:315-324.
  29. 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.
  30. 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.
  31. 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.
  32. Supek, F., M. Bosnjak, N. Skunca, and T. Smuc. 2011. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:e21800.
  33. Trapnell, C., L. Pachter, and S. L. Salzberg. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105-1111.
  34. 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 RNAseq experiments with TopHat and Cufflinks. Nat. Protoc. 7:562-578.
  35. 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.
  36. 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.
  37. 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.
  38. 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 poreforming toxin NetB from Clostridium perfringens. MBio 4:e00019-13.
  39. 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.
  40. 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.
  41. 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.

피인용 문헌

  1. Comparative transcriptome analysis provides clues to molecular mechanisms underlying blue-green eggshell color in the Jinding duck (Anas platyrhynchos) vol.18, pp.1, 2017,
  2. Distribution and differential expression of microRNAs in the intestinal mucosal layer of necrotic enteritis induced Fayoumi chickens vol.30, pp.7, 2017,
  3. Analysis of Differentially Expressed Genes in Necrotic Enteritis-infected Fayoumi Chickens using RNA Sequencing vol.54, pp.2, 2017,
  4. Immunogenetics applied to control salmonellosis in chicken: a review vol.46, pp.1, 2018,
  5. type C vol.6, pp.2167-8359, 2018,
  6. Transcriptome analysis of adipose tissue from pigs divergent in feed efficiency reveals alteration in gene networks related to adipose growth, lipid metabolism, extracellular matrix, and immune response pp.1617-4623, 2018,
  7. Novel Insights reveal Anti-microbial Gene Regulation of Piglet Intestine Immune in response to Clostridium perfringens Infection vol.9, pp.1, 2019,