Polymorphisms and Associations of the NRAMP-1 and iNOS Genes on Newcastle Disease and Salmonella enteritidis Resistances in SenSi-1 Agrinak Chickens

  • M. Ardiyana Department of Animal Production and Technology, Faculty of Animal Science, IPB University
  • A. Gunawan Department of Animal Production and Technology, Faculty of Animal Science, IPB University
  • S. Murtini Department of Medical Microbiology, Faculty of Veterinary Medicine, IPB University
  • T. Sartika Indonesian Research Institute of Animal Production (IRIAP)
  • C. Sumantri Department of Animal Production and Technology, Faculty of Animal Science, IPB University
Keywords: Disease resistance, iNOS gene, NRAMP-1 gene, polymorphism, SenSi-1 Agrinak chicken


NRAMP-1 and iNOS genes were reported to be associated with a defense mechanism against bacteria and virus infections. This study aimed to identify NRAMP-1 and iNOS genes polymorphisms and their associations with the defense mechanisms against Salmonella enteritidis and Newcastle Disease (ND) in SenSi-1 Agrinak chicken. The present study used a total number of 172 SenSi-1 Agrinak chicken. Identifications of NRAMP-1 and iNOS genes polymorphisms were performed by PCR-RFLP method. NRAMP-1 and iNOS genotypes were associated with immunoglobulin Y (IgY) concentration, specific antibodies against S. enteritidis and ND using General Linear Model (GLM). Immunity characteristics were further grouped into high, medium, and low categories. NRAMP-1|SacI exon 11 and iNOS|AluI intron 24 in SenSi-1 Agrinak chickens were polymorphic. TC genotype has a higher immune response to infectious agents compared to TT and CC genotypes. The frequency of C allele was higher than the T allele in the concentration of immunoglobulin Y (IgY), antibodies titers against S enteritidis and ND. The TC genotype of NRAMP-1 gene was significantly associated with ND antibody titers, and the TT genotype of iNOS was significantly associated with S. enteritidis specific antibody. NRAMP-1 and iNOS genes can be used as potential candidate genes for immune traits in SenSi-1 Agrinak chickens.


Download data is not yet available.


Abbas, A.K., A.H. Lichtman, & S. Pilai. 2017. Molecular and cellular immunology. 9th ed. Elsevier, Philadelphia.

Allendorf, F.W., G. Luikar, & S.N. Aitken. 2013. Conservation and the genetics of population. 2nd ed. Wiley-Blackwell, Oxford.

Bauler, T.J., T. Starr, T.A. Nagy, S. Sridhar, D. Scott, C.W. Winkler, O. Steele-Mortimer, C.S. Detweiler, & K.E. Peterson. 2017. Salmonella meningitis associated with monocyte infiltration in mice. Am. J. Pathol. 187: 187-199. https://doi.org/10.1016/j.ajpath.2016.09.002

Botstein, D, R.L. White, M. Skolnick, & R.W. Davis. 1980. Construction of genetic linkage map in human using restriction fragmen length polymorphisms. Amer. J. Hum. Genet. 32: 314-331.

Cellier, M.F.M. 2017. Developmental control of NRAMP-1 (SLC11A1) expression in professional phagocytes. Biology 6: 28. https://doi.org/10.3390/biology6020028

Cherayil, B.J. 2011. The role of iron in the immune response to bacterial infection. Immunol. Res. 50: 1-9. https://doi.org/10.1007/s12026-010-8199-1

Dai, C.H., J.Y. Wu, C.X. Zhao, L.H. Yu, W.B. Bao, & S.L. Wu. 2017. NRAMP-1 gene expression in different tissues of meishan piglets from newborn to weaning. Genet. Mol. Res. 16: 1-10. https://doi.org/10.4238/gmr16019288

Delgado, F., C. Estrada-Chávez, M. Romano, F. Paolicchi, F. Blanco-Viera, F. Capellino, G. Chavez-Gris, & A.L. Pereira-Suárez. 2010. Expression of NRAMP-1 and iNOS in mycobacterium avium subsp. paratuberculosis naturally infected cattle. Comp. Immunol. Microbiol. Infect. Dis. 33: 389-400. https://doi.org/10.1016/j.cimid.2009.03.001

Dincel, G.C. & O. Kul. 2015. eNOS and iNOS trigger apoptosis in the brains of sheep and goats naturally infected with the border disease virus. Histol. Histopathol. 30: 1233-1242.

Geissmann, F., M.G. Manz, S. Jung, M.H. Sieweke, M. Merad, & K. Ley. 2010. Development of monocytes, macrophages and dendritic cells. Science 327: 656-661. https://doi.org/10.1126/science.1178331

Gunawan, A., D. Anggrela, K. Listyarini, M.A. Abuzahra, Jakaria, M. Yamin, I. Inounu, & C. Sumantri. 2018. Identification of Single Nucleotide Polymorphism and Pathway Analysis of Apolipoprotein A5 (APOA5) Related to Fatty Acid Traits in Indonesian Sheep. Trop. Anim. Sci. J. 41:165-173. https://doi.org/10.5398/tasj.2018.41.3.165

Guo, R., S. Geng, H. Jiao, Z. Pan, X. Chen, & X. Jiao. 2016. Evaluation of protective efficacy of a novel inactivated Salmonella pullorum ghost vaccine against virulent challenge in chickens. Vet. Immunol. Immunopathol. 173: 27-33. https://doi.org/10.1016/j.vetimm.2016.03.015

Halbert, N.D., N.D. Cohen, N.M. Slovis, J. Faircloth, & R.J. Martens. 2006. Variations in equid SLC11A1 (NRAMP1) genes and associations with rhodococcus equi pneumonia in horses. J. Vet. Intern. Med. 20: 974-979. https://doi.org/10.1111/j.1939-1676.2006.tb01814.x

Hamida, F., K.G. Wiryawan, & A. Meryandini. 2015. Selection of lactic acid bacteria as probiotic candidate for chicken. Med. Pet. 38:138-144. https://doi.org/10.5398/medpet.2015.38.2.138

Harbison, A.M. & J.N.T. Nguyen. 2017. PCR: Identifcation of genetic polymorphisms. Methods Mol. Biol. 1606: 193-203. https://doi.org/10.1007/978-1-4939-6990-6_13

He, X.M., M.X. Fang, Z.T. Zhang, Y.S. Hu, X.Z. Jia, D.L. He, S.D. Liang, Q.H. Nie, & X.Q. Zhang. 2013. Characterization of chicken natural resistance-associated macrophage proteinencoding genes (Nramp1 and Nramp2) and association with salmonellosis resistance. Genet. Mol. Res. 12: 618-630. https://doi.org/10.4238/2013.January.30.5

Hu, Y., Y.J. Shan, C.H. Zhu, W.T. Song, W.J. Xu, W.Q. Zhu, S.J. Zhang, & H.F. Li. 2015. Up-regulation of NRAMP1 mRNA confirms its role in enhanced host immunity in post-artificial infections of Salmonella enteritidis in chicks. Br. Poult. Sci. 56: 408-415. https://doi.org/10.1080/00071668.2015.1052371

Iskandar, S. 2018. Phenotypic characterization and distributin of SenSi-1 Agrinak chicken. Wartazoa. 28:051-060. https://doi.org/10.14334/wartazoa.v28i2.1673

Jin, Y.C., P. Wei, S. Cui, & L. Huang. 2015. The relationship between the three-dimensional (3D) structures of BF molecules and MHC-related Marek’s disease resistance in chickens. Scand. J. Immunol. 81: 325-327. https://doi.org/10.1111/sji.12278

Kramer, J., M. Malek, & S.J. Lamont. 2003. Association of twelve candidate gene polymorphisms and response to challenge with Salmonella enteritidis in poultry. Anim. Genet. 34: 339-348. https://doi.org/10.1046/j.1365-2052.2003.01027.x

Lamont, S.J., W. Liu, & M. Kaiser. 2002. Natural resistance associated macrophage protein 1 gene polymorphisms and response to vaccine against or challenge with Salmonella enteritidis in young chicks. Poult. Sci. 82: 259-266. https://doi.org/10.1093/ps/82.2.259

Lantier, I., C.R. Moreno, P. Berthon, G. Sallé, F. Pitel, L. Schibler, A.V. Gautier-Bouchardon, R. Boivin, J.L. Weisbecker, & D. François. 2012. Quantitative trait loci for resistance to infection in sheep using a live Salmonella abortusovis vaccine. Anim. Genet. 43: 632-635. https://doi.org/10.1111/j.1365-2052.2011.02291.x

Li, P., H. Wang, X. Zhao, Z. Gou, R. Liu, Y. Song, Q. Li, M. Zheng, H. Cui, N. Everaert, G. Zhao, & J. Wen. 2017. Allelic variation in TLR4 is linked to resistance to Salmonella enteritidis infection in chickens. Poult. Sci. 96: 2040-2048. https://doi.org/10.3382/ps/pex010

Liu, L., X.W. Zhao, Y.M. Song, Q.H. Li, P. Li, R.R. Liu, M.Q. Zheng, J. Wen, & G.P. Zhao. 2016. Difference in resistance to Salmonella enteritidis infection among allelic variants of TLR4 (903, 1832) in SPF chickens. J. Appl. Genet. 57: 389-396. https://doi.org/10.1007/s13353-015-0324-3

Machado, M.A., I. Schuster, M.L. Martinez, & A.L. Campos. 2003. Genetic diversity of four cattle breed using microsatellite markers. Rev. Bras. De Zool. 32: 93-98. https://doi.org/10.1590/S1516-35982003000100012

Mahmud, H.A., H. Seo, S. Kim, M.I. Islam, K.W. Nam, H.D. Cho, & H.Y. Song. 2017. Thymoquinone (TQ) inhibits the replication of intracellular Mycobacterium tuberculosis in macrophages and modulates nitric oxide production. BMC Complement Altern. Med. 17: 279-287. https://doi.org/10.1186/s12906-017-1786-0

Malek, M. & S.J. Lamont. 2003. Association of INOS, TRAIL, TGF-b2, TGF-b3, and IgL genes with response to Salmonella enteritidis in poultry. Genet. Sel. Evol. 1: 99-111. https://doi.org/10.1186/1297-9686-35-S1-S99

Mamutse, J., A. Gunawan, C. Sumantri, S. Murtini, & T. Sartika. 2018. Association of the Toll-Like Receptor 4 (TLR4) and Myxovirus (MX) genes with resistance to Salmonella and Newcastle Disease in selected sentul chickens. Int. J. Poult. Sci. 17: 591-599. https://doi.org/10.3923/ijps.2018.591.599

McPherson, M.J. & S.G. Moller. 2006. PCR. 2nd ed. Taylor & Francis Group, New York. https://doi.org/10.4324/9780203002674

Medapati, R.V., S. Suvvari, S. Godi, & P. Gangisetti. 2017. NRAMP1 and VDR gene polymorphisms in susceptibility to pulmonary tuberculosis among Andhra Pradesh population in India: a case-control study. BMC Pulm. Med. 17: 89-95. https://doi.org/10.1186/s12890-017-0431-5

Muhsinin, M., N. Ulupi, A. Gunawan, I.W.T. Wibawan, & C. Sumantri. 2016. Association of NRAMP1 polymorphisms with immune traits in Indonesian native chickens. Int. J. Poult. Sci. 15: 401-406. https://doi.org/10.3923/ijps.2016.401.406

Nei, M. & S. Kumar. 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York.

Noor, R.R. 2010. Genetika Ternak. Penebar Swadaya, Jakarta.

Pagala, M.A. & L.O. Nafiu. 2012. Identifikasi molekuler sifat anti viral ayam Tolaki melalui deteksi gen Mx sebagai marka genetik. Agriplus. 23:139-144.

Pecoraro, H.L., B. Thompson, & G.E. Duhamel. 2017. Histopathology case definition of naturally acquired Salmonella enterica serovar Dublin infection in young Holstein cattle in the northeastern United States. J. Vet. Diagn. Invest. 29: 860-864. https://doi.org/10.1177/1040638717712757

Pereira-Suárez, A.L., C. Estrada-Chávez, C. Arriaga-Díaz, P. Espinosa-Cueto, & R. Mancilla. 2006. Coexpression of NRAMP1, iNOS, and nitrotyrosine in bovine tuberculosis. Vet. Pathol. 43: 709-717. https://doi.org/10.1354/vp.43-5-709

Psifidi, A., G. Banos, O. Matika, T.T. Desta, J. Bettridge, D.A. Hume, T. Dessie, R. Christley, P. Wigley, O. Hanotte, & P. Kaiser. 2016. Genome-wide association studies of immune, disease and production traits in indigenous chicken ecotypes. Genet. Sel. Evol. 48: 74-90. https://doi.org/10.1186/s12711-016-0252-7

Ramasamy, K.T., M.R. Reddy, & S. Murugesan. 2011. Toll-like receptor mRNA expression, INOS gene polymorphism and serum nitric oxide levels in indigenous chickens. Vet. Res. Commun. 35: 321-327. https://doi.org/10.1007/s11259-011-9472-z

Reventun, P., M. Alique, I. Cuadrado, S. Marquez, R. Toro, C. Zaragoza, & M. Saura. 2017. iNOS-Derived nitric oxide induces integrin-linked kinase endocytic lysosome-mediated degradation in the vascular endothelium. Arterioscler Thromb Vasc. Biol. 37: 1272-1281. https://doi.org/10.1161/ATVBAHA.117.309560

Ryan, K.J. & C.G. Ray. 2014. Sherris Medical Microbiology. 6th ed. McGraw Hill, New York.

Sambrook, J., E.F. Fritsch, & T. Maniatis. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.

Serbina, N.V., T. Jia, T.M. Hohl, & E.G. Pamer. 2008. Monocyte-Mediated Defense against Microbial Pathogens. Annu. Rev. Immunol. 26:421. [PubMed: 18303997]. https://doi.org/10.1146/annurev.immunol.26.021607.090326

Sun, X., S. Li, Y. He, H. Zhao, Y. Wang, X. Zeng, & M. Xing. 2017. Arsenic-induced testicular toxicity in Gallus gallus: Expressions of inflammatory cytokines and heat shock proteins. Poult. Sci. 96:3399-3406. https://doi.org/10.3382/ps/pex073

Sundaresan, N.R., K.A. Ahmed, V.K. Saxena, K.V. Sastry, M. Saxena, A.B. Pramod, M. Nath, K.B. Singh, T.J. Rasool, A.K. DevRoy, & R.V. Singh. 2005. Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response. Vet. Immunol. Immunopathol. 108: 373-385. https://doi.org/10.1016/j.vetimm.2005.06.011

Tambasco, D.D., C.C.P. Paz, M. Tambasco-Studart, A.P. Pereira, M.M. Alencar, A.R. Freitas, L.L. Coutinho, I.U. Packer, & C.A. Regitano. 2003. Candidate genes for growth traits in beef cattle crosses Bos taurus x Bos indicus. J. Anim. Breed Genet. 120: 51-56. https://doi.org/10.1046/j.1439-0388.2003.00371.x

Togashi, K. & C.Y. Lin. 2010. Theoretical efficiency of multiple-trait quantitative trait loci-assisted selection. J. Anim. Breed Genet.127: 53-63. https://doi.org/10.1111/j.1439-0388.2009.00817.x

Tohidi, R., I. Idris, J.M. Panandam, & M.H. Bejo. 2012. The effects of polymorphisms in IL-2, IFN-γ, TGF-β2, IgL, TLR-4, MD-2, and iNOS genes on resistance to Salmonella enteritidis in indigenous chickens. Avian Pathol. 41: 605-612. https://doi.org/10.1080/03079457.2012.739680

Tohidi, R., I.B. Idris, J.M. Panandam, & M.H. Bejo. 2013. The effects of polymorphisms in 7 candidate genes on resistance to Salmonella enteritidis in native chickens. Poult. Sci. 92: 900-909. https://doi.org/10.3382/ps.2012-02797

Ulupi, N., Muladno, C. Sumantri, & I.W.T. Wibawan. 2013. Association of TLR4 gene genotype and resistance against Salmonella enteritidis natural infection in kampung chicken. Int. J. Poult. Sci. 12: 445-450. https://doi.org/10.3923/ijps.2013.445.450

Ulupi, N., Muladno, C. Sumantri, & I.W.T. Wibawan. 2014. Study of kampung chicken resistance against Salmonella enteritidis using TLR4 gene as marker. Int. J. Poult. Sci. 13: 467-472. https://doi.org/10.3923/ijps.2014.467.472

Ulupi, N., Muladno, C. Sumantri, & I.W.T. Wibawan. 2014. Identifikasi keragaman gen Toll-Like Receptor-4 ayam lokal dengan polymerase chain reaction- restriction fragment lenght polymorphism. Jurnal Veterin. 15: 345-352.

Velge, P., A. Cloeckaert, & P. Barrow. 2005. Emergence of Salmonella epidemics : the problems related to Salmonella enterica serotype enteritidis and multiple antibiotic resistance in other major serotypes. Vet. Res. 36:267-288. https://doi.org/10.1051/vetres:2005005

Viljoen, G.J., L.H. Nel, & J.R. Crowther. 2005. Molecular Diagnosis PCR Handbook. Springer, Netherlands.

Webster, R., N. Cox, & K. Stohr. 2002. WHO Manual on Animal Influenza Diagnosis and Surveillance.

Wibawan, I.W.T. & R.D. Soedjono. 2013. Intisari Imunologi Medis. Fakultas Kedokteran Hewan Institut Pertanian Bogor, Bogor

Wils-Plotz, E.L. & K.C. Klasing. 2016. Effects of immunomodulatory nutrients on growth performance and immune-related gene expression in layer chicks challenged with lipopolysaccharide. Poult. Sci. 96: 548-555. https://doi.org/10.3382/ps/pew376

Yan, M., M. Hou, J. Liu, S. Zhang, B. Liu, X. Wu, & G. Liu. 2017. Regulation of INOS-Derived ROS generation by HSP90 and Cav-1 in porcine reproductive and respiratory syndrome virus-infected swine lung injury. Inflammation 40: 1236-1244. https://doi.org/10.1007/s10753-017-0566-9

Yuniwarti, E.Y.W., W. Asmara, W.T. Artama, & C.R. Tabbu. 2013. Virgin coconut oil meningkatkan aktivitas fagositosis makrofag ayam pedaging pasca vaksinasi flu burung. J. Vet. 14: 190-196.

How to Cite
Ardiyana, M., Gunawan, A., Murtini, S., Sartika, T., & Sumantri, C. (2020). Polymorphisms and Associations of the NRAMP-1 and iNOS Genes on Newcastle Disease and Salmonella enteritidis Resistances in SenSi-1 Agrinak Chickens. Tropical Animal Science Journal, 43(2), 95-102. https://doi.org/10.5398/tasj.2020.43.2.95