Veterinary Research Communications

, Volume 43, Issue 4, pp 225–230 | Cite as

Co-administration of toll-like receptor (TLR)-3 and 4 ligands augments immune response to Newcastle disease virus (NDV) vaccine in chicken

  • T. R. KannakiEmail author
  • E. Priyanka
  • M. R. Reddy
Original Article


Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate first line of host defence to pathogens. TLR agonists are potent immunostimulatory agents that help to prime a robust adaptive immune response. In the present study, adjuvant potential of Poly I:C and lipopolysaccharide (LPS) were evaluated with live Newcastle disease virus (NDV) vaccine. Cornish chickens were immunized with live Newcastle disease virus (NDV) vaccine (R2B-mesogenic strain) adjuvanted either with Poly I:C (TLR3 agonist) or LPS-TLR4 agonist and both. Humoral Immune response to ND vaccine was evaluated through haemagglutination inhibition (HI) test and ELISA, while the cellular immune response (CMI) was quantified by lymphocyte transformation test (LTT). IL-1β cytokine mRNA levels in spleen tissue were also quantified by real time PCR. The results suggest that TLR3 and TLR4 agonists are an efficient immune-stimulators separately, as LPS co-administered group has shown significantly higher serum titre on second week post-immunization and Poly I:C group on third week post-immunization both by HI and ELISA (P < 0.01), however, the combined administration of both LPS and Poly I:C did not give any complementary effect on serum titre. There were no significant differences in stimulation indices (SI) and IL-1β cytokine levels between groups at different intervals post-immunization. Hence, TLR agonists LPS followed by Poly I:C could be used as adjuvant to enhance the immune response to NDV vaccine in chicken.


NDV vaccine Immune response TLR agonist Poly I:C LPS 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Adler HE, DaMassa AJ (1979) Toxicity of endotoxin to chicks. Avian Dis 23(1):174–178PubMedCrossRefGoogle Scholar
  2. Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2(8):675–680CrossRefGoogle Scholar
  3. Arsenault RJ, Kogut MH, He H (2013) Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands. Cell Signal 25(11):2246–2254PubMedCrossRefGoogle Scholar
  4. Bashir K, Kappala D, Singh Y, Dar JA, Mariappan AK, Kumar A, Krishnaswamy N, Dey S, Chellappa MM, Goswami TK, Gupta VK, Ramakrishnan S (2019) Combination of TLR2 and TLR3 agonists derepress infectious bursal disease virus vaccine-induced immunosuppression in the chicken. Sci Rep 9(1):8197PubMedPubMedCentralCrossRefGoogle Scholar
  5. Berczi I (1998) Neurohormonal host defense in endotoxin shock. Ann. N.Y. Acad Sci 840:787–802PubMedCrossRefGoogle Scholar
  6. Berczi I, Bertók L, Bereznai T (1966) Comparative studies on the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Can J Microbiol 12:1070–1071PubMedCrossRefGoogle Scholar
  7. Chaung HC, Cheng LT, Hung LH, Tsai PC, Skountzou I, Wang B, Compans RW, Lien YY (2012) Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens. Vet Microbiol 157(1–2):69–77PubMedCrossRefGoogle Scholar
  8. Chen R, Alvero AB, Silasi DA, Mor G (2007) Inflammation, cancer and chemoresistance: taking advantage of the toll-like receptor signaling pathway. Am J Reprod Immunol 57:93–107PubMedCrossRefGoogle Scholar
  9. Dil N, Qureshi MA (2002) Involvement of lipopolysaccharide related receptors and nuclear factor kappa B in differential expression of inducible nitric oxide synthase in chicken macrophages from different genetic backgrounds. Vet Immunol Immunopathol 88(3–4):149–161PubMedCrossRefGoogle Scholar
  10. Fan YF, Hou ZC, Yi GQ, Xu GY, Yang N (2013) The sodium channel gene family is specifically expressed in hen uterus and associated with eggshell quality traits. BMC Genet 14(1):90PubMedPubMedCentralCrossRefGoogle Scholar
  11. Farnell MB, He H, Kogut MH (2003) Differential activation of signal transduction pathways mediating oxidative burst by chicken heterophils in response to stimulation with lipopolysaccharide and lipoteichoic acid. Inflammation. 27(4):225–231PubMedCrossRefGoogle Scholar
  12. Gould AR, Kattenbelt JA, Selleck P, Hansson E, Della-Porta A, Westbury HA (2001) Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000. Virus Res 77(1):51–60PubMedCrossRefGoogle Scholar
  13. Gupta SK, Deb R, Dey S, Chellappa MM (2014 Jul) Toll-like receptor-based adjuvants: enhancing the immune response to vaccines against infectious diseases of chicken. Expert Rev Vaccines 13(7):909–925PubMedCrossRefGoogle Scholar
  14. He H, Crippen TL, Farnell MB, Kogut MH (2003) Identification of CpG oligodeoxynu-cleotide motifs that stimulate nitric oxide and cytokine production in avianmacrophage and peripheral blood mononuclear cells. Dev Comp Immunol 27(6):621–627PubMedCrossRefGoogle Scholar
  15. He H, Genovese KJ, Nisbet DJ, Kogut MH (2006) Profile of toll-like receptor expressions and induction of nitric oxide synthesis by toll-like receptor agonists in chicken monocytes. Mol Immunol 43(7):783–789PubMedCrossRefGoogle Scholar
  16. He H, MacKinnon KM, Genovese KJ, Kogut MH (2011) CpG oligodeoxynucleotide and double-stranded RNA synergize to enhance nitric oxide production and mRNA expression of inducible nitric oxide synthase, pro-inflammatory cytokines and chemokines in chicken monocytes. Innate Immun 17(2):137–144PubMedCrossRefGoogle Scholar
  17. Iqbal M, Philbin VJ, Smith AL (2005) Expression patterns of chicken toll-like receptor mRNA in tissues, immune cell subsets and cell lines. Vet Immunol Immunopathol 104(1–2):117–127PubMedCrossRefGoogle Scholar
  18. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu RevImmunol 20:197–216Google Scholar
  19. Kannaki T, Reddy M, Shanmugam M, Verma P, Sharma R (2010) Chicken toll-like receptors and their role in immunity. World's Poultry Science Journal 66(4):727–738CrossRefGoogle Scholar
  20. Karnati HK, Pasupuleti SR, Kandi R, Undi RB, Sahu I, Kannaki TR, Subbiah M, Gutti RK (2015 Mar) TLR-4 signalling pathway: MyD88 independent pathway up-regulation in chicken breeds upon LPS treatment. Vet Res Commun 39(1):73–78PubMedCrossRefGoogle Scholar
  21. Karpala AJ, Lowenthal JW, Bean AG (2008) Activation of the TLR3 pathway regulates IFNbeta production in chickens. Dev Comp Immunol 32(4):435–444PubMedCrossRefGoogle Scholar
  22. Keestra AM, van Putten JP (2008) Unique properties of the chicken TLR4/MD-2 complex: selective lipopolysaccharide activation of the MyD88-dependent pathway. J Immunol 181(6):4354–4362PubMedCrossRefGoogle Scholar
  23. Kogut MH, Iqbal M, He H, Philbin V, Kaiser P, Smith A (2005) Expression and function of Toll-like receptors in chicken heterophils. Dev Comp Immunol 29(9):791–807PubMedCrossRefGoogle Scholar
  24. Kogut MH, Swaggerty C, He H, Pevzner I, Kaiser P (2006) Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses. Microbes Infect 8(7):1866–1874PubMedCrossRefGoogle Scholar
  25. Linghua Z, Xingshan T, Fengzhen Z (2007) Vaccination with Newcastle disease vaccine and CpG oligodeoxynucleotides induces specific immunity and protection against Newcastle disease virus in SPF chicken. Vet Immunol Immunopathol 115(3–4):216–222PubMedCrossRefGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4):402–408CrossRefGoogle Scholar
  27. Marcus PI, Sekellick MJ (2001) Combined sequential treatment with interferon and dsRNA abrogates virus resistance to interferon action. J Interf Cytokine Res 21(6):423–429CrossRefGoogle Scholar
  28. McAleer JP, Rossi RJ, Vella AT (2009) Lipopolysaccharide potentiates effector T cell accumulation into nonlymphoid tissues through TRIF. J Immunol 182:5322–5330PubMedPubMedCentralCrossRefGoogle Scholar
  29. Mogensen TH (2009) Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev 22(2):240–273PubMedPubMedCentralCrossRefGoogle Scholar
  30. Mosman T (1983) Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J Immunol Meth 65(1):55–63CrossRefGoogle Scholar
  31. Nang NT, Lee JS, Song BM, Kang YM, Kim HS, Seo SH (2011) Induc-tion of inflammatory cytokines and toll-like receptors in chickens infected with avian H9N2 influenza virus. Vet Res 42(64):10–186Google Scholar
  32. Parvizi P, Mallick AI, Haq K (2012) A toll-like receptor 3 ligand enhances protective effects of vaccination against marek’s disease virus and hinders tumor development in chickens. Viral Immunol 25(5):394–401PubMedCrossRefGoogle Scholar
  33. Paul MS, Mallick AI, Read LR et al (2012) Prophylactic treatment with toll-like receptor ligands enhances host immunity to avian influenza virus in chickens. Vaccine 30(30):4524–4531CrossRefGoogle Scholar
  34. Philbin VJ, Iqbal M, Boyd Y, Goodchild MJ, Beal RK, Bumstead N, Young J, Smith AL (2005) Identification and characterization of a functional, alternatively spliced toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens. Immunology. 114(4):507–521PubMedPubMedCentralCrossRefGoogle Scholar
  35. Ramakrishnan S, Annamalai A, Sachan S, Kumar A, Sharma BK, Govindaraj E (2015) Synergy of lipopolysaccharide and resiquimod on type I interferon,pro-inflammatory cytokine, Th1 and Th2 response in chicken peripheral bloodmononuclear cells. Mol Immunol 64(1):177–182PubMedCrossRefGoogle Scholar
  36. Ruan WK, Zheng SJ (2011) Polymorphisms of chicken toll-like receptor 1 type 1 and type 2 in different breeds. Poult Sci 90(9):1941–1947PubMedCrossRefGoogle Scholar
  37. Sachan S, Ramakrishnan S, Annamalai A, Sharma BK, Malik H, Saravanan BC, Jain L, Saxena M, Kumar A, Krishnaswamy N. (2015) Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken. 33(36):4526–4532Google Scholar
  38. Seppala IJ, Makela O (1984) Adjuvant effect of bacterial LPS and/or alum precipitation in responses to polysaccharide and protein antigens. Immunology 53:827–836PubMedPubMedCentralGoogle Scholar
  39. St Paul M, Mallick AI, Haq K, Orouji S, Abdul-Careem MF, Sharif S. (2011) In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen. Vet Immunol Immunopathol 144(3-4):228–237Google Scholar
  40. Tseng LP, Chiou CJ, Chen CC, Deng MC, Chung TW, Huang YY, Liu D (2009) Effect of lipopolysaccharide on intranasal administration of liposomal Newcastle disease virus vaccine to SPF chickens. Vet Immunol Immunopathol 131:285–289PubMedCrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Avian Health LabICAR-Directorate of Poultry ResearchHyderabadIndia

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