Skip to main content Accessibility help

Role of cytosine-phosphate-guanosine-Oligodeoxynucleotides (CpG ODNs) as adjuvant in poultry vaccines

  • M. USMAN ISHAQ (a1), A. RAFIQUE (a2), H.M.N. CHEEMA (a3), M. UMER ASHRAF (a1), S.U. RAHMAN (a1), R. ZAHID ABBAS (a4) and M. SHAHID MAHMOOD (a1)...


Oligodeoxynucleotides (ODNs) containing cytosine-phosphate-guanosine sequence (CpG) is considered as an immune stimulator when it is fed to animals. These synthetic molecules mount different immune responses in the animals including mice, chickens, ducks, dogs and horses. CpG ODNs induce specific antigenic immunity against co-administered vaccines and are well tolerated in healthy individuals and are capable of stimulating toll-like receptors (TLRs) such as TLR-9 to activate innate immunity. The CpG ODNs can be used as an adjuvant in different vaccines synthesised specifically for poultry diseases caused by viruses and bacteria. In chickens, CpG ODNs stimulate TLRs involved in humoral immunity. CpG ODNs have been used as mucosal vaccine adjuvants against several pathogens, including avian influenza and Newcastle disease. The CpG ODNs function to protect the chickens from Newcastle disease by producing plasma dendric cells (pDCs) which ultimately produce interferons (INFs). The inoculation of CpG ODNs along with the cationic microparticles and DNA vaccine for infectious bursal disease virus result in the influx of T cells and a reduction of antigen load. When CpG ODNs are used against avian leucosis, they result in significantly higher antibody titres. In many other vaccines e.g., infectious laryngotracheitis, infectious bronchitis, herpes, viral enteritis, Marek's disease virus, E. coli and Salmonella spp. including CpG ODNs exhibit immunostimulatory effects. In conclusion, CpG ODNs may be used as effective adjuvants in viral, bacterial and parasitic vaccines in poultry.


Corresponding author

Corresponding author:


Hide All
ABBAS, A., IQBAL, Z., ABBAS, R.Z., KHAN, M.K. and KHAN, J.A. (2017) immunomodulatory activity of Pinus radiata extract against Coccidiosis in broiler chicken. Pakistan Veterinary Journal 37: 145-149.
ALKIE, T.N., ST. PAUL, M., BARJESTEH, N., BRISBIN, J. and SHARIF, S. (2015) Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands. Veterinary Immunology and Immunopathology 163: 157-163.
ASLAM, A., SHAHZAD, M.I., PARVEEN, S., ASHRAF, H., NAZ, N., ZEHRA, S.S., KAMRAN, Z., QAYYUM, A. and MUKHTAR, M. (2016) Evaluation of Antiviral Potential of Different Cholistani Plants against Infectious Bursal Disease and Infectious Bronchitis Virus. Pakistan Veterinary Journal 36: 302-306.
BARJESTEH, N., SHOJADOOST, B., BRISBIN, J.T., EMAM, M., HODGINS, D.C., NAGY, E. and SHARIF, S. (2015) Reduction of avian influenza virus shedding by administration of Toll-like receptor ligands to chickens. Vaccine 33: 4843-4849.
BODE, C., ZHAO, G., STEINHAGEN, F., KINJO, T. and KLINMAN, D.M. (2011) CpG DNA as a vaccine adjuvant. Expert Reviews Vaccines 10: 499-511.
CHU, R.S., TARGONI, O.S., KRIEG, A.M., LEHMANN, P.V. and HARDING, C.V. (1997) CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. The Journal of Experimental Medicine 186: 1623-1631.
COFFMAN, R.L., SHER, A. and SEDER, R.A. (2010) Vaccine adjuvants: Putting innate immunity to work. Immunity 33: 492-503.
CUI, N., LI, Y., SU, S., CUI, Z., DING, J., KANG, M., SUN, P. and ZHU, H. (2016) Protection of Chickens against Very Virulent Marek's Disease Virus (MDV) by an Infectious Clone of Meq-Null MDV Vaccination. Pakistan Veterinary Journal 36: 16-20.
DAR, A., TIKOO, S., POTTER, A., BABIUK, L.A., TOWNSEND, H., GERDTS, V. and MUTWIRI, G. (2014) CpG-ODNs induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs. Veterinary Immunology and Immunopathology 160: 209-217.
DOU, W., LI, H., CHENG, Z., ZHAO, P., LIU, J., CUI, Z., LIU, H., JING, W. and GUO, H. (2013) Maternal antibody induced by recombinant gp85 protein vaccine adjuvanted with CpG-ODN protects against ALV-J early infection in chickens. Vaccine 31: 6144-6149.
FATIMA, Z., KHAN, M.A., AHMAD, M.D., MUHAMMAD, K., KHWAJA, K.N., KHAN, A., ANWAR, Z., AHAD, A. and MAHMOOD, A. (2017) Cross sectional survey of live bird markets and zoo birds for circulating influenza subtypes in Pakistan. Pakistan Veterinary Journal 37: 185-189.
FU, J., LIANG, J., KANG, H., LIN, J., YU, Q. and YANG, Q. (2013) Effects of different CpG oligodeoxynucleotides with inactivated avian H5N1 influenza virus on mucosal immunity of chickens. Poultry Science 92: 2866-2875.
GOMIS, S., BABIUK, L., GODSON, D.L., ALLAN, B., THRUSH, T., TOWNSEND, H., WILLSON, P., WATERS, E., HECKER, R. and POTTER, A. (2003) Protection of Chickens against Escherichia coli Infections by DNA Containing CpG Motifs. Infection and Immunity 71: 857-863.
GUL, S.T., KHAN, A., FAROOQ, M., NIAZ, S., AHMAD, M., KHATOON, A., HUSSAIN, R., SALEEMI, M.K. and HASSAN, M.F. (2017) Effect of Sub lethal doses of Thiamethoxam (A Pesticide) on Hemato-Biochemical values in cockerels. Pakistan Veterinary Journal 37: 135-138.
GUNAWARDANA, T., FOLDVARI, M., ZACHAR, T., POPOWICH, S., CHOW-LOCKERBIE, B., IVANOVA, M.V., TIKOO, S., KURUKULASURIYA, S., WILSON, P. and GOMIS, S. (2015) Protection of Neonatal Broiler Chickens Following in ovo Delivery of Oligodeoxynucleotides Containing CpG Motifs (CpG-ODN) Formulated with Carbon Nanotubes or Liposomes. Avian Diseases 59: 31-37.
HÄCKER, G., REDECKE, V. and HÄCKER, H. (2002) Activation of the immune system by bacterial CpG-DNA. Immunology 105: 245-251.
HAN, Q., GAO, X., WU, P., XIAO, S., WANG, X., LIU, P., TONG, L., HAO, H., ZHANG, S., DANG, R. and YANG, Z. (2017) Research in veterinary science re-evaluation the immune efficacy of Newcastle disease virus vaccine in commercial laying chickens. Research in Veterinary Science 111: 63-66.
HARTLEY, C., SALISBURY, A.M. and WIGLEY, P. (2012) CpG oligonucleotides and recombinant interferon-γ in combination improve protection in chickens to Salmonella enterica serovar Enteritidis challenge as an adjuvant component, but have no effect in reducing Salmonella carriage in infected chickens. Avian Pathology 41: 77-82.
HASSAN, K.U., KHALIQUE, A., PASHA, T.N., AKRAM, M., MAHMOOD, S., SAHOTA, A.W., IMRAN, M.S. and SALEEM, G. (2017) Influence of Moringa oleifera decorticated seed meal on broiler performance and immunity. Pakistan Veterinary Journal 37: 47-50.
HE, H., GENOVESE, K.J., SWAGGERTY, C.L., MACKINNON, K.M. and KOGUT, M.H. (2012) Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-gamma and regulatory cytokine IL-10 expression in chicken monocytes. Developmental and Comparative Immunology 36: 756-760.
HE, H., GENOVESE, K.J., SWAGGERTY, C.L., NISBET, D.J. and KOGUT, M.H. (2007) In vivo priming heterophil innate immune functions and increasing resistance to Salmonella enteritidis infection in neonatal chickens by immune stimulatory CpG oligodeoxynucleotides. Veterinary Immunology and Immunopathology 117: 275-283.
HUNG, L., TSAI, P., WANG, C., LI, S., HUANG, C., LIEN, Y. and CHAUNG, H. (2011) Immunoadjuvant efficacy of plasmids with multiple copies of a CpG motif co-administrated wit avian influenza vaccine in chickens. Vaccine 29: 4668-4675.
HUSSAIN, Z., KHAN, M.Z., SALEEMI, M.K., KHAN, A. and RAFIQUE, S. (2016) Clinicopathological effects of prolonged intoxication of aflatoxin B1 in broiler chicken. Pakistan Veterinary Journal 36: 477-481.
KANG, H., WANG, H., YU, Q. and YANG, Q. (2012) Effect of intranasal immunization with inactivated avian influenza virus on local and systemic immune responses in ducks. Poultry Science 91: 1074-1080.
KANG, H., WANG, H., YU, Q. and YANG, Q. (2013) A novel combined adjuvant strongly enhances mucosal and systemic immunity to low pathogenic avian influenza after oral immunization in ducks. Poultry Science 92: 1543-1551.
KEMP, T.J., ELZEY, B.D. and GRIFFITH, T.S. (2003) Plasmacytoid dendritic cell-derived IFN-alpha induces TNF-related apoptosis-inducing ligand/Apo-2L-Mediated antitumor activity by human monocytes following CpG oligodeoxynucleotide stimulation. The Journal of Immunology 171: 212-218.
KHAN, J.M., EL-ASHRAM, S., LIU, H.B., KHAN, S.H., AYAN, A., LIU, X.Y., WANG, H., TANG, X.M., SUO, X. and HASSAN, M.F. (2016) Transgenic Eimeria mitis expressing chicken IL-4 mediated decrease in pathogenicity compared to wild type Eimeria mitis strains in broiler chickens. Pakistan Veterinary Journal 36: 415-420.
KODAMA, S., ABE, N., HIRANO, T. and SUZUKI, M. (2006) Safety and Efficacy of Nasal Application of CpG Oligodeoxynucleotide as a Mucosal Adjuvant. Laryngoscope 116: 331-335.
KOO, B.S., JEON, E.O., BAE, Y.J., MO, J.S., KIM, J.N. and MO, I.P. (2016) An Endogenous Avian Leukosis Virus Element in the Genome of Commercial Chickens Showing Emaciation of Unknown Etiology. Pakistan Veterinary Journal 36: 233-235.
KRIEG, A.M., YI, A.K., MATSON, S., WALDSCHMIDT, T.J., BISHOP, G.A., TEASDALE, R., KORETZKY, G.A. and KLINMAN, D.M. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374: 546-549.
KWANT, A. and ROSENTHAL, K.L. (2004) Intravaginal immunization with viral subunit protein plus CPG oligodeoxynucleotides induces protective immunity against HSV-2. Vaccine 22: 3098-3104.
LI, Y., CAO, H., WANG, N., XIANG, Y., LU, Y., ZHAO, K., ZHENG, J. and ZHOU, H. (2011) A novel antagonist of TLR9 blocking all classes of immunostimulatory CpG-ODNs. Vaccine 29: 2193-2198.
LINGHUA, Z., XINGSHAN, T. and FENGZHEN, Z. (2007) Vaccination with Newcastle disease vaccine and CpG oligodeoxynucleotides induces specific immunity and protection against Newcastle disease virus in SPF chicken. Veterinary Immunology and Immunopathology 115: 216-222.
MALLICK, A.I., PARVIZI, P., READ, L.R., NAGY, E., BEHBOUDI, S. and SHARIF, S. (2011) Enhancement of immunogenicity of a virosomes-based avian influenza vaccine in chickens by incorporating CpG-ODN. Vaccine 29: 1657-1665.
NEGASH, T., LIMAN, M. and RAUTENSCHLEIN, S. (2013) Mucosal application of cationic poly(D,L-Lactide-co-Glycoside) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease. Vaccine 31: 3656-3662.
OXENIUS, A, MARTINIC, M.M., HENGARTNER, H. and KLENERMAN, P. (1999) CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines. Journal of Virology 73: 4120-4126.
PARVIZI, P., ABDUL-CAREEM, M.F., MALLICK, A.I., HAQ, K., HAGHIGHI, H.R., OROUJI, S., HEIDARI, M., BEHBOUDI, S. and SHARIF, S. (2014) The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek's disease in chickens. Vaccine 32: 1932-1938.
PATEL, B.A., GOMIS, S., DAR, A., WILLSON, P.J., BABIUK, L.A., POTTER, A., MUTWIRI, G. and TIKOO, S.K. (2008) Oligodeoxynucleotides containing CpG motifs (CpG-ODN) predominantly induce Th1-type immune response in neonatal chicks. Developmental and Comparative Immunology 32: 1041-1049.
PAUL, M.S., PAOLUCCI, S., READ, L.R. and SHARIF, S. (2012) Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens. Veterinary Immunology and Immunopathology 149: 237-244.
PETROVSKY, N. and AGUILAR, J.C. (2004) Vaccine adjuvants: Current state and future trends. Immunology and Cell Biology 82: 488-496.
QI, Y., YAN, B., CHEN, S., CHEN, H., WANG, M., JIA, R., ZHU, D., LIU, M., LIU, F., YANG, Q., SUN, K., WU, Y., CHEN, X., JING, B. and CHENG, A. (2016) CpG oligodeoxynucleotide-specific goose TLR21 initiates an anti-viral immune response against NGVEV but not AIV strain H9N2 infection. Immunobiology 221: 454-461.
QIN, T., YIN, Y., YU, Q., HUANG, L., WANG, X., LIN, J. and YANG, Q. (2015) CpG Oligodeoxynucleotides Facilitate Delivery of Whole Inactivated H9N2 Influenza Virus via Trans-Epithelial Dendrites of Dendritic Cells in Nasal Mucosa. Journal of Virology 89: 5904-5918.
ROH, H.J., SUNG, H.W. and KWON, H.M. (2006) Effects of DDA, CpG-ODN, and plasmid-encoded chicken IFN-γ on protective immunity by a DNA vaccine against IBDV in chickens. Journal of Veterinary Science 7: 361-368.
ROMAGNANI, S. (2000) T-cell subsets (Th1 versus Th2). Annals of Allergy, Asthma and Immunology 85: 9-18.
SCHEIERMANN, J. and KLINMAN, D.M. (2014) Clinical evaluation of CpG oligodeoxynucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine 32: 6377-6389.
SHAHROKHI, V., RAD, M. and KALIDARI, G.A. (2013) Treatment of newly hatched chicken with CpG oligodeoxynucleotides decreases liver/spleen colonization of Salmonella enteritidis in broiler chickens. Comparative Clinical Pathology 22: 935-939.
SHIROTA, H., TROSS, D. and KLINMAN, D. (2015) CpG Oligonucleotides as Cancer Vaccine Adjuvants. Vaccine 3: 390-407.
SINGH, S.M., ALKIE, T.N., ABDELAZIZ, K.T., HODGINS, D.C., NOVY, A., NAGY, E. and SHARIF, S. (2016) Characterization of Immune Responses to an Inactivated Avian Influenza Virus Vaccine Adjuvanted with Nanoparticles Containing CpG ODN. Viral Immunology 29: 269-275.
SINGH, S.M., ALKIE, T.N., HODGINS, D.C., NAGY, E., SHOJADOOST, B. and SHARIF, S. (2015) Systemic immune responses to an inactivated, whole H9N2 avian influenza virus vaccine using class B CpG oligonucleotides in chickens. Vaccine 33: 3947-3952.
TAGHAVI, A., ALLAN, B., MUTWIRI, G., FOLDVARI, M., VAN KESSEL, A., WILLSON, P., BABIUK, L., POTTER, A. and GOMIS, S. (2009) Enhancement of immunoprotective effect of CpG-ODN by formulation with polyphosphazene against E. coli septicaemia in neonatal chickens. Current Drug Delivery 6: 76-82.
THAPA, S., CADER, M.S.A., MURUGANANTHAN, K., NAGY, E., SHARIF, S., CZUB, M. and ABDUL-CAREEM, M.F. (2015) In ovo delivery of CpG DNA reduces avian infectious laryngotracheitis virus induced mortality and morbidity. Viruses 7: 1832-1852.
WANG, Y., SHAN, C., MING, S., LIU, Y., DU, Y. and JIANG, G. (2009) Immunoadjuvant effects of bacterial genomic DNA and CpG oligodeoxynucleotides on avian influenza virus subtype H5N1 inactivated oil emulsion vaccine in chicken. Research in Veterinary Science 86: 399-405.
WEINER, G.J., LIU, H.M., WOOLDRIDGE, J.E., CHRISTOPHER, E.D. and ARTHUR, M.K. (1997) Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization. Proceedings of the National Academy of Sciences of the United States of America 94: 10833-10837.
WIBOWO, M.H., ANGGORO, D., AMANU, S., WAHYUNI, A.E.T.H., UNTARI, T., ARTANTO, S. and ASMARA, W. (2017) Receptor binding and antigenic site analysis of hemagglutinin gene fragments of avian influenza virus serotype H5N1 isolated from Indonesia. Pakistan Veterinary Journal 37: 123-128.
XIAOWEN, Z., QINGHUA, Y., XIAOFEI, Z. and QIAN, Y. (2009) Co-administration of inactivated avian influenza virus with CpG or rIL-2 strongly enhances the local immune response after intranasal immunization in chicken. Vaccine 27: 5628-5632.
YU, Z., GUO, F., ZHANG, Z., LUO, X., TIAN, J. and LI, H. (2017) Protective effects of Glycyrrhizin on LPS and Amoxicillin/Potassium Clavulanate-Induced liver injury in chicken. Pakistan Veterinary Journal 37: 13-18.
ZAHEER, Z., HUSSAIN, I., RAHMAN, S.U., YOUNAS, T., ZAHEER, I., ABBAS, G. and NASIR, M. (2017) Occurrence and antibiotic susceptibility of Methicillin-Resistant Staphylococcus aureus recovered from Oropharynx of live cockerels. Pakistan Veterinary Journal 37: 108-110.
ZHANG, D., LI, H., ZHANG, Z., SUN, S., CHENG, Z., LIU, J., ZHAO, P., REN, Q. and GUO, H. (2015) Antibody responses induced by recombinant ALV-A gp85 protein vaccine combining with CpG-ODN adjuvant in breeder hens and the protection for their offspring against early infection. Antiviral Research 116: 20-26.
ZHANG, H. and GAO, X.D. (2017) Nano delivery systems for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides. Materials Science and Engineering 70: 935-946.
ZHANG, L., ZHANG, M., LI, J., CAO, T., TIAN, X. and ZHOU, F. (2008) Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo. Research in Veterinary Science 85: 495-502.
ZHANG, T., WANG, T., ZHAO, P., LIANG, M., GAO, Y., YANG, S., QIN, C., WANG, C. and XIA, X. (2011) Antisense oligonucleotides targeting the RNA binding regions of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1. International Immunopharmacology 11: 2057-2061.
ZHANG, Z. and WANG, F-S. (2005) Plasmacytoid dendritic cells act as the most competent cell type in linking antiviral innate and adaptive immune responses. Cellular and Molecular Immunology 2: 411-417.



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed