Aim: The objective of the present study was to prepare a trivalent inactivated vaccine of Newcastle disease virus (NDV), H5N1, and H9N2 viruses.

Materials and Methods: Three monovalent and a trivalent vaccines were prepared by emulsifying inactivated NDV (LaSota strain), reassortant H5N1, and H9N2 viruses with Montanide ISA 71 oil adjuvant. Parameters used for evaluation of the efficacy of the prepared vaccines in specific pathogen-free chickens were cellular immunity assays (blastogenesis, interferon gamma, interleukin 1 [IL1], and IL6), humoral immunity by hemagglutination inhibition, protection percentage, and shedding.

Results: A single immunization with trivalent vaccine-enhanced cell-mediated immunity as well as humoral immune response with 90% protection against challenges with highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic (LP) avian influenza H9N2 viruses with 100% protection after challenge with NDV.

Conclusion: Development and evaluation of the trivalent vaccine in the study reported the success in preparation of a potent and efficacious trivalent vaccine which is a promising approach for controlling HPAI H5N1, LP H9N2, and ND viral infections.

Keywords: avian influenza, immunization, Newcastle disease virus, shedding, trivalent vaccine.

1. Swayne, D.E., Suarez, D.L. and Sims, L.D. (2013) Influenza. In: Swayne, D.E., Glisson, J.R., McDougald, L.R., Nair, V., Nolan, L.K., Suarez, D.L. and Nair, V., editors. Diseases of Poultry. Vol. 13. Willey-Blackwell; Ames, Iowa. p181-218. [Crossref]

2. FAO. (2015) Improvement of Biosecurity and Production Practices in Hatcheries and Parent Flocks. Rome. Available from: http://www.fao.org/3/a-bb033e.pdf. Last accessed on 20-06-2016.

3. Webster, R.G., Webby, R.J., Hoffmann, E., Rodenberg, J., Kumar, M., Chu, H.J., Seiler, P., Krauss, S. and Songserm, T. (2006) The immunogenicity and efficacy against H5N1 challenge of reverse genetics derived H5N3 influenza vaccine in ducks and chickens. Virology, 351: 303-311. [Crossref] [PubMed]

4. Tian, G., Zeng, X., Li, Y., Shi, J. and Chen, H. (2010) Protective efficacy of the H5 inactivated vaccine against different highly pathogenic H5N1 avian influenza viruses isolated in China and Vietnam. Avian Dis., 54: 287-289. [Crossref] [PubMed]

5. Swayne, D.E., Beck, J.R., Garcia, M. and Stone H.D. (1999) Influence of virus strain and antigen mass on the efficacy of H5 avian influenza inactivated vaccines. Avian Pathol., 28: 245-255. [Crossref] [PubMed]

6. Kayali, G., Kandeil, A., El-Shesheny, R., Kayed, A.S., Maatouq, A.M., Cai, Z. and Ali, M.A. (2016) Avian influenza A (H5N1) virus in Egypt. Emerg. Infect. Dis., 22(3): 379-388. [Crossref] [PubMed] [PMC]

7. Afifi, M.A., El-Kady, M.F., Zoelfakar, S.A. and Abdel-Moneim, A.S. (2013) Serological surveillance reveals widespread influenza A H7 and H9 subtypes among chicken flocks in Egypt. Trop. Anim. Health Prod., 45: 687-690. [Crossref] [PubMed]

8. Monne, I., Hussein, H.A., Fusaro, A., Valastro, V., Hamoud, M.M., Khalefa, R.A., Dardir, S.N., Radwan, M.I., Capua, I. and Cattolim, G. (2013) H9N2 influenza a virus circulates in H5N1 endemically infected poultry population in Egypt. Influenza Other Respir Viruses, 7: 240-243. [Crossref] [PubMed]

9. Arafa, A.S., Hagag, N.M., Yehia, N., Zanaty, A.M., Naguib, M.M. and Nasef, S.A. (2013) Effect of circulation of highly pathogenic avian influenza H5N1 subtype with low pathogenic H9N2 subtype on the spread of infections. Avian Dis., 56: 849-857. [Crossref] [PubMed]

10. Kayali, G., Kandeil, A., El-Shesheny, R., Kayed, A.S., Gomaa, M.M., Maatouq, A.M., Shehata, M.M., Moatasim, Y., Bagato, O., Cai, Z., Rubrum, A, Mohamed, A.K, Pamela, P.M., Webster, R.G., Webby, R.J. and Mohamed, A.A. (2014) Active surveillance for avian influenza virus, Egypt, 2010-2012. Emerg. Infect. Dis., 20: 542-551. [Crossref] [PubMed] [PMC]

11. Kandeil, A., El-Shesheny, R., Maatouq, A.M., Moatasim, Y., Shehata, M.M., Bagato, O., Rubrum, A., Shanmuganatham, K., Webby, R.J., Ali, M.A. and Kayali, G. (2014) Genetic and antigenic evolution of H9N2 avian influenza viruses circulating in Egypt between 2011 and 2013. Arch. Virol., 159(11): 2861-2876. [Crossref] [PubMed] [PMC]

12. Khalil, A.A., Hussein, H.A., Tolba, S.K. and El-Sanousi, A.A. (2015) Preparation and evaluation of H9N2 vaccine adjuvant with Montanide ISA 71. Glob. Vet., 14(5): 670-674.

13. El-Naggar, H.M., Madkour, M.S. and Hussein, H.A. (2017) Preparation of mucosal nanoparticles and polymer-based inactivated vaccine for Newcastle disease and H9N2 AI viruses. Vet. World, 10(2): 187-193. [Crossref] [PubMed] [PMC]

14. Miller, P.J. and Koch, G. (2013) Newcastle disease, other avian paramyxoviruses and avian metapneumovirus infections. In: Swayne, D.E., Glisson, J., McDougald, L.R., Nolan, L.K., Suarez, D.L. and Nair, V., editors. Diseases of Poultry. Vol. 13. Wiley-Blackwell, Hoboken, NJ. p89-138.

15. Liu, X.F., Wan, H.Q., Ni, X.X., Wu, Y.T., Liu, W.B. (2003) Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985-2001. Arch. Virol., 148: 1387-1403. [PubMed]

16. Jeon, W.J., Lee, E.K., Lee, Y.J., Jeong, O.M., Kim, Y.J., Kwon, J.H. and Choi, K.S. (2008) Protective efficacy of commercial inactivated Newcastle disease virus vaccines in chickens against a recent Korean epizootic strain. J. Vet. Sci., 9: 295-300. [Crossref] [PubMed] [PMC]

17. Reed, L.J. and Muench, H. (1938) Simple method of estimating 50 percent end point. Amer. J. Hyg., 27: 793-799.

18. OIE. (2014) Avian Influenza OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Ch. 2.3.4. OIE, Paris, France.

19. Allan, W.H. and Cough, R.E. (1976) A comparison between the haemagglutination inhibition and complement fixation tests for new castle disease. Vet. Sci., 20: 101-103.

20. OIE. (2004) Highly Pathogenic Avian Influenza. International Health Code. Ch. 2.7.12. OIE, Paris.

21. OIE. (2012) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Mammals, Birds and Bees, Biological Standards Commission. 7th ed. World Organization for Animal Health, Paris.

22. Reda, I.M. and Sheble, A. (1976) Isolation and Characterization of Local Viscertropic NDV Unpublished Data.

23. SPSS. PC. (2015) Software. Version. 22.0. SPSS Inc., Chicago, IL.

24. Snedecor, G.W., Cochran, W.G. (1989) Statistical Methods. 8th ed. Iowa State Uuniv, Ames, IA, USA.

25. Crevar, C.J. and Ross, T.M. (2008) Elicitation of protective immune responses using a bivalent H5N1 VLP vaccine. Virol. J., 5(131): 131. [Crossref]

26. Fiore, A.E., Timothy, M.U., Karen, B., Lyn, F., James, A.S., Flavia, Z., Analia, B., Ariel, P., Oscar, T. and Elisa, C. (2010) Prevention and control of influenza with vaccines. Recommendations of the Advisory Committee on Immunization Practices (ACIP), 59(RR08): 1-62.

27. Sharma, J.M. (2003) The avian immune system. In: Saif, Y.M., Barnes, H.J., Glisson, J.R., Fadly, A.M., McDougald, L.R. and Swayne, D.E., editors. Disease of Poultry. Iowa State Univ. Press, Ames, Iowa. p5-16.

28. El-Bagoury, G.F., Nasr, M.H.M., El-Habbaa, A.S. and Hala, M.E.M. (2015) A trial to improve stability and immunogenicity of inactivated NDV vaccine with paraffin oil adjuvant using aluminum stearate. Benha Vet. Med. J., 28(1): 199-209.

29. Heggen, C.L., Qureshi, M.A., Edens, F.W. and Barnes, H.J. (2000) Alterations in macrophage-produced cytokines and nitrite associated with poult enteritis and mortality syndrome. Avian Dis., 44: 59-65. [Crossref] [PubMed]

30. Kaiser, P., Rothwell, L., Galyov, E.E., Barrow, P.A., Burnside, J. and Wigley, P. (2000) Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum. Microbiology, 146: 3217-3226. [Crossref] [PubMed]

31. Peschke, T., Bender, A., Nain, M. and Gemsa, D. (1993) Role of macrophage cytokines in influenza A virus infections. Immunobiology, 189: 340-355. [Crossref]

32. Van Reeth, K. (2000) Cytokines in the pathogenesis of influenza. Vet. Microbiol., 74, 109-116. [Crossref]

33. Karpala, A.J., Bingham, J., Schat, K.A., Chen, L.M., Donis, R.O., Lowenthal, J.W. and Bean, A.G. (2011) Highly pathogenic (H5N1) avian influenza induces an inflammatory T helper Type 1 cytokine response in the chicken. J. Interferon Cytokine Res., 31(4): 393-400. [Crossref] [PubMed]

34. Kaiser, L., Fritz, R.S., Straus, S.E., Gubareva, L. and Hayden, F.G. (2001) Symptom pathogenesis during acute influenza: Interleukin-6 and other cytokine responses. J. Med. Virol., 64: 262-268. [Crossref]

35. Svitek, N, Rudd, P.A., Obojes, K., Pillet, S. and von Messling, V. (2008) Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction. Virology, 376(1): 53-59. [Crossref] [PubMed]

36. Lienenluke, B. and Christ, B. (2007) Impact of interleukin-6 on the glucose metabolic capacity in rat liver. Histochem. Cell Biol., 128(4): 371-377. [Crossref] [PubMed]

37. Zhao, J., Wohlford-Lenane, C., Zhao, J., Fleming, E., Lane, T.E., McCray, P.B Jr. and Perlman, S. (2012) Intranasal treatment with poly(I•C) protects aged mice from lethal respiratory virus infections. J. Virol., 86(21): 11416-11424. [Crossref] [PubMed] [PMC]

38. Cornelissen, J.B., Post, J., Peeters, B., Vervelde, L., Rebel, J.M. (2012) Differential innate responses of chickens and ducks to low-pathogenic avian influenza. Avian Pathol., 41(6): 519-529. [Crossref] [PubMed]

39. Cary, A.R., Leonardo, S., Ingrid, C., Corrie, C.B, Darrell, R.K., David, L.S., Daniel, J.K., Patti, J.M. and Claudio, L.A. (2011) Virulent Newcastle disease virus elicits a strong innate immune response in chickens. J. Gen. Virol., 92: 931-939. [Crossref] [PubMed]

40. Zhao, J., Huiming, Y., Hongjun, X., Zengbin, M. and Guozhong, Z.E. (2017) Efficacy of an inactivated bivalent vaccine against the prevalent strains of Newcastle disease and H9N2 avian influenza Virol. J., 14: 56. [Crossref] [PubMed] [PMC]

41. Dong-Hun, L., Jae-Keun, P., Jung-Hoon, K., Seong-Su, Y., Tseren-Ochir, E.O., Yo-Han, J., Baik-Lin, S., Lee, Y.P., Seung-Yong, P., In-Soo, C. and Chang-Seon, S. (2013) Efficacy of single dose of a bivalent vaccine containing inactivated Newcastle disease virus and reasserting highly pathogenic avian influenza H5N1 virus against lethal HPAI and NDV infection in chickens. PLoS One, 8(3), e58186. [Crossref] [PubMed] [PMC]

42. El-Sayed, D.A.A., Abdou A.M., Shalash S.M.M., Safaa, H.M. and Riad S.A. (2011) Productivity and immune response of broiler chickens vaccinated with different avian influenza vaccines at one or seven days of age. Aust. J. Basic Appl. Sci., 5(10): 325-334.

43. OIE. (2012), Disease Immediate Notification. OIE, 25(3): 19.