Effect of In Ovo Injection of VG/GA Vaccine, an Apathogenic Enteric Strain of Newcastle Disease Vaccine and Aluminum Hydroxide as an Adjuvant on Hatchability and Immune Response of Commercial Pullets

Document Type: Original Paper


1 Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

3 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran


Current vaccination strategies for commercial poultry using live attenuated and inactivated Newcastle disease (ND) vaccines have some limitation and difficulties, and new vaccines with distinct features are needed. Recently, in ovo vaccination technology is concerned as a safe, efficacious, and convenient method. Common ND vaccines used in chickens cannot be employed in ovo due to embryo toxicity and high early mortality. One of the agents that may lead to attenuate ND virus (NDV) strains is aluminum hydroxide (AH) as an adjuvant. The objective of this study was to evaluate AH ability to attenuate NDV for in ovo administration of commercial pullets. Three hundred sixty fertile eggs of a Bovans strain as a factorial arrangement of six doses of the ND vaccine (50% egg infectious (EID50) of 0, 102, 103, 104, 105, and 106) with or without AH were ordered into 12 groups. At 18 d of incubation 0.1 mL of the inoculums was injected into the amniotic fluid of eggs. On the farm, each treatment group was further subdivided into two groups and one of these groups received ND-B1 vaccine on day seven post-hatch. Lowest hatchability was recorded in groups vaccinated with doses of 105 and 106 EID50. On day 21, the highest hem agglutination inhibition (HI) was detected for group vaccinated with dose 102 EID50. Furthermore, hatchability and ND-HI titer were found to be up for pullets received AH in ovo on day 42 posthatch. The results of this study indicated that aluminum hydroxide as an adjuvant could significantly improve hatchability and immune efficacy of pullets when used in ovo. Further, lentogenic VG/GA strain-Avinew will have the potential for application as in ovo vaccine against Newcastle disease, if the vaccine is prepared with sufficient dose.


Ahmad J & Sharma JM. 1992. Evaluation of a modified-live virus vaccine administered in ovo to protect chickens against Newcastle disease. AmericanJournal of Veterinary Research, 53:1999-2004.

Arous JB, Deville S, Pal JK, Baksi S, Bertrand F & Dupuis L. 2013. Reduction of Newcastle disease vaccine dose using a novel adjuvant for cellular immune response in poultry. Procedia in Vaccinology, 7:28-33. DOI: 10.1016/j.provac.2013.06.006

Chen C, Han D, Cai C & Tang X. 2010. An overview of liposome lyophilization and its future potential. Journal of Controlled Release, 142:299-311. DOI: 10.1016/j.jconrel.2009.10.024

De Gregorio E, Caproni E & Ulmer JB. 2013. Vaccine adjuvants: mode of action. Frontiers in Immunology, 4:214-220. DOI: 10.3389/fimmu.2013.00214

Dilaveris D, Chen C, Kaiser P & Russell PH. 2007. The safety and immunogenicity of an in ovo vaccine against Newcastle disease virus differ between two lines of chicken. Vaccine, 25:3792-3799. DOI: 10.1016/j.vaccine.2007.01.115

El Sabry MIM, Atta AMM, Tzschentke B, Gharib HBA& Stino FKR. 2012. Potential use of Interleukin-2-rich supernatant adjuvant in Fayoumi hens. Archiv Fur Geflugelkunde, 76:162-167.

Gupta SK, Deb R, Dey S & Chellappa MM. 2014. Toll-like receptor-based adjuvants: enhancing the immune response to vaccines against infectious diseases of chicken. Expert Review of Vaccines, 13:909-925. DOI: 10.1586/14760584.2014.920236

Hogenesch H. 2013. Mechanism of immunopotentiation and safety of aluminum adjuvants. Frontiers in Immunology, 3:406-419. DOI: 10.3389/fimmu.2012.00406

Jafari M, Moghaddam Pour M, Taghizadeh M, Masoudi S & Bayat Z. 2016. Comparative assessment of humoral immune responses of aluminum hydroxide and oil-emulsion adjuvants in Influenza (H9N2) and Newcastle inactive vaccines to chickens. Artificial Cells, Nanomedicine, and Biotechnology, 45:84-89. DOI: 10.3109/21691401.2015.1129626

Kapczynski DR, Martin A, Haddad EE & King DJ. 2012. Protection from clinical disease against three highly virulent strains of Newcastle disease virus after in ovo application of an antibody-antigen complex vaccine in maternal antibody-positive chickens. Avian Diseases, 56:555-560. DOI: 10.1637/9980-110311-Reg.1

Kool M, Fierens K & Lambrecht BN. 2012. Alum adjuvant: some of the tricks of the oldest adjuvant. Journal of Medical Microbiology, 61:927-934. DOI: 10.1099/jmm.0.038943-0

Lowenthal J, Johnson MA, Tyack SG, Hilton LS& Bean AGD. 2005. Oral delivery of novel therapeutics: development of a fowl adenovirus vector expressing chicken IL-2 and MGF. World's Poultry Science Journal, 61:87-94. DOI: 10.1079/WPS200444

Lowenthal JW, Lambrecht B, Van den Berg TP, Andrew ME, Strom ADG & Bean AG. 2000. Avian cytokines—the natural approach to therapeutics. Developmental and Comparative Immunology, 24:355-365. DOI: 10.1016/S0145-305X(99)00083-X

Manna C, Manna S, Das R, Batabyal K and Roy R. 2007. Development of in ovo vaccine against Newcastle disease of birds. Current Science, 93:1305-1309.

Maughan CN, Preston SG & Williams GR. 2014. Particulate inorganic adjuvants: recent developments and future outlook. The Journal of Pharmacy and Pharmacology, 67:426-449. DOI: 10.1111/jphp.12352

Mayo M. 2002. A summary of taxonomic changes recently approved by ICTV. Archives of Virology, 147:1655-1656. DOI: 10.1007/s007050200039

Mebatsion T, Verstegen S, De Vaan LTC, Römer-Oberdörfer A & Schrier CC. 2001. A recombinant Newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos. Journal of Virology, 75:420-428. DOI: 10.1128/JVI.75.1.420-428.2001.

Mishra N, Khatri K, Gupta M & Vyas SP. 2014. Development and characterization of LTA-appended chitosan nanoparticles for mucosal immunization against hepatitis B. Artificial Cells, Nanomedicine, and Biotechnology, 42:245-255. DOI: 10.3109/21691401.2013.809726

Nayan N, Shohaimi SA, Raus RA, Yusof AM & Huai OG. 2015. Effect on immune response and virus shedding in the chicken vaccinated against inactivated local strain of Newcastle disease virus genotype VII. Jurnal Teknologi, 77:89-93. DOI: 10.11113/jt.v77.6745

Ohta H, Ezoe S, Yamazaki K, Kawai T & Honda T. 2009. Application of aluminum hydroxide for an in ovo live Newcastle disease vaccine. Avian Diseases, 53:392-395. DOI: 10.1637/8555-120908-Reg.1

Okwor GO, El-Yuguda A & Baba SS. 2014. Profile of maternally derived antibody in broiler chicks and in-ovo vaccination of chick embryo against Newcastle disease. World Journal of Vaccines, 4:72-80. DOI: 10.4236/wjv.2014.42009 

Ramp K, Topfstedt E, Wäckerlin R, Höper D, Ziller M, Mettenleiter TC, Grund C & Römer-Oberdörfer A. 2012. Pathogenicity and immunogenicity of different recombinant Newcastle disease virus clone 30 variants after in ovo vaccination. Avian Diseases, 56:208-217. DOI: 10.1637/9870-080311-Reg.1

Rautenschlein S, Sharma JM, Winslow BJ, McMillen J, Junker D & Cochran M. 1999. Embryo vaccination of turkeys against Newcastle disease infection with recombinant fowlpox virus constructs containing interferons as adjuvants. Vaccine, 18:426-433. DOI: 10.1016/S0264-410X(99)00254-6

Reed SG, Orr MT & Fox CB. 2013. Key roles of adjuvants in modern vaccines. Nature Medicine, 19:1597-1608. DOI: 10.1038/nm.3409

Ricks C, Avakian A, Bryan T, Gildersleeve R, Haddad E, Ilich R, King S, Murray L, Phelps
P, Poston R& Whitfill C. 1999. In ovo vaccination technology. Advances in Veterinary Medicine, 41:495-515.

Romao JM, De Moraes TGV, Salles RPR, Cardoso WM & Buxade CC. 2011. Effect of in ovo vaccination procedures on Japanese quail embryos (Coturnix japonica) and incubation performance. Ciência Animal Brasileira,12:585-592. DOI: 10.5216/cab.v12i4.5234

Saravanabava K, Nachimuthu K & Padmanaban V. 2005. Effect of tuftsin on embryo vaccination with Newcastle disease virus vaccine. Comparative Immunology, Microbiology and Infectious Diseases, 28:269-276. DOI: 10.1016/j.cimid.2005.03.003

Senne D, King D & Kapczynski D. 2003. Control of Newcastle disease by vaccination. Developments in Biologicals, 119:165-170.

Stone H, Mitchell B & Brugh M. 1997. In ovo vaccination of chicken embryos with experimental Newcastle disease and avian influenza oil-emulsion vaccines. Avian Diseases, 41:856-863. DOI: 10.2307/1592339

Thapa S, Cader MSA, Murugananthan K, Nagy E, Sharif S, Czub M & Abdul-Careem MF. 2015. In ovo delivery of CpG DNA reduces avian infectious laryngotracheitis virus induced mortality and morbidity. Viruses, 7:1832-1852. DOI: 10.3390/v7041832

Wang M, Meng X, Yang R, Qin T, Li Y, Zhang L, Fei C, Zhen W, Zhang K, Wang X, Hu Y & Xue F. 2013. Cordyceps militaris polysaccharides can improve the immune efficacy of Newcastle disease vaccine in chicken. International Journal of Biological Macromolecules, 59:178-183. DOI: 10.1016/j.ijbiomac.2013.04.007.