Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines Stivalis Cardenas-Garcia a, b , Diego G. Diel c , Leonardo Susta a , Eduardo Lucio-Decanini d , Qingzhong Yu a , Corrie C. Brown b , Patti J. Miller a , Claudio L. Afonso a, * a Southeast Poultry Research Laboratory, 934 College Station Rd., Athens, GA 30605, USA b Department of Veterinary Pathology, College of Veterinary Medicine, The University of Georgia, College of Veterinary Medicine, 501 D. W. Brooks Dr., Athens, GA, USA c Department of Veterinary Pathobiology, College of Veterinary Medicine, The University of Illinois, 2001 S Lincoln Ave, Urbana 61802, USA d Investigaci on Aplicada, S.A. de C.V., 7 Norte 416, Col. Centro, Tehuac an, Puebla CP 75700, Mexico article info Article history: Received 8 September 2014 Received in revised form 7 November 2014 Accepted 9 November 2014 Available online xxx Keywords: NDV Vaccine efficacy Newcastle disease Homologous vaccination Mortality abstract While there is typically 100% survivability in birds challenged with vNDV under experimental conditions, either with vaccines formulated with a strain homologous or heterologous (different genotype) to the challenge virus, vaccine deficiencies are often noted in the field. We have developed an improved and more stringent protocol to experimentally evaluate live NDV vaccines, and showed for the first time under experimental conditions that a statistically significant reduction in mortality can be detected with genotype matched vaccines. Using both vaccine evaluation protocols (traditional and improved), birds were challenged with a vNDV of genotype XIII and the efficacy of live heterologous (genotype II) and homologous (genotype XIII) NDV vaccines was compared. Under traditional vaccination conditions there were no differences in survival upon challenge, but the homologous vaccine induced significantly higher levels of antibodies specific to the challenge virus. With the more stringent challenge system (multiple vaccine doses and early challenge with high titers of vNDV), the birds administered the homologous vaccine had superior humoral responses, reduced clinical signs, and reduced mortality levels than those vaccinated with the heterologous vaccine. These results provide basis for the implementation of more sensitive methods to evaluate vaccine efficacy. Published by Elsevier Ltd on behalf of The International Alliance for Biological Standardization. 1. Introduction Newcastle disease (ND) is one of the most important diseases affecting poultry world-wide. It is caused by virulent strains of Newcastle disease virus (vNDV), also known as avian para- myxovirus serotype 1 (APMV-1) [1,2]. NDV belongs to the genus Avulavirus of the family Paramyxoviridae [1,2]. The virus genome consists of a single-stranded, negative sense, non-segmented, RNA molecule with approximately 15.2 kb which encodes six structural proteins: nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion (F), hemagglutinin-neuraminidase (HN), and RNA polymerase (L) [1,2]. Genome sequence analysis of multiple NDV isolates allowed their classification into two major classes (class I and II). Class II is subdivided into at least eighteen genotypes (I to XVIII), and contains most of the vNDV strains circulating in poultry around the world [3e5]. According to the OIE, virulent strains are defined as those NDV containing an F protein cleavage site with at least three basic amino acids betweenposition 113 and 116, and a phenylalanine at position 117, or an intracerebral pathogenicity index 0.7 [6]. Infection with vNDV in countries with endemic disease results in significant economic losses to the poultry in- dustry due to decreased growth rates and to drop in egg production in vaccinated birds, or due to high levels of mortality in naïve or poorly vaccinated birds. Control of ND requires implementation of expensive culling measures, preventive vaccination and biosecurity measures to prevent the disease from spreading [1,7]. Several studies have concluded that classical live or inactivated vaccines made of viruses of genotype I or II (heterologous), when administered to healthy birds in adequate doses, are capable of * Corresponding author. Tel.: þ1 706 546 3642. E-mail addresses: stivaliscgs@gmail.com (S. Cardenas-Garcia), diego.diel@ sdstate.edu (D.G. Diel), leonardo.susta@ars.usda.gov (L. Susta), elucio@grupoidisa. com (E. Lucio-Decanini), qingzhong.yu@ars.usda.gov (Q. Yu), corbrown@uga.edu (C.C. Brown), patti.miller@ars.usda.gov (P.J. Miller), claudio.afonso@ars.usda.gov (C.L. Afonso). Contents lists available at ScienceDirect Biologicals journal homepage: www.elsevier.com/locate/biologicals http://dx.doi.org/10.1016/j.biologicals.2014.11.003 1045-1056/Published by Elsevier Ltd on behalf of The International Alliance for Biological Standardization. Biologicals xxx (2014) 1e10 Please cite this article in press as: Cardenas-Garcia S, et al., Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.11.003