The experimental approach included a determination of the pathological and molecular characteristics of v-NDV strains involved in field outbreaks, preparation of autogenous bivalent v-NDV vaccine from the characterized predominant strains, preparation of different challenges, establishment of a completely randomized experimental design, and evaluation of the immunity and protection provided by the developed vaccine against the different challenges by v-NDV strains. The completely randomized experimental design enabled to address the scientific objectives, aiming at evaluation of the immunity and protection by the vaccine against predominant v-NDV strains.
Pathological and molecular characteristics of v-NDV strains
The intracerebral pathogenicity Index (ICPI) of v-NDV strains was determined by the protocol of Office Internationale des Epizooties (OIE), Manual of Standards for Diagnostic Tests and Vaccines [15]. Briefly, it involved the individual inoculation of each strain of genotype VI or VII into the brain of 10-day-old chicks, offsprings of NDV - free parents, obtained from our institution’s hatchery, and monitoring their mortality at specific times. The reverse transcription –polymerase chain reaction (RT-PCR) was implemented to amplify the fusion protein cleavage site (FPCS) and its surrounding dibasic amino acids (35), while the sequencing of the generated amplicons was accomplished by Big-Dye terminator v.3.1 kit (Applied Systems, USA). The mean mortality percent, presented in Table 1, is obtained from field outbreaks in three respective broiler flocks, caused by each strain of genotype VI and VII.
Preparation of autogenous bivalent v-NDV vaccine
The preparation of an autogenous bivalent v-NDV vaccine, containing the pathologically and molecularly characterized strains of genotypes VI and VII, included the growth of each strain in chicken embryos of 10 day-old, obtained from our institution’s hatchery. The inoculum applied of each strain through the allontoic membrane route was of 100 particles/100 ul/embryo. The harvesting of the propagated virus was at the day of mortality of the embryo (averaging 48 hr post inoculation). Each harvested strain was adjusted to a strength of 128 HA units against 0.1 % of chicken-red blood cells (c-RBC) suspension. The adjusted harvest of each strain was inactivated by an overnight contact with 0.3 % formalin; the absence of viral viability, post this contact, was checked by individual inoculation of each of the two formalized strains in 10 d - old embryos, followed by incubation for 3 d, and testing the allontoic fluid of embryos for absence of agglutination against a 0.1 % of c-RBC suspension. Equal volume of each adjusted strength of the two inactivated strains were pooled and emulsified with equal volume of mineral oil (viscometer reading of 40 SUS), with an inclusion of Arlacel C (Emulsifier) and Tween 20 (Surfactant), at the level recommended by the manufacturer (TCI Development, Shanghai, China). The emulsified preparation was passed in a colloidal mill at the following stator gap of 0.002’’, resulting in water-in-oil micelles with average diameter of 1.2 μm.
Preparation of different challenges
Three different challenges were prepared from the harvested allontoic fluid, deprived of inactivation by formalin. The harvested genotype VI and VII strains was each adjusted to 104 x Embryo Infectious Dose50 (EID50). Each bird assigned to specific challenge-treatment received genotype VI strain (TRTS 1 and 4) or VII strain (TRTS 2 and 5), in 50 μl/bird, through the left pectoral muscle. Each challenged bird in TRTs 3 and 6 received 50 μl of each genotype strain in the left pectoral muscle.
Completely Randomized Experimental design
The completely randomized experimental design is comprised of 8 Treatments (TRTs), with four replicate isolation rooms per treatment, in which each room contained 25 meat birds (Table 2), obtained at one-day old from our institution’s hatchery. The day-old birds had a mean weight of 38 g, and weight range of 37- 40 g. The inclusion of this number of birds is indispensable for providing unbiased data that will allow the implementation of proper statistical analysis for comparison of assessed parameter means among the 8 different treatments. The day-old birds were randomly distributed in their pens, reared on the floor that is previously disinfected and covered with wood shaving; the birds were provided with continuous lighting, controlled temperature and feed formulation, following the instructions of Aviagen Co for Ross 308 broiler management. The birds had a continuous access to feeders and drinkers, while abiding by a welfare-related assessment to the broiler’s environment throughout the whole trial. The specie of the experimental birds was Gallus gallus domesticus, Ross 308 strain, with ratio of males to females equivalent to 1:1. Birds of TRT 1 were vaccinated each with 0.5 ml of the developed vaccine, delivered subcutaneously in the neck at 6 and 21 d of age, followed by challenge with genotype VI strain at 28 d of age. Birds of TRT 2 were similarly vaccinated but challenged with strain of genotype VII. Birds of TRT 3 were similarly vaccinated but challenged with strains of both genotype strains. Birds of TRTs 4, 5, and 6 were the positive controls, deprived of vaccination but respectively challenged with strains of genotype VI, VII, and VI + VII. Birds of TRT 7 were similarly vaccinated, but deprived of all types of challenge, while birds of TRT 8 were the negative controls, deprived of vaccination and challenge. The experiment was terminated at the market age of 40 days by euthanization, using an injection via the pectoral muscle, equivalent to 20 mg/Kg of tiletamine/zolazepam.
Acquired humoral Immunity by Vaccine and Challenge
The acquired humoral immunity in broilers, specific to the hemagglutinin protein of v-NDV present in the vaccine and challenge, was assessed by hemagglutination-inhibition (HI) test [17]. Blood was collected from the brachial vein of each surviving bird at the ages of 21, 28, and 40 d. Sera were separated from the collected coagulated blood; the HI titer of each serum was obtained against an antigen of 4 HA units/50 μl of allontoic fluid containing the individual strain of genotype VI or genotype VII, and including in the test a 0.1% of c-RBC suspension [36]. The HI titer of each sample detected the maximum dilution of the collected serum that still contained enough antibodies specific to H protein in the antigen, preventing this protein from binding to its receptor on the c-RBC, and by that inhibiting agglutination phenomenon.
Acquired CMI to developed vaccine and challenges
The same collected chicken sera were also analyzed for the level of IFN- γ at the ages of 21, 28, and 40 d., reflecting the acquired CMI to developed vaccine and challenges in these birds [37, 38]. The analysis of sera IFN- γ was performed by sandwich-ELISA system, using a commercial kit by Shanhai CrystalDay Biotech Co., Yangpu district, Shanghai, China [39, 40].
Protection by developed vaccine
The evaluation of the acquired protection in differently treated broilers by the developed vaccine, against different challenges, is based on birds’ cumulative mean of mortality, feed conversion, and live body weight by the market age of 40 d. The feed conversion is calculated by the division of the consumed feed/total body weight of broilers in each isolation room, and the mean was calculated from the feed conversion of birds in the four replicate rooms/treatment.
Statistics
The One Way Analysis of Variance (ANOVA) was used to compare the means of HI titers, IFN- γ, feed conversion, and live body weight, among the eight different treatments; however, the means of cumulative mortality percent, up to market age of 40 d, were compared among the 8 treatments by CHI Square (χ2). The significant difference among means of each statistically analyzed parameter is reported at P<0.05 [41].