Evaluation of immunity and protection in chicken administered a developed vaccine against predominant Middle Eastern strains of genotypes VI and VII of velogenic Newcastle Disease Virus CURRENT STATUS: POSTED

Background The velogenic-Newcastle Disease Virus (v-NDV) causes an important disease in chicken, associated with serious economic losses to the global poultry industry. This research evaluated the immunity in broilers administered a developed bivalent vaccine, aiming at protection against predominant Middle Eastern strains of genotypes VI and VII of v-NDV. The completely randomized design implemented in this evaluation included eight treatments, differing in birds being administered or deprived of the developed vaccine, with a difference in type of challenge, either by v-NDV strain(s) of genotype VI, VII, or both. Vaccination was administered subcutaneously at 6 and 21 d of age, followed by an intra-pectoral challenge at the age of 28 d. Results The acquired humoral immunity by vaccinated and challenged birds to Hemagglutinin (H) protein was the highest at market age of 40 d, compared to challenged birds deprived of vaccination, and to vaccinates deprived of challenge (P<0.05). The same statistical difference pattern was obtained by the cell-mediated immunity (CMI), represented by birds’ level of serum IFN- γ . The type of challenge by either strain(s) of genotype VI, VII, or VI+VII did affect statistically the cross reactivity of acquired humoral immunity specific to H protein of homologous versus heterologous strains. The absence of humoral immunity and the low IFN- γ levels at 28 d of age in challenged birds deprived of vaccination lead to highest mortality, and lowest performance compared to vaccinates and challenged, vaccinates and deprived of challenge, and unvaccinated-unchallenged birds (P<0.05). Conclusions The developed bivalent vaccine was able to induce enough humoral and CMI responses, enabling protection of the broilers against production losses by each of the three types of v-NDV challenges. It is recommended to conduct future studies to evaluate such types of vaccines in chicken breeders and commercial layers, reared in various world’s zones with existing endemicity of v-NDV.

Arab Emirates, Iraq, Jordan, Egypt, Lebanon, and Kingdom of Saudi Arabia belong mainly to genotypes VI and VII [6][7][8]. The genotype VI strains have affinity to respiratory, nervous, and gastrointestinal (GI) tract of the birds, causing apparent gross lesions of bluish ulcers in the intestine and petechial hemorrhage in the proventriculus [7]; however, the genotype VII strains have affinity to tissues of the respiratory and nervous system [9] causing apparent torticollosis and pneumonic signs [7].
Global reports are pointing at frequent failure of NDV-commercial vaccines in induction of acceptable immunity and protection against emerging strains of NDV [10]. Reports from the Middle East countries included also documentations of failure of various imported commercial vaccines in protection of poultry against v-NDV [7,11]. Other workers documented the persistent shedding of the NDV, in spite of the administration to poultry of the available vaccines in their market [12][13][14].
The hypothesis of this research predicted that a development of a bivalent autogenous vaccine constructed from predominant v-NDV strains of genotypes VI and VII will acquire enough immunity for protection against velogenic NewCastle Disease.

Results
Pathological and molecular characteristics of v-NDV The pathological and molecular characteristics of v-NDV vaccine seed and challenge strains, included in this study, are shown in Table 1. The intracerebral pathogenicity indices of the genotypes VI and VII strains were 1.86 and 1.92, respectively. This high pathogenicity is confirmed by the high mean broiler's mortality percent, observed in the three field outbreaks caused by each strain of genotypes   [15]. bThe Mean Mortality % is recorded from three field outbreaks in broiler flocks caused by each genotype strain. cThe amplification and sequencing at FPCS were performed according to previously documented protocol by Seal et al [16]. 1Means in a column, followed by the same numerical superscript, are insignificantly different (P > 0.05).

Acquired Humoral Immunity By Vaccine And Challenge
The completely randomized design (CRD) of this work is presented in    aVaccination is administered at 6 and 21 d of age, subcutaneously in the neck, with a vaccine dose equivalent to 0.5 ml/bird bChallenge by either the genotype VI or VII strain, or by both, at an age of 28 d cThe serum HI titer is the Hemaaglutination -Inhibition titer quantifying the humoral antibodies specific to the H protein present in the 4 HA units/50 µL of the genotype VI or VII strains used in the test against 0.1% of c-RBC dNA stands for not applicable, due to the 100% mortality obtained by birds of TRT 6, by the age of 40 days.
1−5Means of HI titer in a column, followed by different numerical superscripts, are significantly different at P < 0.05. e,fMeans of HI titer in numerator and denominator of a fraction located in a row of the last column, followed by different alphabet superscripts, are significantly different at P < 0.05.

Acquired CMI By Developed Vaccine And Challenges
The acquired CMI to vaccination and to challenge, quantified by the IFN-γ levels in sera of the differently treated birds, is shown in Table 4. The pattern of quantified INF-γ is similar to that obtained by the humoral responses to H-protein of the two genotype-strains of v-NDV; more specifically, the highest IFN-γ levels, post the vaccine booster delivered at 28 d of age, was obtained by the vaccinated birds of TRTs 1, 2, 3, and 7, with mean levels that were significantly higher than that obtained by all unvaccinated birds (P < 0.05). This acquiring of higher CMI response at challenge age of 28 days in birds of TRTs 1, 2, and 3 helped to further rise significantly their INF-γ levels at 12 d post challenge (40 d of age), compared to unvaccinated-challenged birds of TRTs 4, 5, and 6 (P < 0.05). It is worth noting that vaccinated birds, deprived of challenge (TRT 7), had a decay in INF-γ level by the market age of 40 d. In addition, the negative control birds of TRT 8 had a similar background levels of INF-γ at the three ages of 21, 28, and 40 d.

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.

Discussion
The obtained intracerebral pathogenicity indices of the genotypes VI and VII strains were skewed towards the high scale of pathogenicity, as documented by the World Animal Health Organization [15]. In addition, the high mortality observed in field outbreaks caused by each genotype strain is in agreement with previous literature related to performance of broilers infected by similar strains [7,16,17]. The FPCS sequence of the two isolates is confirmative of the velogenic nature of the two genotypes of NDV [18].
The acquired humoral response to the second vaccination in TRTs 1, 2, 3, and 7 is most likely due to class switch from IgM to IgG [19]; the high level of IgG isotype is expected to react efficiently against the different nature of challenges, administered at 28 d of age to birds of TRTs 1, 2, and 3. The specific higher significant immune response to H protein of the homologous compared to heterologous strains used in the challenge indicates most likely a difference in the configuration of Hprotein epitopes present in strains of genotype VI and VII, in spite of the typical sequence of their FPCS [20,21]. Differently challenged birds of TRTs 4, 5, and 6, that were deprived of vaccination, had lower immune conversion by the market age, an in agreement data with a documentation confirming that a challenge by v-NDV in absence of sensitization by vaccines will lead to highly virulent challenge due to absence or presence of an unsubstantial immunity [22,23]. The fact that the vaccine-HI titer, at 40 d of age in vaccinates that were deprived of challenge (TRT 7), was significantly lower than the maintained titers obtained by vaccinated and challenged birds of TRTs 1, 2 and 3, presents a proof of the ability of developed vaccine to prime the immune cells, enabling them to respond efficiently to the different challenges [24].
The data of Table 4, related to acquired CMI to vaccination and to challenge, quantified by the IFN-γ levels in sera of the differently treated birds, document the impact of administering the developed vaccine at two different ages on inducing higher levels of IFN-γ; this response is indispensably needed for protection against v-NDV challenges administered at 28 d of age [25,26]. The data of INF-γ levels post challenge is in agreement with documentations confirming an improvement in INF-γ level of vaccinated -challenged birds compared to unvaccinated -challenged ones [27,28]. In addition, a previous report showed that INF-γ decays by time in vaccinated birds that are deprived of a secondary antigen exposure (challenge), as observed in birds of TRT 7, while the negative controls maintain similar low background levels of INF-γ at different ages [28].
The significant higher performance of the vaccinated-challenged broilers (TRTs 1, 2, and 3), in comparison to unvaccinated-challenged ones (TRTS 4, 5, and 6) ( Table 5) is most likely due to the significant difference in the vaccine-acquired humoral and CMI, an obtained data that is in agreement with previous documentations [29,30,31,32,33]. The similar high production parameters of lower mortality percent, lower feed conversion, and high live body weight in birds of TRT 7 and those of the negative controls reflect the safety of the developed killed vaccine on production [34], pending avoidance of stress, by careful handling of the birds during the subcutaneous administration of the vaccine.

Availability of data and materials
The datasets and analysis are available upon requests made to the E-mail address of the manuscript's corresponding author.

Ethics approval and consent to participate
The

Consent for Publication
Not applicable. The manuscript does not contain any individual person's data.