Evaluation of Immune Modulators as Vaccine Enhancers: Comparative Study of Newcastle Disease Virus Vaccine Potency and Immunity

Abdelrahman Maher Abdelmoneim1, Mohamed Ali Zain El-Abideen2, Dalia Mansour Hamed1*, Wael Kamel Elfeil1

1Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt; 2Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute (AHRI), Agricultural Research Center (ARC), P.O. Box, 264, Dokki, Giza 12618, Egypt.

Received | October 22, 2025; Accepted | December 04, 2025; Published | December 12, 2025

*Correspondence | Dalia Mansour Hamed, Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522, Egypt; Email: [email protected]

Citation | Abdelmoneim AM, El-Abideen MAZ, Hamed DM, Elfeil WK (2025). Evaluation of immune modulators as vaccine enhancers: Comparative study of Newcastle disease virus vaccine potency and immunity. Adv. Anim. Vet. Sci., 13(s1):198-210.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.s1.198.210

ISSN (Online) | 2307-8316

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Newcastle disease virus (NDV), classified as avian orthoavulavirus-1 (genus Orthoavulavirus, family Paramyxoviridae), represents one of the most contagious viral pathogens threatening global poultry. The order Mononegavirales includes negative sense single-stranded RNA viruses with helical capsid symmetry, of which NDV remains a key member alongside other avian paramyxoviruses (APMV) (Walker et al., 2020). According to the World Organization for Animal Health (WOAH), NDV continues to constitute a serious impediment to poultry production worldwide, particularly in Asia and Africa, where it remains an endemic control problem (WOAH, 2021). NDV causes tremendous economic losses due to the severe morbidity and mortalities that may reach total loss of flocks , egg production drop and cost of vaccination and prevention (Suarez et al., 2020). Despite intensive vaccination programs and biosecurity measures, the disease continues to circulate globally, with emerging genotypes demonstrating different virulence characteristics and vaccine escape potential.

NDV strains have been classified into two primary classes based on molecular and pathogenic characteristics. Class I includes predominantly avirulent strains characterized by a single genotype, while Class II includes 21 distinct genotypes, including both virulent and avirulent variants (Torres-Velez et al., 2019). Recent epidemiological surveillance data have demonstrated that NDV genotype VII (GVII) strains continue to circulate extensively in field conditions, causing recurrent disease outbreaks and substantial economic losses despite widespread vaccination implementation (Bello et al., 2018). The emergence and persistence of GVII variants highlight the limitations of conventional vaccine strategies and underscore the necessity for enhanced immunological interventions to combat Newcastle disease. Comprehensive immunization programs have been implemented in commercial poultry operations worldwide. Numerous commercially accessible vaccine formulations are available, including live attenuated vaccines, inactivated whole-virus vaccines, recombinant viral vector vaccines, and DNA-based vaccines (Swayne and King, 2003, Dimitrov et al., 2017). However, despite the application of intensive vaccination programs, their protective efficacy remains suboptimal, particularly against emerging field isolates with genetic difference from vaccine strains. The limitations of conventional vaccines require innovative solutions to enhance vaccine-induced protective immunity and increase cross-protective efficacy against diverse circulating NDV genotypes.

Immunostimulants which can be synthetic or of plant origin compounds with a well-established history of enhancing the immune system capacity to encounter infectious agents. These substances represent benefits towards all aspects of animal husbandry, including production performance, disease combating, and overall welfare outcomes (Hamill et al., 2008). The incorporation of immunomodulatory substances (IMS) into vaccine formulations is increasingly recognized as a beneficial strategy for enhancing vaccine efficacy. IMS can play an essential role in initiating and amplifying the immune response, thereby improving the overall effectiveness of vaccination. By modulating the immune system, IMS facilitate faster seroconversion, allowing for quicker protection post-vaccination. Additionally, they help stimulate both humoral and cellular immune pathways, resulting in a more reliable and long-lasting immune response. This two action is particularly important in the development of vaccines against challenging pathogens, where traditional formulations may not elicit sufficient immunity. Overall, the strategic use of IMS not only augments vaccine potency but also accelerates the onset of protective immunity, making them a potent component in modern vaccine design and development.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, possesses previously unrecognized immunomodulatory properties extending beyond neurological functions. Recent evidence demonstrates that GABA can enhance autophagy in immune effector cells, thereby improving host defense mechanisms against intracellular pathogens (Kim et al., 2018). By promoting autophagy-mediated clearance of intracellular viral replicates and enhancing the presentation of processed viral antigens, GABA may potentiate both humoral and cell-mediated immune responses against NDV.

Quercetin, a plant-derived polyphenolic flavonoid abundantly present in various botanical sources including green tea and red wine, exerts pleiotropic immunomodulatory effects. This flavonoid promotes the production of pro-inflammatory cytokines essential for antiviral defense, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) (Li et al., 2016). Simultaneously, quercetin suppresses inappropriate histamine release and modulates critical immune cell signaling pathways, including nuclear factor-kappa B (NF-κB) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), resulting in diminished inflammatory responses and attenuated allergic manifestation (Mlcek et al., 2016). This dual capacity for enhancing antiviral immunity while limiting excessive inflammation makes quercetin an attractive adjuvant candidate for vaccine formulations.

Hesperidin, a citrus-derived flavanone glycoside, possesses potent antioxidant and immunomodulatory properties. Supplementation with hesperidin in preclinical animal models significantly enhances natural killer (NK) cell cytotoxic capacity, increases phagocytic monocyte populations, and enforces T helper cell (Th) numbers in critical lymphoid tissues including peripheral blood, thymus, and spleen (Ganeshpurkar and Saluja, 2020). Moreover, hesperidin prevents immune suppression and aberrant leukocytosis induced by intense physiological stress, suggesting protective effects on both innate and adaptive immune compartments. Additionally, hesperidin modulates the organization and function of gut-associated lymphoid tissue (GALT), increases intestinal immunoglobulin A (IgA) production critical for mucosal immune defense and fortifies lymphocyte populations within the intestinal lamina propria, thereby strengthening the intestinal mucosal immune barrier (Kim et al., 2018; Estruel-Amades et al., 2019).

Transfer factors (TFs) represent naturally occurring molecules derived from cellular sources that possess the capacity to transfer specific cell-mediated immune responses. Recent investigations employing transfer factor supplementation in broiler chickens vaccinated against NDV demonstrated significant amplification of cell-mediated immunity. Transfer factor treatment elevates the expression of key pro-inflammatory and immunoregulatory cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) within primary immune organs including the spleen, thymus, and bursa of Fabricius (Guo et al., 2020). This cytokine enhancement reflects potentiated T cell-mediated immunity and accelerated lymphocyte proliferation, suggesting that transfer factor represents a viable immunological adjuvant for enhancing vaccine-induced protective immunity. While conventional inactivated NDV vaccines provide baseline protection against virulent challenge, their efficacy can be substantially improved through judicious incorporation of immunomodulatory adjuvants. Despite growing interest in vaccine immunomodulation, systematic comparative studies evaluating multiple immunomodulatory substances against contemporary NDV strains are lacking. Most NDV vaccine efficacy assessments focus on mortality protection and antibody titers, with limited quantitative analysis of post-challenge viral shedding a critical determinant of transmission dynamics and field-level disease control. Furthermore, the relationship between accelerated seroconversion kinetics induced by immunomodulators and subsequent viral replication suppression remains poorly understood, particularly for emerging GVII genotypes that exhibit antigenic variation from conventional vaccine strains. These knowledge deficits limit rational design of enhanced vaccine formulations for commercial poultry production. So, this study addresses these gaps by systematically comparing four immunomodulatory substances GABA, quercetin, hesperidin, and transfer factor as adjuvants in an inactivated NDV vaccine containing contemporary GVII 1.1 antigens.

The objectives of the current study were to determine how quickly each immunomodulator helps birds develop protective antibody levels after vaccination, measured at multiple timepoints using standard HI tests ,evaluate protection against post-challenge with virulent NDV GVII 1.1, also to Quantify post-challenge viral shedding to determine transmission-blocking potential. In addition, identify the most effective immunomodulator based on combined assessment of antibody kinetics, clinical protection, and viral shedding suppression.

MATERIALS AND METHODS

Ethical approval and biosafety compliance

Experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Medicine at Suez Canal University (approval number: SCU-VET-REC-2024062) and All conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). All work involving virulent NDV was conducted within a biosafety level-3 (BSL-3) containment facility with appropriate personnel training, engineering controls, and personal protective equipment (PPE) protocols. Humane euthanasia of terminally ill animals was performed using cervical dislocation following brief CO₂ anesthesia, in accordance with American Veterinary Medical Association (AVMA) guidelines.

Viral source and strain selection

A wild-type Newcastle disease virus genotype VII 1.1 isolate (NDV/EGY/CK/291/2024) recovered from naturally infected poultry in Egypt was utilized as the virulent challenge virus. This field isolate was molecularly characterized and deposited in the GenBank repository (accession number: PP516376), confirming genotype VII classification and contemporary circulation status.

Virus propagation

The challenge virus was propagated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECE) obtained from (Nile SPF eggs, Kom Oshiem, Fayoum, Egypt). Eggs were inoculated via the allantoic sac route with 0.1 mL of working viral stock diluted in sterile phosphate-buffered saline (PBS, pH 7.2-7.4). Post-inoculation, embryonated eggs were maintained at 37°C with controlled relative humidity (50–65%) for 72 hours. Allantoic fluid was harvested aseptically by piercing the chorioallantoic membrane under biosafety cabinet conditions.

Virus clarification and titration

Harvested allantoic fluid was clarified by low-speed centrifugation at 2,000 rpm for 10 minutes at 4°C to remove cellular debris and egg components. The clarified viral suspension was subjected to hemagglutination (HA) assay to determine viral hemagglutinin titers expressed as log₂ values. The embryo infective dose 50 (EID₅₀) was calculated using the Reed-Muench method (Reed and Muench, 1938) and converted to TCID₅₀ equivalents for standardized challenge dose preparation. Viral stocks were aliquoted (500 μL per cryovial) and stored at −80 °C until utilization in challenge experiments.

Vaccine seed virus propagation

The vaccine seed virus, a recombinant genotype VII NDV strain (rgNDV6/ME-G7/2024), was propagated following World Organization for Animal Health (WOAH) standards. SPF embryonated chicken eggs (10 days of age) were inoculated via the allantoic sac route with 0.1 mL of vaccine seed virus suspension. Inoculated eggs were incubated at 37°C with 50–65% relative humidity for 72 hours. Allantoic fluid containing replicated vaccine virus was harvested aseptically.

Virus clarification and concentration

Harvested allantoic fluid was clarified by low-speed centrifugation (2,000 rpm, 10 minutes, 4°C) to remove particulate matter. The clarified supernatant was then tittered HA titers and EID₅₀ values were determined using standard virological protocols (Karakus et al., 2018; Reed and Muench, 1938). The concentrated viral antigen underwent formaldehyde-based inactivation compliant with OIE (WOAH, 2021) standards. Formaldehyde solution (37% stock) was added to achieve a final concentration of 0.2% formaldehyde per liter of bulk antigen. The inactivation reaction proceeded at ambient temperature (22–25°C) for a minimum of 48 hours, permitting complete viral protein crosslinking and irreversible inactivation of infectious particles. Complete viral inactivation was confirmed by three consecutive serial passages through 5 SPF embryonated chicken eggs (9–11 days of age) via allantoic sac inoculation (0.1 mL per egg). Eggs were maintained at 37°C for 72 hours, and allantoic fluid from each passage was harvested and subjected to hemagglutination assays using 1% washed chicken erythrocytes. Absence of HA activity in all three passages confirmed complete inactivation and absence of residual infectious viral particles. The inactivated bulk viral antigen (bivalent formulation containing both Lasota-like and GVII 1.1 antigens) was formulated into an oil-in-water emulsion vaccine using Montanid ISA70 (SEPPIC, 2012) as the adjuvant component. The adjuvant consists of 30% aqueous phase and 70% oil phase (v/v). The inactivated antigen and Montanid ISA70 were combined in a 30:70 volumetric ratio and emulsified using a Silverson L5M high-shear laboratory mixer (Silverson Machines, Inc., Buckinghamshire, United Kingdom) at standardized rotational speeds to achieve uniform, stable emulsion formation. Temperature was maintained between 18°C and 22 °C throughout emulsification using jacketed mixing vessels. Following emulsification, vaccine batches were allowed to stabilize at room temperature for a minimum of 24 hours prior to quality control testing.

The base inactivated vaccine formulation was divided into five equal portions and vaccine formulation shown in Table 1:

• Group A (Vaccine Control): Inactivated NDV vaccine (GVII 1.1) without immunomodulator supplementation
• Group B: Inactivated NDV vaccine + γ-aminobutyric acid (GABA) [5 mg/mL final concentration]
• Group C: Inactivated NDV vaccine + quercetin [3 mg/mL final concentration]
• Group D: Inactivated NDV vaccine + hesperidin [2.5 mg/mL final concentration]
• Group E: Inactivated NDV vaccine + transfer factor [50 μg/mL final concentration, bovine colostrum-derived]

All immunomodulator-supplemented vaccines were thoroughly mixed using the Silverson mixer at 2,500 rpm for 5 minutes to ensure uniform distribution. Vaccines were aliquoted into sterile vials (0.5 mL per vial) and stored at 4°C for maximum 2 weeks, with stability verified by HA assay every 3 days.

Experimental animals and housing conditions

Seventy one-day-old SPF chicks (Nile SPF eggs, Kom Oshiem, Fayoum, Egypt). Prior to experimental assignment, all birds were confirmed serologically negative (via HI assays) for Newcastle disease, infectious bursal disease (IBD), infectious bronchitis (IB), avian leukosis (AL), reticuloendotheliosis (REV), and Mycoplasma species. Chicks demonstrated uniform health status with mean body weight of 45 ± 3g, appropriate feathering, clear eyes, and absence of clinical abnormalities or physical defects.

Isolator housing and environmental controls

Birds were individually housed in positive-pressure chicken isolators equipped with HEPA filtration for both incoming and exhaust airstreams, located within a BSL-3 containment facility (MEVAC, Elsalheiya El-gedida, Ismailia, Egypt). Environmental conditions were precisely maintained: photoperiod (12L:12D), ambient temperature (22–24°C ± 1°C via thermostat-controlled heating), relative humidity (50–65%). Bedding material (pine shavings, autoclaved at 121°C, 15 psi for 30 minutes) was changed every 3 days. All birds received ad libitum pathogen-free commercial broiler starter feed (crude protein ≥24%, metabolizable energy ≥3,100 kcal/kg, compliant with NRC 1994 recommendations) and sterile drinking water (distilled, autoclaved at 121°C for 20 minutes). Water quality was verified weekly by microbial culture on blood agar plates.

Personnel biosecurity

Personnel entering the facility wore complete PPE including suits, head covers, gloves, N95 masks, and boot covers, with complete suit changes between experimental groups.

Experimental design and vaccination protocol

Study design overview

A completely randomized experimental design was employed with seventy SPF chicks randomly assigned (using computerized randomization stratified by initial body weight) to seven treatment groups (Figure 1) (n=10 birds per group):

Vaccination protocol

At seven days of age, birds in groups A–E received 0.5 mL of the appropriate vaccine formulation via intramuscular injection into the right pectoral muscle using sterile 25-gauge needles and 1 mL tuberculin syringes. Groups F and G received 0.5 mL sterile PBS (pH 7.2-7.4) via identical route and needle gauge. All vaccination procedures employed strict aseptic technique including skin sterilization with 70% ethanol and sterile needle exchange between birds to prevent iatrogenic pathogen transmission.

Serological monitoring: Blood sample collection and HI assay

Blood sampling protocol

Venous blood samples were collected from all birds via brachial vein puncture at predetermined intervals: day 0 (pre-vaccination baseline), week 2 post-vaccination, week 3 post-vaccination, and week 4 post-vaccination (immediately prior to challenge). Blood samples (500–700 μL per bird) were allowed to clot at room temperature for 2 hours in sterile glass serum separator tubes, then centrifuged at 1,500 × g for 10 minutes at 4°C. Serum was harvested using sterile transfer pipettes, aliquoted into sterile microcentrifuge tubes (250 μL per aliquot), and stored at −20°C until analysis. All samples were coded with unique identifiers to ensure examiner blinding.

Haemagglutination inhibition (HI) assay

Humoral immune responses were quantified using the HI assay, the internationally recognized gold standard for NDV antibody detection in poultry. Serum samples were heat-inactivated at 56°C for exactly 30 minutes in a water bath. Serial two-fold dilutions of each serum sample were prepared in sterile PBS (pH 7.2-7.4), ranging from 1:2 to 1:2¹⁶. To each well of a V-shaped 96-well microtiter plate (Nunc, Thermo Fisher Scientific), 50 μL of serum dilution was added in triplicate. Subsequently, 50 μL of standardized NDV antigen (4 hemagglutinating units [HAU]) was added. The virus-serum mixture was incubated at room temperature (22–25°C) for exactly 20 minutes.

Following incubation, 50 μL of 1% washed chicken erythrocytes prepared fresh from SPF chickens were added to each well. Cells were obtained by cardiac puncture, centrifuged at 1,000 × g for 5 minutes, washed three times in sterile PBS, and resuspended to 1% concentration. Plates were incubated at room temperature for 15–20 minutes to allow erythrocyte sedimentation. Complete agglutination (pellet with clear supernatant) indicated virus-antibody binding; absence of agglutination (dispersed “button”) indicated high antibody concentration. HI titers were expressed as the reciprocal of the highest serum dilution demonstrating complete inhibition, converted to log₂ values.

HI testing

To assess responses against both vaccine antigens, HI assays were performed using (1) Lasota-like strain antigen (4 HAU) and (2) GVII 1.1 strain antigen (4 HAU) prepared from the same isolate used for challenge. Protective immunity was defined as HI titer ≥log₂ (reciprocal 1:16). Seroconversion was defined as a four-fold increase in HI titer between consecutive samplings.

Challenge timing and viral dose

At five weeks of age (35 days post-hatch), all birds in groups A–F underwent virulent viral challenge (Figure 1) with NDV/EGY/CK/291/2024 at 10⁶ TCID₅₀ per 0.5 mL per bird. This dose was selected based on preliminary dose-ranging studies demonstrating consistent 100% mortality in naive controls while permitting differentiation of protective efficacy among vaccinated groups.

Challenge administration

Viral challenge was administered via intramuscular injection into the right pectoral muscle (contralateral to vaccination site) using sterile 25-gauge needles and 1 mL tuberculin syringes. Injection sites were sterilized with 70% ethanol prior to inoculation. This route ensures standardized viral delivery and systemic infection kinetics comparable to natural aerosol-route infection.

Post-challenge monitoring

Following challenge, all birds were observed continuously for clinical signs recorded at 6-hour intervals for 48 hours post-challenge, then at 12-hour intervals through day 10. Parameters recorded included depression, ruffled feathers, conjunctivitis, inappetence, respiratory distress, neurological signs (torticollis, paralysis, tremors, lack of coordination), greenish diarrhea, and mortality. All mortality events were recorded with precise timing and associated clinical observations. Severely affected birds were euthanized humanely. Necropsy was performed on all dead and euthanized birds, with tissue specimens (trachea, lung, intestine, brain, spleen) collected in 10% neutral buffered formalin for immunohistochemistry (IHC) confirmation of NDV-specific pathological changes and viral antigen localization.

Sample collection protocol

Cloacal swabs were collected from all birds in groups A–F at days 2, 4, 6, and 9 post-challenge using sterile cotton-tipped applicators (Puritan Medical Products, Guilford, ME, USA) inserted gently to approximately 2 cm depth and rotated 3–5 seconds for epithelial cell collection. Swabs were immediately placed into sterile 1.5 mL microcentrifuge tubes (Eppendorf, Hamburg, Germany) containing 1.0 mL sterile PBS (pH 7.2-7.4) supplemented with 1× penicillin-streptomycin-amphotericin B antibiotic-antimycotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) to preserve viral RNA integrity and prevent microbial overgrowth.

Sample processing and storage

Samples were transported on ice within insulated containers to the laboratory and stored at −80 °C in sterile cryovials within 30 minutes of collection. No samples underwent more than one freeze-thaw cycle prior to analysis. Group G (negative control) cloacal swabs were collected at identical timepoints for procedural consistency and to confirm absence of viral shedding.

Viral RNA quantification

Viral RNA was extracted from cloacal swab samples using the TRANS EasyPure® Simple Viral DNA/RNA Kit following manufacturer instructions. Briefly, 140 μL of clarified cloacal swab suspensions (centrifuged at 5,000 × g for 15 minutes at 4°C) was combined with 560 μL lysis buffer (AVL buffer) containing carrier RNA and 560 μL absolute ethanol. The lysate was applied to silica-based spin columns and subjected to integrated purification with sequential washes (buffers AWL and AW2). Viral RNA was eluted in two sequential 30 μL aliquots of RNase-free elution buffer (AVE), yielding approximately 60 μL purified RNA. Samples were stored at −80°C for RT-qPCR analysis within 7 days.

Viral RNA Extraction: extraction was done by using TRANS EasyPure® Simple Viral DNA/RNA Kit following the manufacturer’s instructions.

Reverse Transcription Quantitative Real-Time PCR (RT-qPCR): Viral RNA quantification was performed using one-step RT-qPCR targeting the NDV fusion protein (F) gene (101 bp product), a conserved region essential for viral infectivity. The AgPath-ID™ One-Step RT-PCR Reagent Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used with the following primers and probe (Wise et al., 2004):

Forward Primer: 5’-CGS ARG ATM CAA GGG TCT-3’

Reverse Primer: 5’-CTA CAC TGC CAA TAA CRG C-3’

Probe: [FAM] 5’-AGG AGA CRA AAA CGY TTT ATA GGT GC-3’ [BHQ-1]

Each 25 μL reaction in 0.2 mL optical tubes contained: 12.5 μL master mix, 1 μL forward primer (10 pmol), 1 μL reverse primer (10 pmol), 0.5 μL probe (10 pmol), 1 μL RT-PCR Enzyme Mix, 4 μL RNase-free water, and 5 μL extracted RNA template. Reactions were performed in triplicate per sample.

The thermal profile consisted of: (1) reverse transcription at 45°C for 10 minutes (1 cycle); (2) initial denaturation at 94°C for 10 minutes (1 cycle); and (3) 40 amplification cycles of denaturation at 95°C for 20 seconds followed by annealing/extension at 60°C for 45 seconds with continuous fluorescence monitoring using a Biorad CFX960 system (Bio-Rad Laboratories, Hercules, CA, USA).

Ct value determination and viral load quantification

Cycle threshold (Ct) values were automatically determined by Bio-Rad CFX Manager v3.0 software (threshold set at 1000 relative fluorescence units). Lower Ct values correlate with higher viral RNA concentrations; Ct >40 cycles indicate negative result (below 10² copies/mL detection limit). Ct values were converted to viral load estimates using a standard curve from serial dilutions (10⁻¹ to 10⁻⁷) of quantified NDV RNA standard prepared from known-titer viral stock and quantified by spectrophotometry (NanoDrop, 2000, Thermo Fisher Scientific). The standard curve demonstrated linear regression (R² ≥0.99) with 90–110% amplification efficiency. Results are expressed as mean ± standard deviation (SD) of viral RNA copies per milliliter of cloacal swab suspension. Standard curve shown in Figure 4.

Statistical analysis

Data on seroconversion were not normally distributed and were analyzed in SPSS using the Kruskal-Wallis test (Hazra and Gogtay, 2016). The post hoc analysis was done by using the Mann-Whitney test, then graphs were generated by using GraphPad Prism9.

Results

Post-vaccination antibody response and seroconversion kinetics

Baseline Serology (Pre-Vaccination, Day 0): At study initiation (one day of age), all experimental groups (A–G) demonstrated No HI titers against both NDV antigens As it’s SPF Chicks.

 

 

Two-week post-vaccination serology (Week 2 P.V.): At two weeks post-vaccination, significant divergence in antibody responses became apparent between immunomodulator-supplemented and control vaccine groups. Mean HI titers against NDV GVII 2024 antigen in Group B (GABA-supplemented, 6.6±0.64 log₂) demonstrated statistically significant elevation compared to Group A (vaccine alone, 3.4±1.85 log₂; Mann-Whitney U test: p<0.002). Similarly, Group D (hesperidin-supplemented, 6.1±2.13 log₂) demonstrated significantly higher titers than Group A (p<0.0026). Group C (quercetin-supplemented, 5.3±2.13 log₂) showed numerically higher titers than Group A, though this difference approached but did not achieve statistical significance (p=0.082) (Figure 2).

Against Lasota-like strain antigen at week 2 P.V., Group B demonstrated mean HI titers of 7.8±0.89 log₂ compared to Group A 4.3±2.1 log₂ (p<0.00077), representing a 1.8-fold higher titer. Group D (7.5±2.45 log₂) also demonstrated significantly elevated titers compared to

 

Table 1: IMS concentration /dose and vaccineHA titer.

		Dose/Route	N	IMS/Dose
A	Inactivated NDV vaccine (bivalent Lasota/GVII 1.1)300HAU/Dose	0.5 mL, IM	10	N/A
B	Vaccine + GABA (5 mg/mL)	0.5 mL, IM	10	2.5mg/Dose
C	Vaccine + Quercetin (3 mg/mL)	0.5 mL, IM	10	1.5mg/Dose
D	Vaccine + Hesperidin (2.5 mg/mL)	0.5 mL, IM	10	1.25mg/Dose
E	Vaccine + Transfer Factor (50 μg/mL)	0.5 mL, IM	10	25 μg/mL
F	PBS vehicle (positive control) + challenge	0.5 mL, IM	10	N/A
G	PBS vehicle (negative control), no challenge	0.5 mL, IM	10	N/A

 

Table 2: HI Mean of different time points across study.

Time points/log2 titers	A (ND vaccine control)		B (ND+GABA)		C (ND+Quercitin)		D (ND+Hesperidine)		E (ND+Transfer factor)
	NDV lasota like	NDV GVII	NDV lasota like	NDV GVII	NDV lasota like	NDV GVII	NDV lasota like	NDV GVII	NDV lasota like	NDV GVII
W1	0	0	0	0	0	0	0	0	0	0
W2	4.3	3.9	7.8	6.6	6.4	5.4	7.5	6.1	3.9	3.5
W3	8.5	7.5	9.3	8.5	9.1	8.4	8.9	8.1	7.5	5.5
W4	7.5	6.6	8.8	7.6	9.3	8.3	8.8	7.6	6.4	5.4
10 DPCH	9.5	9.3	9.1	8.9	9.5	8.3	9.5	9.0	9.0	8.6

 

Log2≥ 4 Considered protective.

 

 

 

Group A (p<0.011). Group C exhibited intermediate titers (6.4±2 log₂) without achieving statistical significance versus Group A. Three-Week Post-Vaccination Serology: At three weeks post-vaccination, continued intergroup divergence in antibody titers was observed, though differences were generally less pronounced than at week 2. Group B maintained elevated HI titers against both antigens, while Group D demonstrated sustained but comparatively declining titer levels (Figure 3).

 

Four-week post-vaccination serology (Pre-challenge baseline): By week 4 post-vaccination (immediately prior to virulent challenge), mean HI titers had stabilized across all vaccinated groups. Group C (quercetin-supplemented) demonstrated statistically significant elevation of HI titers against NDV GVII 2024 antigen (mean 8.8±1.7 log₂) compared to Group A (6.6±1.6log₂⁴; p<0.0017). Against Lasota-like antigen, Group C showed mean titers of 9.3±0.89 log₂ versus Group A’s 7.5±.93 log₂ (p<0.025). Groups B, D, and E demonstrated maintained elevation of HI titers, though intergroup comparisons did not always achieve statistical Mean HI Titers throughout the study shown in Table 2.

 

Table 3: Shedding positive and percentage by rt-qPCR post challenge.

Days post vaccination	Group A	Group B	Group C	Group D	Group E	Group F
2 D.P.C	3/10	0/10	2/10	1/10	3/10	6/10
	30%	0%	20%	10%	30%	60%
4 D.P.C	4/10	0/10	2/10	3/10	4/10	All chicks died by the 4th day post challenge
	40%	0%	20%	30%	40%
6 D.P.C	4/10	2/10	3/10	2/10	6/10
	40%	20%	30%	20%	60%
9 D.P.C	2/10	2/10	4/10	5/10	5/10
	20%	20%	40%	50%	50%

 

Seroconversion dynamics: Groups B and D demonstrated the most rapid seroconversion kinetics, achieving protective HI titers (≥log₂ [reciprocal 1:16]) against both viral antigens by week 2 post-vaccination. Group C demonstrated slightly delayed seroconversion, achieving protective titers by week 3-4 post-vaccination. Group E (transfer factor-supplemented) demonstrated modest though consistent elevation of HI titers throughout the observation period. Group A (vaccine alone) achieved protective titers against both antigens by week 3-4 post-vaccination, demonstrating 1–2 week delay in seroconversion compared to Groups B and D.

Protective efficacy against virulent challenge: Negative control group (Group G): All ten birds in Group G remained clinically normal throughout the 10-day post-challenge observation period, confirming maintenance of biosafety protocols, absence of environmental contamination, and appropriate negative control conditions. No clinical abnormalities, mortality, or detectable viral shedding was observed in this group.

Positive control group (Group F, nonvaccinated-challenged): All birds in Group F (nonvaccinated control subjected to virulent challenge) displayed characteristic clinical signs of acute Newcastle disease beginning on days 2–3 post-challenge. Clinical manifestations included severe depression with marked reduction in activity, complete inappetence, conjunctivitis with ocular discharge, greenish diarrhea, and progressive neurological manifestations including torticollis (twisted neck), paralysis of lower extremities, and lack of coordination. The disease progression was rapid, with 100% mortality occurring within 3–4 days post-challenge (mean survival time: 3.2±0.4 days). No birds in this group survived to days 4 Newcastle Disease persists as a serious disease in developing country. This ongoing threat poses significant risks on poultry farming. Current vaccines, when used broadly, are essential tools for controlling and preventing such problem. In our study we tested different (IMS) to assess its ability to improve the protective effect of vaccination and the study revealed that such improvement is applicable in improving humoral immune response as the rapid induction of neutralizing antibodies as early as 2 weeks post vaccination in case of vaccines fortified by either GABA or quercetin or hesperidin and maintained protective antibody levels throughout the study, also to protect individuals completely from mortality after being challenged by the virulent circulating strain meanwhile the group with the GABA could express a significant point in preventing shedding until the 4th day of challenge and maintained an acceptable percentage of shedders reaching 9D.P.C. Protection in all vaccinated groups reached 100% and mortality reached 100% in non vaccinated group post challenge.

Our study tried to spotlight the effect of modulating vaccines by substances that may be helpful in competing such contagious disease meanwhile further studies are surely needed to either determining the appropriate doses or experimenting other substances that may be helpful to serve the main purpose of controlling NDV post-challenge sampling time points. Vaccinated Groups (Groups A–E): All vaccinated birds in groups A through E demonstrated complete protection against mortality following virulent challenge, with 0% mortality observed in each group through the complete 10-day post-challenge observation period. Protective efficacy was accordingly calculated as 100% for all vaccinated groups [(100%−0%)/100% × 100 = 100%]. Notably, vaccinated birds demonstrated no clinical signs of Newcastle disease, no visible conjunctivitis, no diarrhea, no neurological manifestations, and normal behavior including normal feeding, drinking, and activity levels comparable to negative control birds. Complete clinical protection was maintained throughout the 10-day observation period in all vaccinated groups.

Day 2 post-challenge (2 D.P.C.) viral shedding: Group A (vaccine alone) demonstrated viral shedding in 3/10 birds (30% shedding prevalence) (Table 3) with viral titers of 2.1, 2.4, and 2.5 log₁₀ copies/mL (mean: 2.33±0.21 log₁₀ copies/mL). Group B (GABA-supplemented) demonstrated complete absence of viral shedding in all 10 birds (0% shedding prevalence), indicating superior early viral replication suppression compared to vaccine-only control. Group C (quercetin-supplemented) demonstrated viral shedding in 2/10 birds (20% shedding prevalence) with titers of 2.1 and 2.8 log₁₀ copies/mL (mean: 2.45±0.49 log₁₀ copies/mL). Group D (hesperidin-supplemented) demonstrated viral shedding in 1/10 birds (10% shedding prevalence) with a titer of 3.3 log₁₀ copies/mL. Group E (transfer factor-supplemented) demonstrated viral shedding in 3/10 birds (30% shedding prevalence) with titers of 1.2, 2.9, and 3.8 log₁₀ copies/mL (mean: 2.63 ±1.34 log₁₀ copies/mL). Group F (nonvaccinated control) demonstrated viral shedding in 6/10 birds (60% shedding prevalence) consistent with early active viral replication in immunologically naive hosts. Day 4 Post-challenge (4 D.P.C.). Group F had 100% mortality, whereas Group B (NDV + GABA) exhibited the greatest reduction in viral shedding with 100% protection.

After 4 D.P.C Viral Shedding from Group A demonstrated increased viral shedding prevalence to 4/10 birds (40%) with titers of( 1.2,3.6,4.4 and 5.7) 4D.P.D (mean: 3.1±1.67 log₁₀ copies/mL). Group B Also demonstrated complete absence of viral shedding in all 10 birds (0% shedding prevalence), indicating superior early viral replication suppression compared to vaccine-only control. Group C (quercetin-supplemented) demonstrated viral shedding in 2/10 birds (20% shedding prevalence) with titers of 4.5 and 4.7 log₁₀ copies/mL (mean: 4.6±0.14 log₁₀ copies/mL). Group D (hesperidin-supplemented) demonstrated viral shedding in 3/10 birds (30% shedding prevalence) with a titer of 1.7,1.1and2.7 log₁₀ (mean 1.4 0.42 log₁₀ copies/mL). Group E shedding increased to 40% with titers of 1.1,1.9,2.6 and 5.7(mean 1.86 0.75 log₁₀ copies/mL).

6 D.P.C Group A shedding remained 40% and the titers were 2.5, 3.1, 3.4 and 4 log10 (mean 3.67 1.4 log₁₀ copies/mL). Group B started to shed viruses by 20 % with titers of 2.2and2.7(mean 2.45 0.35 log₁₀ copies/mL). Group C (quercetin-supplemented) demonstrated viral shedding in 4/10 birds (40% shedding prevalence) with titers of and 1.1,1.2,3.6 and 4.6 log₁₀ copies/mL (mean: 1.96±1.4 log₁₀ copies/mL). Group D (hesperidin-supplemented) demonstrated viral shedding in 2/10 birds (20% shedding prevalence) with a titer of 2.1,2.9 log₁₀(mean 2.5 0.57 log₁₀ copies/mL). Group E shedding increased to 60% with titers of 1.4, 1.5, 3.6, 3.6, 3.6 and 4.8 log10 (mean 2.98 1.48 log₁₀ copies/mL).

Mean shedding of shedding individuals during the after challenge period of all groups summarized in figure 5 also percentage of shedding individuals of all groups in all time points after challenge demonstrated in Table 3.

DISCUSSION

The primary objectives of vaccination regarding Newcastle Disease virus (NDV) are three: first, to reduce the clinical manifestations of the disease; second, to diminish the extent of virulent virus shedding by vaccinated birds; and third, to elevate the infectious dose threshold required for the challenge virus to induce clinical signs (Kapczynski et al., 2013).

This study demonstrates that certain immuno-stimulants can effectively enhance the humoral immunity when administered alongside the NDV vaccine. Notably, groups receiving GABA, quercetin, and hesperidin exhibited significantly higher hemagglutination inhibition (HI) titers at two weeks post-vaccination, indicating an early and efficient antibody-mediated response. These findings suggest that these immuno-stimulants may potentiate the vaccine’s immunogenicity by promoting increased antibody production, which is critical for effective protection. The observed enhancement of HI titers aligns with previous research (Hamill et al., 2008), supporting the notion that using immunostimulants can improve vaccine efficacy by strengthening humoral immunity. Overall, the ability of these immuno-stimulants to raise HI titers highlights their potential role in supporting vaccine-induced protection through the stimulation of humoral immune responses.

The findings clearly demonstrate that immunomodulatory substances (IMS) play a critical role in reducing viral shedding and enhancing immune responses against virulent Newcastle Disease Virus (NDV). The significant decrease or absence of viral shedding in groups B and C, as evidenced by rt-qPCR at 2 and 4 days post-challenge, indicates that IMS can effectively limit viral replication and expression within the host. This reduction not only reflects improved immune control but also has important implications for transmission limitations, potentially decreasing the spread of the virus. Furthermore, the observed correlation between elevated hemagglutination inhibition (HI) titers and decreased viral shedding suggests that IMS boosts humoral immunity, which is critical for neutralizing the virus and preventing infection. Overall, these results highlight the capacity of IMS to enhance both immune responses and viral shedding prevention, thereby providing increased protection against virulent NDV challenge. This highlights the potential of incorporating IMS into vaccination strategies to improve protective efficacy and control disease transmission.

In terms of the Mechanisms, adjuvants or different immuno-stimulants increase antigen presentation and immune activation. For example, using cytokine adjuvants such as IL-12 with an NDV DNA vaccine has been shown to significantly increase neutralizing antibody levels and lymphocyte proliferation, which also appeared as lower viral shedding and faster controlling the challenge (Xie et al., 2020) and this finding is consistent with our study as of increasing humoral immune response and reducing viral shedding in case of addition of GABA as inhibitory neurotransmitter that acts as anti-inflammatory and having signals can enhance autophagy in immune cells, improving host defense against intracellular infections, Similarly, in another study a vaccine candidate with IL-28b as molecular adjuvant significantly improved HI titers compared to vaccine alone, demonstrating that adding immuno-modulatory cytokines helps to enhance early humoral antibody responses (Amoia et al., 2025).

Also the usage of plant-derived saponins or other non-protein immunostimulants or herbal extract. the early humoral immune response detected in our study matched with the use of soyasaponin orally alongside inactivated NDV vaccine that produced significantly higher HI titers at multiple time points (including early time points) compared to vaccine alone and afforded complete protection in challenge trials (Naveed et al., 2020) that finding also goes along with the results of using herbal extracts like thyme and ginseng oil on the improvement of body performance parameters and humoral immune response (Hassanin et al., 2024). Although shedding was not always quantified by PCR in those studies, the absence of disease and lowered histopathological signs suggests reduced virus replication.

Protection against mortality in all vaccinated groups with NDV GVII 1.1 completely in our study contrariwise the complete mortality of chicks in the non-vaccinated challenged group (F) this finding confirms the velogenic nature of NDV GVII 1.1 used as challenge which have the multibasic amino acid sequence in F0 protein the proteolytic cleavage site motifs (112-117) that determines the virulence, the shedding reduction supports the use of Genotype-matched or multivalent vaccines as serological and animal testing found distinguishable antigenic variation between NDV genotype VII and the commercial vaccine strain Lasota, as indicated by 2-fold and 3–6-fold differences in HI of week two post vaccination, respectively (Yang et al., 2017) so when vaccines were formulated from genotype VIId NDV, either alone or combined with genotype II, induced higher HI titers and significantly reduced virus shedding compared to unmatched vaccine formulations (Mahmoud et al., 2019).

Our results suggest that higher HI titers correlate with lower shedding, particularly in (IMS) enhanced groups. This strengthens findings in previous work: e.g. in the IL-12 co-adjuvanted DNA vaccine study, higher neutralizing antibody levels correlated with fewer positive swabs (oropharyngeal, cloacal) post-challenge (Xie et al., 2020). except of group E added to it the transfer factor as the main aim of using it is to significantly increases the cell-mediated immunity, and elevation the expression of key cytokines.

Implicating the finding in our study of early protective immunity. The proven ability of immuno-stimulants to raise humoral responses within two weeks is highly useful in cases where rapid immunity is needed (e.g., outbreak control, short lifespan flocks and endemic areas early challenge). Shedding control and transmission. Even if vaccines prevent disease, shedding allows transmission; reducing viral shedding via immuno-stimulant supplementation helps in reducing environmental load, virus spread, and potential for emergence of more virulent or vaccine escape strains.

In our study we’d like to highlight the limitations of it. Major limitations include the single dose of each IMS, the use of a bivalent vaccine, the statistical approach, the lack of investigation into cell-mediated immunity.

According to vaccine design These findings support the inclusion of immuno-modulatory adjuvants (of plant origin, synthetic or cell extract) and antigenic matching in vaccine formulation. Also, monitoring with molecular assays (PCR, RT-PCR) for shedding is crucial for assessing vaccine efficacy beyond just clinical protection or mortality.

All these benefits achieved by inclusion of (IMS) outweigh the cost of using such modulators to help in the aim of controlling NDV.

CONCLUSION

In our study we tested different (IMS) to assess its ability to improve the protective effect of vaccination and the study revealed that such improvement is applicable in improving humoral immune response as the rapid induction of neutralizing antibodies as early as 2 weeks post vaccination in case of vaccines fortified by either GABA or quercetin or hesperidin and maintained protective antibody levels throughout the study, also to protect individuals completely from mortality after being challenged by the virulent circulating strain meanwhile the group with the GABA could express a significant point in preventing shedding until the 4th day of challenge and maintained an acceptable percentage of shedders reaching 9D.P.C. Our study tried to spotlight the effect of modulating vaccines by substances that may be helpful in combating such contagious disease though further studies are surely needed to either determine the appropriate doses or experiment with other substances that may be helpful to serve the main purpose of controlling NDV.

Recommendations

GABA should be used as an immunostimulant adjuvant for inactivated NDV vaccines based on its demonstrated superiority in enhance humoral immunity and attenuating viral shedding kinetics relative to other immunomodulatory substances evaluated in this study.

ACKNOWLEDGEMENTS

The authors are grateful to the Avian and Rabbit Medicine Department at the Suez Canal University in Ismailia, Egypt, and Middle East for Vaccines (MEVAC) Co. For their technical assistance by all the authors.

NOVELTY STATEMENT

This study aimed to compare adding different immune substances (γ-aminobutyric acid (GABA), quercetin, hesperidin, and transfer factor) to an inactivated Newcastle disease vaccine. Group A received the vaccine alone; groups B–E received the vaccine with γ-aminobutyric acid (GABA), quercetin, hesperidin, or transfer factor; group F served as challenged nonvaccinated control; and group G was nonvaccinated, nonchallenged. Groups were vaccinated at one week of age and challenged at four weeks post vaccination with NDV/EGY/CK/291/2024 (GenBank accession no. PP516376), to detect the immune substance’s ability to enhance vaccine performance and reduce viral transmission between groups. The results demonstrated that adding GABA to the vaccine not only accelerated the development of protective antibodies in chickens but also significantly reduced viral shedding post-challenge. a critical finding for controlling disease transmission within flocks. This approach is practical and cost-effective, making it particularly valuable for poultry farmers in regions where Newcastle disease causes substantial economic losses. Our work bridges the gap between laboratory innovation and field application by combining contemporary strain-matched vaccines with readily accessible immune enhancers.

AUTHORS CONTRIBUTION

The authors shared equally in writing this article, each bringing their technical skills and viewpoints.

Declarations

The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Medicine at Suez Canal University (approval number: SCU-VET-REC-2024062) and All conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Ethical consideration 

Ethical issues (including plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy) have been checked by all the authors.

Generative AI and AI-assisted technology statement

We declare that AI assisted tools used mainly to improve readability, language clarity of the manuscript while all interpretations, conclusions and scientific content made and verified by the authors who take all responsibility for the work.

Conflict of interest

The authors have declared no conflict of interest. 

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