Comparative Serological Effectiveness of Newcastle Disease Vaccines and Vaccination Regimens on Commercial Broilers: A Multisite Study in Iran

Introduction

Newcastle disease (ND) is a significant threat to the global poultry industry, particularly in regions where the virus is endemic. Effective control of ND relies primarily on biosecurity and vaccination (Hassanzadeh et al., 2024). Both live and inactivated oil-adjuvanted ND vaccines are widely applied to induce a strong systemic antibody response (Rauw et al., 2009). Live vaccines, derived from avirulent or lentogenic strains, are often preferred because they stimulate both local and systemic immunity, thereby conferring broader protection (Ghafouri, Ghaniei, moghani, & lotfalizadeh, 2024; Steensels, Soldan, Rauw, Roupie, & Lambrecht, 2025).

It is well established that differences in vaccination programs and vaccine strains can significantly influence protection and be associated with higher HI titers by the chosen threshold (Dimitrov, Afonso, Yu, & Miller, 2017; Ghafouri et al., 2024). The selection of vaccine strain and vaccination schedule often varies depending on different epidemiological conditions (Rehmani, 1996). For example, in high-risk areas, frequent vaccinations at short intervals may be necessary to lower the mortality rate in broilers (Ghafouri et al., 2024; Steensels et al., 2025). Conversely, in regions with lower NDV prevalence, immunization programs are usually designed mainly to reduce airsacculitis in broilers or to prevent sudden drops in egg production in layers and breeders (Patti J Miller, Decanini, & Afonso, 2010; Steensels et al., 2025). Therefore, an appropriately planned vaccination program, combined with effective ND vaccine administration, is crucial not only for protecting against virulent field strains but also for minimizing virus shedding.

The duration of vaccine-induced immunity varies depending on the vaccine formulation and host immune status at the time of vaccination (M Abdoshah et al., 2012; Kapczynski, Afonso, & Miller, 2013). Typically, the primary immune response develops more slowly and is less persistent (approximately 2–3 weeks), whereas a booster vaccination enhances both the magnitude and provides long-lasting immunity. Importantly, once peak antibody levels are achieved, subsequent vaccinations mainly extend the duration of immunity rather than further elevating peak titers. For this reason, repeated administration of live or a combination of live and inactivated vaccines is commonly recommended to achieve reliable protection against virulent NDV challenge.

In Iran, ND has remained endemic and continues to threaten commercial broiler production, particularly in the Alborz and East Azerbaijan provinces (Mohammad Abdoshah et al., 2022; M Abdoshah et al., 2012). Although numerous ND vaccines are widely used, few comparative studies have evaluated their relative efficacy or assessed different vaccination regimens under field conditions. Therefore, the present study was designed to investigate the immunogenicity of several commercially used live and inactivated ND vaccines in two broiler farms located in geographically distinct provinces of Iran.

Materials and Methods

Experimental design and study area. This study consisted of two field experiments and study units conducted in Alborz Province (central Iran) and East Azerbaijan Province (northwest Iran), both recognized as high-risk regions for Newcastle disease outbreaks(M Abdoshah et al., 2012). Two commercial broiler farms were selected: one in Alborz with four houses of 6000 and 24,000 Ross 308 broilers in total, and a farm in East Azerbaijan with four houses of 14500 chicken and a total of 58,000 Ross 308 broilers. Each house received the same regimen but a different vaccine brand. All broilers were reared on the litter with free access to feed and water. Birds received the routine vaccination program for broilers (including infectious bursal disease, infectious bronchitis, and inactivated avian influenza vaccines), in addition to the ND vaccination program under investigation. The ND vaccines were obtained from different manufacturers and anonymized as V1, V2, V3, and Razi (Tables 1 and 2). Each house from the poultry site (either Alborz or Azerbaijan) received a vaccine package of a different Newcastle vaccine strain from a single vaccine brand

Blood sampling and Hemagglutination inhibition (HI) assay. On day 1, maternal-derived antibody (MDA) titers against NDV were determined from 20 randomly selected chicks per site. At 21 and 42 days of age, 20 blood samples were collected from each of the 4 experimental groups. Sera were separated, and specific antibody titers against NDV were measured using the HI assay according to the procedure described by the World Organization for Animal Health. (WOAH, 2021)

Performance indices. Throughout the trial, birds were observed daily for clinical signs of ND or other diseases. Mortality, final body weight, and feed conversion ratio (FCR) were recorded to compare production performance among experimental groups.

Statistical analysis. Data were analyzed using a completely randomized design. Serological data were subjected to analysis of variance (ANOVA) using the GLM procedure of SAS software (version 9.4, SAS Institute, Cary, NC, USA). Results were expressed as mean ± standard deviation (SD). The GENMOD procedure was used to compare the proportion of birds achieving protective HI titers (HI ≥ 4). Tukey’s test was applied for multiple mean comparisons, and statistical significance was set at P < 0.05.

Results

Serum antibody titers

Experiment 1: Alborz farm

Serum HI antibody titers against NDV and the proportion of birds achieving protective titers are summarized in Figure 1 and Table 3, respectively. The average maternal antibody titers (MDA) were 5.58 ± 0.95 log2 titers. At day 21, mean HI antibody titers ranged from 3.60 to 4.20 log2 titers and did not differ significantly between groups. However, the proportion of birds with protective titers was significantly higher in the Razi and V1 groups compared with the V2 group (P < 0.05). By day 42, mean HI titers (4.10 to 4.70 log2 titers) and the proportion of birds with protective titers (65 to 85%) did not differ significantly among groups (P > 0.05). (Fig1. And Table 3)

Figure 1. The HI antibody titers of vaccinated chickens with different commercial Newcastle disease vaccines (V1 to V3 and Razi) in broiler farms located in Alborz province. Note: The birds were inoculated according to the vaccination program reported in Table 1; Error bar= Standard deviation.

Experiment 2: East Azerbaijan farm

Mean MDA on day 1 was 7.1 ± 0.96 log2 titers. On day 21, mean HI titers did not differ significantly, although the highest values were observed in V1 and Razi groups, whereas V3 showed the lowest titers (P > 0.05). The proportion of birds with protective titers on day 21 was higher in V1 and Razi groups compared with V3 (40% and 35% vs. 10%; P< 0.05), while differences involving V2 were not significant (P> 0.05). At day 42, mean HI antibodies were higher in V1 and Razi compared to V3 groups (P<0.05); however, no significant differences were detected among groups regarding the percentage of birds with protective titers (Fig. 2 and Table 4).

Figure 2. The HI antibody titers of vaccinated chickens with different commercial Newcastle disease vaccines (V1 to V3 and Razi) in broiler farms located in East Azerbaijan province. Note: The birds were inoculated according to the vaccination program reported in Table 2; Error bar= Standard deviation. Values with different superscripts (a,b) are significantly different (P<0.05).

Production Performance Indices

Production performance indices for broilers vaccinated with different ND vaccines in Alborz and East Azerbaijan are presented in Tables 5 and 6, respectively. In Alborz, mortality ranged from 6.24% to 9.68%, final body weight ranged from 2.880 to 2.910 kg, and FCR ranged from 1.92 to 1.99. Production indices tended to be higher in the Razi group (~296) compared with other groups (~267–280).In East Azerbaijan, mortality ranged from 2.80% to 4.30%, FCR ranged from 1.99 to 2.00, and production index ranged from ~293 to 335 across groups

Discussion

The control of Newcastle disease (ND) in both commercial and backyard poultry remains essential to minimize economic losses (Hassanzadeh et al., 2024). Vaccination is the cornerstone of ND prevention, and in the endemic regions, it is scheduled by priming within the first week of life, followed by one or more boosters to ensure sustained protective antibody levels.

Live attenuated vaccines are widely used because they stimulate both humoral and cell-mediated immunity. Protection against ND largely depends on the magnitude and persistence of systemic antibodies, primarily IgY, which are routinely measured by serological assays. IgA also contributes importantly to mucosal immunity and can be accessed via tracheal washes or tears (5). Lentogenic vaccinal strains such as LaSota and B1 Hitchner are effective at inducing both local and systemic responses. The ND vaccines manufactured from strains isolated during the 1940s–1960s have been administered for many years; however, their efficacy for achieving HI titers by the chosen threshold, reducing transmission, and controlling outbreaks under high challenge pressure remained controversial(Ambali, Nwoha, & Abdu, 2017; Hassanzadeh et al., 2024; P. J. Miller, King, Afonso, & Suarez, 2007; Samakkhah et al., 2023). Biosecurity measures are still critical, but vaccine type (live vs. inactivated), timing, and manufacturer-specific characteristics strongly influence protective outcomes.

In this study, maternal-derived antibodies (MDA) were at satisfactory levels, indicating efficient transfer from breeder flocks. Since MDA can interfere with live vaccines (Steensels et al., 2025), early ocular administration is preferable to induce local immunity without neutralization. Both experiments applied the first vaccination via eye drop, facilitating early mucosal priming.

Vaccine performance varied by location and schedule. In Alborz, V1 and Razi yielded higher protective rates than V2 and V3 at 21 days, whereas in EastAzerbaijan, V1 and Razi outperformed V3 at day 21; by day 42, titers were comparable across groups. The higher early protection in Alborz likely reflects a more intensive vaccination schedule (B1 at day 1, boosted at day 10 with B1 plus an inactivated ND+AI vaccine). In contrast, in the EastAzerbaijan experiment, vaccination was started by B1 and inactivated ND+AI administration at 7 days of age. Early priming is critical, as primary responses typically take ~2 weeks, explaining stronger antibody levels at 3 weeks in Alborz. The administration of LaSota at 20 days in EastAzerbaijan subsequently resulted in more robust titers at day 42, consistent with its higher immunogenicity.

According to WOAH (WOAH, 2021), an HI titer ≥ 4 log2 and ≥ 85% flock seroconversion are required for effective ND protection. In our study, the Alborz program reached this threshold earlier. Still, the difference between Alborz and Azerbaijan was significant as shown in Figure 1 and 2 only for 2 vaccine brands which showed protective HI titers at 21th days (V1 and Razi groups) more than log₂ 4 with 95% CI in comparison with V1, V2, V3 and Razi in Azerbaijan, But V2 and V3 groups in Alborz were at lower protection rate and titer at 21th day of age and in Azerbaijan experiment too that showed low protection rate and titer at day 21. So our study could not support the idea that vaccination at the first day of age versus the 7th day of age could result in a more explicit protective antibody rate or titer. This is particularly important given that ND outbreaks in Iran typically occur at 4 and/or 5 weeks of age (Mohammad Abdoshah et al., 2022; M Abdoshah et al., 2012). Earlier achieving protective titers could reduce the risk of clinical disease. As of the 42nd day of the two experiments, all vaccination groups and programs had more than mean log₂ 4 and 85%HI≥4 with 95% CI, and no significant differences between brands or regimes were seen, which does not support the preference of a first-day vaccination versus a 7th-day vaccination, either.

Variation in protection between flocks, even with the same vaccine strain, has been reported previously(Ambali et al., 2017; Samakkhah et al., 2023).In our experiments, differences between vaccine groups (e.g., superior performance of V1 and Razi compared to V2 and V3) most likely reflect differences in vaccinal seed properties, manufacturer-related quality, and formulation. Such vaccine intrinsic factors can strongly affect immunogenicity, particularly during the early stages of immune response (Kapczynski et al., 2013; Rauw et al., 2009). Moreover, the discrepancies between the two experimental sites (Alborz vs. East Azerbaijan) are more plausibly attributed to environmental and management-related factors, vaccination schedules, and interactions between vaccine type and field conditions. Variables such as flock management, stocking density, stressors, and MDA levels may modulate the effectiveness of otherwise identical vaccine strains. This highlights that vaccine effectiveness is related to its specification, as well as farm management and production conditions. Additionally, genetic background, stress, or metabolic disorders can delay antibody responses in birds (Oberländer et al., 2020), but booster vaccinations help synchronize immunity across the flock. These influencing factors may therefore explain why the same vaccine can perform differently across geographical or environmental attributes.

Multiple revaccinations enhance and prolong protective immunity: triple LaSota revaccination protected laying hens for 3 months against velogenic NDV (Okechukwu et al., 2020), Boasiako et al. (Boasiako PA., 2024) reported stronger titers with at least three doses. Generally, two to three well-timed vaccinations suffice for broilers, while longer-lived flocks such as breeders or layers require extended programs (Boasiako PA., 2024; Steensels et al., 2025).

Regarding formulations, we used bivalent inactivated ND+AI vaccines alongside live vaccines. Previous studies have shown that bivalent inactivated IB+ND vaccines can induce higher antibody titers than monovalent ND vaccines (El-Dabae et al., 2023; Gough, Allan, & Nedelciu, 1977). However, viral interference should be considered, as IBV infection may impair Harderian gland function. Careful use of well-formulated vaccines can help overcome these limitations.

Beyond immunological outcomes, production indices are a practical measure of vaccination program success. In both experiments, growth performance and feed efficiency were within normal ranges in Iran, suggesting that vaccination did not impair productivity. Although no significant differences in performance parameters were detected among groups, the East Azerbaijan flocks exhibited slightly lower mortality and higher production indices, likely reflecting better management practices or stronger MDA levels. These findings underscore that effective ND vaccination, while essential for disease control, should also be evaluated in relation to production performance to ensure both biological protection and economic viability.

Overall, our findings indicate that both imported and domestic ND vaccines can elicit an effective humoral immune response, but vaccination schedule and management are critical determinants of success. The study showed that priming on the first day of chicken age could lead to a higher humoral antibody titer and proportion rate of positive responses, HI titer ≥ 4 log2, (Alborz experiment) at 3 weeks of age in comparison with priming on the 7th day (Azerbayejan experiment). Therefore, it can be concluded that in high-risk regions for ND outbreaks, earlier immune competency is needed, priming at the first day of age via the ocular-nasal route, even in the presence of maternal antibodies, would be preferred. Also, in case of priming at the 7th day in order to reach more than 85% positive HI titers, using more immunogenic strains such as the Lasota vaccine might be more effective.

Limitations

It is worth mentioning several limitations of the study, which may affect the results, such as observational field setting, confounding by site/management and mixed vaccine components (live + inactivated ND+AI), lack of virology and challenge evaluation, and unit of analysis limitations.

Conclusion

This multisite study demonstrated that both imported and locally produced Newcastle disease vaccines provided broadly comparable levels of serological protection and production performance in commercial broiler flocks. However, some differences were observed in antibody responses at 21 days of age, particularly with V1 and the domestic vaccine (Razi), yielding higher proportions of protected birds; these differences diminished by day 42, indicating that all evaluated vaccines were ultimately capable of inducing protective immunity. The obtained data don’t support a difference between day1 and day7 initiation, nor do they indicate any detectable difference between the two experiments regarding the vaccine initiation time. The vaccination program implemented in Alborz promoted earlier serological responses, while the East Azerbaijan regimen resulted in delayed but stronger antibody titers, likely due to the inclusion of LaSota at midcycle. Production indices remained within acceptable commercial ranges across all groups, with a slight advantage observed for the domestic vaccine (Razi). Overall, the findings suggest that ND vaccines from both foreign and domestic manufacturers, when applied under appropriate vaccination regimens, can provide effective immune response and sustain satisfactory flock performance in Iranian broiler production systems. Razi

Acknowledgements

The authors wish to express their appreciation to everyone who assisted in this study.

Conflict of Interest

The authors declare that they have no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethical Considerations

All ethical procedures were conducted in accordance with accepted veterinary ethical standards and applicable animal welfare regulations. Written informed owner/guardian consent was obtained for all procedures and for publication of this report (including any images, if applicable). The study protocol was reviewed and approved under the ethical approval code IR.RVSRI.REC-1400.003.

Funding

This research did not receive a specific grant from any funding agency in the public, commercial, or not-for-profit sectors.