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. 2024 Mar 22;10(3):e1428. doi: 10.1002/vms3.1428

Evaluation of the immune response of layer chickens to Newcastle disease virus vaccines using the new vaccination regimens

Abel Sorsa Geletu 1, Dereje Tulu Robi 2,✉
PMCID: PMC10959821  PMID: 38519843

Abstract

Background

The study aimed to evaluate the immunological response of layer chickens to live Newcastle disease virus vaccine using a newly developed vaccine schedule administered via the ocular route, as well as assess the persistence of passive antibodies in layer chickens and the effectiveness of protection against strains of the virus.

Methods

A total of 140‐day‐old Lohmann Brown chicks were randomly divided into seven groups, 20 chicks each. Groups 1–3 received a single eye instillation of the vaccine at ages 5, 26 and 54 days, respectively, whereas groups 4–6 received a double eye instillation. Group 7 served as non‐vaccinated control group. Ten days after immunization, samples were taken from hens that had received the vaccine at ages 15, 36 and 64, as well as from control chickens that had not received the vaccine at ages 5, 15, 21 and 31.

Results

A total of 10 serum samples from all chickens exhibited protective antibodies, and booster doses resulted in the highest haemagglutination inhibition titre. No significant change in antibody production was observed among layer hens (p > 0.05). The study found that the La Sota (GMT ± SD: 6.71 ± 4.96), La Sota (GMT ± SD: 8.00 ± 0.00) and thermostable I2 (GMT ± SD: 7.60 ± 6.02), vaccination schedules provided the maximum immune response in single eye instillation, whereas the HB1 (GMT ± SD: 7.11 ± 4.77), La Sota (GMT ± SD: 7.83 ± 5.76) and La Sota (GMT ± SD: 7.60 ± 6.02), combination was the second‐best vaccination schedule in double eye instillation. Furthermore, maternally‐derived antibodies were maintained up to 31 days of age, indicating the level of passive immunity prior to vaccination. Characteristic lesions, such as edematous and diphtheria mucosal membranes of the trachea, along with petechial and necrotic haemorrhages of the proventriculus, were observed during the necropsy of the birds that died from the challenged virus.

Conclusion

This study suggests that subsequent live virus vaccine by ocular route immunization is required to effectively protect against velogenic viscerotropic Newcastle disease infection. The results also highlight the importance of developing effective vaccination schedules and routes to enhance immunity against ND in layer chickens.

Keywords: Haemagglutination inhibition, immunological reactions, protective antibodies, ocular route

Graphical Abstract: This study examined layer immune responses of chickens to Newcastle disease virus vaccines via a novel ocular route. One hundred forty Lohmann Brown chicks were divided into seven groups, receiving single or double eye instillations at various ages. Antibody titres were measured 10 days post‐immunization. The La Sota, La Sota thermostable I2 schedule showed the highest response with single instillation, whereas HB1, La Sota La Sota were effective with double instillation. Maternally‐derived antibodies persisted for 31 days, indicating passive immunity. Necropsy findings emphasized the need for tailored vaccination schedules for enhanced immunity against velogenic viscerotropic Newcastle disease in layer chickens.

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1. INTRODUCTION

Poultry farming plays a vital role in providing sustenance and income for communities worldwide, particularly in low‐ and middle‐income countries where rural households rear a substantial 70% of all chickens (Mujyambere et al., 2022). However, challenges arise in the form of the Newcastle disease (ND), a pervasive threat that significantly impacts chicken populations, leading to high mortality rates during severe outbreaks (Anebo et al., 2014). This disease, a major cause of bird mortality globally, poses a particularly severe risk to chickens, especially in regions like Ethiopia, where it is colloquially known as ‘Yedoro Fengil,’ highlighting its sudden onset and severity (Tulu, 2020; Asfaw et al., 2021).

ND, caused by the Newcastle disease virus (NDV), manifests in various pathotypes, with the velogenic strains, particularly the velogenic viscerotropic (VvND) and velogenic neurotropic subtypes, posing significant threats to chicken health (Samuel et al., 2013; Tulu, 2020). Chickens infected with these strains exhibit diverse symptoms, including respiratory distress, reduced egg production, paralysis and diarrhoea, with postmortem lesions characteristic of VvNDV (Etriwati et al., 2017; Zhang et al., 2023). The economic implications are considerable, as ND not only results in decreased productivity but also demands substantial financial resources for control measures and vaccinations (Ganar et al., 2014; Sedeik et al., 2019).

Despite widespread vaccination efforts, recurrent outbreaks of ND continue to pose challenges to both commercial and rural chicken farms in Ethiopia, and the circulating virulent NDV strains differ genetically from the vaccination strains, raising concerns about the effectiveness of existing immunization strategies (Fentie et al., 2014; Dimitrov et al., 2016). This study aims to address the gaps in understanding by comparing antibody titres in vaccinated chickens using different administration methods, measuring the persistence of maternally generated antibodies, and assessing the protection levels against virus challenge in both vaccinated and unvaccinated layer chicken groups. The findings are crucial for enhancing the efficiency of ND prevention and control strategies in poultry farming contexts.

2. MATERIALS AND METHODS

2.1. Study areas

The study was conducted in the Amibara district, which is situated between Latitude 9° 39′ 59.99″ N and Longitude 40° 19′ 60.00″ E. Werer and Addis Ababa are separated by a driving distance of 291 km. The study was carried out at the Werer Agricultural Research Center, which is located in the Ethiopian region of Afar, at the Institute of Agricultural Research in Ethiopia. The climate in the study area is mainly arid to semi‐arid, with average temperatures ranging from 25 to 42°C and average rainfall of 560 mm per year.

2.2. Experimental house

An experimental house contains 20 pins (2m × 2m), carefully disinfected, provided with teff straw as bedding on the floor. The house was disinfected using a 1% formalin solution. Buckets, feeders and other equipment were cleaned and sterilized before being placed inside the house. The house was left in its current location for 40 days before introducing the chickens.

2.3. Study chickens

A total of 140‐day‐old Lohmann Brown chicks were acquired from Alema farm in Bishoftu, Ethiopia, all of which had a history of being vaccinated against ND by their parent stock. The chicks were raised at the Werer Agricultural Research Center in deep litter with access to water and food, and strict biosecurity measures were implemented. All groups of chicks received the same feed and water ad libitum throughout the experiment.

2.4. Vaccines and antigens

ND vaccines, namely La Sota, Hitchner B1 and thermostable I2, produced by the National Veterinary Institute in Bishoftu, Ethiopia, were administered to vaccinate chickens. Hitchner B1 and La Sota are class II genotype II viruses used as live vaccines worldwide, whereas thermostable I2 is a class II genotype I virus of Australian origin employed as a thermostable live vaccine. These vaccines were frequently distributed upon request to both large‐scale and small‐scale commercial producers but were rarely used to vaccinate rural family chickens in response to outbreaks. The lyophilized La Sota, Hitchner B1 and thermostable I2 vaccines, each with a titre of 107 EID50 per dose, were used after reconstitution into the recommended dilution. Experimental chickens were vaccinated using the eye drop (0.1 mL) method (Ellakany et al., 2018).

2.5. Study design

In the study, 140 day‐old mixed‐sex layer chickens (Lohmann Brown) were distributed into seven groups using the complete random design approach. Each group consisted of 20 randomly assigned chicks. Groups 1–3 were vaccinated with single dose of vaccines at different ages, whereas groups 4–6 received double dose. All vaccinated chickens in groups 1–6 received the NDV vaccine through ocular instillation according to manufacturer guidelines (National Veterinary Institute, Ethiopia). Group 7 served as the unvaccinated control. Table 1 shows the permutations between the three vaccines used. The vaccine administration followed established protocols using a calibrated eye dropper (Alders & Spradbrow, 2001).

TABLE 1.

Experimental design and vaccination schedule.

Vaccination scheduled and vaccine used
Treatments Primary (day 5) Secondary (day 26) Thirdly (day 54) Routes
Group 1 HB1 La Sota I2 Single eye drop
Group 2 La Sota La Sota I2 Single eye drop
Group 3 I2 I2 I2 Single eye drop
Group 4 HB1 La Sota I2 Double dose eye drop
Group 5 La Sota La Sota I2 Double dose eye drop
Group 6 I2 I2 I2 Double dose eye drop
Group 7 Controlled Controlled Controlled Controlled
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Note: HB1, Hitchner B1vaccines,—Thermostable ‐I2 vaccines.

2.6. Serum collection

Ten representative serum samples were collected from each group of chickens for an HI test to measure antibody titres. Samples were collected 10 days after each vaccination on days 15, 36 and 64. Moreover, samples were taken on days 5, 15, 21 and 31 of age from the control group that had not been vaccinated. Blood samples of 1–1.5 mL per bird were obtained using a 3 mL sterile disposable syringe to draw from the wing vein, following the standard methods described by Alders and Spradbrow (2001). The collected blood was allowed to clot overnight at room temperature before being spun at 1000 rpm for 10 min. The separated serum was then kept at −20°C until used to measure the presence of antibodies using HI assays.

2.7. Antibody detection and protection test

A positive outcome for NDV was defined as HI titres of 4 log2 (Office International des Epizooties (OIE), 2013). Each bird was administered a viral suspension of 107 EID50/mL via naso‐ocular routes in doses of 0.1 mL. The NDV strain, known as the Alemaya strain (genotype VI), was used in this study. The EID50 of virus was calculated according to Reed and Muench (1938). The morbidity and mortality of the chickens from each treatment group were monitored in separate pens every day for 15 days.

2.8. Data management and analysis

After being imported into Microsoft Excel for Windows 2010, the data was entered, saved, and then processed using STATA version 14.0 for Windows (Stata Corp.). To analyse (GMT + SD) for both the vaccinated and unvaccinated groups, a Microsoft spreadsheet was used. The ANOVA single‐factor and paired t tests were performed to assess whether there were any significant differences in the HI titres of hens in a group following the first, second and third vaccinations (Beri, 2005). The significance level was set at a p‐value of 0.05 for detecting a significant difference.

3. RESULTS

3.1. Antibody titre for passive immunity

The levels of maternally‐derived antibodies (MDA) in layer chickens were evaluated by measuring antibody titres for passive immunity from days 5 to 31. The mean titre recorded for the chickens was 7.75 ± 5.96 at 5 days old, exhibiting a statistically significant difference (p = 0.002), followed by 6.44 ± 5.30 at 15 days old, although the difference was not statistically significant (p = 0.413). At 21 days old, the mean titre was 6.61 ± 5.05, with a significant difference observed (p = 0.004), and at 31 days old, it was 6.30 ± 4.96, although the difference was not statistically significant (p = 0.119), as shown in Table 2.

TABLE 2.

Haemagglutination inhibition test titres result of passive immunity of layer chicken (GMT ± SD (n = 10).

Age of birds Number of chickens HI titre log2 (GMT ± SD) p‐Value
Day 5 10 7.75 ± 5.96 0.002
Day 15 10 6.44 ± 5.30 0.413
Day 21 10 6.61 ± 5.05 0.004
Day 31 10 6.30 ± 4.96 0.119
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Abbreviations: GMT, geometric mean titre; n, numbers; SD, standard deviation.

3.2. The immune response of vaccinated chickens

The mean and standard deviation of log2 HI titres were 6.00 ± 5.89, 6.71 ± 4.96 and 6.31 ± 6.02 after primary vaccination using single eye instillation in groups 1, 2 and 3, respectively. Notably, significant differences in HI titres were observed in group G‐2 among the groups that received single‐eye drop administration (p = 0.014). Following secondary immunization given after 10 days, the log2 HI titres were 7.74 ± 6.13, 8.00 ± 0.00 and 7.92 ± 5.36 for groups 1, 2 and 3, respectively. After the third vaccination had been given for 10 days, the mean and standard deviation for group one were 6.28 ± 5.86, for group two, they were 7.60 ± 6.02, and for group three, they were 7.49 ± 6.04. For the experimental chicken groups (4–6) receiving two‐eye vaccinations, the log2 HI titres were 7.11 ± 4.77, 6.40 ± 5.76 and 6.50 ± 6.33, respectively. After 10 days of the secondary vaccination, the mean and standard deviation of the log2 HI titres in experimental chicken groups 4, 5 and 6 were 7.83 ± 5.76, 6.92 ± 6.74 and 7.49 ± 6.27, respectively. The mean and standard deviation of the log2 HI titres in groups 4 and 6 after 10 days following the third immunization were 7.60 ± 6.02, 6.32 ± 6.41 and 7.00 ± 6.51, respectively (Table 3).

TABLE 3.

Comparative immunological response of chickens to live Newcastle disease (ND) vaccines using the new vaccination schedules.

Log2 serum HI titre (GMT ± SD) (n = 10)
Groups Routes Post‐1st vaccination Post‐2nd vaccination Post‐3rd vaccination p‐Value
G‐1 Single eye 6.00 ± 5.89 7.74 ± 6.13 6.28 ± 5.86 0.184
G‐2 Single eye 6.71 ± 4.96 8.00 ± 0.00 7.60 ± 6.02 0.014
G‐3 Single eye 6.31 ± 6.02 7.92 ± 5.36 7.49 ± 6.04 0.054
G‐4 Both eye 7.11 ± 4.77 7.83 ± 5.76 7.60 ± 6.02 0.428
G‐5 Both eye 6.40 ± 5.76 6.92 ± 6.74 6.32 ± 6.41 0.483
G‐6 Both eye 6.50 ± 6.33 7.49 ± 6.27 7.00 ± 6.51 0.164
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Note: Groups 1, 2 and 3 (Hitchner B1, La Sota and thermostable I2) had single eye drop admin. Groups 4, 5 and 6 (Hitchner B1, La Sota and thermostable I2) had double eye drop admin. HI testing ages: days 15, 36 and 64.

Abbreviations: GMT, geometric mean titer; G, group; HI, haemagglutination inhibition; n, number; SD, standard deviation.

3.3. Protection results

During the 72‐h (3‐day) period following the challenge, affected birds appeared to be healthy at the day post‐challenge (dpc). Clinical symptoms included reduced feed intake, mild to moderate depression, ruffled feathers, and respiratory distress with gasping and sneezing within 4–15 dpc. Nervous signs, such as wing drop, leg paralysis, prostration and death, were also observed within 4–15 dpc. On the fourth day after the challenge, three chickens in the control group showed signs of morbidity, and seven other chicks began exhibiting further symptoms. The first death was also noted on the fifth day after the test, and all unvaccinated chicks died within 13 days after the challenge. While adopting the new vaccination schedule, vaccines administered through a single eye for groups 1, 2 and 3 and via both eyes (double dose) for groups 4, 5 and 6 at days 5, 26 and 54 of age did not display any usual clinical signs of NDV or death (Table 4).

TABLE 4.

Observed clinical signs after challenge with a virulent virus (Alemaya strain) in layer chicken.

dpc 1–3 dpc 4 dpc 5 dpc 7–9 dpc 10–13
Groups NC NS, LP and Dep Death Death G&S, NS and Dep Death Death Survive (%)
G1 0 0 0 0 0 0 0 100
G2 0 0 0 0 0 0 0 100
G3 0 0 0 0 0 0 0 100
G4 0 0 0 0 0 0 0 100
G5 0 0 0 0 0 0 0 100
G6 0 0 0 0 0 0 0 100
G7 0 3 3 1 4 4 2 000
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Abbreviations: dpc, day post‐challenge; Dep, depression; G&S, gasping and sneezing; LP, leg paralysis; NC, no clinical sign; NS, nervous sign.

3.4. Necropsy results

At necropsy, all of the dead birds had characteristic lesions, such as edematous and diphtheria mucosal membrane in the trachea, petechial and necrotic haemorrhages in the proventriculus, intestine, caecum and caecal tonsils, petechial haemorrhage in the heart and deep‐green contents in the gastrointestinal tract starting from the proventriculus, which ended with green faeces (Figure 1).

FIGURE 1.

FIGURE 1

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Schematic representation of petechial and necrotic haemorrhages in the proventriculus (A), necrotized and coagulated blood in vessels observed in intestinal lesions (B), coagulated blood in the trachea (C), greenish faecal content leaks out on proventriculus (D), haemorrhages, diphtheria mucosal membrane intestine (E) and clotted blood in intestinal wall (F).

4. DISCUSSION

This study aimed to evaluate various vaccination regimens for layer chickens, utilizing ND virus vaccines to establish new, village chicken production system‐friendly vaccine schedules. The results indicated that the La Sota, La Sota and thermostable I2 vaccination schedule produced the best immunological response with single‐eye instillation, whereas the HB1, La Sota and La Sota combination was the second‐best with double‐eye instillation. These results were crucial for enhancing the effectiveness of strategies aimed at preventing and controlling ND in poultry farms.

The birds exhibited the strongest immunological response on the fifth day (7.75 ± 5.96). Subsequently, on days 15 (6.44 ± 5.30), 21 (6.61 ± 5.05) and 31 (6.30 ± 4.96), GMT significantly decreased. The decline in GMT values can be attributed to the waning of maternal antibody levels in the chicks as they grow older. This suggests that the passive immunity acquired from the dam may not be sufficient to protect the chicks from NDV as they continue to grow. Therefore, it is necessary to provide the chicks with active immunization to enhance their immunity against ND. Our findings are consistent with those of Cvetic et al. (2021), who reported that MDA persisted for up to 28 days, and Ezzulddin et al. (2022), Kapczynski et al. (2013) and Oberlander et al. (2020), who also observed that MDA persisted up to 27 days of age. Antibodies in chicks endured for 31 days post‐birth, inherited from parent stock, with peak titres observed during the layer stage. The transfer of abundant passive antibodies began at 5 days, gradually waning but persisting until day 31 in the control group of layer chickens, as evident in the declining levels of MDA.

The highest GMTs were observed in group 2 for single‐eye drop vaccination, with values of 6.71 ± 4.96 for the first vaccination, 8.00 ± 0.00 for the second vaccination and 7.60 ± 6.02 for the third vaccination. Groups 1 and 3 showed lower GMTs but exhibited strong vaccine‐induced immunity. In double‐dose eye drop vaccination, group 4 exhibited the highest GMTs for the first and second vaccinations (7.11 ± 4.77 and 7.83 ± 5.76, respectively), whereas group 6 had the lowest GMT. The results suggest that the route of vaccination (single‐eye drop vs. double‐dose eye drop) had an impact on the immunological response of the chickens to the ND vaccines. The groups vaccinated via double‐dose eye drop had a higher serum HI titre GMT than the groups vaccinated via a single‐eye drop, which is in agreement with the findings of Hassanzadeh et al. (2020). Administering a double dose of vaccination to a flock of chickens addresses the variability in immune responses, ensuring that even birds with weaker immune systems can develop protective immunity (Nedeljkovic et al., 2022; Illango et al., 2005; Sarba et al., 2021). This finding also supports previous study (Hu et al., 2022; Dimitrov et al., 2017; Miller et al., 2013; Ravikumar et al., 2022), which found that a double dose offers a higher concentration of antigens, boosting the possibility of a stronger and more effective immune response in chickens.

The study observed that the antibody titres of layer chickens receiving booster dose vaccines increased after single and double eye instillation. Chickens in group 2, which received their initial vaccination with La Sota at 5 days old and were later boosted with La Sota vaccines at 26 days old, exhibited the highest log2 HI titre (GMT = 8) compared to groups 1 and 3. Notably, statistically significant differences were observed among these groups (p < 0.05). These results correspond with Kokate et al. (2017) findings that the second immunization produced higher HI titres than the first vaccine. These findings are also in agreement with the findings of Umali et al. (2014) and Oberlander et al. (2020). However, the booster dose of live NDV vaccines produced the highest antibody titre in the double‐eyed vaccinated chickens of group 4, and no significant difference was observed (p > 0.05).

Layer chickens primed with double‐dose live thermostable I2 vaccination via ocular route showed the strongest antibody response compared to single‐dose recipients. Birds receiving a single eye drop three times exhibited the highest protective antibody levels. Both trials yielded significant antibody production (p < 0.05). Nega et al. (2012) reported that the I2 thermostable vaccine has been widely used to protect village chickens against ND. This is due to its decreased dependence on the cold chain for transport and storage. Furthermore, Okechukwu et al. (2020) found that triple La Sota revaccinations provided 3 months of protection against a decrease in egg production caused by velogenic viscerotropic NDV infection in laying chickens. Furthermore, Boasiako et al. (2022) discovered that chicks receiving three vaccinations had stronger protective antibody titres.

The chickens that were under control exhibited various signs, such as reduced feed intake, depression, gasping, sneezing and respiratory problems. Furthermore, they showed nervous signs like wing droop, leg paralysis and prostration, which, in some cases, resulted in death. The clinical signs of ND observed in this study are consistent with the findings of Zhang et al. (2023) and Fentie et al. et al. (2014). At necropsy, the dead birds displayed typical lesions, including haemorrhages in the proventriculus and diphtheria mucosal membranes of the trachea. The observed lesions in the trachea and proventriculus are also consistent with previous findings (Fentie et al., 2014; Anjum et al., 2020), where unvaccinated birds exhibited similar pathological changes post‐challenge. This underscores the importance of vaccination in preventing or mitigating such severe clinical outcomes. Hu et al. (2022) and Ananda Kumar et al. (2023) demonstrated that the NDV vaccine induced a robust immune response characterized by protective immunity, likely contributing to the observed protection in our study. Furthermore, the studies by Dimitrov et al. (2017) and Fallah Mehrabadi et al. (2020) highlighted the importance of vaccine coverage in a population for effective control of ND.

A new ocular vaccination method for layer chickens in the study achieved 100% protection with 1:16 antibody titres against VvNDV. Compared to the standard schedule used in Ethiopia, this approach provided superior protection against the virulent ND virus, enhancing layer chicken immunity through eye‐administered live virus vaccines. Anebo et al. (2014) reported similar findings, stating that the new immunization method provided protective immunity that was comparable to the conventional approaches employed in Ethiopia. Furthermore, several studies (Kapczynski et al., 2013; Sedeik et al., 2019; Sedeik et al., 2022) have suggested that vaccination programs employing a progressive strategy, which involves successive booster immunizations with increasingly virulent strains, could offer protection against clinical disease and mortality following infection with virulent NDV. This is provided that vaccinated birds maintain high antibody levels. In the present study, all vaccinated chickens developed protective antibody levels, and a positive correlation was observed between the presence of these levels on the day of challenge in layer birds and protection against the challenging strain.

The study only evaluated the immunological reactions of layer chickens to a live NDV vaccine using a new vaccine schedule and ocular route of administration. Thus, its results may not be widely applicable to other vaccine schedules or routes of administration. Furthermore, the study only assessed the persistence of passive antibodies up to 31 days of age, which may not provide a complete understanding of the long‐term effectiveness of vaccines. Future research could investigate the persistence of passive antibodies over a longer period to determine the duration of immunity conferred by maternally‐derived antibodies. The study also did not evaluate the efficacy of vaccines against other strains of the virus, limiting its applicability to different virus strains. While providing valuable insights, further studies are necessary to broaden the scope and comprehensively evaluate the vaccine efficacy in layer chickens.

5. CONCLUSION

The most effective vaccination schedule for eliciting the strongest immune responses in layer chickens involves a combination of La Sota, La Sota and thermostable I2 vaccines administered as a single eye drop. This regimen includes vaccinations at 5, 26 and 54 days of age, resulting in the highest level of immunity. The second‐best immunization regimen is the administration of Hitchner B1, La Sota and thermostable I2 vaccines in combination with double ocular drops. Although not as effective as the top regimen, it still generates robust immune responses. Surprisingly, a single dose of the thermostable I2 vaccine administered through the ocular route at ages 5, 26 and 54 produces more protective antibodies than a double dose of the thermostable I2 vaccine in combination with the Hitchner B1, La Sota and thermostable I2 vaccination schedules. This information is crucial for the poultry industry in developing effective vaccination strategies to combat ND and safeguard the health and productivity of layer chickens. Further research and improvements in vaccination schedules and routes could continue to enhance immunity against this significant poultry disease.

AUTHOR CONTRIBUTIONS

Conceptualization, methodology, writing – original data preparation and writing – review and editing: Abel Sorsa Geletu. Formal analysis and investigation, project administration and supervision: Dereje Tulu Robi. All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors say they have no conflicts of interest.

FUNDING INFORMATION

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

PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/vms3.1428.

ETHICS STATEMENT

Ethiopian Institute of Agricultural Research (EIAR) carried out all techniques following the experimental standards and procedures that were approved by the ethical committee of EIAR animal health research program (reference number EIAR‐2662/2010) on animal welfare and ethical issues and were in line with the international standards for animal welfare.

CONSENT FOR PUBLICATION

Not applicable

ACKNOWLEDGEMENTS

We appreciate the financial assistance provided by the Ethiopian Institute of Agricultural Research (EIAR). The National Veterinary Institute (NVI) laboratory provided logistic support to the authors. In addition, we appreciate the assistance provided by the Werer Agricultural Research Center during experiment.

Geletu, A. S. , & Robi, D. T. (2024). Evaluation of the immune response of layer chickens to Newcastle disease virus vaccines using the new vaccination regimens. Veterinary Medicine and Science, 10, e1428. 10.1002/vms3.1428

DATA AVAILABILITY STATEMENT

The corresponding author will provide the data used in the current study upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The corresponding author will provide the data used in the current study upon reasonable request.

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