Evaluation of the Efficacy of Three Newcastle Disease Vaccines Produced at the National Veterinary Institute, Bishoftu, Ethiopia, at Different Temperature Storage Conditions

Abstract

Newcastle disease, which affects poultry and is endemic in many nations across the world, is caused by Avian Paramyxovirus-1 (APMV-1). This experimental study was conducted from January to June 2021 at the National Veterinary Institute (NVI) to evaluate the virus viability and antibody titer of Newcastle disease vaccines (Hichner’s B1, Lasota, and ThermostableI2) stored at different temperature storage conditions. Chickens (12 treatment groups and 1 control group) were vaccinated and challenged with the virulent ND virus (0.5 × 10^6.5 embryonic lethal dose fifty (ELD₅₀)). The immune responses (antibody titers) of chickens were evaluated using hemagglutination (HA) and hemagglutination inhibition (HI) assays. The Newcastle disease vaccines (Hachiner’s B1 (ND-HB1), ND-Lasota, and ND-Thermostable I2) stored at +4 °C HI-induced antibody titers of 151 (±103.3), 136 (±53.4), and 145 (±91) on day 14, respectively, whereas on day 21, they increased to 160 (±82) for ND-HB1 and 144 (±74.5) for ND-Lasota. ND-Thermostable I2 showed a decrement to 133 (±44.8). All three vaccines stored at different temperature storage conditions (+4, +23, and +30 °C) used in this experiment induced antibody titers greater than 128 on day 28 post-vaccination, except the Newcastle disease vaccine Thermostable I2 stored at +30 °C. The vaccines collected from private veterinary drugstores (customer vaccines Hachiner’s B1 and ND-Thermostable I2) used in this experiment induced very low antibody titers, less than 128 antibody titers, from days 14 to 21. Statistically significant induced mean antibody titers were observed for chickens that received vaccines stored at different temperature storage conditions for 72 h (p < 0.05), except for the ND-HB1 mean HI-induced antibody titer at days 7 and 28. Further, vaccine protection was confirmed by inoculation of both the vaccinated (treatment groups) and control groups by the virulent ND virus, where the control group started dying three days post-challenge but all chicks that received the vaccines survived. Overall, this study showed the impact of temperature storage conditions on the antibody titer and their effect on the titer of the viable virus in the vaccine, and thereby its protective capacity, warranting appropriate cold chain management of the vaccines along the value chain.

1. Introduction

Newcastle disease (ND), caused by Newcastle disease virus (NDV), an Avian Paramyxovirus-1 (APMV-1), subfamily Avulavirinae, genus Orthoavulavirus, and species Avian orthoavulavirus, is one of the important contagious viral diseases of domestic poultry and wild birds [1,2]. Though it is vaccine-preventable, it is a persistent threat to the poultry industry across the globe in general and Ethiopia in particular. In Ethiopia, a meta-study conducted indicated a pooled seroprevalence of 21.47% (19.54–23.4%) [3]. The control approach used so far in Ethiopia is vaccination using three different vaccines produced by NVI and available vaccines from commercial providers. Though millions of doses are used for vaccinating commercial poultry production year-round, outbreak occurrence is a common phenomenon (anecdotal evidence). The repeated outbreak occurrence results from vaccine failure that arises from changes in the genetic diversity of the circulating strain (mismatched vaccine strains), incorrect vaccine timing, mishandling of vaccines, poor vaccine quality, and low antigen load in the vaccine. Other factors include immune suppression, maternal antibodies, stress, and management practices [4,5].

Some countries in Africa and Asia have implemented regular vaccination programs using live or inactivated vaccines of ND based on lentogenic virus genotypes I and II for effective control of the disease [6]. On the contrary, well-vaccinated commercial farms in many endemic countries have reported outbreaks of ND [7]. The virus is known for its rapid evolution that has brought the genetic divergence between strains responsible for outbreaks and the vaccines available to control the disease [8,9]. On the other hand, these vaccines can still protect chickens from the disease but not viral excretion [10,11,12].

Vaccination is the most cost-effective way to control Newcastle disease (ND) in poultry. Several conventional live vaccines are available internationally and have successfully reduced disease incidence. However, maintaining a cold chain during storage and distribution is a major challenge, especially in tropical regions where ND is widespread. Poor biosecurity and inadequate storage conditions lead to significant economic losses, and in countries with unreliable electricity, vaccines may be transported for hours without refrigeration, further diminishing their efficacy [13].Storage temperature’s impact on vaccine efficacy has been reported by different researchers, among which is the foot-and-mouth disease (FMD) vaccine, and other vaccines are often stored outside the recommended temperature ranges in veterinary drugstores, which reduces vaccine efficacy [14]. Another study indicated that a type O FMD vaccine remained 100% effective after 2 years at 4 °C, 3 weeks at 25 °C, and 1 week at 37 °C. However, efficacy dropped to 80% after 4 weeks of storage at 25 °C or 2 weeks at 37 °C [15].

Though effective evaluation is mandatory to control ND in poultry, evaluating vaccine efficacy using serological tests is effective for assessing immune responses, such as antibody titers. The detection of Newcastle disease virus (NDV) typically relies on HA and HI tests, regarded as the gold standard. However, despite vaccine use in broilers, recent reports of vaccination failures have raised concerns about the effectiveness of current vaccination approaches [16].

The vaccines produced at the National Veterinary Institute (NVI) are used at different ages and production stages, starting from day one. The repeated outbreak occurrence in Ethiopia might arise from vaccine failure as indicated above due to improper storage of the vaccine out of the recommended storage conditions and possibly other factors associated with the value chain of the vaccine.

The optimal storage temperature for the ND vaccine produced at the NVI is indicated on the product description of the vaccine: from +4 °C to −20 °C [17].

Based on the above rationale, we aimed to evaluate the antibody titer of chickens vaccinated using ND-HB1, ND-Lasota, and ND-Thermostable I2 vaccines stored both in and out of the recommended storage temperature conditions intentionally.

2. Materials and Methods

2.1. Vaccine Selection and Storage

This study was conducted at NVI, an animal facility, in Bishoftu, Ethiopia. All vaccines included in this study were produced by the National Veterinary Institute, except the vaccine collected from a private veterinary drugstore that was designated as a customer vaccine afterwards, and all vaccines purchased from the NVI were stored at −20, +4, +23, and +30 °C for 72 h. These vaccines were selected considering their wider use in Ethiopia. The vaccine was collected from the customer; though we did not have the exact temperature storage conditions used during transport and storage (at the private veterinary drugstore), it was stored similarly to the vaccines from the NVI. All the vaccines were checked for the presence of viable virus by an HA test after 48 h of incubation in the respective storage conditions. The three experimental storage temperature conditions (+4, +23, and +30 °C) were chosen based on the availability and environmental conditions. This means +4 °C was chosen for the fact that any customer that buys the ND vaccine has at least a kitchen refrigerator to store the vaccine. The other storage temperature condition in this experiment was +23 °C; this was simply chosen due to the fact that an individual can purchase a vaccine and store their vaccine at room temperature until the vaccinator is available or until arrival at the farm for vaccination. The third storage temperature condition, +30 °C, was also chosen considering the fluctuation in daily temperature, especially the environmental temperature of the low land and Rift Valley area. The duration of incubation was set to 72 h considering the distance of the vaccine-producing institute and that the farms located in the country might take three days to reach by vehicle.

2.2. Animals, Vaccines and Treatment Groups

One hundred and nineteen (119) experimental chicks were hatched at the NVI Research and Development (R&D) Directorate and were brooded together until 14 days in a pen with infrared bulbs as a heat source and teff straw for bedding. On the 14th day, the chicks were randomly split into 13 treatment groups for each vaccine used (ND-HB1, ND-Lasota, and ND-Thermostable-I2) depending on the vaccine storage temperature conditions as described under the study design below (

Table 1. Design of vaccination experiment.

Group	No. of Chickens	Temperature °C	Type of Vaccine	Duration of Storage	Route of Administration
Groups 1–4	10 chicks each	Customer vaccine (−20)	ND-HB1	72 h	Intraocular
		+4
		+23
		+30
Groups 5–8	10 chicks each	−20	ND-Lasota	72 h	Intraocular
		+4
		+23
		+30
Groups 8–12	10 chicks each	Customer vaccine (−20)	ND-TH-I2	72 h	Intraocular
		+4
		+23
		+30
Groups 13	7 chicks	Control

). The chickens were fed on a purchased commercial ration for 2 months. All of them were confirmed to be seronegative to APMV-1 using HI following the standard methods [18] before experimentation. Three types of lyophilized vaccines were used: the conventional freeze-dried ND-HB1 and ND-Lasota with a titer of 10⁻⁹ embryo infective doses (EID₅₀) per dose and the freeze-dried ND-thermostable I2 vaccine containing 10⁻⁹ EID₅₀ per dose produced by NVI, Bishoftu, Ethiopia. Thirteen different experimental groups were studied for three vaccines and four temperature storage conditions (Table 1).

Groups 1–4: Each chick received one dose of vaccine collected from a private veterinary drugstore (customer) stored at −20 °C, and the NVI vaccine stored at +4, +23, and +30 °C. The type of vaccine used here was the ND-HB1, and the route of administration was intraocular (i.o.).

Groups 5–8: Each chick received one dose of the vaccine collected from a private veterinary drugstore (customer) stored at −20 °C and the NVI vaccine stored at +4, +23, and +30 °C. The type of vaccine used here was ND-Lasota, and the route of administration was intraocular (i.o.).

Groups 9–12: Each chick received one dose of the vaccine collected from a private veterinary drugstore (customer) stored at −20 °C and the NVI vaccine stored at +4, +23, and +30 °C. The type of vaccine used here was ND-thermostable I2, and the route of administration was intraocular (i.o.).

Group 13 remained untreated; they received only distilled water through the intraocular (i.o.) route at the time when the other chicks were vaccinated. Each group was composed of ten chicks except for the control group (Group 13), where only thirteen chicks were used.

2.3. Stability of the ND Vaccine

The stability, infectivity, and HA activity of the ND vaccine were tested after reconstituting it with phosphate-buffered saline (PBS). The reconstituted vaccine solution was kept at room temperature for 48 h. Embryonated eggs were inoculated with 0.1 mL (mixture of vaccine diluted in 10 mL of PBS) to check the viability of the vaccine virus.

2.4. Blood Sample Collection

Blood samples (1.5 mL per chick) were collected using a 3 mL sterile disposable syringe from the wing vein following the standard methods described in [19]. The blood samples were collected on days 0, 7, 14, 21, and 28 from each group. The blood samples were labeled with identification aligned to the chicks sampled and allowed to clot overnight at room temperature to facilitate serum separation. Half a milliliter (0.5 mL) of serum was harvested in labeled cry vials and stored at −20 °C until the HI titer was carried out. Collection of the blood for serological testing was carried out by an experienced researcher.

2.5. Hemagglutination Inhibition Test

The HI test as a serological method was used as described [14] and conducted with twofold serum dilutions of 4 units of hemagglutinin (in-house-produced at NVI). This was carried out by inoculating a local virulent strain of Newcastle disease virus into an eleven-day-old embryonated chicken egg; the allantoic fluid containing the virus was harvested. Cell remnants and debris were removed by centrifugation. Then, the supernatant was filtered using 0.22 μL and concentrated using precipitation. The hemagglutinin used for this experiment was from the final precipitate. The range of serum dilution used in this study was twofold dilution (1:2, 1:4, and 1:8), where 25 μL of serum was serially diluted to 25 μL of PBS. This preparation used 4–8 HA units and 1% chicken erythrocyte suspension (prepared in-house). The sera and all other reagents were brought to room temperature before the assay. The sera were inactivated at 58 °C for 30 min in a water bath, and then the virus (in-house-produced at NVI) was added in each well. The plates were shaken and kept for 15 min at room temperature. Finally, 1% RBC suspension was added; it was shaken gently and left to stand for 30 min. In performing this test, reference positive serum (in-house-produced at NVI), negative serum (in-house-produced at NVI), known antigens, and 1% avian red blood cells (ARBCs) were used as controls. The last dilution where there is complete inhibition of hemagglutination represents the titration of the serum. Full agglutination indicates a negative result. The cut-off titer for the ND vaccine is 1:16. Hence, sedimentation of ARBCs at a dilution of serum ≥ 1:16 indicates positive reactions.

2.6. Challenge and Clinical Monitoring of Vaccinated Chickens

All the vaccinated and non-vaccinated chickens were challenged by embryonic lethal dose (0.5  ×  10^6.5 ELD₅₀ based on viral titration) as described by Abdi et al. [20] with minor modification. A local virulent ND virus strain was obtained from the NVI pathogen bank and inoculated via the intramuscular route into the breast muscle. After the challenge, the animals were observed daily for fourteen days, and all cases of morbidity (100%) and mortality (100%) were recorded in the control group. All suffering chickens were humanely sacrificed. Necropsies were carried out on dead or sacrificed birds to evaluate lesions due to NCD and to take samples of the affected organs. Brain, spleen, and tracheal tissue was collected from each chicken after necropsies, and the matrix protein gene of the Newcastle virus was checked by PCR.

2.7. Evaluation of Viral Shade after the Vaccination and Challenge

Spleen, brain, and tracheal swab samples were collected, chopped into small pieces, and ground with sterile sand in 10% PBS using a mortar and pestle in a virology laboratory. The mixture was centrifuged at 3000 rpm at +4 °C for 20 min. Then, the suspension was collected in a separate tube, and viral RNA was extracted using a Qiagen viral RNA mini kit according to the manufacturer’s instructions (Qiagen, Germantown, MD, USA). Polymerase chain reaction (PCR) was performed targeting the viral matrix protein gene using primers (M) M + 4100, FW: 5′-AGTGATGTGCTCGGACCTTC-3′ and M-4220, RV: 5′-CCTGAGGAGAGGCATTTGCTA-3′, as described previously [21]. The reaction was allowed to run for one-step RT-PCR initial cDNA synthesis at 50 °C for 30 min. This was followed by initial denaturation at 95 °C for 15 min, followed by denaturation at 94 °C for 30 s of 40 cycles. Then, annealing dropped to 55 °C for 30 s for 40 cycles. The elongation step was for 30 s for 40 cycles at 72 °C. The final elongation was set at 72 °C for 5 min for one cycle. After gel electrophoresis using 2% agarose gel, visualization was performed by a gel Doc system fitted with a UV light and a camera. The amplified product for the NDV matrix gene was found at 120 bp.

2.8. Data Management and Analysis

The post-vaccination mean-induced HI antibody titers were analyzed using IBM SPSS Statistics (Statistical Package for the Social Sciences) version 23. In this case, mean, standard deviation, and standard error were calculated. In addition, the impact of storage temperature condition on antibody titers at 7, 14, 21, and 28 days post-vaccination was analyzed using simple linear regression, and statistical significance was computed considering storage temperature as an independent variable, whereas the induced antibody titer was considered as a dependent variable.

3. Results

3.1. Mean Induced Antibody Titer of Chickens Vaccinated with Newcastle Disease Vaccine

This ND-Lasota vaccine was stored at three different temperatures in order to see the storage temperature conditions’ effect on the vaccine antibody response in chickens. At day zero, the antibody titer was not detected for unvaccinated chickens. Chickens that received the vaccines stored at 4 °C via the intraocular route (i.o.) at 7, 14, 21, and 28 days post-vaccination showed HI-induced mean antibody titers of 20 (±18.6), 136 (±53.4), 144 (±74.5), and 208 (±66.2), respectively. When the vaccine stored at +23 °C was used, and the HI-induced mean antibody titers detected were 16 (±16), 96 (±67.8), 136 (±74.6), and 199 (±67.4) at 7, 14, 21, and 28 days post-vaccination, respectively. The maximum storage temperature used in this experiment was +30 °C, and chickens that received this vaccine were seen to have induced mean antibody titers of 14.5 (±9.5), 92 (±71.4), 112 (±66.2), and 176 (±66.2) at 7, 14, 21, and 28 days post-vaccination, respectively. In this experiment, the trend of induced antibody titer was seen to increase from 7 to 28 days in a similar fashion for all Newcastle disease vaccines stored at different temperature conditions. A higher HI-induced mean antibody titer was detected in chickens that received the vaccine stored at +4 °C at all days post-vaccination than those stored at +23 and +30 °C (Figure 1). In this study, HI-induced antibody titers greater than 16 were seen in most storage conditions. On the other hand, the vaccine stored at +4 °C induced a better antibody titer of 136 (±53.4) starting from day 14, which was not the case for other storage temperature conditions. In this study, the increased antibody titer of chicks that received vaccines stored at different storage conditions was statistically significant (p < 0.05) (

Table 2. Storage temperature conditions of Newcastle disease vaccines and induced mean antibody titers.

Storage Temperature	Days Post-Vaccination	Mean HI Antibody Titer	SE	CI	p-Value
NCD-HB1 (+4, +23, +30)	7	15.57	6.03	[−14.66, 49.48]	0.14
	14	85.39	19.00	[−4.24, 217.72]	0.05
	21	108.81	22.01	[53.96, 183.92]	0.01
	28	145.72	21.13	[112.62, 341.32]	0.20
NCD-Lasota (+4, +23, +30)	7	16.83	1.64	[20.62, 21.07]	0.00
	14	108.00	14.05	[51.07, 232.53]	0.03
	21	130.37	9.54	[−16.78, 318.20]	0.05
	28	194.34	9.53	[48.24, 381.15]	0.03
NCD-TH-I2 (+4, +23, +30)	7	23.05	3.07	[13.12, 34.92]	0.01
	14	111.76	17.98	[19.99, 188.35]	0.03
	21	133.83	10.48	[72.70, 173.94]	0.01
	28	153.71	19.21	[59.34, 234.79]	0.02

).

The second vaccine used in this experiment was the Newcastle disease vaccine Hachiner’s B1 (ND-HB1) stored at three different temperature conditions (+4, +23, and +30 °C), in addition to the customer vaccine purchased from a private veterinary drugstore believed to be stored at −20 °C. The HI-induced mean antibody titers of chickens that received the vaccine stored at +4 °C at 7, 14, 21, and 28 days post-vaccination were 4 (±5.3), 151 (±103.2), 160 (±82.2), and 244 (±38.9), respectively. For the chickens that received the vaccines stored at +23 °C, HI-induced mean antibody titers of 19 (±10.3), 64 (±35.1), 157 (±82.7), and 174 (±64.5) were recorded at 7, 14, 21, and 28 days post-vaccination. For the group of chickens that received the vaccines stored at +30 °C, the HI-induced mean antibody titers were 33.5 (±76.5), 122 (±72.7), 157 (±66.2), and 157 (±82.7) at 7, 14, 21, and 28 days post-vaccination. The ND-HB1 vaccine (believed to be stored at −20 °C at a private veterinary drugstore) from the customer was also given to chickens and it was found that the HI-induced mean antibody titers were lower than for all vaccines stored at different storage temperature conditions on day 21 and 28 post-vaccination. Generally, in this experiment, an increase in antibody titer was found from day 7 to 28 (Figure 2). In this study, the increased antibody titers of chicks that received vaccines stored at different storage conditions were statistically significant (p < 0.05), except for the antibody titers induced at days 7 and 28 (Table 2).

The third vaccine used in this experiment was Newcastle Thermostable I-2, produced at the NVI and stored at three different storage temperature conditions (+4, +23, and +30 °C). The customer vaccine was believed to be stored at −20 °C. The HI-induced mean antibody titers of chickens that received the vaccine stored at +4 °C on 7, 14, 21, and 28 days post-vaccination were 29 (±15.7), 145 (±90.9), 133 (±44.8), and 177 (±94.2), respectively. For the chickens that received the vaccine stored at +23 °C, the HI-induced mean antibody titers on 7, 21, and 28 days post-vaccination were 21 (±17.5), 84 (±38), 152 (±67.8), and 168 (±76). For the chickens that received the vaccine stored at +30 °C, the HI-induced mean antibody titers were 19 (±12), 105 (±76), 115.7 (±71), and 115.7 (±54.7) on 7, 14, 21, and 28 days post-vaccination. The thermostable Newcastle disease vaccine (believed to be stored at −20 °C at a private veterinary drugstore) from the customer was also given to chickens and it was found that the HI-induced mean antibody titer was lower than that of all vaccines stored at different temperature storage conditions on days 21 and 28 post-vaccination, except for that stored at +30 °C. Generally, this experiment found that the trend of induced antibody titer increased from day 7 to 28 (Figure 3). In this study, the increased antibody titers of chicks that received vaccines stored at different storage conditions were statistically significant (p < 0.05) (Table 2).

In this experiment, it was seen that storage conditions have an impact on vaccine immunogenicity. Therefore, the length of storage of the customer vaccine outside the cold chain could be another possible factor in vaccine failure. Our study only stored vaccines for 72 h in those designated storage conditions.

In our experiment, we checked the induced immune response of chickens vaccinated with three Newcastle disease vaccines stored at three different storage temperature conditions. The mean antibody titers on days 7, 14, 21, and 28 were determined for chickens that received vaccines stored at different storage conditions for 72 h. A statistically significant mean HI-induced antibody titer was observed at different dates of vaccination (p < 0.05), except for the ND HB1 mean HI antibody titer on days 7 and 28 (Table 2).

3.2. Challenge of Vaccinated and Control Chickens

Both (vaccinated and control) chickens were challenged using the virulent Newcastle disease virus at 28 days post-vaccination. The challenged chickens were checked every day for evidence of morbidity and/or mortality for both the vaccinated and control groups. There was no morbidity in chickens who received vaccines stored at different storage conditions or vaccines collected from customers. On the other hand, all control chickens started to show clinical signs of Newcastle disease (morbidity). By the 8th day of the challenge, all the control chickens had surrendered to the challenge and died (8/8, 100% mortality), while all vaccinated chickens survived (100% survival) (

Table 3. Post-challenge and survival rate of chickens vaccinated with vaccine stored at different conditions.

Vaccines and Storage Conditions	Survived	Dead
Control	0	8
ND-HB1 + 23	11	0
ND-HB1 + 30	11	0
ND-HB1 + 4	11	0
ND-HB1 cu	10	0
ND-L + 23	9	0
ND-L + 30	8	0
ND-L + 4	8	0
ND-Thermostable-I2 cu	11	0
ND-Thermostable-I2 + 23	8	0
ND-Thermostable-I2 + 30	13	0
ND-Thermostable-I2 + 4	11	0

).

Dead chickens were also sampled to check if the cause of death was the challenge virus. For this, spleen, brain, and tracheal swab samples were collected from dead birds. These samples were transported in a viral transport medium to the virology laboratory and then to the molecular biology laboratory in a cold chain to perform PCR. The target gene here was a matrix gene and amplification of the gene was found at 120 bp. In this analysis, it was found that all birds died of the virulence virus inoculated in our experiment. The following figure shows that lanes 1–5 and 7 are the samples from dead birds, and lane 8 is a control. The molecular marker used here was DNA Molecular Weight Marker XIV (100 bp ladder) from Sigma Aldrich (St. Louis, MO, USA) (Figure 4).

3.3. Viable Virus Titer of the Vaccines

The ND vaccine’s viable virus titer after 48 h of incubation in the respective storage temperature conditions was evaluated by an HA test. At +4 °C storage temperature conditions, the viable virus titers for ND-HB1, ND-Lasota, and ND-Thermostable-I2 were 64, 32, and 128. For the +23 °C-stored vaccines, the viable virus titers were 32, 32, and 128 for ND-HB1, ND-Lasota, and ND-Thermostable-I2. At +30 °C storage temperature conditions, the viable virus titers were 32, 16, and 128. The viable virus titer of the vaccine collected from customers was 128. The storage duration of the customer vaccine was not considered here as it was collected from a willful customer.

4. Discussions

This research has investigated different storage temperature conditions and their impact on the induced antibody titer of three Newcastle disease vaccines (ND-HB1, ND-Lasota, and ND)-I2 produced at NVI. The storage temperature conditions used were +4, +23, and +30 °C for 72 h. In addition, vaccines collected from a private drugstore (customer vaccine) were also used, considering their storage at −20 °C. This investigation was carried out considering power failure, long-distance travel with the vaccine, and vaccination delay. These conditions have an impact on the viability of the live virus in the vaccine, affecting its immunogenicity. In developing countries like Ethiopia, where infrastructures such as power are poor and cold chain vaccine transportation facilities are not well organized or available, the quality of the vaccine would be in question [22].

The viable virus titers found in ND-HB1 and ND-Lasota were below the standard. This is in agreement with the finding of Sieng et al. [14], who reported a decline in the virus titer under a fluctuating storage temperature. From the three vaccines, the ND-Thermostable-I2 viable virus titer was 128. This was also supported by the finding of Wajad et al. [6], who reported a temperature effect on the stability of the vaccine virus.

The HI-induced mean antibody titers of the vaccine stored at +4 °C on days 14, 21, and 28 post-vaccination with ND-Lasota showed protective titers (136, 144 and 208) that were greater than 16. Similarly, the HI-induced mean antibody titer of the vaccine stored at +4 °C on days 14, 21, and 28 post-vaccination with ND-HB1 showed protective titers (151, 160, and 208) that were greater than 16. The third vaccine-induced mean antibody titer of the vaccine stored at +4 °C at days 14, 21, and 28 post-vaccination with ND-Lasota showed protective titers (145, 133, and 177) that were greater than 16. Our findings are in agreement with the findings of Bordoloi et al. [16], who evaluated ND vaccine antibody titers in broilers and found comparable results.

The customer vaccines used (ND-HB1 and ND-Thermostable-I2) on days 14 and 21 in this experiment had HI-induced mean antibody titers of 89 and 70 for ND-HB1 and 84 and 107 for ND-Thermostabl-I2, which were lower than other vaccines. Our results are in agreement with the findings of Boumart et al. and Zander et al. [13,22] that the storage temperature conditions of vaccines can significantly affect the HI titer of the vaccines and in turn affect the induced antibody titer, leading to vaccine failure. In this experiment, it was found that storage conditions have an impact on vaccine potency, and thereby the vaccine antibody titer. The recommended storage temperature condition should be maintained for the vaccines to remain potent during storage. From this study, it was observed that vaccines stored at +4 °C storage temperature conditions could also give a good protective antibody titer starting from day 14 post-vaccination. There was an increasing trend of HI-induced mean antibody titer on day 28 post-vaccination for all the vaccines stored at +4 °C, whereas this was not the case for other storage temperature conditions. The customer vaccine stored at −20 °C, believed to be in a stable storage temperature condition, had an antibody titer that was far below the value of the antibody titers of other vaccines for the same date of vaccination. Therefore, the length of storage of vaccines outside the cold chain could be another possible factor in vaccine failure. The limitation of our study is that we only stored vaccines for 72 h in those designed storage conditions.

5. Conclusions

In conclusion, Newcastle disease vaccines Hachner’s B1 (ND-HB1), ND-Thermostable-I2, and ND-Lasota stored at different temperature conditions for 72 h and administered via the intraocular route provoked protective antibody titers and survival after infection with the virulent challenge virus. Vaccines stored at +4 °C produced high HI-induced mean antibody titers for all three vaccines used in this experiment. The vaccine stored under the second storage condition (+23 °C) also induced a good antibody titer that started from days 7 to 28 post-vaccination. The +30 °C vaccine- and customer vaccine-vaccinated chickens had lower antibody titers than the above two. It was also seen that there was a tendency to decrease HI-induced mean antibody titers for all storage temperature conditions except the one stored at +4 °C. An important finding here is that +4 °C was found to be an ideal storage temperature condition with a higher antibody titer. Further, the viability of the vaccine virus was also affected by an increase in temperature as well as the length of storage. Overall, this study showed the impact of storage conditions on the induced antibody titer and its effect on the titer of the viable virus in the vaccine, and thereby its potency, warranting the appropriate cold chain management of the vaccines along the value chain.