Comparison of the Impact of Different Administration Routes on the Efficacy of a Thermoresistant Newcastle Disease Vaccine in Chickens

Abstract

Route of vaccine administration has a great impact on immunization and protection outcomes in chickens. This study was conducted to compare the effect of different administration routes on the efficacy of a thermoresistant Newcastle disease (ND) vaccine (ND.TR.IR) in chickens. A total of 100 one-day-old specific pathogen-free chicks were divided into five groups (n = 20 chicks per group) and vaccinated through different routes at 10 and 20 days of age. Treatments included no vaccination (control [C]), 1 dose inoculation through eye drop (ED), 1 dose inoculation through drinking water (DW), 1 dose inoculation through feed (FV₁), and 10 doses inoculation through feed (FV₁₀). At 20 and 34 days of age, antibody titers were measured against ND virus (NDV) in all the chickens by hemagglutination inhibition (HI) test. Chicks immunized with ND.TR.IR vaccine through different routes of administration also were intramuscularly challenged with a local virulent NDV (vNDV) (Ck/ir/Beh/2011) 14 days after booster vaccination (at 34 days of age). Our results showed that in comparison with the FVs groups, the immunized chicks through ED induced a higher HI antibody titers at 20 days of age (p < 0.05). Meanwhile, vaccination through ED induced higher HI antibody titers at day 34 of age compared with all other groups (p > 0.05). The percentages of the protective HI antibody titers (≥log₂ ³) detected in ED and DW groups at 20 days of age were higher than those detected in the FV₁ group (p < 0.05). However, routes of vaccination had no significant effect on the rate of protective titers at day 34 of age (100%, 90%, 75%, and 85% for ED, DW, FV₁, and FV₁₀, respectively). The percentage of post-NDV challenge survived chickens was not affected by the route of vaccination (p > 0.05), but immunization of chicks with ND.TR.IR in FV₁ group provided relatively poorer protection when compared with the other groups (90% vs. 100%, respectively). Altogether, immunization of chicks with ND.TR.IR vaccine through different routes of administration induced protective NDV antibody HI titers, and provided protection against vNDV. However, when the vaccine was administrated through feed, a higher dose of vaccine is recommended.

Introduction

Newcastle disease (ND) is a highly contagious viral disease of poultry with a high level of mortality in unvaccinated chickens (8). ND has affected the poultry industry around the world for many years. Among the viral infections in fowl, ND causes huge economic losses, worldwide (18). The high virulence of the ND virus (NDV) is responsible for the morbidity and severe mortality in many poultry-producing areas (18). Although vaccination is the known major strategy to ND prevention (15), effective control of ND is needed to improve the livelihood of smallholder farmers, particularly in developing countries (25). The backyard flocks are the reservoirs of virulent NDV (vNDV). They are perpetual and potential risk factors of ND transmission to industrial poultry farms.

The safety and efficacy of the current ND vaccines, manufactured from genotypes I and II viruses, have been confirmed during the past 50 years. However, these vaccinal strains are divergent from NDVs that caused outbreaks in the past two decades. It has been shown that genotype V viruses are the predominant isolates from current outbreaks in the United States (28), and genotypes VI (pigeon paramyxovirus type I) and VII caused ND outbreaks in Africa, Asia, and Europe (15). Abdoshah et al. (3) reported that the very vNDVs that currently circulate in Iranian poultry flocks are close to and even more virulent than standard vNDVs. Accordingly, there are several studies showing that the vNDV isolates from chickens in Iran mostly belong to genotype VII (16,19 –22). However, the efficacy of the commercially available vaccines on prevention of genotype VII NDVs is still controversial. Thermoresistant (TR) ND vaccines are recommended to control ND in village chickens. Therefore, the efficacy of these vaccines must be evaluated against predominant wild viruses.

The TR ND vaccines, originating from I-2 or HR-V4 strains (4), have been successfully used against ND in different countries (18,26,31). The thermostability properties of these vaccines have nominated them as the best choice under inappropriate cold chain and storage (7). Although the necessity of a vaccine against ND has grown in smallholder poultry producers, vaccine administration by drinking water (DW) or aerosol sprays is the major concern in a rural area. Hence, the conventional vaccine application methods are not suitable for village poultry production systems (25). Administration of a TR vaccine through feed is a more feasible approach to meet the acceptable immunity (17) under inappropriate cold chain and inoculation difficulties.

The levels of immunity induced by different routes of vaccination are still questionable and may result in a different outcome at the natural challenge (2). Administration of the TR vaccines has resulted in different outcomes in both laboratory and field trial experiments (17), because it is affected by several environmental factors and conditions of vaccine preparation (1,6). The efficacy of different administration routes of the ND.TR.IR, as an I-2 strain originated ND vaccine, has not been evaluated in Iran. The objective of this study was to compare the effect of different routes of immunization with an ND vaccine belonging to genotype I on the induction of antibodies and the protective efficacy against a vNDV genotype VII isolate in specific pathogen-free (SPF) chickens.

Materials and Methods

This study was conducted at Razi Vaccine and Serum Research Institute (RVSRI), Karaj, Iran, with respective latitude and longitude coordinates of 35.83266 and 50.99155, during May to August 2018. The serological and challenge experiments with vNDV were done at biosafety level 3 in BioFlex™ B40 Rigid Body Poultry Isolator (Bell Isolation Systems Ltd, United Kingdom).

Experimental design and immunization

A total of 100 one-day-old SPF chicks were randomly divided into five groups (n = 20 chickens per group), and vaccinated with an I-2-driven vaccine (ND.TR.IR, Razi, Iran) at 10 and 20 days of age through different routes of administration. The chickens were housed at standard environmental conditions, had free access to water and a same diet. Treatments included no virus inoculation (Control [C]), inoculation with 1 dose (10⁶ EID₅₀) through eye drop (ED), inoculation with 1 dose through DW, inoculation with 1 dose through feed (FV₁), and inoculation with 10 doses (10⁷ EID₅₀) through feed (FV₁₀).

Vials of freeze-dried ND.TR.IR vaccine (250 doses per vial) were reconstituted in 125 mL of clean sterile nonchlorinated distilled water. After primary addition of some clean nonchlorinated water (28 mL) to wet the grain (cracked maize), 10 mL of vaccine suspension (0.5 mL per chicken) was sprayed on 100 g of cracked maize (5 g per chick) to deliver one dose (10⁶ EID₅₀) to FV₁ chickens (2). In the same procedure, the vaccine reconstituted in 12.5 mL of water, and a 10 mL of the vaccine suspension was sprayed on 100 g of cracked maize to deliver 10 doses (10⁷ EID₅₀) to FV₁₀ chickens. The cracked maize was chosen as a vaccine carrier because it has shown that it proceeded to a higher antibody in hemagglutination inhibition (HI) test, and it was more suitable than other grains (2). Before vaccination, the chicks in the FVs groups were feed deprived overnight. The chicks in the DW group were vaccinated by addition of 10 mL of vaccine suspension (containing one dose per 0.5 mL per chicken) to the 25% of the daily water consumption, after a 2 h water deprivation. To vaccination chicks through ED, one vial of the vaccine (250 doses per vial) was reconstituted with 6 mL clean and nonchlorinated water and 25 μL of the vaccine suspension was inoculated to the right eye of each chicken.

Serological assays

Blood samples were collected from the brachial vein of all the chickens at 20 and 34 days of age to evaluate the antibodies induced against NDV. Antibody titers against NDV were measured by HI test, according to OIE terrestrial manual (27). Results are reported on the base of log₂.

NDV challenge experiment

To evaluate the protective efficacy of ND.TR.IR vaccine in different groups, all the chickens were intramuscularly challenged with ≥10⁵ LD₅₀ of a local vNDV (Ck/ir/Beh/2011), 14 days after boost immunization (at day 34 of age) as described in OIE terrestrial manual (27). The challenge virus was characterized by intracerebral pathogenicity index of 1.81 (21).

Statistical analysis

In a completely randomized design, data were analyzed using a generalized linear model procedure of SAS 9.2 (30). Normal distribution of the data was tested by UNIVARIATE procedure and Shapiro–Wilk test. To compare the number of chickens with protective antibody titer (HI titer ≥ log₂ ³), data were analyzed by the GENMOD procedure using a binary distribution and a logit odds ratio link. The mathematical model was Y _ij = μ + T _i + e _ij, in which, Y _ij is observations, T _i is treatment effect, and e _ij is a residual random error. Results are reported as mean and standard error (SE). Tukey's test was used for multiple comparisons of the mean and statistical differences and tendencies are declared at p < 0.05 and 0.05 < p < 0.10, respectively.

Results

The effect of different routes of vaccine administrations on mean HI antibody titer (±SE) against NDV is presented in Table 1. At days 20 and 34 of age, all the vaccinated chickens had higher HI antibody titers than the control group (p < 0.05). At day 20 of age, immunization of chickens through the ED route induced higher HI antibody titers than those vaccinated by feed (FV₁ and FV₁₀; p < 0.05), but no significant difference was noted between the DW group and the ED or FV groups. However, at 34 days of age, birds in the ED group showed higher HI antibody titers than the other vaccinated groups (p < 0.05; Table 1).

Table 1.

The Mean of Hemagglutination Inhibition Antibody Titer (± Standard Error) Against Newcastle Disease Virus in Specific Pathogen-Free Chickens Inoculated with a Thermoresistant Newcastle Disease Vaccine Through Different Administration Routes

Route of administration^*	Days of blood sampling
Route of administration^*	20	34
ED	4.05 ± 0.33^a,B	5.09 ± 0.26^a,A
DW	3.40 ± 0.29^ab,A	4.10 ± 0.27^b,A
FV₁	2.30 ± 0.26^b,B	3.65 ± 0.32^b,A
FV₁₀	2.70 ± 0.28^b,B	4.00 ± 0.30^b,A
Control	<1^c,A	<1^c,A

n = 20 chickens per group.

a–c

Values with different superscripts within a column indicate a significant difference, p < 0.05.

A,B

Values with different superscripts within a row indicate a significant difference, p < 0.05.

Treated birds were vaccinated with ND.TR.IR on 10 and 20 days of age.

Control, no vaccine administration; DW, drinking water; ED, eye drop; FV₁, one dose through feed; FV₁₀, 10 doses through feed.

High percentages of chickens having protective titers (HI ≥ log₂ ³) were detected in immunized chickens (Table 2). In comparison with the FV₁ group, the higher percentage of chickens with protective titers was detected in ED and DW groups at day 20 of age (p < 0.05). Routes of administration did not affect the percentage of chickens with protective titers at 34 day of age. In exception of the DW group, proportion of chickens with protective antibody titers was significantly increased at day 34 than that measured at day 20 of age. However, the DW group tended to have a higher (p < 0.10) percentage of chickens with protective antibody titers at 34 days than at 20 days of age.

Table 2.

Effect of Different Vaccination Routes of a Thermoresistant Newcastle Disease Vaccine on the Percentage of Specific Pathogen-Free Chickens with Protective Titer Against Newcastle Disease Virus

Route of administration^*	Days of blood sampling
Route of administration^*	20	34
ED	75% (15/20)^a,B	100% (20/20)^a,A
DW	70% (14/20)^a,A	90% (18/20)^a,A
FV₁	30% (6/20)^b,B	75% (15/20)^a,A
FV₁₀	45% (9/20)^ab,B	85% (17/20)^a,A
Control	0% (0/20)^c,A	0% (0/20)^b,A

n = 20 chickens per group.

a–c

Values with different superscripts within a column indicate a significant difference, p < 0.05.

A,B

Values with different superscripts within a row indicate a significant difference, p < 0.05.

Mean hemagglutination inhibition antibody titer ≥2³ was considered as protective titer (27).

Treated birds were vaccinated with ND.TR.IR on 10 and 20 days of age.

The percentage of chickens survived after challenge with vNDV strain Ck/ir/Beh/2011 is shown in Table 3. Although the immunized chicks with ND.TR.IR vaccine provided protection against vNDV compared with the control birds, the percentage of survived chickens was almost similar in all vaccinated groups (p > 0.05). However, immunization through FV₁ provided relatively less protection than that of other groups (90% vs. 100%, respectively).

Table 3.

Effect of Different Vaccination Routes of a Thermoresistant Newcastle Disease (ND.TR.IR) Vaccine on the Percentage of Protected Specific Pathogen-Free Chickens in a Challenge with a Virulent Newcastle Disease Virus

Route of administration^*	No. of dead birds	Protection %
ED	0	100% (20/20)^a
DW	0	100% (20/20)^a
FV₁	2	90% (18/20)^a
FV₁₀	0	100% (20/20)^a
Control	20	0% (0/20)^b

n = 20 chickens per group.

a,b

Values with different superscripts within the column indicate a significant difference, p < 0.05.

Ck/ir/Beh/2011.

Treated birds were vaccinated with ND.TR.IR on 10 and 20 days of age.

Discussion

Current routes of vaccination, such as ED inoculation, are impractical for mass vaccination of a large number of birds in villages and undeveloped areas (33). In this study, we evaluated the contribution of different routes of administration of TR vaccine on the induction of antibody and the protective efficacy against a local vNDV.

Our results revealed that at least 10⁶ EID₅₀ per dose of ND vaccine was required to achieve satisfactory antibody response and protection against vNDV. Moreover, administration of 10 doses of the ND.TR.IR through feed did not cause any postvaccination reactions or clinical signs. This finding was in agreement with the results reported by Wegdan et al. (33) that showed that double or fourfold I-2 ND vaccine inoculation was absolutely safe. In addition, ED route of administration induced a stronger antibody response and provided a better protection on day 20 postimmunization. This suggests vaccination through ED resulted in better vaccine antigen presentation to the immune system in chickens. Wegdan et al. (33) also showed that in comparison with other methods, ocular route of vaccination elicited higher antibodies and better protection.

There were not significantly differences between the antibody titers detected at day 20 of age in DW and FVs groups. However, the lower percentage of chickens with protective HI antibody titers was detected in the FV₁ group than those of other groups. This is probably due to less uniformity in antigen presentation to the immune system. In comparison with the feed vaccine, administration through ED or DW is more effective in surviving vaccinal virus. Moreover, as water-diluted vaccines pose a better distribution, the DW administration probably resulted in better uniformity in antibody response than that of the FV method. It has been shown that ratio between amount of vaccine-coated feed eaten by chicks and the time required to consume the feed is critical factors affecting outcomes of FV vaccination (1).

Higher antibody titers induced at day 34 of age than at day 20 of age is probably a result of booster vaccination. Mebrahtu et al. (25) also showed that 2 weeks after the first ND vaccination, antibody titer significantly increased to higher levels, resulting in a ∼11%, 30%, and 23% increase in the proportion of chicken with HI antibody titer ≥ log₂ ³ in the vaccinated chickens through DW, ED, and spray, respectively. Meanwhile, lower antibody titers detected in the FV₁ group at day 20 of age than those of other groups indicated that a higher dose of vaccine is required when vaccine is administrated through feed. In consistent with our result, Wegdan et al. (33) reported that a high dose of I-2 ND vaccine is required to achieve appropriate serological protection in chicks. However, in contrast with our results, Abdi et al. (2) demonstrated that there was no significant difference between HI titers induced after the prime vaccination with I-2 ND at 14 and 28 days of age through DW and FV (one dose). It is worth nothing that in comparison with our study, a higher dose of vaccine (10⁷ vs. 10⁶ EID₅₀) was used in a later report; therefore, a significant difference was not detected in their study. This finding supports our results, wherein there was no significant difference between the percentage of chickens with protective antibody titer against NDV in the FV₁₀ group and in the ED or DW groups.

In agreement with results detected for antibody titers at day 20 of age, the highest and lowest antibody titers were recorded for ED and FV₁ chickens at day 34 of age. However, the percentages of chickens with protective antibody levels were not different among chickens inoculated through different routes at 34 days of age. It has been well documented that the antibody titers were progressively increased as the chickens received the booster vaccine (25). At field condition, however, the protective HI antibody titers could be different. For instance, Kapczynski and King (24) suggested that under commercial broiler housing, only chickens with HI titers > log₂ ⁴ will survive after vNDV challenge, while 66% of chickens with unprotective titers died. More commonly, HI titers ≥ log₂ ⁵ are typically suggested as protective level (10,23). As the incidence of ND usually occurs at 4–5 weeks of age in Iranian flocks, the lower proportion of chickens with protective level of antibody observed at FV₁ at day 20 of age may result in a higher morbidity and mortality than other groups. This assumption was supported by the relative lower survived chickens in FV₁ in comparison with other groups (90% vs. 100%). Probably higher number of birds in each experimental group could elicit significant difference between survived chickens at challenge test in FV₁ than the other groups. The 10% unprotection leads to at least 10% economic loss, which is a huge loss at field condition. However, in agreement with the results of previous studies (9,12,32), our findings showed that antibody titer ≥ log₂ ³ was minimal that could be considered as protective against mortality arising from ND. With respect to the lower uniformity commonly observed in antibody titers at farm condition than the controlled experiments, a higher threshold of antibody titers is recommended (2).

In this study, the chickens were intramuscularly challenged with a local vNDV (Ck/ir/Beh/2011). The intramuscular route is usually preferred than contact challenge due to passing the mucosal immunity resulted in receiving an equal dose of challenge virus (2). Our results showed that ND.TR.IR provided 100% protection against Ck/ir/Beh/2011, as a vNDV and local virus belonged to genotype VII, in the immunized chickens through different routes of vaccination even 2-weeks after booster vaccination. However, there are concerns about providing enough protection against challenge with vNDVs due to the existing heterogeneity between vaccinal strains and circulating virulent isolates. The current results showed that SPF chickens vaccinated with ND.TR.IR protected against circulating vNDV belonged to VII genotype, whereas the original strain of the TR vaccine belonged to a class II genotype I virus (14). These findings were not in agreement with the results reported by Van Boven et al. (31), suggesting that current vaccines induced better protection against viruses isolated in previous epizootics than currently circulating viruses. In addition, Cornax et al. (13) reported that SPF chickens vaccinated with an adequate titer of LaSota strain were completely protected from morbidity and mortality after challenge with a genotype VII of vNDV. These findings show that the genetic diversity between vaccine strain and challenge virulent virus most likely does not affect protection level. However, there are studies showing that live ND vaccines can only prevent clinical disease but not virus shedding, especially after heterologous virus challenge (29).

Result of this study showed that when the chickens were inoculated by feed vaccination method, 10-fold administration doses were required to achieve complete protection against vNDV. Mebrahtu et al. (25) also showed that 4 weeks after booster vaccination, all the chickens of the vaccinated groups survived after vNDV challenge. It has also been suggested that immunization of chickens through oral route elicited good protection level, but a booster vaccination is needed 2–4 weeks later (6,11). These pieces of evidence approved the importance of dose of vaccine as well as booster vaccination in through oral route. However, it is worth noting that the protection arising from an FV vaccine is closely associated with type of the carrier and experimental condition. It has been shown that the method of coating (2), type of the coated grain (2,5), and laboratory or village conditions (1,5) caused considerable variations in protection level (60%–90%).

Conclusion

Our results showed that there was no difference among vaccination route outcomes when 10-fold doses of the vaccine were used as a feed vaccine. In addition, inoculation of ND.TR.IR vaccine protected the vaccinated chickens against the local heterologous vNDV. It also did not cause postvaccination reactions. Therefore, ND.TR.IR is a safe and efficacious vaccine against ND in chickens. These findings help smallholder farmers to choose a vaccination method that is more fit to their field conditions.

Ethical Statement

The experiment was conducted under the approval given by Razi Vaccine and Serum Research Institute Ethics Committee.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was funded by Razi Vaccine and Serum Research Institute (Grant No.: 12-18-18-036-95017-950501).

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