The Immune Response of Boschveld Chickens to a Newcastle Disease Vaccination Program Designed for Commercial Layers

doi:10.21203/rs.3.rs-3866661/v1

This study evaluated Boschveld chicken’s antibody response to a Newcastle Disease (ND) vaccination program designed for the Hyline Brown chickens. Both chicken breeds were challenged with the LaSota vaccine at days 1, 69, 111, and 195. A total of 160 sera samples were collected, 80 from each breed. The sera were tested for anti-Newcastle Disease Virus (NDV) antibodies using the Haemagglutination inhibition assay. The anti-NDV antibody titers were expressed in log 2 as geometric mean ± standard deviation. Antibody titers were evaluated and compared pre-infection and 10 days post-infection (dpi). Sera positivity to anti-NDV was tested at 10 dpi. A higher percentage of negative sera (6.875) was recorded in Boschveld than in Hyline Brown chickens (3.75). Hyline Brown chicks had a higher antibody titer (4.95 ± 0.21) than Boschveld (3.21 ± 0.43) before any vaccination. There was a significant association (p < 0.05) between the number of vaccinations and antibody titers in both chicken breeds. Sera from the Boschveld reached a higher antibody titer value (13.98) than that of Hyline Brown chickens (13.85). The Boschveld had a higher immune response peak and immunological memory compared to the Hyline Brown chickens. The candidate genes from Boschveld chickens can be introgressed in highly productive chicken germplasm with less NDV response as an alternative solution to ND. The antibody titers of the Boschveld did not change significantly (p > 0.05) after the 4th vaccination time while those from Hyline Brown chickens continued to increase. The 4th ND vaccination must be ignored or delayed in Boschveld chickens.

Boschveld

Hyline-Brown

Newcastle Disease Vaccine

Haemagglutination inhibition assay

Newcastle Disease (ND) continues to negatively impact poultry farmers and poultry-producing nations by infecting birds worldwide, despite the advances made in diagnosis and vaccination since it was first discovered in 1926 (Mahamud et al., 2022; Moustapha et al., 2023). An aspect that can be part of complementary control strategies but is often neglected is the differences in immune response due to genetic variation (Igwe et al., 2018). In Zimbabwe, vaccination against Newcastle Disease is one of the main control measures of Newcastle Disease Virus (NDV) with its endemic occurrence in poultry farms. The Department of Veterinary Services (DVS) recommends vaccinating chickens against Newcastle Disease with attenuated live vaccines via drinking water or spray on day 1, day 69 day 111, and once every 12 weeks. However, different breeds and lines of chickens have shown different genetic resistance to NDV (Deist et al., 2017) and also have different seroconversions to Newcastle Disease vaccines (Churchil et al., 2023). Genetic variations in ND resistance were also observed among breeds of turkey (Seal et al., 2000) and ducks (Dai et al., 2014). This led to the conclusion that NDV may be better adapted in one species or breed versus the other like what is seen with PPMV1 (pigeon NDV) strains in chickens (Hossain et al., 2023).

The Boschveld chicken is a hybrid of three Southern African native breeds which are the Venda, Matabele, and Ovambo chickens (Okoro et al., 2017). Several researchers concluded that native chicken breeds have higher disease resistance compared to their improved commercial counterparts (El-Safty, 2012; Kokate et al., 2017). Although the Boschveld chicken is the only locally developed indigenous breed in Africa, little is known about its antibody production, and as a result, there is no vaccination program developed specifically for it. The ND vaccination program for commercial layer strains is said to apply to the Boschveld chickens. This research evaluates the antibody response of the Boschveld chicken breed to a Newcastle Disease vaccination program of commercial layer strains.

2.1 Study site

The rearing of chickens was done in the Bioassay Laboratory at the University of Zimbabwe, Department of Livestock Sciences. Antibody analysis was carried out at Central Veterinary Laboratories, which is located in Harare, the capital city of Zimbabwe.

2.2 Ethical approval

The care and use of the animals were approved by and conformed to the guidelines for animal experimentation of the National Animal Research Ethics Committee (NAREC) 18 A Borrowdale Road P.O Box CY 551 Causeway Harare Zimbabwe for biomedical research involving the animals. The following Animal Ethics consideration issues were followed.

Do not harm (pain or discomfort should be minimized)

Only assess relevant components
Reduction in the number of animals used
Minimize the overall impact on the animals used

2.3 Flock management

A total of 200 day-old chicks, 100 from each breed were obtained from Charles Stewart Day Old Chicks Pvt Ltd. The chicks were kept in groups of 10 in both breeds. A completely randomized design (CRD) was used to assign the chicks to their respective cages. Management and hygiene practices were the same in both chicks. The chicks were also given the same type of feed and all biosecurity risk factors were monitored. Feed and water were provided ad libitum while temperature and humidity were also monitored.

2.4 Blood collection

Blood samples were aseptically collected from the randomly selected birds with the help of a Veterinarian. From day 1 to 2 weeks of age, the blood was collected by sacrificing the birds. After 2 weeks of age, the blood was collected from the wing vein. About 2.0 mL of blood was collected from each randomly selected bird using sterile hypodermic needles and a 2 mL syringe. The blood samples were centrifuged to get the plasma/sera which were put in well-labeled sample bottles. The labels had sample number, breed, and vaccination number. The sera were kept in a cooler box with ice during transportation from the bioassay laboratory to the Central Veterinary Laboratory (CVL).

2.5 Preparation of Newcastle Disease virus antigen

The Newcastle Disease virusantigen was prepared from the NDV-LaSota vaccine obtained from the National Veterinary Distributors (NVD) Private Limited. Each 200-dose vial of the NDV-LaSota vaccine was reconstituted in 2 mL of phosphate buffer solution (PBS) (pH 7.4). The latter was then mixed with seven liters of water. The chickens were starved for 2 hours and then given the vaccine in water for an average of 2 hours. The birds were vaccinated on days 1, 69, 111, and 195.

2.6 Data collection

The blood samples were aseptically collected pre and post-infection from 10 birds per breed. Sera were transported to the Central Veterinary Laboratory for the Hemagglutination inhibition (HI) assay. The HI was used according to the OIE Terrestrial Manual (2023). Anti-Newcastle Disease Virus Antibody titers were quantified and then expressed as log 2.

2.7 Statistical analysis

The serum HI titers were tested for normality, linearity, and homoscedasticity before data transformation. The transformed antibody titers were analyzed using IBM SPSS 25.0 (SPSS IBM Corp. 2017) for Windows. The Chi-square test for independence was carried out to test for associations between the number of vaccinations and antibody titers. Statistical significance was set at p < 0.05.

2.1 Study site

The rearing of chickens was done in the Bioassay Laboratory at the University of Zimbabwe, Department of Livestock Sciences. Antibody analysis was carried out at Central Veterinary Laboratories, which is located in Harare, the capital city of Zimbabwe.

2.2 Ethical approval

The care and use of the animals were approved by and conformed to the guidelines for animal experimentation of the National Animal Research Ethics Committee (NAREC) 18 A Borrowdale Road P.O Box CY 551 Causeway Harare Zimbabwe for biomedical research involving the animals. The following Animal Ethics consideration issues were followed.

Do not harm (pain or discomfort should be minimized)

Only assess relevant components
Reduction in the number of animals used
Minimize the overall impact on the animals used

2.3 Flock management

A total of 200 day-old chicks, 100 from each breed were obtained from Charles Stewart Day Old Chicks Pvt Ltd. The chicks were kept in groups of 10 in both breeds. A completely randomized design (CRD) was used to assign the chicks to their respective cages. Management and hygiene practices were the same in both chicks. The chicks were also given the same type of feed and all biosecurity risk factors were monitored. Feed and water were provided ad libitum while temperature and humidity were also monitored.

2.4 Blood collection

Blood samples were aseptically collected from the randomly selected birds with the help of a Veterinarian. From day 1 to 2 weeks of age, the blood was collected by sacrificing the birds. After 2 weeks of age, the blood was collected from the wing vein. About 2.0 mL of blood was collected from each randomly selected bird using sterile hypodermic needles and a 2 mL syringe. The blood samples were centrifuged to get the plasma/sera which were put in well-labeled sample bottles. The labels had sample number, breed, and vaccination number. The sera were kept in a cooler box with ice during transportation from the bioassay laboratory to the Central Veterinary Laboratory (CVL).

2.5 Preparation of Newcastle Disease virus antigen

The Newcastle Disease virusantigen was prepared from the NDV-LaSota vaccine obtained from the National Veterinary Distributors (NVD) Private Limited. Each 200-dose vial of the NDV-LaSota vaccine was reconstituted in 2 mL of phosphate buffer solution (PBS) (pH 7.4). The latter was then mixed with seven liters of water. The chickens were starved for 2 hours and then given the vaccine in water for an average of 2 hours. The birds were vaccinated on days 1, 69, 111, and 195.

2.6 Data collection

The blood samples were aseptically collected pre and post-infection from 10 birds per breed. Sera were transported to the Central Veterinary Laboratory for the Hemagglutination inhibition (HI) assay. The HI was used according to the OIE Terrestrial Manual (2023). Anti-Newcastle Disease Virus Antibody titers were quantified and then expressed as log 2.

2.7 Statistical analysis

The serum HI titers were tested for normality, linearity, and homoscedasticity before data transformation. The transformed antibody titers were analyzed using IBM SPSS 25.0 (SPSS IBM Corp. 2017) for Windows. The Chi-square test for independence was carried out to test for associations between the number of vaccinations and antibody titers. Statistical significance was set at p < 0.05.

The large number of negative sera in chicks implies that antibody development due to NDV vaccination or infection was slow in some of the chicks and increased with age as described by Parmentier et al, (2014). This indicates that some of the vaccinated chicks were still vulnerable to NDV. With the increase in the number of vaccinations, the number of negative sera declined and went to zero, meaning that both chicken breeds had fully developed anti-ND antibodies and were protected from NDV. A higher number of negative sera in Boschveld chicks might mean that there is a lot of variation in the immune response of that particular breed.

The Hyline Brown chicks had higher Ab titers than Boschveld chickens. This could be due to the differences in the levels of maternal-derived antibodies. According to Liu et al, (2023) vaccinated hens can transfer antibodies to their offspring through the egg yolk. Maternally derived antibodies can provide passive protection against diseases but can also interfere with vaccination efficacy early in life (Shrestha et al., 2022). The Hyline Brown parent flock could have been vaccinated against ND. The presence of maternal-derived antibodies for other diseases such as infectious bronchitis (IB) was also observed by Isham et al, (2023) in day-old broiler chicks. The huge variation in both breeds might be due to the presence of variable amounts of Maternal Derived Antibodies in chicks as was also observed by (Okuliarova et al 2014). Antibody titers of both chicken breeds exceeded 12 log 2 after the third vaccination time and this is closely related to the findings of Anebo et al, (2014) who concluded that repeated vaccinations enhance the level of immunity developed in a group. Despite having lower Ab titers in day-old chicks, the Boschveld chickens had higher figures after the third vaccination. This significant difference in Ab titers after the third vaccination means that Boschveld chickens have better immune systems compared to the Hyline Brown chickens. This is closely related to findings by Kokate et al, (2017) who concluded that native chicken breeds have better immunity than the improved breeds. The differences in immune response among the different chicken breeds were also observed by Bílková et al, (2017)and while Ojiezen et al, (2014) concluded that local chicken breeds have higher antibody titers than exotic commercial breeds. According to Kokate et al, (2017), the higher and longer immune-responsive local chickens can be utilized for selective introgression of their candidate genes in highly productive chicken germplasm with less NDV response.

The second vaccination resulted in a more rapid and effective immune response than the first vaccination due to immunologic memory as was also described by Wang et al, (2015). The Boschveld had a higher immune response implying that they have a higher immunological memory compared to Hyline Brown chickens. High immune response in native chickens was also seen by Kokate et al, (2017). The non-responsiveness of the Boschveld chickens to the fourth vaccination could be the start of over immunisation which leads to adverse conditions such as greenish diarrhea, respiratory problems and a drop in egg production as was also seen in indigenous chickens by Orajaka et al, (2004). Wang et al, (2015) explained the non-responsiveness of chickens to vaccines as the adaptation to repeated NDV immunization.

4.2 Recommendations

Parental flocks should be vaccinated against ND to attain maternally derived antibodies to chicks. One vaccination is not enough to secure protective immunity in the two chicken breeds. Repeated vaccinations enhance the level of immunity developed in a group. The third vaccination time must not be ignored in both breeds of chickens. The 4^th vaccination is not necessary or must be delayed in Boschveld chickens since antibody titers did not change significantly and could result in adverse effects such as greenish diarrhea, respiratory problems, and a drop in egg production.

Author’s contribution

All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Pride Hodzi, Takudzwa Charambira, and Blessed Masunda. The first draft of the manuscript was written by Tonderai Mutibvu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors are thankful to the National Animal Research Ethics Committee (NAREC) and the Central Veterinary Laboratory (CVL) for their contribution in making the research a success.

Conflict of interest statement

The authors declare that they had no conflict of interest.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

The data that support the findings of this study are available on request from the corresponding author, [PRIDE HODZI].

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Table 1

	Number of sera tested	Number of sera tested negative	Overall percentage of negative sera
Boschveld	80	11	6.875
Hyline Brown	80	6	3.750
Total	160	17	10.625

Table 2

Vaccination number (Vn)	Boschveld	Hyline Brown
V1	8	4
V2	2	1
V3	1	1
V4	0	0
Total	11	6

Table 3

Age (days)	Immune response of birds 10 dpi (Geometric Mean Ab titer ± SD) Log ₂
Age (days)	Boschveld	Hyline Brown
1 (1^st vaccination)	3.21±0.43^a_p0	4.95±0.21^b_p0
11 (10dpi)	7.59 ±0.28^a_p1	8.67 ±0.01^b_p1
69 (2^nd vaccination)	6.03±0.22^a_p1	6.43±0.07^b_p1
79 (10dpi)	11.02±0.25^a_p2	11.00 ±0.02^a_p2
111 (3^rd vaccination)	10.46±0.22^a_p2	9.57±0.04^b_p2
121 (10dpi)	13.96 ±0.24^a_p2	13.28 ±0.02^b_p2
195 (4^th vaccination)	13.72±0.12^a_p2	12.62±0.09^b_p2
205 (10 dpi)	13.98 ±0.02^a_p2	13.85 ±0.31^a_p2

SD = Standard deviation; Different superscripts (^aand ^b) within the rows show significant differences (p < 0.05). _P0= the chickens that are biologically protected against the ND virus but are still at risk if the vNDV virus strikes. _P1=the chickens that are biologically protected against ND virus but at less risk if vNDV virus strikes. _P2= the chickens are fully protected against ND even if vNDV strikes.

The Immune Response of Boschveld Chickens to a Newcastle Disease Vaccination Program Designed for Commercial Layers

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Results

Discussion and Conclusion

4.2 Recommendations

Declarations

References

Tables

Status:

Version 1