In ovo Vaccination in Ducks against Duck Virus Enteritis

Background: One of the major diseases affecting ducks is Duck Virus Enteritis causing heavy mortality and high economic losses in duck farming. Methods: In this study, in ovo vaccination technology was adopted using a Duck Embryo Fibroblast passaged Duck Virus Enteritis vaccine virus. This would be the first reported use of in ovo vaccination in ducks against a viral disease. Virus Neutralisation Test was used to measure the immune response after standardisation of the technique using hyperimmunised Barred Plymouth Rock rooster. An optimised dose of the Duck Virus Enteritis vaccine virus was computed using Embryo Lethal Dose50 assessment of hatchability. In ovo vaccination was done in 2 groups of 40 embryos each, one group acting as negative control and the other group as virus treatment group. A booster dose was given to the treatment group on 14 th day post hatch. Result: Virus Neutralisation test using a beta method was carried out to measure the immune response. The treatment group showed an immune response of 3.33±0.21 Log 2 Antibody titre. This opens up a plethora of options of application of in ovo vaccination in ducks against viral diseases which would be a boon to the duck farming industry.


INTRODUCTION
Ducks are an important component of poultry industry being more resistant to most infectious diseases.They are more productive as they lay eggs even during second and third year.As they are also foragers, feed cost is reduced in semiintensive rearing (Rajput et al., 2014).However, Duck Virus Enteritis (DVE) or duck plague as it is popularly known due to the high mortality caused, is a serious threat to duck farming.Sudden death and haemorrhages and necrosis on post mortem examination are typical (Neher et al., 2018).Young ducklings are highly susceptible.It is caused by Anatid alphaherpesvirus-1 from the genus Mardivirus under Herpesviridae family (Li et al., 2009).Liver and kidney specific biomarkers were reported to be elevated due to the viral infection (Chabukdhara et al., 2023).Vaccination in India is carried out using chicken embryo adapted live virus which has drawbacks (Konwar et al., 2019).There is latency of the virus in the Trigeminal nerve which can cause an outbreak due to reactivation (Shawky and Schat, 2002).This also causes a problem in vaccination in live vaccines while inactivated vaccines are not efficient in generating an immune response (Aravind et al., 2015;Samia and Sandhu, 1997).
In ovo vaccination provides early immunity, uniform and fast delivery as well as reduced stress and labour costs (Souza, 2008).In ovo vaccination in ducks against aflatoxicosis was done in our previous research (Sarmah et al., 2023).Other than that, there are no reports of in ovo vaccination in ducks, however in ovo feeding of amino acids and vitamins has been carried out (Hussian et al., 2019;Syahruddin et al., 2019).In this study, in ovo vaccination was carried out on 25 th day of embryonation in Khaki Campbell ducks using a duck embryo fibroblast adapted vaccine at an optimised dose to generate immune response against DVE.The vaccine virus was passaged 4 times in chicken embryo fibroblast (CEF) cells to observe whether the virus was replication competent.Then, it was passaged 4 times in duck embryo fibroblast (DEF) cells as well.Titration of the virus was done as previously described (Reed and Muench, 1938).Virus Neutralisation Test (VNT) was standardised using serum of a hyperimmunised Barred Plymouth Rock rooster.The immune response was checked by Virus Neutralisation Test.The results revealed that immune response was generated which could be a major breakthrough as there are no reports of in ovo vaccination in ducks against DEV.
Dulbecco's Minimum Essential Medium (DMEM) as growth media and 1% FBS concentration in DMEM as maintenance media. of virus (10 μl).The virus inoculated media were collected after 3 freeze thaw cycles and centrifuged at 10000G at 4C for 15 minutes and the viral supernatants collected and kept at -20C.

Passage of vaccine virus in DEF
The CEF passaged virus was diluted 1:100 times and then passaged 4 times in DEF and the viral inoculated media were collected using same conditions as before after appearance of cytopathic effects (CPE) after 96 hours post inoculation.

Titration of DEF passaged virus
This was done as per Reed and Muench (1938) in 96 well cell culture plates (Tarsons).Fixing was done with 4% paraformaldehyde (Merck) and staining by 0.1% crystal violet (Loba Chemie).

Hyperimmunization of Barred Plymouth Rock rooster
Hyperimmunization of a Barred Plymouth Rock rooster was done as per Table 1.

Standardisation of VNT
A beta method using 10 2.43 Tissue Culture Infective Dose (TCID 50 ) CEF passaged virus was used with two-fold dilutions of decomplemented hyperimmune sera raised from the Barred Plymouth Rock rooster which remained apparently healthy even after intravenous virus inoculation.

Embryo Lethal Dose 50 (ELD50) assessment of DEF passaged virus
ELD50 was assessed by hatchability of duck eggs after viral inoculation by in ovo route on 25 th day of embryonation at different dilutions from 10 -2 to 10 -5 of the DEF passaged virus.

In ovo vaccination of duck embryos
In ovo vaccination of duck embryos were carried on 25 th day of incubation in 2 different groups of 40 embryos.0.5 ml was the total amount injected via in ovo route.Group 1 was negative control receiving only Phosphate Buffered Saline (PBS) and Group 2 was the treatment group receiving the optimised dose of DEF passaged DEV virus.

Booster dose after 14 days post hatch
A booster dose of 100 TCID 50 of DEF passaged DEV virus was given subcutaneously on 14 th day post hatch to the DEV treatment group.

Virus Neutralisation Test
VNT was done by beta method using constant 1000 TCID 50 DEF passaged virus with two-fold dilutions of decomplemented sera from six ducklings from each group collected on 10 th , 21 st and 28 th day post hatch.

RESULTS AND DISCUSSION
The present study was aimed with the purpose of introducing a novel technology i.e., in ovo vaccination which is the standard for vaccination against Marek's disease in chickens (Sharma and Burmester, 1982), in the duck farming industry to combat a fatal disease like DEV.Firstly, the DEV vaccine virus was passaged 4 times in CEF to assess its replication competence.Following that after 4 passages of the DVE virus in DEF, the virus remained replication competent as observed by the CPE like rounding of cells, blebbing and vacuole formation.The DEV vaccine virus was passaged in DEF instead of CEF prior to vaccination so that there would be no immune response to chicken proteins present in CEF.Also, it was to check the replication competence of the virus in duck cells so that the virus would replicate when given by in ovo route.This would help in generating the required immune response specifically against DVE in the ducklings.
Titre of the DEV virus per ml was 10 5.43 /ml TCID 50 .Following the ELD 50 assessment by hatchability of the embryos, 100 TCID 50 was found to be the safe dose for in ovo vaccination as all of the ducklings hatched at this dilution.
The log 2 antibody titre to CEF passaged virus used for standardisation of VNT was observed to be 8.82±0.22.The negative control cells showed a normal monolayer cell architecture with which the monolayers of virus neutralized wells were comparable.CPE was observed 96 hours post inoculation The hatch of the in ovo vaccinated groups were comparable i.e., 32 for the negative control group while it was 34 for the virus treatment group.Therefore, it can be said that there was no mortality due to the virus.Serum collected from the ducklings (6 per group) on 10 th , 21 st and 28 th day post hatch was decomplemented at 56C for 30 minutes prior to use in VNT.The VNT results are shown in Table 2.The treatment group had a constant immune response of 3.33±0.21Log 2 Antibody titre even after booster dose of DEV was given.
In ovo vaccination technology is a novel approach to offer protection to diseases in the embryonic stage itself so as to confer protection during the post hatch stage.It In ovo Vaccination in Ducks against Duck Virus Enteritis has been reported to result in increased immune response in chickens which was evident by higher lymphoproliferative ability of splenocytes, percentage of CD4 T cells and expression of MHC-II in splenic T cells when herpesvirus of turkey was injected (Boone et al., 2020).Furthermore, inactivated fowl adenovirus 8b given along with poly [di(sodium carboxylatoethylphenoxy)] phosphazene and avian beta defensin as adjuvants stimulated strong immune response in chickens (Sarfraz et al., 2017).
From Table 2, it can be seen that there was no neutralising antibody titre in the negative control while in the virus treatment, an immune response can be observed.The VNT Log 2 antibody titre remained constant in the virus treatment group even after booster dose suggesting the virus was replication competent and that the immune response was skewed towards Th1 system.DEV virus skews the immune response towards Th1 (Rauw et al., 2010).It is important to note that an immune response is generated with a replication competent DVE virus thus creating vast possible avenues of utilisation of in ovo vaccination in ducks against DVE.Ducklings are susceptible to DVE prior to the standard vaccination age of 2 -6 weeks which could be solved by adopting in ovo vaccination as industry standard.

CONCLUSION
From the above results, it can be inferred that in ovo vaccination can be adopted in ducks against the dangerous disease of DVE which causes high mortality.This would provide protection against the disease during the post-natal stage of the ducklings at which they are most susceptible.Also, there has been no report of in ovo vaccination in ducks till the compilation of this article thus this provides a novel approach which is safe, less laborious and cheaper compared to post hatch vaccination.Adoption of the in ovo vaccination technology in duck farming would be a boon to the poultry industry.