Comparative Efficacy of Different Inactivated Hydro-Pericardium Syndrome Vaccines Prepared from Infected Liver and Vero Cell Line Adapted Adeno Type 4 Virus

Hydro-Pericardium Syndrome (HPS) is viral problem of commercial poultry caused by aviadeno virus type-4. In Pakistan the problems have been controlled by administering inactivated infected liver homogenate vaccine (ILHV). The use of liver based HPS vaccines remained potential threat for having hypersensitivity reactions in poultry. The current study was carried out to compare the serological potency of HPS ILHV to vero cell line adopted vaccine in term of anti HPS-ELISA antibody titers. 14 HPS virus vaccines were prepared based on different concentration of antigen, type of adjuvants and source of virus substrate. Total of 160 birds were divided into 16 groups each containing 10 birds. At day of 14 th age each bird of every group was injected with 0.3 ml dose of respective vaccine. It was observed that HPS infected liver based vaccine having 1 × 10 5.6 , 1 × 10 5.6 and 1 × 10 3.6 bird lethal dose 50 induced 1092.10, 875.25 and 702.2 anti-HPS ELISA antibody titer respectively. The 20, 25 and 30 doses/gm HPS infected liver vaccine induced 110.4, 1071.9 and 1037.8 anti-HPS ELISA antibody titer respectively.


Introduction
Hydro-pericardium syndrome (HPS) associated hepatitis is a viral illness of poultry especially 3 to 6 week old chicks caused by fowl adenovirus serotype-4. It was first reported in 1987 at Angara Goth near Karachi, Pakistan. Poultry industry is considered to be a most effective growing industry in the commercial sector due to increasing demand of its value added products. Although, the industry in Pakistan is highly organized and well established but still it suffers from highly infectious outbreaks of Hydro-pericardium Syndrome, Infectious Avian Bronchitis, Newcastle Disease, Infectious Bursal Disease and Chronic Respiratory Disease. The HPS has made massive economic losses in past to the Pakistan poultry industry [1] [2] [3].
The serotype 4 of fowl adenovirus belongs to family adenoviridae genus aviadenovirus. The virus is non-enveloped, 70 -90 nm size, icosahedral particles with linear, double standard DNA genome. The antigen of fowl adenovirus was isolated from geese, ducks and turkeys [4]. The disease is characterized by impulsive onset desolation, mortality upto 70%, and accumulation of straw color fluid in the pericardial sac, liver is marked with necrotic foci, and intra-nuclear inclusion bodies in hepatocytes are the cardinal lesion observed during postmortem [5] [6]. The pericardial fat may exhibit yellowish discoloration with petechial hemorrhages and the heart appears misshapen and flabby at its apex floating in the pericardial sac.
Against most of the poultry diseases, standard vaccines are available but ample literature is still scanty regarding the standard protocols for development and evaluation of avian HPS virus vaccines from infected liver homogenate with and without different adjuvants. Single dose formalized liver homogenate vaccine prepared from infected HPS virus is develop to induce immunity in broilers against HPS and double dose of formalized liver homogenate is more efficacious in broilers [7]. So, extensive use of infected liver homogenate vaccine minimized production of cell culture based HPS vaccines [8]. Due to emerging viral resistance against various antiviral drugs, it becomes difficult to control viral problems that may result into heavy economic losses. Currently, there are few commercial vaccines available for immunophylaxis against HPS. All these vaccines are being manufactured in the country from local isolates by using conventional technique based on infected liver homogenate. In such scenario non-specific proteins such as (arginine, ornithine) of liver homogenate vaccine (HPS-ILHV) would have been showed allergic reactions after injection. Therefore, current study has been designed to develop standard protocols for production of cell free

Reactivation of HPS Infected Liver
40 percent weight/volume (W/V) infected liver homogenate was prepared by adding 40 gms of infected liver in 100 ml normal saline (0.85% sodium chloride aqueous solution (pH 7.2). The mixture was homogenized @ 1000 rpm for 5 minutes using sharp blade electric homogenizer. The homogenate was centrifuged at 3000 rpm for 10 minutes and sediment was decanted. The clear supernatant was admixed with gentamycin @ 200 µgms/ml, penicillin @ 10,000 u/ml and streptomycin @ 100 µgms/ml [9].
26 days old 10 HPS susceptible chicks were given infection with one ml of the HPS infected liver homogenate inoculum through intramuscular route (IM). At postmortem morbid samples were educed and stored in sterile container having label "HPS infected liver". These HPS infected livers were stored at −20˚C till further processing.

Calculation of BLD50
Broilers of 26 days old (unvaccinated against HPS vaccine) were given infective dose for production of the disease. The dead birds were opened and HPS infected liver removed. The liver was homogenized in nine times volume of normal saline. Antibiotics such as gentamycin 200 µgms /ml, penicillin 1000 u/ml, streptomycin 1 mg/ml were added in the liver homogenate. The homogenate was diluted as 10-fold in the antibiotic containing normal saline (such as 1:1000, 1:10,000, 1:100,000, and 1:1,000,000). One ml of each dilution was injected to each of the 5 birds of the respective group.
The birds were marked with black dye and observed critically for mortality and morbidity for next 72 -90 hrs for any mortality.

Preparation of Vaccine from HPSV Infected Liver Homogenate
The liver homogenate with known units of infectivity titer (LD 50 ) was processed for preparation of vaccines as described by Rabbani (1987). Ten grams of the liver was homogenized in 35 ml sterilized saline solution. 0.2% of 37% formaldehyde (Scharlau-UK), antibiotics (Streptomycin @ 1 mg/ml, penicillin @ 1000 units per ml and gentamycin @ 200 ug per ml) (Univet-Ireland) were added in the HPS infected liver homogenate. The suspension was incubated at 37˚C for 24 hours and placed for overnight. The clear supernatant was decanted from the homogenate. The volume was made up to 45 ml using the saline solution ( Figure 2).

Vero Cell Line Adapted HPS Virus Vaccines
In vitro vero cell line was cultivated in DMEM media (Caisson-USA) with additional 5% fetal calf serum (Gibco-UK) in T-175 disposable tissue culture flask as described previously by (Malik et al., 2018).

HPS Virus Inoculation
The confluent monolayer of vero cell line was inoculated with one ml of HPS virus inoculum (1 × 10 TCID 50 ) and incubated at 37˚C for 45 minutes. After viral adsorption, the infected vero cell line monolayer was supplemented with 8 ml of DMEM containing 5% of FCS as maintenance media. One flask having confluent monolayer was kept as uninoculated/negative control. The monolayer was routinely observed for 3 -5 days for appearance of any cytopathic effects (CPE) as compared with the normal cells in the control flask. When 90% of the infected cells were damaged due to CPE, both the infected and control flasks were freeze-thawed thrice by alternately placed them at −20˚C in freezer and then at 25˚C at room temperature.
The virus suspension was centrifuged at 500 g for 10 minutes and the cell free

Effect of Adjuvants
Oil base montanide vaccine was prepared as directed by the manufacturer    Figure 4).

ELISA (Enzyme Linked Immunosorbent Assay) Testing
Indirect ELISA was performed on all serum samples in 96 well plates following the procedures as described by the manufacturer (BioChek ELISA-Germany).    (Figure 6).

Statistical Analysis
The data on the challenge protection test were subjected to the statistical analysis for the interpretation of results by using one way analysis of variance.     Figure 8, Figure 9).

All
Adjuvants play a vital role in the stimulation and augmentation of immune response but chiefly act as depot effect at injection site. On 20 th day post vaccination   Figure   10).      [17]. Mehmood reported that the protection titer in vaccinated birds was found to be the highest for montanide based HPS virus vaccine (100%), followed by aluminum hydroxide gel based vaccine (80%), whereas the vaccine without adjuvant provided 40% protection when challenged with virulent virus at 28 days post vaccination [13]. These findings are congruent with the observations of Hussain and Roy [7] [18]. The variation in the protection percentage induced by montanide and lanolin based vaccine could be due to instability of the latter during storage.

Discussion
The results of the current study revealed infected liver homogenate vaccine induced significantly higher anti-HPS ELISA antibody titer (2009.3) to that of primary culture of liver hepatocytes adapted virus vaccine (1082.5) regardless of its composition due to production of heavy antigenic count per volume is well supported by substrate and the environment. The findings are in line with Jabeen who reported that in his study that efficacy of liver culture based inacti-vated adjuvanted vaccines was significantly higher at 2 week post vaccination (P < 0.05) than groups tissue homogenate based inactivated vaccines [19].
During earlier days of investigation many attempts were made for the control of HPS in broilers by using formalin inactivated liver homogenate vaccines and there have been a lot of contrary findings regarding the efficacy of such liver homogenate vaccines. The results of the present study suggested that tissue culture based inactivated vaccines performed best in experimental conditions as compared to liver homogenate vaccine. Our results are in close agreement with already reported work.
The objective of the present study was to develop cell culture based efficacious vaccine against HPS in poultry. Cell culture based inactivated montanide adjuvanted vaccine performed better in experimental conditions and showed protective anti-HPS ELISA antibody titers whereas, HPS infected liver homogenate based technology could be considered as better tool for the production of inactivated vaccines. However, it is recommended that further trials may be designed to get fully adapted aviadenovirus with optimum growth in limited time which shall replace commercial tissue homogenate based vaccine.

Conclusion
It is observed that the HPS vaccine containing more than 10 5.6 units of immunogen is effective for broilers to achieve the required level of resistance to field challenge. Less than 20 doses per gram of the HPS infected liver homogenate containing 10 5.6 units of immunogen can be prepared for effective immuno-prophylaxis. Addition of the oil base montanide in the HPS infected liver homogenate vaccine improves its efficacy and induces immunity for longer period of time.

Recommendations
• HPS liver homogenate vaccine shall contain more than 10 4.6 units of immunogen for broilers as compare to tissue cultured HPS vaccine where minimum immunogen count would be 10 5.6 . • Always prepare 20 doses per gram from HPS infected liver having 10 5.6 BLD 50 .
• Montanide oil can be better alternative adjuvant for breeders and layers which can induces immunity for longer period of time.
• Tissue culture adopted fowl adeno virus vaccine is free of non-specific liver cells and other extraneous agents. Hence, could be used for effective immuno prophylaxis without any ill effect.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.