Detection of hydropericardium syndrome virus by direct fluorescent antibody technique in broilers

Poultry industry is the second leading industry of Pakistan which plays an important role in fulfilling the burgeoning demand of meat (Hussain et al., 2015). Unfortunately, the poultry industry has continuously been hit by economically important viral infectious (Newcastle disease) (Nisa et al., 2017) and zoonotic bacterial infections i.e. salmonellosis (Arora et al., 2009). Hydropericardium syndrome (HPS) is one of the most severe diseases associated with heavy economic losses to the poultry farmers worldwide. The disease was first reported in Karachi, Pakistan in 1987 near Angara Goth, hence named “Angara Disease” (Matsushima et al., 2011). The disease was then reported from different parts of country and later on spread in commercial broiler flocks across Pakistan (Mase et al., 2010). HPS is an acute infectious disease that causes high mortality up to 80% in broiler chickens. Mostly young birds (3-6 weeks old) are more susceptible to HPS infection (Vairamuthu et al., 2002; Ganesh and Raghavan, 2000). HPS is caused by Fowl adenovirus serotype 4 (FAdV-4). FAdV-4 is non-enveloped and icosahedral in shape containing a linear dsDNA (Zhang et al., 2016). The disease is characterized by distended pericardial sac due to accumulation of transparent jelly-like fluid of amber, white or sometimes green color fluid in pericardium sac of heart. Multifocal hepatic necrosis with enlarged, pale friable liver and congested kidneys were also observed in affected birds (Zia et al., 2001). HPS infected birds usually undergo sudden death without showing any apparent clinical signs. Along with gross and microscopic changes in liver, various serological tests i.e. counter immuno electro phoresis, agar gel immunodiffusion, indirect hemagglutination and ELISA have been used to diagnose HPS (Asthana et al., 2013). However, a little contribution has been made to develop and optimize direct fluorescent antibody technique (DFAT) for detection of HPS in broilers. The present study was aimed to evaluate and standardize DFAT to determine the post-infection tissue dissemination pattern of HPS virus.

Isolation and purification of field HPS virus
 
A total of 100 liver samples from HPS diseased broilers (based on history, clinical signs and postmortem finding) were collected from the poultry farm in and around Faisalabad district. Liver samples were chopped and homogenized by making 30% w/v suspension of chopped liver in PBS containing antibiotics. Homogenized suspension was centrifuged at 5000 rpm for 20 minutes and resultant supernatant was filtered through syringe filter (0.2 micron). Supernatant was treated with chloroform in 2:1 ratio followed by centrifugation at 5000 rpm for 20 minutes. Clear supernatant above the band of liver proteins was stored at -20^oC (Anjum, 1990).
 
Raising of hyper immune serum against HPS antigen
 
Hyper immune serum against HPS antigen was raised in rabbits according to method of (Kumar et al., 2003). Fifteen days post last immunization with HPS antigen, rabbits were slaughtered and serum was separated followed by inactivation at 56^oC in water bath. Inactivated hyper immune serum was then further employed in agar gel precipitation test (AGPT) and DFAT.
 
Confirmation of field HPS isolate by agar gel precipitation test
 
Confirmation of HPS virus in field samples collected from diseased birds was done by AGPT (Kumar et al., 2003).
 
Titration of virus
 
A total of thirty embryonated eggs were taken and divided into five groups each with six eggs. For determination of egg infectivity dose 50 (EID₅₀) of isolated virus, 10-fold serial dilutions of virus was prepared in PBS (pH: 7.2) from 10-1 to 10-5 and 0.1 ml of each dilution was inoculated into each embryonated egg through chrorioallontoic membrane (CAM) route. Mortality during the first 24 hours was considered nonspecific. The infectivity was checked for 48 hours and EID₅₀ was calculated by Reed and Muench (1938).
 
Experimental design
 
A total of 100 broiler chicks (one day old) were procured from a commercial source. At day 15, all broiler chicks were tested for the presence of anti-HPS antibodies by AGPT. About 50 birds were found negative for anti-HPS Abs which were further used in the experimental design. At day 16, all 50 birds having no Abs against HPS were divided into group A (comprising 20 birds) and group B (30 birds). Group A birds were inoculated with normal saline (control), while group B birds were injected with 0.1 ml of the virus inoculums by subcutaneous (s/c) route. Each group was observed for 10 days PI, after which they were killed and examined. Mortality and gross lesions due to HPS were also recorded during this experiment. Tissue samples of birds from both groups were collected immediately on their death and by slaughtering at 2nd, 6th and 8th days post inoculation and further proceeded for histopathology and DFAT.
 
Histopathology
 
Histopathology was performed on different tissues according to the method of Cruz-Coy et al., (1993). Briefly, collected tissues were fixed in neutral buffered formalin solution followed by dehydration through a series of gradually increasing percentage (%) of alcohol. Alcohol was then removed from tissues by using Xylene. A 5 µm FAdV-4 infected liver section was made with microtome followed by tissue embedding in paraffin wax and subsequent floating of sections in warm water at 50^oC. The sectioned tissues were mounted on the glass slides with Mayer’s egg albumin. The slides were deparaffinized and stored at -20^oC.
 
Preparation of fluorescin isothiocyanate (FITC) conjugated antibody
 
i) Fractionation of serum
 
The major protein component of the sera (raised initially against HPS antigen) was separated by ammonium sulphate precipitation. Globulins were transferred to dialysis tubing and dialyzed at 4oC against 0.85% NaCl (pH-8.0). The total immunoglobulin (Ig) of the rabbit serum was determined by Biuret method (Sing, 2012) (Chang and Zhang, 2017).
 
ii) Conjugation of serum globulin with FITC
 
Protein solutions were placed in a beaker with stir bar. Amount of fluorescein needed was calculated (0.035-0.05 mg fluorescein/mg protein) and added to beaker, stirred at moderate speed for 12-24 hours. After conjugation, free dye was removed by dialysis against 0.01 M phosphate-buffered saline (PBS), pH-7.6 for about 18-24 hours. Conjugate was then collected in several small fractions.
 
Direct fluorescent antibody technique (DFAT)
 
For the detection of viral antigen from various organs of the inoculated birds, DFAT was standardized (Kumar et al., 2003).
 
Immunofluorescence staining and observation
 
A total of 0.4 ml of the conjugate was applied to the slide and incubated for 45 minutes at 37^oC in a moist chamber. Slides were air dried, mounted with 90% glycerol and 10% PBS solution and observed under Leica DM RXA Epifluorescent Microscope at 40X. Blue filter having wavelength of 200-400 nm (as according to visual adjustment) was used.
 
Hematoxylin and eosin staining
 
Some slides were stained with Hematoxylin and Eosin (H&E) stain to make visible cell organelles and tissue components and examined under light microscope as per standard procedures (Bancroft et al., 2013).

The present study was aimed to determine post infection tissue dissemination pattern of hydropericardium syndrome virus through DFAT. The first part of the research comprises on isolation of HPS virus from the diseased birds. Based on clinical signs and PM findings, liver samples were collected, homogenized and processed for HPS virus isolation. Confirmation of HPS virus was done by AGPT test. Based on AGPT test, 84% samples were found positive for HPS virus. Furthermore, embryo infecting dose (EID₅₀) of the field HPS isolate was calculated in 9 day old embryos by inoculating HPS virus through chrorioallontoic membrane. Mortality occurred between 3^rd and 5^th day of post infection and lesions developed were noted in infected embryos. Our results showed that 0.1 ml of inoculum contains 103.36EID₅₀.

After confirmation and titration, field HPS virus was inoculated into experimental group B birds through s/c route as reported by Ahmad et al., (2011). Moreover, (Anjum, 1990) have also reported s/c route as an effective route for HPS induction in birds. Complete observation was made on morbidity and mortality of birds injected with HPS virus. An increase in mortality was observed at 4^th, 5^th and 6^th day post inoculation with the HPS virus. Maximum mortality was found on 6^th day PI suggesting that disease was at peak on this day (Table 1). After this, on 7^th and 8^th day PI, a deceases in disease severity was observed as only one bird was found dead on 7^th day PI, while no mortality was noted on 8^th day PI. Various PM lesions observed on different time intervals are categorized in Table 2. Group B birds died due to HPS infection showed a number of clinical manifestations including hydropericarditis, pale and enlarged friable liver, enlarged kidneys with distended tubules. On the other hand, group A birds did not develop any disease and no mortality was observed.



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Tissue samples of liver from experimental and control groups were also collected on 2^nd, 6^th and 8^th day PI in order to perform DFAT. Different histopathological changes and gross lesions in liver were seen during different time intervals (Table 2). Stained tissues specimens of liver from both groups were observed under epifluorescent microscope. The specimens from experimentally inoculated birds when examined by DFAT indicated presence of virus in liver. This suggests that virus mainly resides and multiplies in liver. Similar findings have also been reported by Kumar et al., (2003a) in which HPS virus was detected in different major organs of infected birds by fluorescent antibody technique (FAT). During pathogenesis of virus, intranuclear inclusion bodies and viral antigens were observed in various organs including liver (Kumar et al., 2003a). The fluorescent nuclei indicated the presence of intranuclear inclusion bodies in the cells. Presence of intranuclear inclusion bodies seen by H&E staining also supported DFAT results. Major histopathological changes that were observed in liver and other tissue specimens were intranuclear inclusion bodies in hepatocytes. Similar kind of findings were also reported by (Anjum, 1989).

On 2^nd post infection day (PID), fluorescence was observed in only 10% of the liver cells with bright granules (Fig 1a). About 60-70% cells showed fluorescence in liver specimens on 6th PID (Fig 1b) indicating the maximum viral titer. After that fluorescence was reduced which was very little on 8^th PID (Fig 1c). In the control birds, stained liver specimens showed no fluorescence suggesting the absence of viral antigen (Fig 1d). These findings correlate with Saffudin and Wilks (1991) who investigated that during dissemination of HPS, viral antigen (FAdV-4) was detectable at 2^nd day of PI and reaches to peak level at 6^th day of PI. Deepak (1998) while studying the pathogenesis of HPS in 2 week old chickens detected an immunofluorescent FAdV-4 antigen in the liver from 3 to 14 days PI and in heart up to 5 days PI. Takase et al., (1990) also demonstrated intranuclear inclusion bodies in liver and other tissue in chicks experimentally inoculated with inclusion body hepatitis by FAT.



Our results indicated that early and reliable HPS virus detection can be achieved through DFAT. Our results also correlate with Kumar et al., (2004) who worked on detection of viral antigen from the broiler chicks infected with hydropericardium syndrome using FAT and suggested it a reliable method for early detection of HPS virus. Virus detection done by Lembo et al., (2006) was also based on immunohistochemical approaches and suggests that fluorescent detection of virus is useful and cost effective. Similar findings were also reported by Kumar et al., (2013) for detection of HPS virus and use of immunofluorescence techniques for pathological studies. Roy et al., (2001) worked on pathological studies for detection of HPS virus using various techniques and one of those was immuno fluorescence. 

Moreover DFAT diagnosis of HPS virus in liver samples was more sensitive and rapid when compared to some other methods such as virus isolation, AGPT and electron microscopy (may prove less sensitive and is readily not available in each and every diagnostic laboratory). Virus isolation results in tissue have revealed the presence of virus in liver tissue for longer period of time when compared with the DFAT, but there is one disadvantage that isolation and identification of virus required a considerably longer period of time.

The result of present study reveals that use of DFAT is a reliable method for early detection of viral and other antigens from different infected organs. As non-clinical HPS is apparently prevalent, it is felt that significance of DFAT should be considered in conjunction with histological lesions in the infected birds.

The author would like to acknowledge all the co-authors. Special thanks to Dr. Faisal Rasheed Anjum for his guidance and help in editing and shaping the final version of manuscript.