The disease causes sudden onset of symptoms with high mortality of up to 80% when it occurs in healthy broiler flocks of 3 to 5 weeks old. The predominant gross lesions are accumulation of 5 to 20 ml of clear or amber coloured, watery or jelly-like fluid in the pericardial sac and mis-shapen, flabby heart. Other changes include discolouration of liver with focal hepatic necrosis, petechial and ecchymotic haemorrhages in heart muscles, congestion and oedema in lungs, and pale kidneys with prominent tubules. The liver is the major organ showing histopathological changes and the presence of intranuclear basophilic inclusions in the
The incubation period is reported to vary from 5 to 18 days and the duration of the disease is 10-15 days. The incubation period varied between 2 to 4 days in experimental reproduction of the disease in broilers using both liver homogenate and purified virus prepared from the field samples (1).
The physical properties of the virus and demonstration of basophilic intranuclear inclusion bodies in hepatocytes, characteristic hexagonal virions in electron microscopic preparations of liver homogenates were suggestive of involvement of a DNA virus. Later, fowl adenovirus belonging to serotype 4 named as PARC 1 isolate was characterized and propagated in cell-culture and in embryonated eggs.
All isolates from field outbreaks of HHS have been serotyped as FAV4 strains and some biotypes of FAV4 causing HHS (2). The virus isolated from liver samples of HHS cases was neutralized by FAV4 antiserum. Voss (3) isolated an adenovirus (K31/89) from field outbreaks of HHS in Pakistan and later Voss et al. (4) identified the isolate as adenovirus 4.
Isolates recovered from different outbreaks of HHS in Pakistan differed in their pathological characteristics although no significant differences were recorded in their biological properties or in their protein profiles (5).
The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan was investigated in day-old to 15-month-old specific-pathogen-free (SPF) chickens (6). With this, HPS lesions were reproduced in SPF chicks and in adult chickens.
In Chile, three fowl adenovirus (FAV) isolates (341, 344, and 215) obtained during 1996-97 from field outbreaks of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) affecting broilers and broiler breeders were characterized by virus neutralization tests (VNTs) and restriction enzyme analysis of a DNA fragment (7). The results confirmed the classification of all three isolates as FAV4.
Three Indian isolates of FAV recovered from inclusion body hepatitis/hydropericardium syndrome of poultry from different geographical locations were typed as serotype 4 (8). The virus associated with HHS was purified by sucrose density gradient ultracentrifugation using the infected liver tissue homogenate to avoid an additional step of virus adaptation to an indicator system and verified by double immunodiffusion and virus neutralization tests using 1 to 12 FAV type specific antisera. The syndrome could be experimentally reproduced in broilers using liver homogenate or purified virus preparations from the field samples. These experiments were suggestive that a fowl adenovirus serotype 4 was responsible for HHS (1,9).
The DNA isolated separately from purified virus and from infected liver tissue were subjected to PCR. The HHS virus specific 0.7 kb DNA was amplified from the viral DNA and from DNA extracted directly from the HHS positive liver samples (1). PCR amplified DNA was labeled with Digoxigenin (DIG-11-dUTP) and the labeled DNA was used as a probe for the detection of virus specific sequences in the PCR amplified products after Southern blotting. The DNA probe detected the presence of the virus by dot blot hybridization of the viral genome (1,10,11).
In order to study the existence of possible serotype variations in the 0.7 kb variable region of hexon gene encoding L1 and part of P1 was amplified and sequenced (11). Both nucleotide and the derived amino acid sequences, corresponding to the variable region, were compared with the published fowl adenovirus sequences (FAV serotypes 10, 1 and 8). As expected the 0.7 kb sequence showed a single open reading frame (ORF). There was a nucleotide sequence variation of 8.2, 28.1 and 40.3%, respectively, with FAV serotypes 10, 1 and 8. The dendrogram constructed with the nucleotide sequences showed that HHS virus and FAV10 are closer to each other but differed from FAV1 and FAV8. However, the derived amino acid sequence showed variations as high as 28.8, 38 and 45.1% with FAV serotypes 10, 1 and 8, respectively. Such a high degree of variation has been found due to a shift in the reading frame caused by deletions indicating that the FAV4 associated with HHS was unique and different from FAV10.
Physico-chemical properties of HHS virus
The virus is resistant to acid and alkaline range of pH 3 and 9. Unlike other adenoviruses, HHS virus in the liver preparations withstood heating at 60°C for 30 min. and at 50°C for 1 hr. However, heating at 60°C for 1hr, 80°C for 10 min. and 100°C for 5 min. inactivated the agent. Treatment with chloroform (5%) or ether (10%), that normally inactivates adenovirus, eliminated infectivity of the HHS virus. Also they are inactivated with formaldehyde (1:1000) and inhibited by DNA synthesis inhibitors IudR and BudR.
Purified virus samples from liver homogenate by negative staining electron microscopy showed isometric, roughly spherical particles resembling adenovirus morphology, measuring 83-93 nm in diameter inside the nucleus of hepatocytes from HHS outbreaks (1,12,13).
HHS is contagious and is transmitted horizontally among broilers by mechanical means and contaminated litter. Anjum (14) failed to reproduce the disease by oral inoculation or by contact with affected birds whereas Abdul-Aziz and Hassan (15) successfully transmitted the disease to experimental birds by inoculating infected liver suspension intramuscularly, orally or by contact.
The syndrome was reproduced in broilers of up to 3 weeks of age by inoculation of liver homogenate from infected birds and the virus pellet obtained by ultracentrifugation of homogenates. Mortality was 100% when infected liver suspension was inoculated intramuscularly as compared to oral inoculation where mortality was 30% (15). The disease however, could not be reproduced experimentally with fluid collected from the pericardial sac or homogenates from other organs including bursa, lungs, heart and kidneys (16). Further studies have shown that homogenate of lymphoid organs reproduced the disease when inoculated into healthy chickens (17).
Gowda and Satyanarayana (18) experimentally reproduced the disease in broilers. They have described hydropericardium with accumulation of large volume (up to 12 ml) of straw coloured fluid, pale enlarged livers, oedematous and congested lungs, pale and tumified kidneys and an icteric carcass. Other changes such as spleenomegaly, necrotic foci on the myocardium, hypertrophy of bursa of Fabricius and poorly developed musculature have also been described.
Cowen et al. (19) observed that an isolate, LA/C serotype 4 from Chile produced classical lesions of HHS and the virus could be recovered from rectal swabs of the infected birds and they reported an increase in mortality after oral injection of 2-day-old SPF chicks.
Filtered homogenates of liver tissue from affected cases when inoculated on to the chorioallantoic membrane or into the yolk sac of 8-day-old SPF embryos resulted in embryonic death within 4 to 7 days. The agent produced small syncytia on both chick kidney and liver monolayers (20).
Pathogenicity study on 6 isolates from different field outbreaks of HHS in Ecuador (K1013 number 1 to 6) on embryo and chicken revealed high embryo and chick mortality (2). The disease was reproduced in day-old SPF chicks by oral route. The results showed that some strains of FAV4 caused HHS following oral or intra-muscular infection.
Day-old to 15-month-old SPF chickens were inoculated with liver homogenates from HPS chickens or serotype 4 adenovirus (6). There were no clinical signs before death. The mortality rate in all the groups of day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates in 7-month-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25% respectively. In 15-month-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%.
The chicks inoculated with the inclusion body hepatitis and HPS strains exhibited similar histologic and immunohistochemical changes. Neither mortality nor pathologic changes occurred in 3-week-old SPF chicks inoculated with IBH strains, although HPS strain induced specific lesions in them (21). This study indicated that IBH strains of adenovirus could also reproduce HPS lesions and caused mortality in day-old SPF chicks and that IBH and HPS strains may have similar
On the basis of epidemiologic and experimental data of the virulence of Chilean FAV isolates, and the pathogenicity results with isolate 341, Toro et al. (7) speculated that Chilean FAV strains may require an immunosuppressive agent to induce IBH/HPS outbreaks in the field.
FAV4 could be grown in various tissue cultures namely chicken kidney, chicken embryo and MDCC-MSB1 cells (22,23), chicken embryo liver cells (24).
The disease was first observed in Pakistan among broiler flocks of 3 to 5 weeks of age with flock mortality exceeding 50% (16). Occasional outbreaks were also reported outside the 3 to 5 week-age range among breeder pullets, and in layers. Asrani et al. (25) also noticed high mortality in 2 to 6-week-old broilers. Occasional outbreaks were also recorded in broiler breeder-flocks aged 32 weeks and among commercial layers of 17 weeks with approximately 5 to 8 per cent mortality. Fast growing birds in a flock were the most affected and a sudden mortality reaching its peak within 3 to 4 days followed by an almost constant death rate for 5 to 7 days before declining. According to Asrani et al. (25) the average mortality in an affected flock was between 15 and 60%.
In Russia, HHS was recorded in chickens aged between 2 and 13 weeks with mortality ranging from 3.5 to 30% in broilers and 2.6 to 15.29% in layers (26). Most of the workers have reported that different strains of broilers are equally susceptible in the field and in experimental cases. However, Khan (27) has noticed that the Hubbard broiler strain to be relatively more susceptible followed by Indian River and Lohmann.
Haematological studies indicated a significant decrease in total leukocyte count, total erythrocyte count, haemoglobin, haematocrit, ESR, and lymphocyte values and an increase in heterophils and MCV in HPS cases as compared to normal birds (28,29,30). Sreenivasa Gowda (31) and Gowda and Satyanarayana (18) recorded decreased total leukocyte count, total erythrocyte and haemoglobin values and increased heterophil percentage in spontaneous as well as experimental cases of HPS in broilers. Voss et al. (4) and Cowen et al. (19) reported decreased haematocrit values of HPS affected birds as compared to the control birds.
The most predominant and consistent gross lesions in the field cases of HPS included accumulation of upto 20 ml of clear, watery or jelly like fluid in the pericardial sac with misshapen and flabby heart (12,16,32). Other lesions comprised of petechial and ecchymotic haemorrhages in heart musculature and other organs, congestion and oedema in lungs, swollen pale friable enlarged liver and pale kidneys with prominent tubules (12,15).
Cheema et al. (33) and Muneer et al. (34) described lesions of hydropericardium, splenomegaly, necrotic foci on myocardium, hypertrophy of bursa of Fabricius and poorly developed musculature in experimentally induced HPS cases. Abdul Aziz and Al-Attar (35) reported a marked distention of pericardial sac with clear yellowish fluid, enlarged friable, dark to yellow mottled livers with foci of necrosis, haemorrhage and enlarged kidneys and icteric carcass.
Gowda and Satyanarayana (18) described hydropericardium with accumulation of a large volume of straw coloured fluid thus giving the heart a Litchi fruit appearance, pale enlarged livers, oedematous and congested lungs, pale and tumified kidneys and icteric carcass in experimentally infected HPS cases. Similar gross lesions have been described by others (25,36) in spontaneous cases of HPS.
Abe et al. (22) reported the common characteristic histologic lesions as necrosis of hepatocytes, accompanied by intranuclear inclusions in hepatocytes and haemorrhages. In the spleen, there were activation of macrophages in splenic sinus and ellipsoids and erythrophagocytosis in the splenic sinus. The interlobular interstitium of the lung showed marked oedema. The air and blood capillary areas of parabronchi included many macrophages with yellow pigments. Intranuclear inclusion bodies within degenerating hepatocytes stained by immunoperoxidase technique positively reacted for group I adenovirus antigen. Ultrastructurally, numerous viral particles (65-70 nm in diameter) were demonstrated in the intranuclear inclusions of hepatocytes. Group I adenovirus (serotype 4) was isolated from liver samples of adult broiler breeders and broiler chicks with HPS. This study suggests that HPS may be caused by group I adenovirus.
Anjum et al. (16) described oedema and infiltration of mononuclear leukocytes in the myocardium of HPS cases. Cheema et al. (12) reported multiple necrotic areas on the ventricles of heart especially involving the papillary muscles. Histologically they noticed shrunken, eosinophilic, fragmented and calcified myocardial fibers. Deposition of proteinacious material and extravasation of blood were the additional features.
The other microscopic changes described both in spontaneous and experimentally infected cases of HPS included multifocal areas of coagulative necrosis, mononuclear cell infiltration and presence of intranuclear inclusions in liver; congestion, oedema and leukocytic infiltration in lungs and tubular epithelial degeneration in the kidneys (12,16,32,34).
Lymphocytolytic activity leading to poor cellularity has also been described in cases of HPS involving lymphoid organs like bursa of Fabricius, spleen and thymus (15,18,25,36).
Day-old to 15-month-old specific-pathogen-free chickens were inoculated with liver homogenates from HPS chickens or isolated serotype 4 adenovirus (6). Histologically, the liver had diffuse or multifocal hepatic necrosis and haemorrhage with intranuclear inclusion bodies within the hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at day-old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, caecum, kidney, and lung of the chicks inoculated at day-old.
Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS.
Immunosuppression by HHS
The reports on pathogenicity of Chilean FAV isolates suggest that synergism with other viruses or prior immunosuppression is necessary to reproduce IBH/HPS in chickens. The susceptibility of chickens to the FAV isolates by oral infection varies throughout the course of chicken anemia virus infection (37). Also there were reports of infectious bursal disease (IBD) and chicken infectious anaemia (CIA) viruses in areas where HHS occurred (38). FAV4 associated with HHS also had a predilection for lymphoid tissues, which resulted in immunosuppression (17). Ahmad (39) reported that subclinical infection with HHS showed low titer against NDV vaccinated chicks indicating that HHS produced immunosuppression in birds.
Clinical diagnosis of the disease before the symptoms appeared, is difficult since the birds do not show specific clinical signs. Sudden mortality at the third week of age and necropsy findings such as hydropericardium and demonstration of basophilic intranuclear inclusion bodies in hepatocytes are considered as
Serological tests such as indirect HA test, ELISA and AGPT, counter immunoelectrophoresis using filtrate from infected liver homogenate as crude antigen have been done. These studies indicate that the specificity of the currently available serological tests for diagnosis needed improvement. The electron microscopic demonstration of the virus particles in hepatocytes (13) and virus neutralization test (5,8) using specific antiserum have also been used in HHS diagnosis. A polymerase chain reaction and nucleic acid hybridization techniques have been standardized for the diagnosis of HHS (1).
An autogenous formalin inactivated vaccine prepared from infected liver homogenate has been used in prophylaxis. Cell culture vaccine developed by growing the virus in chicken embryo liver cells (24) and a killed oil emulsified vaccine was prepared from Indian isolates of HHS propagated in chick-embryo liver cell cultures (23) are being considered on an experimental basis. Roy et al. (40) further confirmed the efficacy of inactivated oil emulsion vaccine against hydropericardium syndrome in broilers and found it to be effective in both experimental and field trials. Shamin (41) also reported the preparation of vaccine using HHS virus adapted to chicken embryos and chicken kidney and liver cells. Ahmad (39) used PR1-10 strain and showed that the vaccination against HHS with aqua-base liver organ and oil-base tissue culture gave protection against subclinical infection.
- Ganesh, K. (1998). “Molecular Studies on hexon gene fragment of fowl adenovirus associated with hydropericardium hepatitis syndrome” Ph.D. thesis. University of Agricultural Sciences, Bangalore, India.
- Mazaheri, A. et al. (1998). Avian Pathology, 27: 269.
- Voss, M. (1989). Lohman symposium on the hydropericardium syndrome, Cuxhaven, Germany.
- Voss, M. et al. (1996). Proc. Int. Symp. adenovirus and reovirus infections in poultry. p. 75. June 24-27. Rauischholzhausen, Germany.
- Rabbani, M. and Naeem, K. (1996). Proc. Int. Symp. adenovirus and reovirus infections in poultry. p. 26. June 24-27. Rauischholzhausen, Germany.
- Nakamura, K. et al. (1999). Avian Diseases, 43: 414.
- Toro, H. et al. (1999). Avian Diseases, 43: 262.
- Jadhao, S.J. et al. (1997). Indian J. Comp. Microbiol. Immunol. Infect. Dis., 18: 33.
- Ganesh, K. et al. (2002). Trop. Anim. Hlth. Prod., 33: (In press).
- Ganesh, K. et al. (2002). Vet. Res. Comm., 26: (In press).
- Ganesh, K. et al. (2001). Vet. Microbiol., 78: 1.
- Cheema, A.H. et al. (1989). Rev. Sci. Tech. Off. Int. Epizoot., 8: 789.
- Chandra, R. et al. (1997). Vet. Rec., 138: 70.
- Anjum, A.D. (1990). Avian Pathology, 19: 655.
- Abdul-Aziz, T.A. and Hassan, S.Y. (1995). Res. vet. Sci., 59: 219.
- Anjum, A.D. et al. (1989). Vet. Rec., 124: 247.
- Naeem, K. et al. (1995). Avian Diseases, 39: 723.
- Gowda, R.N.S. and Satyanarayana, M.L. (1994). Indian J. vet. Path., 18: 159.
- Cowen, B.S. et al. (1996). Proc. Int. Symp. adenovirus infection in poultry, p. 79. Jun. 24-27. Rauischholzhausen, Germany.
- Shane, S.M. (1996). Zootecnica Int., 29: 20.
- Nakamura, K. et al. (2000). Avian Diseases, 44: 192.
- Abe, T. et al. (1998). Avian Diseases, 42: 606.
- Kataria, J.M. et al. (1997). Indian J. Comp. Microbiol. Immunol. Infect. Dis., 18: 38.
- Naeem, K. et al. (1995). Pakistan Vet. J., 15: 150.
- Asrani, R.K. et al. (1997). Vet. Rec., 141: 271.
- Borisov, V.V. et al. (1997). Proc. 10th Int. Cong. WVPA. p. 258. Aug. 18-22. Budapest, Hungary.
- Khan, S.A. et al. (1995). Asian-Australasian J. Anim. Sc. B., 4: 325.
- Bhatti, B.M. et al. (1988). Proc. Natl. Seminar on hydro-pericardium syndrome in chickens in Pakistan. p. 121. Jul. 4. Rawalpindi, Pakistan.
- Papasalmontos, P. (1988). Proc. Seminar organized by Hubbard Poultry, U.K. Oct. 1. Lahore, Pakistan.
- Niazi, A.K. et al. (1989). Vet. Rec., 124: 400.
- Sreenivasa Gowda, R.N. (1994). Poultry Adviser, 27: 53.
- Khan, M.Z. et al. (1988). Proc. Natl. Seminar on hydro-pericardium syndrome in chickens in Pakistan. p. 105. Jul. 4. Rawalpindi, Pakistan.
- Cheema, A.H. et al. (1988). Proc. Natl. Seminar on hydro-pericardium syndrome in chickens in Pakistan. p. 41. Jul. 4. Rawalpindi, Pakistan.
- Muneer, M.A. et al. (1989). Zootecnica Int., 5: 46.
- Abdul-Aziz, T.A. and Al-Attar, M.A. (1991). Vet. Rec., 129: 272.
- Oberoi, M.S. et al. (1996). Indian J. Virol., 12: 123.
- Toro, H. et al. (2000). Avian Diseases, 44: 51.
- Shane, S.M. and Jaffery, M.S. (1997). Diseases of poultry. p. 1019. 10th edn. Eds. B.W. Calnek, H.J. Barnes, C.W. Beard, L.R. McDougald and Y.M. Saif. Iowa State University Press, Ames, USA.
- Ahmad, K. (2000). Poster p. 17.01. World Poultry Conference, Canada.
- Roy, P. et al. (1999). Vet. Rec., 145: 458.
- Shamin, S. (2000). Poster p. 17.22. World Poultry Conference, Canada.