- Email: [email protected]
Hydropericardium hepatitis syndrome of broiler poultry: current status of research
Research in Veterinary Science 2000, 68, 201–206 doi:10.1053/rvsc.1999.0365, available online at http://www.idealibrary.com on
REVIEW
Hydropericardium hepatitis syndrome of broiler poultry: current status of research K. GANESH*, R. RAGHAVAN† *Indian Veterinary Research Institute, Bangalore-560024, India, †Department of Veterinary Microbiology, Veterinary College, University of Agricultural Sciences, Bangalore-560024, India
A PREVIOUSLY unknown emerging disease with some clinical similarities to inclusion body hepatitis but characteristically causing severe hydropericardium resulting in heavy mortality of broiler poultry was reported in specific areas of the world. Studies undertaken at various laboratories (Rabbani and Naeem 1996, Voss et al 1996, Mazaheri et al 1998) have indicated that the causative agent is a fowl adenovirus (FAV) group I, serotype 4. Sudden deaths among broilers in the age group of 3 to 5 weeks with mortality levels reaching up to 75 per cent, hydropericardium, hepatitis with focal necrosis, pulmonary oedema, and nephritis are the highlights of the episodes. Although liver damage was also seen in inclusion body hepatitis (IBH), severe hydropericardium in 3-week-old chicks, due to an adenovirus infection, was not a feature noticed until very recently. In order to distinguish hydropericardium hepatitis syndrome (HHS) from classical IBH and to emphasise its infectious nature, the new disease was named as ‘infectious hydropericardium IH’ (Abdul-Aziz and Hassan 1995, Mazaheri et al 1998). The disease is variously termed as ‘Angara disease’ in Pakistan, after the place Angara Goth near Karachi (Akhtar 1994), ‘Leechy’ or ‘Litchi disease’ in India, after the look of heart floating in pericardial fluid, simulating a de-skinned Leechy fruit (Gowda and Satyanarayana 1994), or ‘inclusion body hepatitis–hydropericardium syndrome IBH–HPS’ (Jadhao et al 1997) and also as ‘hydropericardium hepatitis syndrome HHS’ (Shane 1996, Ganesh 1998) or ‘hydropericardium hepatopathy syndrome HHS’ (Asrani et al 1997) or ‘hydropericardium syndrome HPS’ (Naeem et al 1995a).
INCIDENCE AND DISTRIBUTION OF THE DISEASE HHS was first reported in late 1987 in Angara Goth near Karachi, Pakistan (Khawaja et al 1988, Cheema et al 1989, Hasan 1989). It has been subsequently reported in Iraq (Abdul-Aziz and Al-Attar 1991), India (Gowda and Satyanarayana 1994), Mexico, Ecuador, Peru, Chile (cited
*Corresponding author: Tel: 91-80-3418021, 3410729, Fax: 91-803412509, E-mail: [email protected] 0034-5288/00/030201 + 06 $35.00/0
by Voss et al 1996, Cowen et al 1996), South and Central America (Shane 1996), and Russia (Borisov 1997). A few of the Indian states which recorded HHS between 1994 and 1996 include Punjab, Uttar Pradesh, Haryana, Jammu Delhi, Madhya Pradesh, Maharashtra, Tamilnadu, Andhra Pradesh and Karnataka (Gowda and Satyanarayana 1994, Asrani et al 1997, Kumar et al 1997, Shukla et al 1997). The study group on poultry diseases, appointed by the Technology Mission on Dairy Development on the advice of the National Advisory Committee on the Control of Animal Diseases in March 1996, reported the percent mortality due to HHS ranging from 10 to 80 (Anonymous 1996) and identified the HHS as an emerging problem of the broiler poultry industry.
DISEASE SYMPTOMS AND LESIONS Sudden onset and high mortality of up to 75 per cent in well-grown, clinically healthy broiler flocks in the age group of 3 to 5 weeks are the salient features of the outbreaks (Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994, Voss et al 1996, Asrani et al 1997). Generally there are no apparent clinical signs associated with the disease. Individual birds occasionally are depressed, huddle in corners and have ruffled feathers before death. However the necropsy findings are quite striking. The most predominant and consistent gross lesions were accumulation of clear or amber-coloured, watery or jelly-like fluid in the pericardial sac with the quantity of fluid ranging from 5 to 20 ml and a misshapen and flabby heart (Cheema et al 1988, Anjum et al 1989, Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994, Asrani et al 1997, Kumar et al 1997). Other changes observed were a discoloured and swollen reticulated friable liver with focal hepatic necrosis, petechial and ecchymotic haemorrhages in heart musculature and other organs, congestion and oedema in lungs, and pale kidneys with prominent tubules (Cheema et al 1989, Abdul-Aziz and Hasan 1995, Oberoi et al 1996). The liver was identified as the major organ showing histopathological changes such as small multifocal areas of coagulative necrosis, mononuclear cell infiltration and the presence of intranuclear basophilic inclusions in the hepatocytes (Cheema et al 1989, Asrani et al 1997). Infected © 2000 Harcourt Publishers Ltd
K. Ganesh, R. Raghavan
202
chickens showed reduced serologic response to Newcastle disease vaccination compared with that of the controls (Naeem et al 1995a).
INCUBATION PERIOD AND DURATION Akhtar (1992) reported a range of 5 to 18 days of incubation with a mean of 10 days in controlled experiments, and in another study (Akhtar 1995) reported the incubation period of 9.5 to 14.5 days. Duration of the disease was 10 to 15 days. Aliev et al (1997) observed the incubation period as 48 to 72 hours in experimental infection while the duration was 7 days. The syndrome was experimentally reproduced in broilers using both liver homogenate and purified virus prepared from the field samples. The incubation period varied between 2 to 4 days after inoculation (Ganesh 1998). Anjum (1990), Afzal et al (1991) and Gowda and Satyanarayana, (1994) in their experiments showed that the clinical signs/ sudden deaths due to HHS were observed during 2 to 5 days post infection. However, in their infectivity trials with liver homogenate by intramuscular route in SPF birds, Voss et al (1996) observed mortality during 6 to 17 days post infection. Reasons for such variation in the incubation period were unsatisfactorily explained by any of the workers.
TRANSMISSION HHS is a contagious disease and is transmitted horizontally among broilers by mechanical means including vaccinators (Akhtar et al 1992) and litter contaminated with infected faeces (Shafique and Shakoori 1994). Anjum (1990) failed to reproduce the disease by oral inoculation or by contact with naturally affected birds. However, Abdul-Aziz and Hassan (1995) successfully transmitted the disease to experimental birds by inoculating infected liver suspension intramuscularly, orally or by contact. The results of Cowen et al (1996), who demonstrated HHS virus in the rectal swabs of infected birds, showed the possible mechanism of spread of the disease under field conditions.
30 per cent (Abdul-Aziz and Hassan 1995). The disease, however, could not be reproduced experimentally with fluid collected from the pericardial sac or homogenates from other organs including bursa, lungs, heart, kidneys (Anjum et al 1989). Subsequent studies have shown that the virus has a predilection for lymphoid organs; homogenate of these organs reproduced the disease when inoculated into healthy chickens (Naeem et al 1995a). Gowda and Satyanarayana (1994) experimentally reproduced the disease in broilers. They have described hydropericardium with accumulation of about 12 ml strawcoloured fluid thus giving the heart a litchi fruit appearance; the other changes were pale enlarged livers; oedematous and congested lungs; pale and tumified kidneys and an icteric carcass. Lesions of hydropericardium, spleenomegaly, necrotic foci on the myocardium, hypertrophy of bursa of Fabricius and poorly developed musculature have also been described in experimentally induced HHS cases (Cheema et al 1988, Muneer et al 1989, Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994). Cowen et al (1996) in their pathogenicity study of fowl adenoviruses observed that an isolate, LA/C serotype 4 from Chile produced classical lesions of HHS and they could recover the virus from rectal swabs of the infected birds and reported an increase in mortality after oral injection of 2day-old specific pathogen free (SPF) chicks with the pathogenic fowl adenovirus serotype 4 (FAV4) strain. Filtered homogenates of liver tissue from affected cases when inoculated onto 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 (Shane 1996). A pathogenicity study on six isolates from different field outbreaks of HHS in Ecuador (K1013 number 1 to 6) on embryos and chickens revealed high embryo and chick mortality (Mazaheri et al 1998). The disease was reproduced in day-old SPF chicks by the oral route of inoculation and subsequently the agent was re-isolated. The results showed that some strains of FAV4 caused HHS following oral or intramuscular infection.
EPIDEMIOLOGICAL STUDIES EXPERIMENTAL REPRODUCTION The syndrome was reproduced in broilers of up to 3 weeks of age by inoculation of a bacteria-free liver homogenate from infected birds (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Afzal et al 1991, Gowda and Satyanarayana 1994, Asrani et al 1997, Chandra et al 1997, Satyanarayana 1998). HHS was reproduced using liver homogenate extract and the pellet obtained thereof, from ultracentrifugation at 145 000 g for 90 minutes. The disease was transmitted to broilers by liver homogenate filtered through 0.22µ (and 0.1µ) membrane filters, indicating the virus was less than 100 nm in size and it is the aetiological agent of HHS (Afzal et al 1991). Mortality was observed after 3 to 5 days post infection. Generally, birds dying before 60 hours did not show any clinical sign (Kumar et al 1997). Mortality was 100 per cent when infected liver suspension was inoculated intramuscularly as compared to oral inoculation where mortality was
The disease was first observed in Pakistan in 1987 among broiler flocks of 3 to 5 weeks of age with flock mortality exceeding 50 per cent (Anjum et al 1989). Occasional outbreaks were also reported outside the 3- to 5-week age range (Ahmad et al 1989, Muneer et al 1989,- Akhtar and Cheema 1990), among breeder pullets (Akhtar 1992), and in layers (Ahmad et al 1988, Jaffery 1988, Asrani et al 1997). Asrani et al (1997) 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 was followed by an almost constant death rate for 5 to 7 days before declining. The average mortality in an affected flock was between 15 to 60 per cent (Asrani et al 1997). In Russia, HHS was recorded in chickens aged between 2 and 13 weeks with mortality ranging from 3.5 to 30 per cent
Hydropericardium hepatitis syndrome of broiler poultry: current status of research
in broilers and 2.6 to 15.29 per cent in layers (Borisov et al 1997). In a number of broiler poultry farms, HHS was recorded as an acute disease with hydropericarditis in 3- to 5-week-old broilers (Aliev et al 1997). Most of the workers have reported that different strains of broilers are equally susceptible in the field (Anjum et al 1989, Akhtar and Cheema 1990) and in experimental cases (Afzal and Hussain 1993). However, Khan et al (1995) have noticed the Hubbard broiler strain to be relatively more susceptible followed by Indian River and Lohmann respectively.
AETIOLOGY HHS was initially suspected to be caused by toxicity or by nutritional deficiency. The possible causative factors investigated were mycotoxins, toxic fat agent, polychlorinated biphenyl, sodium chloride, chlordane and phytotoxins, all of which were associated with hydropericardium (Jaffery 1988, Qureshi 1989). Attempts to reproduce the disease experimentally with feed samples from farms having natural outbreaks of HHS or feed containing 100 ppm mycotoxins or interaction of the two, however, failed (Anjum 1988, 1990). Anjum et al (1989) and Anjum (1990) successfully transmitted HHS to young broilers by subcutaneous injection of liver homogenates from affected birds. Subsequent studies confirmed the association of a virus with the condition and an adenovirus was identified as the aetiological agent (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Afzal et al 1991). The physical properties of the virus and demonstration of basophilic intranuclear inclusion bodies in hepatocytes, and characteristic hexagonal virions in EM preparations of liver homogenates (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Niazi et al 1989, Abdul-Aziz and Al-Attar 1991) were suggestive of involvement of a DNA virus. Later, fowl adenovirus belonging to serotype 4 named as PARC1 isolate was characterised and propagated in cell culture and in embryonated eggs. The isolation of an adenovirus in chicken embryo liver cells, reproduction of the disease with isolated virus (PARC1) and its use as a vaccine (Naeem et al 1995b) confirmed these observations. It is true that so far the correlation of FAV with specific disease has been difficult because fowl adenoviruses have been isolated from both healthy and diseased chickens. But there was growing evidence to show fowl adenoviruses as primary pathogens in the case of HHS. All isolates from field outbreaks of HHS have been serotyped as FAV4 strains and it was shown that some biotypes of FAV4 caused the HHS (Mazaheri et al 1998). Fowl adenovirus was isolated from liver samples of HHS cases and the virus was neutralised by antiserum against KR5 strain, which is a representative FAV serotype 4 strain of group 1 adenovirus. No other viral agent was isolated from liver samples by chicken kidney, chicken embryo, and MDCC-MSB1 cells (Abe et al 1998). Afzal et al (1990) in their study showed that the pellet after centrifugation of liver homogenate was infectious, but not the supernatant. Addition of an inclusion body hepatitisproducing adenovirus to the supernatant restored infectivity, suggesting the presence of an additional viral agent other than the primary adenovirus. In another study with liver
203
homogenate in Mexico in 1994, an RNA virus was implicated as an additional agent based on lack of inhibition by 5-bromodeoxyuridine (Shane and Jaffery 1997). This work pertains to treatments with liver homogenate, wherein the agent was not isolated. Moreover the physico-chemical properties of the HHS virus vary in comparison to other group 1 avian adenoviruses. Subsequent work clearly isolated the viral agent and characterised it as FAV4, and the disease was reproduced by the FAV4 isolates. Also there were reports showing the presence of infectious bursal disease (IBD) and chicken infectious anaemia (CIA) viruses in areas where HHS occurs (Shane and Jaffery 1997). IBD and CIA are known for their immunosuppressive effects and fowl adenoviruses require an impairment of the immune response to express their pathogenic potential in domestic fowl (Monreal 1996). FAV4 associated with HHS also has a predilection for lymphoid tissues, which resulted in immunosuppression (Naeem et al 1995a). Therefore the presence of IBD and CIA viruses can predispose for the HHS condition or HHS may predispose for other viral infections. The HHS was reproduced in SPF chicks using isolated and purified virus from field cases of HHS (Cowen et al 1996, Mazaheri et al 1998) thus proving the FAV4 associated with HHS is the sole agent responsible for the disease. Reference has been made to serotype 8 (European serotype 5) in the reproduction of the HHS syndrome, associated with IBH in Mexico (Shane and Jaffery 1997). The liver damage seen in HHS is also seen in IBH but the hydropericardium in chicks of 3 weeks of age, due to an adenovirus infection, was not a feature noticed until relatively recently. IBH is also seen in young broilers, but the high rate of mortality is characteristic of HHS (60 to 70 per cent in Pakistan, 10 to 30 per cent in Iraq, and 10 to 60 per cent in India (Abe et al 1998). Hydropericardium is a prominent symptom of HHS and has never been noted in many reports of IBH field and experimental cases (Gallina et al 1973). Fowl adenoviruses were isolated and characterised from several cases of HHS in Ecuador and Pakistan. Antibodies against serotypes 4 and 10 (European 11) neutralised these isolates. However, cross neutralisation tests and restriction enzyme (RE) analysis strengthened their grouping under serotype 4 (Mazaheri et al 1998). It was shown by immunological and molecular methods that FAV4 and FAV11 are very closely related (Enry et al 1995). The classification of HHS field isolates as FAV4 strains was confirmed by restriction enzyme analysis. Digestion with Bam H I, Dra I, and Eco R I demonstrated similar restriction fragment patterns between FAV4 strain and field isolates (Mazaheri et al 1998). In Pakistan and in several South American countries, inclusion body hepatitis (IBH–HPS) virus in many cases associated with hydropericardium was predominant and the condition was caused by FAV serotype 4 (Voss et al 1996). Voss,(1989) described the isolation of an adenovirus (K31/89) from field cases of HHS in Pakistan. Later, Voss et al (1996) identified the isolate as adenovirus serotype 4 and as a causative agent of HHS. Fowl adenovirus serotype 4 isolates recovered from different outbreaks of HHS in Pakistan have been found to differ in their pathological characteristics, although no significant differences were recorded regarding their biological properties or in their protein profiles (Rabbani and Naeem 1996).
204
K. Ganesh, R. Raghavan
Three Indian isolates of FAV recovered from IBH–HPS of poultry from different geographical locations were typed as serotype 4 (Jadhao et al 1997). 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, thereby avoiding further manipulation of the field isolate (Ganesh 1998). The presence of the virus in the samples collected was verified by performing double immunodiffusion and virus neutralisation tests with antiserum against all 1 to 12 FAV serotypes (Ganesh 1998). The syndrome was experimentally reproduced in broilers using both liver homogenate and purified virus prepared from the field samples (Ganesh 1998). The experimental reproduction of the disease in susceptible chicks accompanied with serological and electron microscopic evidences is suggestive that fowl adenovirus serotype 4 is solely responsible for causation of HHS (Ganesh 1998). Six isolates from different field outbreaks of HHS in Equador (K1013 NO 1–6), FAV reference strains [KR5 (FAV4), C2B (FAV11) and Pakistan isolate (K31)] were analysed by restriction enzymes. Identical restriction patterns were noticed between KR5, K1013 NO.2 and K31, while PstI digestion differentiated field isolates from reference strains. This study concluded that some specific strains of FAV4 caused the disease (Mazaheri et al 1998). The DNA isolated from either purified virus or from infected liver tissue was subjected to PCR using FAVH(L) and (R) primers (Ganesh 1998). Two oligonucleotides, FAVH(L) and FAVH(R), from the consensus region of hexon genes (corresponding to the nucleotide positions 722–747 and 1433–1452 respectively of the hexon gene of FAV10) were designed to amplify a 0.7 kb stretch variable region of HHS virus DNA. 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 (Ganesh 1998). PCR-amplified DNA was labelled with Digoxigenin (DIG-11-DUTP) and the labelled DNA was used as a probe for the detection of virusspecific sequences in the PCR-amplified products after Southern blotting. The DNA probe could detect the presence of the virus by dot blot hybridisation of the viral genome (Ganesh 1998). A 728 bp sequence of HHS viral DNA was obtained by sequencing the 0.7 kb PCR-amplified DNA (Ganesh 1998). A comparative study of the 0.7 kb fragment of the hexon gene of HHS with those of FAV10 and FAV1 indicated homology of 91.8 and 71.9 per cent respectively showing that the HHS virus is closer to FAV10 than to FAV1 (Ganesh 1998). In comparing corresponding amino acid sequences among the serotypes (FAV10, FAV1 and HHS), the variations were found as high as 28.8 and 38 per cent with FAV10 and FAV1 respectively (Ganesh 1998). Though nucleotide sequences showed a difference of only 8.2 per cent between HHS and FAV10 sequences, the amino acid sequence was found to be as high as 38 per cent. A marked difference existed in the variable regions of the hexon gene of the two serotypes (Ganesh 1998). The aetiological agent of HHS may be a variant of a non-pathogenic FAV type 4 that was reported earlier among poultry flocks (Ganesh 1998).
PHYSICO-CHEMICAL PROPERTIES OF THE VIRUS The sensitivity of HHS agent to pH 3 to 10 is variable. Unlike other adenoviruses, HHS virus in the liver preparations withstood heating at 60°C for 30 minutes and at 50°C for 1 hour. However heating at 60°C for 1 hour; 80°C for 10 minutes; and 100°C for 5 minutes inactivated the agent. Treatment with chloroform (5 per cent) or ether (10 per cent) which normally inactivates adenovirus eliminated infectivity of the HHS virus (Afzal et al 1991).
ELECTRON MICROSCOPY Purified samples of virus from liver homogenate from cases of HHS when examined by negative staining electron microscopy showed virus particles with the characteristic morphology of adenovirus (Cheema et al 1989, Ganesh 1998). Isometric, roughly spherical particles resembling adenovirus morphology, measuring 83–93 nm in diameter, were demonstrated inside the nucleus of hepatocytes from HHS outbreaks by transmission electron microscopy and in negative staining electron microscopy of virus pellets obtained from liver homogenates. The pellet when inoculated into experimental chicks reproduced the disease (Chandra et al 1997, Ganesh 1998).
DIAGNOSIS AND CONTROL Clinical diagnosis of the disease before the symptoms appear 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 pathognomonic for diagnosis (Akhtar 1994). Certain serological tests, such as indirect HA test (Rahman et al 1989, Hassan et al 1993, Maskoor et al 1994a) ELISA (Hassan et al 1993), AGPT (Hassan et al 1993, Kumar et al 1997) and counter immunoelectrophoresis (Kumar et al 1997), using filtrate from infected liver homogenate as a crude antigen have been done. These studies indicate that the specificity of the currently available serological tests for diagnosis needs improvement. Dot-immunobinding assay with virus purified from infected liver homogenate was used for the study of immunosuppressive potential and pathogenicity of HHS (Naeem et al 1995a). The electron microscopic demonstration of the virus particles in hepatocytes (Chandra et al 1997) and virus neutralisation test (Rabbani and Naeem 1996, Jadhao et al 1997) using specific antiserum have also been used in HHS diagnosis. Polymerase chain reaction and nucleic acid hybridisation techniques were developed for the diagnosis of HHS (Ganesh 1998). Flocks were protected with an autogenous formalin inactivated vaccine prepared from infected liver homogenate (Cheema et al 1989, Chishti et al 1989, Afzal and Ahmad 1990, Ahmad et al 1990, Anjum 1990, Mashkoor et al
Hydropericardium hepatitis syndrome of broiler poultry: current status of research
1994b, Shane 1996, Kumar et al 1997). Experimentally, a cell culture vaccine was developed by growing the virus in chicken embryo liver cells (Naeem et al 1995b) and a killed oil emulsified vaccine was prepared from Indian isolates of HHS propagated in chick-embryo liver cell cultures (Kataria et al 1997).
REFERENCES ABDUL-AZIZ, T.A. & AL-ATTAR, M.A. (1991) New Syndrome in Iraqi chicks. Veterinary Record 129, 272 ABDUL-AZIZ, T.A. & HASSAN, S.Y. (1995) Hydropericardium syndrome in broiler chickens: its contagious nature and pathology. Research in Veterinary Science 59, 219–221 ABE, T., NAKAMURA, K., TOJO, H., MASE, M., SHIBAHARA, T., YAMAGUCHI, S. & YUASA, N. (1998) Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks. Avian Diseases 42, 606–612 AFZAL, M. & AHMAD, I. (1990). Efficacy of an inactivated vaccine against hydropericardium syndrome in broilers. Veterinary Record 126, 59–60 AFZAL, M., MUNEER, R., STEIN, JR, G. & COWEN, B.S. (1990) Aetiology and control of hydropericardium syndrome in broilers of Pakistan. Proceedings of the third International Veterinary Congress, Islamabad, Pakistan. pp. 218–228 AFZAL, M. & HUSSAIN, A. (1993). Age susceptibility of different commercial broiler strains to Angara disease (Hydropericardium syndrome). Pakistan Journal of Agricultural Research 14, 387–392 AFZAL, M., MUNEER, R. & STEIN, G. (1991) Studies on the etiology of hydropericardium syndrome in broilers. Veterinary Record 128, 591–593 AHMAD, I., AFZAL, M., MALIK, M.I., HUSSAIN, Z. & HANIF, W. (1988) Studies on the disease pattern and etiology of hydropericardium syndrome (Angara Disease) in broiler chickens in Pakistan. Pakistan Journal of Agricultural Research 10, 195–199 AHMAD, I., AFZAL, M., MALIK, M.I., HUSSAIN, Z. & HANIF, W. (1989) Disease patterns and etiology of hydropericardium syndrome (Angara disease) in broiler chickens in Pakistan. Pakistan Journal of Agricultural Research 10, 195–199 AHMAD, I., MALIK, M.I., IQBAL, K., AHMED, K. & NAZ, S. (1990) Efficacy of formalinised liver organ vaccine against Angara disease in broilers. Veterinarski Arhiv 60, 131–138 AKHTAR, S. (1992) Studies on the rate of lateral spread of hydropericardium syndrome agent(s). In: Etiology, pathogenesis, and control of hydropericardium syndrome in poultry. Board on Science and Technology for International Development (BOSTID), Washington, USA AKHTAR, S. (1994) Hydropericardium syndrome in broiler chickens in Pakistan. World’s Poultry Science Journal 50, 177–182 AKHTAR, S. (1995). Lateral spread of the etiological agent(s) of hydropericardium syndrome in broiler chickens. Veterinary Record 136, 118–120 AKHTAR, S. & CHEEMA, A.H. (1990) Some epidemiological characteristics of hydropericardium syndrome outbreaks among commercial broiler flocks in Karachi. Proceedings of the third International Veterinary Congress, Islamabad, Pakistan. Pp. 228–234 AKHTAR, S., ZAHID, S. & KHAN, M.I. (1992) Risk factors associated with hydropericardium syndrome in broiler flocks. Veterinary Record 131, 481–482 ALIEV, A.S., DJAVADOV, E.D. & NIKITINA, N.V. (1997) Etiology of hydropericarditis in broiler chicks. Proceedings of the tenth international congress of the WVPA 18–22 Aug, Budapest, Hungary, p. 257 ANJUM, A.D. (1988) Study of pathogenicity of the hydropericardium syndrome agent. Proceedings of national seminar on hydropericardium syndrome in chickens in Pakistan, Rawalpindi, 4 Jul, pp. 111–115 ANJUM, A.D. (1990) Experimental transmission of hydropericardium syndrome and protection against it in commercial broiler chickens. Avian Pathology 19, 655–660 ANJUM, A.D., SABRI, M.A. & IQBAL, Z. (1989). Hydropericarditis syndrome in broiler chickens in Pakistan. Veterinary Record 124, 247–248 ANONYMOUS (1996). Report of the study group on poultry diseases appointed by technology mission on dairy development on the advice of National Advisory Committee on Control of Animal Diseases, New Delhi, India ASRANI, R.K., GUPTA, V.K., SHARMA, S.K., SINGH, S.P. & KATOCH, R.C. (1997) Hydropericardium hepatopathy syndrome in Asian poultry. Veterinary Record 141, 271–273 BORISOV, V.V., BORISOV, A.V. & GUSEV, A.A. (1997) Hydropericardium syndrome in chickens in Russia. Proceedings of the tenth international congress of the WVPA, 18–22 Aug, Budapest, Hungary, p. 258 CHANDRA, R., SHUKLA, S.K., KUMAR, M. & GARG, S.K. (1997) Electron microscopic demonstration of adenovirus in the hepatocytes of birds experimentally infected with hydropericardium syndrome. Veterinary Record 138, 70–71 CHEEMA, A.H., AFZAL, M. & AHMAD, J. (1988) Studies on the causation of hydropericardium syndrome in Pakistan. Proceedings of national seminar on hydropericardium syndrome in chickens in Pakistan. Rawalpindi, 4 Jul, pp. 41–48. CHEEMA, A.H., AHMAD, J. & AFZAL, M. (1989) An adenovirus infection of poultry in Pakistan. Rev. Sci. Tech. Off. Int. Epi. 8, 789–795
205
CHISHTI, M.A., AFZAL, M. & CHEEMA, A.H. (1989) Preliminary studies on the development of hydropericardium syndrome vaccine of poultry. Off. Int. Epi. Report 8, 797–801 COWEN, B.S., LU, H., WEINSTOCK, D. & CASTRO, A.E. (1996) Pathogenicity studies of fowl adenoviruses isolated in several regions of the world. International symposium on adenovirus infection in poultry, Rauischholzhausen, 24–27 Jun, Germany, pp. 79–88 ERNY, K.M., PALLISTER, J. & SHEPPARD, M. (1995). Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10. Arch. Virol. 140, 491–501 GANESH, K. (1998) Molecular studies on hexon gene fragment of fowl adenovirus associated with hydropericardium hepatitis syndrome. Ph.D. thesis submitted to University of Agricultural Sciences, Bangalore, India GALLINA, A.M., WINTERFEILD, R.W. & FADLY, A.M. (1973) Adenovirus infection and disease. II. Histopathology of natural and experimental disease. Avian Diseases 343–353 GOWDA, R.N.S. & SATYANARAYANA, M.L. (1994) Hydropericardium syndrome in poultry. Indian Journal Veterinary Pathology 18, 159–161 HASAN, S.A. (1989) Pakistan is mystified by hydropericardium syndrome. PoultryMisset 5, 35–37 HASSAN, N.U., AFZAL, M., HAMEED, A. & KHAN, A.R. (1993) Immune response to inactivated hydropericardium syndrome vaccine in broilers. Pakistan Veterinary Journal 14, 5–10 JADHAO, S.J., KATARIA, J.M., VERMA, K.C. & SAH, R.L. (1997). Serotyping of Indian isolates of fowl adenovirus recovered from IBH-HPS (Litchi disease) in chickens. Indian Journal Comparative Microbiology Immunology and Infectious Diseases 18, 33–37 JAFFERY, M.S. (1988) A treatise on Angara disease (hydropericardium-pulmonary oedema hepatonephritis syndrome). Pakistan Veterinary Medical Association, p. 34 KATARIA, J.M., VERMA, K.C., JADHAO, S.J., DEEPAK, J.N. & SAH, R.L. (1997). Efficacy of inactivated oil emulsified vaccine against IBH-HPS in chickens prepared from cell culture propagated fowl adenovirus. Indian Journal Comparative Microbiology Immunology and Infectious Diseases 18, 38–42 KHAN, S.A., ZAIDI, F.H., CHAUDHRY, R.A., ASHRAF, S.K., MIAN, M.S. & QURESHI, I.R. (1995). Comparative study on pathology of haemic system of three broiler chicken strains suffering from experimental hydropericardium syndrome. Asian-Australasian Journal of Animal Sciences B(4), 325–328 KHAWAJA, D.A., AHMED, S., RAUF, A.M., ZULFIGAR, M., MAHMOOD, S.M. & HASSAN, M. (1988) Isolation of an adenovirus from hydropericardium syndrome in broiler chicks. Pakistan Journal Veterinary Research 1, 2–17 KUMAR, R., CHANDRA, R., SHUKLA, S.K., AGARWAL, D.K. & KUMAR, M. (1997) Hydropericardium syndrome in India: A preliminary study on the causative agent and control of disease by inactivated autogenous vaccine. Tropical Animal Health and Production 29, 158–164 MASHKOOR, S., HAMEED, A., AHMAD, K. & QURESHI, M.S. (1994a) Improved angara disease vaccine for broiler chicks (abstract). Veterinarski Arhiv 64, 27–33 MASHKOOR, S., HAMEED, A., AHMAD, K. & QURESHI, M.S. (1994b) Immune response of Angara disease (hydropericardium syndrome) vaccines in broilers (abstract). Veterinarski Arhiv 64, 103–107 MAZAHERI, A., PRUSAS, C., VO, M. & HESS, M. (1998) Some strains of serotype 4 fowl adenoviruses cause Inclusion body hepatitis and hydropericardium syndrome in chickens. Avian Pathology 27, 269–276 MCFERRAN, J.B. (1991) Adenovirus infections. In: Diseases of poultry, Eds. BW Calnek, HJ Barnes, CW Beard, WM Reid, HW Yoder JR. 10th edn. Iowa State University Press, Ames England. pp 607–620 MONREAL, G. (1996) History and development of research about avian adenoviruses. International symposium on adenovirus infection in poultry, Rauischholzhausen, 24–27 Jun, Germany, pp. 4–9 MUNEER, M.A., AJMAL, M., ARSHAD, M., AHMAD, M.D., CHAUDHRY, Z.I. & KHAN, T. M. (1989) Preliminary studies of hydropericardium syndrome in broilers in Pakistan. Zootechnique International 5, 46–48 NAEEM, K., NIAZI, T., MALIK, S.A. & CHEEMA, A.H. (1995a) Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers. Avian Diseases 39, 723–728 NAEEM, K., RABBANI, M., HUSSAIN, M. & CHEEMA, A.H. (1995b) Development of cell culture vaccine against hydropericardium syndrome in poultry. Pakistan Veterinary Journal 15, 150–15 NIAZI, A.K., KHAN, M.Z. & SIDDIQUE, M. (1989) Hematological studies on naturally occurring hydropericardium syndrome in broiler chicks. Veterinary Record 124, 400 OBEROI, M.S., SINGH, A. & SINGH, B. (1996) Isolation of avian adenovirus from outbreaks of IBH-HPS in poultry. Indian Journal Virology 12, 123–124 QURESHI, A.A. (1989) Hydropericardium and ascitis. Poultry International 28, 44–48 RABBANI, M. & NAEEM, K. (1996) In vitro and in vivo evaluation of avian adenovirus isolates from outbreaks of hydropericardium syndrome. Proceedings of the international symposium on adenovirus and reovirus infections in poultry. Rauischholzhausen, 24–27 Jun, Germany, pp. 26–31 RAHMAN, S.U., ASHFAQUE, M., ANJUM, A.D. & SINFHU, T.A. (1989) Indirect hemagglutination for detecting Angara disease (Hydropericardium) agent antibody. In: Proceedings of international conference and trade show on poultry production, 27 Feb-March, pp. 73–74
206
K. Ganesh, R. Raghavan
SATYANARAYANA, M.L. (1998) Etiopathology and pathogenesis of hydropericardium syndrome in broilers. Ph.D. thesis submitted to University of Agricultural Sciences, Bangalore, India SHAFIQUE, M. & SHAKOORI, A.R. (1994) Transmission of hydropericardium syndrome in poultry. Pakistan Journal of Zoology 26, 145–148 SHANE, S.M. (1996) Hydropericardium hepatitis syndrome, the current world situation. Zootechnique International 29, 20–27 SHANE, S.M. & JAFFERY, M.S. (1997) Hydropericardium-hepatitis syndrome (Angara disease). In: Diseases of poultry, Eds. BW CALNEK, HJ BARNES, CW BEARD, WM REID, HW YODER JR. 10th edition, Iowa State University Press, Ames, England. pp. 1019–1022 SHUKLA, S.K., CHANDRA, R., KUMAR, M. & DIXIT, V.P. (1997) Hydropericardium syndrome in tarai of Uttar Pradesh: a report. Indian Journal Animal Sciences 67, 766–767
VOSS, M. (1989) Evaluation of field serology and isolation and identification of an agent from Pakistan field cases of hydropericardium syndrome. Lohman symposium on the hydropericardium syndrome, Cuxhaven, Germany VOSS, M., VIELITZ, E., HESS, M., PRUSAS, C.H. & MAZAHERRI, A. (1996) Aetiological aspects of Hepatitis and HPS caused by pathogenic adenoviruses in different countries. International symposium on adenovirus and reovirus infections in poultry, Rauischholzhausen, Germany, 24–27 Jun, pp. 75–78
Accepted November 25, 1999
REVIEW
Hydropericardium hepatitis syndrome of broiler poultry: current status of research K. GANESH*, R. RAGHAVAN† *Indian Veterinary Research Institute, Bangalore-560024, India, †Department of Veterinary Microbiology, Veterinary College, University of Agricultural Sciences, Bangalore-560024, India
A PREVIOUSLY unknown emerging disease with some clinical similarities to inclusion body hepatitis but characteristically causing severe hydropericardium resulting in heavy mortality of broiler poultry was reported in specific areas of the world. Studies undertaken at various laboratories (Rabbani and Naeem 1996, Voss et al 1996, Mazaheri et al 1998) have indicated that the causative agent is a fowl adenovirus (FAV) group I, serotype 4. Sudden deaths among broilers in the age group of 3 to 5 weeks with mortality levels reaching up to 75 per cent, hydropericardium, hepatitis with focal necrosis, pulmonary oedema, and nephritis are the highlights of the episodes. Although liver damage was also seen in inclusion body hepatitis (IBH), severe hydropericardium in 3-week-old chicks, due to an adenovirus infection, was not a feature noticed until very recently. In order to distinguish hydropericardium hepatitis syndrome (HHS) from classical IBH and to emphasise its infectious nature, the new disease was named as ‘infectious hydropericardium IH’ (Abdul-Aziz and Hassan 1995, Mazaheri et al 1998). The disease is variously termed as ‘Angara disease’ in Pakistan, after the place Angara Goth near Karachi (Akhtar 1994), ‘Leechy’ or ‘Litchi disease’ in India, after the look of heart floating in pericardial fluid, simulating a de-skinned Leechy fruit (Gowda and Satyanarayana 1994), or ‘inclusion body hepatitis–hydropericardium syndrome IBH–HPS’ (Jadhao et al 1997) and also as ‘hydropericardium hepatitis syndrome HHS’ (Shane 1996, Ganesh 1998) or ‘hydropericardium hepatopathy syndrome HHS’ (Asrani et al 1997) or ‘hydropericardium syndrome HPS’ (Naeem et al 1995a).
INCIDENCE AND DISTRIBUTION OF THE DISEASE HHS was first reported in late 1987 in Angara Goth near Karachi, Pakistan (Khawaja et al 1988, Cheema et al 1989, Hasan 1989). It has been subsequently reported in Iraq (Abdul-Aziz and Al-Attar 1991), India (Gowda and Satyanarayana 1994), Mexico, Ecuador, Peru, Chile (cited
*Corresponding author: Tel: 91-80-3418021, 3410729, Fax: 91-803412509, E-mail: [email protected] 0034-5288/00/030201 + 06 $35.00/0
by Voss et al 1996, Cowen et al 1996), South and Central America (Shane 1996), and Russia (Borisov 1997). A few of the Indian states which recorded HHS between 1994 and 1996 include Punjab, Uttar Pradesh, Haryana, Jammu Delhi, Madhya Pradesh, Maharashtra, Tamilnadu, Andhra Pradesh and Karnataka (Gowda and Satyanarayana 1994, Asrani et al 1997, Kumar et al 1997, Shukla et al 1997). The study group on poultry diseases, appointed by the Technology Mission on Dairy Development on the advice of the National Advisory Committee on the Control of Animal Diseases in March 1996, reported the percent mortality due to HHS ranging from 10 to 80 (Anonymous 1996) and identified the HHS as an emerging problem of the broiler poultry industry.
DISEASE SYMPTOMS AND LESIONS Sudden onset and high mortality of up to 75 per cent in well-grown, clinically healthy broiler flocks in the age group of 3 to 5 weeks are the salient features of the outbreaks (Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994, Voss et al 1996, Asrani et al 1997). Generally there are no apparent clinical signs associated with the disease. Individual birds occasionally are depressed, huddle in corners and have ruffled feathers before death. However the necropsy findings are quite striking. The most predominant and consistent gross lesions were accumulation of clear or amber-coloured, watery or jelly-like fluid in the pericardial sac with the quantity of fluid ranging from 5 to 20 ml and a misshapen and flabby heart (Cheema et al 1988, Anjum et al 1989, Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994, Asrani et al 1997, Kumar et al 1997). Other changes observed were a discoloured and swollen reticulated friable liver with focal hepatic necrosis, petechial and ecchymotic haemorrhages in heart musculature and other organs, congestion and oedema in lungs, and pale kidneys with prominent tubules (Cheema et al 1989, Abdul-Aziz and Hasan 1995, Oberoi et al 1996). The liver was identified as the major organ showing histopathological changes such as small multifocal areas of coagulative necrosis, mononuclear cell infiltration and the presence of intranuclear basophilic inclusions in the hepatocytes (Cheema et al 1989, Asrani et al 1997). Infected © 2000 Harcourt Publishers Ltd
K. Ganesh, R. Raghavan
202
chickens showed reduced serologic response to Newcastle disease vaccination compared with that of the controls (Naeem et al 1995a).
INCUBATION PERIOD AND DURATION Akhtar (1992) reported a range of 5 to 18 days of incubation with a mean of 10 days in controlled experiments, and in another study (Akhtar 1995) reported the incubation period of 9.5 to 14.5 days. Duration of the disease was 10 to 15 days. Aliev et al (1997) observed the incubation period as 48 to 72 hours in experimental infection while the duration was 7 days. The syndrome was experimentally reproduced in broilers using both liver homogenate and purified virus prepared from the field samples. The incubation period varied between 2 to 4 days after inoculation (Ganesh 1998). Anjum (1990), Afzal et al (1991) and Gowda and Satyanarayana, (1994) in their experiments showed that the clinical signs/ sudden deaths due to HHS were observed during 2 to 5 days post infection. However, in their infectivity trials with liver homogenate by intramuscular route in SPF birds, Voss et al (1996) observed mortality during 6 to 17 days post infection. Reasons for such variation in the incubation period were unsatisfactorily explained by any of the workers.
TRANSMISSION HHS is a contagious disease and is transmitted horizontally among broilers by mechanical means including vaccinators (Akhtar et al 1992) and litter contaminated with infected faeces (Shafique and Shakoori 1994). Anjum (1990) failed to reproduce the disease by oral inoculation or by contact with naturally affected birds. However, Abdul-Aziz and Hassan (1995) successfully transmitted the disease to experimental birds by inoculating infected liver suspension intramuscularly, orally or by contact. The results of Cowen et al (1996), who demonstrated HHS virus in the rectal swabs of infected birds, showed the possible mechanism of spread of the disease under field conditions.
30 per cent (Abdul-Aziz and Hassan 1995). The disease, however, could not be reproduced experimentally with fluid collected from the pericardial sac or homogenates from other organs including bursa, lungs, heart, kidneys (Anjum et al 1989). Subsequent studies have shown that the virus has a predilection for lymphoid organs; homogenate of these organs reproduced the disease when inoculated into healthy chickens (Naeem et al 1995a). Gowda and Satyanarayana (1994) experimentally reproduced the disease in broilers. They have described hydropericardium with accumulation of about 12 ml strawcoloured fluid thus giving the heart a litchi fruit appearance; the other changes were pale enlarged livers; oedematous and congested lungs; pale and tumified kidneys and an icteric carcass. Lesions of hydropericardium, spleenomegaly, necrotic foci on the myocardium, hypertrophy of bursa of Fabricius and poorly developed musculature have also been described in experimentally induced HHS cases (Cheema et al 1988, Muneer et al 1989, Abdul-Aziz and Al-Attar 1991, Gowda and Satyanarayana 1994). Cowen et al (1996) in their pathogenicity study of fowl adenoviruses observed that an isolate, LA/C serotype 4 from Chile produced classical lesions of HHS and they could recover the virus from rectal swabs of the infected birds and reported an increase in mortality after oral injection of 2day-old specific pathogen free (SPF) chicks with the pathogenic fowl adenovirus serotype 4 (FAV4) strain. Filtered homogenates of liver tissue from affected cases when inoculated onto 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 (Shane 1996). A pathogenicity study on six isolates from different field outbreaks of HHS in Ecuador (K1013 number 1 to 6) on embryos and chickens revealed high embryo and chick mortality (Mazaheri et al 1998). The disease was reproduced in day-old SPF chicks by the oral route of inoculation and subsequently the agent was re-isolated. The results showed that some strains of FAV4 caused HHS following oral or intramuscular infection.
EPIDEMIOLOGICAL STUDIES EXPERIMENTAL REPRODUCTION The syndrome was reproduced in broilers of up to 3 weeks of age by inoculation of a bacteria-free liver homogenate from infected birds (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Afzal et al 1991, Gowda and Satyanarayana 1994, Asrani et al 1997, Chandra et al 1997, Satyanarayana 1998). HHS was reproduced using liver homogenate extract and the pellet obtained thereof, from ultracentrifugation at 145 000 g for 90 minutes. The disease was transmitted to broilers by liver homogenate filtered through 0.22µ (and 0.1µ) membrane filters, indicating the virus was less than 100 nm in size and it is the aetiological agent of HHS (Afzal et al 1991). Mortality was observed after 3 to 5 days post infection. Generally, birds dying before 60 hours did not show any clinical sign (Kumar et al 1997). Mortality was 100 per cent when infected liver suspension was inoculated intramuscularly as compared to oral inoculation where mortality was
The disease was first observed in Pakistan in 1987 among broiler flocks of 3 to 5 weeks of age with flock mortality exceeding 50 per cent (Anjum et al 1989). Occasional outbreaks were also reported outside the 3- to 5-week age range (Ahmad et al 1989, Muneer et al 1989,- Akhtar and Cheema 1990), among breeder pullets (Akhtar 1992), and in layers (Ahmad et al 1988, Jaffery 1988, Asrani et al 1997). Asrani et al (1997) 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 was followed by an almost constant death rate for 5 to 7 days before declining. The average mortality in an affected flock was between 15 to 60 per cent (Asrani et al 1997). In Russia, HHS was recorded in chickens aged between 2 and 13 weeks with mortality ranging from 3.5 to 30 per cent
Hydropericardium hepatitis syndrome of broiler poultry: current status of research
in broilers and 2.6 to 15.29 per cent in layers (Borisov et al 1997). In a number of broiler poultry farms, HHS was recorded as an acute disease with hydropericarditis in 3- to 5-week-old broilers (Aliev et al 1997). Most of the workers have reported that different strains of broilers are equally susceptible in the field (Anjum et al 1989, Akhtar and Cheema 1990) and in experimental cases (Afzal and Hussain 1993). However, Khan et al (1995) have noticed the Hubbard broiler strain to be relatively more susceptible followed by Indian River and Lohmann respectively.
AETIOLOGY HHS was initially suspected to be caused by toxicity or by nutritional deficiency. The possible causative factors investigated were mycotoxins, toxic fat agent, polychlorinated biphenyl, sodium chloride, chlordane and phytotoxins, all of which were associated with hydropericardium (Jaffery 1988, Qureshi 1989). Attempts to reproduce the disease experimentally with feed samples from farms having natural outbreaks of HHS or feed containing 100 ppm mycotoxins or interaction of the two, however, failed (Anjum 1988, 1990). Anjum et al (1989) and Anjum (1990) successfully transmitted HHS to young broilers by subcutaneous injection of liver homogenates from affected birds. Subsequent studies confirmed the association of a virus with the condition and an adenovirus was identified as the aetiological agent (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Afzal et al 1991). The physical properties of the virus and demonstration of basophilic intranuclear inclusion bodies in hepatocytes, and characteristic hexagonal virions in EM preparations of liver homogenates (Khawaja et al 1988, Anjum et al 1989, Cheema et al 1989, Niazi et al 1989, Abdul-Aziz and Al-Attar 1991) were suggestive of involvement of a DNA virus. Later, fowl adenovirus belonging to serotype 4 named as PARC1 isolate was characterised and propagated in cell culture and in embryonated eggs. The isolation of an adenovirus in chicken embryo liver cells, reproduction of the disease with isolated virus (PARC1) and its use as a vaccine (Naeem et al 1995b) confirmed these observations. It is true that so far the correlation of FAV with specific disease has been difficult because fowl adenoviruses have been isolated from both healthy and diseased chickens. But there was growing evidence to show fowl adenoviruses as primary pathogens in the case of HHS. All isolates from field outbreaks of HHS have been serotyped as FAV4 strains and it was shown that some biotypes of FAV4 caused the HHS (Mazaheri et al 1998). Fowl adenovirus was isolated from liver samples of HHS cases and the virus was neutralised by antiserum against KR5 strain, which is a representative FAV serotype 4 strain of group 1 adenovirus. No other viral agent was isolated from liver samples by chicken kidney, chicken embryo, and MDCC-MSB1 cells (Abe et al 1998). Afzal et al (1990) in their study showed that the pellet after centrifugation of liver homogenate was infectious, but not the supernatant. Addition of an inclusion body hepatitisproducing adenovirus to the supernatant restored infectivity, suggesting the presence of an additional viral agent other than the primary adenovirus. In another study with liver
203
homogenate in Mexico in 1994, an RNA virus was implicated as an additional agent based on lack of inhibition by 5-bromodeoxyuridine (Shane and Jaffery 1997). This work pertains to treatments with liver homogenate, wherein the agent was not isolated. Moreover the physico-chemical properties of the HHS virus vary in comparison to other group 1 avian adenoviruses. Subsequent work clearly isolated the viral agent and characterised it as FAV4, and the disease was reproduced by the FAV4 isolates. Also there were reports showing the presence of infectious bursal disease (IBD) and chicken infectious anaemia (CIA) viruses in areas where HHS occurs (Shane and Jaffery 1997). IBD and CIA are known for their immunosuppressive effects and fowl adenoviruses require an impairment of the immune response to express their pathogenic potential in domestic fowl (Monreal 1996). FAV4 associated with HHS also has a predilection for lymphoid tissues, which resulted in immunosuppression (Naeem et al 1995a). Therefore the presence of IBD and CIA viruses can predispose for the HHS condition or HHS may predispose for other viral infections. The HHS was reproduced in SPF chicks using isolated and purified virus from field cases of HHS (Cowen et al 1996, Mazaheri et al 1998) thus proving the FAV4 associated with HHS is the sole agent responsible for the disease. Reference has been made to serotype 8 (European serotype 5) in the reproduction of the HHS syndrome, associated with IBH in Mexico (Shane and Jaffery 1997). The liver damage seen in HHS is also seen in IBH but the hydropericardium in chicks of 3 weeks of age, due to an adenovirus infection, was not a feature noticed until relatively recently. IBH is also seen in young broilers, but the high rate of mortality is characteristic of HHS (60 to 70 per cent in Pakistan, 10 to 30 per cent in Iraq, and 10 to 60 per cent in India (Abe et al 1998). Hydropericardium is a prominent symptom of HHS and has never been noted in many reports of IBH field and experimental cases (Gallina et al 1973). Fowl adenoviruses were isolated and characterised from several cases of HHS in Ecuador and Pakistan. Antibodies against serotypes 4 and 10 (European 11) neutralised these isolates. However, cross neutralisation tests and restriction enzyme (RE) analysis strengthened their grouping under serotype 4 (Mazaheri et al 1998). It was shown by immunological and molecular methods that FAV4 and FAV11 are very closely related (Enry et al 1995). The classification of HHS field isolates as FAV4 strains was confirmed by restriction enzyme analysis. Digestion with Bam H I, Dra I, and Eco R I demonstrated similar restriction fragment patterns between FAV4 strain and field isolates (Mazaheri et al 1998). In Pakistan and in several South American countries, inclusion body hepatitis (IBH–HPS) virus in many cases associated with hydropericardium was predominant and the condition was caused by FAV serotype 4 (Voss et al 1996). Voss,(1989) described the isolation of an adenovirus (K31/89) from field cases of HHS in Pakistan. Later, Voss et al (1996) identified the isolate as adenovirus serotype 4 and as a causative agent of HHS. Fowl adenovirus serotype 4 isolates recovered from different outbreaks of HHS in Pakistan have been found to differ in their pathological characteristics, although no significant differences were recorded regarding their biological properties or in their protein profiles (Rabbani and Naeem 1996).
204
K. Ganesh, R. Raghavan
Three Indian isolates of FAV recovered from IBH–HPS of poultry from different geographical locations were typed as serotype 4 (Jadhao et al 1997). 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, thereby avoiding further manipulation of the field isolate (Ganesh 1998). The presence of the virus in the samples collected was verified by performing double immunodiffusion and virus neutralisation tests with antiserum against all 1 to 12 FAV serotypes (Ganesh 1998). The syndrome was experimentally reproduced in broilers using both liver homogenate and purified virus prepared from the field samples (Ganesh 1998). The experimental reproduction of the disease in susceptible chicks accompanied with serological and electron microscopic evidences is suggestive that fowl adenovirus serotype 4 is solely responsible for causation of HHS (Ganesh 1998). Six isolates from different field outbreaks of HHS in Equador (K1013 NO 1–6), FAV reference strains [KR5 (FAV4), C2B (FAV11) and Pakistan isolate (K31)] were analysed by restriction enzymes. Identical restriction patterns were noticed between KR5, K1013 NO.2 and K31, while PstI digestion differentiated field isolates from reference strains. This study concluded that some specific strains of FAV4 caused the disease (Mazaheri et al 1998). The DNA isolated from either purified virus or from infected liver tissue was subjected to PCR using FAVH(L) and (R) primers (Ganesh 1998). Two oligonucleotides, FAVH(L) and FAVH(R), from the consensus region of hexon genes (corresponding to the nucleotide positions 722–747 and 1433–1452 respectively of the hexon gene of FAV10) were designed to amplify a 0.7 kb stretch variable region of HHS virus DNA. 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 (Ganesh 1998). PCR-amplified DNA was labelled with Digoxigenin (DIG-11-DUTP) and the labelled DNA was used as a probe for the detection of virusspecific sequences in the PCR-amplified products after Southern blotting. The DNA probe could detect the presence of the virus by dot blot hybridisation of the viral genome (Ganesh 1998). A 728 bp sequence of HHS viral DNA was obtained by sequencing the 0.7 kb PCR-amplified DNA (Ganesh 1998). A comparative study of the 0.7 kb fragment of the hexon gene of HHS with those of FAV10 and FAV1 indicated homology of 91.8 and 71.9 per cent respectively showing that the HHS virus is closer to FAV10 than to FAV1 (Ganesh 1998). In comparing corresponding amino acid sequences among the serotypes (FAV10, FAV1 and HHS), the variations were found as high as 28.8 and 38 per cent with FAV10 and FAV1 respectively (Ganesh 1998). Though nucleotide sequences showed a difference of only 8.2 per cent between HHS and FAV10 sequences, the amino acid sequence was found to be as high as 38 per cent. A marked difference existed in the variable regions of the hexon gene of the two serotypes (Ganesh 1998). The aetiological agent of HHS may be a variant of a non-pathogenic FAV type 4 that was reported earlier among poultry flocks (Ganesh 1998).
PHYSICO-CHEMICAL PROPERTIES OF THE VIRUS The sensitivity of HHS agent to pH 3 to 10 is variable. Unlike other adenoviruses, HHS virus in the liver preparations withstood heating at 60°C for 30 minutes and at 50°C for 1 hour. However heating at 60°C for 1 hour; 80°C for 10 minutes; and 100°C for 5 minutes inactivated the agent. Treatment with chloroform (5 per cent) or ether (10 per cent) which normally inactivates adenovirus eliminated infectivity of the HHS virus (Afzal et al 1991).
ELECTRON MICROSCOPY Purified samples of virus from liver homogenate from cases of HHS when examined by negative staining electron microscopy showed virus particles with the characteristic morphology of adenovirus (Cheema et al 1989, Ganesh 1998). Isometric, roughly spherical particles resembling adenovirus morphology, measuring 83–93 nm in diameter, were demonstrated inside the nucleus of hepatocytes from HHS outbreaks by transmission electron microscopy and in negative staining electron microscopy of virus pellets obtained from liver homogenates. The pellet when inoculated into experimental chicks reproduced the disease (Chandra et al 1997, Ganesh 1998).
DIAGNOSIS AND CONTROL Clinical diagnosis of the disease before the symptoms appear 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 pathognomonic for diagnosis (Akhtar 1994). Certain serological tests, such as indirect HA test (Rahman et al 1989, Hassan et al 1993, Maskoor et al 1994a) ELISA (Hassan et al 1993), AGPT (Hassan et al 1993, Kumar et al 1997) and counter immunoelectrophoresis (Kumar et al 1997), using filtrate from infected liver homogenate as a crude antigen have been done. These studies indicate that the specificity of the currently available serological tests for diagnosis needs improvement. Dot-immunobinding assay with virus purified from infected liver homogenate was used for the study of immunosuppressive potential and pathogenicity of HHS (Naeem et al 1995a). The electron microscopic demonstration of the virus particles in hepatocytes (Chandra et al 1997) and virus neutralisation test (Rabbani and Naeem 1996, Jadhao et al 1997) using specific antiserum have also been used in HHS diagnosis. Polymerase chain reaction and nucleic acid hybridisation techniques were developed for the diagnosis of HHS (Ganesh 1998). Flocks were protected with an autogenous formalin inactivated vaccine prepared from infected liver homogenate (Cheema et al 1989, Chishti et al 1989, Afzal and Ahmad 1990, Ahmad et al 1990, Anjum 1990, Mashkoor et al
Hydropericardium hepatitis syndrome of broiler poultry: current status of research
1994b, Shane 1996, Kumar et al 1997). Experimentally, a cell culture vaccine was developed by growing the virus in chicken embryo liver cells (Naeem et al 1995b) and a killed oil emulsified vaccine was prepared from Indian isolates of HHS propagated in chick-embryo liver cell cultures (Kataria et al 1997).
REFERENCES ABDUL-AZIZ, T.A. & AL-ATTAR, M.A. (1991) New Syndrome in Iraqi chicks. Veterinary Record 129, 272 ABDUL-AZIZ, T.A. & HASSAN, S.Y. (1995) Hydropericardium syndrome in broiler chickens: its contagious nature and pathology. Research in Veterinary Science 59, 219–221 ABE, T., NAKAMURA, K., TOJO, H., MASE, M., SHIBAHARA, T., YAMAGUCHI, S. & YUASA, N. (1998) Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks. Avian Diseases 42, 606–612 AFZAL, M. & AHMAD, I. (1990). Efficacy of an inactivated vaccine against hydropericardium syndrome in broilers. Veterinary Record 126, 59–60 AFZAL, M., MUNEER, R., STEIN, JR, G. & COWEN, B.S. (1990) Aetiology and control of hydropericardium syndrome in broilers of Pakistan. Proceedings of the third International Veterinary Congress, Islamabad, Pakistan. pp. 218–228 AFZAL, M. & HUSSAIN, A. (1993). Age susceptibility of different commercial broiler strains to Angara disease (Hydropericardium syndrome). Pakistan Journal of Agricultural Research 14, 387–392 AFZAL, M., MUNEER, R. & STEIN, G. (1991) Studies on the etiology of hydropericardium syndrome in broilers. Veterinary Record 128, 591–593 AHMAD, I., AFZAL, M., MALIK, M.I., HUSSAIN, Z. & HANIF, W. (1988) Studies on the disease pattern and etiology of hydropericardium syndrome (Angara Disease) in broiler chickens in Pakistan. Pakistan Journal of Agricultural Research 10, 195–199 AHMAD, I., AFZAL, M., MALIK, M.I., HUSSAIN, Z. & HANIF, W. (1989) Disease patterns and etiology of hydropericardium syndrome (Angara disease) in broiler chickens in Pakistan. Pakistan Journal of Agricultural Research 10, 195–199 AHMAD, I., MALIK, M.I., IQBAL, K., AHMED, K. & NAZ, S. (1990) Efficacy of formalinised liver organ vaccine against Angara disease in broilers. Veterinarski Arhiv 60, 131–138 AKHTAR, S. (1992) Studies on the rate of lateral spread of hydropericardium syndrome agent(s). In: Etiology, pathogenesis, and control of hydropericardium syndrome in poultry. Board on Science and Technology for International Development (BOSTID), Washington, USA AKHTAR, S. (1994) Hydropericardium syndrome in broiler chickens in Pakistan. World’s Poultry Science Journal 50, 177–182 AKHTAR, S. (1995). Lateral spread of the etiological agent(s) of hydropericardium syndrome in broiler chickens. Veterinary Record 136, 118–120 AKHTAR, S. & CHEEMA, A.H. (1990) Some epidemiological characteristics of hydropericardium syndrome outbreaks among commercial broiler flocks in Karachi. Proceedings of the third International Veterinary Congress, Islamabad, Pakistan. Pp. 228–234 AKHTAR, S., ZAHID, S. & KHAN, M.I. (1992) Risk factors associated with hydropericardium syndrome in broiler flocks. Veterinary Record 131, 481–482 ALIEV, A.S., DJAVADOV, E.D. & NIKITINA, N.V. (1997) Etiology of hydropericarditis in broiler chicks. Proceedings of the tenth international congress of the WVPA 18–22 Aug, Budapest, Hungary, p. 257 ANJUM, A.D. (1988) Study of pathogenicity of the hydropericardium syndrome agent. Proceedings of national seminar on hydropericardium syndrome in chickens in Pakistan, Rawalpindi, 4 Jul, pp. 111–115 ANJUM, A.D. (1990) Experimental transmission of hydropericardium syndrome and protection against it in commercial broiler chickens. Avian Pathology 19, 655–660 ANJUM, A.D., SABRI, M.A. & IQBAL, Z. (1989). Hydropericarditis syndrome in broiler chickens in Pakistan. Veterinary Record 124, 247–248 ANONYMOUS (1996). Report of the study group on poultry diseases appointed by technology mission on dairy development on the advice of National Advisory Committee on Control of Animal Diseases, New Delhi, India ASRANI, R.K., GUPTA, V.K., SHARMA, S.K., SINGH, S.P. & KATOCH, R.C. (1997) Hydropericardium hepatopathy syndrome in Asian poultry. Veterinary Record 141, 271–273 BORISOV, V.V., BORISOV, A.V. & GUSEV, A.A. (1997) Hydropericardium syndrome in chickens in Russia. Proceedings of the tenth international congress of the WVPA, 18–22 Aug, Budapest, Hungary, p. 258 CHANDRA, R., SHUKLA, S.K., KUMAR, M. & GARG, S.K. (1997) Electron microscopic demonstration of adenovirus in the hepatocytes of birds experimentally infected with hydropericardium syndrome. Veterinary Record 138, 70–71 CHEEMA, A.H., AFZAL, M. & AHMAD, J. (1988) Studies on the causation of hydropericardium syndrome in Pakistan. Proceedings of national seminar on hydropericardium syndrome in chickens in Pakistan. Rawalpindi, 4 Jul, pp. 41–48. CHEEMA, A.H., AHMAD, J. & AFZAL, M. (1989) An adenovirus infection of poultry in Pakistan. Rev. Sci. Tech. Off. Int. Epi. 8, 789–795
205
CHISHTI, M.A., AFZAL, M. & CHEEMA, A.H. (1989) Preliminary studies on the development of hydropericardium syndrome vaccine of poultry. Off. Int. Epi. Report 8, 797–801 COWEN, B.S., LU, H., WEINSTOCK, D. & CASTRO, A.E. (1996) Pathogenicity studies of fowl adenoviruses isolated in several regions of the world. International symposium on adenovirus infection in poultry, Rauischholzhausen, 24–27 Jun, Germany, pp. 79–88 ERNY, K.M., PALLISTER, J. & SHEPPARD, M. (1995). Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10. Arch. Virol. 140, 491–501 GANESH, K. (1998) Molecular studies on hexon gene fragment of fowl adenovirus associated with hydropericardium hepatitis syndrome. Ph.D. thesis submitted to University of Agricultural Sciences, Bangalore, India GALLINA, A.M., WINTERFEILD, R.W. & FADLY, A.M. (1973) Adenovirus infection and disease. II. Histopathology of natural and experimental disease. Avian Diseases 343–353 GOWDA, R.N.S. & SATYANARAYANA, M.L. (1994) Hydropericardium syndrome in poultry. Indian Journal Veterinary Pathology 18, 159–161 HASAN, S.A. (1989) Pakistan is mystified by hydropericardium syndrome. PoultryMisset 5, 35–37 HASSAN, N.U., AFZAL, M., HAMEED, A. & KHAN, A.R. (1993) Immune response to inactivated hydropericardium syndrome vaccine in broilers. Pakistan Veterinary Journal 14, 5–10 JADHAO, S.J., KATARIA, J.M., VERMA, K.C. & SAH, R.L. (1997). Serotyping of Indian isolates of fowl adenovirus recovered from IBH-HPS (Litchi disease) in chickens. Indian Journal Comparative Microbiology Immunology and Infectious Diseases 18, 33–37 JAFFERY, M.S. (1988) A treatise on Angara disease (hydropericardium-pulmonary oedema hepatonephritis syndrome). Pakistan Veterinary Medical Association, p. 34 KATARIA, J.M., VERMA, K.C., JADHAO, S.J., DEEPAK, J.N. & SAH, R.L. (1997). Efficacy of inactivated oil emulsified vaccine against IBH-HPS in chickens prepared from cell culture propagated fowl adenovirus. Indian Journal Comparative Microbiology Immunology and Infectious Diseases 18, 38–42 KHAN, S.A., ZAIDI, F.H., CHAUDHRY, R.A., ASHRAF, S.K., MIAN, M.S. & QURESHI, I.R. (1995). Comparative study on pathology of haemic system of three broiler chicken strains suffering from experimental hydropericardium syndrome. Asian-Australasian Journal of Animal Sciences B(4), 325–328 KHAWAJA, D.A., AHMED, S., RAUF, A.M., ZULFIGAR, M., MAHMOOD, S.M. & HASSAN, M. (1988) Isolation of an adenovirus from hydropericardium syndrome in broiler chicks. Pakistan Journal Veterinary Research 1, 2–17 KUMAR, R., CHANDRA, R., SHUKLA, S.K., AGARWAL, D.K. & KUMAR, M. (1997) Hydropericardium syndrome in India: A preliminary study on the causative agent and control of disease by inactivated autogenous vaccine. Tropical Animal Health and Production 29, 158–164 MASHKOOR, S., HAMEED, A., AHMAD, K. & QURESHI, M.S. (1994a) Improved angara disease vaccine for broiler chicks (abstract). Veterinarski Arhiv 64, 27–33 MASHKOOR, S., HAMEED, A., AHMAD, K. & QURESHI, M.S. (1994b) Immune response of Angara disease (hydropericardium syndrome) vaccines in broilers (abstract). Veterinarski Arhiv 64, 103–107 MAZAHERI, A., PRUSAS, C., VO, M. & HESS, M. (1998) Some strains of serotype 4 fowl adenoviruses cause Inclusion body hepatitis and hydropericardium syndrome in chickens. Avian Pathology 27, 269–276 MCFERRAN, J.B. (1991) Adenovirus infections. In: Diseases of poultry, Eds. BW Calnek, HJ Barnes, CW Beard, WM Reid, HW Yoder JR. 10th edn. Iowa State University Press, Ames England. pp 607–620 MONREAL, G. (1996) History and development of research about avian adenoviruses. International symposium on adenovirus infection in poultry, Rauischholzhausen, 24–27 Jun, Germany, pp. 4–9 MUNEER, M.A., AJMAL, M., ARSHAD, M., AHMAD, M.D., CHAUDHRY, Z.I. & KHAN, T. M. (1989) Preliminary studies of hydropericardium syndrome in broilers in Pakistan. Zootechnique International 5, 46–48 NAEEM, K., NIAZI, T., MALIK, S.A. & CHEEMA, A.H. (1995a) Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers. Avian Diseases 39, 723–728 NAEEM, K., RABBANI, M., HUSSAIN, M. & CHEEMA, A.H. (1995b) Development of cell culture vaccine against hydropericardium syndrome in poultry. Pakistan Veterinary Journal 15, 150–15 NIAZI, A.K., KHAN, M.Z. & SIDDIQUE, M. (1989) Hematological studies on naturally occurring hydropericardium syndrome in broiler chicks. Veterinary Record 124, 400 OBEROI, M.S., SINGH, A. & SINGH, B. (1996) Isolation of avian adenovirus from outbreaks of IBH-HPS in poultry. Indian Journal Virology 12, 123–124 QURESHI, A.A. (1989) Hydropericardium and ascitis. Poultry International 28, 44–48 RABBANI, M. & NAEEM, K. (1996) In vitro and in vivo evaluation of avian adenovirus isolates from outbreaks of hydropericardium syndrome. Proceedings of the international symposium on adenovirus and reovirus infections in poultry. Rauischholzhausen, 24–27 Jun, Germany, pp. 26–31 RAHMAN, S.U., ASHFAQUE, M., ANJUM, A.D. & SINFHU, T.A. (1989) Indirect hemagglutination for detecting Angara disease (Hydropericardium) agent antibody. In: Proceedings of international conference and trade show on poultry production, 27 Feb-March, pp. 73–74
206
K. Ganesh, R. Raghavan
SATYANARAYANA, M.L. (1998) Etiopathology and pathogenesis of hydropericardium syndrome in broilers. Ph.D. thesis submitted to University of Agricultural Sciences, Bangalore, India SHAFIQUE, M. & SHAKOORI, A.R. (1994) Transmission of hydropericardium syndrome in poultry. Pakistan Journal of Zoology 26, 145–148 SHANE, S.M. (1996) Hydropericardium hepatitis syndrome, the current world situation. Zootechnique International 29, 20–27 SHANE, S.M. & JAFFERY, M.S. (1997) Hydropericardium-hepatitis syndrome (Angara disease). In: Diseases of poultry, Eds. BW CALNEK, HJ BARNES, CW BEARD, WM REID, HW YODER JR. 10th edition, Iowa State University Press, Ames, England. pp. 1019–1022 SHUKLA, S.K., CHANDRA, R., KUMAR, M. & DIXIT, V.P. (1997) Hydropericardium syndrome in tarai of Uttar Pradesh: a report. Indian Journal Animal Sciences 67, 766–767
VOSS, M. (1989) Evaluation of field serology and isolation and identification of an agent from Pakistan field cases of hydropericardium syndrome. Lohman symposium on the hydropericardium syndrome, Cuxhaven, Germany VOSS, M., VIELITZ, E., HESS, M., PRUSAS, C.H. & MAZAHERRI, A. (1996) Aetiological aspects of Hepatitis and HPS caused by pathogenic adenoviruses in different countries. International symposium on adenovirus and reovirus infections in poultry, Rauischholzhausen, Germany, 24–27 Jun, pp. 75–78
Accepted November 25, 1999