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Outbreaks of Inclusion Body Hepatitis (IBH) in Chickens: Pathological Studies

and Isolation of Fowl Adenovirus

Article  in  Advances in Animal and Veterinary Sciences · January 2013

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Advances in Animal and Veterinary Sciences. 1 (3S): 21 – 24
Special Issue–3 (Epidemiology and Animal Disease Investigations)
http://www.nexusacademicpublishers.com/journal/4

Short Communication
Outbreaks of Inclusion Body Hepatitis (IBH) in Chickens; Pathological
Studies and Isolation of Fowl Adenovirus
Vipan Kumar1, Rajesh Kumar2*, Rajesh Chandra3, Prakash Bhatt4, Kuldeep Dhama5
1
Department of Veterinary Microbiology, Khalsa College of Veterinary and Animal Sciences, Amritsar, Punjab; 2Department of
Veterinary Microbiology, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar-
263145, Uttarakhand; College of Veterinary & Animal Sciences, Central Agricultural University, Selseih, Aizawal; 4Veterinary Clinics,
College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar, Uttarakhand; 5Division of
Pathology, Indian Veterinary Research Institute, Izatnagar (UP), India
*Corresponding author: rajeshvet@rediffmail.com

ARTICLE HISTORY ABSTRACT

Received: 2013–09–26 Fowl adenoviruses (FAdV) are known to cause many diseases in birds including poultry and
Revised: 2013–12–31 wild birds including inclusion body hepatitis (IBH). The IBH is endemic in many states of
Accepted: 2013–12–31 India. The disease outbreaks of IBH in poultry farms have been investigated through clinical
and histopathological examination of affected birds, isolation via chicken embryo liver cell
(CEL) and detection of virus using immunofluorescent test. Clinically, the birds showed
Key Words: Inclusion body depression and greenish diarrhea. Necropsy showed jaundice, enlargement of kidneys,
hepatitis, Fowl adenovirus, greyish white area of necrosis on liver and atrophy of bursa of Fabricius. Histopathology
Chicken, Pathology, revealed large intracellular inclusion bodies in hepatocyte, degeneration and congestion of
Fluorescent antibody test sinusoids and degeneration of real tubules. Virus infected CEL cells revealed intranuclear
fluorescence in indirect fluorescent antibody test (IFAT) using FAdV specific antisera.
Present study demonstrated the involvement of fowl adenovirus in inclusion hepatitis
outbreaks and adds to the epidemiological data available in India.
All copyrights reserved to Nexus® academic publishers
ARTICLE CITATION: Kumar V, Kumar R, Chandra R, Bhatt P and Dhama K (2013). Outbreaks of inclusion body hepatitis (IBH) in
chickens; pathological studies and isolation of fowl adenovirus. Adv. Anim. Vet. Sci. 1 (3S): 21 – 24.

Fowl adenoviruses are known to cause many diseases in disease occurs in hot and humid weather (Shah et al., 2011;
poultry birds like Inclusion body hepatitis (IBH), Shahzad et al., 2011). The disease, inclusion body hepatitis is
hydropericardium syndrome (HPS), and respiratory characterized by sudden onset of mortality, severe anemia
infections in chickens and quail bronchitis. Among these and necrotic hepatitis with basophilic or eosinophillic
conditions, Inclusion body hepatitis has a worldwide intranuclear inclusion bodies in hepatocytes (Kim et al.,
distribution, and there are indications that its incidence is 2008; Shahzad et al., 2011; Dar et al., 2012). Present study
increasing in many poultry industries (Mase, 2012; Kataria deals with investigation of outbreaks of Inclusion body
et al., 2013). All eleven serotypes of fowl adenovirus (FAdV) hepatitis, histopathology of naturally infected birds and
have been isolated from natural infections of poultry birds isolation of causative agent and its identification by IFAT.
(Kim et al., 2008; Choi et al., 2012; Asthana et al., 2013) and Disease outbreaks of Inclusion body hepatitis (IBH)
wild birds like wild black kites (Kumar et al., 2010) and were suspected in broiler poultry birds in two private
pigeons (Schrenzel et al. 2005; Catroxo et al., 2011). The poultry farms of Bilaspur region (28.80N/ 79.00E) of Rampur
disease causes high morbidity among broiler birds leading to district, Uttar Pradesh and Kashipur (29.220N/ 78.950E) of
production losses although average mortality is low (5– Uttarakhand on the basis of clinical signs and post mortem
10%) but 30% mortality has been reported from Australia examination carried out in dead and sacrificed birds. The
(McFerran and Smyth, 2000). Recently, some of FAdVs flock size of Bilaspur farm was 2200 and mortality rate
have been implicated in immunosuppression (Hussain et al., recorded was 10%. In Kashipur farm, flock size was 4000
2012). The disease was first reported from USA as a and mortality was 3%. Morbidity in both farms reached
necrotizing hepatitis in 7–weeks–old chickens (Helmboldt 80% and average age of affected birds was between 6–8
and Frazier, 1963). Since then, the disease has been reported weeks. Clinical samples of liver, kidneys and bursa of
from all continents. In India, many outbreaks of this disease Fabricius were collected from dead and necropsied birds for
have been investigated by earlier workers (Garewal et al., histopathological and virus isolation studies. The liver
1981; Sandhu et al., 1994; Deshmukh et al., 2000; Kumar et samples were collected in 50% glycerol saline for virus
al., 2003a; Kataria et al., 2013). isolation. Tissue samples of liver, kidneys and bursa of
In domestic poultry, the disease usually affects birds at the Fabricius were collected in 10% formal saline for
age of 2 to 6 weeks (Asthana et al., 2013). The onset of histopathological examination.

21
Kumar et al (2013). IBH in Chickens; Pathology and Virus Isolation
ISSN: 2307–8316 (Online); ISSN: 2309–3331 (Print)
Advances in Animal and Veterinary Sciences. 1 (3S): 21 – 24
Special Issue–3 (Epidemiology and Animal Disease Investigations)
http://www.nexusacademicpublishers.com/journal/4

The tissues (liver, kidneys and bursa of Fabricius) were monolayer was kept as negative control and processed in
processed for histopathological examination as per standard similar way.
protocol, fixed in 10% formal saline, washed in running tap In the present disease investigation, naturally affected
water overnight and then dehydrated for one hour in birds did not show any clinical signs at initial stages, except
different concentrations of ethanol, 50%, 60%, 70%, 80%, greenish watery diarrhea. However at terminal stages birds
90% and absolute alcohol for dehydration of tissues in same were dull depressed and reluctant to move and showed
order. Then the tissues were cleared in xylene and typical changes in posture. Kumar et al. (2009) also
embedded in paraffin wax. Sections of 4–5 µ thickness were reported varying degree of dullness, depression and diarrhea
cut and stained with Haemotoxylin and Eosin (H & E) leading to prostration and death. Grgic et al. (2006) also had
staining procedure as described by Kumar et al. (2003a). similar observations. Absence of clinical signs and sudden
Twenty percent (w/v) liver homogenates of both mortality may be attributed to relatively short course of
isolates were prepared in phosphate buffered saline (PBS) disease and acute damage to vital organs like liver and
from the clinical samples collected from both the outbreaks. kidney. In this investigation, the mortality rate was about 3–
Virus isolation was done on 14 day–old chicken embryo liver 10%. Earlier workers have also reported low mortality rates
(CEL) cell culture (Kumar and Chandra, 2009). A total of in adult broiler birds in IBH infections (McFerran and
six passages were done for isolation of the virus and the Smyth, 2000; Kumar et al., 2003).
virus was identified by observing characteristic cytopathic The predominant post mortem lesions included
effects in CEL cell cultures. enlarged livers with friable texture and pin point or white
The presence of fowl adenovirus (FAdV) was detected necrotic foci (Figure 1). In some cases, petechial and
by indirect immunofluorescent antibody technique (Kumar ecchymotic haemorrhages were also present. Kidneys were
et al., 2003b). Briefly, CEL cells were grown on cover slips congested and contained haemorrhagic patches on the
and infected with both the virus isolates obtained from the surface (Figure 2). Haemorrhages were also evident on thigh
two disease outbreaks. After 48 hours of infection cover muscles. Lungs were congested and odematous. Previously
slips were flooded with chilled acetone for 30 min for fixing many investigators have noticed these findings (Sandhu et
of cells of monolayers. Then the cells were rehydrated with al., 1994; Dar et al., 2012). In some cases, livers were also
PBS (pH 7.2). Single washing of infected cells was done covered with a fibrinous mass of exudates as reported by
with wash buffer (Triton X–100 0.01% v/v in PBS). After 5 other workers (Sandhu et al., 1994; Nayak et al., 1990; Mitra
min. of incubation, the detergent was removed and blocking et al., 1996).
buffer (10% foetal calf serum in wash buffer) was added to The histopathological examination of liver revealed
block unreactive sites on monolayers. Then, after three necrosis of hepatocytes, vacuolar degeneration and
washings with wash buffer, FAdV specific antiserum was infiltration of mono nuclear cells. Many hepatocytes
added and incubated for 30 min at 37˚C again after three revealed large basophilic intranuclear inclusion bodies,
washings with PBS, rabbit anti–chicken FITC conjugate which were round and compact and occupied almost entire
(Sigma) was added to detect the binding of anti adenoviral nucleus. Large ‘bird eye’ basophilic intranuclear inclusion
antibodies with adenoviral antigens present in infected cell bodies and sinusoidal congestion were also observed in this
cultures. The coverslips were washed thrice as mentioned study (Figure 3).
above with wash buffer, air–dried and mounted in glycerol.
The stained coverslips were examined under U.V. light in
fluorescent microscope. The un–infected CEL cell culture

Figure 1: Photograph of liver from dead bird showing Figure 2: Photograph of kidney from dead birds showing
necrosis and pin point haemorrhages enlargement and congestion with focal area of necrosis

22
Kumar et al (2013). IBH in Chickens; Pathology and Virus Isolation
ISSN: 2307–8316 (Online); ISSN: 2309–3331 (Print)
Advances in Animal and Veterinary Sciences. 1 (3S): 21 – 24
Special Issue–3 (Epidemiology and Animal Disease Investigations)
http://www.nexusacademicpublishers.com/journal/4

Figure 3: Photomicrograph of liver showing basophilic Figure 4: Photomicrograph of kidneys showing

intranuclear inclusion bodies in hepatocytes with degeneration and necrosis of tubular epithelium with
infiltration of mononuclear cells. (H&E stain x 400).area intranuclear inclusion bodies, focal haemorrhages and
of necrosis mononuclear cell infiltration. (H &E x 400)

Figure 5: Photomicrograph of bursa of Fabricius showing Figure 6: Photograph of infected CEL cells showing
degeneration and necrosis of bursal follicles, focal areas of intranuclear fluorescence. (X 400)
haemorrhages and mononuclear cell infiltration. (H&E x
400)

Theses findings are in accordance with those observed by Fabricius. Increased vascular permeability may be
earlier workers (Kaur et al., 2003; Kim et al., 2008). Kidneys attributed to deposition of serofibrinous exudates in the
showed congestion and haemorrhages, degeneration of renal parenchyma of liver and kidney.
epithelium and infiltration of mononuclear cells. Large
basophilic intranuclear inclusions were occasionally On CEL cell cultures, the cytopathic effects (CPE)
observed (Figure 4). These findings were also reported by were characterized by rounding and degeneration of cells
many earlier workers (Singh et al., 1996; Kaur et al., 2003). from first passage where microplaques were evident after 72
Bursa of Fabricius also had varying degree of degenerative hours. By second passage, the CPE appeared within 24
changes and reduction in number of follicles (Figure 5). hours. After 72 hours, almost 80% of the cell layer detached
These findings are in agreement with earlier findings of from its surface. Successive passaging showed more distinct
Nayak et al., 1990 and Sandhu et al., 1994. However, and stable CPEs. Chicken embryo liver cell culture and
Inclusion bodies were not demonstrated in the bursal chicken kidney cell culture are reported to support the
tissues. The lesions in different organs were suggestive of growth of adenoviruses (Kumar and Chandra, 2004;
acute extensive damage in the liver, kidneys and bursa of Nakamura et al., 2002; Kaur et al., 2003). The cytopathic

23
Kumar et al (2013). IBH in Chickens; Pathology and Virus Isolation
ISSN: 2307–8316 (Online); ISSN: 2309–3331 (Print)
 Advances in Animal and Veterinary Sciences. 1 (3S): 21 – 24
Special Issue–3 (Epidemiology and Animal Disease Investigations)
http://www.nexusacademicpublishers.com/journal/4

effects during isolation of the virus in the present study are Kataria JM, Dhama K, Nagarajan S, Chaktaborty S, Kaushal A and Deb R
(2013). Fowl adenoviruses causing hydropericardium syndrome in
in accordance with those reported by earlier workers (Kaur poultry. Adv. Anim. Vet. Sci., 1(4S): 5–13.
et al., 2003; Kumar et al., 2009). Micro and macro plaque Kaur G, Maiti NK and Oberoi MS (2003). Biological and immunological
formation is suggestive of cytolytic properties of virus. characterization of fowl adenovirus–4 isolates from inclusion body
For confirmation of adenoviruses, different diagnostic hepatitis–hydropericardium syndrome. Indian J. Anim. Sci. 73(1): 51–52.
Kim JN, Byun SH, Kim MJ, Kim JJ, Sung HW and Mo IP (2008). Outbreak of
tests are used including molecular tools, and among these, hydropericardium syndrome and molecular characterization of Korean
immunofluorescence detection of viral antigens during virus fowl adenoviral isolates. Avian Dis. 52(3):526–30.
isolation has high diagnostic value because of the high Kumar R and Chandra R (2009). Isolation and characterization of fowl
degree of accuracy of detection of group specific antigens by adenovirus associated with hydropericardium syndrome. Indian Vet. J.
86(10): 1084–1086.
using specific antibodies, and is in use since many years Kumar R, Kumar V, Asthana M, Shukla SK and Chandra R (2010). Isolation
(Leland and Ginocchio, 2007). In the present study, the and identification of Fowl adenovirus from Wild Black Kites (Milvus
presence of viral antigens in CEL cell culture was migrans). J. Wild Dis. 46(1): 272–276.
demonstrated by indirect immunofluorescence antibody Kumar R, Shukla SK, Chandra R and Agarwal DK (2003a). Outbreaks of
inclusion body hepatitis in domestic fowl. Indian J. Anim. Sci. 73(5):
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Kumar, R. and Chandra, R. (2004). Studies on structural and immunogenic
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Kumar et al (2013). IBH in Chickens; Pathology and Virus Isolation
ISSN: 2307–8316 (Online); ISSN: 2309–3331 (Print)

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