D9087.PDF

Rev. sci. tech. Off. int. Epiz., 1996, 15 (3), 1053-1060
Epidemiology of inclusion body hepatitis
in poultry in northern India
from 1990 to 1994
A. SINGH *, M.S. OBEROI **, S.K. JAND ** and B. SINGH ***
Summary: The epidemiology of inclusion body hepatitis (IBH) was studied in
poultry in northern India, from April 1990 to March 1994, to evaluate the
various factors responsible for causing and determining the severity of the
disease. Broiler chicks and Japanese quail (Coturnix coturnix japonica) were
the species examined. The factor observed to be most commonly associated
with IBH was the presence of aflatoxins in the feed at higher than permissible
levels, i.e. 20 parts per billion. Avian adenovirus-1 was isolated from the livers
of affected birds. In the final year of the study, a number of outbreaks of IBH
caused heavy mortalities among three to five-week-old broiler chicks, which
displayed typical IBH lesions in addition to hydropericardium.
KEYWORDS: Aflatoxin - Avian adenoviruses - Epidemiology -
Hydropericardium - Inclusion body hepatitis - India - Poultry diseases -
Viral diseases.
INTRODUCTION
Avian adenoviruses have been shown to cause various diseases in different species
of birds. In India the diseases caused by avian adenoviruses, particularly inclusion
body hepatitis (IBH), threaten the viability of the poultry industry. This is particularly
important in view of the fact that poultry farming has become an important livestock
industry in India. India is ranked fifth in the world for egg production, and the
production of broiler chickens has increased from four million in 1971 to 275 million
in 1994. Thus, from April 1990 to March 1994, studies were conducted in northern
India on the epidemiology of IBH, to evaluate the roles of various field conditions in
the causation and severity of the disease, in order to develop and recommend various
preventive measures.
* Department of Veterinary Public Health and Epidemiology, College of Veterinary Science,
Punjab Agricultural University, Ludhiana-141 004, India.
** Veterinary Bacteriology and Virology, College of Veterinary Science, Punjab Agricultural
University, Ludhiana-141 004, India.
*** Veterinary Pathology, College of Veterinary Science, Punjab Agricultural University,
Ludhiana-141 004, India.

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MATERIALS AND METHODS
Dead birds which had been brought for diagnosis were given detailed post-mortem
examinations. Liver tissue was collected and processed for histopathological
examination to confirm cases of IBH. Poultry farms with outbreaks of IBH were
examined and a detailed record was made of management practices (floor space and
feeding and watering conditions). Meteorological data for the region during the study
period, i.e. the atmospheric temperature and relative humidity, were obtained from the
Department of Agricultural Meteorology, Punjab Agricultural University, Ludhiana,
India. Feed samples were collected from affected poultry farms and screened for the
presence of aflatoxins (2, 17).
When an outbreak of IBH was confirmed by histopathology, attempts were made to
isolate the virus from the samples of liver tissue by using chicken-embryo liver (CEL)
cell culture. The CEL cell culture was prepared from twelve-to-thirteen-day-old
chicken embryos as per McFerran et al. (11). The trypsenised cell suspension of
1 X 106 cells/ml was seeded in Hank's balanced salt solution (HBSS) containing 10%
calf serum. The maintenance medium consisted of 2% calf serum. The infected CEL
cell cultures were observed for six to seven days for any evidence of cytopathic effect
(CPE). At the end of the incubation period, the cells were frozen and thawed three
times, then harvested, along with the culture fluid, for further passage. Each sample
was subjected to three blind passages, after which the culture fluid was tested by
counterimmunoelectrophoresis (CIE) (14), dot enzyme-linked immunosorbent assay
(ELISA) and double antibody sandwich ELISA (16) for the presence of avian
adenovirus (AAV) group antigens. Serotyping was performed on ten isolates, using the
microneutralisation test in a 96-well tissue culture plate as described by Grimes et al.
(8). All the isolates were tested against standard AAV-1 rabbit serum.
RESULTS AND DISCUSSION
During the study period, forty-seven outbreaks of IBH were confirmed by the
presence of intranuclear inclusion bodies in hepatocytes. Table I summarises the
epidemiological factors recorded for these outbreaks. IBH outbreaks occurred among
broiler chicks of two to seven weeks of age. The largest number of outbreaks (40/47)
occurred in the three-to-five-week age group. Earlier outbreaks of IBH have been
recorded among broiler chicks of the same age group by a number of researchers (3,
6, 11, 15). Dhillon et al. demonstrated that serotypes of AAV were pathogenic for
three-week-old specific-pathogen-free (SPF) chicks, which were inoculated
intratracheally (4).
The majority of IBH outbreaks within the study (26/47) were associated with
mycotoxicosis (aflatoxicosis). The presence of anatoxin in the feed could be
established in twenty outbreaks, with levels ranging from 200 parts per billion (ppb)
to 1,000 ppb, both of which are well above the permissible level of 20 ppb. Other
diseases diagnosed in these chicks, by necropsy and histopathology, were
hydropericardium, infectious bursal disease, chronic respiratory disease, airsacculitis,
rickets, coryza, coccidiosis, encephalomalacia and vitamin A deficiency. These
diseases appeared either alone or (in seventeen outbreaks) in combination with
IBH.

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TABLE I
Year
No. of
confirmed
outbreaks Flock size Age
of birds
(weeks)
Space/bird
(sq. ft.) Mortality (% age) Anatoxins in feed * Virus
isolated **
1990-1991 10 500-13,000 2-5
0.3-0.8 3.3-20.0 5/7 10/10
1991-1992 10 500-10,000 2-7 0.4-2.5 0.43-17.5 2/3 8/10
1992-1993 8 400-3,000 2-6
0.5-1.8 0.26-12.5 4/7 5/7
1993-1994 19 850-40,000 3-5 0.4-1.6 0.75-21.0 9/10 8/19
* Nominator: number of outbreaks in which feed samples tested positive for anatoxins with a range of 200
parts per billion (ppb) to 1,000 ppb. Denominator: number of outbreaks from which feed samples were
processed
** Nominator: number of outbreaks which yielded avian adenovirus (AAV). Denominator: number of
outbreaks from which isolation of AAV was attempted
Mortality in the IBH-affected flocks varied from 0.26% to 21% with an average of
5.77 ± 0.86. The average mortality rate in an outbreak of IBH, where there was no
other concurrent disease, was 3.34 ± 1.35. However, the average mortality rate of an
outbreak of IBH associated with aflatoxicosis was 6.58 ± 1.37. When IBH was
associated with a disease other than aflatoxicosis, the mortality rate was 4.51 ± 1.78.
In IBH outbreaks where aflatoxicosis was found in conjunction with another disease,
the mortality rate was 8.08 ± 2.26. It is evident that aflatoxicosis and other diseases
play a major role in increasing the mortality rate of an IBH outbreak.
The observations of this study have been substantiated by other experimental studies
of broiler chicks, which also suggest that the presence of the aflatoxin B1 in feedstuffs
is a significant risk factor for an outbreak of IBH, caused by AAV-1 (20). Moreover,
Jand et al. have also recently found a close association between aflatoxins in feed and
the occurrence of IBH (9). Because aflatoxins not only damage the liver but also
suppress the immune system, chicks are more likely to suffer from other concurrent
diseases, resulting in a higher mortality (5).
Table II shows the monthly incidence of IBH outbreaks alongside the mean
temperature and relative humidity for each month. Most outbreaks (27/47) were
recorded during the months of October, November and December. There is a clear
correlation between the high number of outbreaks during these months and the high
temperatures and relative humidities of the preceding two months, August and
September, which provided suitable conditions for the growth of toxin-producing
moulds in the feed ingredients (1, 18). Such feed ingredients are usually fed to the
chicks during subsequent months (i.e., October to December), resulting in a
predisposition to IBH. Furthermore, it was found that, in the majority of outbreaks
which had a mortality rate of more than 10%, the birds were provided with less space
than normal (one square foot per bird), in addition to less space for watering and
feeding (Table I). This would indicate that over-intensive husbandry practices and poor
management also predisposed the birds to higher mortalities.
Epidemiological factors recorded in histopathologically confirmed outbreaks
of inclusion body hepatitis from 1990 to 1994

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TABLE II
Month
No. of outbreaks Mean atmospheric
temperature (°C) Relative humidity
(%)
April 2 24.85 49.3
May 2 31.10 40.7
June 1 32.03 51.6
July 4 30.14 72.4
August 0 29.64 77.4
September 3 28.52 73.2
October 8 23.88 59.1
November 10 18.88 63.3
December 9 13.85 72.0
January 1 12.56 76.0
February 3 14.73 72.8
March 4 18.74 64.7
Total 47
Avian adenovirus was isolated from the tissues of chicks involved in thirty-one
outbreaks. The isolates showed cytopathic effects with evidence of intranuclear
inclusion bodies in the cell culture. The presence of AAV in all the isolates was
confirmed by positive reactions from CIE, dot ELISA and double antibody sandwich
ELISA tests. Grewal et al. and Nagal et al. have also reported the isolation of AAV
from cases of IBH in this region (6, 12). The results of titration and virus neutralisation
of ten isolates and standard AAV-1 (chicken embryo lethal orphan: CELO) with 1:50
dilution of standard type-1 rabbit serum are shown in Table III. There was a decrease
of more than four-fold in the titres of all ten isolates and the standard strain. The
serotyping of these isolates indicates that they were all neutralised with standard
type-1 rabbit serum, in turn indicating that these isolates were all AAV-1 (CELO) type.
The isolates of Grewal et al. and Nagal et al. were also found to be closely related to
AAV-1 (6, 13).
AAV-1 was also found to play a major role in the IBH-hydropericardium syndrome
observed in the broiler chicks. During the study period, four outbreaks of IBH-
hydropericardium syndrome were recorded from July 1993 onwards. AAV-1 was
isolated from three of these outbreaks. The rate of mortality in these outbreaks varied
from 12.5% to 21%. Since IBH-hydropericardium syndrome is proving very damaging
to the poultry industry in India, further work is being done in order to determine the
parts played by various aetiological agents, and to find suitable preventive measures.
To this end, experimental trials in broiler chicks with a live AAV-1 (IBH isolate) virus
vaccine have yielded encouraging results, according to virus neutralisation and
challenge tests (10).
Consolidated data of monthly incidence of confirmed outbreaks
of inclusion body hepatitis and meteorological data from 1990 to 1994

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TABLE III
Log10 values of
Serial No. Isolate No. Virus titre
(VT) Serum-virus
titre (ST) Neutralisation index (VT-ST)
1 Standard
CELO virus 7.5 0 7.5
2 4935 7.5 0 7.5
3 4944 7.0 2.5 4.5
4 5088 7.3 1.0 6.3
5 5091 7.0 1.5 5.5
6 10897 5.5 0 5.5
7 12883 7.3 0 7.3
8 12996 6.5 2.0 4.5
9 13901 5.5 1.5 4.0
10 14295 5.6 0 5.6
11 14522 7.5 2.5 5.0
CELO: chicken embryo lethal orphan
In December 1991, an outbreak of IBH was recorded in ten-week-old Japanese quail
(Coturnix coturnix japonica), which experienced a heavy mortality rate (57.25%), due
to necrotic hepatitis, characterised by the presence of large basophilic intranuclear
inclusion bodies in hepatocytes. AAV-1 was isolated from the livers of these birds (7).
During experimental transmission studies, this isolate reproduced IBH in broiler
chicks and Japanese quail of more than three weeks of age (19).
The conclusion of this present study, which spanned more than four years, is that
IBH in broilers is primarily caused by AAV-1 and is accompanied by a low degree of
mortality. However, the mortality range, as well as the number of outbreaks, increases
when the feed has more than the permissible levels of aflatoxin. The mortality becomes
even greater when other diseases are concurrent.
ACKNOWLEDGEMENTS
This research has been financed by a grant made by the United States Department
of Agriculture under the Co-operative Agricultural Research Grant Programme
(PL-480). Thanks are also due to P.W. Chang, Department of Fisheries, Animal and
Veterinary Science, University of Rhode Island, Kingston, United States of America,
who kindly supplied the AAV-1 rabbit serum.
*
* *
Results of microneutralisation test of avian adenovirus (AAV)
isolates with standard AAV type-1 antibody