Avian Pathology

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Bursa atrophy at 28 days old caused by variant

infectious bursal disease virus has a negative
economic impact on broiler farms in Japan

Ohnmar Myint , Mathurot Suwanruengsri , Kenji Araki , Uda Zahli Izzati ,

Apisit Pornthummawat , Phawut Nueangphuet , Naoyuki Fuke , Takuya
Hirai , Daral J. Jackwood & Ryoji Yamaguchi

To cite this article: Ohnmar Myint , Mathurot Suwanruengsri , Kenji Araki , Uda Zahli Izzati , Apisit
Pornthummawat , Phawut Nueangphuet , Naoyuki Fuke , Takuya Hirai , Daral J. Jackwood &
Ryoji Yamaguchi (2021) Bursa atrophy at 28 days old caused by variant infectious bursal disease
virus has a negative economic impact on broiler farms in Japan, Avian Pathology, 50:1, 6-17, DOI:
10.1080/03079457.2020.1822989

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AVIAN PATHOLOGY
2021, VOL. 50, NO. 1, 6–17
https://doi.org/10.1080/03079457.2020.1822989

ORIGINAL ARTICLE

Bursa atrophy at 28 days old caused by variant infectious bursal disease virus
has a negative economic impact on broiler farms in Japan
Ohnmar Myinta, Mathurot Suwanruengsria, Kenji Arakib, Uda Zahli Izzati a, Apisit Pornthummawata,
Phawut Nueangphueta, Naoyuki Fukea, Takuya Hiraia, Daral J. Jackwoodc and Ryoji Yamaguchia
a
Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan; bBoehringer Ingelheim Animal
Health Co. Ltd, Tokyo, Japan; cFood Animal Health Research Program, The Ohio State University/OARDC, Wooster, OH, USA

ABSTRACT ARTICLE HISTORY

Infectious bursal disease (IBD), caused by IBD virus (IBDV), is highly contagious, Received 11 May 2020
immunosuppressive and causes a negative economic impact on poultry industry. IBDV- Accepted 9 September 2020
vaccinated broiler farms at south Kyushu, Japan had a bursa-to-bodyweight ratio (BB ratio)
KEYWORDS
reduction at 28 days (d) old, followed by high mortality 30 d later. We analysed the Variant infectious bursal
influence of the IBDV on atrophy of the bursa of fabricius (BF) and the subsequent mortality disease (vIBD); broiler; bursa
after 30 d. Ten broilers were sampled at each timepoint from the farm with high mortality to body weight ratio;
at 21, 25, 28 and 35 d. A second flock from the same farm was sampled at 14, 21, 25, 28, 35 mortality; histopathological
and 42 d. IBDV was detected in BF samples at 25, 28 and 35 d and at 21, 25, 28 and 35 d in tests; molecular test
the first and second flocks, respectively, using immunohistochemical staining and RT-PCR.
IBDV isolates from both flocks were closely related to the China KM523643 strain.
Histopathology and TUNEL assay indicated apoptosis, severe lymphoid depletion, vacuoles
within follicles, lymphoid follicle atrophy and fibrosis in the BF. We observed 75% of the
polyserositis and 10% of the airsacculitis at 30 D in dead broilers. The antigenic variant IBDV
infection was appeared to be the main influencing factor on BF atrophy and BB ratio
reduction in the broilers. High mortality in the broilers after 30 d could be due to secondary
infection. The disease caused by IBDV had a negative economic impact in the farm.

RESEARCH HIGHLIGHTS

. New variant IBDV caused bursa atrophy and reduced BB ratio in 28-day-old broilers.
. After vIBDV had infected broilers, at 21 days old they became immunosuppressed.
. High mortality at 30 days old in broilers was due to secondary infection.
. New vIBDV has a negative economic impact on broiler farms in Japan.

Introduction
used. There are two distinct serotypes: serotype 1 viruses
Infectious bursal disease (IBD), known as Gumboro are pathogenic in chickens and serotype 2 viruses are
disease, is a highly contagious infectious disease that avirulent for chickens (Müller et al., 2003; Eterradossi
usually occurs in young chickens at 3–6 weeks of age & Saif, 2008). The pathotypes can be classified as classic
after maternal antibody has waned (Kumar et al., virulent (cvIBDV), subclinical (scIBDV) & very virulent
2000; Eterradossi & Saif, 2008; Badji et al., 2016). (vvIBDV) (van den Berg, 2000). Michel & Jackwood
IBD virus (IBDV) belongs to the family Birnaviridae (2017) described the classification of seven major gen-
and genus Avibirnavirus. The primary target cells for ogroups of IBDV. There are six genogroups: 1 (classical
IBDV are lymphocytes in the bursa of Fabricius viruses), 2 (antigenic variant viruses), 3 (very virulent
(BF), resulting in lymphoid depletion and destruction IBDV), 4 (distinct IBDV), 5 (recombination between
in the BF. Consequently, the chickens become immu- classical and variant viruses), 6 (93% identity to the
nosuppressed and more susceptible to other viral and ITA genotype observed in Italy), and 7 (IBDV from
bacterial infections. IBDV infections can cause high Australia as well as from Russia). The degree of patho-
mortality in chicken flocks and a negative economic genicity of IBDV for chickens depends on the virus
impact on the poultry industry (Müller et al., 2003; strain (Tsukamoto et al., 1992).
Eterradossi & Saif, 2008). In Japan, the first IBD outbreak occurred in 1967,
IBDV strains have been classified into serotype, and vvIBDV outbreaks with high mortality occurred
pathotype and genotype based on the detection methods in many broiler and layer flocks from 1990 to 1991

CONTACT Ryoji Yamaguchi [email protected] Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, 1-1
Gakuenkibanadai-Nishi, Miyazaki 889-2192, Japan
Supplemental data for this article can be accessed at https://doi.org/10.1080/03079457.2020.1822989
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrest-
ricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 AVIAN PATHOLOGY 7

(Tsukamoto et al., 1992). Yamaguchi et al. (2007) at 27–29 d and the subsequent high mortality at 35 d
showed that IBDV from the samples of BF collected of age, one of new LPB farms was chosen for further
during 1996–2004 was of cvIBDV and vvIBDV analysis. A chronological investigation was conducted
types. IBDV infection in commercial poultry farms on two flocks: the first flock in November 2018 and the
throughout Japan is a major economic problem. second flock in January 2019. Taishu vaccine (Kyoto-
In Japan, some IBDV-vaccinated broiler farms at biken, Uji, Japan) was used at 14 and 21 d for IBD pre-
south Kyushu had atrophy of the BF lymphoid follicles vention in both flocks on this farm.
and remarkable reduction of bursa-to-bodyweight
ratios (BB ratios) (less than 0.03) at 28 days (d) old.
Sample collection
The subsequent mortality on these farms became high
after 30 d of age. Consequently, these farms became Samples of 10 vaccinated broiler chickens (Ross 308
low performance broiler (LPB) farms. The BB ratios breed) were collected at 21, 25, 28 and 35 d from the
of broilers from good performance broiler (GPB) first flock and at 14, 21, 25, 28, 35 and 42 d from the
farms were 0.2 or higher at around 28 d of age and second flock on the LPB farm. The 10 vaccinated broi-
the mortality rate was low in the high BB ratio farms. ler chickens collected at each sampling included five
There are several causes for BF atrophy and BB normal growth (NG) and five poor growth (PG) broi-
ratio reduction on poultry farms. In addition to ler chickens. These chickens were necropsied after
IBDV, chicken infectious anaemia virus, Marek’s dis- euthanasia and examined for gross lesions. BF samples
ease virus, Reovirus, Newcastle disease virus, Escheri- from the chickens were collected for both histopatho-
chia coli (E.coli) and Mycotoxicosis infections were logical and molecular tests. Thus, 40 broilers from the
also associated with BF atrophy and lymphoid first flock and 60 broilers from the second flock were
depletion in the BF (Hoerr et al., 1982; Nakamura used in this study. In addition, 100 dead broiler chick-
et al., 1986; Wang et al., 2007; Eterradossi & Saif, ens were also necropsied for gross lesions between 32
2008; Jones, 2008; Schat & Santen, 2008; Adi et al., and 46 d of age, at the time high mortality was
2010; Cazaban et al., 2015; Berthault et al., 2018). observed in the second flock. The Chicken Pathologic
A new antigenic variant IBDV (vIBDV) strain was Autopsy Ethics Committee of the broiler company,
considered as one of the main causes for the BB ratio Japan (2019-1) approved this experiment procedure.
reduction and BF atrophy because the vIBDV was
detected at 28 d of age during a preliminary survey
Histopathological examination
of broiler farms. We investigated the involvement of
IBDV infection in the LPB farms. The aim of this The BF samples were fixed with 10% buffered formalin
study was to investigate the negative impact of IBDV and then processed for paraffin embedding. After
infection-induced BF atrophy, BB ratio reduction making 4 µm paraffin sections, these tissues were
and the subsequent high mortality in broilers after stained with haematoxylin and eosin (H&E). The his-
30 d of age on a LPB farm using histopathological topathological lesion scoring system for the BF lesions
and molecular techniques. Genetic sequence analysis was determined based on the degree of lymphoid
was also conducted to determine the genotype of the depletion: 0 = no significant lesions; 1 = less than 24%;
IBDV strain. This study has demonstrated through 2 = between 25% and 49%; 3 = between 50% and 74%;
detailed pathological and genetic analyses that the 4 = between 75% and 100% lymphoid depletion in
new vIBDV invasion causes BF atrophy and affects affected follicles; 5 = atrophy of follicles, BF plicae and
mortality on the LPB farm. Information on this vacuoles within follicles with lymphoid depletion.
virus will hopefully help with the control and preven-
tion of the infection.
Terminal deoxynucleotidyl transferase-
mediated dUTP nick-end labelling (TUNEL)
assay
Materials and methods
For the detection of apoptotic lesions in the BF, the
Farm information for surveillance of BB ratio
TUNEL assay was conducted by using a commercial
The 2012–2017 surveillance of BB ratios and mortality kit (Apoptosis in situ detection kit, Wako, Japan)
was carried out on IBDV-vaccinated broiler farms, according to the manufacturer’s instructions. After
which are located at south Kyushu, Japan. Based on the deparaffinizing and rehydrating steps, these slides
the BB ratios and mortality, six broiler farms, which were immersed in the protein digestion enzyme sol-
included three GPB farms (Farm D, E, F) and three ution at 37°C for 10 min. After washing with phos-
LPB farms (Farm A, B, C), were chosen for this phate buffered saline (PBS), the slides were labelled
study. IBDV detection from BF samples of the six with Terminal deoxynucleotidyl transferase solution
broiler farms was done by RT-PCR and sequence at 37°C for 10 min. After washing with phosphate
analysis. Based on the results of BB ratio reduction buffered saline, they were inactivated with 3%
8 O. MYINT ET AL.

hydrogen peroxide solution for 5 min, washed with procedure was performed, as previously described by
PBS again and labelled with horseradish peroxidase- Diep et al. (2015).
conjugated antibodies at 37°C for 10 min. Colour
development for apoptotic cells was done by using
3,3′ -diaminobenzidine substrate solution at room Data analysis
temperature for 5 min. Haematoxylin was then used The formula of BB ratio = [BF weight in gram/body
for counter-staining. The scoring system for TUNEL weight in gram] × 100 was described by Cazaban
assay was as follows: 0 = no stained cells; 1 = less et al. (2015). The difference in mortality between the
than 50%; 2 = greater than 51% stained cells within low and high BB ratio farms was analysed by Student’s
the cortex or medulla of follicles; 3 = less than 30%; T-test. The difference in number of broilers with air-
4 = 31–50%; 5 = above 51% stained cells within both sacculitis between 21, 25, 28 and 35 d groups was ana-
the cortex and medulla of follicles. lysed by Chi-square test. All analyses were performed
using computer programming language R (version
Immunohistochemistry (IHC) test procedure 3.4.3; R development core team, Vienna, Austria).

The BF tissue slides were deparaffinized and dehy-

drated. Antigen retrieval was done in citrate buffer sol- Results
ution (pH 6) for 12 min at 110°C using a microwave. Low BB ratio and high mortality in IBDV-
After blocking endogenous peroxidase activity with vaccinated broiler farms from 2012 to 2017
3% hydrogen peroxide solution, these slides were incu-
bated with Blocking One solution (Nacalai Tesque, The BB ratio was examined in broilers raised on farms
Kyoto, Japan) to avoid non-specific reactions. The where IBD vaccines were used at south Kyushu, Japan.
monoclonal IBDV antibody (IBDV9, HyTest Ltd, Some broiler farms used intermediate virulence (IV)
Turku, Finland) was used as a primary antibody and vaccines at 14 or 18 d of age and some broiler farms
the Histofine® Simple Stain MAX PO (Multi) (Nichirei used mild virulence (MV) vaccines at 14 and 21 d
Biosciences, Tokyo, Japan) was used as a secondary for IBD prevention. The BB ratio on some MV-vacci-
antibody. Diaminobenzidine solution was applied as a nated broiler farms was found to be significantly
substrate and haematoxylin as a counter-stain. The reduced compared to that of IV-vaccinated broiler
viral antigen positive cells were identified using light farms, as shown in Figure 1. The average BB ratios
microscopy. The scoring system of IHC was as follows: at 27–29 d in IBDV-vaccinated broiler farms are
negative: no lymphocytes stained; 1+: one to two lym- shown in Figure 1. In 2017, BB ratio of MV-vaccinated
phocytes stained; 2+: less than 30% lymphocytes stained broiler farms dropped severely from at least 0.2 in
within lymphoid follicles; 3+: above 31% lymphocytes 2012 to 0.03 with BF atrophy. Figure 2 shows the broi-
stained within lymphoid follicles. ler farm with low BB ratio did not show high mortality
at 28 d demonstrating BF atrophy, but the mortality
was significantly increased at 30 d.
Reverse transcription-polymerase chain
reaction (RT-PCR) and nucleotide sequence
analysis Different BB ratios and mortality on broiler
farms
Total RNA extraction was done from 250 µl of sample
homogenate by using ReliaPrep™ RNA Cell Miniprep Six different broiler farms (Farms A, B, C, D, E and F)
kits (Promega Corporation, WI, Madison, USA) were chosen for the analysis of mortality and BB
according to the manufacturer’s instructions. IBDV ratios. We also determined the genotype of the
was detected by AccessQuickTM RT-PCR kit (Promega IBDV strains detected on these farms. Farm A had
Corporation). The IBDV forward primer BB ratios of 0.03, severe BF atrophy and high mor-
(5ʹACCTTCCAAGGAA GCCTGAGTG-3ʹ) and tality. Farms B and C were located near Farm A
reverse primer (−5ʹATCAGCTCG AAGTTG where the TY2 variant IBDV strain was isolated in
CTCACC-3ʹ) were used and the amplicon size was 2002. These farms had low BB ratios and high mor-
720 base pairs (Pikula et al., 2017). The RT-PCR pro- tality. Both Farms D and E showed good performance.
cedure was modified from Diep et al. (2015). The Farm F had high BB ratios and low mortality and was
reverse transcription step for IBDV detection was con- GPB farm. The broiler populations and distances
ducted at 45°C for 45 min followed by incubation at between these six broiler farms are shown in Table 1.
94°C for 2 min. For the PCR, 35 cycles of 94°C for The new antigenic variant IBDV (vIBDV) strains
30 s, 61°C for 1 min and 72°C for 1 min were con- like China KM 523643 were identified from Farms
ducted. The terminal extension of PCR was 72°C for A, B, C in 2017 and Farm D in 2018. One classical
10 min (Pikula et al., 2017). The RT-PCR products IBDV strain, the same as Canada EF138968, was
were visualized on a 1.2% agarose gel. The sequencing identified from Farms E and F in 2017 and 2018,
 AVIAN PATHOLOGY 9

Figure 1. The BB ratio on IBDV-vaccinated broiler farms at 14, 18 and/or 21 d old from 2012 to 2017. The oval shape around the
2017 data points indicates that mild virulence IBDV-vaccinated broiler farms showed severe BB ratio reduction to a value of 0.03.

age were analysed, the mortality of the low BB ratio

broiler farms was significantly higher than that of
the high BB ratio broiler farms at 31–49 d (P < 0.05)
(Table 2). One of the new LPB farms with low BB
ratios and high mortality was located near Farm A
and was chosen for further investigation in detailed
chronological analysis as the LPB farm. This new
selected LPB farm was another separated farm and
not involved in Farms A, B, C, D, E and F.

Chronological comparison for mortality and BB

Figure 2. Low BB ratio broiler farm showing high mortality at ratios between GPB farm and the LPB farm
35–49 d old in the farm vaccinated with mild virulence IBDV in
2017. Each bar represents the mortality in each day of this Two flocks on the LPB farm were examined in compari-
farm. The solid dot is the BB ratio which reduced at 28 d old son with a GPB farm, Farm F. The BB ratios of the first
broilers. The figure shows the mortality on this farm increased and second broiler flocks at 28 d of age were 0.11 and
35 d later after BB ratio had reduced significantly at 28 d old. 0.06, respectively, in comparison with 0.27 BB ratio in
The standard minimum BB ratio of broilers is 0.11 (Cazaban the GPB farm. The mortalities at 30–50 d of the first
et al., 2015).
and second flocks were 5.7% and 10.4%, respectively,
in comparison with 1.06% low mortality in the GPB
respectively. One representative IBDV strain from farm. The first and second flocks of the LPB farm
each of Farms A, B, C, D, E and F was selected for phy- with low BB ratios encountered a higher mortality
logenetic analysis of IBDV. than the GPB farm at the 35–50 d. The differences in
When three GPB farms with high BB ratios and the mortality and BB ratios between the GPB farm
three LPB farms with low BB ratios at 27–30 days of and the LPB farm were compared in Figure 3.

Table 1. Six different broiler farms for comparison of BB ratio and mortality by the impact of IBDV infection.
Broiler Number of broilers in Number of broilers in GPB/LPB
farm Different or same house/farm the house the farm Distance farm
A Different house in the same farm which showed 0.03% BB ratio 26,000 269,000 - LPB
and high mortality after 30 d
B House near the farm where TY2 was isolated in 2002 18,000 72,000 15 km LPB
C Another house near the farm where TY2 was isolated in 2002 20,000 80,000 14 km LPB
D New farm (first chick placement) 27,000 188,000 6 km GPB
E House in small-scale farm 10,000 50,000 6 km GPB
F House in high rank farm (small-scale farm) 11,000 33,000 12 km GPB
Note: LPB farm - Low performance broiler farm, GPB farm - Good performance broiler farm.
10 O. MYINT ET AL.

Table 2. Broiler farms with low BB ratio showed high and follicular atrophy were not seen in 14 D old broilers
mortality. of the second flock. Severe apoptosis of lymphocytes in
Broiler farm BB ratio at 27–30 d Mortality at 31–49 d (%) the BF occurred at 25 and 21 d of the first and second
A 0.04 23.6 flock, respectively. Apoptotic lesions were gradually
B 0.06 12.5
C 0.04 18.3 reduced in older age birds of both flocks. Severe lym-
Average 0.05 18.1A phoid depletion in the BF started at 25 d and was
D 0.15 3.0
E 0.17 2.9 most severe at 35 d of the first flock. In the second
F 0.27 1.6 flock, lymphoid depletion in the BF was found at 21–
Average 0.20 2.5B
A,B
42 d, where lymphoid depletion was most severe at
The data within the same column with the different superscripts are sig-
nificantly different at (P < 0.05). 28 d.
In both flocks, reticular cells and macrophages
replaced lymphocytes in the lymphoid follicles
Clinical signs and gross lesions on the LPB farm during lymphocyte depletion. Then, vacuoles and
cystic cavities appeared within the lymphoid follicles.
The broilers showed slight sneezing at 21 d of age in Follicular atrophy and loss of lymphoid follicles were
the LPB farm. Only BF atrophy, and atrophy with seen from 28 to 35 d in the first flock and from 21 to
some congestion were grossly found in the first and 42 d in the second flock. Infolding epithelium into
second flock, respectively. damaged follicles of the BF was found from 28 to
Airsacculitis, which indicates inflammation caused 35 d in the first flock and from 25 to 42 d in the
by the secondary bacterial infection, usually E. coli, second flock. Fibrous tissue infiltrations between fol-
was seen at 25, 28 and 35 d in the first flock and at licles of the BF were seen from 25 to 35 d in the first
14, 21, 25, 28, 35 and 42 d in the second flock. In flock and between 21 and 42 d in the second flock.
the first flock, the number of broilers with airsacculitis The data and scores for histopathological evaluation
at 28 d was significantly higher (P < 0.05) than at 21 of the BF from both flocks are shown in Table 3. The
and 25 d. In the second flock, there was no significant histopathology of the BF from different age groups
difference in the number of broilers with airsacculitis for the second flock is shown in Figure 4.
between the age groups (P > 0.05). The number of Comparing the severity of the lesions in each group
broilers with airsacculitis from the two flocks is of NG and PG broiler chickens, PG chickens had more
shown in Supplementary Table 1. severe damage to lymphoid follicles than NG chickens
of both flocks.
Histopathological findings and evaluation of
the BF TUNEL assay results
The histopathological evaluation of the BF showed that The lymphocyte apoptotic lesions of the BF were
there were no obvious lesions in 21-d-old broilers of the examined by the TUNEL assay in both flocks. Two
first flock. Lymphoid depletion, vacuoles within follicles tissue sections from each group were selected for

Figure 3. Comparison for BB ratio at 28 d and mortality at 35 d between good and low performance broiler farms.
 AVIAN PATHOLOGY 11

Table 3. Histopathological evaluation of BF.

Flocks
First flock Second flock
Histopathological lesions 21 * 25* 28* 35* 14* 21 * 25* 28* 35* 42*
Apoptosis 0/10 7/10 10/10 10/10 5/10 10/10 9/10 9/10 8/10 8/10
Average apoptosis score 0 3.2 2.3 1.8 1.5 3.1 2.2 2.3 1.6 0.9
Lymphoid depletion 0/10 8/10 10/10 10/10 0/10 9/10 10/10 10/10 10/10 9/10
Average lymphoid depletion score 0 2.7 4.6 4.9 0 3.5 3.9 4.5 3.8 3.4
Infolding epithelium into damaged lymphoid follicles 0/10 1/10 5/10 8/10 2/10 1/10 6/10 9/10 9/10 7/10
Vacuoles within follicles 0/10 0/10 7/10 9/10 0/10 3/10 6/10 7/10 3/10 3/10
Cystic cavity within follicles 0/10 4/10 1/10 5/10 1/10 1/10 6/10 6/10 6/10 6/10
Lymphoid follicle atrophy 0/10 1/10 9/10 10/10 0/10 5/10 4/10 6/10 5/10 4/10
Note: No. of IBD histopathological lesions present in broilers/no. of detected broilers.
*days of age.

Figure 4. Histopathological appearance (H&E) of the BF in different age groups at 14, 21, 25, 28, 35, and 42 d old in the second
flock broilers. (a) 14 d; no obvious change; inset: no obvious change in lymphoid follicles, (b) 21 d; lymphoid depletion and fibrosis;
inset: apoptosis of lymphocytes, lymphocyte necrosis with pyknotic nuclei within follicles, (c) 25 d; lymphoid depletion, lymphoid
follicle atrophy, vacuoles within follicles and fibrosis; inset: apoptosis of lymphocytes, lymphocyte necrosis, macrophages and
vacuoles within follicles, (d) 28 d; lymphoid depletion, lymphoid follicle atrophy, vacuoles within follicles and infolding surface
epithelium into damaged follicles and fibrosis; inset: vacuoles, necrotic lymphocytes, macrophage and reticular cells within the
follicles, (e) 35 d; lymphoid depletion, loss of some lymphoid follicles, vacuoles within follicles, infolding surface epithelium
into damaged follicles and fibrosis; inset: vacuoles, necrotic lymphocytes, macrophage and reticular cells occupied the follicles,
(f) 42 d; cystic cavities, lymphoid depletion in some follicles, some regenerative lymphoid follicles and some fibrosis; inset: regen-
erative lymphocytes (right), necrotic lymphocytes (left) and reticular cells were seen.

the TUNEL assay. The apoptotic lesions of the BF in of the BF. The IHC-positive score for IBDV in the
the TUNEL assay appeared clearer than with H&E BF was highest at 28 and 35 d.
staining. The TUNEL assay-stained apoptotic cells In the second flock, IBDV antigen was detected in
were found in all broiler groups of both flocks. The lymphocytes and macrophages of the BF at 21, 25,
highest lymphocyte apoptotic lesion scores were 28 and 35 d. The IHC-positive scores for IBDV in
found at 25 d in the first flock and at 21 d in the the BF were highest at 21 d. However, there were no
second flock. The TUNEL assay results for apoptotic immunolabelled cells in the BF at 14 and 42 d. The
cells in the BF from the second flock are shown in IHC staining of the BF for the different age groups
Figure 5. is shown in Figure 6.

Detection of IBDV in BF by RT-PCR

IBDV detection by IHC
In the first flock, IBDV was detected in the BF samples
In the first flock, there were no IHC-labelled cells in by RT-PCR at 25 and 28 d of age, and at a low level at
the BF at 21 d. At 25, 28 and 35 d, IBDV-positive 35 d, but was absent at 21 d. In the second flock, IBDV
cells were observed in lymphocytes and macrophages RT-PCR detected positive samples at 21, 25 and 28 d,
12 O. MYINT ET AL.

Figure 5. TUNEL assay results for the second flock. (a) 14 d bursa; less than 30% of cells stained in the cortex and medulla of
follicles, (b) 21 d bursa; above 50% of cells stained in the cortex and medulla of follicles, (c) 25 d bursa; above 50% of cells stained
in the cortex and medulla of follicles, (d) 28 d bursa; less than 30% of cells stained in the cortex and medulla of follicles, (e) 35 d
bursa; less than 30% of cells stained in the cortex and medulla of follicles, (f) 42 d bursa; less than 50% of cells stained in the cortex
of follicles.

and faint positive at 35 d. However, samples from 14 seven IBDV isolates from both flocks, 16 IBDV iso-
and 42 d were negative (Supplementary Figure 3). lates from Farms A, B, C, D, E, F, and the Taishu
The number of IBDV-positive samples by RT-PCR IBDV vaccine strain were registered into GenBank.
and IHC in each group from both flocks is shown in The genotype, sequence accession numbers and isolate
Table 4. or strains of reference for the IBDV, Taishu IBDV vac-
cine strain, six representative IBDV isolates from the
preliminary survey LPB Farms A, B, C, D, E, F and
seven IBDV isolates from this study are shown in
Phylogenetic analysis of IBDV
Table 5.
Phylogenetic analysis of IBDV isolates from the BF Phylogenetic analysis demonstrated that the IBDV
samples was carried out by following the genogroup strain from the preliminary survey of LPB Farms A,
classification of Michel & Jackwood (2017). The B, C and D (Accession numbers from MT 215072 to

Figure 6. The immunohistochemical staining of BF in different age groups in second flock broilers. (a) no staining at 14 d, (b)
above 31% lymphocytes and macrophage stained within lymphoid follicles at 21 d, (c) less than 30% lymphocytes and few macro-
phages stained within lymphoid follicles at 25 d, (d) few lymphocytes stained at 28 d, (e) few lymphocytes stained at 35 d, (f) no
staining in BF at 42 d.
 AVIAN PATHOLOGY 13

Table 4. IBDV detection in BF by IHC and RT-PCR tests. Based on the partial viral protein (VP2) gene sequences
No. of IBDV-positive broilers of the isolates from the LPB farm, these isolates were
14 * 21 * 25 * 28 * 35 * 42 * highly homologous to China isolates, KM523643,
First flock IHC - 0/10 9/10 10/10 10/10 - JX134483, MH879092 and MN485882 strain, sharing
RT-PCR - 0/10 9/10 9/10 6/10 -
Second flock IHC 0/10 10/10 10/10 10/10 3/10 0/10 98.5–99.1%, 96.4–97.1%, 96.8–97.5% and 97.1–97.7%
RT-PCR 0/10 10/10 10/10 10/10 4/10 0/10 nucleotide identity, respectively. The IBDV isolates
Note: No. of IBDV IHC and RT-PCR test positive broilers/no. of tested broi- from the LPB farm were not similar to the vaccine
lers.
*days of age. strain (MN700903), which was used on this farm.
This vaccine strain was clustered into the classical
IBDV genogroup. The phylogenetic analysis, based on
MT 215077, and from MT 215080 to MT 215086) was the partial VP2 gene for IBDV, is shown in Figure 7.
placed in the antigenic vIBDV genogroup and its
nucleotide identity was similar to the China
KM523643 strain. The IBDV strain from Farms E Pathological findings of 100 dead broilers
and F (Accession numbers MT 215078, MT215079, collected at the time of high mortality in the
MT215087) was placed in the classical IBDV gen- second flock
ogroup, which shares nucleotide identity with the In gross findings, perihepatitis and pericarditis were
Canadian strain (EF138968). seen in 75% and ascites was found in 10% of the 100
IBDV isolates from 25, 28 and 35 d birds of the first dead broilers from the second flock at 32–46 d of age.
flock, and 21, 25, 28 and 35 d birds of the second flock
from the LPB farm (Accession numbers from
MN171470 to MN171485) were placed in the same Discussion
phylogenetic genogroup and clustered with vIBDV. During 2017, some broiler farms vaccinated with a
mild-virulent IBDV from south Kyushu, Japan, had
severely reduced BB ratios around 28 d of age and
Table 5. IBDV strains from this study and reference strains for
the phylogenetic tree. then high mortality followed at 30 d of age. When dis-
Accession ease diagnosis after 35 d of age was carried out by RT-
No. Genogroup no. Isolates/strain PCR during the time of high mortality, the involve-
1. Classical AF498631 Bursine 2 ment of IBDV disappeared in these broilers. However,
2. Classical AY321953 F52-70
3. Classical D00499 STC antigenic vIBDV-like KM523643 strain was isolated
4. Classical MN700903 Taishu IBDV vaccine from a preliminary survey of LPB broiler farms
5. Classical AJ878891 Burs706
6. Classical MH329180 D78
(Farms A, B and C) before a high mortality was
7. Classical EF138968 99-40488-1 observed. The BB ratio of LPB farms A, B and C was
8. Classical MT215078 Kagoshima MO IBDV severely reduced at 27–30 d. Although vIBDV infec-
E1
9. Classical MT215087 Kagoshima MO IBDV tion was also found in Farm D, which was a GPB
F farm, a severe decrease of BB ratio with BF atrophy
10. Antigenic variant KM523643 IBD11XJo1
11. Antigenic variant JX134483 GX-NNZ-11 at 28 d was not observed because IBDV introduction
12. Antigenic variant JF736011 AL-2 in this farm occurred after 28 d.
13. Antigenic variant AF133904 Variant E
14. Antigenic variant MH879092 SHG19 When the mortality, based on BB ratio of the six
15. Antigenic variant MN485882 ZD-2018-1 preliminary surveys in farms A, B, C, D, E and F,
16. Very virulent EU835861 Br/03/DB
17. Very virulent AF092943 HK46
was analysed, the mortality for low BB ratio farms
18. Very virulent KT884486 Henan (HN) was significantly higher than that for high BB ratio
19. Very virulent NC004178 UK661 farms between 31 and 49 d. Therefore, the factors
20. Distinct IBDV LC136880 TY2
21. Variant and classical DQ916210 Mexico04M101 causing BF atrophy, reduced BB ratios and high mor-
recombinant tality after 30 d of age in broilers were investigated in
22. Italy JN852986 ITA-02
23. Australia HM071991 V877-W two flocks from one of the LPB farms.
24. Antigenic variant MN171479 Kagoshima MO 2B-21 This study indicated that the new antigenic vIBDV
25. Antigenic variant MN171480 Kagoshima MO 1B-25
26. Antigenic variant MN171481 Kagoshima MO 2B-25 in Japan was the main influencing factor for BF atro-
27. Antigenic variant MN171482 Kagoshima MO 2B-28 phy, and BB ratio reduction, and may be followed by
28. Antigenic variant MN171483 Kagoshima MO 1B-28
29. Antigenic variant MN171484 Kagoshima MO 1B-35
immunosuppression resulting in high mortality after
30. Antigenic variant MN171485 Kagoshima MO 2B-35 30 d of age in broilers. Our results showed that
31. Antigenic variant MT215072 Kagoshima MO IBDV vIBDV caused not only severe bursa damage resulting
A1
32. Antigenic variant MT215075 Kagoshima MO IBDV in reduced BB ratio but also immunosuppression
B1 resulting in high mortality in broilers (Kurukulsuriya
33. Antigenic variant MT215080 Kagoshima MO IBDV
C1 et al., 2016).
34. Antigenic variant MT215084 Kagoshima MO IBDV McMullin (2004) described that the normal weight
D1
of the BF in broilers was about 0.3% of the body
14 O. MYINT ET AL.

Figure 7. Phylogenetic tree based on the alignment of partial VP2 gene sequence for IBDV infection. It represents the IBDV isolates
from the LPB farm, and the IBDV isolates from the preliminary survey of Farms A, B, C, D, E and F. The phylogenetic tree was
constructed by the maximum likelihood method.

weight. Cazaban et al. (2015) showed that the mini- and 21 d of age in the first and second flocks in this
mum BB ratio standard of broilers at 7–42 d of age study. The apoptotic cells were frequently present
was 0.11. The BB ratio from the first and second broi- near the IBDV antigen-presenting cells because apop-
ler flocks of this study was lower than the new mini- tosis-inducing factor, like interferon (IFN), would
mum standard BB ratio. In this study, the mortality induce inhibition of protein synthesis and cause apop-
of the second flock was also higher than the first tosis in double-stranded RNA virus infections (Lee &
flock and this is correlated with a lower BB ratio Esteban, 1994; Jungmann et al., 2001). Therefore, if
observed in the second flock. IBDV replication increases in the B-lymphocytes of
The clinical signs of IBDV-infected broilers are the BF, the apoptotic cell proportion would also be
soiled vent feathers, diarrhoea, anorexia, depression, expected to increase. The highest apoptotic cells due
ruffled feathers, trembling, severe prostration, dehy- to IBDV infection were demonstrated at 48 hr post-
dration and mortality (Eterradossi & Saif, 2008; Mah- infection in chicken embryo cells (Jungmann et al.,
goub, 2012). However, diarrhoea, dehydration and 2001) and after 2 d post-infection in the BF (Nieper
soiled vent feathers were not seen in our study. Eterra- et al., 1999). The apoptotic lesions indicated that
dossi and Saif (2008) described that the severity of the IBDV was introduced before 25 and 21 d in the first
lesions depends on the pathogenicity of the IBDV and second flocks, respectively. The TUNEL assay
strains. The vvIBDV causes the most severe lesions. gave a clearer indication of apoptosis lesions com-
The variant IBDV causes no obvious clinical signs or pared to the H&E staining in this study.
death, although BF atrophy, thymus atrophy and mus- IBDV destroyed the lymphoid follicles, and lym-
cular haemorrhage were occurred in chickens (Fan phoid depletion occurred from 25 to 35 d of age in
et al., 2019; Xu et al., 2020) The clinical signs and the broilers of the first flock, and from 21 to 42 d of
gross findings for broilers were not severe in our the second flock. After nearly 100% lymphoid
study, presumably because the IBDV strain from this depletion had occurred, atrophy of lymphoid follicles
study may be a less pathogenic vIBDV strain. followed. Many vacuoles and cystic lesions were
Although many studies on IBDV infection with observed in the follicles and infolding epithelium
histopathological findings have been conducted exper- into the damaged lymphoid follicles was a distinct
imentally, there is limited information on the histo- indicator of a severe BF damage. The most severe lym-
pathological lesions from week-to-week in broilers phoid depletion was found at 35 and 28 d in the first
naturally infected with IBDV. The lymphocyte apop- and second flock, respectively. During this stage, we
totic lesions in the BF were most pronounced at 25 observed the most severe lymphoid depletion and it
 AVIAN PATHOLOGY 15

would be the peak period of BF damage. This suggests from China. This vIBDV may be circulating not only
that the broilers were immune suppressed. IBD histo- in this LPB farm but also neighbouring farms. These
pathological lesions of the BF, such as numerous vacu- IBDV isolates from both flocks were new antigenic
oles and cysts, infolding epithelium within follicles vIBDV in Japan.
and atrophy of lymphoid follicles, were more severe Although the clinical signs, gross findings and
in PG broilers than NG broilers. PG broilers may be inflammatory cell infiltration in histopathological
more susceptible to IBDV because BB ratios of PG lesions were not severe, the vIBDV strain was a likely
broilers were slightly lower than those of NG broilers. cause of the immunosuppression and high suscepti-
Apoptosis occurs in BF due to pathological and phys- bility to secondary infections. Therefore, airsacculitis
iological stimuli, chemical and physical agents (Arai in both types of flocks, 75% perihepatitis, pericarditis
et al., 2000). Some apoptosis, cystic lesions and infold- and 10% ascites in the 100 dead birds of the second
ing epithelium into lymphoid follicles were also seen flock were caused by secondary infections in this
at 14 d in PG broilers of the second flock before study and were most likely due to immunosuppression
IBDV introduction. It may be due to the physiological in the broilers. The number of broilers with airsaccu-
stimuli and other pathological agents. litis in the first flock at 28 d was significantly higher
Rauf et al., (2011) observed that the BF damage and than that of the others in the first flock, and 25 d
inflammatory response to classical IBDV were more from the second flock was numerically higher in the
pronounced than vIBDV. However, the inflammatory incidence of airsacculitis than 21 d. It is probable
response of heterophils and plasma cells was not seen that the higher susceptibility to secondary bacterial
in the study. Severe lesions due to vIBDV infection infections started after 1 week of IBDV infection (Sup-
were lymphoid follicle atrophy, connective tissue pro- plementary Table 1).
liferation, infiltration of macrophages and reduced Many studies reported that airsacculitis and fibri-
lymphocyte population in the BF (Fan et al., 2019; nous polyserositis in broilers were found as the main
Xu et al., 2020). Our findings were similar to those gross lesions of colibacillosis (El-Sukhon et al., 2002;
of Sharma et al., (1989), Fan et al. (2019), and Xu Ask et al., 2006; Lau et al., 2010). Therefore, airsaccu-
et al. (2020), but histopathological findings from litis and polyserositis in this study also could be due to
week-to-week were more briefly described in this secondary infections such as E.coli. The cause of high
study. The vIBDV caused extensive lesions in the BF mortality in the LPB farm after 30 d of age may be due
but did not cause an inflammatory response. to secondary bacterial infections such as E.coli,
IBDV antigens were detected in the lymphocytes especially at the time of immunosuppression in these
and macrophages by the IHC test in this study. Pre- broilers. Studies by Müller et al. (2003) and Eterra-
viously, it was reported that IBDV antigen was dossi & Saif (2008) support our findings that IBDV
detected in the cytoplasm of lymphocytes, epithelial caused immunosuppression and then consequently
cells and inflammatory cells, mostly macrophages, of high mortality due to airsacculitis, vaccination failure
the BF in IBDV-infected chickens (Nunoya et al., and colisepticaemia in 6–8 week-old broilers. Kuru-
1992; Tanimura et al., 1995; Oladele et al., 2009). kulsuriya et al. (2016) reported that pre-exposure
IHC and RT-PCR test results for IBDV antigen detec- with variant IBDV (SK09) in broilers caused a higher
tion in the BF samples were similar. According to mortality due to immunosuppression.
these results, IBDV entered around 25 d in the first Our study clearly describes the role that a new
flock and 21 d in the second flock. After that, IBDV vIBDV infection played as a main influence factor
gradually disappeared at 35 d in both flocks. There- on BF atrophy and reduced BB ratios in the commer-
fore, IBDV was not detected after 35 d at high mor- cial broiler farms in Japan. After vIBDV infection at
tality on the preliminary survey farms. The about 21 d of age, the broilers most likely became
introduction of IBDV in the second broiler flock was immune suppressed. Consequently, high mortality
earlier than that of the first flock. It is probably due was observed at 30 d and later, and was most likely
to environmental factors and insufficient IBDV due to secondary infections. The new vIBDV infection
maternal immunity in the second flock of broilers. in Japan should be considered as a primary factor in
Müller et al. (2003) reported that chickens were highly the case of lower BB ratios at 27–29 d and high mor-
susceptible to IBDV at 3–6 weeks of age because the tality at 30 d and later in commercial broiler farms.
BF developed to a maximum size at this age.
The IBDV isolates from both the flocks and the A,
B, C and D farms were clustered into the antigenic Disclosure statement
vIBDV genogroup and were closely related to Chinese No potential conflict of interest was reported by the authors.
strains of KM523643. This suggests that this antigenic
vIBDV strain had been circulating in this LPB farm
and is not related to the vaccine strain being used. ORCID
This strain may have originated by transmission Uda Zahli Izzati http://orcid.org/0000-0001-6635-7561
16 O. MYINT ET AL.

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