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Histopathological and immunohistochemical diagnosis of infectious bursal

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Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE

Available at www.veterinaryworld.org/Vol.8/November-2015/11.pdf Open Access

Histopathological and immunohistochemical diagnosis of infectious

bursal disease in poultry birds
J. Singh, H. S. Banga, R. S. Brar, N. D. Singh, S. Sodhi and G. D. Leishangthem

Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana, Punjab, India.
Corresponding author: J. Singh, e-mail: [email protected], HSB: [email protected],
RSB: [email protected], NDS: [email protected], SS: [email protected], GDL: [email protected]
Received: 27-06-2015, Revised: 04-10-2015, Accepted: 15-10-2015, Published online: ***

doi: 10.14202/vetworld.2015.1331-1339 How to cite this article: Singh J, Banga HS, Brar RS, Singh ND, Sodhi S,
Leishangthem GD (2015) Histopathological and immunohistochemical diagnosis of infectious bursal disease in poultry
birds, Veterinary World 8(11): 1331-1339.

Abstract
Aim: The aim of the present study was to diagnose infectious bursal disease (IBD) using gross, histopathological, and
immunopathological approaches and to compare efficacy of immunohistochemical techniques with conventional diagnostic
techniques.
Materials and Methods: A total of 33 samples were collected from the six different poultry farms from Ludhiana and the
nearby districts. Upon gross analysis of the necropsied birds, the relevant tissue samples such as bursa, kidney, junction of
proventriculus and gizzard, heart, and muscles were then processed for histopathological and immunohistochemical studies.
Results: Varied macroscopic changes were noted in bursa, characterized as swollen, hemorrhages to atrophy in size.
Nonetheless, hemorrhages over thigh muscles were rarely seen. Histologically, the bursa showed prominent fibrotic and
atrophic changes. Rarefaction of bursal follicles with intermittent infiltration of lympho-mononuclear cells with chronic
cystic changes was additional changes, considered to be paramount for IBD. Expression and localization of IBD specific
viral antigens were noticed mainly intracellular to the rarefied areas of bursal follicle section(s), in conjunction to inner
lining of the cystic cavities of affected follicles. In addition, the junction of proventriculus and gizzard, the heart muscle,
respiratory ciliated epithelium, and proventriculus also revealed positive expression to IBD virus (IBDV) antigen. Advanced
immunopathological techniques, i.e., immunofluorescence further testified the evidence of antigen as positive green signal
within affected follicles. Further consideration to the reliability of various techniques employed, positive correlation
(r=0.64623) was emerged out with conventional pathological scoring.
Conclusion: It is concluded that the bursa acts as an organ of choice for demonstrating IBDV antigen for specific diagnosis
of disease using immunohistochemistry (IHC), and IHC staining is a precise, specific, rapid, and reliable method to
demonstrate the IBDV antigen in the altered tissues due to IBDV infection.
Keywords: histopathology, immunohistochemical, infectious bursal disease.
Introduction mutation rate of the IBD virus (IBDV) genome, the
Poultry farming has always been an integral com- virus changes its properties such as antigenic variation
ponent of livestock production. Besides providing egg with increased virulence [5].
and meat, it proves to be a good and reliable source of The disease manifests as acute and subclinical
income in rural areas [1]. There are several viral, bac- form(s) in chicks of age 0-3 weeks as immunosup-
terial, parasitic, and managemental diseases of poul- pression or also in clinical form depending on the
try that cause direct financial loss to farmers. Among age of the bird. The chicks become anorectic, become
these, infectious bursal disease (IBD) ranks high. reluctant to move, and show ruffled feathers with
IBD, also known as Gumboro disease [2], based watery diarrhea, trembling and severe prostration.
on the area of its first identification (in Gumboro, The characteristic gross lesions of the disease include
Delaware United States of America, USA); avian dehydration of the muscles with ecchymotic hemor-
nephrosis [3] and avian infectious bursitis, is classi- rhages, enlargement, and orange discoloration of kid-
fied as an economically important disease of poul- neys. The bursa of Fabricius becomes enlarged and
try. It is caused by a virus that is member of genus shows pale yellow discoloration. Intra-follicular hem-
Avibirnavirus of family Birnaviridae with two sero- orrhages may be found and pin point hemorrhages on
types of this virus, which have been recognized so the skeletal muscles are usually prominent [6].
far, i.e., serotype 1 and serotype 2 of which serotype 1 The acute phase of the disease lasts for 6-10 days
is considered pathogenic [4]. On account of the high and is characterized by atrophy of bursa along with
depletion of B-cells [7] in bursal follicles, the other
Copyright: The authors. This article is an open access article licensed
lymphoid organs such as spleen and cecal tonsils are
under the terms of the Creative Commons Attributin License (http:// also affected. Immunosuppression occurs in clinical
creative commons.org/licenses/by/2.0) which permits unrestricted
use, distribution and reproduction in any medium, provided the
and subclinical form where both humoral and cellular
work is properly cited. immune responses are compromised and thus making
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birds more vulnerable to other secondary infection(s) University [GADVASU]) as well as from owners of
and reduced response to vaccination [8]. respective farms.
Due to this, IBD is a major threat to the poul- The mortality recorded in these farms ranged
try industry both at the national and international from 3% to 18%. Morbidity was although high. Stress
level; as evidenced by the concluded submis- was put on considering only the representative sam-
sion of Farooq et al. [9], who calculated losses of ples from each outbreak. 33 birds suspected for dis-
Rs. 4523.99±447.56/- and Rs. 18,276.96±2388.91/- as ease were necropsied and gross lesions were noted.
amount of rupees loss per flock and per year for 1000 The relevant representative tissue samples such as
broilers, respectively, due to IBD in Mirpur and Kotli bursa, kidney, junction of proventriculus and gizzard,
districts of Kashmir. The potential for economic and heart were subsequently collected in 10% neutral
losses in the poultry industry also exists when any- buffered formalin.
time a new antigenic or pathogenic strain of IBDV is The tissues were processed and the 4 μ thick
introduced into a country or geographic region [10]. tissue sections were cut out of the paraffin embed-
Internationally, IBD is also reported endemic in cer- ded tissue blocks and stained with hematoxylin and
tain areas [11-13]. A total monetary loss of over three eosin staining as per the protocol of Bancroft and
billion Nigerian currency was reported by Musa Gamble [20] for routine HP.
et al. [14] over a period of 3-year recurrent outbreaks IHC
during years 2009, 2010, and 2011. List of OIE, For IHC studies, section(s) were taken on poly-L-
2015 [15], has included the disease in the list of noti- Lysine coated slide and then subjected to clearing and
fiable diseases. then rehydration. The antigen retrieval was carried out
Thus, regular vaccination and adoption of proper in citrate buffer using EZ-Retriever® (Biogenex, USA).
prophylactic measures are mandatory to diminish These slides were then washed in phosphate buffer
the occurance of this disease. In this context, study saline for 20 min. Serum blocking was done using nor-
by Fantay et al. [16] showed that the proper time for mal goat serum and subsequently non-specific binding
administration of the vaccine is 18 days post hatch and endogenous peroxidase blocking was followed by
with the management conditions in place at the farm. overnight incubation with chicken polyclonal to IBDV
As this disease has been focused in recent reviews (Abcam, United Kingdom). It was then followed by
regarding its causative agent [17] and the vaccina- 20 min incubation with horseradish peroxidase-conju-
tion [18,19], the present study is, therefore, mainly gated goat polyclonal secondary antibody to chicken
inclined toward the timely and reliable diagnosis of IgY-Fc (Abcam, UK). Color was developed with sub-
this threatening disease using gross, histopathology strate diaminobenzidine (vector) and counterstained
(HP), immunohistochemistry (IHC), and immunoflu- with Mayer’s hematoxylin stain. Omission of primary
orescent methods. antibodies was used for negative control.
Materials and Methods Immunofluorescence technique
Ethical approval For IHC-fluorescent, tissue sections on
The present study was conducted after the poly-L-Lysine coated slides were deparaffinized and
approval of the research committee and the Institutional rehydrated, and antigen retrieval was done in citrate
Animal Ethics Committee. buffer solution. Blocking of non-specific protein
AQ2 A total of six poultry farms were visited in and binding and endogenous peroxide was followed by
around the areas of Ludhiana and the other districts overnight incubation in primary antibody (Chicken
of Punjab, as shown in Table-1. Prior to the visits to polyclonal to IBDV, Abcam, UK). It was followed by
various farms as mentioned, necessary permission incubation with fluorescein isothiocyanate-conjugated
was sought with regards to collection of samples goat polyclonal secondary antibody to chicken IgY-Fc
from diseased birds and generation of data either from (Merck, India) for 20 min in the dark. Counterstaining
the competent authority of university administration was done using diamino phenylindole (5 mg/ml,
(Guru Angad Dev Veterinary and Animal Sciences Sigma, USA) for 10 min and after washing mounted

Table-1: Various farms visited for sample collection for suspected IBD outbreaks.

S. no. District of Punjab Number of Total birds Birds affected Age group of affected
samples collected (~%) group (weeks)
Farm I Khanna, Ludhiana 15 20,000 4 6
Farm II Chaukiman village 2 16,000 8 4
Farm III Fatehgarh Sahib 4 100,000 6 8
Farm IV Gauhar, Ludhiana 7 25,000 10 10
Farm V Kot Gangurai, Ludhiana 4 10,000 4 3
Farm VI Moga 1 Backyard farming 2 died out of 10 Variable
Total 33
IBD=Infectious bursal disease

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in an aqueous glycerol mounting media. Omission which led to erosion of the bursal epithelium. Sellaoui
of primary antibodies was used for negative control. et al. [27] also described similar degenerative changes
The slides were viewed under fluorescent microscope of the coating epithelium.
(Nikon eclipse). The grossly atrophied bursa(s) exhibited the
The antibody was standardized at the dilution of fibrosis in between the bursal follicles as evidenced
1:200 for the above two techniques. by the fibrous tracts running between them (Figure-3)
Statistical analysis
that was evidenced by pseudolobulation in histopatho-
The Graphpad Prism software was employed logical examination. Hemorrhages in the bursa along
to statistically ascertain the relationship between the
histopathological changes in the bursas from affected
birds and the immunohistopathological scoring of the
histopathologically positive samples. This immuno-
histopathological scoring was done using the scoring
scale of 0-3, as applied by Oladele et al. [21] where,
three sites in each tissue section were observed under
the microscope and scored.
Results and Discussion
A total of 33 cases during 6 outbreaks were
collected, of which 26 were suspected to be having
affected with the disease. It is worth to mention here
that the stress was put on considering only the rep- Figure-1: Correlation between histopathology (HP) score
resentative samples from each outbreak, although the and immunohistochemistry (IHC) score.
number of the birds affected was more.
Gross
The bursa of Fabricius in the affected birds aged
4-8 weeks were found showing significant lesions
in the birds, which were necropsied in five different
farms and showed the atrophic changes. These obser-
vations are in accordance with those reported by
Juranova et al. [22] and Khan et al. [23], while in the
sixth farm most of the necropsied birds were found to
have an enlarged and hemorrhagic bursa (Figure-1).
Besides the bursa, the junction of proventriculus and
gizzard were also hemorrhagic, as found by Islam and
Samad [24]. Renomegaly with congestion and hem-
orrhages (Figure-1) was noticed in most cases which
coincided with the finding of Khan et al. [23]; however,
the hemorrhages on thigh muscles were scarcely found Figure-2: Grossly enlarged and hemorrhagic bursa. The
in three cases. Haghighi et al. [5] also reported the kidneys are also enlarged and hemorrhagic in bird affected
similar lesions as hemorrhages in cecal tonsils, hyper- with infectious bursal disease.
emia of the thymus, bursal edema, and hyperemia with
enlarged and hyperemic kidneys in gross examination
of the experimentally IBDV infected birds.
HP
The histopathological changes in the bursa of
Fabricius mainly showed the fibrotic and atrophic
type of changes, which were in the consonance with
the gross observations, in which the bursa were found
atrophic. Histopathologically, there was rarefaction
of the bursal follicles along with the mixed cellular
infiltration in the bursal follicles. The cystic changes
in the bursal follicles (Figure-2) were also evident in
few cases. However, these findings were not in agree-
ment to the findings of Rosenberger [25] and Sharma
et al. [26], who reported absence of any serious
inflammatory bursal lesions. The desquamation and Figure-3: Cystic changes evident in the medullary region
sloughing of bursal epithelium was a frequent finding, in the bursal follicles (H and E, ×10).

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with the development of cystic cavities were quite dis- of proventriculitis with IBD found in three out of six
cernible either in bursal follicles and/or in the bursal studied outbreaks goes in accordance with the previ-
epithelium. The presence of cystic cavities along with ous findings by Pantin-Jackwood and Brown [35] and
the cellular accumulation in the bursal follicles was Grau-Roma et al. [36], who also reported that naturally
also reported by Guvenc et al. [28], whereas Murmu occurring proventriculitis can occur in the absence of
et al. [29] reported bursal lesions in vaccinated chick- IBDV and that the IBDV strains tested do not directly
ens, which were histopathologically characterized as produce proventriculitis.
either normal follicles with or without mild to mod- The section(s) from kidney(s) showed marked
erate lymphoid depletion and without follicular atro- congestion in the cortex and the medullary area along
phy or with the development of cystic follicles. In with the vacuolar tubular degeneration. The kidney
the present study, some of the bursal follicles showed showed eosinophilic fluid accumulation, suggest-
the degeneration of the core of the follicles along ing the proteinaceous nature of fluid along with the
with the presence of pink stained fluid in some of lymphomononuclear infiltration in the interstitium
the degenerating follicles. Typical histopathological and glomeruli. The cerebrum revealed congestion
lesions of the bursa of Fabricius was found similar to along with the mild perivascular lymphomononuclear
as reported by Hoque et al. [30], Islam et al., [31] and cellular cuffing around the blood vessel. The lungs
Rudd et al. [32] that included mild to severe lymphoid revealed congestion and desquamation of the epithelia
depletion in bursal follicles, follicular atrophy, cystic of parabronchi. The lungs here showed the infiltrative
formation of follicles, and bursal hemorrhage, but the changes in the parabronchi leading to its thickening.
hemorrhages in bursa were not seen in majority of the The liver samples of most of the birds from this
cases which was attributed to the collection of samples outbreak were normal except for some showing some
at the time of post-peak period of occurrence of out- degenerative changes as evidenced by congestion and
break, when majority of bursas had already undergone perivascular cellular infiltration. It was also interest-
atrophy. Ignjatovic et al. [33] reported widespread ing to find that the liver(s) from one outbreak to be
acute lymphoid necrosis, follicular hemorrhage and specifically associated with excessive severe fatty
stromal edema, indicative of acute IBD, the main change with lymphoid cellular infiltration was seen
findings reported by the scientists were lacking in as aggregate, this further suggests the involvement of
the present study (follicular hemorrhage and stromal mycotoxin-induced injury putatively to be attributed
edema),which is an innuendo to the chronic nature of to immunosuppressive effect [37]. The liver was also
the present outbreaks. found hyperemic with the round edges by Haghighi
The scoring of the various IBD lesions in bursa et al. [5].
of Fabricius was done, as shown in Table-2, based
on the criterion and method as adopted by previous Immunohistopathology

researchers such as Moraes et al. [34]. The affected bursal sections revealed viral anti-
As per the analysis of the bursal lesions in these gens in the lesion sites identified in tissue sections,
six different outbreaks, the lymphoid depletion was especially inside the cells in the rarefied areas of bursal
seen the highest in the fourth outbreak (2.86±0.899), follicles along with the inner lining of the cystic cav-
followed by fifth (2.75±1.89), and first (2.785±0.579) ities in affected follicles. In various previous exper-
outbreak, while it was least in the third outbreak imental studies, IBDV antigens have been detected
(0.75±1.5). In the similar way, the extent to which bur- in macrophages within follicles, the interstitium,
sas got fibrosed was also highest in the fourth outbreak and the lymphoid cells of bursa of Fabricius, e.g., by
(2.57±0.786), but unlike lymphoid depletion, it was Oladele et al. [21], but in the present study, the viral
followed by fifth (2.5±1.73) and then first outbreak antigens were found mainly associated with bursal
(2.285±1.20). The presence of cysts was highest in the lymphoid cells and in bursal epithelium in some cases
fifth outbreak (1.25±1.5) followed by first (0.571±1.22) (Figure-4). The findings can be related to the findings
and third (0.5±1). On statistical analyses of the extent of Tippenhauer et al. [38] who reported higher IBDV
of lymphoid depletion, fibrosis, cystic spaces, and con- antigen load in bursa of Fabricius of layer type birds,
gestion/hemorrhage in the first and third outbreaks, it in addition to the clinical signs and death rate when
is proposed that these four histopathological changes compared to the broiler type birds, in their differential
follow a parallel trend in the pathogenesis of the dis- immunopathogenic study. The coinciding fact in the
ease. The extent of proventriculitis although followed present study is being the consideration of mainly layer
unrelated trend to these four changes in bursa, it was type birds. Nunoya et al. [39] and Tanimura et al. [40]
not only concomitantly found in outbreaks which were detected antigen in cytoplasm of lymphocytes, epithe-
marked by extensive bursal histopathological changes, lial cells, and inflammatory cells (mostly macrophages)
i.e., first (0.929±1.141) and fifth (2±0), but also in in the bursa of Fabricius of IBDV infected chickens, but
the outbreak that exhibited minimal bursal lesions, Jonsson and Engstrom [41], however, observed antigen
i.e., outbreak three (2±0). Thus, it is suggested that the only in bursal lymphocytes, which goes in exact accor-
IBDV act as a contributor to proventriculitis, rather than dance with the findings of the present study. In addition
being solely responsible for the same. The association to this, the findings of the present study also coincided
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Table-2: The scoring of the bursal lesions in six outbreaks.

Case no. Lymphoid Fibrosis Cystic Edema Congestion/ Proventriculitis

depletion spaces hemorrhage
OUTBREAK 1
JB02A 2 0 0 0 2 2
JB05B 3 2 0 0 0 2
JB08A 2 3 0 0 2 0
JB10A 3 2 0 2 0 0
JB11A 3 3 4 2 2 2
JB13B 3 3 0 0 0 0
JB17C 3 4 0 0 0 0
JB18A 3 2 2 0 0 3
JB19A 3 2 0 0 0 0
JB10B 2 0 2 0 0 2
JB22B 4 3 0 0 1 2
JB23B 3 2 0 0 0 0
JB25b 3 4 0 0 0 0
JB27 2 2 0 0 0 0
2.785±0.579 2.285±1.20 0.571±1.22 0.285±0.0726 0.5±0.855 0.929±1.141
OUTBREAK 2
JB34 4 3 0 0 0 0
OUTBREAK 3
JB39B 3 3 2 0 0 2
JB40 0 0 0 0 0 2
JB41 0 0 0 0 0 2
JB42 0 0 0 0 0 2
0.75±1.5 0.75±1.5 0.5±1 2±0
OUTBREAK 4
JB50B 3 3 0 0 2 0
JB50C 2 2 0 0 0 0
JB51B 4 4 0 0 0 0
JB51C 2 2 0 0 0 0
JB52 2 2 0 0 0 0
JB53B 4 3 0 0 0 0
JB53A 3 2 0 0 0 0
2.86±0.899 2.57±0.786 0.285±0.756
OUTBREAK 5
JB62A 4 3 3 3 0 2
JB62B 3 3 2 0 2 2
JB63A 4 4 0 0 0 2
JB65 0 0 0 0 0 2
2.75±1.89 2.5±1.73 1.25±1.5 0.75±1.5 0.5±1.0 2±0
OUTBREAK 6
JB71A 2 2 0 0 0 0

tissues, where the viral antigens were found dispersed

as fine to coarse granules within the infected or degen-
erated cells.
The presence of viral antigen was also appreciated
in the bursal interfollicular connective tissue (Figure-5)
as well as the bursal epithelium which was also
reported by Oladele et al. [21]. The distribution of viral
antigen varied from being localized (Figure-6) to focal
to diffuse. In addition, the junction of proventriculus
and gizzard (Figure-7) was also found to be expressing
the IBDV antigen. The IBDV antigens were also local-
ized in the respiratory ciliated epithelium (Figure-8) at
the affected sites in the proventricular sections and in
the heart muscle (Figure-9), whereas Pantin-Jackwood
and Brown [35] did not report any IBDV antigen in the
Figure-4: Ratification of the follicular region in the bursa
along with the inter-follicular fibrosis (Masson trichrome, proventriculus. Hemalatha et al. [42] also reported the
×20). similar IHC results in 100 1-day-old experimentally
infected white leghorn male chicks, where the reac-
with the findings of Haghighi et al. [5] in context to the tion of varying intensities was seen in isolated lym-
viral antigen distribution within the cells of different phoid cells in cortex and surface epithelium of bursa
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of Fabricius. The similar reaction was also seen in the Besides this, the immunofluorescent technique
cortical lymphocytes of thymus and in few glandular was also employed that further confirmed/supple-
epithelial cells of Harderian gland depending on the mented the presence of IBDV in the cystic lesions in
days post-infection. bursal follicles (Figure-10), which was in consonance

Figure-5: The affected bursal section showing infectious Figure-8: The junction of proventriculus and gizzard
bursal disease viral antigens in the lesion sites identified in expressing the infectious bursal disease virus antigens
bursal follicles (Immunohistochemistry, ×4). (Immunohistochemistry, ×4).

Figure-6: Bursal interfollicular connective tissue as well as

Figure-9: Respiratory ciliated epithelium showing
the bursal epithelium showing the presence of infectious
the infectious bursal disease virus antigens
bursal disease viral antigen (Immunohistochemistry, ×20).
(Immunohistochemistry, ×20).

Figure-7: A bursal section demonstrating the focally

extensive localization of infectious bursal disease viral Figure-10: The infectious bursal disease virus antigen in
antigens (Immunohistochemistry, ×40). the heart muscles (Immunohistochemistry, ×20).

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with the findings of Mahgoub [17] and Van den Berg Statistical analysis
et al. [43]. In addition, Hemalatha et al. [42] detected To statistically ascertain the relationship
the viral antigens by specific fluorescence in lym- between the histopathological changes in the bursas
phoid cells, plical epithelium, macrophages in bursa from affected birds and the immunohistopathologi-
of Fabricius, and Kupffer cells in liver and proventric- cal scores, the immunohistopathological scoring was
ular glandular epithelial cells. done using the scoring scale of 0-3, as applied by
Oladele et al. [21], as shown in Table-3. For a statisti-
cal comparison to be drawn, the mean and the standard
deviation of the respective histopathological changes
were also calculated, as shown in Table-4. This mean
represented the overall damage to the histo-architec-
ture, which was then compared with the IHC scoring
of the 27 histopathologically positive samples (out of
33 total samples) using the Graphpad Prism software.
The correlation coefficient between the mean
histopathological scoring and the IHC scoring was
also found to be positive, 0.64623 for the respective
cases, which proves the reliability on IHC for the pos-
itive cases. Further, it can also be represented graph-
ically, as depicted in Figure- 1, where both of the
curves follow similar trend: At 95% confidence inter-
AQ2 Figure-11: Infectious bursal disease virus antigen in the
val, the slope: 0.1219-0.4220, with R²=0.3580 and at
cystic lesions in bursal follicles (IFT, ×20). p<0.001, as shown in Table-5.
Conclusion
Table-3: Immunohistopathological scoring criterion.

IHC positive cell (per×40 objective Score

It is concluded that the bursa is the main organ
lens of the light microscope) involved in the pathogenesis of IBD and thus also
<5 stained cells 0
acts as an organ of choice for demonstrating IBDV
5-50 stained cells 1 antigen for specific diagnosis of disease using IHC.
50-150 cells 2 Although IHC method of diagnosis has previously
Over 150 stained cells 3 been compared with other methods as well [44], yet
IHC=Immunohistochemistry here it yielded obvious results. It is concluded that
Table-4: Overall histopathological scoring and the IHC scoring, as per Oladele et al.[21].

Slide no. Lymphoid depletion Fibrosis Cysts Edema Congestion Mean Standard deviation IHC score
JB02A 2 0 0 0 2 0.8 1.095445 0
JB05B 3 2 0 0 0 1 1.414214 0
JB08A 2 3 0 0 2 1.4 1.341641 2
JB10A 3 2 0 2 0 1.4 1.341641 2
JB11A 3 3 4 2 2 2.8 0.83666 3
JB13B 3 3 0 0 0 1.2 1.643168 3
JB17C 3 4 0 0 0 1.4 1.949359 2
JB18A 3 2 2 0 0 1.4 1.341641 1
JB19A 3 2 0 0 0 1 1.414214 0
JB10B 2 0 2 0 0 0.8 1.095445 0
JB22B 4 3 0 0 1 1.6 1.81659 3
JB23B 3 2 0 0 0 1 1.414214 2
JB25b 3 4 0 0 0 1.4 1.949359 2
JB27 2 2 0 0 0 0.8 1.095445 2
JB34 4 3 0 0 0 1.4 1.949359 3
JB39B 3 3 2 0 0 1.6 1.516575 3
JB50B 3 3 0 0 2 1.6 1.516575 2
JB50C 2 2 0 0 0 0.8 1.095445 0
JB51B 4 4 0 0 0 1.6 2.19089 3
JB51C 2 2 0 0 0 0.8 1.095445 1
JB52 2 2 0 0 0 0.8 1.095445 2
JB53B 4 3 0 0 0 1.4 1.949359 2
JB53A 3 2 0 0 0 1 1.414214 2
JB62A 4 3 3 3 0 2.6 1.516575 3
JB62B 3 3 2 0 2 2 1.224745 3
JB63A 4 4 0 0 0 1.6 2.19089 2
JB71A 2 2 0 0 0 0.8 1.095445 2
IHC=Immunohistochemistry

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Table-5: Correlation between HP score against infectious bursal disease in broilers in Mirpur and Kotli dis-
immunohistopathological score in different samples of IBD. tricts of Kashmir. Int. J. Poult. Sci., 2: 267-270.
10. Jackwood, D.J. and. Sommer-Wagner, S.E. (2010) Detection
Pearson’s correlation Slope p value R2 and characterization of infectious bursal disease viruses in
coefficient (r) (95% CI) broilers at processing. Prev. Vet. Med., 97: 45-50.
0.64623 1.392±0.3288 0.0003 0.4176 11. Lawal, J.R., Jajere, S.M., Bello, A.M., Mustapha, M.,
(0.7148-2.069) Wakil, Y., Ndahi, J.J., Mustapha, F.B., Paul, B.T.,
Gulani, I.A., Ibrahim, U.I., Geidam, Y.A., Ambali, A.G. and
CI=Confidence interval, IBD=Infectious bursal disease,
Waziri, I. (2014) Prevalence of infectious bursal disease
HP=Histopathology
(Gumboro) antibodies in village chickens in Gombe state,
northeastern Nigeria. Int. J. Poult. Sci., 13: 703-708.
IHC staining is a precise, specific, rapid, and reli- 12. Mbuko, I.J., Musa, W.I., Ibrahim, S., Saidu, L., Abdu, P.A.,
able method to demonstrate the IBDV antigen in the Oladele, S.B. and Kazeem, H.M. (2010) A retrospective
analysis of infectious bursal disease diagnosed at poultry
altered tissues due to IBDV infection, which is in con- unit of Ahmadu Bello University, Nigeria. Int. J. Poult. Sci.,
currence with gross or microscopic lesions and sup- 9: 784-790.
plemented by immunofluorescent technique. 13. Anosa, G.N. and Eze, J.I. (2010) A sero-epidemiological
survey of infectious bursal disease in scavenging village
Authors’ Contributions chickens in Enugu State. Niger. Vet. J., 3: 267-270.
14. Musa, I.W., Saidu, L. and Abalaka, E.S. (2012) Economic
JS and HSB initiated research concept and impact of recurrent outbreaks of gumboro disease in a com-
design, collection and analysis of data was com- mercial poultry farm in Kano, Nigeria. Asian. J. Poult. Sci.,
piled by JS, NDS, SS, and GDL. The interpretation 6: 152-159.
of HP was done by HSB and JS. Slide’s photography 15. OIE. (2015) List. Available from: http://www.oie.int/en/ani-
mal-health-in-the-world/oie-listed-diseases-2015. Accessed
was done by JS and GDL. HSB and RSB critically on 23-06-2015.
reviewed the article, while the final approval of the 16. Fantay, H., Balcha, E., Tesfay, A. and Afera, B. (2015)
article was done by JS, HSB, SS, NDS, and RSB. Determining optimum time for administration of live inter-
mediate vaccine of infectious bursal disease to chickens at
Acknowledgments Mekelle farm. J. Vet. Sci. Technol., 6: 223-227.
Authors are sincerely thankful to the Director of 17. Mahgoub, H.A. (2012) An overview of infectious bursal
disease. Arch. Virol., 157: 2047-2057.
Research, GADVASU, for facilitation and funding of 18. Rekha, K., Sivasubramanian, C., Chung, I. and
this work. The Dean, College of Veterinary Science, Thiruvengadam, M. (2014) Growth and replication of infec-
GADVASU, is also acknowledged for providing nec- tious bursal disease virus in the DF-1 cell line and chicken
essary facilities toward completion of this research embryo fibroblasts. Biomed. Res. Int., 2014: 6.
19. Muller, H., Mundt, E., Eterradossi, N. and Islam, M.R.
project. (2012) Current status of vaccines against infectious bursal
Competing Interests disease. Avian Pathol., 41: 133-139.
20. Bancroft, J.D. and Gamble, M. (2002) Theory and Practice
The authors declare that they have no competing of Histological Techniques. 5th ed. Churchill Livingstone
interests. Publisher, Edinburgh. p172-175, 593-620.
21. Oladele, O.A., Adene, D.F., Obi, T.U. and Nottidge, H.O.
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