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CLINICO PATHOLOGICAL STUDY AND MOLECULAR
DETECTION OF IBD INFECTED BROILERS IN BASRAH
PROVINCE
Dheyaa abbas abdali , Waleed Majeed Seger Almayahi
Department of Pathology and poultry diseases., College of Veterinary Medicine,
University of Basrah,Basrah,Iraq.
(Received 22 August 2020 ,Accepted , 22 September 2020 )
Keywords; IBD, Broilers, Clinical Finding.
Corresponding author: [email protected]
ABSTRACT
IBD is a highly contagious viral disease which inhibit the immune system of
chickens, which is responsible for major economic losses within the poultry industries
worldwide. A total of 80 samples were collected from 8 different broiler farms from
Qurna, Midyanah, Qarmat Ali, Zubair. Upon the clinical finding and postmortem
lesions of the necropsied birds; bursa of fabricius, kidney, Junction between gizzard
and proventriculus and thigh muscles were processed for histopathological and
molecular detection with PCR. The clinical signs were depression, pasty vent and
white dropping. Various overall changes are observed in the bursa, such as swelling,
hemorrhages to atrophy in size; In addition, hemorrhages were seen in the thigh
muscles. The histopathological changes of Bursa of fabricius showed follicular
vacuolation and vascular congestion ;multiples degrees of hemosiderin deposition,
and the edema accumulate between follicles and basement membrane ; also, showed
moderate infiltration of inflammatory cells and accumulation of inflammatory cells
lymphocytes macrophage , plasma cells as well to showed a congestion of the bursal
capsule, kidney microscopic findings renal vascular congestion in the cortex and also
the medullary area along with the vacuolar degeneration. The suspected tissue
samples were assayed using RT-PCR for IBDV targeting VP2 gene. Out of the tested
samples 15 were Positives. In spite of using IBD vaccines in different farms of the
studied areas; the present study was detected IBD in different areas of Basrah
province by PCR and mentioned clinical and histopathological finding, therefore it's
necessary to study the sequence analyses of such disease in future.
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INTRODUCTION
Infectious bursal disease is a highly contagious viral disease, which affects young
chickens. It is immunosuppressive disease of young chickens (1). Which belongs to
the Avibirnavirus genus within the Birnaviridae family, this disease involves two
types of serotypes: (serotype one and serotype two) (2). It is conjointly known as
'Gumboro disease' per the place wherever its 1st unfold in Gumboro, Delaware, USA.
This disease was at the start delineated as avian nephrosis due to the symptoms seen
within the kidneys however was later selected infectious bursal disease (IBD), per the
structural and histological changes that was seen within the bursa of Fabrice's (3).
The IBD causes distraction of lymphoid organs, especially differentiating
lymphocytes in the bursa (4).
This disease causes significant direct and indirect economic losses to the
poultry industry and poultry farmers around the world (5). Signs of
immunosuppression include the inability to respond to vaccines adequately antibody
and an increase of the secondary infections (6). Classical forms of disease outbreak
may lead to a mortality rate of 50% and in broilers from 3-6 weeks of age that may
exceed 3%. Include the main clinical signs depression, watery diarrhea, ruffled
feathers, tremors, loss of appetite and death after 2-3 days after the clinical signs
onset (7). The preferred method of controlling this disease is through timely
vaccination (8).
MATERIALS AND METHODS
Samples collection: The suspected samples were taken from broiler chicks which
showed clinical signs related to that of IBD like depression ,pasty vent ,white
dropping and lesions as hemorrhage of bursa ,enlargement or edematous bursa and
enlarged kidney such samples collected from different areas of Basra province
;Qurna , Midyanah , Qaramt Ali ,Zubair , during the period of extend ( August ,
2019 – February , 2020). The suspected samples were included bursa of fabricius,
kidney, junction between gizzard and proventriculus, thigh muscles then subjected to
histopathological preparation and molecular detection with PCR.
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IBDV detection by RT-PCR
RNA Extraction: The kit for genomic RNA was used, the favor prep tissue
Genomic RNA Extraction Mini Kit (Viral Nucleic Acid Extraction Kit II) animal
tissue according to the instructions of manufacturers with modification (Geneaid
Company). A set of primers (table 1) were used for the RT-PCR reaction, such
primers used to amplification of a 523 bp fragment within vp2 gene.
(https://blast.ncbi.nlm.nih.gov/Blast.cgi)
Table (1): The sequence of primers.
Name of
primer Sequence Tm(c)
Length
of
primer
VP2-FWD (5’ – GGG GAG AAC TCG TGT TTC AA – 3’) 55
523 bp
VP2-REV (5’ – CTT GTT GGC CAT ACG GTC TT – 3’) 55
PCR amplification
Amplification of PCR was used the AccuPower® PCR PreMix kit that contains
DNA polymerase, (Bioneer company), dNTPs and reaction buffer in a premixed
format that is vacuum-dried into an individual packet. Briefly, the 50 μl of PCR
reaction mixture was used in the current study as shown in table (2) .A fragment of
523 bp of the vp2 gene of IBDV amplified by PCR thermo cycling using
(Appliedbiosystem) as explained in table (3). Seven μl of PCR products were
analyzed by electrophoresis from amplified sample on 2 %agarose gel containing 0.5
ul/25ml ethidium bromide, and run at eighty V for one hour, Images of the gels were
photographed on BioDoc Analyze Digital Systems (Biometra, Germany
Table (2): The Reaction Mixture (50 μl) for PCR amplification of (IBDV).
No. Chemical Amount
1 Master Mix 5.0 μl.
2 Primer Forward ( VP2 ) 1.0 μl.
3 Reverse primer (VP2) 1.0 μl.
4 Template of DNA 10.0 μl.
5 D.W. 33.0 μl.
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Table (3): The PCR amplification for
Steps
Pre- Denaturation
Denaturation
Annealing
Extension
Final Extension
Histopathological Study
The Histopathological
according to (9).
1-Clinical finding and postmortem:
studied areas in Basrah province were
common clinical signs were
watery white diarrhea. While
edematous bursa and enlarged kidney, hemorrhage in thigh muscle and junction
between gizzard and proventriculus.
Figure (1): Gross section of infected bird showed depression and ruffled
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VP2 gene
Temp. Time
95 ºC 5 mins.
95 ºC 20 sec.
55 ºC 20 sec.
72 ºC 30 sec.
72 ºC 3 mins
pathological preparation of the IBDV infected tissues
RESULTS
Clinically, some of the visited broiler farms in
showed clinical signs related to IBD .
anorexia, depression. Ruffled feathers, pasty vent and
the lesions were hemorrhage of bursa, enlargement or
Figures (1,2)
ross PCR cycles
1 cycle
35 cycle
1cycle
was
The most
feathers.
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Figure (2): gross section of infected bird showed swelling and enlargement of bursa
Molecular Diagnosis
The suspected samples,
reverse transcriptase-polymerase chain reaction (RT
using partial VP2 gene, all (15) RT
523 bp on agarose gel figure (
Figure (3): Agarose gel electrophoresis of PCR product
8) positive simple from infected farms, positive control (7).
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of fabricius (black arrows).
like bursa of fabricius and kidney were
RT-PCR) for IBDV detection,
RT-PCR positive samples showed specific bands at
3)
L= DNA ladder line (1, 4, 5,
subjected to
by
Bas.J.Vet.Res.Vol.19, No.2, 2020.
Histopathological results:
different pathological changes in different organs related to chicken infected with
IBDV which showed variable changes included in kidney like renal vascular
congestion in the cortex and also the medullar
degeneration (figure: 4); The histopathological results of Bursa of
different types of severity of the pathological changes vary from a follicular
vacuolation and vascular congestion (figure:
inflammatory cells and accumulation of inflammatory cells lymphocytes macrophage
, plasma cells as well to showed a congestion of the bursal capsule (figure:
Histopathological results of the junction between proventricul
marked area of muscular tissue vascular congestion (figure:
Figure: (4): Histopathologic section of
arrow), interstitial hemorrhage (white
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The histopathological results of IBD chicken showed
medullary area along with the vacuolar
fabricius
5), also, showed moderate infiltration of
proventriculus and gizzard showed a
7).
Kidney. Renal vascular congestion (black
arrow) H&Estain 125X
y showed
6 ). The
us
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Figure (6). Histopathologic section of Bursa. Bursa reveal vascular congestion (red
arrow), accumulation of inflammatory cells, including; lymphocytes (blue arrow),
macrophage (green arrow) plasma cells (black arrow). (Score+2) H&E stain 500X
Figure (5). Histopathologic section of Bursa. Bursa reveal vascular congestion (red
arrow), Edema in the basement membrane (yellow arrow) (score +1) H&Estain
500X
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DISCUSSION
IBDv causes the massive economic losses through high mortality and
immunological disorder in poultry. The current study showed the infected chicks
become loss of Appetite, depressed, ruffled feathers, pasty vent and Watery White
diarrhea. The characteristic gross lesions of IBD infected chicks were dehydration of
the muscles with ecchymosis hemorrhages and enlargement of kidneys.
The bursa becomes enlarged or edematous and shows pale yellow
discoloration. Intra-follicular hemorrhages could also be found and pin point
hemorrhages on the thigh muscles, the present results were related to that of (10).who
found clinical signs and lesions of IBD infected chicks like depression , pasty vent
and changes of bursa of fabricius . these results occur due to the IBV which cause
increase in the body temperature leading to decrease movement and depression, also
the viral infection causing pasty vent and white dropping may related to nephrosis
with enlarged kidneys theses idea agreed with (11) who reported that infectious
bursal disease in the suspected birds showed trembling, ruffled feathers, depression,
and droopy appearance.
Figure (7): Histopathologic section of Junction between proventriculus and gizzard;
muscular tissue shows mild vascular congestion (black arrow) H&E stain 125X
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The target organ of IBDV is the bursa of fabricius, the current study showed
hemorrhage of bursa, enlargement or edematous bursa, these result occur due to cells
infiltration; heterophils and macrophages in the interfollicular space that’s may agree
with (12). Who reported that hemorrhagic atrophied bursa and enlarged edematous
bursa.
The molecular part of our study that performed by using PCR technique
which regarded most powerful, tools for diagnosis of IBD, also it is, very sensitive
and specific technique to detect such infection in the chicken, were detect presence
of IBD in the studied farms ; such idea in line with (2) that reported Several different
tests are used to detect the IBD, which do not have ability to detect low levels of
IBDV antigens in tissues, while the a fast, specific and sensitive method to detect
IBDV is RT-PCR .The current results of of PCR detection were showed positive
VP2 gene in the suspected IBD samples, these occur due to the VP2 gene is the
major structural protein that builds the viral capsid and used for molecular &
epidemiology and phylogenic studies, these result in agree with (13).
The identification of the IBDV genotype was performed per a and therefore
the molecular represented RT-PCR aimed toward the hypervariable region of VP2
(14). As a part of the pathological process of IBD that may primarily be explained in
histopathological changes in relation to the impact of the virus in many organ among
others, liver and excretory organ (15).
Our results were included histopathological changes ; renal vascular
congestion in the cortex and also the medullary area along with the vacuolar
degeneration &interstitial necrosis; these occur due to aggregation of inflammatory
cells in the renal parenchyma as a result of viremia, these result agreed with (16) ,who
reported Kidneys vacuolar degeneration of the tubules, glomerular shrinkage and
hemorrhages of IBD diseased chicks ,also these results were related to (17) who
showed enlargement of kidney in some cases of infectious bursal diseased chicken.
The current histopathological study of Bursa of fabricius showed different types of
pathological changes vary from a follicular vacuolation, vascular congestion &
follicular vacuolation , that may be occur due to site of replication and its target organ
of IBDV ; these results were related to the ( 18 ) who mentioned Rarefaction of
bursal follicles with intermittent infiltration of lympho-mononuclear cells; also the
histopathological changes like necrosis and hemorrhages in bursa of fabricius were
detected by (15).our results were in line with that of (19), who showed mild
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lymphocyte depletion from bursal follicles, congestion of blood vessels،edema &
infiltration of mononuclear cells in interfollicular spaces. Also the junction between
proventriculus and gizzard showed a marked area of muscular tissue vascular
congestion and present of moderate intravascular infiltration of inflammatory cells,
these occur duo to viral infection of the chicken causing viremia and then distribution
of the virus in the thigh muscle and junction between proventriculus and gizzard
leading to replication of the virus these causing dilation of blood vessels as a result of
infiltration of inflammatory cells against the viral infection lead to damage of blood
vessels, these result agreed with (20), who showed the junction of proventriculus and
the gizzard, includes congestion, hemorrhage and infiltration of heterophils at the
junction.
دراسة سریریة نسیجیة مرضیة والتحری الجزیئی لفیروس التھاب جراب فابریشیا المعدی فی
افراخ فروج اللحم فی محافظة البصرة.
ضیاء عباس عبد علی , ولید مجید صکر المیاحی
فرع الامراض وامراض الدواجن، کلیة الطب البیطری , جامعة البصرة ،البصره ،العراق.
الخلاصة
التھاب جراب فابریشیا المعدی ھو مرض شدید العدوى ومثبط للجھاز المناعی فی افراخ فروج اللحم ،
وھو المسؤول عن الخسائر الاقتصادیة الکبیرة فی صناعات الدواجن فی جمیع أنحاء العالم.. تم جمع ٨٠ عینة
من ٨ مزارع دجاج تسمین مختلفة من القرنة ، المدینھ ، کرمة قرمة علی ، الزبیر. اعتمادا على النتائج السریریة
والآفات بعد الوفاة للطیور التی تم تشریحھا ، تم اخذ العینات من انسجة مختلفة متمثللھ ؛جراب فابریشیا ، الکلى
، منطقة الربط بین المعده العضلیة والغدیھ ، والعضلات للکشف عن التغیرات النسیجیھ والتحدید الجزیئی
العلامات السریریة تضمنت الخمول واسھال مائی ابیض مع تبلیل ریش نقطة المجمع. .PCR بواسطة
ولوحظت ایضا تغیرات مرضیة مختلفة فی جراب فابریشیا ، مثل تورم ، نزیف إلى ضمور فی الحجم ؛ کما
شوھد نزیف على عضلات الفخذ. أظھرت التغیرات المرضیة النسیجیة لجراب فابریشیا فجوة فی الجریبات
واحتقان الأوعیة الدمویة ، وتتضاعف درجات ترسب الھیموسیدیرین ، وذمة تتراکم بین الجریبات والغشاء
القاعدی. أیضا ، أظھر ارتشاح تسلل معتدل للخلایا الالتھابیة وتراکم الخلایا اللمفاویة الضامھ، وخلایا البلازما
أیضا لإظھار احتقان لجراب فابریسیوس، ونتائج التغیرات المرضیة النسیجیة للکلیة اظھرت احتقان الأوعیة
الکلویة فی القشرة ومنطقة النخاع جنبا إلى جنب مع تنکس أنبوبی فجوی. تم فحص عینات الأنسجة المشتبھ بھا
من بین العینات التی تم اختبارھا ، .VP یستھدف الجین 2 IBDV تفاعل تسلسلی ل .(RT-PCR) باستخدام
على بعض الطیور فی المناطق المدروسھ؛ تم تسجیل IBD کانت ١٥ إیجابیة. على الرغم من استخدام لقاحات
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وذکر التقنیات السریریة (PCR) فی مناطق مختلفة من محافظة البصرة بواسطة IBD الدراسة الحالیة
والمختبریة ، لذلک من الضروری دراسة التحلیلات التسلسلیة لمثل ھذا المرض فی المستقبل.
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