CLINICO PATHOLOGICAL STUDY AND MOLECULAR DETECTION OF IBD INFECTED BROILERS IN BASRAH PROVINCE

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


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).

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.

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)

Histopathological Study
The Histopathological according to (9).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).whofound 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.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).ourresults were in line with that of (19), who showed mild

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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 ):The
Figure (1): Gross section of infected bird showed depression and ruffled

Figure ( 2 )
Figure (2): gross section of infected bird showed swelling and enlargement of bursa

and
run at eighty V for one hour, Images of the gels were photographed on BioDoc Analyze Digital Systems (Biometra, GermanyTable (2):The Reaction Mixture (50 μl) for PCR amplification of (IBDV).Table (3): The PCR amplification forSteps Pre-