الفهرس 
Acknowledgment
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Abstract
Infectious bursal disease (Gumboro) is an acute, highly contagious and economically important immunosuppressive viral disease of poultry that is found worldwide. The present study was conducted to investigate the pathological changes caused by local strain of IBDV in lymphoid and non-lymphoid organs of broiler chicken using gross and microscopic examinations. TUNEL assay used to determine the mechanism of lymphocyte cell death due to IBDV. To evaluate the immunopathological response in IBDV infected broiler chicken we used immunohistochemical technique.
To fulfill our goals, 160 broiler chicks (17-35 days old) from outbreaks of suspected infectious bursal disease were collected from 18 farms in Al-Minya, Assiut, Sohag and Quina governorates. Collection of samples was conducted during the period between (January/2016 to September/2016). The collected chicks showed sudden onset of high mortality and revealed haemorrhages and gelatinous exudates in bursa of Fabricius at necropsy. Detailed history of the flocks regarding number of birds, age and vaccination schedule was obtained. Clinical signs, mortality and morbidity rates were also recorded. All freshly dead or slaughtered chicken were examined for the presence of gross lesions of IBDV infection. In addition, 10 broiler chicks (three weeks old) were collected from healthy farms and used as control. Tissue samples from various organs including; Bursa of Fabricius, thymus, spleen, cecal tonsils, kidney, proventriculus, intestine, heart, lungs and liver were quickly removed and fixed immediately in 10% neutral buffered formalin for histopathological, immunohistochemical studies as well as TUNEL assay.
All farms had a history of mortality rates ranged from 15% to 30% and high morbidity rates. Clinically, infected birds showed depression, trembling, anorexia, white and watery diarrhea, prostration, ruffled feather, staining of pericloacal feathers with white matter and death.
The gross findings were expressed by the presence of petechial and ecchymotic haemorrhages in the breast and thigh muscles. Swollen, congested, haemorrhagic or atrophied bursa and spleen samples were also described. There were petechial and ecchymotic haemorrhages in the cecal tonsils and at the junction between proventriculus and gizzard. Congested thymus, swollen pale kidney and pale liver were also observed.
Upon histopathological examination, bursal lesions observed in all examined samples were assigned a lesion severity score (+, ++, +++). Based on the severity of these lesions, bursal samples were divided into three lesion categories (mild, moderate, severe).
Histopathologically, the main findings reported in the bursa of Fabricius from IBDV infected birds were; congestion of stromal and follicular blood vessels, swollen endothelial, vacuolation of tunica media, stromal edema, inflammatory cellular infiltration, reticuloepithelial cells hyperplasia, necrosis of the follicular cells with karyorrhexis of lymphocytic nuclei, depletion of lymphocytes, proliferation of subepithelial and interfollicular connective tissue and bursal follicular atrophy. Each of these observations took mild, moderate and severe lesion severity scores in mild, moderate and severe lesion categories respectively. Marked hyperplasia of the follicular epithelium with extensive epithelial infolding and epithelial cysts formation were characteristic findings in mild lesion category. Moderate lesion category showed necrosis and sloughing of the lining epithelium in some cases and epithelium hyperplasia with epithelial cysts in others. Severe lesion category characterized by the presence of follicular medullary cysts lined by hyperplastic reticuloepithelial, intrafollicular acinar structures formed of metaplastic reticuloepithelial cells and lined by pseudostratified like epithelium, massive follicular and stromal haemorrhage, complete loss of the bursal architecture, ulceration of the lining epithelium, replacement of the bursal follicles by hyperplastic epithelium in some cases. In addition, haemorrhage, necrotic tissue debris and heterophil cells exudate were seen in the bursal lumen in severe lesion category.
Histopathological examination of thymus from diseased birds revealed that thymuses followed bursa of Fabricius of the same birds in severity of the lesion scores. Therefore, all thymus samples were also divided into three categories (mild, moderate, severe). Thymic sections revealed vascular changes in cortical and medullary blood vessels which represented by congestion, edema, swollen endothelial cells and vacuolation of tunica media and haemorrhage. Necrosis and depletion of cortical and medullary lymphocytes associated with karyorrhexis of lymphocytic nuclei, medullary heterophils cellular infiltration and reticuloepithelial cells hyperplasia were also demonstrated. Similar to bursa, all of these observations present in all lesion categories and expressed mild, moderate and severe scores in mild, moderate and severe lesion categories respectively. Apparently increased number of Hassles corpuscles was demonstrated in moderate and severe lesion categories.
Similar to thymus, histopathological examination of the spleen samples revealed that spleens followed BF of the same birds in severity of the lesion scores and divided into three categories (mild, moderate, severe). Spleen sections showed vascular changes, inflammatory cellular infiltration, and exhaustion of lymphocytes in both white red pulps. Similar to bursa and spleen, all of these observations present in all lesion categories and expressed mild, moderate and severe scores in mild, moderate and severe lesion categories respectively.
Unlike thymus and spleen, cecal tonsils didn’t follow the bursa of the same birds in the severity of histopathological lesions. Some cases exhibited only mild focal depletion of lymphoid cells. Other cases showed moderate lymphocytic necrosis and depletion, while the remaining samples revealed massive destruction of lymphoid cells with complete loss of the cecal tonsilar structure.
Histopathological examination of non-lymphoid organs showed nearly similar lesions in all examined birds regardless the different bursal lesion category. Kidney from IBDV infected chicken showed angiopathic changes, and focal lympho-plasmocytic cellular infiltration between the degenerated tubules with marked lymphocytic karyorrhexis. Swollen renal glomeruli with obliteration of Bowman’s space, glomerular necrosis, eosinophilic casts in the tubular lumen, cystic dilatation of the renal tubules, necrobiotic changes in renal tubular epithelium, heterophil cells infiltrating the interstitial tissues and in lumen of some renal tubules were observed.
In proventriculus of IBDV infected birds, angiopathic changes in mucosal and submucosal blood vessels, necrosis and sloughing of mucosal epithelium, mucosal lymphocytic cellular infiltration associated with lymphocytic karyorrhexis and apoptosis as well as necrotic and fibrinous proventriculitis were characteristic findings.
Intestine showed congestion of mucosal and submucosal blood vessels, severe necrosis and desquamation of the lining epithelium, necrobiotic changes in the submucosal glandular structures as well as fibrinonecrotic enteritis in which large fibrin cast and necrotic debris were detected in the intestinal lumen.
Heart of IBDV infected broiler birds showed angiopathic changes in interstitial blood vessels, perivascular haemorrhage and lymphoid cellular infiltration.
Lung sections revealed bronchiolitis, congestion of interalveolar capillaries, perivascular lymphoid cellular infiltration and thickening of interlobular septa with edema and mononuclear cellular infiltration.
Histopathological examination of the liver from IBDV infected birds showed angiopathic changes represented by swollen endothelial cells, vacuolation of tunica media, perivascular edema and lymphoid cellular infiltration as well as multiple thrombi. Necrobiotic changes in hepatocytes, kuppfer cells activation as well as multiple areas of infarction were seen.
The TUNEL assay results demonstrated that, the highest number of apoptotic cells was observed in bursa of Fabricius from moderate lesion category followed by severe and then mild lesion category. In thymus and spleen higher apoptotic cells were seen in severe lesion category followed by moderate and then mild category.
Immunohistochemical investigation of IBDV antigen in bursa of Fabricius revealed the highest number of positive cells in moderate lesion category followed by severe and then mild lesion category. Spleen showed higher numbers of viral antigen positive cells in moderate and severe lesion categories followed by mild lesion category. Viral protein positive cells were recorded also in cecal tonsils and kidney, while no specific reactions were detected in thymus, proventriculus, heart, lungs and liver.
In bursa of Fabricius and spleen, the number of B lymphocytes was significantly increased in mild lesion category and decrease in moderate and severe lesion categories. T cells populations were significantly increased in bursal sections from moderate and severe lesion categories. In spleen and thymus, it was significantly increased in mild lesion category and significantly decreased in moderate and severe lesion categories.