The present research was undertaken to study the gross and pathomorphological alterations due to infectious bursal disease virus infection in broilers. A total of 29 outbreaks were recorded from Srinagar and adjoining districts during the study period. Out of the 29 recorded outbreaks, 22 (75.86%) were clinical outbreaks and the rest 7 (24.13%) were subclinical outbreaks. The average size of the affected flocks ranged between 2500 to 6000 birds. The affected birds were mostly between 17 and 35 days of age. Among the affected flocks morbidity varied from 30% to 60% with mortality ranging from 9 % to 25%. On postmortem examination the carcasses were found to be emaciated, dehydrated with darkened discoloration and presence of petechial and ecchymotic hemorrhages in thigh and breast muscles. Histopathological alterations mostly comprised of depletion of B- lymphocytes with RE cell hyperplasia. Indirect immunoperoxidase staining of the tissues revealed viral antigen in the form of coarsely brown deposits.
Infectious bursal disease (IBD) is a highly contagious acute viral disease of young chickens characterized by massive damage to bursa of Fabricius and immunosuppression (Islam, 2005). The disease causes high mortality in 3–6-week-old birds, but a less acute or subclinical disease in other age group birds. The disease is a major setback to profitability of the poultry industries in both developing and industrialised nations due to mortality, morbidity and impaired growth of young chickens (Sainsbury, 2000). The etiological agent of infectious bursal disease virus is a single-shelled, non-enveloped double stranded RNA virus which belongs to genus Avibirna virus of the family Birnaviridae (Muller and Nitschke, 1987). There are two serotypes of IBDV, serotype I and II. Strains of serotype I IBDV are pathogenic in chickens and are further classified as classical virulent IBDV (cv IBDV), antigenic variant IBDV (av IBDV) and attenuated IBDV (at IBDV). (Van den Berg et al., 2004).
Materials and Methods
Samples comprised of mortalities from various poultry farms operating mainly in Srinagar, Ganderbal, Pulwama, Baramulla and Bandipora districts along with their adjoining areas and those which were brought to Division of Veterinary Pathology for post-mortem examination. The outbreaks suspected for Infectious bursal disease in broiler chicken were identified based on the history, clinical signs and lesions, after following a thorough post mortem examination of birds. History of each suspected flock also included flock size, mortality and total number of birds per outbreak. Representative samples (Bursa of Fabricius, spleen, thymus, harderian gland, caecal tonsils) were collected and stored in 10% buffered formalin for histopathological studies. The antiserum required for detection of the virus was raised in a grey giant rabbit using a live attenuated vaccine strain of IBDV. Appropriate quantity of the vaccine virus was mixed with Freund’s complete/ incomplete adjuvant (FCA/FIA) and three doses were injected subcutaneously on 01st, 21st and 30th day. Before injection zero day serum was collected as pre-vaccination control.
The carcasses were generally emaciated, dehydrated and revealed darkened discoloration of the pectoral and thigh muscles. Many carcasses revealed presence of petechial and ecchymotic hemorrhages in thigh and breast muscles (Fig. 1). In some of these cases hemorrhages in thigh and pectoral region gave typical paint brush appearance. On opening of carcasses the most severe lesions were observed in the bursa of Fabricius. Generally bursa of Fabrici was swollen, oedematous and hemorrhagic in severe cases (Fig. 2).
The bursae were enlarged and revealed cheesy exudates in the form of core (acute cases) and atrophy, whitish or creamy appearance revealing with gelatinous exudate on the serosa (chronic cases). In some cases the bursa was filled with catarrhal exudates that usually formed casts taking the shape of mucosal folds. The spleen was slightly enlarged and often revealed small gray foci uniformly dispersed on the surface. Hepatomegaly and spleenomegaly were observed in most cases. Severely affected birds revealed nephrosis in the form of swollen and pale kidneys along with prominent lobulations. In a few cases ureters were distended and lodged with urates, giving them whitish wormy appearance (Fig. 3). The thymus, caecal tonsils and harderian gland usually revealed no appreciable gross changes, however in severely affected birds thymus revealed slight enlargement, congestion, imparting it a dark discoloured appearance. The caecal tonsils were severely congested and slightly enlarged in severe outbreaks.
Bursa of Fabricius
There was marked congestion, heterophilic infiltration and lymphoid depletion both in clinical and subclinical cases (Fig. 4). The lymphoid necrosis and depletion was observed both in the medulla and cortex of the follicles. In advanced cases the lymphoid cells in the bursal follicles were severely reduced and replaced with vacuolation and cysts, which at times contained clear to pinkish fluid. In some cases medulla was devoid of lymphoid elements with degeneration of interfollicular connective tissue. Degeneration and loss of columnar epithelial tuft was also evident. Some of the cases revealed inter-follicular and intra-follicular oedema, inter-follicular connective tissue proliferation and congestion. In few cases Bursal tissue revealed necrosis of lymphoid elements with pyknotic nuclei along with the presence of both degenerating and normal heterophils and severe fibrosis. Severe interfollicular and intrafollicular oedema with heavy infiltration of heterophils and macrophages was also observed in such cases. When the bursal tissues from these samples were stained with indirect immunoperoxidase, the lymphoid cells and macrophages of the cortices of a few lymphoid follicles of the bursa showed IBD viral antigen in the form of brownish granular deposits (Fig. 5).
Spleen in most of the cases revealed lymphocytic pyknosis and moderate depletion around adenoid sheath arteries. In severe cases there was hyperplasia of reticular cells around adenoid sheath arteries (Fig.6). Some of the cases revealed severe lymphocytic depletion both in sub-clinical and clinical case. In some cases repopulation of lymphocytes was also observed.
Harderian gland of the infected chickens had 5-10 fold fewer plasma cells than those of uninfected chickens However, lymphoid follicles and heterophil populations in the harderian gland were not affected by IBDV infection, nor could necrotic or degenerative changes be found in the acini or excretory ducts (Fig. 7).
In early and sub-clinical cases there was mild to moderate lymphocytic necrosis along with depletion and macrophage infiltration. In more severe cases there was massive lymphocytic necrosis in the germinal follicles and other lymphoid tissue. Blood vessels were generally congested. When the tissues from these samples were stained with indirect immunoperoxidase, the lymphoid cells and macrophages revealed the presence of IBD viral antigen in the form of coarsely granular brown deposits (Fig. 8).
In early and sub-clinical cases, there was mild lymphocytic necrosis and lymphoid depletion. In advanced and clinical cases the blood vessels were congested and there was depletion of the lymphoid elements, infiltration of the RE cells in the cortical and medullary area with necrosis (Fig. 9).
In sub-clinical and mild cases of IBD there was generally congestion and degeneration of renal epithelial cells. In advanced cases kidneys revealed tubular degeneration, congestion, cellular infiltration and necrosis of renal epithelial cells (Fig. 10).
Dehydration and emaciation as observed in present study can be due to diarrhoea which is common with IBDV infection (Cosgrove, 1962). Haemorrhagic signs were also reported in poultry by Baxendale (2002). The changes as observed in bursa of Fabrici like edematous bursa with catarrhal exudates along with haemorrhages are in agreement with those which have been reported earlier by other workers (Vanda den Berg, 2000). Histopathologically bursa of Fabricus revealed lymphoid necrosis, edema and macrophagic infiltration. Indirect immunoperoxidase staining of the bursal tissue revealed the presence of IBDV antigen in the lymphoid cells of bursal follicles. Such findings are similar to earlier studies on bursa in IBDV infected poultry (Okoye and Uzuokwu, 2001; Siavosh et al., 2009) .
The appearance of lesions on spleen is indicative of infection by very virulent strains of IBDV. The ability to cause histological lesions in the non-bursal lymphoid organs, such as spleen or bone marrow has been reported as a potential characteristic of hypervirulent IBDV strains (Cheville, 1967; Lukert and Saif, 2003). The thymus, caecal tonsils and harderian gland usually revealed no appreciable gross changes, however in severely affected birds thymus and caecal tonsils (severe out breaks) revealed slight enlargement, congestion and dark discoloured appearance. Histopathologically, changes in thymus accounted for congestion, mild lymphocytic necrosis in early cases to severe necrosis and depletion of lymphocytes in advanced cases, whereas the changes in caecal tonsils were similar comprising of mild to moderate lymphoid necrosis in early cases to massive lymphocytic necrosis of germinal follicles together with infiltration of macrophages. Harderian gland of the infected chickens had 5-10 fold fewer plasma cells than those of uninfected chickens. The above histopathological results regarding thymus, caecal tonsils and harderian glands were similar to earlier studies (Dhoms et al., 1981; Baxandale, 2002; Siavosh et al., 2009). The gross lesions in kidneys observed in the present study are in agreement with the findings of other workers. The lesions in kidneys are non-specific and could be due to secondary complications. In sub-clinical and mild cases of IBD, there was generally congestion and degeneration of renal epithelial cells. In advanced cases kidneys revealed severe degeneration, necrosis of renal epithelial cells and congestion. Histopathological lesions appearing in the kidneys are nonspecific and resulted from dehydration (Singh et al., 2015).
It was concluded that observation of gross and patho-morphological alterations and their proper interpretation is of paramount importance in diagnosis of Infectious bursal disease. There is a difference in the severity of lesions in clinical and sub-clinical cases of IBD. Histopathological examinations alone are not sufficient for a definitive diagnosis of IBD infections, particularly in birds with subclinical form of the disease. Therefore, Immuno-peroxidase techniques employing antibodies specific for IBDV are required and proven to be a good tool for a definitive diagnosis both in clinical and sub-clinical form of IBD.