Diagnosis of infectious bursal disease by immunoperoxidase technique*

Faculty of Veterinary Medicine, University of Ondokuz Mayıs, Samsun, Turkey; 2Faculty of Veterinary Medicine, University of Ankara, Ankara, Turkey; 3Faculty of Veterinary Medicine, University of Kirikkale, Kirikkale, Turkey; 4Faculty of Veterinary Medicine, University of Kafkas, Kars, Turkey
Summary: The diagnosis of infectious bursal disease of naturally infected 21-day-old chickens was investigated with im-munoperoxidase method. Viral antigens were observed in the macrophages and lymphocytes of the bursa of Fabricius. It was ob-served that the method was sensitive and specific and it could be used in the diagnosis of this disease.
Key words: Immunohistochemistry, infectious bursal disease, polyclonal antibody,

Infectious bursal disease (IBD) virus is the causa-tive agent of a highly contagious disease of young chick-ens. The disease is widespread in chickens and has a great economic importance for both broiler and pullet growers (4,6,7). IBD is normally diagnosed in veterinary laboratory by the isolation of causative virus in eggs and/or cell cultures and demonstration of bursal lesions. However, these procedures can be relatively time con-suming and some strains either can not adapt themselves or not show characteristic changes on embryos. There-fore, diagnosis of the disease might be difficult and dem-onstration of viral antigen by immunoperoxidase (IP) method useful for differential diagnosis and confirmation of histopathological changes (2,3,5). Present study describes detection of IBD virus in the bursa of Fabricius (bF) within naturally infected 21 days old chickens by the indirect IP method using polyclonal antibodies. Twenty one days old 30 chickens showing clinical signs of IBD virus infection were collected from com-mercial flocks. All chickens were euthanasied by cervical dislocation and systemically necropsied. Tissue speci-mens were collected from bF, spleen, liver, cecal tonsil, kidney, lung, heart and brain, than fixed in buffered for-malin and processed for paraffin embedding. Two sec-tions were cut from each block, one for IP method and the other for hematoxyline and eosin staining. The con-trol group was sampled at the same intervals and in-cluded 30 specific pathogen free chickens.
For the IP method, formalin fixed paraffin embedded tissue sections from field cases and SPF chickens were deparaffinized and hydrated. Hydrated samples were reacted with 3% hydrogen peroxide in methanol for 30 minutes at room temperature to quench endogenous peroxidase activity and blocked with normal goat serum for 30 minutes. Serum was blotted and slides were incubated with polyclonal chicken anti serum to IBD virus (1/100) for 30 minutes in a humidified chamber. The slides were incubated with peroxidase labelled rabbit anti-chicken IgG (1/1000 sigma) for 30 minutes and reacted for 5 minutes in DAB, counterstained with hematoxyline. Macroscopically, no significant gross lesions were observed in organs except bF. The basic finding was a slight increase in the weight of bF (bF mean weight was 3,1 gr and 2,8 gr, in naturally infected chickens and control group, respectively). Microscopically, the main pathological alterations were degenerative and multifocal necrotic changes in the lymphocytes belonging to the medullar zone of bF. In the interstitial tissue, acute in-flammation characterised by heterophil accumulation and edema was observed. Cystic cavities were found in the medulla of some follicles. In control chickens, no mac-roscopic and microscopic lesions were seen bF or the other organs.
Immunohistochemically, IBD virus antigens were detected in lymphoid cells in the cortex and medulla of lymphoid follicles of the bF (Figure 1). Strong stain intensity and great numbers of positive cells were ob-served. In addition of these findings, antigens were found mainly in macrophages within all follicles and the inter-stitium. No antigen was observed in the other organs and the control chickens.
In the experimental studies, IBD virus antigens have been detected in macrophages within follicles, the interstitium and the lymphoid cells of bF (1,6,8). This antigen localisation was confirmed by our study. IBD virus antigens have also been observed in lymphocytes of spleen, thymus and cecal tonsil (8). However, in the present study, antigens could not been observed in these organs of naturally infected chickens. This may be due to the field cases of IBD virus have a high virulence and/or acute infections.
It has been recorded that the indirect IP method with polyclonal antibodies were used for detecting viral antigens in the bursae of chickens experimentally in-fected with IBD virus (6). These method findings were confirmed by the present study in naturally infected chickens. The protocol used in this work was verified high sensitivity of the IP test. It can be performed very rapidly, a large number of specimens can be processed quickly. Moreover, subclinical infection of IBD may be diagnosed by this technique.
References

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Geliş tarihi: 31.10.2003 / Kabul tarihi: 16.02.2004
Yazışma Adresi:
Doç.Dr.Tolga Güvenç
Ondokuzmayıs Üniversitesi,
Veteriner Fakültesi, Patoloji Anabilim Dalı,
Kurupelit Kampüsü, Samsun.