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Detection of infectious bursal disease virus from formalin-fixed paraffin-embedded tissue by immunohistochemistry and real-time reverse transcription-polymerase chain reaction

Correspondence: ¹Corresponding Author: Pedro Villegas, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, e-mail: pedrov{at}uga.edu

Formalin fixed paraffin embedded tissues blocks are used routinely to diagnose the economically important immunosuppressive infectious bursal disease virus (IBDV) in chickens. Immunohistochemical detection of viruses in tissue blocks has been done with varying results between laboratories. Extraction of IBDV RNA from tissue blocks allows IBDV strain identification at a molecular level. This allows correlation between virus identity and histological lesions present in the tissue. Experimentally reverse transcription-polymerase chain reaction (RT-PCR) detectable IBDV RNA could always be extracted from tissue blocks with acute +3 or higher histological lesion scores. However, many blocks from diagnostic field cases did not yield detectable IBDV RNA, in spite of having severe IBDV histological lesion scores. The reason for this can be the effect different formalin fixation conditions have on RNA detection from tissue blocks. To study the effect of various fixation parameters on RNA extraction and immunohistochemical detection of IBDV, bursas with maximum histological lesion score of 4 for IBDV were fixed in formalin under various conditions (different pH levels, temperatures, concentrations of formalin, and fixation duration). Only tissues fixed in formalin with a pH of 7.0, concentration of 5 or 10% formaldehyde, storage temperature of 25°C or less, and kept for up to 2 weeks in formalin yielded detectable IBDV RNA upon extraction. No RNA could be detected from tissues fixed under extreme temperature, pH, or formalin concentrations. Optimal fixation conditions for IHC detection of IBDV were 10% formalin concentration, pH 7.0, and temperature of 4°C, where maximum intensity of immunostaining was observed.

Key Words: Chicken • formalin fixation • immunohistochemistry • infectious bursal disease • paraffin embedded tissue • RNA extraction