Detection of Infectious Bursal Disease virus from Formalin-Fixed Paraffin-Embedded Tissue by Immunohistochemistry and Real-Time Reverse Transcription-Polymerase Chain Reaction

Abstract

Abstract. 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 25uC 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 4uC, where maximum intensity of immunostaining was observed.

Evaluation of infectious bursal disease virus stability at different conditions of temperature and pH

“…In addition, identical results were observed when RT was performed using 3 0 end gene specific primer in order to predict the stability of genomic RNA. The finding suggests that the stability of RNA decreases with increase in temperature and completely degraded above 72 C. The findings strengthen the earlier report, which showed degradation of IBDV RNA in tissue samples at extreme temperature [45]. Treatment of the IBDV at different temperatures showed infectivity only up to 42 C (Fig.…”

Livestock Drugs and Disease: The Fatal Combination behind Breeding Failure in Endangered Bearded Vultures

“…Specific immunocytochemical procedures were used for detection of mielodepressive virus, including the alphaherpesvirus causing Marek disease [41] in kidney and bursa of Fabricius, the gyrovirus causing infectious chicken anaemia [42] in thymus and bone marrow, the birnavirus causing infectious bursal disease (IBD, [43]) in bursa of Fabricius, and the coronavirus causing chicken infectious bronchitis in kidney [44]. In addition, we conducted a specific immunocytochemical procedure for West Nile virus antigen detection [45] in brain, spinal medulla, thymus and thyroid.…”

Immunohistochemical Detection of Hepatitis C Virus (Genotype 4) in B-cell NHL in an Egyptian Population

“…It has been reported that formalin fixation conditions might have an effect on RT-PCR detectable RNA from tissue blocks. 34 …”