In situ hybridization, immunohistochemistry, and. in situ reverse transcription-polymerase chain reaction for detection of infectious bursal disease virus

Development: of molecular techniques for the detection of infectious bursal disease virus (IBCV) is an important area of research. An in situ hybridiz ation (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled wi th incorporation of digoxigenin-11-dUTP in a nested polymerase chain reacti on (PCR) assay. The resulting digoxigenin-labeled 491-bp nested PCR product was used as probe for ISH to detect and localize IBDV RNA in formalin-fixe d, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill com mercial broilers suspected to be IBDV infected were examined by ISH and imm unohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bu rsae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBD V stained cells were in RNase treated sections from infected birds, uninfec ted chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previ ously published procedures. We also described a direct in situ reverse tran scription-polymerase chain reaction method for the amplification and detect ion of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11-deox yuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with the ir important modifications represent improved detection of IBDV.