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 hybridization (ISH) test was developed with a 491-bp cDNA fragment derived from the VP2 gene of IBDV. The fragment was amplified and simultaneously labeled with incorporation of digoxigenin-11-dUTP in a nested polymerase chain reaction (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-fixed, paraffin-embedded bursae of Fabricius from chickens both experimentally infected as well as commercially reared. Bursae from six clinically ill commercial broilers suspected to be IBDV infected were examined by ISH and immunohistochemistry. In two samples, IBDV infection was detected by both ISH and immunohistochemistry, whereas in the other two histologically normal bursae, IBDV was detected only by ISH. Two commercial chickens with atrophied bursae were negative by both ISH and immunohistochemistry. No positive IBDV stained cells were in RNase treated sections from infected birds, uninfected chickens, or reovirus-infected chickens. The ISH test developed herein resulted in important modifications, which makes it superior to other previously published procedures. We also described a direct in situ reverse transcription-polymerase chain reaction method for the amplification and detection of IBDV genome in formalin fixed, paraffin-embedded bursae of Fabricius with a single primer pair with direct incorporation of digoxigenin-11-deoxyuraciltriphosphate (dUTP) into the amplicon. Both molecular tests with their important modifications represent improved detection of IBDV.
Liu
X
X
Department of Poultry Science, Alabama Agricultural Experimental Station, Auburn University, 36849-5416, USA.
Giambrone
J J
JJ
Hoerr
E J
EJ
D000818:Animals; D018175:Birnaviridae Infections / Q000662:veterinary* / Q000821:virology; D002645:Chickens; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000662:veterinary; D007243:Infectious bursal disease virus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011201:Poultry Diseases / Q000821:virology*; D015678:Viral Structural Proteins / Q000032:analysis
C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins