Detection and strain differentiation of infectious bronchitis virus in tracheal tissues from experimentally infected chickens by reverse transcription-polymerase chain reaction. Comparison with an immunohistochemical technique

Abstract

Oligonucleotide pairs were constructed for priming the amplification of fragments of nucleocapsid (N) protein and spike glycoprotein (S) genes of avian infectious bronchitis virus (IBV) by reverse transcriptionpolymerase chain reaction (RT-PCR). One oligonucleotide pair amplified a common segment of the N-gene and could detect various strains of IBV in allantoic fluid from inoculated chicken embryos, and in tracheal tissue preparations from experimentally infected chickens. Four pairs of oligonucleotides selectively primed the amplification of the S1 gene of Massachusetts/Connecticut, D1466, D274/D3896 and 793B strains of IBV, respectively. Groups of specific pathogen free chickens were experimentally inoculated with the Massachusetts (H120, M41), the D1466 and the 793B strains of IBV, and tracheal tissue preparations were made from each bird for RT-PCR and for immunohistochemistry (IHC) up to 3 days post-inoculation. The N-gene RT-PCR detected IBV in 82% of the chickens, while IHC only detected IBV in 60%. This difference was significant (P<0.02). The detection rate by N-gene RT-PCR varied from 67 to 100% for the various strains of IBV inoculated. The S1 gene oligonucleotide pairs were applied to the same tissue preparations and they detected specifically the Massachusetts (M41 and H120), the D1466 and the 793B strains of IBV at rates varying between 58 and 92%. When the mixtures of the primers were applied, the detection rate in tissue preparations was reduced to the level of 50 to 67%. It is concluded that the direct detection of IBV in tracheal tissues by RT-PCR is more sensitive than IHC and that the RT-PCR technique is able to distinguish between types of IBV.