In Situ Hybridization Detection of Bovine Respiratory Syncytial Virus in the Lung of Experimentally Infected Lambs

Abstract

We studied the distribution of bovine respiratory syncytial virus (BRSV) RNA in lungs of experimentally inoculated lambs by in situ hybridization at different times postinoculation. The probe used for in situ hybridization was prepared by reverse transcription of BRSV RNA, followed by polymerase chain reaction (PCR) amplification of the cDNA. Twenty-five Merino lambs of both sexes with a live weight of 17 +/- 3 kg received an intratracheal inoculation of 20 ml saline solution containing 1.26 X 10(6) TCID50 BRSV (strain NMK7)/ml. Lambs were slaughtered 1, 3, 7, 11, and 15 days postinoculation (PID). Bronchial and bronchiolar epithelial cells were positive for BRSV nucleic acid by ISH at 1, 3, 7, and 11 PID. However, alveolar epithelial cells contained positive cells at 1, 3, and 7 PID. Cells containing viral RNA were detected from 1 to 11 PID in exudate within bronchial and bronchiolar lumina and from 3 to 7 PID in alveolar exudates. Positive hybridization signals were identified in interstitial mononuclear cells and in bronchi-associated lymphoid tissue from 3 to 11 PID. Mononuclear cells were located in peribronchiolar tissue and interalveolar septa. The highest signal intensity in positive cells was observed at 3 and 7 PID, coinciding with the most important histopathological findings.