Diagnosis of enzootic pneumonia in Danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle.

Abstract

A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for detection of bovine respiratory syncytial virus (BRSV) in lung tissue of naturally and experimentally infected cattle. Primers were selected from the gene coding the F fusion protein, which is relatively conserved among BRSV isolates. The RT-PCR assay was highly specific, it yielded positive reactions only when performed on BRSV-infected cell cultures or tissues. The detection limit of the RT-PCR assay was assessed as 5 TCID50. BRSV was detected in tissues of the respiratory tract and in the tracheobroncheal lymph node of calves euthanized 2-8 days after experimental infection with BRSV, whereas samples of other tissues and samples from mock-infected animals were negative at all time points. Examination of lung samples from 8 different regions of the lungs revealed that although the virus was most often found in the cranioventral lobules, it was frequently present in all lung lobules. Microbiologic examinations of all acute fatal cases of pneumonia (135 animals) in cattle submitted for diagnostic purposes during 1 year revealed that Actinomyces pyogenes (11%), Haemophilus somnus (10%), Pasteurella sp. (7%), and Pasteurella haemolytica (7%) were the most common bacterial agents found in the lungs. BRSV was identified using a conventional antigen enzyme-linked immunosorbent assay (ELISA) in 23 (17%) animals. The established BRSV-specific RT-PCR assay yielded positive results for the same 23 animals. In addition, 10 animals that were negative with the ELISA were positive with the RT-PCR assay. These results indicates that the RT-PCR assay can be a sensitive, reliable alternative to conventional diagnostic procedures.