An endpoint multiplex PCR/RT-PCR for detection of viral agents of bovine respiratory disease

Bovine respiratory disease (BRD) is a clinicopathological syndrome caused by complex interactions among host and environmental factors, viruses and bacteria. The multi-agent BRD etiology makes laboratory diagnosis challenging, as various pathogens should be investigated for an accurate results. Herein, we describe an endpoint multiplex PCR/RT-PCR for detection of major BRD-associated viruses: bovine herpesvirus 1 (BoAHV-1), bovine pestiviruses (BVDV-1, BVDV-2, HoBiPeV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). We designed high-coverage and target-specific primers to be properly combined in the multiplex assay. Following standardization of individual reactions, the multiplex PCR/RT-PCR was optimized for reaction conditions and mix composition. The amplicons of the five targets were easily differentiated in agarose gel: 121 bp (BPIV-3), 157 bp (BoAHV-1), 204 bp (pestiviruses), 282 bp (BRSV) and 323 bp (BCoV). The limits of detection (LoDs) of our multiplex were 38 TCID 50 (BoAHV-1 and BPIV-3), 16 TCID 50 (pestiviruses), 13 TCID 50 (BRSV) and a 10 − 2 dilution of a pool of five BCoV positive samples. No amplification of the main bacteria ( Pasteurella multocida ; Mannheimia haemolytica and Histophilus somni ) potentially involved in BRD was observed. Finally, 289 respiratory specimens from animals with respiratory/nasal signs were evaluated by individual and multiplex PCR/RT-PCR tests to assess the diagnostic performance of the assay. Both tests resulted in 14.8% positive samples (43/289) being 19 for BPIV-3 (19/43), ten for BCoV (10/43), seven for BRSV (7/43), six for BoAHV-1 (6/43) and one for pestiviruses (1/43). Overall, our multiplex PCR/RT-PCR assay represents an alternative for an easier, faster and more practical laboratory diagnosis of viruses associated with BRD.