Abstract
Bovine respiratory disease complex (BRDC) occurs widely in cattle farms. The main viral pathogens include bovine viral diarrhea virus (BVDV), Bovine herpesvirus 1 (BoHV-1), bovine parainfluenza virus type 3 (BPIV3), and bovine respiratory syncytial virus (BRSV), and the newly emerged influenza D virus (IDV). In this study, we have developed a one-step multiplex real-time Polymerase Chain Reaction (PCR) capable of simultaneously detecting these five viral pathogens causing BRDC. The established assay could specifically detect targeted viruses without cross-reaction with others. The detection limit was ~10 copies/reaction for single real-time PCR and 100 copies/ reaction for multiplex real-time PCR assay. A total of 213 nasal samples from cattle with signs of respiratory tract disease were then collected for performance evaluation of the established platform, proving that the method has good specificity and sensitivity. The surveillance data suggested that BVDV and BoHV-1 infections are the dominant cause of BRDC in the herd, whereas the detection rate of IDV, BIPV3, and BRSV is relatively lower. In summary, the established assay provides technical support for rapid clinical detection of BRDC associated viral pathogens to guide the formulation of BRDC prevention and control measures.
Highlights
Bovine respiratory disease complex (BRDC) has posed a great threat to the dairy and beef industries throughout the world [1]
To experimentally evaluate the specificity of the selected primer/probe sets, we isolated the viral genomes from the cultured viruses including bovine viral diarrhea virus (BVDV) NADL strain, Bovine herpesvirus 1 (BoHV-1) isolate HLJ07, bovine parainfluenza virus type 3 (BPIV3) strain HQ510351, bovine respiratory syncytial virus (BRSV) field isolate HLJ01, influenza D virus (IDV) strain D/bovine/Mississippi/C00046N/2014, Bovine coronavirus (BCoV) isolate HM and Bovine rotavirus (BRV) strain NCDV as well as the bacterial genomes from Pasteurella multocida and Mannheimia haemolytica for testing
To further verify the sensitivity of the multiplex detection method, we generated the recombinant plasmids containing the five target fragments amplified from IBPV3 [M gene (40394194)], BoHV-1 [glycoprotein E (gE) gene (120290-123438)], BVDV-1b [5’-UTR [64-494]], BRSV [N gene (1342-2289)], and IDV [PB1gene (129488)] and cloned into pLVX-IRES-zsGreenI vector created by Clontech Laboratories Inc
Summary
Bovine respiratory disease complex (BRDC) has posed a great threat to the dairy and beef industries throughout the world [1]. The disease is usually resulted from stress, primary viral infection and secondary bacterial infection, leading to high mortality and morbidity in cattle [2, 3]. The viral pathogens including Bovine Viral Diarrhea Virus (BVDV), Bovine herpesvirus 1 (BoHV-1), Bovine Parainfluenza Virus Type 3 (BPIV3), Bovine Respiratory Syncytial Virus (BRSV) and Influenza D Virus (IDV) have been proposed to be directly associated with BRDC [5]. Secondary bacterial infections such as Mannheimia haemolytica, Pasteurella multocida, and Haemophilus somnus during or after viral infections potentially cause fatal complication of viral infections [6, 7]. Early diagnosis of viral pathogens associated with each outbreak could be beneficial to timely managing and controlling BRDC [1]
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