Bovine respiratory disease (BRD) causes considerable economic losses in North America. The pathogenesis involves interactions between bacteria, viruses, environment and management factors. Primary viral infection can increase the risk of secondary fatal bacterial infection. The objective of this study was to use metagenomic sequencing to characterize the respiratory viromes of paired nasal swabs and tracheal washes from western Canadian feedlot cattle, with or without BRD. A total of 116 cattle (116 nasal swabs and 116 tracheal washes) were analysed. The presence of influenza D virus (IDV), bovine rhinitis A virus (BRAV), bovine rhinitis B virus (BRBV), bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) was associated with BRD. Agreement between identification of viruses in nasal swabs and tracheal washes was generally weak, indicating that sampling location may affect detection of infection. This study reported several viruses for the first time in Canada and provides a basis for further studies investigating candidate viruses important to the prevention of BRD.

The aetiology and pathogenesis of bovine respiratory disease (BRD) are complex and involve the interplay of infectious agents, management and environmental factors. Previous studies of BRD focused on ante‐mortem samples from the upper respiratory tract and identified several unconventional viruses. The lung, however, is the primary location where significant BRD lesions are usually found and is a common post‐mortem diagnostic specimen. In this study, results of high‐throughput virome sequencing, bacterial culture, targeted real‐time PCR and histological examination of 130 bovine pneumonic lungs from western Canadian cattle were combined to explore associations of microorganisms with different types of pneumonia. Fibrinous bronchopneumonia (FBP) was the predominant type of pneumonia (46.2%, 60/130) and was associated with the detection of Mannheimia haemolytica. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. Sixteen viruses were identified, of which bovine parvovirus 2 (BPV2) was the most prevalent (11.5%, 15/130) followed by ungulate tetraparvovirus 1 (UTPV1, 8.5%, 11/130) and bovine respiratory syncytial virus (BRSV, 8.5%, 11/130). None of these viruses, however, were significantly associated with a particular type of pneumonia. Unconventional viruses such as influenza D virus (IDV) and bovine rhinitis B virus (BRBV) were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples. Taken together, our results show that while virus detection in post‐mortem lung samples is of relatively little diagnostic value, the strong associations of H. somni and M. haemolytica with SBP and FBP, respectively, indicate that histopathology can be useful in differentiating bacterial aetiologies.

Bovine respiratory disease (BRD) is the most common and economically important disease of beef cattle. Its high morbidity and mortality necessitate the use of therapeutic and metaphylactic antibiotics, reducing performance in the cattle industry all over the world (Fulton, 2009;Griffin, 1997;Hilton, 2014). Concerns are increasingly being raised about the variable efficacy of metaphylaxis in reducing BRD morbidity and mortality, and its relationship to antimicrobial resistance in bovine and human pathogens (Portis et al., 2012). The aetiology of BRD is multifactorial, and bacterial infection is thought to be secondary, facilitated by viral damage to the mucociliary respiratory epithelium and lung parenchyma (Griffin et al., 2010;Mosier, 2014). Bovine herpesvirus 1 (BHV1), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus 3 (BPIV3) and bovine respiratory syncytial virus (BRDV) are considered to be primary BRD viruses, and vaccines for these viruses are commercially available (Bowland & Shewen, 2000;Fulton, 2009). Surprisingly, no reduction in prevalence of BRD due to immunization and antimicrobial usage has been observed, which has led to more recent studies using viral metagenomics to identify other viruses as

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What’s In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

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