Application of metagenomic sequencing to characterization of the virome in bovine respiratory disease
Date
2020-12-29
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Degree Level
Doctoral
Abstract
Bovine respiratory disease (BRD) is the most concerning disease in the cattle industry worldwide due to the enormous economic losses it causes, and concerns regarding antimicrobial resistance that is emerging due to use of massive amounts of antimicrobial agents to control the disease. The concept of primary environmental, management or viral factors with ensuing secondary bacterial infections is widely accepted, however, morbidity and mortality of feedlot cattle due to BRD are not reduced despite viral vaccination and antimicrobial usage. This indicates that the etiology and pathogenesis of BRD are not yet fully understood, and that all of the infectious agents and environmental causal factors may not have been identified. The advent of high throughput sequencing (HTS) using Illumina MiSeq and Oxford Nanopore sequencing technologies has revolutionized the genomics field, particularly microorganism identification, antimicrobial resistance prediction and microbiome analysis. The main objective of my study was to use HTS to identify unconventional viruses (those viruses are not included in BRD vaccine and no available diagnostic assays) and characterize their association with BRD.
We first characterized and compared the upper and lower respiratory tract viromes of Canadian feedlot cattle with or without BRD through metagenomic sequencing on the Illumina MiSeq platform. 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 these viruses in nasal swabs and tracheal washes was generally weak, indicating that sampling location may affect detection of infection.
We next characterized the virome of bovine pneumonia lungs collected in western Canada using metagenomic sequencing on the Illumina MiSeq, and combined this information with bacterial culture and targeted real-time PCR results from the same samples to determine the relationships between the microorganisms detected and different types of pneumonia defined by histopathological examination. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. None of these viruses, however, was significantly associated with a particular type of pneumonia. Unconventional viruses such as IDV and BRBV were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples.
In the third chapter, we used IDV as a representative BRD-associated virus to examine the feasibility of using metagenomic sequencing for detection of viruses in clinical bovine respiratory samples. We compared results of long-read sequencing on the Oxford Nanopore GridION platform and previously generated Illumina MiSeq data to the results of an IDV-specific qPCR. We concluded that both MiSeq and Nanopore sequencing were capable of detecting IDV in clinical specimens with a range of Cq values.
In the last chapter, we applied Nanopore metagenomic sequencing to characterize the viromes of cattle upon arrival at nine feedlots in Western Canada, and related the findings to health outcomes of these cattle to determine if the composition of the virome of individual animals could be used to predict the likelihood of their development of BRD. No relationship was found between BRD development and the number of viruses detected, the presence of any specific individual virus, or combination of viruses.
In summary, results of these studies demonstrate the diversity of viruses in bovine respiratory tracts, and highlight the need for further research into prevention and control of BRD development in the context of mixed infections. Meanwhile, our results also demonstrate the potential of metagenomic sequencing on the Illumina MiSeq and Oxford Nanopore platforms for detection of viruses in clinical samples from naturally infected animals with a wide range of viral loads.
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Keywords
nasal virome, bovine coronavirus, bovine respiratory disease (BRD), bovine respiratory syncytial virus, bovine rhinitis virus, influenza D virus, metagenomic sequencing,nanopore sequencing,
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Pathology
Program
Veterinary Pathology