Investigating the effect of bovine respiratory disease (BRD) associated viruses on the host transcriptome and respiratory microbiome in dairy calves

Bovine respiratory disease (BRD) is a multifactorial syndrome affecting cattle of all ages both here in Ireland and worldwide. Responsible for high levels of morbidity and mortality, BRD represents a major economic burden for the cattle industry. Both bacterial (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis) and viral (bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BoHV-1), bovine viral diarrhoea virus, bovine coronavirus, bovine parainfluenza virus 3 and bovine adenovirus agents can be responsible for disease onset, either solely or through co-infections. The ‘traditional’ model of BRD pathogenesis proposed a primary role for viral agents (and some bacterial pathogens) disabling host defences thereby facilitating secondary bacterial proliferation and associated lung pathology. In addition to these infectious agents, host factors such as age, breed and genetics can influence disease onset, as well as psychological stress resulting from environmental and management factors. Disease diagnosis often relies largely on the observation of clinical signs of infection; however, this methodology is often limited with factors such as subjectivity as well as disease severity influencing the results. This can ultimately result in delayed or even failed diagnosis in the case of sub-clinical infections. The diagnosis of BRD using evaluation of clinical respiratory signs or auscultation is insufficient to detect sub-clinical BRD. The type, severity, and extent of BRD cannot always be determined by clinical examination alone and this may lead to misinterpretation of respiratory symptoms and ineffective therapy. Despite the use of vaccines, BRD remains a major issue. The advancements in next-generation sequencing technologies, for example RNA-Seq, have allowed the investigation of the host response of animals to both natural and experimentally induced BRD. Despite these efforts, a paucity of data remains surrounding the response of dairy calves to a key BRD virus, BoHV-1. A double-stranded DNA alphaherpesvirus, BoHV-1 is a key viral pathogen associated with BRD, that following initial infection, can remain latent within the animal and re-emerge during times of stress or unfavourable conditions. Despite the absence of clinical signs, animals can continue to shed the virus during latency spreading infection throughout a herd, making it particularly dangerous. Therefore, the overall aim of this thesis was to investigate the response of dairy calves experimentally challenged with a specific BRD virus. An additional objective was to characterise the respiratory microbiome of these calves following an experimental challenge with BoHV-1. The challenge model consisted of two groups of Holstein-Friesian bull calves, control (n=6) and BoHV-1 challenged (n=12), which were monitored, using clinical scoring of signs of infection, from the day of the challenge (day (d) 0) until euthanasia (d6). Whole blood and nasal swabs were collected each day for use in downstream analysis. At post-mortem (d6) tissue samples were collected for further analysis. In the first study (Chapter 2) RNA-Seq and differential expression analysis was used to identify genes differentially expressed in whole blood in response to BoHV-1 infection. Here, 488 genes were identified as differentially expressed between the BoHV-1 and control calves. Enriched biological pathways were related to the innate immune and inflammatory responses. The second study (Chapter 3) was similar but examined lymphoid and lung tissue collected at post-mortem. Here, differential expression analysis, followed by a weighted gene co-expression network analysis was used to identify differentially expressed genes (DEGs) as well as modules (networks) of co-expressed genes within each tissue type. These results were then cross-analysed with those reported in Chapter 2. Findings from this study identified DEGs in each tissue type in response to BoHV-1, with 33 DEGs common to all tissues. Modules enriched for antiviral and innate immune processes were also identified for each tissue. The third study (Chapter 4) utilised small RNA-Seq to examine the changes in miRNA in lung and lymphoid tissue following the BoHV-1 challenge. Here, analyses revealed DE miRNAs in each tissue type in response to BoHV-1 infection. DE miRNAs were predicted to target 51, 22, 30 and 1 gene(s), which were previously found to be DE in bronchial lymph node (BLN), mediastinal lymph node (MLN), healthy lung (LH) and lesioned lung (LL) tissue between BoHV-1 challenged and control calves. The final study (Chapter 5) involved the use of long—read 16S rRNA sequencing to characterise the respiratory microbiome of the upper and lower respiratory tracts of the control and BoHV-1 challenged calves. Here DNA was extracted from nasal swabs as well as lung and pharyngeal tonsil tissue. Libraries were then prepared and sequenced on an Oxford Nanopore Technologies MinION MK1C device. Data were then analysed using the EPI2ME software and R. Unexpectedly, there was no difference (P > 0.05) in the abundance of any genera between the control and challenged calves for any tissue type. However, a numerical difference in certain genera such as Pasteurella and Mycoplasma, were seen between the control and BoHV-1 challenged calves. This is the first study to characterise the microbiomes of these sites in dairy calves in response to BoHV-1. Furthermore, the use of the MinION device in this study demonstrates the potential for use as a pen-side diagnostic. Overall, the use of a controlled experimental challenge allows for an improved understanding of the immune response of dairy calves to a BoHV-1 infection, with such data aiding in the improvement of BRD diagnostic and treatment options.

Funder

Department of Agriculture, Food and the Marine (DAFM), Ireland
Department of Agriculture, Environment and Rural Affairs (DAERA), Northern Ireland

Publisher

University of Galway

Rights

CC BY-NC-ND

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Collections

University of Galway Theses (PhD Theses)