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Epidemiology and pathogenesis of canine adenovirus type 1 in red foxes (Vulpes vulpes) David Walker Thesis presented for the degree of Doctor of Philosophy Royal (Dick) School of Veterinary Studies and The Roslin Institute The University of Edinburgh, 2017 Declaration I declare that the thesis and the work presented within is my own, except where the contributory work of others (e.g. as part of co-authored publications) is clearly stated and acknowledged. This work has not been submitted for any other degree or professional qualification. David Walker, 2017 Declared contributions to chapters The work presented in chapter 2 was previously published in D. Walker, E. Abbondati, A. L. Cox, G. B. B. Mitchell, R. Pizzi, C. P. Sharp, A. W. Philbey, Veterinary Record, 178, 421, 2016. DW contributed to all aspects of the work, except the undertaking of the post-mortem examinations (since these preceded DW’s scholarship). EA, ALC, GBBM and RP provided the specimens and access to the original clinical notes (2011-2013). Slides were prepared by the histopathology section, Royal (Dick) School of Veterinary Studies (RDSVS). DW is grateful to AWP and CPS for assistance with histopathological interpretations and molecular work respectively. In chapter 3, the slides were prepared by the histopathology section, RDSVS, who also developed the immunohistochemical staining. The work presented in chapter 4 was previously published in D. Walker, S. A. Fee, G. Hartley, J. Learmount, M. J. H. O’Hagan, A. L. Meredith, B. M. de C. Bronsvoort, T. Porphyre, C. P. Sharp, A. W. Philbey, Scientific Reports, 6, 36051, 2016. DW is grateful for the contributions of others in this large, national-scale project. BMdeCB and TP provided the advanced coding scripts and guidance in statistical analyses (and relevant statistical methods and Table 4.3). CPS assisted with the initial design of the detection PCR/qPCR primers. SAF and MO’H performed/organised the post-mortem examinations of the red foxes from Northern Ireland. GH provided some fox carcasses and tissues from Scotland. AM provided tissues from archived Scottish samples. AWP assisted with collection of foxes from wildlife centres and performed some post-mortem examinations. Hannah Willetts performed post-mortem examinations on foxes from England. Nor-Abdul Azlina Aziz, Eric Morgan and JL provided sera and data from a previous study. Tiggywinkles Wildlife Hospital also collected sera samples from England. The work presented in chapter 5 is an extended version of the article D. Walker, W. F. Gregory, D. Turnbull, M. Rocchi, A. L. Meredith, A. W. Philbey, C. P. Sharp, Novel adenoviruses detected in British mustelids, including a unique Aviadenovirus in the tissues of pine martens (Martes martes), Journal of Medical Microbiology, 66, 1177-1182, 2017, and uses tables and figures directly from the publication. AWP performed the otter post-mortem examinations as part of an unrelated project. Pine marten post-mortem examinations were conducted jointly by DW, AWP, ALM and Gidona Goodman, RDSVS as part of additional projects with these specimens. The Centre for Genomic Research, University of Liverpool performed the Illumina sequencing and provided the initial consensus reads using the stated methods. In chapter 6, the ultrahigh-density peptide microarray was performed as a commercial service by Schafer-N (Denmark, Copenhagen). This work was funded by the Innovation Initiative Grant, University of Edinburgh. In chapter 7, the histopathology section, RDSVS prepared the FFPE tissue blocks, H&E and unstained slides as a commercial service. Mary Flook, RDSVS performed the post-mortem examination of the dog used for the RT-qPCR. Abstract Canine adenovirus type 1 (CAV-1) causes severe, and often fatal, disease in domestic dogs and red foxes (Vulpes vulpes). It has also been reported to infect ursids, mustelids and other free-ranging canids. The disease caused by CAV-1 was first reported as ‘fox encephalitis’ and came to prominence when it caused major losses in farmed silver foxes, a colour variant of the red fox, in North America in the early 20th century. The disease caused by CAV-1 soon appeared to ‘spillover’ into the domestic dog population and was described independently as ‘infectious canine hepatitis’ (ICH), by which it is mainly known in all species. ICH became a serious problem among unvaccinated and susceptible pets, along with distemper and other ‘rife’ infectious diseases at the time. Canine adenovirus (in the form of canine adenovirus type 2; CAV-2) is now included in the recommended vaccination schedule of all pet dogs in the UK and, more recently, CAV-1 has been fully sequenced and annotated. This PhD re-investigates the pathology caused by CAV-1 in red foxes compared to domestic dogs. There is a suggestion that neurological disease in foxes may predominate and be the cause of high mortality rates and a rapid course of disease. Outbreaks of ICH in wildlife rehabilitation centres were investigated, and novel immunohistochemical techniques employed, to investigate this possibility. It was shown that CAV-1 causes severe systemic pathology in many animals. In the dog, it was noted that, although central nervous system (CNS) pathology exists, hepatic pathology was often severe and may predominate. Vascular endothelial cells in the red fox CNS were more heavily infected with CAV-1 than in dogs, and lysis of these cells is likely to be the direct cause of the multifocal haemorrhages observed histologically, and possibly a major contributor to the cause of death. New cell types, previously not reported to be permissible for CAV-1 infection, were detected to be positive for CAV-1. It is hypothesised that there are multiple manifestations of the disease caused by CAV-1, which is evident interspecifically. It is recommended that these manifestations are taken into account when describing disease because CAV-1 does not only cause a ‘hepatitis’ and does not infect only canine species. The current epidemiological state of CAV-1 was also investigated using a large sample of red foxes from across the UK. It was found that a large proportion of free- ranging foxes had been exposed to the virus and a proportion of animals remain infected, in multiple tissues and in the absence of disease; these may represent persistent infections. For the first time, some red foxes were demonstrated to shed CAV-1 in high titres, as quantified by quantitative polymerase chain reaction (qPCR). This suggested that red foxes are likely to be a wildlife reservoir of CAV-1 in the UK, and could be a source of infection for dogs. The ‘persistence of infection’ is also a characteristic of human adenoviruses (HAds) and it was hypothesised that these related viruses share common mechanisms to persist in infected tissues. It was also hypothesised that British mustelid species may also be a source of CAV-1 infection and that species is also persistently infected with adenoviruses. New adenoviruses were detected in British pine martens and otters, which also demonstrated persistent infections, as suggested by detection by the polymerase chain reaction (PCR). One adenovirus, tentatively named marten adenovirus type 1 (MAdV-1), partially sequenced by high throughput sequencing (HTS), appeared to be an Aviadenovirus, which is an unusual finding. It is hypothesised that this could be evidence of a host switch from an infected avian prey to a new mammalian host. This raises new questions on the capability of adenoviruses, usually regarded as very host specific, to opportunistically switch hosts. The molecular mechanisms employed by adenoviruses to persist in their hosts have been poorly studied to date. Therefore, a final aim of the project was to investigate the cell types in red foxes which could be capable of maintaining a persistent infection with CAV-1, and to investigate the dynamics of CAV-1 infection in cell cultures and clinically affected tissues by utilising reverse transcriptase qPCR (RT- qPCR). Overall, the study and the techniques developed could provide a basis for future research to investigate how HAds, which persistently infect some human tissues, can re-activate under a state of immunosuppression and cause severe, systemic disease in some patients. Lay abstract Canine adenovirus type 1 (CAV-1) causes severe, and often fatal, disease in domestic dogs and red foxes (Vulpes vulpes). The disease ‘fox encephalitis’ first came to prominence when it was noted to cause high mortality rates in farmed silver foxes, a colour variant of the red fox, in North America in the early 20th century. The disease caused by CAV-1 soon appeared to ‘spill over’ into the domestic dog population and was described as ‘infectious canine hepatitis’ (ICH), by which it is now known. ICH became a serious problem among unvaccinated and susceptible pets, along with distemper and other ‘rife’ infectious diseases at the time. Canine adenovirus is now included in the recommended vaccination schedule of all pet dogs in the UK and, more recently, the virus itself has been genetically characterised. This PhD re-investigates the pathology caused by CAV-1 in red foxes compared to domestic dog.