title: An epidemiological and molecular study of equine sarcoids at The Donkey Sanctuary UK
Creators: name: Jones, Sarah Elizabeth
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: Equine sarcoids, donkey, bovine papillomavirus, epidemiology, risk factors, prevalence, transmission, phylogenetics, transcriptomics, virology, oncology, veterinary.

description: Equine sarcoids are tumours of dermal fibroblasts affecting all equid species including horses/ponies, donkeys, and mules. Their UK prevalence is estimated at ~5% (National Equine Health Survey) and The Donkey Sanctuary UK has identified that sarcoids are a significant problem in their animals. Bovine papillomavirus types 1 and 2 (BPV-1/2) are widely accepted to be causative agents of these tumours, with other factors also required for tumour development. There is a contradiction in the current evidence regarding viral/tumour transmission; there are reports suggesting spread between equines, but infective virus has not been isolated from sarcoids. This PhD project used a range of interdisciplinary methods to investigate both epidemiological and molecular aspects of sarcoids. Methods The sanctuary population demographics were described, and a text mining approach (WordStat) was used to identify animals with several categories and subcategories of disease. The number of animals in each category/subcategory per 100 animal years at risk (AYAR) was calculated to assess the prevalence of sarcoids, compared to other diseases. Univariable and multivariable binary logistic regression modelling (Stata) were performed to identify risk factors for sarcoid development. Details on “friendships” between animals were included and, to validate these, an observational behavioural study was carried out by collecting nearest neighbour data. DNA extracted from sarcoid lesions and Stomoxys calcitrans flies from the sanctuary was used in PCR reactions to amplify the E5 (Early 5) gene or LCR (Long Control Region) section of the viral genome to confirm whether BPV-1/2 DNA was present, and these results were compared to lesions’ histopathology diagnoses. Amplified LCRs were sequenced to determine the BPV type and subtype present and phylogenetic trees were created using FastTree (Geneious). Antibody to the major viral capsid protein was used to capture viral particles in donkey sarcoid and fly samples. DNA attached to the capsid protein was then amplified by PCR (immune capture-PCR assay (IC-PCR)) to determine whether the samples contained potentially infective viral particles. RNA was extracted from normal skin and sarcoid samples from horses and donkeys, and from established cell lines (normal equine fibroblasts, BPV-1-infected equine fibroblasts and sarcoid fibroblasts). microRNA (miRNA) and messenger RNA (mRNA) sequencing were performed by the University of Liverpool and reads were mapped to the horse (EquCab3.0) and BPV-1/2 genomes. The host mRNA and miRNA profiles of samples were normalised and analysed by Principal Components Analysis. The viral copy number was calculated for each sample and the BPV expression profile determined for BPV positive samples using normalised viral read counts, to determine any correlations between viral and host variables. Evidence for the existence of papillomaviral miRNAs was explored.