Equine sarcoids and bovine papillomavirus : unravelling the viral pathogenesis.
PhD thesis, University of Glasgow.
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The equine sarcoid is the most commonly detected skin tumour in equidae worldwide and has been reported in horses, donkeys, mules and zebra. Sarcoids can be defined as locally aggressive, fibroblastic, benign tumours of the equine skin and although they do not metastasise, they exhibit variable clinical presentations ranging from small alopecic areas to rapidly growing, ulcerated fleshy masses. Clinical behaviour may also vary, from aggressive infiltrative growth to spontaneous regression. Diverse treatment modalities have been reported, and these have been used separately, or have been combined, with variable efficacy. No single therapeutic approach has been found to be universally successful. Although rarely life-threatening, equine sarcoids can have important welfare and economic implications. There is a large body of evidence now supporting the hypothesis that bovine papilloma virus (BPV) is the aetiological agent of equine sarcoids and our understanding of the general pathophysiology of the disease continues to progress; however, several fundamental aspects of the disease remain unclear.
In the first chapter, the clinical aspects of the disease are discussed and evidence to support a viral aetiology is presented, along with background information regarding papilloma virus infections in the natural host. An epidemiological overview of the disease that considers the most current theories and understanding of equine sarcoids is also given.
Chapter II describes the materials and methods used in the course of carrying out the investigations detailed in Chapters III, IV and V.
The aim of Chapter III was to investigate the potential role of flies as vectors in transmission of equine sarcoids between infected and susceptible animals. We found that BPV-1 DNA can be detected in flies trapped from areas where animals with BPV-1 infected sarcoids are housed. This study provides evidence to support the hypothesis that flies may therefore be significant in disease transmission. Such evidence will be of benefit in formulating management control strategies for fly control measures that will reduce the incidence and spread of equine sarcoids.
The work described in Chapter IV was carried out to investigate the hypothesis that the development of the sarcoid tumour arises primarily through inhibition of apoptotic pathways by viral gene products. Assessment of DNA-damage-induced apoptosis in BPV transformed equine fibroblasts revealed that sarcoid derived cells and BPV-1 transfected fibroblasts are less resistant to apoptosis than normal, BPV-negative control equine fibroblasts, but are more likely to recover from DNA damage and continue to grow and divide. Further research was directed towards identifying the viral genes responsible for conferring resistance to apoptosis through siRNA knock-down and monitoring expression of endogenous cellular proteins known to be involved in apoptotic pathways. Using an siRNA targeted to a particular viral protein, we were able to reduce the ability of the BPV transformed cells to survive after DNA damage. Potentially, this information can now be used to develop novel therapeutic strategies.
The final chapter describes the results of a study in to the expression of the p53 protein in sarcoids in vivo and in BPV transformed cells in vitro in tissue culture. In vivo, analysis of 51 equine sarcoid lesions showed that 48% of tumours are associated with nuclear p53 protein expression and that high levels of p53 were associated with clinically aggressive tumour types (fibroblastic). Interestingly, we observed high levels of cytoplasmic p53 staining with no nuclear staining in one tumour in vivo. In vitro, p53 mRNA levels were assessed in BPV transformed and BPV negative fibroblasts. The half-life of the p53 protein, the cellular location and functionality were also investigated in vitro. The results showed that BPV transformed equine fibroblast cells also exhibit increased nuclear p53 protein expression and one cell line (EqS04b) showed a cytoplasmic staining pattern similar to that observed in vitro. However, the abnormal level and location of the p53 does not appear to affect the transactivational functionality of p53 since p21 expression was induced by exposing the cells to UVB irradiation.
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