Bovine papillomavirus type 2 infection in the pathogenesis of the Bovine Enzootic Hematuria : related oncological process

Tese de Doutoramento em Ciências Veterinárias, especialidade de Sanidade AnimalBovine Enzootic Hematuria (BEH) is a disease that affects cattle and water buffalos in specific regions of the globe, mainly associated with the chronic ingestion of bracken fern (Pteridium aquilinum) whose major characteristic is the development of urinary bladder tumors. One of the regions where this disease is endemic is the Azores Archipelago, Portugal. In these Atlantic islands bracken fern finds the appropriate conditions for continuous growth in the farmlands, and thus this toxic plant is frequently available for cattle consumption. The involvement of the Bovine papillomavirus type 2 (BPV2) in the genesis of this disease has been pointed out to be paramount, but some aspects involving the association between bracken fern and Papillomavirus remain unclear. This work aimed to study the role of this infectious agent in the oncological process observed in BEH, and is divided into seven chapters. In the first chapter, the major features, the implications and the etiology of this disease, with a special focus on ptaquiloside and BPV2, are reviewed. Additionally, urinary bladder tumors in man, and the genetic alterations associated to the disease, are briefly revised. The second chapter contains the specific objectives that were sought with the present work, and that resulted in the four experimental chapters that follow. In the third chapter, a detailed histopathological characterization of the urinary bladder lesions found in BEH-affected cattle from the Azores Archipelago which were used in the experimental work, is presented. Furthermore, an immunohistochemical study of the expression of cytokeratins in the epithelial neoplasms is also presented. Our results support previous findings that most urinary bladder BEH-associated lesions are neoplastic, and have epithelial origin. The decrease in the number of neoplastic cells expressing cytokeratin 7 in urinary bladder urothelial tumors was associated with increasing pathological grade and stage, whereas the decrease in the expression of cytokeratin 20 was only associated with increasing stage. The fourth chapter is dedicated to the prevalence and transcriptional activity of BPV2 in the urinary bladder lesions of BEH-affected cattle from the Azores Archipelago. The results obtained show that, though BPV2 is widely distributed within the bovine vii population of the Azores, the viral loads determined are very low, suggesting that BPV2 could be inactive, since no transcriptional activity was detected. In the fifth chapter a quantitative and qualitative gene expression study is presented, comparing the expression levels of cell cycle controlling genes and of a growth factor receptor gene in BPV2 positive and negative epithelial and endothelial bovine urinary bladder tumors of BEH-affected cattle. The expression of TP53, MDM2 and CCND1 genes was above normal, but no differences between BPV2 positive and negative epithelial and endothelial tumors were found. The expression of the EGFR gene was lower both in BPV2 positive epithelial and endothelial tumors when compared with BPV2 negative ones. This possible association between BPV2 infection and lower EGFR should be further investigated in future studies. The sixth chapter presents the preliminary in vitro study through which the effects of BPV2’s oncoproteins in a bovine cell line were assessed. Neoplastic transformation was not achieved, but changes in the cellular growth rate and gene expression patterns were observed, suggesting viral oncoprotein activities yet unknown. The main conclusions of this work are pointed out and discussed in the seventh chapter. The oncological process found BEH and its association with BPV2 are still a matter for further research. The results presented within this thesis provide a better insight into this subject but also open and support new questions worth investigating in future research.RESUMO - Infeção por Papilomavírus Bovino tipo 2 na patogenia do processo oncológico associado à Hematúria Enzoótica Bovina - A Hematúria Enzoótica Bovina (HEB) é uma doença que afeta bovinos e búfalos em regiões específicas do Globo particularmente relacionada com a ingestão do feto comum (Pteridium aquilinum), sendo um dos aspetos mais relevantes o desenvolvimento de tumores na bexiga dos animais afetados. Uma dessas regiões onde a HEB é endémica é o Arquipélago dos Açores, Portugal. O feto comum encontra nestas ilhas Atlânticas as condições apropriadas para o crescimento constante nas pastagens, fazendo com que esta planta tóxica esteja facilmente acessível aos animais criados em regime extensivo. A participação do Papilomavírus bovino tipo 2 (BPV2) também tem sido apontada como crucial na génese da HEB. Porém, alguns dos aspetos relacionados com a associação entre o feto comum e o Papilomavírus permanecem por esclarecer. O presente trabalho teve como objetivo estudar o papel deste agente infecioso no processo oncológico associado à HEB, estando dividido em sete capítulos..