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..

Identificação de proteínas estruturais em tumores de bexiga de bovinos

Dissertação de Mestrado Integrado em Medicina VeterináriaA Hematúria Enzoótica Bovina (HEB) é uma doença de evolução arrastada associada à ingestão do feto comum (Pteridium aquilinum), em que a principal característica é o desenvolvimento de tumores da bexiga. Esta doença é endémica em diferentes zonas do Globo onde se encontram condições favoráveis ao crescimento da planta, incluindo o Arquipélago dos Açores. Os objectivos do presente trabalho foram os de (i) pesquisar através de métodos de imuno-histoquímica a expressão das proteínas estruturais das células uroteliais, uroplaquina III (UPIII) e citoqueratina 7 (CK7), em carcinomas do urotélio da bexiga de animais afectados pela HEB, e a sua possível associação com o grau e estadio tumorais, (ii) pesquisar a expressão dos factores reguladores do ciclo celular Ciclina D1 e p53, e a sua possível associação com o grau e estadio tumorais, (iii) analisar o possível valor dos tumores da bexiga de bovinos como modelo para os tumores de bexiga de outros animais e dos humanos. Os resultados demonstram que a expressão da UPIII é perdida em carcinomas do urotélio de alto grau e invasivos, tendo esta característica sido associada estatisticamente com estes dois parâmetros. Do mesmo modo, a perda da expressão de CK7 observada em carcinomas de alto grau e invasivos foi associada estatisticamente com ambos os parâmetros. A sobre-expressão da Ciclina D1 foi identificada em todos os graus e estadios tumorais, não tendo sido observadas diferenças estatisticamente significativas. Devido a problemas de ordem técnica a avaliação da sobre-expressão da p53 reduziu a casuística apenas a 4 casos, não sendo possível propor associações estatisticamente válidas. Este trabalho traz novos dados que auxiliam na compreensão das alterações moleculares envolvidas no desenvolvimento de tumores de bexiga de bovinos com origem no epitélio de transição, podendo ser utilizados no âmbito da Patologia Comparada, quer para os Animais Domésticos quer para o Homem.ABSTRACT - Identification of Structural Proteins in Bovine Urinary Bladder Tumours - Bovine Enzootic Hematuria (BEH) is a disease characterized by a chronic progress associated with the ingestion of bracken fern (Pteridium aquilinum), which main characteristic is the development of urinary bladder tumours. This disease is endemic in different parts of the World where the appropriate conditions are settled for bracken fern upgrowth, including the Azores Archipelago. The objectives of this research were (i) the characterization by immunohistochemical methods of the expression of the uroplakin III (UPIII) and cytokeratin 7 (CK7), which are structural proteins of the urothelium, in carcinomas of the urinary bladder of animals affected by BEH, and its possible association with tumour grade and stage, (ii) the survey of the altered expression of the cell cycle regulating factors Cyclin D1 and p53 and its possible association with tumour grade and stage, (iii) evaluation of the applicability of bovine urinary bladder tumours as a model for urinary bladder tumours in Domestic Animals and in Humans. The results showed that loss of UPIII expression was seen in high grade and invasive urothelial carcinomas, and that this loss was statistically associated with both parameters. Similarly, the loss of CK7 expression was seen in high grade and invasive tumours and was statistically associated with high grade and invasion. The overexpression of Cyclin D1 was identified in urothelial carcinomas of all grades and stages and no statistical association was identified. Due to technical problems the evaluation of p53 overexpression was reduced to only 4 cases. Therefore, it was not possible to draw statistical significant associations. This research brings new data to the understanding of urinary bladder tumours allowing a better knowledge of the molecular changes involved and may be useful in the framework of Comparative Pathology, either for Domestic Animals or Humans

Biocide Use for the Control of Non-Typhoidal <em>Salmonella</em> in the Food-Producing Animal Scenario: A Primary Food Production to Fork Perspective

Biocides are a group of substances commonly used in food production settings to destroy or control a wide range of microorganisms, which can be present in food of animal origin, since contamination can occur in the several steps of the food production chains. In order to achieve the desired results, the users of biocides must first understand the diverse characteristics of such compounds, mainly the usage requirements, limitations, and the factors affecting the activity of biocides. Food-producing animals and their products, namely meat and eggs, represent a major source of non-typhoidal Salmonella for humans and are associated with foodborne outbreaks worldwide. The prevention of cross-contamination, which can occur in any step of the food production chain, is essential for the ultimate objective of producing safe food products. The correct use of biocides, along with good hygiene and manufacturing practices, is one of the pillars of Salmonella spp. control and should be implemented in all steps of the food production chain. The present chapter reviews the accumulated knowledge on the use of biocides to control non-typhoidal Salmonella, from a farm to fork standpoint, along with the possible impacts on human health arising from improper use

Pheno and genotyping of Salmonella from slaughtered pigs in a Portuguese abattoir reveal differential persistence ability

Microbiology / Veterinary SciencesABSTRACT - Pork is one of the most common vehicles of non-typhoid foodborne Salmonella, with the slaughterhouse representing a key point for the infection of pigs and carcass contamination. By comparing matching samples taken from animals at the dirty (skin) and clean (inner and outer carcass surface) areas of the slaughterline, this study aimed to assess potential Salmonella contamination routes of pig carcasses within a Portuguese abattoir. Forty-four Salmonella isolates were retrieved from 120 pigs, and further characterized through pheno and genotypical methods. Most frequent serotypes found were Salmonella 4, [5],12:i:- (47.7%), Salmonella Rissen (40.9%) and Salmonella Derby (11.4%). Isolates were most commonly collected from the skin of pigs sampled at the dirty area (59.1%), followed by the inner (38.1%) and outer (9.1%) carcass surface sampled at the clean area. Most isolates (79.5%) were considered to be multidrug resistant and all harbored the virulence associated genes invA, invH, sopB, stn, slyA, phoP, phoQ and agfA. PFGE analysis revealed that most bacterial isolates belonging to the same serotype, recovered from animals from different farms, and slaughtered at separate days were genetically undistinguishable. Furthermore, our findings suggest that Salmonella Rissen might have an increased ability to endure on the slaughterhouse environment when compared with the other serotypes. Concluding, this study shows that the slaughterhouse may be a key point for the dissemination of resistant and virulent Salmonella strains, which stresses the importance of the implementation of good hygiene practices at the slaughterhouse and of the application of corrective measures to avoid cross-contamination.info:eu-repo/semantics/publishedVersio