Aspectos da patogenia da piómetra na cadela : fímbrias tipo 1 de escherichia coli e mecanismos de defesa no endométrio
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2017
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Piómetra é uma doença do foro reprodutivo comum em cadelas sendo Escherichia coli a bactéria mais frequentemente isolada. Neste estudo estabelecemos dois objectivos distintos: a) avaliar a capacidade de E. coli associada a piómetra para transcrever os genes fim e expressar fímbrias tipo 1; b) estudar a resposta imune inata do endométrio, sob a forma de explantes, após estimulação com E. coli.
De modo a determinar se os genes fim são transcritos, a orientação (On/Off) do elemento de ADN que contém o promotor e conduz à transcrição dos genes fim, foi determinada por amplificação da região do promotor por PCR, seguida da digestão com a enzima HinfI. Para tal, foram efectuados ensaios em três condições de cultura; agar, arejada, estática. Os resultados mostraram que a maioria dos isolados tem a capacidade de mudar da posição Off para a posição On. Em agar, a posição Off foi predominante (60% dos isolados) e 40% dos isolados apresentaram uma mistura de células na posição On e Off. Na cultura arejada, 80% dos isolados tinham células em posições On e Off e 20% estavam na posição Off. Na cultura estática, a maioria dos isolados (67%) tinha células nas posições On e Off e 7% mudaram completamente para a posição On. Neste estudo demonstrámos ainda que os isolados de E. coli associados a piómetra têm a capacidade de expressar fimbrias tipo 1, uma vez que aglutinam células de levedura. No que se refere ao primeiro objectivo, concluímos que os isolados de E. coli de piómetra têm a capacidade de transcrever genes fim e expressar fimbrias tipo 1. No entanto, a transcrição e expressão estão dependentes de estímulo ambiental. Quanto ao segundo objectivo, mostrámos que os explantes endometriais estimulados com E. coli transcrevem genes que codificam para mediadores de inflamação. Os níveis de transcrição dos genes que codificam para o TNFα, IL-6 e CXCL10 foram significativamente mais elevados em explantes estimulados com E. coli., comparativamente a explantes não estimulados. Por outro lado, a transcrição do gene IL-1β foi menor nos explantes estimulados. Estes resultados demonstram que os explantes de endométrio foram capazes de montar uma resposta imunitária.
Pyometra is a common reproductive disease of bitches and Escherichia coli is the most frequently isolated bacteria. In this study we establish two distinct objectives: a) evaluate the capacity of E. coli isolated from pyometra to transcribe fim genes and express type 1 fimbriae; b) study the innate immune response of endometrial explants to E. coli stimulation. Although, fimA and fimH genes are the most prevalent adhesin genes in E. coli pyometra isolates, their capacity to express type 1 fimbriae is unclear. To determine whether type 1 fimbriae genes are transcribed, the orientation (On or Off) of the invertible promoter element, that drives the transcription of the fim genes, was assessed by PCR amplification, followed by HinfI digestion. The capacity of E. coli isolates to transcribe fim genes was evaluate in three culture conditions: agar, aerated, static. Results showed that the majority of the isolates have the ability to switch from Off to On position. In agar, the Off position was predominant (60% of the isolates) and 40% of isolates had a mixture of cells in the On and Off position. In the aerated culture, 80% of the isolates had cells in On and Off positions and 20% of the isolates were in the Off position. In static culture most of the isolates (67%) had cells on On and Off positions and 7% of the isolates switch completely to On position. In addition, we demonstrate that pyometra E. coli isolates had the capacity to express type 1 fimbriae, as they agglutinate yeast cells. Concerning the first objective, we conclude that pyometra E. coli isolates had the ability to transcribe fim genes and express type 1 fimbriae. However, transcription and expression are dependent on environmental stimulus. Regarding the second objective, we show that the endometrial explants stimulated with E. coli transcribe inflammatory mediators. Differential cytokine gene transcription (qRT-PCR) was also observed in endometrial explants stimulated with E. coli and non-stimulated endometrial explants. The transcription levels of TNFα, IL-6 and CXCL10 genes were significantly higher in E. coli stimulated explants. In contrast, IL-1β gene transcription was lower in the stimulated endometrial explants. These results show that the endometrial explants were capable of mounting immune responses.
Pyometra is a common reproductive disease of bitches and Escherichia coli is the most frequently isolated bacteria. In this study we establish two distinct objectives: a) evaluate the capacity of E. coli isolated from pyometra to transcribe fim genes and express type 1 fimbriae; b) study the innate immune response of endometrial explants to E. coli stimulation. Although, fimA and fimH genes are the most prevalent adhesin genes in E. coli pyometra isolates, their capacity to express type 1 fimbriae is unclear. To determine whether type 1 fimbriae genes are transcribed, the orientation (On or Off) of the invertible promoter element, that drives the transcription of the fim genes, was assessed by PCR amplification, followed by HinfI digestion. The capacity of E. coli isolates to transcribe fim genes was evaluate in three culture conditions: agar, aerated, static. Results showed that the majority of the isolates have the ability to switch from Off to On position. In agar, the Off position was predominant (60% of the isolates) and 40% of isolates had a mixture of cells in the On and Off position. In the aerated culture, 80% of the isolates had cells in On and Off positions and 20% of the isolates were in the Off position. In static culture most of the isolates (67%) had cells on On and Off positions and 7% of the isolates switch completely to On position. In addition, we demonstrate that pyometra E. coli isolates had the capacity to express type 1 fimbriae, as they agglutinate yeast cells. Concerning the first objective, we conclude that pyometra E. coli isolates had the ability to transcribe fim genes and express type 1 fimbriae. However, transcription and expression are dependent on environmental stimulus. Regarding the second objective, we show that the endometrial explants stimulated with E. coli transcribe inflammatory mediators. Differential cytokine gene transcription (qRT-PCR) was also observed in endometrial explants stimulated with E. coli and non-stimulated endometrial explants. The transcription levels of TNFα, IL-6 and CXCL10 genes were significantly higher in E. coli stimulated explants. In contrast, IL-1β gene transcription was lower in the stimulated endometrial explants. These results show that the endometrial explants were capable of mounting immune responses.
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MESTRADO EM ENGENHARIA BIOTECNOLÓGICA, ENGENHARIA BIOTECNOLÓGICA, MEDICINA VETERINÁRIA, PIÓMETRA, CÃES, DOENÇAS DO SISTEMA REPRODUTIVO, BIOTECHNOLOGY ENGINEERING, VETERINARY MEDICINE, PYOMETRA, DOGS, REPRODUCTIVE DISEASES