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Browsing EM - Artigos Científicos by Field of Science and Technology (FOS) "Ciências Agrárias::Ciências Veterinárias"
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- Assessing in vivo and in vitro biofilm development by Streptococcus dysgalactiae subsp. dysgalactiae using a murine model of catheter-associated biofilm and human keratinocyte cellPublication . Alves-Barroco, Cinthia; Botelho, Ana Maria Nunes; Américo, Marco Antonio; Fracalanzza, Sérgio Eduardo Longo; Matos, António P. Alves de; Guimaraes, Márcia Aparecida; Ferreira-Carvalho, Bernadete Teixeira; Figueiredo, Agnes Marie Sá; Fernandes, Alexandra R.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is an important agent of bovine mastitis. This infection causes an inflammatory reaction in udder tissue, being the most important disease-causing significant impact on the dairy industry. Therefore, it leads to an increase in dairy farming to meet commercial demands. As a result, there is a major impact on both the dairy industry and the environment including global warming. Recurrent mastitis is often attributed to the development of bacterial biofilms, which promote survival of sessile cells in hostile environments, and resistance to the immune system defense and antimicrobial therapy. Recently, we described the in vitro biofilm development on abiotic surfaces by bovine SDSD. In that work we integrated microbiology, imaging, and computational methods to evaluate the biofilm production capability of SDSD isolates on abiotic surfaces. Additionally, we reported that bovine SDSD can adhere and internalize human cells, including human epidermal keratinocyte (HEK) cells. We showed that the adherence and internalization rates of bovine SDSD isolates in HEK cells are higher than those of a SDSD DB49998-05 isolated from humans. In vivo, bovine SDSD can cause invasive infections leading to zebrafish morbidity and mortality. In the present work, we investigated for the first time the capability of bovine SDSD to develop biofilm in vivo using a murine animal model and ex-vivo on human HEK cells. Bovine SDSD isolates were selected based on their ability to form weak, moderate, or strong biofilms on glass surfaces. Our results showed that SDSD isolates displayed an increased ability to form biofilms on the surface of catheters implanted in mice when compared to in vitro biofilm formation on abiotic surface. A greater ability to form biofilm in vitro after animal passage was observed for the VSD45 isolate, but not for the other isolates tested. Besides that, in vitro scanning electron microscopy demonstrated that SDSD biofilm development was visible after 4 hours of SDSD adhesion to HEK cells. Cell viability tests showed an important reduction in the number of HEK cells after the formation of SDSD biofilms. In this study, the expression of genes encoding BrpA-like (biofilm regulatory protein), FbpA (fibronectin-binding protein A), HtrA (serine protease), and SagA (streptolysin S precursor) was higher for biofilm grown in vivo than in vitro, suggesting a potential role for these virulence determinants in the biofilm-development, host colonization, and SDSD infections. Taken together, these results demonstrate that SDSD can develop biofilms in vivo and on the surface of HEK cells causing important cellular damages. As SDSD infections are considered zoonotic diseases, our data contribute to a better understanding of the role of biofilm accumulation during SDSD colonization and pathogenesis not only in bovine mastitis, but they also shed some lights on the mechanisms of prosthesis-associated infection and cellulitis caused by SDSD in humans, as well.
- Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosisPublication . Zhou, Ershun; Silva, Liliana M. R.; Conejeros, Iván; Velásquez, Zahady D.; Hirz, Manuela; Gärtner, Ulrich; Jacquiet, Philippe; Taubert, Anja; Hermosilla, CarlosBesnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.
- fHER2, PR, ER, Ki-67 and cytokeratin 5/6 expression in benign feline mammary lesionsPublication . Soares, Maria; Correia, Assunção N.; Batista, Mariana R.; Correia, Jorge; Ferreira, FernandoBiomarkers are essential in the characterization of neoplastic lesions and aid not only in the classification of the nature of the lesions, but also in the understanding of their ontogeny, development and prognosis. In cats, while mammary carcinomas are increasingly being characterized, information on their benign lesions is still scarce. Indeed, a better characterization of benign lesions could have an important role in unravelling mammary oncogenesis, similar to that in human breast cancer. Thus, in this study, the expression of five markers was analyzed in 47 benign mammary lesions (hyperplasia, dysplasia and benign tumors) collected from 27 queens. Dysplastic and hyperplastic lesions were the most common (41/47, 81.7%). Most of the lesions were classified as ER positive (43/47, 91.5%), PR negative (30/47, 63.8%), fHER2 negative (29/47, 64.4%), CK 5/6 negative (36/47, 76.6%) and with a low Ki-67 index (37/47, 78.7%). Statistical analysis revealed a correlation between younger ages and ER positivity (p = 0.013) and between larger lesions and negative PR status (p = 0.038). These results reinforce the importance of evaluating the expression of the ER status, prevalent in benign lesions, as a putative precursor in cancer progression.