Multiyear surveillance of Influenza A virus in wild birds in Portugal

This report presents the results of a multiyear (2005 to 2009) study of avian influenza virus (AIV) occurrence in wild birds in Portugal. A total of 5691 samples from wild birds belonging to 13 different orders were examined. Ninety-three samples tested positive for AIV by matrix reverse transcriptase-polymerase chain reaction, giving a total prevalence of 1.63%. Twenty-one viruses were successfully cultured in embryonated chicken eggs, which represent a rate of viral infectivity of 22.6% in the samples. Nine subtypes of haemagglutinin (H1, H3 to H7, H9 to H11) and eight subtypes of neuraminidase (N1 to N4, N6 to N9) were identified in 20 different combinations. The most prevalent subtypes of haemagglutinin detected were H5, H1 and H4, while for neuraminidase subtypes N2 and N6 were the most common. The subtype combinations H4N6 and H1N1 were predominant (15.1%). All H5 and H7 viruses detected in the present study were low pathogenic for poultry as determined by the sequence of amino acids at the cleavage site of haemagglutinin. The full-length nucleotide sequences of five H5, one H7 and five N3 genes were analysed phylogenetically. The Bayesian analysis revealed that all but one of the strains analysed were closely related to isolates detected in the same period in North and Central European countries. Three H5N3 isolates, all from 2007, formed a separate cluster in both H5 and N3 phylogenetic trees. This study provides evidence that various subtypes of AIV, including subtypes H5 and H7, circulate in Portugal, which may pose a risk to industrial poultry

Resultados serológicos que demonstram a ocorrência de infecções pelo vírus do Nilo Ocidental (WNV) em equinos e aves em Portugal (2004-2011)

O vírus West Nile (WNV) foi originalmente identificado no distrito West Nile no Uganda, tendo sido isolado pela primeira vez em 1937. No ciclo biológico deste flavivírus estão envolvidos mosquitos e aves que servem de multiplicadores e reservatório de vírus. Os humanos e equinos são hospedeiros acidentais e finais. Os primeiros surtos epidémicos de encefalite provocados pelo WNV em humanos e equinos registaram-se nos anos 50 e 60 do século passado em Israel e França. Em consequência dos casos de doença e morte registados em humanos, aves e equinos, particularmente nos deltas dos rios Danúbio e Volga e após emergência do vírus em 1999 nos EUA, o WNV passou a ser considerado um problema de saúde pública e animal. Nos últimos anos (2004-2010) registaram-se numerosos surtos da doença nos países da bacia mediterrânica. Em Portugal, o vírus foi isolado pela primeira vez em 1971, em mosquitos, pelo Prof. Armindo Filipe. Para o rastreio desta virose, o LNIV utiliza testes ELISA e de seroneutralização para detecção e confirmação de anticorpos WNV, respectivamente. A pesquisa de vírus é efectuada com recurso ao isolamento do vírus em cultura de células e a técnicas moleculares de diagnóstico (RT-PCR). Os rastreios serológicos realizados entre 2004 e 2011 em equinos e aves, revelaram a presença de anticorpos para o WNV em ambas as espécies. Das 116 aves estudadas, 23 (19,8%) foram positivas, com títulos de anticorpos neutralizantes relativamente baixos (20-40). Entre as espécies de aves seropositivas encontram-se águias, corujas, cegonhas, flamingos, avestruzes, nandus e íbis. Em cavalos, das 1509 amostras testadas, 44 (2,9%) foram positivas a anticorpos neutralizantes com títulos que variaram entre 16 e 2560. Todas as amostras analisadas foram colhidas de animais aparentemente saudáveis, com excepção de duas provenientes de éguas pertencentes a uma exploração situada na região de Setúbal. Estes dois animais apresentavam sintomas neurológicos e no exame serológico foram detectados anticorpos IgM para o WNV, indicando uma infecção recente. Uma égua morreu e a outra recuperou, registando-se nesta uma subida muito significativa do título de anticorpos neutralizantes. O WNV não foi detectado por RT-PCR nas amostras de sangue de equinos (n=558) e aves (n=886) incluídas neste estudo. Contudo, os dados serológicos obtidos demonstram a presença e circulação do WNV no nosso país

Serological evidence of West Nile virus circulation in Portugal

The circulation of West Nile virus in Portugal was assessed by serological surveys conducted during 2004–2010 in horses and birds. The detection of WNV antibodies in both species in all the years covered by the study as well as the presence of anti-WNV IgM in symptomatic horses that had not traveled outside the country, support the notion that WNV circulates in Portugal

Proteção de soft targets: a ameaça de terrorismo na área do Bairro Alto

Nos últimos anos, os soft targets têm vindo a ser alvos cada vez mais recorrentes para a prática de atos terroristas devido às vulnerabilidades típicas que apresentam. Quer seja pela falta de medidas de segurança robustas, pela facilidade de acesso, pelos aglomerados de pessoas, pelo mediatismo, entre outros, os soft targets compreendem um conjunto de caraterísticas que se consubstanciam como mais valias para os objetivos de quem pretende realizar um ataque terrorista e esse facto não pode ser negligenciado. As zonas turísticas enquadram-se nessa tipologia de alvos, onde existe a possibilidade de ocorrência de um ataque terrorista e deve ser considerada a implementação de medidas de prevenção para que se possam diluir as vulnerabilidades desses locais. Na presente dissertação, foi considerada a área do Bairro Alto como objeto de estudo, analisando-se as medidas de segurança em implementação e qual o nível de preparação para dar resposta a um ataque terrorista, face às vulnerabilidades existentes. Para esse efeito, foi realizado um estudo qualitativo com a concretização de 13 entrevistas, envolvendo elementos policiais e indivíduos civis, todos com conhecimento privilegiado no âmbito do terrorismo ou da segurança da área. O estudo demonstra que apesar de existirem algumas medidas a serem implementadas no âmbito da prevenção, estas são ainda insuficientes para fazer face à imprevisibilidade e variedade de métodos para a realização de um ataque terrorista. Os entrevistados revelam algumas vulnerabilidades, designadamente a disposição das infraestruturas na área e do estado degradado em que se encontram, assim como a formação providenciada ao efetivo. A dissertação apresenta ainda algumas propostas de melhoria a implementar, tanto para o efetivo, como para as condições de segurança da área.In recent years, soft targets have become increasingly recurrent targets for terrorist acts due to the typical vulnerabilities they present. Due to the lack of robust security measures, ease of access, crowds of people, media exposure, among others, soft targets comprise a set of characteristics that are added value to the objectives of those who intend to carry out a terrorist attack and that fact cannot be overlooked. Tourist areas, due to their particularities, fit into this typology of targets, being some of the places where the possibility of a terrorist attack occurring cannot be denied and the implementation of preventive measures must be considered so the vulnerabilities existing in these locations can be diluted. In this dissertation, the Bairro Alto area was considered the object of study, by analyzing the security measures being implemented and the level of preparation to respond to a terrorist attack, given the vulnerabilities that exist in the area. For this purpose, a qualitative study was carried out with the completion of 13 interviews, involving police elements and civilian individuals, all with privileged knowledge in the field of terrorism or in the security in the area. The study showed that although there are already some measures to be implemented in the scope of prevention, these are still insufficient to face the unpredictability and variety of existing methods for carrying out a terrorist attack. The participants also reveal some vulnerabilities, whether in the area, through the layout of the infrastructures and the degraded state in which they are found, or in the training provided to the agents. That said, some proposals for improvement to be implemented are also presented, both for the agents and for improving the conditions of the area

Imagem corporativa positiva duradoura: identidade corporativa, estratégias, comunicação e a sua importância

A dissertação apresentada centra-se na importância de uma imagem corporativa positiva e a forma como esta surge. Apresenta-se uma relação entre identidade corporativa e imagem corporativa, ou seja, a imagem corporativa advém de uma identidade, composta por vários elementos, que traduzem uma determinada personalidade corporativa para o exterior, formando assim uma imagem corporativa na mente dos receptores. Uma identidade corporativa possui muitos elementos mas a sua observação tendo em conta a estrutura da organização e importância das estratégias organizacionais, a cultura organizacional, missão e valores, comunicação interna e identidade visual, permite compreender a forma como esta exerce influência na imagem corporativa. Este trabalho académico pretende mostrar a forma como uma imagem corporativa surge, os elementos que contribuem para tal e a importância de se ter em conta o papel do comportamento do ser humano em todo o processo

Molecular study of Porcine Circovirus type 2 circulating in Portugal

Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the Circoviridae family and has been associated with a disease named postweaning multisystemic wasting syndrome (PMWS). In Portugal the disease caused severe problems mainly between 1995 and 2003, but is nowadays less evident probably as the result of vaccination programmes initiated in 2007. This study reports the molecular analysis of 22 PCV2 strains obtained from domestic pigs originated from different regions of Portugal. One hundred and seventy-seven samples from PCV2 suspected cases received between 2003 and 2010 were analysed, 79 (44.6%) of which tested PCV2 PCR-positive. The entire genomes of 22 viruses were amplified and sequenced. Two major groups were observed, according to the genome length and in single nucleotide polymorphisms. The nucleotide similarity found among the PCV2 strains ranged between 94% and 99%. The Bayesian inference of phylogeny based on the genome of the strains studied revealed the existence of two distinct genetic groups consistent with the nucleotide sequence observations. The majority of the strains branched in genotype PCV2b, while six strains with a common geographical origin obtained between 2007 and 2009, were included in genotype PCV2a. The analysis performed with cap and rep genes confirmed that cap is a reliable alternative to full-length genome based phylogenetic studies of PCV2

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E. coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E. coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 lM Cu and 50 and 200 lM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

Abstract Background: Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results: A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions: Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.This work was supported by the project “Development of ultra-sensitive detection methods and plant nano-vaccines for the fungi Fusarium spp. using nanotechnological devices” Iberian Capacitation Program in Nanotechnologies: Call 2006/2007”

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

<p>Abstract</p> <p>Background</p> <p>Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility.</p> <p>Results</p> <p>A plant cell suspension culture of <it>Medicago sativa </it>was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by <it>Medicago sativa </it>cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, <it>Medicago sativa </it>cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H₂DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation.</p> <p>Conclusions</p> <p>Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for <it>Medicago sativa </it>cells, a safe range of 1-5 nM should not be exceeded for biological applications.</p