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Andreoletti O.,National Veterinary School of Toulouse | Orge L.,Laboratorio Nacional Of Investigacao Veterinaria | Benestad S.L.,National Veterinary Institute | Beringue V.,French National Institute for Agricultural Research | And 11 more authors.
PLoS Pathogens | Year: 2011

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed. © 2011 Andréoletti et al.


Goncalves R.,Laboratorio Nacional Of Investigacao Veterinaria | Mariano I.,Novo A/S | Nunez A.,Veterinary Laboratories Agency Weybridge | Branco S.,University of Évora | And 2 more authors.
Veterinary Journal | Year: 2010

An outbreak of severe respiratory disease in a goat herd was associated with Mycoplasma ovipneumoniae, Mycoplasma arginini, Mannheimia haemolytica and Pasteurella multocida with mortality rates exceeding 20% in kids. Post mortem features in affected kids included severe pleuropneumonia, lung consolidation, large quantities of pleural fluid and pericarditis. This is the first report of atypical proliferative pneumonia in goats in Portugal. © 2009.


Duarte I.M.,Polytechnic Institute of Coimbra | Almeida M.T.M.,University of Minho | Duarte M.M.,Laboratorio Nacional Of Investigacao Veterinaria | Brown D.J.F.,Central Laboratory of General Ecology | Neilson R.,Scottish Crop Research Institute
Plant Pathology | Year: 2011

A PCR-RFLP assay was developed for the identification of trichodorid nematodes belonging to Nanidorus, Paratrichodorus and Trichodorus genera. Using the variability of the 18S SSU rDNA gene, this method provides a new molecular diagnosis tool which allows identification within mixed samples of trichodorid and non-trichodorid species, differentiation of juveniles, and represents an alternative to the difficult and time consuming phenotyping of similar species. Based on the alignment of previously obtained 18S rDNA nucleotide sequences of trichodorids from Portugal, a pair of selective primers was designed in conserved regions to allow the amplification of a variable region located at the 3' end of the gene in all known Portuguese species. The 615bp PCR product showed nucleotide variability enabling the generation of restriction fragment patterns which were consistent among populations of the same species but allowed discrimination of trichodorids at the species level. The proposed protocol was tested and proved effective with 12 trichodorid species from Portugal (N. minor, P. allius, P. anemones, P. divergens, P. hispanus, P. pachydermus, P. porosus, T. beirensis, T. lusitanicus, T. primitivus and two other Trichodorus species, A and B) and six non-indigenous trichodorid populations (N. minor, P. allius, P. anemones, P. pachydermus, P. porosus and T. primitivus). © 2010 The Authors. Plant Pathology © 2010 BSPP.


Henriques A.M.,Laboratorio Nacional Of Investigacao Veterinaria | Fagulha T.,Laboratorio Nacional Of Investigacao Veterinaria | Barros S.C.,Laboratorio Nacional Of Investigacao Veterinaria | Ramos F.,Laboratorio Nacional Of Investigacao Veterinaria | And 3 more authors.
Avian Pathology | Year: 2011

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. © 2011 Copyright Houghton Trust Ltd.


Freitas A.,Laboratorio Nacional Of Investigacao Veterinaria | Barbosa J.,Laboratorio Nacional Of Investigacao Veterinaria | Ramos F.,University of Coimbra
Food Analytical Methods | Year: 2012

An amoxicillin stability study was performed under different pH (1, 3 and 5) and temperature (4 °C, 22 °C, 37 °C and 55 °C) conditions and for incurred samples stored at -20 °C with the goals of better understanding amoxicillin degradation and characterising its main degradation products (amoxicilloic acid and amoxicillin diketopiperazine). The analytical methodology used consisted of a simple extraction using phosphate buffer (pH 8) with sodium chloride followed by a sample clean-up using OASIS® HLB cartridges and a liquid chromatography-tandem mass spectrometric analysis. Amoxicillin was found to be greatly unstable at temperatures above 22 °C for all pH values studied, so it was recommended that biological samples should be frozen at temperatures below -70 °C until analysis of the amoxicillin residues. © 2011 Springer Science+Business Media, LLC.


Barbosa J.,Laboratorio Nacional Of Investigacao Veterinaria | Freitas A.,Laboratorio Nacional Of Investigacao Veterinaria | Mourao J.L.,Royal University | Noronha Da Silveira M.I.,University of Coimbra | Ramos F.,University of Coimbra
Journal of Agricultural and Food Chemistry | Year: 2012

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2- oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment. © 2012 American Chemical Society.


Freitas A.,Laboratorio Nacional Of Investigacao Veterinaria | Barbosa J.,Laboratorio Nacional Of Investigacao Veterinaria | Ramos F.,University of Coimbra
Meat Science | Year: 2014

A multi-residue quantitative screening method covering 41 antibiotics from 7 different families, by ultra-high-performance-liquid-chromatography tandem mass spectrometry (UHPLC-MS/MS), is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected after a simple sample preparation of bovine muscle optimized to achieve the best recovery for all compounds. A simple sample treatment was developed consisting in an extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA), followed by a defatting step with n-hexane. The methodology was validated, in accordance with Decision 2002/657/EC by evaluating the required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. Precision in terms of relative standard deviation was under 20% for all compounds and the recoveries between 91% and 119%. CCα and CCβ were determined according the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when required. © 2014 Elsevier Ltd.


Barbosa J.,Laboratorio Nacional Of Investigacao Veterinaria | Freitas A.,Laboratorio Nacional Of Investigacao Veterinaria | Moura S.,Laboratorio Nacional Of Investigacao Veterinaria | Mourao J.L.,University of Trás os Montes e Alto Douro | And 2 more authors.
Journal of Agricultural and Food Chemistry | Year: 2011

Nitrofurans were broadly used as an extremely effective veterinary antibiotic especially in pig and poultry production farms. Because of fears of the carcinogenic effects on humans, the nitrofurans were banned from use in livestock production in many countries, including the European Union. The present study examines the accumulation, distribution, and depletion of furaltadone and nifursol and of their tissue-bound metabolites [3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 3,5-dinitro-salicylic acid hydrazine (DNSAH), respectively, in poultry edible tissues (muscle, liver, and gizzards) following administration to chickens of therapeutic and subtherapeutic concentrations of both compounds. Nitrofurans determination was performed by high-performance liquid chromatography-diode array detection and liquid chromatography-tandem mass spectrometry, respectively, for feeds and for poultry tissues. Furaltadone and nifursol, in very low concentrations, were found in samples of muscle, liver, and chicken's gizzard collected from slaughtered animals after 5 weeks of treatment and no withdrawal time period. When a withdrawal time period of 3 weeks was respected, no detectable nitrofuran parent compounds was observed in all of the studied matrices. For AMOZ, concentrations of 270 μg/kg in meat, 80 μg/kg in liver, and 331 μg/kg in gizzard were determined after administration of a medicated feed with furaltadone (132 mg/kg), 3 weeks after withdrawal of treatment. For DNSAH, the concentration values obtained are much lower than those observed for AMOZ. For meat, liver, and gizzard, DNSAH concentrations of 2.5, 6.4, and 10.3 μg/kg, respectively, were determined, after administration of a medicated feed with nifursol (98 mg/kg), 3 weeks after withdrawal of treatment. The gizzard could be considered a selected matrix for nitrofuran residues evaluation in poultry, due to its capacity of retaining either nitrofuran parent compounds or metabolites in higher concentrations, regardless of the administered dose or of the respected withdrawal time period. © 2011 American Chemical Society.


Gomes-Neves E.,Abel Salazar Biomedical Sciences Institute | Gomes-Neves E.,University of Porto | Antunes P.,University of Porto | Tavares A.,Laboratorio Nacional Of Investigacao Veterinaria | And 7 more authors.
International Journal of Food Microbiology | Year: 2012

In this study the occurrence of Salmonella in swine, pork meat and meat handlers along with their clonal relatedness is evaluated at abattoir level. Samples from the lymph nodes, carcass surface and meat of 100 pigs and 45 meat handlers were collected in eight abattoirs (July 2007-August 2008). Salmonella isolates were serotyped and genotyped by pulsed-field gel electrophoresis (PFGE). From the pigs tested, 42 produced at least one positive sample. A relatively high frequency of Salmonella occurrence was found in the ileoceacal lymph node samples (26.0%), followed by carcass (16.0%) and meat samples (14.0%). However, ileoceacal lymph nodes that test positive for Salmonella are not found to be a predictor of positive test results further on in the process. Besides the slaughterhouse environment, meat handlers were identified as a possible source of subsequent contamination, with 9.3% of the sample testing positive. Diverse Salmonella enterica serotypes were detected, mainly S. Typhimurium and the monophasic variant S. 4,[5],12:i:-, but also S. Derby, S. Rissen, S. Mbandaka, S. London, S. Give, S. Enteritidis and S. Sandiego, in total corresponding to 17 PFGE types. Our results demonstrate that besides a high level of Salmonella swine contamination at pre-harvest level, slaughtering, dressing, cutting and deboning operations are contributing to the occurrence of clinically relevant clones (e.g. S. Typhimurium DT104 and S. 4,[5],12:i:-) in pork products. This study also highlights the possibility of an ongoing Salmonella community being spread by abattoir workers. © 2012 Elsevier B.V.


PubMed | Laboratorio Nacional Of Investigacao Veterinaria, Marine and Environmental science Center and University of Coimbra
Type: | Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences | Year: 2016

Together with fish, algae reared in aquaculture systems have gained importance in the last years, for many purposes. Besides their use as biofilters of effluents, macroalgaes rich nutritional profiles have increased their inclusion in human diets but also in animal feeds as sources of fatty acids, especially important for the fish industry. Nonetheless, algae are continuously exposed to environmental contaminants including antibiotics and possess the ability for bioaccumulation of such compounds. Therefore, the present paper describes the development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of antibiotics in the green macroalgae Ulva lactuca. This multi-residue method enables the determination of 38 compounds distributed between seven classes and was fully validated according to EU Decision 2002/657/EC.

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