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Wu G.,Veterinary Laboratories Agency VLA Weybridge | AbuOun M.,Veterinary Laboratories Agency VLA Weybridge | Hackl E.,AIT Austrian Institute of Technology | La Ragione R.M.,Veterinary Laboratories Agency VLA Weybridge | And 6 more authors.
Environmental Microbiology Reports | Year: 2010

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid. © 2009 Crown.


Whelan C.,Enfer Scientific | Whelan A.O.,Veterinary Laboratories Agency VLA Weybridge | Shuralev E.,Enfer Scientific | Kwok H.F.,Fusion Antibodies | And 3 more authors.
Clinical and Vaccine Immunology | Year: 2010

Rapid, simple, and accurate antemortem tests for tuberculosis (TB) in cattle need to be developed in order to augment the existing screening methods. In particular, as cattle vaccines are developed, such tests would allow the continuation of test-and-slaughter policies alongside vaccination. Therefore, the development of an assay that distinguishes infected from vaccinated animals (a DIVA test) is an urgent research requirement. In this study, we assessed the performance of a novel multiplex serological test with sera collected from 96 skin-tested animals with bovine tuberculosis, 93 TB-free animals, and 39 cattle vaccinated with Mycobacterium bovis BCG. Our results indicate that the test has a relative sensitivity range of 77.0% to 86.5% at corresponding specificity levels of 100.0% to 77.6%. Comparison with the Bovigam gamma interferon antemortem test revealed that this serology test was significantly more sensitive at specificities above 97.9%, while the Bovigam test was, on average, about 10% more sensitive when the test specificity was set below 97%. Importantly, this serological multiplex assay does not react with sera from BCG-vaccinated calves and is therefore suitable as a DIVA test alongside BCG-based vaccine strategies. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Nunes S.F.,University of Cambridge | Murcia P.R.,University of Cambridge | Tiley L.S.,University of Cambridge | Brown I.H.,Veterinary Laboratories Agency VLA Weybridge | And 3 more authors.
Influenza and other Respiratory Viruses | Year: 2010

Background: The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives: We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods: Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co-ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real-time RT-PCR. Results: Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion: The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. © 2009 Blackwell Publishing Ltd.


Slomka M.J.,Veterinary Laboratories Agency VLA Weybridge | Densham A.L.E.,Veterinary Laboratories Agency VLA Weybridge | Coward V.J.,Veterinary Laboratories Agency VLA Weybridge | Essen S.,Veterinary Laboratories Agency VLA Weybridge | And 8 more authors.
Influenza and other Respiratory Viruses | Year: 2010

Background: There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods. Objectives: First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections. Methods: RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v. Results: The " perfect match" M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a " gold standard" , while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. " Perfect match" M gene RRT PCR had 100% sensitivity and 95·2% specificity for swabs, 93·6% and 98·6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99·1%, respectively, for the swabs, and 100% and 100% for the tissues. Conclusions: Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated. © 2010 Blackwell Publishing Ltd.


Perez De Val B.,Autonomous University of Barcelona | Lopez-Soria S.,Autonomous University of Barcelona | Nofrarias M.,Autonomous University of Barcelona | Martin M.,Autonomous University of Barcelona | And 7 more authors.
Clinical and Vaccine Immunology | Year: 2011

Caprine tuberculosis (TB) has increased in recent years, highlighting the need to address the problem the infection poses in goats. Moreover, goats may represent a cheaper alternative for testing of prototype vaccines in large ruminants and humans. With this aim, a Mycobacterium caprae infection model has been developed in goats. Eleven 6-month-old goats were infected by the endobronchial route with 1.5 × 10 3 CFU, and two other goats were kept as noninfected controls. The animals were monitored for clinical and immunological parameters throughout the experiment. After 14 weeks, the goats were euthanized, and detailed postmortem analysis of lung lesions was performed by multidetector computed tomography (MDCT) and direct observation. The respiratory lymph nodes were also evaluated and cultured for bacteriological analysis. All infected animals were positive in a single intradermal comparative cervical tuberculin (SICCT) test at 12 weeks postinfection (p.i.). Gamma interferon (IFN-γ) antigen-specific responses were detected from 4 weeks p.i. until the end of the experiment. The humoral response to MPB83 was especially strong at 14 weeks p.i. (13 days after SICCT boost). All infected animals presented severe TB lesions in the lungs and associated lymph nodes. M. caprae was recovered from pulmonary lymph nodes in all inoculated goats. MDCT allowed a precise quantitative measure of TB lesions. Lesions in goats induced by M. caprae appeared to be more severe than those induced in cattle by M. bovis over a similar period of time. The present work proposes a reliable new experimental animal model for a better understanding of caprine tuberculosis and future development of vaccine trials in this and other species. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


PubMed | Veterinary Laboratories Agency VLA Weybridge
Type: Journal Article | Journal: The Veterinary record | Year: 2011

A real-time PCR was developed to detect Coxiella burnetii (the cause of Q fever) in ruminant placentas and aborted fetuses. Primer and probe sets previously developed for human tissue studies were used to target the insertion sequence IS1111 gene for C burnetii. The assay was highly sensitive, with a limit of detection of 10 copies of template, theoretically equating to a single bacterium, and did not cross-react with a panel of other bacteria. To determine sensitivity on field samples submitted for the diagnosis of abortion, results using the IS1111 PCR assay were compared with a com1 PCR assay. When applied to ruminant abortion material, including placental cotyledons and fetal samples, the IS1111 and com1 assays yielded positive results in 23 (25 per cent) of 93 and 19 (20 per cent) of 93 samples, respectively. One infected goat herd was monitored for 31 months: 57 (92 per cent) of 62 placental cotyledon samples from aborting and non-aborting goats, and 10 (30 per cent) of 33 fetal samples were positive by the IS1111 PCR assay.


Weeks M.E.,Veterinary Laboratories Agency VLA Weybridge
Methods in molecular biology (Clifton, N.J.) | Year: 2010

Proteomic methodologies have been at the forefront of cancer research for several years. The use of proteomic strategies to study all expressed genes aims to discover biomarkers indicative of the physiological state of cancer cells at specific time points, enabling early diagnosis, following cancer development/progression, screening and monitoring the efficacy of new therapeutic agents. Onco-proteomics has the potential to impact on oncology practice by delivering individualised highly selective clinical care. 2D-DIGE (2D difference in gel electrophoresis) enables simultaneous examination and comparison of multiple samples using cyanine dyes to label amino acid residues that are then separated based on charge and mass. These advantages combined with universal availability have until recently made 2D-DIGE a first method of choice in cancer proteome analysis of diverse specimens, including tissues, cell lines, blood and other body fluids.


PubMed | Veterinary Laboratories Agency VLA Weybridge
Type: Comparative Study | Journal: Influenza and other respiratory viruses | Year: 2010

There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods.First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections.RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v.The perfect match M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a gold standard, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. Perfect match M gene RRT PCR had 100% sensitivity and 95.2% specificity for swabs, 93.6% and 98.6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99.1%, respectively, for the swabs, and 100% and 100% for the tissues.Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.

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