Abstract Book of EAVLD2012 - eavld congress 2012

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S1 - P - 16 EVALUATION OF AN ELISA ASSAY AS AN ANCILLARY METHOD IN A HERD WITH A NATURAL TUBERCULOSIS INFECTION I. Arnaiz 1 , C. Eiras 1 , M.L. Luaces 2 , J. Fraga 3 , M. Rodríguez 3 , C. Calvo 1 1 Laboratorio de Sanidade e Produción Animal de Galicia, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 2 Área Veterinaria de Lugo, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España 3 Área Veterinaria de Vilalba, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España Keyword: Tuberculosis, ELISA, IFN-g, SIT, isolation Introduction The single intradermal tuberculin (SIT) test and the Interferongamma assay (IFN-g) are the tuberculosis diagnostic tools in the tuberculosis eradication program currently used in Spain. These tests based on cell-mediated response can detect animals in the early stages of infection (1), although not all affected animals are detected with these tests. Humoral immune response is found in cattle with natural infection and this response could be detected by the use of an ELISA assay. ELISA testing is not routinely used in bovine tuberculosis control programs mainly due to a reduced sensitivity (2) although it has been suggested that it might be used as a complement to the tuberculin test, especially for the detection of anergic tuberculous cattle (3). The aim of this study is the evaluation of the use of SIT test and the IFN-g assay together with an ELISA assay in order to obtain the most complete detection of tuberculosis infected animals in a herd previously confirmed to have tuberculosis. Material & methods 314 animals from a tuberculosis infected beef herd from Galicia, in north-west Spain, were tested using the SIT test. The IFN-g detection assay (BovigamTM, Prionics AG, Switzerland) was used in parallel with the SIT test. The 16 positive SIT animals (of which 15 animals were also detected by IFN-g assay) and 17 cattle that were only detected by IFN-g assay were slaughtered and their tissues were collected for Mycobacterium tuberculosis complex (MTC) isolation. At the same time, 20 cows older than 10 years were slaughtered too. Tissues from the 53 slaughtered animals were inoculated into the selective media (4). The MTC was recovered and confirmed by real-time PCR (5) in 18 cattle. The plasma collected for the IFN-g assay was also used on the ELISA assay for M. bovis antibody (IDEXX Laboratories). 18 cattle were detected with the ELISA assay. The agreement and kappa statistic measurements were calculated by comparing the MTC isolation with SIT test, IFN-g and ELISA assays respectively. References 1. Rua-Domenech R, Goodchild AT, Vordermeier HM, Hewinson RG, Christiansen KH, Clifton-Hadley RS: Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques. Res Vet Sci 2006, 81:190-210. 2.Ritacco V, Lopez B, Barrera L, Nader A, Fliess E, de Kantor: Further evaluation of an indirect enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis. Zentralbl Veterinarmed B 1990, 37:19- 27. 3. Plackett P, Ripper J, Corner LA, Small K, de Witte K, Melville L, Hides S, Wood PR: An ELISA for the detection of anergic tuberculous cattle. Aus Vet J 1989, 66:15-19. 4. Idigoras P., Beristain X., Iturzaeta A., Vicente D., Pérez-Trallero E. 2000. Comparison of the automated nonradiometric BACTEC MGIT 960 system with Löwestein-Jensen, Coletsos, and Middlebrook 7H11 solid media for recovery of mycobacteria. European Journal of Clinical Microbiology Infections Diseases 19: 350-354. 5. Huard R.C., Oliveira-Lazzarini L.C., Butler W.R., van Soolingen D., Ho J.L. 2003. PCR-Basaed method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. Journal of Clinical Microbiology, vol 41, n 4: 1637-1650. Results 10 bovine with MTC recovered were SIT test positive. 16 were positive in the IFN-g assay and the ELISA detected 13 of the 18 cattle with MTC recovered (one of them was negative in SIT test and IFN-g assay). One bovine with MTC recovered was negative in the three tests; this animal was slaughtered because it was more than10 years old. The kappa statistic shows a lower agreement for the SIT test (0.39) and the IFN-g assay (0.36) with the MTC isolation than the ELISA assay (0.58). Discussion & conclusions The results suggest that the use of IFN-g and ELISA assays as ancillary techniques might help to detect a larger number of tuberculosis infected animals in a TB-confirmed herd. These assays complement each other because they may detect animals in different stages of tuberculosis. Acknowledgements We thanks IDEXX Laboratories, Inc., for providing the ELISA test for detection of M. bovis antibodies. We also would like to thank the Official Veterinary Services from Vilalba for assisting with abattoir sampling, the Regional Animal Health Veterinary Services for their support and the Laboratory of Animal Health of Galicia.
S1 - P - 17 VIROLOGICAL SURVEILLANCE AND CHARACTERIZATION OF AVIAN-LIKE REASSORTANT H1N2 SWINE INFLUENZA VIRUSES IN SWEDEN Giorgi Metreveli , Eva Emmoth, Lena Renström National Veterinary Institute, Department of Virology, Immunobiology and Parasitology, Uppsala, Sweden Introduction The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In 2009 and 2010 there was the first reported isolations and demonstrations of natural reassortants of H1N2 viruses in pigs in Sweden (1,2). Characterized Swedish isolates possessed avian-like SIV H1N1 HA and European H3N2 SIV-like NA. They were compared regarding their molecular composition and biological characteristics. Materials & methods Virus was isolated from the clinical material by infecting Madin Darby Canine Kidney (MDCK) (ATCC CCL-34) cells, following standard cell culture procedures. Viruses were analyzed by endpoint titration through cpe using 96-well plates containing MDCK cells, in 10-fold dilutions assaying eight replicates of 50 µl per dilution, and the virus titres after 6-8 days were calculated according to Kärber (3). NA enzyme activity and drug inhibition assays were performed with methylumbelliferone nacetylneuraminic acid (MUNANA) as the substrate. Genetic analyses were conducted on the clinical material and on the MDCK cell isolates. Total RNA was prepared from virus-infected MDCK cells by the QiagenRNeasy Mini kit, according to the manufacturer’s instructions (Qiagen, Hilden Germany). Sequencing templates were generated by amplification of each gene segment using one-step RT-PCR (QIAGEN OneStep RT- PCR Kit). The PCR products were purified using the ‘Purification Kit from Promega’ and sequenced using the fluorescent dye terminator method with an ABI PRISM® Big Dye Terminator Cycle Sequencing v3.1 Ready Reaction kit (Perkin Elmer, Waltham, MA, USA) on an ABI PRISM® 310 genetic analyzer according to the manufacturer’s recommendations (Applied Biosystems).Sequence data analyses, multiple alignments of the DNA sequences of each gene, were performed using CLC, Main Workbench 5.0.2 (CLC bio A/S, Aarhus, Denmark). Results The most remarkable result was a truncated coding region for PB1-F2 in the earlier isolates and a full length coding region in the more recent isolates. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. Figure 1: Always put legend below the figure Discussion & conclusions Recent serological investigations in Swedish pig population has also shown that this uncommon avian-like reassortant H1N2 SIV variant appears to be gaining a stronger foothold among Swedish pig populations and producing more clinical disease. The molecular genomic differences found here indicate that the virus population is steadily evolving. In order to determine the effect on the swine industry and influenza ecology, further surveillance investigations and detailed analyses are needed. References 1 Bálint, A., et al., The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant. Virology Journal, 2009. 6 Oct 28(180). 2. Metreveli, G., et al. Comparison of two H1N2 swine influenza A viruses from disease outbreaks in pigs in Sweden during 2009 and 2010.Virus Genes, 2011. Apr;42(2):236-44 3. Kärber, G., Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv für experimentelle Pathologie und Pharmacologie, 1931. 162: p. 480-483. Influenza A, swine, genetic characterization
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2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Welcome Dear colleagues, Welcome to
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worked with many diagnostic compani
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Products and services for scientist
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16:4518.15 20:0023:00 [S1.]Oralpres
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Day4 Wednesday,4July2012 4 th sessi
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Oral presentations “General Sessi
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antigens or attenuated vaccines can
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conditions, e.g. Staphylococcus aur
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S1 - O - 1 ORAL FLUIDS - SAMPLE MAT
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S1 - O - 03 INTRODUCTION RATE OF A
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S1 - O -05 VALIDATION OF A COMPETIT
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S1 - O - 07 SUBTYPING OF SWINE INFL
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S3 - P - 22 COMPARISON OF THE PERFO
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S4 - P - 01 MOLECULAR CHARACTERIZAT
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S4 - P - 11 EFFECTIVE TESTING STRAT
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List of Abstracts (in order of numb
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S1-P-03 BALLAGI ANDREA S1-P-04 BENI
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S3-P-06 KELLER SELINA S3-P-07 KÖNI
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Abstract Book of EAVLD2012 - eavld congress 2012

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