Abstract Book of EAVLD2012 - eavld congress 2012

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S1 - O - 06 DIAGNOSTIC PERFORMANCE OF A COMMERCIAL PRRS SERUM ANTIBODY ELISA ADAPTED TO ORAL FLUID SPECIMENS: FIELD SAMPLES Apisit Kittawornrat 1 , John Prickett 1 , Chong Wang 1, 2 , Chris Olsen 1 , Yaowalak Panyasing 1 , Andrea Ballagi 3 , Anna Rice 3 , Sergio Lizano 3 , John Johnson 1 , Rodger Main 1 , Chris Rademacher 4 , Marlin Hoogland 4 , Jeffrey Zimmerman 1 1 Department of Veterinary Diagnostic and Production Animal Medicine, 2 Department of Statistics, Iowa State University, Ames, Iowa, United States, 3 IDEXX Laboratories, Westbrook, Maine, United States, 4 Murphy-Brown LLC, Ames, Iowa, United States Oral fluid, ELISA, Field samples, PRRSV Introduction Oral fluid samples are of interest because of their ease of collection and documented use in surveillance of PRRSV and other pathogens (1,2). Previous work showed that a commercial PRRS ELISA (HerdChek® PRRS X3 ELISA) could be adapted to detect anti-PRRSV IgG in oral fluid specimens (IgG ELISA). The objective of the current study was to evaluate the ability of the assay to detect anti-PRRSV IgG antibody in pen-based oral fluid field samples. References 1. Kittawornrat A, et al. 2010. PRRSV in serum and oral fluid samples from individual boars. Virus Res 154:170-176. 2. Prickett, J et al.: 2011. Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation. Transbound Emerg Dis 58:121-127 3. Ramirez et al. 2012. Efficient surveillance of pig populations using oral fluids. Prev Vet Med (Epub ahead of print). Materials & methods Positive samples were derived from a longitudinal field study in 10 wean-to-finish barns (3). At each site, oral fluid samples were collected from the same 6 pens at 2-week intervals (total of 10 sampling points per barn). Positive oral fluid samples were defined as all samples collected from a pen after the first PRRSV PCR positive oral fluid sample from that pen (n = 250). Negative oral fluid (n = 284) field samples were diagnostic samples submitted to the ISU VDL for PRRSV qRT-PCR testing from expected-negative herds. Results The cumulative S/P results for defined positive and negative oral fluid samples are given in Figure 1. Of 284 expected-negative field samples, all were negative on the IgG ELISA (S/P
S1 - O - 07 SUBTYPING OF SWINE INFLUENZA VIRUSES BY MULTIPLEX REAL-TIME PCR Kees van Maanen 1 , Ingrid Wiggers 1 , Chris Schouten 3 , Tom Duinhof 1 , Paolo Cordioli 2 , Remco Dijkman 1 1 GD-Animal Health Service, Diagnostics,Research and Epidemiology,Deventer , the Netherlands 2 Istituto Zooprofilattico Esperimentale Brescia, the Netherlands 3 DAP Aadal, Erp, the Netherlands Swine Influenza Virus; real-time PCR; subtyping; lung lavage samples; diagnosis Introduction Swine influenza is an acute respiratory disease of swine caused by type A influenza viruses. In Europe, swine influenza is considered one of the most important primary pathogens of swine respiratory disease and infection is primarily with H1N1, H1N2 and H3N2 influenza A viruses. Avian-like H1N1 and human-like H3N2 swine influenza viruses (SIV) have been considered widespread among pigs in Western Europe since the 1980s, and a novel H1N2 reassortant with a human-like H1 emerged in the mid 1990s. These viruses have remained endemic in European pig populations but significant differences in the circulation of these strains occur at a regional level across Europe (1,2). For diagnosis of swine influenza infections serological and virological tests can be used. For detection of subtype-specific antibodies HI-tests are commonly used. However, crossreactions can occur, and the interpretation can be difficult depending on the vaccination and infection history of the herd. For diagnosis of recent infections paired serum samples are required collected with a 2-3 week interval. For rapid detection and discrimination of the different swine influenza subtypes several PCRs have been described. However, these methods were either conventional PCRs, targetted only the Haemagglutinin gene, or the coverage for European SIV strains was too low according to our BLAST analysis results. Therefore we decided to develop an in-house subtype-specific SIV real-time PCR. In the course of the project a prototype realtime PCR for detection and differentiation of SIV (Life Technologies) became available and was also evaluated. Materials & methods In this study two RNA extraction methods (High Pure nucleic acid kit, Roche Applied Science, and MagMax AM1836, Life Technologies) were compared in combination with either the inhouse designed primer/probe sets + AgPath-ID multiplex onestep RT-PCR kit (Life Technologies) or a prototype SIV Screening qRT-PCR kit (Life Technologies). All tests were run on an ABI7500 fast platform. Primers and probes were designed in silico using GenBank SIV sequences of the last 15 years. Primers and probes should have a coverage of at least 95%, and were used in working dilutions of 10 and 5 µM, respectively. Primers and probes were designed to enable all sorts of combinations (singleplex, multiplex H+N, multiplex N and multiplex H in parallel). Test results were compared with the results of an influenza A real-time PCR targetting the M gene that had been validated and implemented several years ago. Detection limits and efficiency were determined for decimal dilution series of H1N1, H3N2, and H1N2 reference viruses, as compared with the influenza A matrix real-time PCR. Subtypespecificity was evaluated by testing 22 different SIV strains provided by the IZSLER institute, Brescia, Italy. Diagnostic performance for lung lavage samples was evaluated by testing influenza A PCR positive pooled lung lavage samples (n=17) of 4, 7 and 10 week old piglets. For some pools the lung lavage samples that contributed to the pool were also tested individually (n=25) in order to compare the reproducibility of subtyping results and the presence of dual infections. Results For the haemagglutinin genes of H1N1, H1N2, and H3N2 viruses 81, 36, and 61 entries were aligned for the development of primer/probe sets. For the neuraminidase genes of the same subtypes 67, 28, and 25 entries were aligned for the development of primer/probe sets. Both for the in-house PCRs and for the prototype SIV Screening qRT-PCR kit RNA extraction with the MagMax AM1836 kit yielded 10-100 times lower detection limits than the High Pure nucleic acid kit. Using the MagMax AM1836 kit for RNA extraction, the detection limits of the in-house PCRs and the prototype commercial kit were comparable for the neuraminidase genes, whereas detection limits showed more variation for the haemagglutin genes. Overall, the detection limits of the subtypespecific primer/probe sets were equivalent to 10 times higher than the detection limit of the influenza A real-time PCR targeting the matrix gene. Subtype-specificity of the in-house PCR and the commercial kit were investigated by blind testing of a panel of Swine influenza virus isolates (H1N1 n=10; H1N2 n=4; H3N8 n=8) generously provided by the IZSLER institute, Brescia, Italy. The in-house PCRs failed to subtype several strains and also one probe appeared to be labile. In contrast the prototype commercial kit subtyped 21/22 strains correctly (for one sample a dual infection was reported, whereas this sample only contained H3N2). All strains were detected with the influenza A matrix PCR with Ctvalues ranging from 20-33. A first diagnostic evaluation of the commercial kit was performed by testing influenza A positive (results from another laboratory) pooled lung lavage samples (n=17) from piglets of 4-10 weeks of age. All pools were also tested in our in-house influenza A PCR and results were confirmed. In the subtype-specific PCR 15/17 pools yielded a single subtype (H1N2: n=9; H1N1: n=5; H3N2: n=1), whereas 2 pools yielded 2 or 3 subtypes. Retesting of the individual lung lavage samples that contributed to the latter pools confirmed the presence of dual infections in the majority of these piglets. Retesting of the individual lung lavage samples of three pools that were subtyped unequivocally yielded exactly the same subtype in all individual samples. Discussion & conclusion Influenza viruses are highly variable RNA viruses that show rapid evolution of especially the haemagglutinin and neuraminidase genes. Therefore, development of reliable PCR protocols for the subtyping of these viruses is not easy. Despite the effort we spent on selection of primers and probes, the results of our inhouse PCR were disappointing and inferior when compared to a prototype commercial real-time PCR kit. The latter kit showed a good performance when combined with RNA extraction on a MagMax AM1836 platform. Detection limits were comparable to tenfold higher than the influenza A matrix PCR, 95% of well-defined SIV strains were subtyped correctly, and the test also demonstrated a good diagnostic performance for subtyping of influenza A positive lung lavage samples. Although some practices in the Netherlands are very experienced in collecting lung lavage samples, the method is cumbersome; collecting other types of samples like nasal swabs or chewing ropes is much less invasive laborious. However, nasal and oral virus excretion is often short-lived. In the next study we will evaluate the diagnostic performance of real-time influenza A PCR and subtype-specific PCRs for nasal swabs and chewing ropes in fattening pigs with acute respiratory disease. Acknowledgements We thank Life Technologies for generously providing the prototype SIV Screening qRT-PCR kit for this study. References 1. Van Reeth, K, Brown, IH, Dürrwald, R, Foni, E, Labarque, G, Lenihan, P, Maldonado, J,,Markowska-Daniel, I, Pensaert, M, Pospisil, Z, Koch, G (2008). Seroprevalence of H1N1, H3N2 and H1N2 influenza viruses in pigs in seven European countries in 2002-2003. Influenza Other Respi Viruses, 2(3), 99-105. 2: Brown, IH (2012). History and Epidemiology of Swine Influenza in Europe. Curr Top Microbiol Immunol Jan 11. [Epub ahead of print].
Page 2 and 3: 2 nd CONGRESSOFTHEEUROPEAN ASSOCIAT
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Abstract Book of EAVLD2012 - eavld congress 2012

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