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World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 INTERNATIONAL VALIDATION OF A NEW 3 ABC COMPETITIVE ELISA FOR FMD SEROLOGY C. J. Morrissy 1 , J. McEachern 1 , A. Colling 1 , L. Wright 1 , Ngo Thanh Long 3 , W. Goff 1 , J. Hammond 1 , M. Johnson 1 , J. Crowther 2 , 3 Dong Manh Hoa 3 and P. Daniels 1 1 CSIRO Livestock Industries, Australian Animal Health Laboratory (AAHL), Geelong, Victoria, Australia. 2 International Atomic Energy Agency (IAEA), Vienna, Austria 3 Regional Animal Health Centre, Ho Chi Minh City (RAH0 - 6, HCMC), South Vietnam Introduction Foot and Mouth Disease (FMD) is an economically important disease of cloven-hoofed species. It is prevalent throughout Asia and Africa and is present in South America. North America, Japan, Australasia, Indonesia and Europe are considered free. FMD virus occurs as seven main serotypes; O, A, Asia 1, C, and SAT 1, 2 and 3. The disease is usually controlled by vaccination against the prevailing serotypes where it is endemic, or by stamping out where introduced into disease free areas. Infected animals produce antibodies to both the serotype specific structural proteins and also the non-structural (NS) proteins of the virus, which are conserved among the serotypes. Vaccinated animals will produce antibodies predominantly to structural proteins, depending on the purity of the vaccine. A diagnostic assay based on the non-structural polyprotein, 3ABC, was developed to differentiate animals infected with FMDV from vaccinates, the DIVA strategy. Routine FMD ELISAs measure antibodies to virus structural proteins, and if positive do not indicate whether serum is from an animal that has been infected or vaccinated. Another disadvantage of these ELISAs is that a separate assay is required to measure antibody to each of the seven serotypes of FMD virus. The assay described here is a competitive ELISA (c-ELISA) that detects antibody to 3ABC regardless of the infecting serotype. The use of DIVA tests will be important in FMD free countries such as Australia if vaccines are used to control any future FMD outbreaks. Materials & methods Recombinant 3ABC protein was produced in both a baculovirus expression system and an E. coli expression system. Although the baculovirus system is considered to yield a protein with a more natural conformation than the E coli expressed protein, the E.coli expressed 3ABC was easier and quicker to grow up in large amounts than the baculovirus expressed protein, and was more easily purified. Laying chickens were inoculated with the purified E. coli expressed 3ABC to obtain antibodies from the egg yolk. The more purified protein was chosen for immunisation to help reduce non-specific reactions in the ELISA. The unpurified baculovirus expressed 3ABC protein was used as the antigen to coat ELISA plates. The polyclonal antibody obtained from the chickens egg yolk was used as the competing antibody in the assay. A buffer containing skim milk powder and normal horse serum was also used to reduce non-specific reactions in the ELISA. Field sera from FMD disease cases necessary to validate FMD diagnostics were obtained from international projects and collaborations. The FMD CARD AusAID project has been valuable in improving AAHL diagnostic capability for detection of antibody and antigen. This project in Vietnam has used the NS c-ELISA for serosurveillance. Results The assay has been validated using a panel of experimental and field sera derived from naïve, vaccinated and post-infected animals of various species. All vaccinated and naïve animals tested thus far are negative for antibodies to 3ABC and antibodies to 3ABC have been detected as early as seven days in experimentally infected animals. The preliminary results indicate that the c-ELISA is sensitive and highly specific. Conclusions Validation of new FMD tests such as this C-ELISA for detection of NS antibodies is difficult in Australia without access to live virus. Samples required for validation of serology tests are obtained through a number of international collaborations This ELISA is simple to perform and can differentiate post-vaccination sera from post-infection sera. The current validation data suggests that this c-ELISA will prove to be a reliable diagnostic assay for post-FMD outbreak surveillance and the assay also offers an additional advantage for such surveillance in that antibodies to all seven serotypes can be detected in the one test. Hence it can be used as a simple sero-surveillance tool in FMD free countries. More importantly, because of its DIVA potential, it could also allow vaccination to be used to control the disease in traditionally disease free areas to avoid mass slaughtering of animals during an outbreak. Mon 12 November Mon 12 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 1330 - 1455 Concurrent Session 2.2 - Avian Influenza RESULTS OF 2006 WILD BIRD SURVEILLANCE FOR THE DETECTION OF HIGHLY PATHOGENIC AVIAN INFLUENZA IN THE UNITED STATES Janice Pedersen 1 , Dennis Senne 1 , Mary Lea Killian 1 , Nichole Hines 1 , Brundaban Panigrahy 1 , Seth Swafford 2 , Tom Deliberto 2 , Hon Ip 3 1 U. S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, IA, USA 2 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services, National Wildlife Disease Program, Fort Collins, CO, USA 3 U.S. Geological Survey, National Wildlife Health Center, Madison, WI, USA The potential role played by migratory waterfowl and water birds in the spread of H5N1 highly pathogenic avian influenza (HPAI) virus from Asia to Europe, the Middle East, and Africa prompted a large-scale surveillance to detect HPAI H5N1 in wild aquatic birds in the United States. The program was a cooperative effort between the U.S. Departments of Agriculture (USDA) and Interior (DOI) and State Wildlife Agencies. Between April 2006 and March 2007, more than 148,000 cloacal (CL) and fecal swabs were collected from dead birds as well as apparently healthy and hunter-killed birds in all 50 states and tested for presence of avian influenza virus (AIV) by the real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay at National Animal Health Laboratory Network (NAHLN) laboratories, the National Wildlife Research Center, and the National Wildlife Health Center. Cloacal swab pools were screened for AI virus by the matrix rRT-PCR assay. Positive matrix pools were subsequently tested by the H5 and H7 rRT-PCR subtyping assays, and individual specimens from H5 and H7 positive pools were shipped to the National Veterinary Services Laboratories (NVSL) for confirmatory testing, virus isolation (VI), and characterization. A total of 2210 specimens were submitted to the NVSL for rRT-PCR screening or confirmatory testing. Of these 1413 specimens, 340 CL pools and 21 fecal pools were H5 presumptive positive (PP), 745 were surveillance and mortality event swabs, and 31 were viral isolates. Of the 340 H5 PP pools, 250 were confirmed as H5 rRT-PCR positive, giving the H5 subtyping assay a diagnostic sensitivity and specificity of 74% and 100%, respectively for wild bird CL swab specimens when compared to the AI matrix rRT-PCR assay. When compared to VI, the H5 assay showed a diagnostic sensitivity and specificity of 79% and 89%, respectively (64/304 H5 rRT-PCR positive specimens yielded H5 virus and 17 H5 rRT-PCR negative specimens yielded H5 virus). A total of 97 H5 subtype AI viruses, including 5 H5N1, were isolated and characterized as low pathogenicity AI (LPAI) of North American lineage by nucleotide sequence analysis. The H5 subtypes identified were H5N2 (80), H5N1 (5), H5N3 (5), H5N8 (3), H5N9 (3), and H5N4 (1). Other subtypes isolated included H1-H6, as well as H10 and H11. No highly pathogenic viruses were found and no H7 rRT-PCR positive specimens were detected, although H7 AI subtype viruses have subsequently been isolated from rRT-PCR negative swabs. Diagnostic challenges posed by the surveillance included presence of PCR inhibitors, low virus recovery rate from rRT-PCR positive specimens, mixed viral infections and the 0% detection rate of H7 wild bird North American lineage viruses by the USDA H7 rRT-PCR assay.
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 CANADA’S INTER-AGENCY WILD BIRD INFLUENZA SURVEILLANCE PROGRAMME 2005-2007 *I.K. Barker, Canadian Cooperative Wildlife Health Centre, University of Guelph, Guelph, ON; S. Lair, Centre canadien coopératif de la santé de la faune, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, QC; P.-Y. Daoust, Canadian Cooperative Wildlife Health Centre, University of Prince Edward Island, Charlottetown PEI; J. Pasick, National Centre for Foreign Animal Diseases, Canadian Food Inspection Agency, Winnipeg MB; P.A. Buck, Foodborne, Waterborne and Zoonotic Infections Division, Public Health Agency of Canada, Ottawa, ON; E.J. Parmley, Centre for Coastal Health, Nanaimo, BC; E.J. Jenkins, Science and Technology Branch, Environment Canada, Saskatoon, SK; C. Soos, Science and Technology Branch, Environment Canada, Saskatoon; F.A. Leighton, Canadian Cooperative Wildlife Health Centre, University of Saskatchewan, Saskatoon, SK. Introduction Stimulated by a major outbreak of avian influenza (AI) in British Columbia poultry in 2004, and by the dissemination of H5N1 highly pathogenic avian influenza virus from Asia to Europe and Africa in 2005, Canada initiated an avian influenza surveillance programme in wild birds in 2005. Organized, under the oversight of a federal-provincial inter-agency committee, by the Canadian Cooperative Wildlife Health Centre (CCWHC), it initially involved active surveillance for influenza A viruses in populations of wild birds, mainly waterfowl, supplemented by scanning surveillance in dead wild birds beginning in September 2005. Active surveillance was to determine the influenza A viruses circulating in wild bird populations; monitor for those of animal or human health concern; and establish in Canada an integrated multiagency field, laboratory, regulatory and communications capacity to carry out Influenza A virus sampling, identification, and molecular characterization on many specimens under emergency conditions, from any species. Formally initiated in 2006, passive surveillance for highly pathogenic avian influenza in dead wild birds was aimed mainly at detection of H5N1 influenza virus, but it also contributes to monitoring its global and continental dissemination, and to the science base for risk assessment. Materials & methods Oropharyngeal and/or cloacal swabs were collected into transport medium from live and hunter-harvested waterfowl and selected other species, sampled at multiple regional sites across Canada in 2005, 2006 and 2007, and in Iceland in 2006. For scanning surveillance oropharyngeal and cloacal swabs were collected at necropsy from birds of a wide array of species found dead across Canada, emphasizing those using wetland or aquatic habitat, or involved in mortality events unusual in their circumstances, size or species composition. In one of seven collaborating regional veterinary laboratories, swabs were initially tested by real-time reverse transcriptase PCR (RRT-PCR) for Influenza M1 (matrix protein) gene sequences; positive samples were then tested by RRT-PCR for H5 and H7 protein gene sequences, and any samples positive for these were forwarded immediately to the National Centre for Foreign Animal Disease, CFIA for further identification, characterization and virulence testing. In 2005 and 2006, samples testing matrix protein positive and H5/H7 negative were cultured in regional laboratories to attempt virus isolation; in 2007 they were retained frozen. Isolates were deposited in a national influenza strain archive at NCFAD CFIA. Data of all types were recorded in the CCWHC national Internet web-interfaced relational database, for rapid communication of results among laboratories and agencies and to decision-makers. Results Through Canada’s Inter-agency Wild Bird Influenza Survey, over 15,000 live wild birds have been sampled and tested for avian influenza since the fall of 2005. Of these, over 3,000 (>20%) have tested positive for avian influenza by RRT-PCR. About 3% of over 5400 dead wild birds have been AI matrix protein sequence positive. The survey results have revealed wide variation in detection of AI viruses among regions and between species sampled in different years. To date all AI viruses detected have been of low pathogenicity North American strains. In addition to the Canadian birds tested, over 700 trans-Atlantic migrants (Eastern High Arctic Brant and Red Knot) were sampled in Iceland and tested in Canada for the presence of avian influenza in 2006/07. Only 1 has been positive for avian influenza by RRT-PCR. Discussion & conclusions The infrastructure and laboratory capacity-building objectives of the programme largely have been met. A firm base for understanding the species in which AI viruses predominantly circulate has been established, though highly pathogenic AI viruses have yet to be identified in wild birds in Canada. References Parmley, E.J., et al. Influenza viruses in wild ducks in Canada: PCR results from the 2005 Wild Bird Influenza Survey. Emerging Infectious Diseases. Accepted for publication, 2007. Mon 12 November Mon 12 November World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007 PERFORMANCE EVALUATION OF FIVE DETECTION TESTS FOR AVIAN INFLUENZA ANTIGEN WITH VARIOUS AVIAN SAMPLES Tze-Hoong Chua, AB* Trevor M. Ellis, AC Chun W. Wong, D Yi Guan, EF Sheng Xiang Ge, F Geng Peng, F Chinta Lamichhane, G Con Maliadis, H Sze-Wee Tan, I Paul Selleck, J and John Parkinson K (A)School of Veterinary and Biomedical Sciences, Murdoch University, South Street, Murdoch, Western Australia, 6150, Australia (B) Agri-Food and Veterinary Authority, 5 Maxwell Road, Singapore 069110 (C) Senior author (D)Tai Lung Veterinary Laboratory, Agriculture Fisheries and Conservation Department, Lin Tong Mei, Sheung Shui, Hong Kong SAR, China (E) Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China (F) National Institute of Diagnostics and Vaccine Development in Infectious Disease, Xiamen University, Xiamen, Fujian, China (G) Synbiotics Corporation, 11011 Via Frontera, San Diego, CA 92127 (H) Bio-Mediq DPC (Diagnostic Products Corporation) Pty. Ltd., 1 Williamson Road, Doncaster, Victoria, 3108, Australia (I) Rockeby Biomed (Singapore) Pte. Ltd., 350 Orchard Road, Shaw House, Singapore 238868 (J) CSIRO Australian Animal Health Laboratory, 5 Portarlington Road, Geelong, 3220, Australia (K) Department of Agriculture and Food, 3 Baron Hay Court, South Perth, Western Australia, 6151, Australia INTRODUCTION. In this paper, we report on the evaluation of five influenza antigen detection tests by avian influenza H5N1 virus–positive swab samples to estimate their diagnostic sensitivity. The tests included two chromatographic immunoassays, an H5 avian influenza–specific antigen detection enzyme-linked immunosorbent assay (ELISA), an influenza antigen detection ELISA, and anH5 rapid immunoblot assay. METHODS & RESULTS. The results showed that the overall sensitivities of these tests ranged from 36.3% to 51.4% (95% confidence interval ranging from 31.0% to 57.0%), which were comparable to Directigen_Flu A antigen detection tests but substantially lower than genome detection methods. Diagnostic sensitivity performance is a function of the concentration of antigens in samples and the analytical sensitivity of the individual test. The test sensitivities were significantly higher for sick and dead birds by cloacal, tracheal, or tissue swabs than for fecal swabs from apparently healthy birds, and these tests would not be suitable for surveillance testing of clinically healthy birds. Furthermore, the sensitivity for testing tracheal and cloacal swabs from waterfowl and wild birds was not as good as for chickens. This was most likely to be associated with variation in virus titers between specimens from different bird species. However, the tests showed good sensitivities for testing brain swabs from clinically affected waterfowl species. CONCLUSION. The results indicate that these antigen detection tests could be used for preliminary investigations of H5N1 outbreaks as a low-cost, simple flock test in sick and dead birds for the rapid detection of H5N1 infection. However, the relatively low sensitivity of the tests as individual bird tests means that they should be used on optimal clinical specimens from diseased birds, testing birds on a flock basis, or testing samples as close to the onset of disease as possible before viral titers diminish. They should be followed up by confirmatory tests, such as reverse transcription polymerase chain reaction or viral culture, wherever possible but could assist in facilitating rapid investigations and control interventions. *Presenting author email: T.Chua@murdoch.edu.au
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