Highly pathogenic avian influenza and low pathogenic avian influenza (poultry)
OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / Highly pathogenic avian influenza and low pathogenic avian influenza (poultry)Address of laboratory: / National Veterinary Services Laboratories
USDA, APHIS, VS
PO Box 844, 1920 Dayton Ave.
Ames, IA 50010
UNITED STATES OF AMERICA
Tel.: / +1-515 337-7551
Fax: / +1-515 337-7348
e-mail address: /
website: /
Name (including Title and Position) of Head of Laboratory (Responsible Official): / Dr ElizabethA. Lautner
Director, National Veterinary Services Laboratories
Name(including Title and Position) of OIE Reference Expert: / Ms Janice C. Pedersen
Microbiologist, Avian Viruses Section
Name (including Title and Position) of writer of this report
(if different from above):
Part I:Summary of general activities related to the disease
1.Test(s) in use/or available for the specified disease/topic at your laboratory
Test / For / Specificity / TotalAgar gel immunodiffusion / Antibody / Type / 835
Hemagglutination-inhibition / Antibody/Virus / Sub type / 49,829
Neuraminidase-inhibition / Antibody/Virus / Subtype / 25,776
Virus Isolation
/ Virus / Subtype / 9049Real-time RT-PCR (matrix, H5, H7, N1)
/ RNA / Type/subtype / 2277Intravenous Pathogenicity Index in SPF chickens
/ Virulence / Subtype / 26HA cleavage site sequencing
/ Virulence / Subtype / 1242.Production and distribution of diagnostic reagents
The National Veterinary Services Laboratories (NVSL) produces virus, inactivated antigen and antiserum for avian influenza (AI) subtypes H1-16 for internal use, research, and distribution to other countries.These reagents are used in a variety of tests such as hemagglutination, hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI).In addition, antigen and antiserum are produced and quality control tested for use in the agar gel immunodiffusion (AGID) test.The NVSL also produces proficiency tests for HI, NI, AGID and real-time RT-PCR (rRT-PCR) assays, and distributes positive amplification control (PAC) (transcribed RNA) as well as positive and negative extraction control (PEC, NEC) for rRT-PCR and positive, weak positive and negative reference serum for the AGID test.
In 2011, a total of 12,928sets of AGID reagents (antigen and antiserum) were distributed to domestic laboratories, including Puerto Rico, and 738 sets were distributed to 10 international laboratories (Argentina, Brazil, Chile, Costa Rica, El Salvador, Republic of Korea, Laos, Nicaragua, Panama, and Peru).Each set will test approximately 120 serums.Additionally, 3127 sets of AGID reference serum (weak positive, strong positive and negative) were distributed to domestic laboratories and 312 sets to international laboratories.
The NVSL produced 18 lots (approximately 16.9 liters) of AGID antiserum and reference sera and prepared 12,961 sets of AI AGID reagents (antigen and antiserum).This represents 3,163 vials of AI AGID reference sera (weak positive, strong positive and negative).Also, HI and NI subtyping reagents were produced: 10 lots of reference antiserum (total of 2.1 liters), nine lots of inactivated reference antigen (total of 1.4 liters), and 211 vials of reference virus (pH1N1, H5N2, H7N3, H5N8, and H5N1). The NVSL distributed 79 AGID proficiency test panels (10 serums each) to domestic laboratories in 36 states and to 2 international laboratories (Brazil and Chile) and 338 influenza rRT-PCR proficiency panels (15 specimens) to 55 domestic laboratories and 3 international laboratories for avian and pandemic H1N1 swine influenza.
Type of reagent / Amount supplied nationally(including for own use) / Amount supplied to other countries
AGID antigen and antiserum sets / 12,935 / 738
AGID strong positive reference serum / 974 (2.0 ml vials) / 104 (2.0 ml vials)
AGID weak positive reference serum / 1285 (2.0 ml vials) / 104 (2.0 ml vials)
Type of reagent / Amount supplied nationally
(including for own use) / Amount supplied to other countries
AGID negative reference serum
/ 907 (2.0 ml vials) / 104 (2.0 ml vials)HI/NI antigen
/ 316 (2.0 ml vials) / 147 (2.0 ml vials)HI/NI antiserum
/ 315 (2.0 ml vials) / 189 (2.0 ml vials)AI reference virus
/ 26 (0.6 ml vial) / 49 (0.6 ml vial)AGID proficiency panels
/ 79 / 2Real-time RT-PCR proficiency panels
/ 338 / 3Real-time RT-PCR positive matrix, H7 and H7 amplification control
/ 138 vials / 10 vialsReal-time RT-PCR positive extraction control
/ 324 (1.0 ml vial) / 2 (1.0 ml vial)Real-time RT-PCR negative extraction control
/ 408 (1.0 ml vial) / 0 (1.0 ml vial)Part II:Activities specifically related to the mandate
of OIE Reference Laboratories
3.International harmonisation and standardisation of methods for diagnostic testing or the production and testing of vaccines
a)Establishment and maintenance of a network with other OIE Reference Laboratories designated for the same pathogen or disease and organisation of regular inter-laboratory proficiency testing to ensure comparability of results.
The NVSL is a member of the North American Animal Health Laboratory Network (NAAHLN) Security and Prosperity Partnership [SPP] (Canada, Mexico and the United States) formed to enhance detection of avian influenza, Newcastle disease, vesicular diseases and tuberculosis.The goal of the network is to identify gaps in diagnostic testing, harmonize testing protocols, share reagents, provide training where and when necessary and exchange proficiency test samples to ensure reliability of diagnostic testing between the network laboratories.The NVSL distributed rRT-PCR proficiency tests to both the National Centre for Foreign Animal Disease (NCFAD) Canadian Food Inspection Agency (CFIA) and the Mexican CPA laboratory and participated in HI proficiency testing.The exchange of proficiency tests and the comparison of results is part of a continued effort to harmonize and standardize rRT-PCR and subtyping procedures.
The NVSL participated with the LANAGRO-SP Brazilian national reference laboratory, Campinas, Brazil in an OIE twinning project for avian influenza.Proficiency testing and comparison of testing results were conducted for AGID, HI NI and rRT-PCR.NVSL actively participated in the standardization of methods for diagnostic testing for AI and for the harmonisation of nucleotide sequencing methods for the Lanagro-SP.In addition, the NVSL participated in the First Annual South American Workshop for Avian Influenza and Newcastle Disease, presenting on proficiency testing, reagent production and quality control testing.
The NVSL participated with theServicioAgrícola y Ganadero (SAG) Ministerio de Agricultura, Santiago, Chile in an OIE twinning project for avian influenza.The project involves hands-on training in the methods and procedures for the diagnosis, subtyping and pathotying of avian influenza virus and antibodies as well as the procedures for avian influenza reagent production, specifically AGID and HI antigens and antiserum. Improvement of BSL-3 biosecurity and biosafety laboratory and animal facilities procedures is a major objective of the twinning project as well.An on-site visit at the SAG was conducted to provide guidance in the development of BSL3 biosecurity and biosafety procedures for working with influenza viruses.Harmonization of standard procedures and proficiency testing for the demonstration of competency are in progress.
Members of the NVSL staff are coordinating an OFFLU technical group to develop recommendations for international proficiency tests used to evaluate performance of avian influenza testing at reference laboratories. The technical group is working to increase the standardization of diagnostic testing procedures, develop guidelines for proficiency tests and to distribute proficiency tests.Proficiency tests have been distributed to 9 laboratories (USA, Germany, Italy, Australia, Canada, United Kingdom, Japan and India).
Participated in discussions involving the harmonization of global surveillance of influenza in swine and the development of a global network of laboratories for swine influenza diagnostics and research at the annual OFFLU meeting in Paris, France.OFFLU is a global influenza working group that was jointly developed by the OIE and the FAO.
The NVSL provided hands-on training and lectures at a workshop on Avian Influenza Diagnostic Testing Procedures at the Central Laboratory of National Animal Health Center in Vientiane, Lao PDR.Training was provided to 23 field veterinarians and laboratory technicians from nine providences throughout Lao PDR. Harmonization of methods and procedures as well as the implementation of those methods wereaddressed.
NVSL participated in the North American (Mexico, Canada, US) PROCINORTEworkshopon Influenza A Virus Molecular Diagnostic Techniques which was held at the National Centre for Foreign Animal Disease Canadian Food Inspection Agency, Winnipeg, Canada.The workshop focused onharmonization of nucleotide sequencing methods and procedures for molecular subtyping and pathotyping of avian and swine influenza.
b)Organisation of inter-laboratory proficiency testing with laboratories other than OIE Reference Laboratories for the same pathogens and diseases to ensure equivalence of results
The NVSL prepares and distributes rRT-PCR proficiency panels (15 specimens) for avian and swine pandemic H1N1 (pH1N1) influenza.The panels are distributed on an annual basis to National Animal Health Laboratory Network (NAHLN) technical staff.The results of the proficiency panels are evaluated statistically by the NVSL, and those whom have successfully completed the proficiency test are approved to conduct diagnostic testing.All diagnostic testing within the NAHLN must be conducted according to the official USDA avian influenza and swine influenza standard operating procedures.Suspect positive rRT-PCR specimens are confirmed by the NVSL prior to reporting.
4.Preparation and supply of international reference standards for diagnostic tests or vaccines
The NVSL has distributed AI transcribed RNA for use as a positive amplification control (PAC) for the real-time RT-PCR testing (Spackman et al.) directly to domestic laboratories in 19 states (82 vials) and 2 international laboratories (Canada and Mexico) (9 vials).
The NVSL distributed positive amplification control for rRT-PCR testing directly to domestic laboratories in 31 states (208 vials) and to 2 international laboratories (Canada and Mexico) (4 vials).
The NVSL has distributed negative extraction control for the rRT-PCR assay (Wise et al.) to domestic laboratories in 25 states (166 vials) and to no international laboratories.
The NVSL has provided H1-16 hemagglutination-inhibition (HI) and N1-9 neuraminidase-inhibition (NI) reference antigens and antiserum to Argentina, and India as well as HI and NI reagents for requested subtypes to Israel, Laos, and Yemen.
The NVSL has provided reference controls for the AI AGID test to Argentina, India, Japan, Laos, Nicaragua, Panama, Peru, and Puerto Rico.
5.Research and development of new procedures for diagnosis and control
The NVSL participated with the USDA Southeast Poultry Research Laboratory, Athens, GA in a comparison of 5 and 11 cloacal and tracheal/oropharyngeal swab pools.Real-time RT-PCR and virus isolation testing was conducted on 5 swab and 11 swab pools for the detection of avian influenza and Newcastle disease. Evaluation of the data is in progress at this time to determine if 11 swab pools are appropriate specimens for rRT-PCR and virus isolation testing.Study results will be used to determine if the surveillance for AI can be conducted with 11 swab pools.
NVSL conducted a study on the comparison of virus isolation and rRT-PCR for testing domestic duck and poultry cloacal swab specimens.Real-time RT-PCR results were compared to virus isolation, the currently approved test for the surveillance of influenza for cloacal swab specimens.The limit of detection was statistically determined for VI and rRT-PCR for cloacal swabs from commercial domestic ducks, poultry and mixed species live bird market ducks.With magnetic bead RNA extraction (Ambtion MagMAX AI/ND, Life Technologies, Austin, TX) the difference was determined to be <1 log10 LOD for both chicken and domestic duck CL swabs with AI and ND.A difference of <1 log10 LOD was considered to be comparable to VI.Cloacal duck specimens were approved as a sample matrix for rRT-PCR testing with the USDA SOPAV1510 rRT-PCR procedures when the RNA is extracted with magnetic bead RNA extraction procedures.
Co-author on the publication of a novel and fast strategy for the molecular pathotyping of H5/H7 AIVs.Leijon M, et al. Rapid PCR-based molecular pathotyping of H5 and H7 avian influenza viruses. J. Clinc Microbiol, 2011. Nov;49 (11): 3860-73.
Participation in the USDA National Animal Health Laboratory Network (NAHLN) Methods Technical Working Group (MTWG) Assay Comparison and Validation subgroup for the development of standardized procedures for the comparison or validation of new procedures for diagnostic testing of AI and ND, with an emphasis on those procedures used for molecular detection.NVSL also participated in the subcommittee drafting a good laboratory practices for molecular testing reference manual.
A surveillance system for influenza from swine was initiated for production swine.Surveillance testing includes rRT-PCR and virus isolation.A national repository for swine influenza viruses has been created at the NVSL, and the nucleotide sequences of the hemagglutinin, neuraminidase and matrix genes are deposited in the National Center for Biotechnology Information’s (NCBI) GeneBank.
6.Collection, analysis and dissemination of epizootiological data relevant to international disease control
The official U.S. Department of Agriculture and Interior and State Wildlife agency wild bird surveillance for highly pathogenic avian influenza virus in wild aquatic birds in the United States has been discontinued and was not conducted in 2011.The USDA and DOI continue to conduct passive surveillance for highly pathogenic avian influenza in dead and sick birds involved in mortality events.Specimens are screened for the detection of avian influenza by the AI matrix rRT-PCR and virus isolation tests at the NVSL and DOI National Wildlife Health Laboratory in Madison, WI.
Conducted the initial hemagglutinin and neuraminidase subtyping for the first H14 avian influenza viruses isolated in North America.Co-author on publication of the characterization of the H14 viruses and their genetic and phylogenetic relationship to the 1982 Caspian Sea H14 influenza viruses.J. Nolting, A.C. Fries, R. Slemons, C. Courtney, N. Hines, M.L. Killian and J. Pedersen. Recovery of H14 influenza A virus isolates from sea ducks in the Western Hemisphere. PLoS Currents, in publication.
Conductedhemagglutinin and neuraminidase subtyping for viruses isolated from North American wild birds.The viruses are submitted to the NVSL for subtype characterization by several wild bird surveillance programs conducted in the United States by independent researchers or state surveillance programs.
Conductedhemagglutinin and neuraminidase subtyping for research projects conducted within the United States.The research projects focus on the surveillance of avian influenza in mammalian hosts.
In-vivo pathotyping and nucleotide sequence characterization for an H3N8 virus recently isolated from Harbor Seals involved in a 2011 mortality event in the US New England coastal waters. Co-authorship of the paper reporting the isolation, pathotyping and phylogenetic characterization of the virus.The emergence of new strains of influenza virus are always of great public concern, particularly because the infection of a new mammalian host has the potential to result in a widespread outbreak of disease.This is the first report of H3N8 avian influenza in Harbor Seals.
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogenand the disease concerned
The National Veterinary Services Laboratories’ Quality Management System is accredited to ISO/IEC 17025 by an independent accreditation body recognized by the International Laboratory Accreditation Cooperation. Audits and surveillance visits are an ongoing part of maintaining ISO/IEC 17025 accreditation.
Biosafety and biosecurity at the NVSL comply with the U. S. Department of Agriculture and U. S. Department of Health and Human Services Select Agent Program and as such meet or exceed guidelines established in “Biosafety in Microbiological and Biomedical Laboratories”.
8.Provision of consultant expertise to OIE or to OIE Member Countries
NVSL staff conducted a workshop in quality control testing and biosecurity/biosafety procedures and practices for a BSL3 laboratory at the SAG laboratory, Santiago, Chile as part of the OIE twinning project.In addition, a laboratory assessment was conducted for biosecurity/biosafety procedures and BSL3 facilities under current use at the SAG virology laboratory.
NVSL staff participated in the First Annual South American Workshop on Avian Influenza and Newcastle Disease in Campinas, Brazil.Presentations were given at the workshop on Quality Control for Reagent Production, Production and Evaluation of Proficiency Tests and NVSL’s Experience as a Parent OIE Laboratory.
9.Provision of scientific and technical training to personnel from other OIE Member Countries
The NVSL provided technical training in avian influenza diagnostic techniques at the Central Laboratory of National Animal Health Center in Vientiane, Lao PDR. Training was provided to 23 field veterinarians and laboratory technicians from nine providences throughout Lao PDR on the following topics: background of avian influenza (AI) virus, biosafety,agar gel-immunodiffusion (AGID), hemagglutination (HA), hemagglutination-inhibition (HI), and neuraminidase-inhibition (NI) procedures, specimen handling and collection, enzyme linked immunosorbent assay (ELISA), antigen detection, rRT-PCR and virus isolation.
The NVSL is participating with the Servicio Agrícola y Ganadero (SAG) Ministerio de Agricultura, Santiago,Chile in an OIE twinning project for avian influenza (AI) and Newcastle disease (ND).As part of the agreement the NVSL has provided scientific and practical training to staff from the SAG laboratory.In 2011 a practical training session was conducted at the NVSL for 2 scientists.The practical training included hands-on training in neuraminidase-inhibition (NI), genomic sequencing, molecular viral characterization, real-time RT-PCR, AGID and HI antigen and antiserum production and BSL3 laboratory biosafety and biosecurity procedures.In addition, an on-site training session and workshop was conducted at the SAG laboratory.The work-shop included lectures on biosecurity and biosafety and reagent quality control testing.Practical training and exchange of standard operating procedures and reference reagents was conducted for the harmonization and standardization of diagnostic methods for the diagnosis of AI and the production of reagents.Monthly conference calls are held to provide assistance and technical expertise as needed.
10.Provision of diagnostic testing facilities to other OIE Members
During 2011, the NVSL provided primary diagnostic assistance to seven OIE member countries: Barbados (4specimens, 4 submissions), Belize (19 specimens,10submissions), Brazil (12 specimens, 1 submission), Chile (2specimens,1 submission), Dominican Republic (1 specimen, 1 submission), Nicaragua (23 specimens, 3 submissions) and Puerto Rico (10 specimens, 4 submissions).Diagnostic testing included virus isolation, rRT-PCR, hemagglutination-inhibition, neuraminidase-inhibition (subtyping), chicken inoculation and nucleotide sequencing and molecular pathotyping interpretation.