Avian influenza
OIE Reference Laboratory Reports
Activities in 2010
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / Avian influenzaAddress of laboratory / National Veterinary Services Laboratories
USDA, APHIS, VS
P.O. Box 844, 1920 Dayton Avenue
Ames, IA 50010 USA
Tel.: / (+1-515) 337.72.66
Fax: / (+1-515) 337.73.97
e-mail address: /
website: / http://www.aphis.usda.gov/animal_health/lab_info_services
Name of Head of Laboratory (Responsible Official): / Dr Elizabeth A. Lautner
Name of OIE Reference Expert: / Ms Janice C. Pedersen
Name 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 / 667
Hemagglutination-inhibition / Antibody/Virus / Sub type / 46,811
Neuraminidase-inhibition / Antibody/Virus / Subtype / 24,696
Virus Isolation
/ Virus / Subtype / 9659Real-time RT-PCR (matrix, H5, H7, N1)
/ RNA / Type/subtype / 2952Intravenous Pathogenicity Index in SPF chickens
/ Virulence / Subtype / 25HA cleavage site sequencing
/ Virulence / Subtype / 1352. 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). The NVSL also produces proficiency tests for HI and real-time RT-PCR (rRT-PCR) assays, and distributes positive amplification control (PAC) (transcribed RNA) and extraction control (PEC) for rRT-PCR.
In 2010, a total of 13,709 sets of AGID reagents (antigen and antiserum) were distributed to domestic laboratories and 309 sets were distributed to 11 international laboratories (Australia, Canada, Costa Rica, Dominican Republic, El Salvador, Haiti, Nicaragua, Panama, Saudi Arabia, Uzbekistan, and West Indies). Each set will test approximately 120 serums. Additionally, 2,756 sets of AGID reference serum (weak positive, strong positive and negative) were distributed to domestic laboratories and 113 sets to international laboratories.
The NVSL produced 6 lots (approximately 12 liters) of AGID antiserum and reference sera and prepared 13,655sets of AI AGID reagents (antigen and antiserum) and 4,265 sets of AI AGID reference sera (weak positive, strong positive and negative). Also, following subtyping reagents were produced: nine lots of reference antiserum (400ml each), nine lots of inactivated reference antigen (170 ml each), and 250 ml of reference virus (one subtype). Also, the NVSL distributed 79 AGID proficiency test panels (10 serums each) to domestic laboratories.
Country / Reagent(s) / No. vialsAustralia / AGID antigen and antiserum / 6
Brazil / Reference viruses of the H1-16 and N1-9 subtypes, positive amplification control (PAC) (transcribed RNA) and positive extraction control ,(PEC) for rRT-PCR / 64
Canada / AGID antigen and antiserum, strong positive, weak positive and negative AGID reference serums / 22
Chile
/ AI AGID proficiency test / 1Country / Reagent(s) / No. vials
Columbia
/ rRT-PCR PAC and PEC / 10Costa Rica
/ AGID antigen and antiserum and rRT-PCR PAC and PEC / 84Dominican Republic
/ AGID antigen and antiserum, rRT-PCR PAC and PEC and H5, H7 and H9 HI antigens and antiserum / 228El Salvador
/ AGID antigen and antiserum / 50Guatemala
/ rRT-PCR PAC and PEC / 4Haiti
/ AGID antigen and antiserum, rRT-PCR PAC and PEC, AGID strong positive, weak positive and negative reference serum, H5, H7 and H9 HI reference antigens and antiserum / 229Israel
/ H5 and H7 HI reference antigens and antiserum / 50Korea
/ rRT-PCR PAC / 1Kuwait
/ H7 and H9 HI reference antigens and antiserum / 8Mexico
/ rRT-PCR proficiency test / 3Nicaragua
/AGID antigen and antiserum
/ 58Paraguay
/ rRT-PCR PAC / 3Panama
/ AGID antigen and antiserum, AGID reference strong positive, weak positive and negative reference serum, rRT-PCR PAC and PEC / 59Saudi Arabia
/ AGID antigen and antiserum, H5 and H9 reference antiserum, H9 reference antigen / 18Singapore
/ H1, H6 and H16 reference antigens and antiserum / 10Sweden
/ H5 and H7 reference viruses / 20Kazakhstan and Georgia
/ rRT-PCR PAC / 18Uzbekistan
/ AGID antigen and antiserum, AGID reference strong positive, weak positive and negative reference serum / 15West Indies
/ AGID antigen and antiserum / 4Part 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
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. In cooperation with the National Centre for Foreign Animal Disease (NCFAD) Canadian Food Inspection Agency (CFIA) a panel of viruses were distributed to the NVSL, CFIA and CPA, Mexico City, Mexico for participation in harmonization and standardization of virus isolation, identification, subtyping and pathotyping procedures.
The NVSL is participating with the Laboratôrio Nacional Agropecuário (Lanagro-SP), Campinas, Brazil in an OIE twinning project for avian influenza (AI) and Newcastle disease (ND). 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. Procedures and reagents have been exchanged for the AGID, HI, rRT-PCR, NI, genomic sequencing and virus isolation and identification tests.
The NVSL is participating with the Servicio Agicola Y Ganadero (SAG) Laboratory, Santiago, Chile in an OIE twinning project for AI and ND. As part of the project practical training on HI and NI procedures for subtyping, hemagglutination and neuraminidase antibody and virus subtype along with the exchange of HI and NI reference reagents was conducted.
Members of the NVSL staff are working with the OFFLU technical group to develop recommendations for international proficiency tests used to evaluate performance of avian influenza and pandemic H1N1 testing. The technical group is working to increase the standardization of diagnostic testing procedures and develop guidelines for proficiency tests for harmonization purposes. These procedures would complement the OIE Manual’s chapter on Laboratory Proficiency Testing.
The NVSL participated in the 3rd OFFLU meeting of influenza technical experts, Rome, Italy (November 2010) to develop guidelines for HPAI H5 and pandemic H1N1 testing.
Participation in an Animal Research Service (ARS) National Program, Procinorte, in collaboration with the Canadian Food Inspection Agency and Mexican counterparts. The objective of Procinorte is to expand research and diagnostics for pH1N1 for both avian and swine.
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) and inactivated virus prepared for use as a positive extraction control (PEC) for the real-time RT-PCR assay (Spackman et al.) directly to eight international laboratories (Brazil, Columbia, Costa Rica, Dominican Republic, Guatemala, Haiti, Korea, and Paraguay) as well a broker service which supplies international laboratories in Kazakhstan and Gerogia. The PAC and PEC reagents were also distributed to 51 domestic laboratories (183 vials in 40 states).
Continued participation in the international effort (coordinated by VLA, Weybridge) to develop an H5 reference serum. The NVSL is providing stock reagent for two of the international reference serums.
5. Research and development of new procedures for diagnosis and control
Participation in field studies with Life Technologies Corporation, Applied Biosystems (Carlsbad, CA) for the licensing of a commercial rRT-PCR kit detection of the matrix gene of influenza virus with an emphasis on avian influenza.
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.
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 sequence of the hemagglutinin, neuraminidase and matrix genes is deposited in the National Center for Biotechnology Information (NCBI) GeneBank.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
Wild bird surveillance for highly pathogenic avian influenza virus in wild aquatic birds in the United States continued in 2010. The program is a cooperative effort between the U.S. Departments of Agriculture and Interior and State Wildlife Agencies. Approximately 36,061 specimens were collected from dead as well as healthy and hunter-killed birds in all 36 states and tested for presence of avian influenza virus by real-time RT-PCR (rRT-PCR) at National Animal Health Laboratory Network (NAHLN) laboratories, the National Wildlife Research Center and the National Wildlife Health Center. Positive matrix specimens were subsequently tested by the H5 and H7 rRT-PCR subtyping assays and H5 and H7 positive specimens were shipped to the NVSL for confirmatory testing, virus isolation (VI) and characterization. A total of 535 H5/H7 presumptive positive specimens were tested. These studies did not detect a HPAI virus but provided basic information about avian influenza viruses circulating in wild aquatic birds. A summary of the surveillance can be accessed at http://wildlifedisease.nbii.gov/ai/.
7. Provision of consultant expertise to OIE or to OIE Members
NVSL staff conducted a workshop in the production and quality control testing of reference antigens and antiserum at the Lanagro-SP, Campinas, Brazil as part of the OIE twinning project. In addition, a laboratory assessment was conducted for antigen and antiserum production and testing.
8. Provision of scientific and technical training to personnel from other OIE Members
In February 2010, a member of the NVSL staff provided lecturers and clinical training in disease recognition of HPAI to approximately 30 Canadian Food Inspection Agency veterinarians at the National Centre for Foreign Animal Diseases, Winnipeg, Canada.
The NVSL is participating with the Laboratôrio Nacional Agropecuário (Lanagro-SP), Campinas, Brazil 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 Lanagro-SP laboratory. Two separate practical training sessions have been conducted at the NVSL for a total of 5 scientists, one of which was conducted in 2010. The practical training has included hands-on training in virus isolation, virus characterization by the hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI), genomic sequencing and molecular viral characterization, real-time RT-PCR, antibody detection by HI and NI and in vivo pathotyping by the intravenous pathogenicity index (IVPI) test. Two separate on-site training sessions and workshops were conducted at the Campinas Lanagro-SP laboratory. The first work-shop included lectures on AI and the diagnostic tests used to diagnose or detect the disease. The workshop was a full day in length. The second workshop focused on antigen and antiserum production and quality control testing for evaluation of those reagents. 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. Several NVSL staff members trained 3 scientists from Brazil at the NVSL in avian influenza and Newcastle classical and molecular diagnostic procedures and reagent production (May 2010).
As part of the Lanagro-SP/NVSL OIE twinning project two members of the NVSL staff provided on-site training in quality control testing for AI and HI reference reagents at the Lanagro-SP laboratory in Campinias, Brazil.
Several (approximately 5) members from the NVSL provided antigen and antiserum production, HI, NI and quality control training (5 days) for a scientists from Pakistan.
The NVSL is participating with the Servicio Agicola Y Ganadero (SAG) Laboratory, Santiago, Chile in an OIE twinning project for AI and ND. This project started in June 2010. Several members of the NVSL staff provided training at the NVSL (2 weeks) in biosecurity and biosafety procedures for HPAI, HI and NI diagnostic testing and quality control and management to two scientists from the SAG laboratory. In addition training was conducted for virus characterization using classical procedures and real-time RT-PCR tests. An additional training session will be held at the NVSL in 2011/2012 and on-site training, lectures and workshops will be held at the SAG facility.
Two members of the NVSL staff participated in a FAO pH1N1 training in Panama in August 2010. Practical laboratory training on diagnostic testing procedures (rRT-PCR and virus isolation and identification) for influenza viruses was provided in Panama for 5 scientists from Panama, Guatemala and Costa Rica. In addition, one scientists from Panama received training at the NVSL for influenza genomic sequencing.
9. Provision of diagnostic testing facilities to other OIE Members
During 2010, the NVSL provided primary diagnostic assistance to six OIE member countries: Belize (10 submissions and 66 specimens), Chile (2 submissions and 4 specimens), Dominican Republic (7 submissions and 29 specimens), Honduras (2 submissions and 14 specimens), Nicaragua (4 submission and 4 specimens), and Puerto Rico (1 submission and 4 specimens).
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
A member of the NVSL staff is serving as vice-president of the OIE Biological Standards Commission.
11. Participation in international scientific collaborative studies
None.
12. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)
¡ Presentations at international conferences and meetings
¡ Pedersen JC. (2010). Influenza Surveillance in Poultry and Overview of Diagnostic Tests. Pressented at Live Bird Market System Continuing Education Training.