OIE Reference Laboratory Reports
Activities in 2010
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Avian influenza
Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe),
Viale dell’Università, 10 – 35020 Legnaro (Padua), Italy
Tel.:
(+39-049) 808.43.79
Fax:
(+39-049) 808.43.60
e-mail address:
icapua@izsvenezie.it
website:
Name of Head of Laboratory
(Responsible Official):
Name of OIE Reference Expert:
Ilaria Capua
Name of writer of this report
(if different from above):
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
1
Avian influenza
Part I: Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
ELISA
Antibody
Group
559
DIVA test (iIFA)
Antibody
Subtype
161
Haemagglutination inhibition test (HI)
Antibody
Subtype
40373
Haemagglutination inhibition test (HI)
Viral typing
Subtype
1685
Neuraminidase inhibition test (NI)
Viral typing
Subtype
90
ELISA
Viral antigen
Group
757
Inoculation of SPF embryonated chicken eggs
Virus isolation
Group
2987
rRT-PCR
(reverse transcriptase- polymerase chain reaction)
Virus
Group
24847
Sequencing
Virus
Pathotype
96
Production and distribution of diagnostic reagents
In 2010, 26,595 ml of reference antigens and 3,375 ml of reference sera were produced. The list of reagents
produced is shown in detail below:
Avian influenza antigens (H1-H16)
Infectious bronchitis antigens
Newcastle disease antigen
Infectious bursal disease antigen
Avian Paramyxovirus antigens (APMV-1, APMV-9)
Infectious laryngotracheitis antigen
Reovirus antigen
Negative serum
Positive sera for avian influenza virus (H1-H16)
Positive serum for Newcastle disease virus
Positive Avian Paramyxovirus serum (APMV-1, APMV-9)
Infectious bursal disease serum
Infectious laryngotracheitis serum
Reovirus serum
Hev
Hepatitis E serum


A total of 2,314 ml of antigens were distributed, of these 1,139 ml nationally and 1,175 ml internationally.
A total of 1,492 ml of sera were supplied, of these 660 ml nationally and 832 ml internationally.
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
Ring trials in which the OIE Italian laboratory participated during 2010
EU serological and virological identification ring trial.
2
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Avian influenza
Ring trials organized by the OIE Italian laboratory during 2010
National Ring-test
In 2010 a serological and virological ring test was organised by the Italian National Reference Centre to assess the
capability of Italian public laboratories to identify selected antigens and to detect antibodies in avian sera to avian
influenza viruses and Newcastle disease and to standardize the diagnostic procedures used by the different
laboratories.
All the laboratories were able to detect H5 and H7 subtypes viruses by virological, serological and molecular
methods. Furthermore, most laboratories were able to detect the H1N1 pandemic virus, although without specific
reagents to detect this viral subtype. The number of reagents will be increased in order to enable different
laboratories to enlarge the diagnostic efforts to identify influenza viruses in general.
International Ring-test
In addition to the National Ring-Test, the Reference Centre has organized a Ring-test for avian influenza and
Newcastle disease for African reference laboratories, and for laboratories in the Balkan region and in the Middle
East. This initiative was funded by FAO, and by FAO ECTAD of Mali.
All the laboratories were able to detect antibodies against H7 and H5 subtypes by HI test and to detect H5 viral
RNA by molecular methods (RT-PCR and RRT-PCR). Areas of improvement have been identified in the
serological assay (AGID) and in molecular methods (e.g. RT-PCR and RRT-PCR) to identify H7 subtype viruses.
Corrective actions are being implemented to improve the accuracy of these tests in specific laboratories. This
proficiency test was essential in providing the necessary feedback to FAO, donors and reference laboratories to
better design future training courses and provide technical support of the network
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
See point 2 above.
5.
Research and development of new procedures for diagnosis and control
Development of an antigen capture ELISA for the rapid detection of avian influenza virus in the feathers of
poultry
It has been recently reported that feathers, together with faeces and respiratory secretions, could represent a
potential source of infection by avian influenza viruses (AIV). In this study, we have developed a double antibody
sandwich enzymed-linked immunosorbent assay (DAS-ELISA), to detect AIV in feathers of experimentally
infected poultry. Briefly, two separate monoclonal antibodies against the nucleoprotein (NP) of AIV were used to
develop an ELISA assay, one as a capture antibody and the other as a detection antibody. The test has been applied
to feathers collected from experimentally infected ducks and chickens. The statistical analysis, to assess the
specificity and sensitivity of the test, is currently ongoing.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
The results of all diagnostic investigations related to Avian influenza (AI) have been notified to the OIE, according
to its guidelines. This diagnostic effort contributed to improve knowledge on the global spread of avian influenza
viruses. IZSVe was also actively involved in epidemiological and molecular studies of avian influenza viruses of
H5N1 and H9N2 subtypes.
In particular, IZSVe investigated the impact of vaccination on the genetic evolution of H5N1 viruses in Egypt.
Since 2006 H5N1 HPAI viruses belonging to clade 2.2.1 have been extensively circulating in the poultry
population in Egypt. In order to control the disease in poultry and reduce the risk for human health, stamping out
and vaccination have been applied on a large-scale. Despite this, the virus became endemic in Egypt. The results
obtained, suggest that vaccines and vaccination practices may impact the evolutionary rate and the occurrence of
mutations in H5N1 viruses circulating in poultry. This should be taken into account when considering vaccination
as a tool for controlling this infection. Proper vaccine strain selection, the use of good quality vaccines,
implementation of rigorous vaccination practices and monitoring of field vaccination coverage and vaccine
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
3
Avian influenza
efficacy must all be seriously considered to reduce the risk of vaccine failures and major antigenic drift.
IZSVe also performed a molecular epidemiological study of the HA gene of H9N2 viruses isolated in the Middle
East, from 2004-2009. Genetic data were generated in order to elucidate the molecular characteristics and the
phylogenetic relationships of 21 H9N2 viruses identified in 4 distinct Middle Eastern Countries, namely Jordan,
Iraq, Saudi Arabia and the United Arab Emirates (UAE between 2004 and 2009). Analysis of deduced amino acid
sequences of the HA glycoprotein revealed that 6 viruses from Saudi Arabia, two viruses from Dubai and all the
viruses isolated in Kurdistan-Iraq contained the substitution Q226L (H3 numbering) at the receptor binding site.
This mutation is associated with a preferential receptor binding specificity for sialic acid 2,6-linked galactose and
displays human virus-like cell tropisms. Molecular analysis allowed us also to identify a Saudi Arabian strain,
exhibiting 3 out of 4 amino acid residues at the receptor binding site previously associated with respiratory
transmission in ferrets (Sorrel et al., 2009). The genetic variability identified between the H9N2 viruses used in the
present study combined with the identification of specific substitutions which are believed to modify the efficiency
of the replication in non-avian species, highlight the need to constantly monitor the evolution of this potentially
pandemic strain in the Middle East
7.
Provision of consultant expertise to OIE or to OIE Members
OIE Twinning project between the Italian OIE/FAO reference laboratory for Avian Influenza and Newcastle
Disease and National Centre for Animal and Plant Health (Censa) of Cuba (final workshop, November 2010). This
twinning had the aim of implementing capacity building of the Cuban laboratory in order to bring it closer to OIE
reference laboratory status by improving standards in specific selected areas.
Collaboration Agreement between IZSVe e IVO (Iranian Veterinary Organization) with the aim of giving
scientific and technical support on Avian influenza and Newcastle disease to the Iranian veterinary service.
8.
Provision of scientific and technical training to personnel from other OIE Members
During 2010 IZSVe continued and increased the training activities on avian influenza (AI) and Newcastle disease
(ND). In particular, it managed training courses within the framework of European funded projects (EPIZONE,
FLUTRAIN), organizing training courses on avian influenza diagnosis and epidemiology, for veterinarians and
technicians. The list of visitors is reported in Table 2.
During 2010, IZSVe’s staff members carried out assessment missions on laboratories which applied for such
activity. The list of the missions is reported below:

20-23 March 2010 (Qatar), OIE meeting for a possible twinning project between IZSVe and National
veterinary laboratory

March 2010 Cairo (Egypt), Meeting with IFT Corporation to create a collaboration for the management of
the H5N1 crisis

October 2010 Cairo (Egypt), Workshop on the surveillance activity for Avian influenza in Egypt
Table 2: List of people attending courses at IZSVe during 2010
4
Name
Laboratory
Country
Training subject
Period
Rosa-Stella Mbulu
Central Veterinary Laboratory,
Ministry of Agriculture, Water
and Forestry
Namibia
Diagnosis of AI and NDV
15/02/2010
26/02/2010
Canaan Tinashe
Department of Veterinary
Technical Services, Ministry of
Agriculture
Zimbabwe
Diagnosis of AI and NDV
15/02/2010
26/02/2010
Wegdan Hassan Ali Abdel
El Mageed
Central Veterinary Research
Laboratories, Department of
Virology
Sudan
Diagnosis of AI and NDV
15/02/2010
26/02/2010
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Avian influenza
Name
Laboratory
Country
Training subject
Maria Lucia Mandola
Istituto Zooprofilattico
Sperimentale del Piemonte,
Liguria e Valle d’Aosta
Italy
Diagnosis of major avian
diseases-Laboratory quality
assurance
07/04/2010
08/04/2010
Tony Joannis
National Veterinary Research
Institute
Nigeria
Research studies
01/09/2009
30/05/2010
Jeffrey Christ
Virginia-Maryland Regional
College of Veterinary Medicine
United
States
Diagnosis of AI and NDV
01/06/2010
08/08/2010
Rabab Amin Ahmed Khalifa
Cairo Poultry Company (CPC)
Laboratory
Egypt
Molecular diagnosis of AI
07/06/2010
11/06/2010
Dina Moustafa Ali Hamed
Cairo Poultry Company (CPC)
Laboratory
Egypt
Diagnosis of AI
07/06/2010
11/06/2010
Eleonora Molesti
Medwey School of Pharmacy
University of Greenwich and
Kent a Medway
Italy
Pseudoparticles
neutralization assay for AIV
01/10/2010
22/10/2010
Kateri Bertran Dols
Centre de Recerca en Sanitat
Animal (CReSA)
Spain
Diagnosis of AI
10/10/2010
31/01/2011
Hesamodin Emadi Chashmi
Department of Clinical Sciences,
Faculty of Veterinary Medicine
Iran
Diagnosis of AI and major
avian diseases
18/10/2010
18/04/2011
Laìs Pancotto
Universidade Luterana Do
Brasil
Brasil
Diagnosis of AI and major
avian diseases
25/10/2010
24/12/2010
Mamoona Chaudhry
University of Edimburg (UK)
Pakistan
8/11/2010
17/11/2010
9.
Molecular techniques for AI
diagnosis and sequencing
Period
Provision of diagnostic testing facilities to other OIE Members
IZSVe has supported the Veterinary Services of foreign countries by providing official confirmatory diagnosis of
routine cases identified during surveillance activities in domestic and wild birds.
Table 3 indicates the number of samples from foreign countries submitted for Avian Influenza diagnosis in 2010.
Table 3: Samples from foreign Countries submitted for Avian Influenza diagnosis at IZSVe
Country
Serological tests
Virological tests
Molecular tests
Total
BANGLADESH
0
17
56
73
BULGARIA
0
0
1
1
BURKINA FASO
12
0
50
62
EGYPT
74
212
712
898
JORDAN
0
7
14
21
IRAN
0
10
149
159
KYRGYZSTAN
2
0
0
2
MYANMAR
80
23
468
571
PAKISTAN
0
30
368
398
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
5
Avian influenza
Country
Serological tests
Virological tests
Molecular tests
Total
TURKEY
0
239
2
241
ZIMBAWE
0
1
425
426
LITHUANIA
0
0
308
308
UK
12
112
0
124
Total
180
651
2,832
3,866
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
In 2010 IZSVe organized the following conference:
27-29 April, 2010 Verona (Italy): “2nd OIE/FAO/WHO Scientific consultation on influenza and other emerging
infectious diseases at the human-animal interface”. The meeting gathered a group of experts in Verona, Italy, to
focus on emerging zoonoses including influenza and identify commonalities at the human-animal interface. The
main purpose of the meeting was to provide a technical basis for developing or modifying policies and strategies
to more effectively prepare for and respond to the next emerging event.
11. Participation in international scientific collaborative studies
Working groups
 Dr. Ilaria Capua and Dr. Isabella Monne – Members of EFSA working group on “scientific opinion on the
monitoring for emergence of possible new pandemic strains of influenza in animals”, Panel on Animal Health
and Welfare (AHAW) European Food Safety Authority (EFSA)”.
Research projects
 CARIPLO-“Novel Strategies of vaccine design to prevent emerging and pandemic influenza virus infections
NoFlu”
 NEW FLUBIRD – “Network for early warning of influenza viruses in migratory birds in Europe”
 FLUPATH-“Avian Influenza: impact of virus-host interactions on pathogenesis and ecology”
 FLUTRAIN – “Training and technology transfer of Avian Influenza diagnostics and disease management
skills”
 FLURESIST-“Studies on Avian Influenza virus survival in poultry commodities, poultry manure and the
environment”
 FLU-LAB-NET-“Development and enhancement of laboratory networks for avian influenza”
 FLUPIG-“Pathogenesis and transmission of influenza in pigs”
 EPIZONE-“Network of Excellence for Epizootic Disease Diagnosis and Control”
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)
1.
2.
3.
4.
5.
6
Anderson T.C., De Benedictis P., Perez A., Viale E., Veggiato C., Tiozzo Caenazzo S., Crawford C., Capua I.
(2010)."A Diagnostic Algorithm for Detection of Antibodies to Influenza A in Dogs"; Proceding of The
International Symposium on Neglected Influenza Viruses; Amelia Island (Florida) 3-5 February.
Anderson T., Capua I., Dauphin G., Donis R., Fouchier R., Mumford E., Peiris M., Swayne D., Thiermann A.
(2010).“FAO-OIE-WHO Joint Technical Consultation on Avian Influenza at the Human-Animal Interface”
Influenza Other Respi Viruses, 4 Suppl 1:1-29.
Beato MS., Monne I., Mancin M., Bertoli E., Capua I. (2010). “A proof-of-principle study to identify suitable
vaccine seed candidates to combat introductions of Eurasian lineage H5 and H7 subtype avian influenza viruses.
Avian Pathol., 39(5):375-82.
Capua I., Alexander D. (2010). “Perspectives on the global threat: the challenge of avian influenza viruses for the
world's veterinary community” Avian Dis., 54(1 Suppl):176-8.
Capua I. (2010). "Patogeni emergenti nell'era globalizzata"; Proceedings 44 th Congresso Nazionale SIRM - Verona
11-15 Giugno 2010.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Avian influenza
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7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Capua I., Cattoli G. (2010). “One flu for One Health” Emerg Infect Dis., 16(4):719.
Cecchinato M., Ceolin C., Busani L., Dalla Pozza M.,Terregino C., Moreno A., Bonfanti L., Marangon S. (2010).
"Low Pathogenicity Avian Influenza in Italy During 2007 and 2008: Epidemiology and Control"; Avian Diseases
54(1):323-328.
Cecchinato M., Comin A., Bonfanti L. , Terregino C., Monne I, Lorenzetto M., Marangon S. (2010).
“Epidemiology and control of Low Pathogenicity Avian Influenza infections in rural poultry in Italy. Avian
Diseases in press.
Cilloni,F., Toffan,A.; Giannecchini,S.; Clausi,V.; Azzi A.; Capua I.; Terregino C. (2010). "Increased pathogenicity
and shedding in chickens of a wild bird-origin LPAI virus of the H7N3 subtype following multiple in vivo passages
in quail and turkey"; Avian Diseases 54:555-557.
Dauphin G., Hamilton K., Kim L.M., Choudhury B., Capua I., Edwards S. (2010). “Main achievements of the
World Organisation for Animal Health/United Nations Food and Agriculture Organization network on animal
influenza” Avian Dis., 54(1 Suppl):380-3. 9.
Dauphin G., Mumford L., Thiermann A., Capua I. (2010) “Opening letter. Avian influenza at the human animal
interface” Influenza Other Respi. Viruses, 4 Suppl 1:i.
De Benedictis P., Anderson T. C., Perez A., Viale E., Veggiato C., Tiozzo Caenazzo S., Crawford P. C., Capua I.
(2010). “A diagnostic algorithm for detection of antibodies to influenza A viruses in dogs in Italy (2006–2008)”
J Vet Diagn Invest., 22: 914-920.
Di Lullo G.; Soprana E.; Panigada M.; Palini A.; Agresti A.; Comunian C.; Milani A.; Capua I.; Erfle V.; Siccardi
G.A. (2010). "The combination of marker gene swapping and fluorescence-activated cell sorting improves the
efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use"; Journal of
Virological Methods, 163(2):195-204.
Dundon W.G., De Benedictis P., Veggiato C., Capua I. (2010). "Serological evidence of H1N1 infection in dogs in
Italy"; Proceding of The International Symposium on Neglected Influenza Viruses; Amelia Island (Florida) 3-5
February 2010.
Dundon W.G., De Benedictis P., Viale E., Capua I.(2010). "Serological evidence of H1N1 infection in dogs in
Italy"; Emerging Infectious Diseases, 16(12):2019-21.
Fereidouni S.R., Werner O., Starick E., Beer M., Harder T.C., Aghakhan M., ModirroustaH., AminiH.,
Moghaddam M.K., Bozorghmehrifard M.H., Akhavizadegan M.A., Gaidet N., Newman S.H., Hammoumi S.,
Cattoli G., Globig A., Hoffmann B., Sehati M.E., Masoodi S., Dodman T., Hagemeijer W., Mousakhani S.,
Mettenleiter T.C. (2010). "Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007; Virol J.
19;7:43.
Fusaro A., Monne I., Cattoli G., De Nardi R., Salviato A., Moreno Martin A., Capua I., and Terregino C. (2010)
“Gene segment reassortment between Eurasian and American clades of avian influenza virus in Italy”. Arch Virol.,
155(1):77-81.
Fusaro A., Nelson M.I., Joannis T., Bertolotti L., Monne I., Salviato A., Olaleye O., Shittu I., Sulaiman L., Lombin
L.H., Capua I., Holmes E.C., Cattoli G. (2010). “Evolutionary dynamics of multiple sublineages of H5N1 influenza
viruses in Nigeria from 2006 to 2008”. J Virol., 84(7):3239-47.
Giannecchini S., Clausi V., Di Trani L., Falcone E., Terregino C., Toffan A., Cilloni F. Matrosovich M.,
Gambaryan A.S., Bovin N.V., Delogu M., Capua I., Donatelli I., Azzi A.(2010). “Molecular adaptation o an H7N3
wild duck influenza virus following experimental multiple passages in quail and turkey”. Virology, 408:167-173.
Keiner B., Maenz B., Wagner R., Cattoli G., Capua I., Klenk HD. (2010). "Intracellular distribution of NS1
correlates with the infectivity and interferon antagonism of an Avian Influenza virus (H7N1). Journal of Virology,
84(22), 11858-11865.
Rigoni, M. , Toffan, A., Viale, E. , Mancin, M. , Cilloni, F. , Bertoli, E., Salomoni, A., Marciano, S. , Milani, A.,
Zecchin, B. , Capua, I., Cattoli G., (2010). “The mouse model is suitable for the study of viral factors governing
transmission and pathogenesis of highly pathogenic avian influenza (HPAI) viruses in mammals”, Veterinary
Research, 41:66.
Smietanka K., Fusaro A., Domanska-Blicharz K., Salviato A., Monne I., Dundon W. G., Cattoli G., Minta Z.
(2010). “Full-length Genome Sequencing of the Polish 1 HPAI H5N1 Viruses Suggests Separate Introductions in
2006 and 2007”. Avian Dis., 54(1 Suppl):335-9.
Terregino C., Toffan A., Cilloni F., Monne I., Bertoli E., Castellanos L., Amarin N., Mancin M., Capua I.; (2010).
"Evaluation of the protection induced by avian influenza vaccines containing Mexican H5N2 LPAI seed strain
against a 2008 Egyptian H5N1 virus belonging to clade 2.2.1 by means of serological and in vivo tests"; Avian
Pathology, vol 39(3), 215-222.
Toffan A.; Olivier A.; Mancin M.; Tuttoilmondo V.; Facco D.; Capua I.; Terregino C. (2010). "Evaluation of
different serological tests for the detection of antibodies against highly pathogenic avian influenza in
experimentally infected ostriches (Struthio camelus)"; Avian Pathology, 39 (1), 11-15.
Uttenthal A., Parida S., Rasmussen T.B., Paton D.J., Haas B., Dundon W.G. “Strategies for differentiating infection
in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza” Expert Rev.
Vaccines. 9(1):73-87.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
7
Avian influenza
26. Zecchin, B., Munir, M., Zohari, S., Cattoli, G. Berg, M., Capua, I., Dundon W.G. (2010). Analysis of the interferon
abrogation ability of the non-structural 1 protein from influenza A. Fourth Annual EPIZONE meeting June 7-10, St
Malo, France.
27. Zecchin, B, Munir, M., Zohari, S., Cattoli, G., Berg, M., Capua, I., Dundon W.G. (2010). The interferon abrogation
of the Non-structural I proetin from influenza A is not effected by carboxy terminal length variations. 4th European
congress of Virology, 7-11, April, Cernobbio, Italy.
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
_______________
8
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010