Last update: 17/06/2022
After taking up the role as new European Union Reference Laboratory (EURL) for Avian Influenza and Newcastle Disease, starting from January 2019 the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) has been entrusted with making sure that the recommended protocols for the detection, identification and typing of AI and AOAV-1 are fit for purpose.
This process has been and is carried out by testing the efficacy of the existing methods using newly received viruses or by comparing the performance of the new available methods with the ones in use, in agreement with the WOAH guidelines on the validation of diagnostic tests. The diagnostic procedures have been developed according to the Diagnostic Manual for Avian Influenza (2006/437/EC) as provided for in Council Directive 2005/94/EC, which sets out the guidelines to carry out the collection of samples, tissue material to be examined, transport of samples, laboratory protocols, and criteria required to evaluate the results of laboratory tests for a proper diagnosis of AI. This document will continue to be technically valid also after the abrogation of Directive 2005/94/CE with the forthcoming entry into force of Regulation (EU) 2016/429 on transmissible animal diseases (‘Animal Health Law’).
In particular, sampling and diagnostic procedures as described in the diagnostic manual for AI can be considered efficient for gallinaceous poultry, whereas additional sampling is advised for Anseriformes. Revisions of EFSA and the AHAW Panel of experts are reported in the Scientific Opinion on the assessment of the control measures of category A diseases of Animal Health Law: Highly Pathogenic Avian Influenza and refer to: (1) clinical and laboratory sampling procedures, (2) monitoring period and (3) the minimum radius of the protection and surveillance zone, as well as the minimum length of time the measures should be applied in these zones. Several scenarios for which these control measures had to be assessed are reported in this document. Specific guidelines have been produced for the weekly pool sampling (“bucket sampling”) in poultry species that may not manifest clinical signs when infected with HPAI.
Given the similarity between Avian influenza and Newcastle disease in terms of target species and tissue tropism, the collection of samples, tissue material to be examined and transport of samples as described in the aforementioned documents and summarized here are considered applicable in the management of both diseases.
Detailed procedures to carry out the diagnostic methods which are currently made available by the EURL IZSVe and the former EURL APHA are itemized below.
Interpretation of results is reported in each procedure and in this document. Please note that any substantial modification of the recommended procedures or submission of new diagnostic tests by a third party laboratory should be supported by proper validation data confirming that the method is fit for purpose. The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Chapter 1.1.6. – Principles and methods of validation of diagnostic assays for infectious diseases and Section 2.2. – Validation of diagnostic tests), as well as the requirements of the ISO/IEC 17025 provide guidance for validating diagnostic methods to ensure they are compliant with the purpose they are intended for.
As foreseen in article 6 of Commission Delegated Regulation (EU) 2020/689, the competent authority of each Member State shall ensure that the collection of samples, the techniques, validation and interpretation of the diagnostic methods for the purposes of surveillance shall comply with the specific legislation adopted in accordance with Regulation (EU) 2016/429 and that the relevant details and guidance are made available on the websites of the European Union Reference Laboratories (EURL) and of the Commission.
A diagnostic method is considered compliant with the ones recommended by the EURL for AI/ND if it is:
- A procedure based on a method acquired directly from the EURL for AI/ND;
- A method whose sensitivity and specificity have been demonstrated as being effective following a comparative test organized by the EURL for AI/ND;
- A method for which no evaluation has been organized by the EURL for AI/ND and for which the procedure and validation dossier were considered compliant after a review of any relevant information by the EURL for AI/ND validation unit.
Please contact the IZSVe-EURL staff for technical advice on the procedures and information you may need for the official evaluation of your diagnostic methods.
|Isolation and characterization of Avian influenza viruses using SPF embryonated chicken eggs and haemagglutination inhibition test (Released on 12/10/21)||IZSVe||SOP VIR 005|
|Isolation and characterization of Newcastle disease viruses using SPF embryonated chicken eggs and haemagglutination inhibition test (Released on 12/10/21)||IZSVe||SOP VIR 007|
|Influenza – Neuraminidase Inhibition Test||APHA|
|Detection of antibodies to type A influenza virus by agar gel immunodiffusion assay (AGID) (Released on 30/12/20)||IZSVe||SOP IMM 063|
|Detection of subtype-specific antibodies to Avian influenza virus by haemagglutination inhibition test (Released on 29/12/20)||IZSVe||SOP IMM 064|
|Detection of antibodies to Newcastle disease virus by haemagglutination inhibition test (Released on 30/12/20)||IZSVe||SOP IMM 065|
|Sample preparation and nucleic acids isolation for the detection and typing of Avian influenza virus and Avian Orthoavulavirus type 1 by molecular methods (Released on 12/10/21)||IZSVe||SOP VIR 1000|
|Detection of type A Avian influenza virus by real-time RT-PCR (Heine et al., 2015) (Released on 09/04/21)||IZSVe||SOP VIR 018|
|Detection of type A influenza virus by real-time RT-PCR (Nagy et al., 2021) (Released on 07/04/21)||IZSVe||SOP VIR 1003|
|Detection of influenza A matrix gene by real-time Taqman® RTPCR||APHA|
|Detection of Eurasian H5 Avian influenza virus by real-time RT-PCR (Slomka et al., 2007) (Released on 09/04/21)||IZSVe||SOP VIR 143|
|Detection of Eurasian H5 Avian influenza virus by One-step RT-PCR and Sanger sequencing of the hemagglutinin cleavage site (Slomka et al., 2007) (Released on 21/02/22)||IZSVe||SOP VIR 125|
|Detection of Eurasian H7 Avian influenza virus by real-time RT-PCR (Slomka et al., 2009) (Released on 09/04/21)||IZSVe||SOP VIR 144|
|Detection of Eurasian H7 Avian influenza virus by real-time RT-PCR (Van Borm et al., 2010 modified) (Released on 07/04/21)||IZSVe||SOP VIR 1001|
|Detection of Eurasian H7 Avian influenza virus by One-step RT-PCR and Sanger sequencing of the hemagglutinin cleavage site (Slomka et al., 2007) (Released on 21/02/22)||IZSVe||SOP VIR 126|
|Detection of H9 Avian influenza virus by real-time RT-PCR (Panzarin et al., 2022)(Released on 17/06/22)||IZSVe||SOP VIR 014|
|HA and NA subtyping of Avian influenza virus by real-time RT-PCR (Hoffmann et al., 2016; James et al., 2018) (Released on 08/04/21)||IZSVe||SOP VIR 1004|
|Avian Influenza virus (AIV) typing by reverse transcription PCR using a universal primer set (Released on 06/19)||IZSVe|
|Detection of Avian Orthoavulavius type 1 (AOAV-1) by real-time RT-PCR (Sutton et al., 2019) (Released on 09/04/21)||IZSVe||SOP VIR 151|
|Molecular pathotyping of Avian Avulavirus type 1 by real time PCR||APHA|
|Detection and pathotyping of Avian Orthoavulavirus type 1 (AOAV-1) by one-step RT-PCR and Sanger sequencing of the fusion protein cleavage site (De Battisti et al., 2013) (Released on 09/04/21)||IZSVe||SOP VIR 063|
|Detection and pathotyping of Avian Orthoavulavirus type 1 (AOAV-1) by one-step RT-PCR and Sanger sequencing of the fusion protein cleavage site (Kant et al., 1997) (Released on 07/04/21)||IZSVe||SOP VIR 1002|