Diagnostic protocols

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 OIE 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.

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:

  1. A procedure based on a method acquired directly from the EURL for AI/ND;
  2. A method whose sensitivity and specificity have been demonstrated as being effective following a comparative test organized by the EURL for AI/ND;
  3. 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.

Protocol Laboratory Download
Isolation and characterization of Avian influenza viruses using SPF embryonated chicken eggs and haemagglutination inhibition test IZSVe SOP VIR 005
Isolation and characterization of Newcastle disease viruses using SPF embryonated chicken eggs and haemagglutination inhibition test IZSVe SOP VIR 007
Influenza – Neuraminidase Inhibition Test APHA PDF
Detection of antibodies to type A influenza virus by agar gel immunodiffusion assay (AGID) IZSVe SOP IMM 063
Detection of subtype-specific antibodies to Avian influenza virus by haemagglutination inhibition test IZSVe SOP IMM 064
Detection of antibodies to Newcastle disease virus by haemagglutination inhibition test 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 IZSVe SOP VIR 1000
Detection of type A Avian influenza virus by real-time RT-PCR (Heine et al., 2015) IZSVe SOP VIR 018
Detection of type A influenza virus by real-time RT-PCR (Nagy et al., 2021) IZSVe SOP VIR 1003
Detection of influenza A matrix gene by real-time Taqman® RTPCR APHA PDF
Detection of Eurasian H5 Avian influenza virus by real-time RT-PCR (Slomka et al., 2007) 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) IZSVe SOP VIR 125
Detection of Eurasian H7 Avian influenza virus by real-time RT-PCR (Slomka et al., 2009) IZSVe SOP VIR 144
Detection of Eurasian H7 Avian influenza virus by real-time RT-PCR (Van Borm et al., 2010 modified) 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) IZSVe SOP VIR 126
Detection of H9 Avian influenza virus by real-time RT-PCR (Monne et al., 2008) 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) IZSVe SOP VIR 1004
Avian Influenza virus (AIV) typing by reverse transcription PCR using a universal primer set IZSVe PDF
Detection of Avian Orthoavulavius type 1 (AOAV-1) by real-time RT-PCR (Sutton et al., 2019) IZSVe SOP VIR 151
Molecular pathotyping of Avian Avulavirus type 1 by real time PCR APHA PDF
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) 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) IZSVe SOP VIR 1002