FLUTEST - Project Objectives

The objective of this project is to develop and validate laboratory tests that can be used as tools in early warning systems and surveillance programmes for avian influenza (AI), in the presence and absence of vaccination. Protocols will be harmonised and applicable to surveillance of wild birds and different compartments of the poultry industry. AI is a highly contagious trans-boundary animal disease (TAD), able to spread in a susceptible population in a short period of time. Therefore, the prompt identification of infected animals is crucial for control and eradication purposes. Diagnostic tests must be appropriate for the setting in which they will be used, be properly validated and ‘fit for purpose’.

FLUTEST project has two collaborative and complementary strands focused on addressing the main objectives of the FP6-2005-SSP-5B-Influenza Call, namely:

1. Evaluation and development of suitable validated methods for the diagnosis of avian influenza, in the presence and absence of vaccination (including the ‘DIVA’ vaccination strategy).

2. Evaluation and development of appropriate surveillance systems and risk-based models for avian influenza with the application of diagnostic tools in surveillance and early-warning systems for AI.

The research programme outlined comprises a matrix of six, themed and synergistic Work Packages (WP), each managed by a WP leader. The nature of the project also places a strong emphasis on horizontal interactions, both within Task 2, FLUTEST, itself and also with the other Tasks elaborated in the SSP-5B-Influenza Call. This approach favours maximum and timely integration, exchange and dissemination of methodologies, data, know-how and protocols and provides for high-level complementarities between the WP’s and Call Tasks.

Work Package 1: Task Coordination & Logistics

Has been established to achieve maximum integration between WP’s in this project. Also to ensure strong interaction and coordination with other EU projects that have outputs relevant to this task e.g. AVIFLU, FLUAID, EPIZONE, ESNIP2, LAB-ON-SITE, Healthy Poultry and PORTCHECK, and also with other projects selected within this call, FLUPATH and FLURESIST in order not to duplicate activities and to seek maximum synergies. In addition, this work package will coordinate interactions between EU partners and partners from the U.S.A who have been invited to contribute to the project because of their wealth of experience in AI surveillance and outbreak management.

Work Package 2: Establish comprehensive, harmonised, validated AI surveillance, ‘early warning’ and diagnosis protocols

Based on an intelligent framework, spanning different poultry populations and industry sectors in the EU. This will provide an appropriate surveillance system model for the EU, with capabilities extending beyond the immediate remit of establishing optimal surveillance strategies. Such a dynamic surveillance model can also be utilised to provide decision-support mechanisms for disease control policies and enhanced analysis of novel, emerging test technologies and emerging threats or variables that may be encountered in the future. Deliverables for this WP will include a report describing the quality of the current EU surveillance programme and blueprints for improvement of surveillance programmes. This will be achieved through a step-wise process of information gathering and analysis. A description of the poultry population and production sector demographics across European Union Member States will be provided and the average time between virus introduction and detection and the average number of infected flocks at the time of detection, given the current EU surveillance programme, will be established. Then we will determine the influence that factors such as vaccination, sensitivity and specificity of the test, sample sizes, sampling interval and frequency of clinical inspection have on these parameters. In addition, factors such as the efficient capture and use of data in the face of an outbreak and the optimum distribution of resources for surveillance and for action on disclosure of the first detected case will be evaluated. This will enable us to establish the optimal lay-out of surveillance programmes, developed “fit-for-purpose” for different conditions between EU-member states, using different tests available and/or developed in the other WP’s.

Work Package 3: Molecular diagnostics, antigen detection and novel technologies.

It will build upon the outputs from AVIFLU (EU funded project contract QLRT-CT2002-01454), FLUAID and LAB-ON-SITE (SSP3-513 645: “New and emerging technologies: improved laboratory and on-site detection of OIE List A viruses in animals and animal products.”) in which a wide range of rapid, high throughput, novel diagnostic assays were developed. The assays are being standardised following the five steps of OIE validation, harmonisation, national and international ring-tests, as it is shown on the homepages of the project: http://www.labonsite.com/. FLUTEST will extend this work by evaluating and further developing novel molecular based tests; for example Loop-mediated isothermal amplification (LAMP), that may be applied under field conditions or in simply equipped laboratories, novel real-time RT-PCR chemistries and rapid sequencing. The purpose is to focus efforts on the development, evaluation, application and harmonisation of these novel molecular techniques, for detection and differential diagnosis of AI virus infections in domestic and free-living avian populations. The assays will be internationally standardised and validated, by following the OIE rules of assay validation, in order to harmonise and standardise the diagnosis of avian influenza across the EU and to provide test to the standard specified in the EU diagnostic manual. Where appropriate the assays developed will be transferred to portable formats, trialled between partners and ‘in field’.

Work Package 4: Serological surveillance for AI

Experience has shown that introduction of HPAI strains to poultry may not be reported immediately and the situation is exacerbated for LPAI strains. Serological surveillance programs are not primarily suited to detect an infected flock at a very early stage based on the time lag for detectable seroconversion to occur following exposure and challenge. However, a high-test frequency can counter this disadvantage. Using blood samples at high frequency of testing is unpractical and not cost-effective, but the measurement of AI antibodies in egg yolk could offer many advantages. A laboratory test to detect AI antibodies in eggs will be developed, validated and automated to make it cost-effective for use in surveillance programmes. At present there is no robust scientific data reported in the scientific literature regarding the serological response of Ratites, Anseriformes, or other waterfowl, following infection with HPAI viruses. Equally there are no validated diagnostic tests for this purpose; hence sensitivity and specificity values are not available from published literature. Such technical gaps must be filled as quickly as possible to improve the efficiency of surveillance measures. A profile for seroconversion and protective antibody levels will be generated experimentally in ducks and a serum bank will be established with specimens collected in AI outbreak areas from domestic and wild Anseriformes, Charadriiformes, Ratites and other species where possible. These sera will be used to assess and compare a range of serological tests (e.g. HI, VNT, ELISA, blocking LFD) with the aim of making recommendations with regard to the most appropriate test methodologies for these species that form significant compartments for the introduction and ecology of influenza viruses in avian species. Many laboratories experience difficulties when subtyping AI isolates with success, influenced by the reagents and the expertise required interpreting the test results. In an extension of work in FLUAID (QLRT-CT2001-01454/SSPE-022417: ‘Generation of information and tools to support the management of the avian influenza crisis in poultry’), a competitive lateral flow device (LFD) will be developed that will subtype the virus isolates. This will involve the use of recombinant antigens and phage display antibodies. An LFD capable of detecting virus specific antibodies will be developed in conjunction with an antigen sub-typing LFD, as both benefit from utilisation of the same recombinant antigens/peptides can be used for this purpose. This test could have applications for DIVA surveillance when vaccination strategies are employed.

Work Package 5: Innovative technologies for there applicability as avian influenza diagnostic tests.

DNA microarrays are powerful tools for the detection and comprehensive characterisation of viruses. They enable multi-agent detection and hence differential diagnosis. However, the optical detection is expensive and difficult to implement in portable instrumentation. An AI biosensor based on microarrays but using label-free electrical detection of DNA hybridisation and a novel sensing mechanism employing low-temperature polycrystalline silicon-thin film transistors (LTPS-TFTs) technology will be developed. The advantages of this technology are cost, €2-3 per device, portability and rapid results, typically in less than 15 minutes. Thus it would be ideal for field use and point-of-care diagnosis By working with the model developed in WP1 a set of minimum performance standards will be developed by which to measure the efficacy and suitability of diagnostic tests and their application in different settings e.g. surveillance of wild birds or poultry, before, during and after an outbreak of H5 or H7 influenza virus (either HPAI or LPAI), or in the face of a vaccination strategy. As well as the tests developed within this project, AVIFLU and FLUAID, commercial kits will also be measured against these standards. A further key aspect will be the transfer of technology to end-users. This will be achieved through links with projects supported under task 6 (FLU-LAB-NET) and task 7 (FLUTRAIN).

Work Package 6: Foster collaborations

Foster collaborative interactions between EU partners and INCO or non-EU partners who have been invited to contribute to the project because of their wealth of experience in AI surveillance and outbreak management, expertise in environmental testing or access to material not available to EU partners. This will enable the sharing of reagents and field material as well as expertise and it will enable the groups to share and coordinate research results so that unnecessary duplication of effort is avoided.