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WP 1 PROJECT MANAGEMENT

OBJETIVES.

  1. To manage the project administratively and financially.
  2. To secure a robust consortium agreement with special emphasis on IPR issues and results commercial exploitation.
  3. To manage the communication among the consortium partners.
  4. To manage the communication with the EC.
  5. To follow up the Project Results Dissemination and Exploitation.

DESCRIPTION OF WORK

Leader: Partner 1. P1 has experience in the project management at national and international level.

The Coordination of the project will be shared by the Coordinator (Partner 1) and the Scientific Coordinator (Partner 2). They will manage the project, including the Consortium Agreement, Ethical, Gender, Financial and Administrative Management. The Steering Committee, integrated by the Partner Leaders and Coordinators, will help to the Coordinators on their decisions.

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WP 2 Real-time monitoring livestock on line

Objectives

Within this WP, an early warning system for animal diseases will be developed based on a computer-aided, non-invasive real-time monitoring system-on line (RTMS-ON). The aim is to detect and thus limit infections at the earliest possible time point through parameters such as body temperature, food intake and water consumption.
As non-invasive tools for assessment of body temperature, infrared thermography and thermal chips will be implemented. The aims will be achieved through five tasks:

  1. Development of a prototype monitoring system.
  2. Implementation of the prototype in a BSL3 laboratory.
  3. Establishment of baseline parameters and use with experimentally infected animals.
  4. Adaptation and establishment of the RTMS-ON in sentinel farms.
  5. Monitoring of sentinel farms with RTMS-ON.

Description of work and role of partners

Leader: Partner 8. P8 has more than 30 years of experience in the field and P4 has very intensive connection with large-scale industrial poultry and swine units and is able to work as a sentinel observation station.
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WP 3 Collection and preparation of samples

Objectives

  1. To develop simple-to-use methods that can be used in the field or in simple field laboratories for the collection, pre-assay processing and storage of diagnostic samples. Where possible, the approaches developed will be homogeneous and generic ensuring that they have broadest application across the wide range of different biological sample types, veterinary species and diseases targeted by RAPIDIA-FIELD.
  2. Evaluation of the developed simple methods.
  3. To establish a virtual database of samples that can be shared amongst the project consortium for assay validation purposes for the selected pathogens.


Description of work and role of partners

Leader: Partner 6. P6 has previous experience in the establishment of data base for biological collections. All participant partners have capability of collecting samples and developing new protocols for the preparation of samples.

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WP 4 Antibody detecttion in the field

Objectives

The aim of this WP is to develop assays able to detect antibodies to multiple pathogens in the field. The main characteristics of these assays will be: user-friendly performance, sensitivity, specificity and low costs. Here, mainly LFDs or related test systems will be in the focus.

The goals will be achieved through the following tasks:

  1. Development of a duplex LFD for detection of BTV and EHDV antibodies
  2. Development of a duplex LFD for detection of ASFV and CSFV antibodies

Description of work and role of partners

Leader: Partner 1. P1 is an SME with more than 25 years of experience in the development of diagnostic assays in veterinary field. P1 has also proved expertise in the production of the necessary reagents for the aforementioned development, such as recombinant antigens expressed in heterologous systems (E.coli or baculovirus) and monoclonal antibodies. Most of the reagents are already available among the partners (P1, P3 and P9). This fact will facilitate the progress of the proposed tasks and will permit to set new product on the market.

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WP 5 Pathogen detection in the field

Objectives

The aim of this WP is to develop rapid multiplex methods for field diagnostic settings such as low-cost field detection devices, which will provide diagnosis with proper preservation of diagnostic sensitivity and specificity/discrimination. The specific objectives are the following:

  1. To adapt and implement a novel amplification technology on portable PCR systems that was recently developed and patented at SVA for AIV and NDV pathotyping in the field application.
  2. To develop multiplex LAMP assays for field detection of three swine disease clusters: vesicular diseases, various pestivirus infections, and hemorrhagic diseases.
  3. To adapt the LFD and LFD-COMB technology for the detection of nucleic acid targets amplified by PCR or LAMP in the above two subtasks.
  4. To develop multiplex LFD for the detection and differentiation of orbiviruses.
  5. To adapt a RT-PCR assay developed at IAH for FMDV to use on the Enigma FL instrument and conduct a validation/equivalence study.

Description of work and role of partners Leader: Partner 10. P10 has a broad expertise in diagnostic in the field and has been leader in many other European projects. P1, 7 and 10 will provide reagents for the development of LFD assays. P2, P6, P9 P10 and p12 will participated on development of molecular techniques.

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WP 6 Antibodies detection in small field labs

Objectives

The aim of this WP is to develop innovative multiplex serology for the detection and quantification of antibodies to multiple pathogens in small field laboratories. The goal will be achieved through the following specific tasks:

  1. Development of a prototype multiplex LFD-Comb for the detection of ASFV/CSFV antibodies
  2. Development of a Q-ELISA test for the detection of companion animal diseases (CPV, CDV, FeLV/FCV)
  3. Development of a quantitative LFD for BTV and EHDV antibodies
  4. Utilization of low density antigen microarray technology for the detection of antibodies against multiple pathogens
  5. Description of work and role of partners Leader: Partner 3. P3 is an SME with experience in the development of diagnosis assays. Similarly to WP4, most of the reagents are available and will be provided by the other partners. This will allow a prompt starting on the different tasks.
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WP 7 Pathogen detection in small field lab

Objectives

The overall objective of this WP is the adaptation and validation of multiplex assays for simple and reliable pathogen detection in small field laboratories. Both direct genome and antigen detection methods will be employed. Moreover, methods that allow for basic typing of selected viruses will be tested.

The following diseases have been selected as the most important and reasonable for this working package:

  1. Cattle cluster “vesicular diseases” simultaneous detection and differentiation of BTV/EHDV/FMDV and BVDV
  2. Pig cluster respiratory and hemorrhagic diseases simultaneous detection and differentiation of PRRSV, PCV2, ASFV, and CSFV
  3. Chicken cluster “fowl plague” simultaneous detection and differentiation of AIV and NDV
  4. Honey bee virus cluster

All disease clusters combine important notifiable, but also endemic diseases, and take especially the importance of differential diagnosis in screening programs and outbreak situations into consideration. For some methodologies, restrictions to certain pathogens will apply.

Based on capacity considerations, the methods will be divided into direct genome detection methods (FLI leader)
and techniques for direct antigen detection (INTA leader)

In detail, the following tasks will be executed:

  1. Evaluation, adaptation and validation of direct genome detection methods including necessary extraction steps utilizing low density DNA chip systems, and LAMP assays.
  2. Development and validation of direct antigen detection methods using low density microarrays, digitally-encoded micro-particles, multiplex antigen-ELISA, Q-ELISA, and AlphaLISA technology.
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WP8 Confirmation and reference techniques

Objectives

The overall objective of this WP is to develop and implement techniques for confirmatory reasons and as a basis
for adaptations especially of molecular tools.

The detailed aims of this Work package are the following:

  1. To develop high technology strategies that can be applied to confirm the status of a sample irrespective of gold standard technologies, without prior knowledge. At the genome detection level: identification of sample status without prior knowledge. Methods will include sequencing (with sequence independent access methods, including next generation sequencing with 454/Roche technology) and highly multiplexed genome detection methods including microarray systems. These technologies will be of use in diagnostically difficult situations like (re-)emergence of unexpected pathogens or pathogen variants (including “diagnostic escape mutants”).
  2. To improve our sequence knowledge with aim to improve test design and evaluation. To achieve this: development of more efficient and economic methodologies to sequence viral genomes (“towards the 100 euro viral genome?”) and their application to generate sequence information. Economic and efficient access to sequence information will enhance on the long term the available knowledge in public databases needed for efficient design and evaluation of molecular diagnostics.
  3. To use multiplex immunoassay for viral diagnosis with a Luminex platform for multiplexed Ab detection. The design of the method here would not be sequence/pathogen independent. The available reagents (antigens, monoclonal antibodies) will be collected from the partners and used to develop the confirmatory technology.

Multiplex immunoassay for viral diagnosis (e.g. FMDV, BTV, West Nile Virus) by using the Luminex liquid array technology will be developed/tested and used as confirmatory technology. This assay will allow e.g. the simultaneous detection of antibodies or antigens, in a single reaction, to multiple viral antigens coupled to microspheres labelled with different proportions of fluorescent dyes.

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WP 9 Standardization

Objectives

Newly developed diagnostic tests have to fulfil the requirements laid down in the “Manual of Standards for Diagnostic Tests and Vaccines” (OIE, 2008). Therefore, a first estimation of analytical and diagnostic sensitivity and specificity as well as repeatability will be done for each test by the partners during test development. For this purpose they will use a set of own samples. Once the tests have passed this first step of quality control their further evaluation will be done by a Proficiency Test (PT) organized by partner 9 in close cooperation with the partners of the FNG and WP3.

The partners in charge of the development of the assays will educate the members of the field net group how to use the tests.

The aim of this PT is to demonstrate the sensitivity and specificity as well as repeatability and robustness of the tests in other laboratories than the developing laboratory. Where necessary the results will be used to improve the tests. The PT will be organized according to the ISO 17043. The PT provider will put all the results of all participants in their central Laboratory Integrated Management System (LIMS). Also the participants are asked (via clear instructions: see subtask 9.2) to put their results and to report to the PT provider via their LIMS.

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WP 10 Dissemination of results

Objectives

The aim of this WP is the dissemination of the objectives of this project and their results through publication and presentation in scientific magazines, at congresses, symposia, forums, and through direct contact with government agencies and international agencies of animal health (OIE, FAO, DG SANCO, and European CVO) and animal producers.