Following the first occurrence of highly pathogenic

avian influenza (HPAI) in the human population in 1997, the migratory pathways of wild birds have become a topic of growing interest. By using nuclear technologies — specifically, stable isotope analyses — it is possible to trace the origin of individual birds and to identify their migration patterns during a specific period. Nuclear technologies can also be used for early and rapid diagnosis of avian influenza, to trace the origin of the virus or the avian carrier, and to identify traits related to resistance to the virus. The IAEA is providing support in these areas through regional technical cooperation project RER/5/015, Supporting Early Warning and Surveillance of Avian Influenza Infection in Wild and Domestic Birds and Assessing Genetic Markers for Bird Resistance.

On the trail of avian influenza: Using nuclear technologies to support early warning and surveillance

counterpart laboratories were matrix gene detection, and H5 and H7 subtype differentiation (as the last two are notifible for the World Organisation for Animal Health – OIE). Additionally, the counterparts were trained to differentiate high pathogenic from low pathogenic subtypes of the AI viruses using translation of the haemaglutinine (HA) genome sequences to amino acid sequences.

Resistance trials were conducted in which Mx gene sequences in domestic poultry from different countries were analysed to determine if there are differences between them. The secrets of the Mx gene may contribute to the development of chicken lines resistant to AI.

With stable isotope analyses, it is possible to trace the origin of individual birds by measuring the ratio of one or more stable isotopes in the feathers or beaks of migratory birds. This is because diverse geographical regions have different, but relatively constant, stable isotope patterns. The isotopes build up in a bird depending on its food and water intake, and can be measured to give important information on the bird's origin and path of migration. Moreover, as the feathers and beak are constantly growing, sequential measurement can provide information on where the particular bird has been during a certain period. The stable isotopes most frequently used as tracers are

2 13 15hydrogen ( H), carbon ( C), nitrogen ( N), oxygen 18 34 87 208( O), sulphur ( S), strontium ( Sr) and lead ( Pb,

207 206Pb and Pb).

The main goal of the project was to harmonize the diagnostic procedures for both detection and characterization of AI viruses. The methods of detection to implement/reinforce in the counterpart

The project aims to: (i) improve the early and rapid diagnosis of avian influenza (AI); (ii) assess genetic markers to identify advantageous traits related to bird resistance using nuclear related, molecular technologies; and (iii) use stable isotope analysis for tracing the migratory movements of waterfowl that might be involved in the spread of AI.

Tracking migratory movements

Diagnosing avian influenza

Identifying virus resistance traits

Fellowships and trainings

To ensure sustainable improvement in the early and

rapid diagnosis of AI, it was essential that each

laboratory participating in this technical

cooperation project have staff that are adequately

trained in the relevant technologies. The project

therefore focused on providing individual fellowship

training and group training courses. A first

coordination and planning meeting took place

26–30 January 2009 in Vienna, during which

counterparts defined a detailed work plan for the

project. Eighteen participants from 15 countries in

Europe and Asia (Albania, Armenia, Austria, Bosnia

and Herzegovina, Bulgaria, Croatia, Greece,

Hungary, Kazakhstan, Montenegro, Republic of

Moldova, Republic of Serbia, Romania, Russian

Federation and The Former Yugoslav Republic of

Macedonia) took part.

Practical classes during the training courses in

the IAEA Laboratory in Seibersdorf.

Under the project, five fellowships were awarded to two participants from Armenia, two from Bulgaria and one from Croatia to attend a course on ‘Theoretical and Practical Training on Early and Rapid Molecular Diagnostic Technologies, with Special Focus on Poultry Diseases’ held at the Federal Centre for Animal Health in Vladimir, Russian Federation. In addition, three regional training courses were organized at the Animal Production and Health Laboratory, Seibersdorf, Austria, to address two key elements in the project: the use of technologies to enable the early and rapid diagnosis of AI; and the value of genetic markers in identifying advantageous traits related to bird resistance to the disease.

As a result of the fellowships and training courses, a significant number of trained scientists are now capable of performing the whole diagnostic cycle for detection and identification of the AI viruses, using molecular diagnostic methods. These methods include conventional RT-PCR, real time RT-PCR, LAMP PCR, DNA sequencing of the HA gene and its translation into an amino acid sequence. The interpretation of the amino acid sequence is particularly important, as it enables the scientists to discriminate between low and high pathogenic AI viruses (as described in appropriate OIE standards).

The Animal Production and Health Section of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture assists Member States to improve livestock productivity through the efficient use of locally available feed resources, adequate management practices, and efficient reproductive and breeding programmes, and helps in the development and transfer of proactive disease prevention and control measures using nuclear and nuclear-related technologies.

For more information please contact: Animal Production and Health Section IAEA Vienna International Centre, P.O. Box 100 1400 Vienna, Austria www.iaea.org

The first regional training course, on genomic DNA preparation, microsatel l ite analyses and sequencing, took place 7–18 December 2009, and was attended by 19 participants from 14 countries. The second regional training course, on animal genetics in bioinformatics tools and microsatellite analyses and sequencing, took place 22 November–3 December 2010, and brought together 15 participants from 11 countries. The third regional training course, on ‘Animal Health in Molecular Diagnosis, Genotyping and Phylogenetic Analyses of Avian Influenza (Bird Flu) and Other Mammalian Influenza A Subtypes’, was held 20 September–1 October 2010, with the involvement of 13 participants from 10 countries.

To ensure a common standard of technical capacity and to support harmonization of results, quality control in the different laboratories was an important target of the diagnostic component of the project. The status of each individual laboratory in terms of its implementation of ISO 17025 Standard and its incorporation of the molecular methods for detection of AI (matrix gene, H5 and H7 protocols for detection of AI viruses) is currently being evaluated.

The evaluation of the microsatellite markers associated with bird resistance was carried out in only three of the counterpart countries: Bulgaria, Greece and Republic of Moldova. However, collection of blood samples has begun and optimization of the analytical protocol is in progress. Supported by the project, stable isotopes have been analysed at the Austrian Institute of Technology GmbH-AIT, Seibersdorf. Samples from Albania, Bulgaria and Mongolia have been analysed for content and ratios of hydrogen, carbon, sulphur and nitrogen isotopes. Only the samples from Mongolia have shown a pattern of very high, high and low altitudes of bird migration, while the Albanian and Bulgarian samples have indicated a pattern of local bird origin.

Trainees in the first training course in front of

the IAEA Laboratory in Seibersdorf.