On the trail of avian influenza: using nuclear technology to support early warning and surveillance

Following the first occurrence of highly pathogenic avian influenza (AI) in the human population in 1997, the migratory pathways of wild birds have become a topic of growing interest. Using nuclear technology – specifically, stable isotopes analyses – it is possible to trace the origin of individual birds and to identify their migration patterns during a specific period. Nuclear technology can also be used for early and rapid diagnosis of avian influenza, and to identify traits related to resistance to the virus. The IAEA is providing support to Member States 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’. The project aims to (i) improve the early and rapid diagnosis of 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

With stable isotopes 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 the beak 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 hydrogen (2H), carbon (13C), nitrogen (15N), oxygen (18O), sulphur (34S), strontium (87Sr) and lead (208Pb, 207Pb and 206Pb).

Diagnosing avian influenza

The main goal of the project was to harmonize the diagnostic procedures in both, detection and characterization of AI viruses. The methods of detection to implement/reinforce in the counterpart laboratories were matrix gene detection, H5 and H7 subtype differentiation (as the last two are notifiable for The World Organisation for Animal Health (OIE)). Additionally, the counterparts were trained to differentiate highly pathogenic from low pathogenic subtypes of the AI viruses using translation of the haemaglutinine (HA) genome sequences to amino-acid sequences.

Identifying virus resistance traits

Resistance trials were conducted to determine if there are differences between the Mx gene sequences in the domestic poultry from different countries by analysing the genome sequence of the Mx gene of domestic poultry samples collected from different countries. This should provide answers why some chickens are more susceptible and other are less susceptible to the AI virus. The secrets of the Mx gene may contribute to development of chicken lines resistant to AI.

Fellowships and trainings under RER/5/015

To ensure a sustainable improvement in the early and rapid diagnosis of AI, it was essential that each laboratory participating in RER/5/015 have staff that are adequately trained in the relevant technologies. On the trail of avian influenza: using nuclear technology to support early warning and surveillance The project therefore focused on providing TC funded individual fellowship trainings and group training courses. A first coordination and planning meeting took place 26–30 January 2009 in Vienna, during which the countries participating in the country defined a detailed work plan for the project. Eighteen participants from 15 countries of the Balkans and Asia (Albania, Armenia, Austria, Bosnia and Herzegovina, Bulgaria, Croatia, Greece, Hungary, Kazakhstan, Macedonia, Moldova, Montenegro, Romania, Russia and Serbia) took part.

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, Russia. In addition, three regional training courses were organized at the Animal Production and Health Laboratory, Seibersdorf, Austria, to address two key elements in RER/5/015: 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. The lecture courses and the practical sessions were provided by invited international lecturers and by experts based at the Seibersdorf laboratory.

On the trail of avian influenza: using nuclear technology to support early warning and surveillance The first regional training course, on ‘Genomic DNA preparation, microsatellite 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.

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 to 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).

On the trail of avian influenza: using nuclear technology to support early warning and surveillance To ensure a common standard of technical capacity and to support results harmonization, quality control in the different laboratories was an important target of the diagnostic component of RER/5/015. The status of each individual laboratory, in terms of its implementation of ISO 17025 standards and its incorporation of the molecular methods for detection AI (Matrix gene, H5 and H7 protocols for detection of AI viruses) is currently being evaluated. The evaluation of this activity is still in progress and will be published upon reception of the status of each participating country/laboratory.

The evaluation of the microsatellite markers associated with bird resistance was carried out in only three of the counterpart countries; Bulgaria, Greece and Moldova. However, collection of blood samples has begun and optimization of the analytical protocol is in progress. Supported through RER/5/015, 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 Albanian and Bulgarian samples have indicated a pattern of local bird origin.