Use of Stable Isotopes to Trace Bird Migrations and Molecular Nuclear Techniques to Investigate the Epidemiology and Ecology of the Highly Pathogenic Avian Influenza

Rationale and Background:

Integrating information on wild waterfowl migration through stable isotope analysis and detecting avian influenza viruses in faecal and environmental samples will contribute to understanding the epidemiology and ecology of the long range transmission of avian influenza viruses, using non-invasive methods.

Background Situation Analysis

Influenza is one of the most common infectious diseases in animals and man. There are three genera, two of which, Influenza Types B and C, are predominantly human pathogens, while the third, Influenza A occur in domesticated animal species including pigs, horses and poultry and a wide variety of wild birds as well as humans. Over 100 species of wild migratory birds, particularly ducks, swans, geese and various wading birds, harbor AIVs. Infections are transmitted amongst WWF by shedding of the virus and faecal contamination of water. Most of the influenza strains found in wild birds are of low pathogenicity producing only mild disease in domesticated birds, whilst wild birds are unlikely to become sick. What has been of considerable concern over the last few years is the emergence of a highly pathogenic strains of Influenza A virus in birds. AIVs threaten animal health, livestock productivity and food security in poor countries, but they can also evolve into dangerous human pathogens. This has been seen with the emergence of HPAI.

Its main impact has been on domesticated poultry, with over 300 million birds killed or destroyed, but in addition, a number of fatal, human infections have also occurred. In March 2010, the International Scientific Task Force on AI and Wild Birds reported that waning attention to HPAI was reducing opportunities for surveillance and research, thereby affecting efforts to understand the epidemiology of the disease. The disease continues to be a major problem in Egypt and parts of Asia and recently outbreaks have occurred in poultry in Romania and in wild birds in Russia, China and Mongolia. Among several important issues that need addressing is the need to improve knowledge of the role WWF populations might play in the dissemination of infection. Tracing the movements of WWF in relation to where they originated as well as their stopover points in their migration between breeding and non-breeding grounds is a particularly challenging task.

There has been some progress in this field by using satellite tracking, geo-locators or external markers of WWF, but these procedures have only been applied to relatively few species and individuals, and it will be necessary to utilize methods that can be used on a larger scale and not biased to initial capture location if we are to fully comprehend the role of migratory birds in the spread of avian influenza. A suitable technique that has already been used to trace migrants is based on the SI signatures of tissues of birds, especially those in feathers. Certain SIs are involved in important biological and ecological processes and there is a strong correlation between levels of these SIs in the environment and the concentration of the same SIs in avian tissues. Of most interest are deuterium (δD) ratios in tissues that reflect those in surface (lakes, rivers, oceans) and in ground waters. Since hydrogen isotope composition of environmental water varies spatially across the globe in a predictable manner and its presence relayed to animal tissues, δD analyses of animal tissues provides a way of linking SIA data on water isoscapes with those in biological tissues such as feathers. These SIA data have been shown to successfully reveal migration patterns and enable identification of the breeding areas of birds sampled at non-breeding grounds and disease outbreak sites.

The measurement of naturally occurring SIs in avian tissues to provide information on origins of individuals is based on matching knowledge of spatial isotopic patterns in foodwebs (isoscapes) with those in tissues such as feathers. In North America and Europe, deuterium feather isotope measurements reflect those expected from the long-term IAEA GNIP dataset. By combining SI measurements of bird tissues and assays of AI and other pathogens, it will be possible to investigate spatial patterns of transmission. For Africa and Asia, correlations between the GNIP precipitation deuterium isoscape and avian tissues have not been established. This CRP will calibrate the feather-precipitation δD relationship for these two continents for WWF and provide a critical tool in the use of an isotopic forensic tool to investigate AI transmission involving wild birds. This will be accomplished by measuring waterfowl tissues known to be grown at specific sites along their flyway.

To date, through efforts of FAO and partners, over 2500 waterbird feather samples have been collected for SIA in Asia and Africa. Approximately 200 samples will be analyzed in the USA with partner labs associated with Oklahoma University and USGS. An additional 400 samples will be analyzed in Queens University, Belfast. Sixteen bar-headed geese (3 feathers per bird) have already been analysed in Austria by the Austrian Institute of Technology. This series of sample analyses from previously collected feathers will contribute to the project by starting the assessment of “where waterfowl have come from”, and by focusing on opportunistic sampling conducted in the context of the FAO HPAI field surveillance programme. For historical analyses of samples, analysis of 35 samples from the same species, over the time period when they are arriving to their overwintering site, is the goal. Analyses should focus on δD measurements of feathers of known moult chronology and potentially claws. Analysis of faecal samples to simultaneously detect the bird species and the carrier status of particular bird using non-invasive analysis of faecal samples will generate the epidemiological link between migration pathways (obtained by SIA) and the transmission of the virus to certain geographical area. Fecal samples should be collected randomly at the same sites where feathers are collected. The samples will undergo two test procedures:

DNA barcoding (species identification), has been developed at the Avian Disease Laboratory; College of Veterinary Medicine; Konkuk University; 1 Hwayang-dong, Gwangjin-gu; Seoul 143-701; Republic of Korea (Dr. Dong-Hun Lee). The technique is based on detection of short gene sequence from a standardized region of the genome as a diagnostic “biomarker” for species. The target sequence has been the 648-bp region of the mitochondrial gene, cytochrome C oxidase I (COI), already optimized as a DNA barcode for the identification of bird species. The optimization of a DNA barcoding technique for faecal samples has been performed by comparing DNA from the faecal samples with the DNA from tissue samples (muscle, feather, and blood) from already known bird species (domestic poultry and WWF), collected from live-bird markets, Conservation Genome Resource Bank for Korean Wildlife, and from the Seoul Grand Park Zoo. The results of bird species identification, using COI gene sequences from tissues matched the fecal samples of the same individuals.

Detection of the AIV in the fecal samples using optimized protocol in five phases: i) detection of M gene to detect the presence of influenza A viruses using PCR technique (Positive samples should be inoculated in SPF eggs for virus isolation), ii) Positive samples should be tested using H5 or H7 protocol on PCR, iii) H5 and H7 positive samples should undergo molecular pathotyping (cleavage site sequencing), iv) M gene positive and H5 and H7 negative, should be further typed in order to differentiate the subtype using conventional (HI-test) and/or molecular methods, v) Test all the positive samples and a portion of negatives using LAMP PCR protocol.

The main pathway of AIV transmission is faecal contamination. Natural water reservoirs are the media where WWF faeces is excreted in the water and contaminating it randomly. However, the survival of the AIV in the natural water reservoirs depends on numerous environmental, physical and chemical influences, as well as on the period between excretion by an infected and infection of a healthy WWF. Testing of natural water reservoirs will generate information on the level of (eventual) contamination and the risk of AIV transmission via these media at different geographical and environmental conditions. Water samples should be collected from different points of each selected area, in an amount of 1 liter per sample. Each sample should be tested for the presence of AIV, using PCR with previous concentration of the virus. Using a standardized protocol, it is possible to quantitatively evaluate the level of contamination, based on comparison with known titrated virus isolate.

Nuclear Component

Stable isotope analysis of feather samples (δD and potentially other SI) of WWF and environmental water in breeding and stopover areas of WWF. Nuclear related molecular diagnostic methods for DNA barcoding, AIV detection in faecal and water samples.

CRP Overall Objective

  • Evaluate the potential of precipitation deuterium isoscapes based on the IAEA GNIP program for determining origin of waterfowl in Africa/Asia by calibrating this isoscape with feathers (and potentially claws) grown at known origins.
  • Utilize species-specific telemetry and other marking data to identify waterfowl flyways including stopover, breeding, non-breeding and moult locations in Africa/Asia and use these data to inform isotopic sampling strategies.
  • Evaluate tissues from waterfowl (feather, blood and claws) samples previously collected and identify key samples for isotope analysis to support objectives 1 and 2.
  • Evaluate the implementation of non-invasive methods for AI surveillance in WWF, based on simultaneous DNA barcoding and detection of the AIV in faecal samples.
  • Evaluate the level of eventual contamination and the risk of AIV transmission in WWF via natural water reservoirs.

Specific Research Objectives

  • Determine the potential use of stable isotope analysis in tracing migratory pathways of wild water fowl.
  • Improve the existing isoscapes for δD and other essential isotopes, especially in the regions of Asia and Africa.
  • Develop validated SOP for DNA barcoding for differentiation of WWF species using faecal samples.
  • Develop validated SOPs for detection and typing of the AIV in WWF using faecal samples.
  • Develop validate SOPs for detection and quantitative evaluation of the AIV in natural water reservoirs.

Expected Research Outputs

  • Stable isotope analysis in tracing migratory pathways of wild water fowl established.
  • Isoscapes for δD and other essential isotopes, at observation areas in the regions of Asia and Africa established.
  • SOP for DNA barcoding for differentiation of WWF species using faecal samples developed.
  • SOPs for detection and typing of the AIV in WWF using faecal samples developed.
  • SOPs for detection and quantitative evaluation of the AIV in natural water reservoirs developed.

CRP Expected Research Outcomes

  • Tracing migratory birds using stable isotopes established and implemented.
  • Non-invasive methods for AIV surveillance using simultaneous detection of wild water fowl species and AIV carrier status established and implemented.
  • A system for estimation of the level of contamination of natural water reservoirs established.


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