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2 Centre de Coopération Internationale en Recherche Agronomique pour le Développement, TA 30/E, Campus International de Baillarguet, 34398 Montpellier, France
3 Wetlands International, Box 471, 6700 AL Wageningen, The Netherlands
4 Istituto Zooprofilattico Sperimentale delle Venezie, OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle Disease, Viale dell’Università 10, 35020 Legnaro, Italy
5 Food and Agriculture Organization of the United Nations, Animal Production and Health Division, Viale delle Terme di Caracalla, 00100 Rome, Italy
6 Office National de la Chasse et de la Faune Sauvage, Box 236, 75822 Paris cedex 17, France
7 Corresponding author (email: nicolas.gaidet{at}cirad.fr)
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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Migratory waterfowl generally are considered to be the natural reservoir of avian influenza virus (Olsen et al., 2006). Large numbers of waterbirds that breed in the Palearctic overwinter on the African continent. In the context of the spread of the highly pathogenic avian influenza (HPAI) Asian lineage H5N1 virus through Eurasia during summer 2005, concerns arose that this virus could be spread southward toward Africa in wild birds during fall migration. In November 2005, the Food and Agriculture Organization (FAO) set up five regional Technical Cooperation Programmes (TCP) of Emergency Assistance for Early Detection and Prevention of avian influenza in five regions of Eastern Europe, the Middle East, and Africa. These programs were developed to provide support for strengthening emergency preparedness against the potential introduction and progressive spread of HPAI H5N1 virus within these regions, on a country basis, specifically in relation to migration of and trade in wild birds and the interface between wild birds and domestic poultry.
The FAO has been collaborating with national veterinary services, national wildlife institutions, and international collaborating centers (Centre de Coopération Internationale en Recherche Agronomique pour le Développement [CIRAD]; Istituto Zooprofilattico Sperimentale delle Venezie; Royal Veterinary College, University of London; and Wetlands International) to strengthen field surveillance and laboratory diagnostic capacities through training and capacity building. A risk analysis procedure was implemented for the development of contingency action plans to strengthen early warning of and early reaction to HPAI introduction. These TCPs also aimed to promote the development of capacity for sharing HPAI disease intelligence through the establishment of information and technology network linkages within and between the regions, in relation to the development of a global system for HPAI surveillance.
Within the framework of these TCPs, a surveillance study was launched in early 2006 to evaluate if the HPAI H5N1 virus could be perpetuated in wild bird populations in countries where HPAI H5N1 outbreaks occurred or may occur considering the movement of wild birds. The objective also was to provide technical support to national surveillance programs through capacity building and to standardize field procedures.
Implementation of the field surveillance campaign was coordinated by CIRAD and Wetlands International, in partnership with national wildlife and veterinary services. The investigations targeted natural sites where waterbirds from various breeding grounds congregate and mix, where there is opportunity for avian influenza virus to be transmitted among various host populations and spread over extensive geographical ranges. Study sites were selected in accordance with national surveillance programs and field logistic constraints. Operations were conducted during 7 to 10-day sampling periods. With the spread of HPAI H5N1 over the TCP region in the course of the survey period, complementary sampling sites were identified in the proximity of the latest notified outbreaks (in particular Egypt, Niger, and Burkina Faso; Table 1
and Fig. 1).
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Samples were collected by teams of national and international experts in collaboration with international conservation and research organizations (AFRING, Oiseaux Migrateurs du Paléarctique Occidental [OMPO], Office National de la Chasse et de la Faune Sauvage [ONCFS], Dutch Centre for Field Ornithology [SO-VON], Wildfowl & Wetlands Trust [WWT]), local ornithological organizations, and national park departments, as well as national hunting associations and safari operators. Cloacal swabs were collected from recently-killed birds provided by hunters and from live-caught birds. In countries with hunting restrictions (Ethiopia) and in countries where emergency surveillance operations were implemented following notification of HPAI H5N1 outbreaks in Nigeria (Burkina Faso, Niger), birds were shot through special permits for sample collection (n = 732). Fresh faecal samples were collected on some occasions at roosting areas for gulls and terns (Laridae) and ducks (Anatinae). Duplicate sampling was performed in the field in order to submit samples to both national and international reference laboratories. The transport medium consisted of an isotonic phosphate buffered saline (PBS), pH 7.0–7.4, containing the antibiotics penicillin (10,000 units/ml), streptomycin (10 mg/ml), amphotericin B (25 µg/ml), and gentamycin (250 µg/ml) supplemented with 10% glycerol. Samples were stored in the field in liquid nitrogen containers or on ice and then stored at <2 70 C after a few hours (generally <4 hr, maximum 24 hr). They were shipped in dry ice in cryopacks until processed.
Most samples were processed at Istituto Zooprofilattico Sperimentale delle Venezie-Italia, while some samples were analyzed in other laboratories (Table 1
). The samples were all screened by real-time RT-PCR specific for type A influenza viruses (Spackman et al., 2002), and positive samples were then tested by RT-PCR specific for H5 subtype. The molecular pathogenicity of all H5-positive samples was determined by sequencing the haemagglutinin gene segment. Sequences were performed using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) in a 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). On the other hand, all type A positive samples were subsequently processed for virus isolation using standard methods. Briefly, 100 µl of the original sample was inoculated into the allantoic cavity of 9 to 10 day embryonated specific-pathogen-free (SPF) eggs for virus isolation attempts according to European Union Directive 92/40. Hemagglutinating isolates were characterized by the hemagglutination-inhibition test (HI) and the neuraminidase-inhibition (NI) test using specific hyperimmune chicken antisera to the reference strains of influenza virus (Alexander and Spackman, 1981).
A total of 5,256 samples was collected in 14 countries, mostly on the African continent, and mostly between mid-January and mid-March (Table 1 and Fig. 1). Field surveillance operations were postponed in Romania due to severe weather conditions and in Iran and Turkey because of delayed official approval from national authorities.
Samples were collected from 87 bird species, with 17 species representing 90% of all samples collected. A majority of these samples originated from Anatidae, including both Afro-tropical ducks (30% of all samples collected, mostly whitefaced whistling duck: Dendrocygna viduata) and Eurasian ducks (29%, mostly garganey: Anas querquedula). Other species consisted mostly of waders (16%), gulls and terns (11%), rails (9%), and cormorants (Phalacrocoracidae) (3%) (Table 2).
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Low pathogenic avian influenza (LPAI) viruses were detected in 14 species of 5 bird families (Anatidae, Rallidae, Scolopacidae, Laridae, Sternidae), including both Eurasian and Afro-tropical bird species (Table 2) from eight countries (Chad, Ethiopia, Mali, Mauritania, Morocco, Niger, Senegal, and Tunisia). LPAI viruses were detected in Palearctic migratory waterbirds in their overwintering sites in Africa, including ducks (garganey, northern pintail Anas acuta, green-winged teal A. crecca, and northern shoveler A. clypeata), waders (curlew sandpiper Calidris ferruginea, ruff Philomachus pugnax, spotted redshank Tringa erythropus), gulls (lesser black-backed gull Larus fuscus), as well as in some Afro-tropical waterbirds, including ducks (white-faced whistling duck, knob-billed duck Sarkidiornis melanotos), gulls (slender-billed gull Larus genei), and rails (purple swamphen Porphyrio porphyrio). In Anatidae, a higher prevalence was detected in Eurasian ducks (6.5%) than in Afro-tropical ducks (2.8%) (chi-square test, p < 0.001).
Five viruses were isolated in embryonated eggs from the 159 RT-PCR–positive samples. Three distinct isolates were obtained from garganey in the Inner Niger Delta in Mali, and two isolates were recovered from white-faced whistling duck in Ethiopia and in Senegal (Table 3).
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No evidence was found of HPAI H5N1 virus circulating in wild birds, including samples collected in countries that had experienced recent avian influenza outbreaks, some of which were ongoing at the time of the surveys. However, this absence of H5N1 viruses among thousands of samples investigated must be interpreted in relation to the millions of waterbirds gathering in African wetlands during the northern winter. This outcome is coherent with the absence of H5N1 virus reported from recent surveillance programs in European countries (EFSA, 2006; Pitman et al., 2007) and with the very limited detection rate of H5N1 virus so far from healthy wild bird populations (Chen et al., 2006). However, results from experimental infection in ducks indicate that, contrary to other avian influenza viruses, HPAI H5N1 virus concentration may be higher in the trachea than in the cloaca (Sturm-Ramirez et al., 2004; Hulse-Post et al., 2005), suggesting that HPAI H5N1 virus could have potentially remained undetected in cloacal and fecal samples used in our survey.
Avian influenza virus was detected from cloacal swabs and fresh faeces collected from white-faced whistling ducks, including samples originating from the same study site (i.e., the Senegal delta). Similar to temperate regions, the collection of freshly deposited faecal droppings can provide a valid method for monitoring LPAI virus presence in tropical regions.
An unusually low virus isolation rate was, however, obtained from the type A RT-PCR–positive samples. Major attention was given to appropriate storage of all samples at < –70 C and to the preservation of the cold chain from the field to the laboratory. Nevertheless, logistic constraints in some remote field sampling areas and unexpected international shipment delays may have accounted for this low recovery rate.
Measurements of avian influenza virus prevalence in wild birds in Africa provide new insights into the host ecology of avian influenza viruses in tropical regions. LPAI viruses were detected in both Palearctic and Afro-tropical waterbirds in several sampling sites, indicating that viruses were circulating in Africa during the northern winter (Gaidet et al., 2007).
The detection of LPAI viruses in Eurasian ducks in several of their major overwintering sites in West Africa (i.e., Lake Chad, Inner Niger and Senegal river deltas) supports the hypothesis that avian influenza viruses persist in wild duck populations through a continuous circulation in a proportion of birds. The different viruses isolated from garganey sampled in the Inner Niger Delta in Mali also indicate that various subtypes are circulating at the same time in a single wintering population, in agreement with patterns observed in Europe and North America (Fouchier et al., 2003; Krauss et al., 2004).
The detection of viruses in some Eurasian wader species contrasts with the apparent absence of avian influenza viruses reported in previous studies of waders in Europe (Fouchier et al., 2003) but is consistent with results found in surveillance in North America (Krauss et al., 2004). Several Afro-tropical bird species from various bird families also were found positive for LPAI viruses, raising the possibility of a potential persistence of avian influenza viruses in the tropical environment all year round.
Results from this large-scale surveillance study provide evidence that LPAI viruses circulate in wild birds in subtropical environments during the northern winter, including in Eurasian waterbirds wintering in sub-Saharan Africa before their northward spring migration. This suggests a potential role of tropical regions for the perpetuation of some avian influenza viruses and in their potential intercontinental transmission. At the same time, no evidence was found for the transmission of HPAI between Eurasia and Africa through bird migration. Such findings stress the need to improve our understanding of the host ecology of avian influenza viruses, in particular in subtropical and tropical regions, which should contribute to the prevention and control of HPAI. During fall 2006 and in winter 2007, this surveillance program implemented within the framework of the TCPs will be replicated and extended over Eastern Europe, the Middle East, and southern Africa.
We would like to acknowledge the participation of and permissions granted by numerous national and local agencies in the participating countries, without which this work would not have been possible. We also are grateful to the numerous ornithologists and veterinarians who collaborated by collecting bird samples, as well as to the various laboratory teams for processing the samples (a detailed list of partners is available from http://wildbirds-ai.cirad.fr/campaign-2006.php). This extensive survey on avian influenza surveillance in wild birds was coordinated by the Food and Agriculture Organization of the United Nations through its Technical Cooperation Programmes, and was made possible by additional financial resources received by the government of France.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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CHEN, H., G. J. SMITH, K. S. LI, J. WANG, X. H. FAN, J. M. RAYNER, D. VIJAYKRISHNA, J. X. ZHANG, L. J. ZHANG, C. T. GUO, C. L. CHEUNG, K. M. XU, L. DUAN, K. HUANG, K. QIN, Y. H. LEUNG, W. L. WU, H. R. LU, Y. CHEN, N. S. XIA, T. S. NAIPOSPOS, K. Y. YUEN, S. S. HASSAN, S. BAHRI, T. D. NGUYEN, R. G. WEBSTER, J. S. PEIRIS, AND Y. GUAN. 2006. Establishment of multiple sublineages of H5N1 influenza virus in Asia: Implications for pandemic control. Proceedings of the National Academy of Sciences of the United States 103: 2845–2850.
EUROPEAN FOOD SAFETY AUTHORITY (EFSA). 2006. Migratory birds and their possible role in the spread of highly pathogenic avian influenza. Annex to the European Food Safety Authority Journal 357: 1–46, http://www.efsa.eu.int/science/ahaw/ahaw_opinions/catindex_en.html.
FOUCHIER, R. A., B. OLSEN, T. M. BESTEBROER, S. HERFST, L. VAN DER KEMP, G. F. RIMMELZWAAN, AND A. D. OSTERHAUS. 2003. Influenza A virus surveillance in wild birds in northern Europe in 1999 and 2000. Avian Disease 47: 857–860.[Medline]
GAIDET, N., T. DODMAN, A. CARON, G. BALANA, S. DESVAUX, F. GOUTARD, G. CATTOLI, F. LAMARQUE, W. HAGEMEIJER, AND F. MONICAT. 2007. Avian influenza viruses in water birds, Africa. Emerging Infectious Diseases 13: 626–629.[Medline]
HULSE-POST, D. J., K. M. STURM-RAMIREZ, J. HUM-BERD, P. SEILER, E. A. GOVORKOVA, S. KRAUSS, C. SCHOLTISSEK, P. PUTHAVATHANA, C. BURANATHAI, T. D. NGUYEN, H. T. LONG, T. S. NAIPOSPOS, H. CHEN, T. M. ELLIS, Y. GUAN, J. S. PEIRIS, AND R. G. WEBSTER. 2005. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proceedings of the National Academy of Sciences of the United States 102: 10682–10687.
KRAUSS, S., D. WALKER, S. P. PRYOR, L. NILES, L. CHENGHONG, V. S. HINSHAW, AND R. G. WEBSTER. 2004. Influenza A viruses of migrating wild aquatic birds in North America. Vector-Borne Zoonotic Diseases 4: 177–189.
OLSEN, B., V. J. MUNSTER, A. WALLENSTEN, J. WALDENSTROM, A. D. OSTERHAUS, AND R. A. FOUCHIER. 2006. Global patterns of influenza A virus in wild birds. SCIENCE 312: 384–388.
PITMAN, M., A. LADDOMADA, R. FREIGOFAS, V. PIAZZA, A. BROUW, AND I. H. BROWN. 2007. Surveillance, prevention, and disease management of avian influenza in the European Union. In Proceedings of the FAO and OIE International Scientific Conference on Avian Influenza and Wild Birds, Rome, Italy, 30 and 31 May 2006. Supplement of the Journal of Wildlife Diseases, In press.
SPACKMAN, E., D. A. SENNE, T. J. MYERS, L. L. BULAGA, L. P. GARBER, M. L. PERDUE, K. LOHMAN, L. T. DAUM, AND D. L. SUAREZ. 2002. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Journal of Clinical Microbiology 40: 3256–3260.
STURM-RAMIREZ, K. M., T. ELLIS, B. BOUSFIELD, L. BISSETT, K. DYRTING, J. E. REHG, L. POON, Y. GUAN, M. PEIRIS, AND R. G. WEBSTER. 2004. Reemerging H5N1 influenza viruses in Hong Kong in 2002 are highly pathogenic to ducks. Journal of Virology 78: 4892–4901.
Received for publication 15 December 2006.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
