Avian influenza viruses in water birds, Africa.

We report the first large-scale surveillance of avian influenza viruses in water birds conducted in Africa. This study shows evidence of avian influenza viruses in wild birds, both Eurasian and Afro-tropical species, in several major wetlands of Africa.

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Wild water birds are considered to be the major natural reservoir for avian influenza viruses (AIV) (1). Large numbers of Eurasian breeding water birds overwinter in the sub-Saharan region of the African continent (2), where the survival of AIV is considered to be restricted by the tropical environment (3). Although the first reported isolation of AIV from wild birds (A/Tern/S.A./61 [H5N3]) was in Africa (4), a knowledge gap exists in the ecology of AIV in tropical regions (1,5). Whether AIV circulate in waterbird communities in Africa and whether tropical ecosystems can play a role in the perpetuation of AIV among waterfowl remain unknown. We report results from large-scale surveillance of water birds in 12 countries in Africa (Figure).

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The Study

This surveillance program was implemented in early 2006 within the framework of the Food and Agriculture Organization (FAO)'s Technical Cooperation Programs of Emergency Assistance for Early Detection and Prevention of Avian Influenza. Field sampling operations were coordinated by Centre de cooperation Internationale en Recherche Agronomique pour le D6veloppement and by Wetlands International, in partnership with wildlife and veterinary national services, international organizations (1), local ornithologic nongovernment organizations, as well as national hunting associations and safari operators. Study species were selected among bird families recognized as major AIV reservoirs (notably among the orders Anseriformes and Charadriiformes), in both Eurasian and Afro-tropical bird communities. Study sites important for congregatory water birds were selected in accordance with national surveillance programs and field logistic constraints and included sites where palearctic and Afro-tropical birds mix.

From mid-January to early March 2006 (and early May in Tunisia), we collected cloacal swab samples from captured birds and from freshly killed birds provided by hunters. Samples of fresh droppings were also collected at roosting areas for gulls, terns, and some ducks. In Ethiopia, which has hunting restrictions, and in countries in which emergency surveillance operations were implemented after notification of influenza A (H5N1) outbreaks in Nigeria (Burkina Faso, Niger), special permits were obtained to shoot birds for sample collection (n = 732).

Materials used and storing procedures were standardized among field teams. The transport medium consisted of an isotonic phosphate-buffered saline, pH 7.0-7.4, containing antimicrobial agents (penicillin 10,000 U/mL, streptomycin 10 mg/mL, amphotericin B 25 [micro]g/mL, and gentamycin 250 [micro]g/mL) supplemented with 10% glycerol. Samples were stored in liquid nitrogen containers or on ice and then stored at <-70[degrees]C after a few hours (generally <4 h, maximum of 24 h). They were shipped in dry ice in cryopacks until processed.

Samples were analyzed at the Istituto Zooprofilattico Sperimentale delle Venezie (Italy), except for samples from Egypt that were analyzed at the US Naval Medical Research Unit-3 (Egypt), samples from Kenya and Malawi which were analyzed at the Agricultural Research Council Onderstepoort Veterinary Institute (RSA), and samples from Tunisia which were analyzed at the Southeast Poultry Research Laboratory (USA). The samples were all screened by real-time reverse transcription (RT)-PCR specific for type A influenza viruses (6), and positive samples were tested by RT-PCR specific for H5 subtype. All type A-positive samples were subsequently processed for virus isolation by using standard methods (inoculation into the allantoic cavity of 9- to 10-day-old embryonated specific-pathogen-free eggs, EU directive 92/40). Isolates were characterized by hemagglutination and neuraminidase-inhibition tests by using specific hyperimmune chicken antisera to the reference strains of influenza virus (7). Molecular pathogenicity of H5 subtype-positive samples was determined by sequencing the hemagglutinin gene segment (BigDye Terminator v3.1 cycle sequencing kit, Applied Biosystems, Foster City, CA, USA).

A total of 4,553 birds (Table 1), consisting mostly of Afro-tropical and Eurasian ducks (32% and 31% of samples, respectively), were tested. The overall protion of AIV detected was 3.5% (n = 159 RT-PCR-positive samples, including both cloacal swabs and fresh droppings). Low-pathogenicity AIV were detected in 14 species of ducks, waders, gulls, terns, and rails, including both Eurasian and Afro-tropical species (Table 1). Positive samples were obtained from 8 countries (Chad, Ethiopia, Mali, Mauritania, Morocco, Niger, Senegal, and Tunisia). In the 2 most frequently sampled species, Eurasian ducks (garganey [Anas querquedula], n = 1,329) and Afro-tropical duck (white-faced whistling ducks [Dendrocygna viduata], n = 1,157), AIV were detected from most surveyed countries but with a highly variable prevalence (Table 2). Neither influenza A (H5N1) viruses nor any highly pathogenic AIV were detected. A total of 11 samples were positive for H5 subtype, mostly from garganey ducks (H5 prevalence of 0.7%). Finally, 5 low-pathogenicity AIV were isolated: 3 distinct isolates that originated from garganey ducks sampled in the Inner Niger Delta in Mali (H5N3, H11N9, H12N5) and 2 isolates that originated from white-faced whistling ducks sampled in Ethiopia (H8N4) and Senegal (H1N1).

Conclusions

The African continent, in particular its sub-Saharan region, constitutes a seasonal shelter for a large number of Eurasian water birds, including an estimated 5.4 million ducks that gather in western and eastern Africa during the northern winter (8). In their overwintering sites, these birds congregate and mix with a wide variety of Afro-tropical water birds, some of them with large populations widespread over Africa.

AIV have been isolated in wild ducks on wintering grounds in both Europe and North America (9,10). Results from this surveillance program established that AIV are also present in wild birds in Africa during the northern winter. Low-pathogenicity AIV were detected and isolated in several species from several major wetlands of northern, western, and eastern Africa, which indicates that environmental conditions in Afro-tropical ecosystems are favorable for the persistence and transmission of AIV.

We detected and isolated AIV in Eurasian and Afrotropical species. This finding shows that AIV circulate in migratory water birds originating from Eurasia and in African species that remain in the continent throughout the year. Moreover, the detection of viruses in some Eurasian wader species during wintering (in January in Mall) and during migration (in May in Tunisia) contrasts with the apparent absence of AIV reported from previous studies of waders in Europe (5,11). Since waders form the most abundant group of African-Eurasian migratory water birds (12), these shorebirds may play a role in maintaining some AIV in waterbird communities at wintering and stopover sites.

The detection of AIV in Eurasian ducks in several of their major overwintering sites in West Africa (e.g., the Inner Niger Delta, the Senegal River Delta, and Lake Chad) supports the hypothesis that AIV can persist in wild duck populations year-round through a continuous circulation in a proportion of birds (1). Variability in the prevalence observed might be related to differences in local logistical constrains but also to differences between African regions in their waterbird assemblage and connectivity with European breeding grounds. The different isolates obtained from garganey from the Inner Niger Delta also indicate that various subtypes are circulating at the same time in a population, a finding that agrees with patterns observed in Europe and North America (11,13).

Various AIV subtypes were isolated from apparently healthy garganey and white-faced whistling ducks, which indicates that both Eurasian and Afro-tropical ducks may serve as reservoirs of AIV. These results not only suggest that some Eurasian ducks could carry AIV on their northward spring migration but also raise the possibility that AIV could persist in the tropical region and be disseminated over Africa through intra-African migratory ducks. The presence of AIV at African wintering and stopover sites, where birds from various geographic origins congregate and mix, provides opportunities for transmission of AIV between different populations and spread of AIV over extensive areas in both Eurasia and Africa.

Acknowledgments

We acknowledge the participation of and permissions granted by numerous national and local agencies in the participating countries. We are also grateful to the numerous ornithologists and veterinarians who collaborated in this surveillance program by collecting bird samples, as well as to the various laboratory teams who processed the samples (see a detailed list of partners in Gaidet and Dodman [14]). We also thank Akiko Kamata, Felix Njeumi, Arnaud LeMenach, Astrid Tripodi, and Vincent Martin for assistance in preparing and coordinating these campaigns; Catherine Cetre-Sossah for technical assistance; Camille Danes for her help in database management and illustration; and Vittorio Guberti for advice in the preparation of field operations.

This extensive survey has been coordinated by FAO through its Technical Cooperation Program and has been made possible by additional financial resources from the government of France.

Dr Gaidet is an ecologist in the Animal Production and Veterinary Department at the French Agricultural Research Centre for International Development. His primary research interests include the host ecology of avian influenza and West Nile viruses.

References

(1.) Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev. 1992;56:152-79.

(2.) Del Hoyo J, Elliot A, Sargatal J. Handbook of the birds of the world. Vols. 1 and 3. Barcelona: Lynx Editions; 1996.

(3.) Stallknecht DE, Shane SM, Keamey MT, Zwank PJ. Persistence of avian influenza viruses in water. Avian Dis. 1990;34:406-11.

(4.) Becker WB. The isolation and classification of tern virus: influenza virus A/tern/South Africa/1961. J Hygiene. 1966;64:309-20.

(5.) Olsen B, Munster VJ, Wallensten A, Waldenstrom J, Osterhaus AD, Fouchier RA. Global patterns of influenza a virus in wild birds. Science. 2006;312:384-8.

(6.) Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256-60.

(7.) Alexander DJ, Spackman D. Characterization of influenza A viruses isolated from turkeys in England during March May 1979. Avian Pathol. 1981;10:281-93.

(8.) Dodman T. Waterbird family estimates in Africa. Waterbird population estimates. 4th edition. Wageningen (the Netherlands): Wetlands International; 2006.

(9.) De Marco MA, Foni AGE, Campitelli BL, Raffini CE, Di Trani DL, Delogu EM, et al. Circulation of influenza viruses in wild waterfowl wintering in Italy during the 1993 99 period: evidence of virus shedding and seroconversion in wild ducks. Avian Dis. 2003;47:861-6.

(10.) Hanson BA, Swayne DE, Senne DA, Lobpries DS, Hurst J, Stallknecht DE. Avian influenza viruses and paramyxoviruses in wintering and resident ducks in Texas. J Wildl Dis. 2005;41:624-8.

(11.) Fouchier RA, Olsen B, Bestebroer TM, Herfst S, van der Kemp L, Rimmelzwaan GF, et al. Influenza A virus surveillance in wild birds in northern Europe in 1999 and 2000. Avian Dis. 2003;47:857-60.

(12.) Stroud DA, Davidson NC, West R, Scott DA, Haanstra L, Thorup O, et al. (compilers) on behalf of the International Wader Study Group 2004. Status of migratory wader populations in Africa and Western Eurasia in the 1990s. International Wader Studies. 2004; 15:1-259.

(13.) Krauss S, Walker D, Pryor SP, Niles L, Chenghong L, Hinshaw VS, et al. Influenza A viruses of migrating wild aquatic birds in North America. Vector Borne Zoonotic Dis. 2004;4:177-89.

(14.) Gaidet N, Dodman T. Influenza surveillance in wild birds in Eastern Europe, the Middle East and Africa: preliminary results from an ongoing FAO-led survey. Rome: Food and Agriculture Organization; 2006. Available from http://wildbirds-ai.cirad.fr

Address for correspondence: Nicolas Gaidet, CIRAD, UR 22 Gestion Integree de la Faune, TA 30/E Campus International de Baillarguet, 34398 Montpellier, France; email: [e-mail]

All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required.

(1) African Waterbird Ringing Scheme (AFRING), Oiseaux Migrateurs du Palearctique Occidental (OMPO), Office National de la Chasse et de la Faune Sauvage (ONCFS), Dutch Centre for Field Ornithology or Stichting Openbaar Voortgezet Onderwijs Noord (SOVON), and Wildfowl and Wetlands Trust (WWT).

Nicolas Gaidet, * Tim Dodman, ([dagger]) Alexandre Caron, * Gilles Balanca, * Stephanie Desvaux, * Flavie Goutard, * Giovanni Cattoli, ([double dagger]) Francois Lamarque, ([section]) Ward Hagemeijer, ([dagger]) and Francois Monicat *

* Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement, Montpellier, France; ([dagger]) Wetlands International, Wageningen, the Netherlands; ([double dagger] Viale dell'Universita, Legnaro, Italy; and ([section]) Office National de la Chasse et de la Faune Sauvage, Paris, France

Table 1. Prevalence of avian Influenza virus in wild birds *

                                                         PCR positive,
Bird group               Species tested           No.       no. (%)

African ducks     9 species (total, including    1,455     41 (2.8)
                         4 named below)
                      Dendrocygna viduata        1,181     38 (3.2)
                     Sarkidiornis melanotos        117      3 (2.6)
                           D. bicolor               88         0
                    Plectropterus gambensis         32         0
Eurasian ducks             10 species            1,409     93 (6.6)
                        Anas querquedula         1,335     87 (6.5)
                            A. acuta                24      2 (8.3)
                           A. crecca                24      3 (12.5)
                          A. clypeata                6      1 (16.7)
Eurasian waders            13 species              409      6 (1.5)
                       Philomachus pugnax          115      2 (1.7)
                        Tringa glareola             74         0
                        Calidris minuta             60         0
                         C. ferruginea              45      2 (4.4)
                     Himantopus himantopus          45         0
                      Gallinago gallinago           30         0
                         T. erythropus              23      2 (8.7)
Rails                      8 species               438      3 (0.7)
                        Porphyrio alleni           187         0
                    Amaurornis flavirostris         88         0
                        Fulica cristata             80         0
                      Gallinula chloropus           31      2 (6.5)
                      Porphyrio porphyrio           10      1 (10)
Gulls                      3 species               366     14 (3.8)
                          Larus genei              156     13 (8.3)
                           L. fuscus               129      1 (0.8)
                       L. melanocephalus            81         0
Terns                      7 species               159      2 (1.3)
                     Sterna sp. ([dagger])         150      2 (1.3)
Cormorants                 2 species               148         0
                      Phalacrocorax carbo          130         0
Other                      36 species              196         0
Total                      87 species            4,553     159 (3.5)

Bird group               Species tested             Positive country

African ducks     9 species (total, including
                         4 named below)
                      Dendrocygna viduata        TD, ET, ML, MR, NE, SN
                     Sarkidiornis melanotos              ML, NE
                           D. bicolor
                    Plectropterus gambensis
Eurasian ducks             10 species
                        Anas querquedula           TD, ML, MR, NE, SN
                            A. acuta                       ML
                           A. crecca                       MA
                          A. clypeata                      MA
Eurasian waders            13 species
                       Philomachus pugnax                  ML
                        Tringa glareola
                        Calidris minuta
                         C. ferruginea                     TN
                     Himantopus himantopus
                      Gallinago gallinago
                         T. erythropus                     ML
Rails                      8 species
                        Porphyrio alleni
                    Amaurornis flavirostris
                        Fulica cristata
                      Gallinula chloropus                  ML
                      Porphyrio porphyrio                  ML
Gulls                      3 species
                          Larus genei                      SN
                           L. fuscus                       MR
                       L. melanocephalus
Terns                      7 species
                     Sterna sp. ([dagger])                 MR
Cormorants                 2 species
                      Phalacrocorax carbo
Other                      36 species
Total                      87 species

* Detected by reverse transcription--PCR (RT-PCR), for all
RT-PCR-positive species and in species with >30 individuals sampled.
Lower numbers in individual species are included in the total for
each bird group. Countries where RT-PCR-positive samples were obtained
are indicated (TD, Chad; ET, Ethiopia; ML, Mali; MR, Mauritania;
NE, Niger; SN, Senegal; MA, Morocco; TN, Tunisia).

([dagger]) Unidentified fresh dropping samples from a multispecies
flock of Sterna caspia, S. maxima, and S. sandvicensis.

Table 2. Reverse transcription PCR-based detection of influenza
A virus in 2 wild duck species sampled in different countries

                                No. samples      No. PCR
Species             Country       tested      positive (%)

Garganey (Anas       Chad           381         11 (2.9)
querquedula)         Kenya          104             0
                     Mali           411         22 (5.4)
                  Mauritania        225         33 (14.7)
                     Niger           87          4 (4.6)
                    Senegal         121         17 (14.0)
White-faced         Burkina         167             0
whistling duck       Faso
(Dendrocygna         Chad           232          1 (0.4)
viduata)           Ethiopia          76         10 (13.2)
                    Malawi           59              0
                     Mali            36          1 (2.8)
                  Mauritania        183          7 (3.8)
                     Niger          232          8 (3.4)
                    Senegal         172         11 (6.4)