Wild bird species sampled and tested for AIVs in Nigeria in 2007, and results of molecular tests and sequence analyses.

a<p>Evaluated by means of real-time RT-PCR specific for type A influenza viruses (M gene).</p>b<p>Evaluated by genomic sequencing.</p>c<p>Birds that breed in the Western Palearctic and migrate to Africa.</p>d<p>Potentially either an Afro-tropical breeding or Eurasian wintering individual.</p

Summary of the cleavage site motif in selected African H5 HPAI and LPAI viruses.

<p>Cleavage site motif of the HA sequence phylogenetically most closely related to the Nigerian H5N2 HA (A/mallard/Bavaria/1/2005) is included for comparison.</p>a<p>Representatives of all the sequenced African H5N1 HPAI viruses circulating in 2006–2007 (exceptions were observed in the Sudanese isolates and in 2 Egyptian isolates).</p

Phylogenetic tree based on the sequence analysis of the entire segment encoding for NA proteins.

<p>The phylogenetic tree includes selected N2 sequences of influenza viruses isolated in Asia, Europe and Africa. Viral sequences analyzed in this study are marked with a circle.</p

Phylogenetic tree based on the sequence analysis of the entire segment encoding for HA proteins, with representative H5N1, H5N2, and H5N3 influenza A viruses isolated in naturally infected wild and domestic birds in Asia, Europe, and Africa.

<p>Viral sequences analyzed in this study are marked with a circle.</p

Movement paths of one White-faced Whistling Duck (<i>Dendrocygna viduata</i>) fitted with a satellite transmitter, from Hadejia-Nguru Wetlands in northern Nigeria to Lake Chad in western Chad, during February–March 2007.

<p>Dates and minimum distances between main staging areas (orange circles) are indicated on the satellite image.</p

List of potential hotspots of AIV infection detected in our study.

<p>These sites correspond to sampling occasions (top) at which the number of AIV-positive birds was above the threshold number of birds for which the hypothesis that prevalence is lower than 10% could not be rejected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone-0046049-g002" target="_blank">Figure 2</a>). Sampling conducted in different years at the same sites during the same months and on the same species (below) detected a low number of AIV-positive birds.</p>a<p>. African jacana <i>Actophilornis africana</i>, Kittlitz's plover <i>Charadrius pecuarius</i>, Little stint <i>Calidris minuta</i>, Blacksmith lapwing <i>Vanellus armatus</i>, Slender-billed gull <i>Chroicocephalus genei</i>, Curlew sandpiper Calidris ferruginea, Wood sandpiper <i>Tringa glareola</i>.</p

Detection of potential hotspots of AIV infection in shorebirds sampled at various sites across Eurasian and Afro-tropical regions (<b>Figure 1</b>-B; and supporting information Table S2).

<p>The number of AIV-positive birds detected in relation to the number of birds sampled per sampling occasion is here compared to the threshold number of positive birds (solid line) below which the prevalence is unlikely (probability <0.05) to be greater than 10% for a sample of the same size. Points on or above the line represent potential AIV hotspots, i.e. sampling occasions (n = 5) for which the number of positive birds was too large for rejecting the hypothesis that prevalence could be >10% (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone-0046049-t003" target="_blank">Table 3</a>). Only sampling occasions (n = 47) that had at least 28 birds sampled were considered in this analysis.</p

Mean seroprevalence of AIV antibodies among closely related shorebird species in relation to the mean latitude of their breeding range.

<p>Seroprevalence were measured in West Africa (the Banc d'Arguin, Mauritania and the Inner Niger Delta, Mali) and the Delaware Bay, USA (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Stallknecht1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Brown2" target="_blank">[31]</a>) using the same commercial bELISA kit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#s2" target="_blank">Methods</a>). The mean species breeding latitude was computed from the northern and southern limits of the breeding distribution of the populations present at each site using distribution maps from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Jourdain1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049-Poole1" target="_blank">[32]</a>. Species include ruddy turnstone (Δ), red knot (□), dunlin (o), sanderling (◊), short-billed dowitcher (×), ruff and wood sandpiper (−). Error bars represent the binomial exact 95% confidence interval.</p

Location of the world's largest congregation sites of waders (sandpipers, plovers and allies), i.e. sites where at least 500,000 birds congregate annually.

<p>Among these sites where birds have been tested for AIV infection (black symbols - from the literature; grey symbols - this study) an AIV hotspot has been reported only at the Delaware Bay (no. 7). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049.s007" target="_blank">Table S6</a> (supporting information) for detailed information on each site. 1-Yukon-Kuskokwim Delta, 2-Copper River Delta, 3-Fraser' River Estuary, 4-Gray's harbour estuary, 5-Bay of Fundy, 6-Great Salt Lake, 7-Delaware Bay, 8-Cheyenne Bottoms, 9-San Francisco Bay, 10-Bahia de Santa Maria, 11-Upper Bay of Panama, 12-Suriname coast, 13-Laguna Mar Chiquita, 14-Wadden Sea, 15-Rhine-Maas-Schelde Delta, 16-Azov Sea, 17-Sea of Okhotsk, 18-Tengiz-Korgalzhyn Lakes, 19-Yellow Sea coast, 20-Arabian Sea off Oman, 21-Banc d'Arguin, 22-Senegal River Delta, 23-Bijagos Archipelago (map by M. Gély ©Cirad).</p

Location of the study sites.

<p>(A) The Banc d'Arguin (Mauritania) and the main shorebirds migratory flyways across Western Eurasia and Africa. (B) All shorebird sampling sites considered in our study (list of sites ranked by latitude: Ukraine - Eastern Sivash, Romania-Danube Delta, Turkey - Kizilirmak Delta and Yumurtalik Lagoons, Iran - Fereydoon Kenar marshes, Morocco - Marais du Bas Loukkos and Sidi Moussa-Oualidia Lagoon, Tunisia - Thyna salt pans, Egypt - Nile River Delta and Lake Qarun, Mauritania - Banc d'Arguin National Park, Senegal/Mauritania - Senegal River Delta, Republic of Sudan - El Saggay Island, Mali - Inner Niger Delta, Niger - Kurfunkura pond and Gaya, Chad - Lake Chad, Nigeria - Hadejia-Nguru wetlands, Burkina Faso - Lake Kompienga, Ethiopia - Lake Debre Zeit, South Sudan - Bargel wetland, Kenya - Lakes around Nairobi, Tanzania - Lake Manyara, Malawi - Lake Chilwa, Zambia - Kafue Flats, Zimbabwe - Lakes Manyame-Chivero, Botswana - Lake Ngami, Mozambique - Massingir Dam and Lake Chuali, South Africa - Barberspan wetland and Strandfontein). A detailed list of sampling sites is provided in the supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046049#pone.0046049.s003" target="_blank">Table S2</a> (map by M. Gély ©Cirad).</p