Qinghai Lake in central China has been at the center of debate on whether wild birds play a role in circulation of highly pathogenic avian influenza virus H5N1. In 2005, an unprecedented epizootic at Qinghai Lake killed more than 6000 migratory birds including over 3000 bar-headed geese (Anser indicus). H5N1 subsequently spread to Europe and Africa, and in following years has re-emerged in wild birds along the Central Asia flyway several times.
To better understand the potential involvement of wild birds in the spread of H5N1, we studied the movements of bar-headed geese marked with GPS satellite transmitters at Qinghai Lake in relation to virus outbreaks and disease risk factors. We discovered a previously undocumented migratory pathway between Qinghai Lake and the Lhasa Valley of Tibet where 93% of the 29 marked geese overwintered. From 2003–2009, sixteen outbreaks in poultry or wild birds were confirmed on the Qinghai-Tibet Plateau, and the majority were located within the migratory pathway of the geese. Spatial and temporal concordance between goose movements and three potential H5N1 virus sources (poultry farms, a captive bar-headed goose facility, and H5N1 outbreak locations) indicated ample opportunities existed for virus spillover and infection of migratory geese on the wintering grounds. Their potential as a vector of H5N1 was supported by rapid migration movements of some geese and genetic relatedness of H5N1 virus isolated from geese in Tibet and Qinghai Lake.
This is the first study to compare phylogenetics of the virus with spatial ecology of its host, and the combined results suggest that wild birds play a role in the spread of H5N1 in this region. However, the strength of the evidence would be improved with additional sequences from both poultry and wild birds on the Qinghai-Tibet Plateau where H5N1 has a clear stronghold.
Citation: Prosser DJ, Cui P, Takekawa JY, Tang M, Hou Y, Collins BM, et al. (2011) Wild Bird Migration across the Qinghai-Tibetan Plateau: A Transmission Route for Highly Pathogenic H5N1. PLoS ONE 6(3): e17622. https://doi.org/10.1371/journal.pone.0017622
Editor: Yan Ropert-Coudert, Institut Pluridisciplinaire Hubert Curien, France
Received: October 28, 2010; Accepted: February 3, 2011; Published: March 9, 2011
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was funded by the United States Geological Survey (Patuxent Wildlife Research Center, Western Ecological Research Center, Alaska Science Center, and Avian Influenza Program); the United Nations FAO, Animal Production and Health Division, EMPRES Wildlife Unit; National Science Foundation Small Grants for Exploratory Research (No. 0713027); and the Chinese Academy of Sciences (No. 2007FY210700, INFO-115-D02, KSCX2-YW-N-063 and 2005CB523007). The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Highly pathogenic avian influenza (HPAI) subtype H5N1 (hereafter H5N1) first emerged in domestic geese of south-eastern China in 1996 ,  and has since become endemic in poultry across much of Eurasia , . Despite extensive eradication and vaccination campaigns, the virus continues to persist, re-emerging across much of its range. Waterfowl belonging to the family Anatidae (ducks, geese, and swans) are natural reservoirs for low pathogenic forms of avian influenza  and are rarely observed with HPAI infection . H5N1 is unique, however, being the first HPAI virus to repeatedly cross the poultry species barrier back to wild birds. The first reports of spillover to wild birds occurred in low numbers of captive waterbirds at a Hong Kong waterfowl park in 2002 . However, it was not until April 2005 that the first large epizootic (more than 6000 birds) occurred in wild species on the remote breeding grounds of Qinghai Lake (QHL), north-western China , . Infections began in bar-headed geese (Anser indicus) soon after their migratory return to QHL, and were followed by infections in great black-headed gulls (Larus ichthyaetus), brown-headed gulls (Larus brunnicephalus), and great cormorants (Phalacrocorax carbo) 10 days later, and ruddy shelducks (Tadorna ferruginea) within 3 weeks . Over half of the reported cases were in bar-headed geese. The QHL outbreak was significant not only because it was the first major H5N1 epizootic in wild populations, but also because it occurred in a region generally lacking poultry , which raised questions about the source of the virus and the ability of wild birds to transport virus over long distances. Following the QHL epizootic, H5N1 expanded beyond Asia, spreading northward and westward into Europe and Africa. All viruses isolated from these regions were subsequently traced back to QHL (clade 2.2) ,  which fueled debate regarding the role wild birds play in H5N1 spread , , , .
QHL is a critical breeding ground and staging area for migratory waterbirds, supporting 150,000 migrants each year , including 15% of the global breeding population of bar-headed geese . It is situated at the intersection of two major flyways: the Central Asian Flyway which extends from western Siberia through central Asia and south to India, and the East Asian Flyway which ranges from Russia through eastern China and south to Australia . QHL holds multiple international designations of ecological importance: Important Bird Area , Key Staging Site for migrating Anatidae (waterfowl) , Wetland of International Importance , and national nature reserve of China. Despite its importance for migratory birds, little is known about wintering and breeding locations for species using the lake during different seasons .
If wild birds had played an integral role in transporting H5N1 to QHL, we hypothesized that the following conditions would be observed: (1) exposure to H5N1 virus on their wintering grounds or migratory stopovers during the northern hemispheric spring migration, (2) significant migration distances and viral shedding before becoming physiologically compromised, and (3) high sequence similarity between virus isolates from areas of exposure and those from QHL. To examine these questions, we sought to develop an improved understanding of the migratory movements, geographic range, habitat use, and overlap of bar-headed geese with poultry in zones of infection , , . In 2007 and 2008, we tracked the migratory movements of 29 bar-headed geese from QHL using GPS satellite telemetry. We compared the spatial ecology of the host with the trajectory of H5N1 across the Qinghai-Tibet Plateau using phylogenetic analysis. This interdisciplinary approach used traditionally disparate tools – satellite telemetry and virological analysis - to assess the role of bar-headed geese in the transmission dynamics of H5N1.
Materials and Methods
We sampled birds at the Qinghai Lake National Nature Reserve (QLNNR), Qinghai Province, in north-western China (36.82 N, 99.81 E). QHL is China's largest saltwater lake (4500 km2) and is located 3200 m above sea level on the eastern edge of the Qinghai-Tibet Plateau. The lake remains frozen November–March, and has a short rainy season from June–August (0.35 m annual average rainfall) . The lake is a closed basin fed by 25 freshwater streams, the majority of which have intermittent flow .
Marking and Virology Sampling
Bar-headed geese return to QHL in late March when temperatures are below freezing and the saline lake is still frozen. During this time, geese use freshwater springs and wetlands surrounding the lake before moving to one of three breeding colony sites in early April. We captured geese during the pre-breeding season at four non-colony sites to reduce disturbance to breeding colonies. Capture and marking occurred in late March 2007–2008 at QLNNR using monofilament noose sets and a net launcher (Coda Enterprises, Mesa, Arizona, USA). We recorded standard morphometrics (mass, flat wing chord, and diagonal tarsus ), age, and sex. Individuals were sampled for avian influenza (cloacal and tracheal swabs, blood serology) following standard procedures . Virology samples were analyzed by the Chinese Academy of Sciences, Wuhan Institute of Virology with the following methods: (1) type A influenza with an ELISA test (Optical Density 630 above 0.23 as positive), (2) H5 subtype with RT-PCR , and (3) H5, H7, H9, and H10 antibodies with hemagglutinin inhibition .
Each bird was equipped with a 45-g, solar-powered GPS platform transmitter terminal (PTT; Microwave Telemetry, Columbia, Maryland, USA). PTTs measured 57×30×20 mm and were attached dorsally with a double-threaded backpack harness made of Teflon ribbon (Bally Ribbon Mills, Bally, Pennsylvania, USA). Transmitter packages weighed on average <2.1% of the bird's body mass. Capture, handling, and marking procedures were approved by the United States Geological Survey Patuxent Wildlife Research Center Animal Care and Use Committee and University of Maryland Baltimore County Institutional Animal Care and Use Committee (Protocol EE070200710).
The PTTs were programmed to take GPS locations every 2 hours, and data were uploaded to satellites every 2 days (CLS America Inc., Maryland, USA). We used ArcGIS 9.3 (Environmental Systems Research Institute, Inc., Redlands, California, USA) and Google Earth 5.0 (Google, Mountain View, California, USA) to plot and analyze telemetry locations.
Telemetry, Movements, and H5N1 Risk Factors
We evaluated chronology, movement rates, seasonal habitat use, wintering locations, and migration stopover sites of the geese. Four seasons of the life cycle of the geese were defined: breeding (including post-breeding molt), fall migration, wintering, and spring migration. Migratory stopovers were defined as areas used during migration in which a goose moved no more than 20 km in a 24 h period. Stopover boundaries were drawn with minimum convex polygons (MCP) since we lacked enough locations to apply kernel home range methods. We calculated cumulative distance and time to complete each migration leg (the path between two consecutive stopover locations) for all geese that completed a minimum of one fall migration. The longest migration leg (km) for each goose was used to estimate the greatest distance travelled between stationary periods. Total migration distance for each individual was defined by the Euclidian distance between its northernmost and southernmost location. We used ArcGIS 9.3 and Hawth'sTools  to conduct these analyses.
Habitat use was evaluated by extracting land cover variables from telemetry locations according to season, pooled across individuals. Habitat use (expressed as a percentage) was based on the number of telemetry locations recorded at each land cover type divided by the total number of locations recorded in a season. Habitat features were derived from 1-km land-cover data produced by the Chinese Academy of Sciences , . We reduced 25 land-cover classes into six for analysis: urban, cropland, grassland, wetland, woodland and other. Unlike conventional land cover data which represent the landscape as a single class per pixel, continuous fields in this dataset included the percent cover of all classes present within a pixel. Spatial overlap between goose movements and poultry farming was examined based on 1-km poultry densities from UNFAO Geonetwork .
Brownian bridge utilization distributions.
To examine relationships between goose movements, landscape features, and potential risk factors associated with H5N1 transmission, we created Brownian Bridge utilization distributions (BBUD)  to describe goose migration patterns. Advantages of using the BBUD model over kernel density estimates for migration analyses is that the BBUD method avoids issues of serial correlation between points, assumes locations are temporally dependent, and explicitly includes length of time between locations in the model. This approach removes subjectivity in estimating temporal weights between intervals of unequal length and uses observed movements and measurable location error to model probability of occurrence. This effect is critical for migration when animals are moving long distances in short periods of time . We created fall and spring migration BBUDs for each individual with Animal Space Use software . An estimate of spring and fall migration routes were calculated as the mean probability of occurrence across individuals . This required defining consistent seasonal dates across individuals from earliest departure and latest arrival dates of marked birds.
Fixed kernel home ranges were created for breeding and winter seasons. Least squares cross-validation was applied to obtain kernel smoothing parameters ,  and utilization distribution models were developed for breeding and wintering with Animal Space Use . Geese were weighted equally by including the same number of locations selected randomly for each individual. Probabilistic utilization distribution (UD) models for the group were created for all birds as a group with a Brownian bridge movement model for the spring and fall migration and fixed kernel home range models for breeding and non-breeding seasons.
HPAI H5N1 outbreaks.
HPAI H5N1 outbreak data for 2003–2009 were obtained from two databases: the People's Republic of China Ministry of Agriculture Prevention and Control of Avian Influenza database  and the UNFAO's Emergency Prevention System for Transboundary Animal and Plant Pests and Diseases (EMPRES-i) database. Outbreak events were cross-checked between the two databases (Table S1) and imported into a GIS framework for analysis.
We conducted a geospatial analysis of bar-headed goose movements in relation to H5N1 risk factors and outbreak locations. This is the first effort we are aware of to compare separate analyses of risk factors for poultry and wild bird outbreaks. Following an information theoretic approach (AIC) to compare outbreak and non-outbreak (random) locations under two sets of a priori defined logistic regression models , we hypothesized that poultry outbreaks would be explained by anthropogenic factors (poultry density, etc.) and wild bird outbreaks by habitat and goose utilization distributions. Covariate predictors in the poultry models included latitude (Lat), bar-headed goose utilization distribution (BHGO UD), poultry density (PD), and cropland (Crop). Wild bird model covariates included Lat, BHGO UD, PD, grassland (Grass), and wetland (Wetl). Non-outbreak locations were represented by 10 random locations for each outbreak  and were drawn proportionally from the spatial extent of outbreaks in each season. A separate a priori univariate logistic regression (BHGO UD) was conducted to examine goose exposure to H5N1 outbreaks. Analyses were performed with the R statistical package  ‘glm’ function (family = binomial, link = −logit).
A classification and regression tree (CART) identified significant model covariates with the Rpart package in R , . Binary trees were built by recursively partitioning explanatory variables into high and low categories that significantly contributed to the prediction of outbreak versus random locations .
Temporal aspects of outbreaks were examined for wild birds and domestic poultry by (a) comparing seasonal differences in numbers of outbreaks of each type (poultry or wild bird) and (b) comparing the observed and expected number of outbreaks based on the length of the season for each outbreak type using Fisher's Exact Tests , . Expected numbers were calculated under the assumption that outbreaks were proportional to the number of days within a seasonal period.
We conducted a phylogenetic analysis to determine genetic relatedness of H5N1 viruses isolated from wild birds and poultry with a focus on the Qinghai-Tibet Plateau. Sequences of HPAI H5N1 were obtained from the Genbank database hosted by the National Center for Biotechnology Information (NCBI) as of 30 September 2010. All phylogenetic analyses were performed with MEGA version 4.0 . A total of 38 sequences of the HA (hemagglutinin) gene were trimmed to a length of 1550 bp and aligned with ClustalW following default settings. A phylogenetic tree was created by applying the neighbor-joining method  and evolutionary distances were computed with the Kimura 2-parameter method . Bootstrapping (×1000) was used to assess the reliability of the tree topology. The tree was rooted to A/goose/Guangdong/1/96 and structured according to the World Health Organization system of avian influenza cladistics.
Marking and Virology
We marked 29 bar-headed geese at QHL; 14 in 2007 and 15 in 2008 (Table S2) including 25 adults (14 male, 11 female) and 4 juveniles (2 male, 2 female). We obtained 20,150 Argos Doppler and 45,021 GPS locations (Table S2). Average transmitter lifespan was 10 months, but some PTTs performed for more than 2 years. GPS data was used for our analyses because they provided a large number of locations and lower spatial error compared with Argos data (15 m versus >100 m error). Twenty-two of the 29 marked geese were sampled for avian influenza virus (sampling materials were unavailable for the first seven birds marked). However, none of the sampled birds tested positive for avian influenza virus.
Migration from Qinghai Lake
We defined the seasonal periods as: breeding (23 May–26 September; 126 days); fall migration (27 September–9 December; 74 days); wintering (10 December–5 March; 87 days); and spring migration (6 March–22 May; 78 days). We mapped migration routes, stopover sites, breeding locations, and wintering locations (Fig. 1).
Migration routes are shown for the 15 individuals (each in unique color) that completed at least one fall migration. White polygons represent stopover areas. Individual 82081 (red path, full path in inset) wintered in India. Individuals 74898 (orange) and 82084 (yellow) are highlighted in Figure 3.
Twenty-seven geese remained at QHL through the breeding season while 2 moved farther north (Table 1): #82084 moved to Hala Lake and #67693 moved to Terkhiin Tsagaan Lake, Mongolia (within 200 km of H5N1 outbreaks in 2005, 2006, and 2009). Short movements (200–250 km) from the breeding grounds to molting locations occurred from 13 June to 19 September with most geese departing QHL in late June. The Zhaling-Eling Lake Region, a high-elevation (4290 m) wetland area 250 km southwest of Qinghai Lake was the most important post-breeding site (Table 1, Fig. 1).
Mean fall departure date was 13 October (range 27 September–30 October). Thirteen geese initiated their migration from Zhaling-Eling Lake region while 2 left from QHL. All geese followed similar routes: in general, they flew along a southwesterly path with stopover locations at Zhamucuo wetland, Selincuo Black-necked Crane Reserve, and Namucuo Lake (Table 1, Fig. 1).
Geese arrived at wintering areas from 29 October–9 December (mean = 9 November). All birds wintered within 100 km of Lhasa except for #82081 which wintered south of the Himalaya at Bhitarkonika National Park in Orissa, eastern India. Lhasa wintering areas included the Pengbo River Basin (25 km northeast of Lhasa), the Lhasa River Basin (south of the cityflowing southwest for 50 km before meeting the Yarlung River), and the Yarlung River Basin (flowing eastward through Gongga then south becoming the Brahmaputra River) (Fig. 1).
The mean spring departure date for the geese was 1 April (range 6 March–April). Spring stopover locations included the Namucuo Lake region and Zhaling-Eling Lakes (Table 1, Fig. 1). The mean breeding arrival date was 29 April (range 28 March–22 May). Seven geese returned to breeding locations used the previous year (six to Qinghai Lake and one to Hala Lake). Two geese used different breeding locations in 2008: #74901 used Longbaotan wetland 570 km southwest of QHL; and #74902 used the Hala Lake region, 260 km northwest of QHL. The spring migration pathway (n = 9, orange line) was broader than the fall route (n = 15, yellow line) despite the fact that it was represented by fewer individuals.
Migration rates and stopover duration.
Ninety-four migration legs were recorded (fall: 65 legs flown by 15 geese; spring: 29 legs flown by 9 geese). Migration was rapid – the longest leg (1158 km) was completed within 5.1 days (Table 2). Individual migrations included an average of two stopovers and were completed in <1 month (fall migration = 26 d; spring migration = 29 d). Migration rates (including time at stopovers) for the fall and the spring did not differ significantly (17 and 14 km/h, respectively; p = 0.23). Distance between QHL and the wintering grounds was 1300 km for 14 geese wintering near Lhasa and 2300 km for goose #82081 that wintered in India.
HPAI H5N1 outbreaks on the Qinghai-Tibet Plateau
Sixteen outbreaks were reported on the Qinghai-Tibet Plateau from 2003–2009; nine in wild birds and seven in poultry (Table S1). Fourteen outbreaks (87.5%) were located within the BHGO UD (Fig. 2a). All poultry cases occurred near Lhasa, whereas wild bird outbreaks were clustered around QHL and between Lhasa and QHL (Fig. 2a). Poultry outbreaks occurred in chickens and wild bird outbreaks occurred in several waterbird species including bar-headed geese, brown-headed gulls, great black-headed gulls, great cormorants, ruddy shelducks and great-crested grebes (Podiceps cristatus). However, the bar-headed goose was the primary species infected during the outbreaks, both in the total numbers killed and the frequency of outbreaks.
Brownian bridge utilization distributions (A) describe fall (yellow; 27 Sep–9 Dec) and spring (orange; 6 Mar–22 May) goose migrations. Fixed kernel home ranges depict (B) population level breeding and post-breeding areas (C) and wintering areas, with only locations near outbreaks shown. Brown shading indicates poultry densities. H5N1 outbreak events in wild birds (white) and poultry (black) are indicated for 2003–2009. Two shading levels indicate isopleths containing 95% (red) and 99% (yellow-orange) of total locations.
Habitat use and overlap with poultry and captive-reared geese.
Habitat use varied seasonally (Table 3). Breeding season habitats included natural wetlands (54% of locations) and grasslands (35%). Grasslands were primarily used during migration (78% fall, 77% spring). In the winter, a combination of natural wetlands (34%) and agricultural fields (39%) were used by the geese (Table 3, Fig. 3). Poultry densities at goose locations were near zero during all seasons except the winter which averaged 35.8 poultry head per km2 (Table 3). Potential exposure of geese to H5N1 occurred on the wintering grounds as was evidenced by their spatial and temporal overlap with a poultry outbreak in January 2008 (Fig. 3a) and with a bar-headed goose captive-rearing facility in 2009 (Fig. 3b and 4).
(A) Winter movements for goose 74898 (3 November 2007–2 April 2008; 1205 locations) in relation to a confirmed HPAI H5N1 outbreak in chickens on 21 January, 2008 (black circle). (B) Winter movements (9 November 2008–5 April 2009; 961 locations) for goose 82084 in relation to a captive bar-headed goose farm (red circle).
(A) Captive bar-headed geese in Gonggar County, Tibet as shown in a December 2007 China Tibet Information Center article (Wu 2007). (B) Wild bar-headed geese (foreground) shown in close proximity to a captive bar-headed goose farm (blue building in background) in a January 2007 photo from an anonymous source. Approximately 250 bar-headed geese were counted in outdoor net pens attached to the building (out of view in photo B). Approximate location of this farm is shown in a red circle (Fig. 3b).
Poultry outbreak risk factors.
The AIC analysis for poultry outbreaks indicated two competing top models (Table 4): Model 1 included poultry density and cropland (AIC weight wi = 0.72) while Model 2 included poultry density, cropland, latitude, and BHGO UD (wi = 0.26). The top model indicated that domestic outbreak locations were explained by measures of poultry density and cropland area. The CART analysis separated outbreaks into those above 66 poultry per km2 and above 0.3 ha of cropland per km2 (Fig. 5a). Although the BHGO UD was not a strong indicator in predicting locations of poultry outbreaks, exposure of BHGO to H5N1 virus via outbreaks in poultry on the wintering grounds was confirmed (a priori univariate logistic regression, BHGO UD, p = 0.032).
Numbers of random points (numerator), outbreak locations (denominator), and percentage of total sample are reported at each terminal node.
Wild bird outbreak risk factors.
Two models best fit the data to explain locations of H5N1 outbreaks in wild birds on the Qinghai-Tibet Plateau. The top model (wi = 0.70) included latitude and wetlands while the second-ranked model (wi = 0.22) included latitude only (Table 4). CART results indicated differences at 36.14°N with wetland area larger than 0.3 ha (Fig. 5b). Contrary to our hypothesis, BHGO UD was not a significant predictor for wild bird outbreaks which tended to occur in remote regions. However, outbreaks were concentrated north of 36°N (near QHL), with fewer outbreaks located between QHL and Lhasa (Fig. 2a) where the BHGO UD is geographically broad and with lower use per pixel. In this region, the BHGO UD does not provide probabilities sensitive to predicting outbreak versus non-outbreak locations given the low number of total outbreaks. Thus, we conclude that while bar-headed geese are an important species in H5N1 events , , , , the BHGO UD is not a strong predictor across the Qinghai-Tibet Plateau, except near QHL where there are high concentrations of this species on the breeding grounds and greater number of outbreaks.
Timing of outbreaks.
We found a temporal lag between peak seasons for poultry and wild bird outbreaks on the Qinghai-Tibet Plateau (Fisher's Exact Test, p = 0.008, Fig. 6a). Poultry outbreaks occurred during the winter, spring and breeding seasons, whereas wild bird outbreaks were not reported until the spring and breeding months (Fig. 6b, Table S1). More poultry outbreaks were found during the winter and the spring than expected (p = 0.079) and subsequently during the spring and the breeding seasons for wild birds (p = 0.017). In contrast, fewer outbreaks than expected occurred in the breeding and the fall for poultry, and the fall and the winter for wild birds (Fig. 6b). The first H5N1 outbreak reported on the Qinghai-Tibet Plateau occurred in poultry near Lhasa in February 2004, followed in the spring (Apr–Jun) of 2005 with the epizootic at QHL. Both results from Fig. 6 and timing of the first two outbreaks reported on the Qinghai-Tibet Plateau suggest initial outbreaks in poultry, followed by outbreaks in wild birds.
(A) Observed versus expected values differed for both poultry and wild bird outbreaks when tested separately (Fisher Exact Test, poultry P = 0.079, wild P = 0.017). (B) Temporal distribution of wild bird outbreaks differed from domestic bird outbreaks (Fishers Exact Test, P = 0.008). Expected numbers were calculated under the assumption that outbreaks are proportional to the number of days within the seasonal period (Winter = 87 d, Spring Migration = 78 d, Breeding = 126 d, and Fall Migration = 74 d).
Phylogenetic analyses indicated that HPAI H5N1 isolates from clades 2.2 and 2.3 infected migratory waterfowl including the bar-headed goose and poultry on the Qinghai-Tibet Plateau. The directionality of virus transmission from domestic to wild birds or vice-versa was however, difficult to ascertain based on the limited number of sequences available within this geographic region. Viruses isolated from bar-headed geese formed distinct sub-clades based on year of outbreak. For instance, isolates from 2005 grouped within sub-clade 2.2.1, and similarly isolates from 2006 and 2008 grouped within sub-clade 2.2.2 and 2.3.2, respectively (Fig. 7). This pattern suggested reintroduction of the virus into bar-headed goose populations, rather than continuous circulation or persistence. Isolates from A/bar-headed goose/Tibet/8/06 and the A bar-headed goose/Qinghai/F/06 formed a monophyletic grouping within sub-clade 2.2.2 and showed high bootstrap support (80%). The evolutionary distance between these two isolates was 0.008 base substitutions per site, lower than the overall average (0.046) for the 38 isolates representative of HPAI H5N1 divergence since 1996.
We hypothesized that if bar-headed geese were a significant contributor in the spread of H5N1 to QHL, certain conditions would have had to occur: (1) exposure of geese to virus sources prior to arriving at QHL, (2) onset of migration and viral shedding before individuals became physiologically compromised, and (3) evidence of genetic relatedness between isolates from virus source regions frequented by geese and QHL. Our findings indicate that these conditions were met; however, we discuss caveats in our findings based on the most current H5N1 disease ecology.
Both the timing of migratory movements and distribution of wintering habitat used by geese around Lhasa exposed them to H5N1 sources (Condition 1) as was evidenced in four ways: (a) location of outbreaks within the BHGO UD (Fig. 2a); (b) detailed movements showing proximity in timing and spatial distribution between marked geese, H5N1 outbreak in chickens, and a captive bar-headed goose farm (Fig. 3 and 4); (c) temporal analysis showing peaks in poultry outbreaks followed by peaks in wild bird outbreaks (Fig. 6); (d) and a significant a priori logistic regression between BHGO UD and poultry outbreaks (Table 5a). The evidence for goose exposure to H5N1 sources on the Lhasa wintering grounds is consistent. What was unexpected is the discovery of Lhasa as a major wintering area for geese from QHL and the potential of the region as an important H5N1 transmission route – both of which have conservation implications for this species. Prior to this work, little was known about migration of breeding and staging waterbirds at QHL , . Of 800 bar-headed goose banding records from the lake in 1987 and 1988, only three returns have been reported (one each in northeast and southwest India  and one in Chittagong, southern Bangladesh ). Bishop et al.  reported that 25% of the global bar-headed goose population winters on the southern Qinghai-Tibet Plateau, however, breeding connectivity for these individuals was previously unknown. Hypotheses regarding the epidemiology of the event at QHL suggested by previous authors , , ,  were based on inadequate migration data available at the time, and associated models therefore did not examine Tibet as a potential H5N1 transmission link to QHL. For example, Chen et al.  and Liang et al.  hypothesized that wild birds transported the virus to QHL from Poyang Lake, southeastern China, where 6 apparently healthy wild ducks (species not reported) tested positive for HPAI H5N1. Results from our study and related work at Poyang Lake , which examined migration patterns of 9 waterfowl species (n = 62), showed no evidence of migratory connectivity between the 2 lakes located in separate flyways. These findings affirmed the importance for including knowledge of host ecology to inform the debate on wild birds and HPAI transmission.
A clear understanding of poultry farming in this remote region is also critical to assessing H5N1 transmission potential. There has been confusion over whether captive bar-headed goose farms exist at QHL, and whether such farms provided introduction of H5N1 virus to this region generally lacking poultry . Through a combination of local investigation, communication with experts, and detailed review of the original blog postings (in Chinese) regarding the farms, we have learned that the farms are not located at QHL, but instead exist 1200 km south of QHL near Lhasa. These conclusions are independently supported by . The largest of the captive bar-headed goose breeding facilities discussed above is included in our study (Fig. 3b and 4), where one of our marked geese spent the winter foraging in the local fields and wetlands.
We found evidence suggesting that migratory movement could occur before the virus impaired a bird's ability to migrate (Condition 2). Recent challenge studies identified the average duration of asymptomatic virus shedding to be 6.5. days in experimentally infected bar-headed geese (A/whooper swan/Mongolia/244/2005H5N1) . Geese from QHL averaged 380 km per day (range 294 km in 1.3 days to 1158 km in 5.1 days; Table 2), indicating a capacity to move the distance between QHL and Lhasa within the asymptomatic period of virus shedding. Migration for most individuals, however, did not occur without time spent at stopover locations, which increased total migration time to an average of one month. Thus, while movement of virus the entire distance (within the asymptomatic period) is possible, as was observed for one bar-headed goose, a more likely mode of transmission is through a relay effect . This would occur if a virus is transported to stopover locations where it can spread among individuals within concentrated areas of feeding and roosting ,  or through environmental persistence , , and then be forwarded to newly infected individuals along the migratory pathway. We have evidence that stopover locations used by marked geese in this study (Table 1, Fig. 1) are important both for bar-headed geese as well as a number of other important waterbird species (Y.S. Hou, unpubl. data). In addition, recent studies ,  have indicated that mallards (Anas platyrhynchos) with previous exposure to homologous LPAI viruses may remain healthy enough to migrate, and this might apply to other waterfowl such as bar-headed geese. The first H5N1 outbreak on the Plateau occurred in chickens in Lhasa in February, 2004 (Table S1), and was followed by the QHL wild bird epizootic in spring of 2005. Unfortunately, none of the seven reported poultry outbreaks have sequences available in open sources such as GenBank. Release of these data would improve our analyses by allowing us to test relationships between isolates from poultry and wild birds from the 2 regions. However, Li et al. , in an updated analysis of H5N1 virus evolution in China, includes one sequence from a 2008 outbreak in chickens from a live-bird market (CH/TB/6/08). Here they discovered high sequence similarity between the 2005 QHL epizootic (bar-headed goose isolates, BHG/QH/3/05) and chicken isolates from Tibet in one of the eight H5N1 genes (PB2), supporting a connection between the Lhasa and QHL outbreaks (Condition 3). In addition, our phylogenetic analysis of publicly available sequences included 2 isolates from wild birds in Tibet: a bar-headed goose (A/BHG/TB/8/06) and great cormorant (A/GC/TB/12/06), both from 2006. The analysis indicated that geese were first infected by HPAI H5N1 belonging to clade 2.2 that emerged at QHL during 2005. Since this time, the virus has continued to infect bar-headed geese periodically within the Central Asian Flyway, most recently during the 2009 outbreak in Mongolia. Goose isolates from the QHL and Tibet outbreaks in 2006 formed a monophyletic group within sub-clade 2.2.2 and were closely related, providing additional evidence that migratory birds were agents of transmission between these two outbreak sites in China. Further analysis of isolates in combination with remaining unreleased sequences would help elucidate the route by which viruses moved from southeastern China to QHL including the directionality of virus transmission between poultry and wild birds , , , .
Lhasa provides a unique situation for intermixing and potential transfer of disease between wild and domestic birds. Each winter, the Lhasa region experiences increased concentrations of humans, poultry, and wild birds when nomadic herders return to populated centers, chicken production peaks preceding the Chinese New Year festivities in late January , and up to 50% of the global population of bar-headed geese winter in sheltered river valleys surrounding Lhasa , , . H5N1 outbreaks in domestic birds spiked in frequency under such winter conditions  followed by an increase in wild bird outbreaks during the spring and breeding seasons (Fig. 6).
The H5N1 situation involving wild birds in the Central Asian Flyway is unique relative to results from studies in the East Asian Flyway (EAF) along the Pacific coast , . The EAF boasts some of the world's most productive poultry systems including rice-paddy duck farming in China and parts of Southeast Asia . Whooper swans marked in eastern Mongolia within the EAF demonstrated spatial proximity to poultry outbreaks in Korea and north-eastern China; however, a lack of correspondence in timing and micro-habitat use precluded the likelihood of transmission between the two groups . Eight duck species marked at Poyang Lake in south-eastern China also showed a temporal mismatch between H5N1 outbreaks and arrival of wild ducks to the wintering grounds . Poultry production in the Central Asian Flyway is extensive in the south, particularly in India and Bangladesh, and limited to absent in the central and northern sections (Tibet and northward). H5N1 outbreaks along the flyway mirror human and poultry densities whereby more domestic bird outbreaks occur in the south and wild bird outbreaks in the sparsely populated north . The Qinghai-Tibet Plateau lies within the transition zone along this gradient, and it appears that bar-headed geese may be an important vector in H5N1 spread as evidenced by the size of the infected population at Qinghai Lake , . Our study identifies QHL and Lhasa as important linkages between wild and domestic transmission of H5N1 and provides new supporting information regarding the role of wild birds in long distance spread of this virus. Further investigation of wild birds and H5N1 transmission within the Central Asian Flyway will increase our understanding of how wild birds may contribute to virus circulation and the unique pattern of outbreaks in this remote region.
Confirmed HPAI H5N1 outbreaks in wild birds and domestic poultry on the Qinghai–Tibet Plateau, 2003–2009.
The authors thank Sue Haseltine, Patti Bright, Steve Schwarzbach, and Judd Howell of USGS and Joseph Domenech and Juan Lubroth of FAO for support of this project. We are grateful to the QLNNR staff, Qinghai Forestry Bureau (Sandan Li), Chinese Academy of Sciences (Xudong Hu, Lianling Hu, Ning Kong, Ze Luo), U. S. Embassy (Bill Chang, Dan Jassem, Qihong Qian), and USGS (Shane Heath, Kyle Spragens) for field and logistical support. We thank Chris Hamilton and Julio Pinto for providing outbreak data from the EMPRES-i database; and Julie Yee (USGS WERC) and Shay Howlin (West-Inc) for statistical advice. Mike Erwin and anonymous reviewers provided helpful comments to strengthen earlier versions of this manuscript. We thank Mary Anne Bishop (International Crane Foundation) for providing local information on the Lhasa region. The use of trade, product, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the U.S. Government.
Conceived and designed the experiments: DJP JYT SHN. Performed the experiments: DJP PC JYT MT YH BMC BY TL YL FL SG ZX YH YZ DCD WMP. Analyzed the data: DJP BMC NJH DCD WMP. Contributed reagents/materials/analysis tools: DJP PC JYT MT YH BMC BY TL YL FL SG ZX YH YZ DCD WMP SHN. Wrote the paper: DJP NJH JYT SHN.
- 1. Guo Y, Xu X, Wan X (1998) Genetic characterization of an avian influenza A (H5N1) virus isolated from a sick goose in China. Zhonghua Shi Xan He Lin Chaung Bing Du Xue Za Zhi 12: 322–325.Y. GuoX. XuX. Wan1998Genetic characterization of an avian influenza A (H5N1) virus isolated from a sick goose in China.Zhonghua Shi Xan He Lin Chaung Bing Du Xue Za Zhi12322325(in Chinese). (in Chinese).
- 2. Xu XY, Subbarao K, Cox NJ, Guo YJ (1999) Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: Similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261: 15–19.XY XuK. SubbaraoNJ CoxYJ Guo1999Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: Similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong.Virology2611519
- 3. OIE (2010) Update on highly pathogenic avian influenza in animals: Type H5 and H7. OIE website. OIE2010Update on highly pathogenic avian influenza in animals: Type H5 and H7. OIE website.Available: http://www.oie.int/downld/AVIAN%20INFLUENZA/A_AI-Asia.htm. Accessed 2010 Oct 5. Available: http://www.oie.int/downld/AVIAN%20INFLUENZA/A_AI-Asia.htm. Accessed 2010 Oct 5.
- 4. Sims LD, Brown IH (2008) Multicontinental epidemic of H5N1 HPAI virus (1996–2007). In: Swayne DE, editor. Avian Influenza. Ames, Iowa, USA: Blackwell Publishing. pp. 251–286.LD SimsIH Brown2008Multicontinental epidemic of H5N1 HPAI virus (1996–2007).DE SwayneAvian InfluenzaAmes, Iowa, USABlackwell Publishing251286
- 5. Alexander DJ (2000) A review of avian influenza in different bird species. Veterinary Microbiology 74: 3–13.DJ Alexander2000A review of avian influenza in different bird species.Veterinary Microbiology74313
- 6. Alexander DJ, Capua I (2008) Avian influenza in poultry. Worlds Poultry Science Journal 64: 513–531.DJ AlexanderI. Capua2008Avian influenza in poultry.Worlds Poultry Science Journal64513531
- 7. Ellis TM, Bousfield RB, Bissett LA, Dyrting KC, Luk GS, et al. (2004) Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathology 33: 492–505.TM EllisRB BousfieldLA BissettKC DyrtingGS Luk2004Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002.Avian Pathology33492505
- 8. Chen H, Smith GJ, Zhang SY, Qin K, Wang J, et al. (2005) Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436: 191–192.H. ChenGJ SmithSY ZhangK. QinJ. Wang2005Avian flu: H5N1 virus outbreak in migratory waterfowl.Nature436191192
- 9. Liu J, Xiao H, Lei F, Zhu Q, Qin K, et al. (2005) Highly pathogenic H5N1 influenza virus infection in migratory birds. Science 309: 1206.J. LiuH. XiaoF. LeiQ. ZhuK. Qin2005Highly pathogenic H5N1 influenza virus infection in migratory birds.Science3091206
- 10. Chen HL, Li YB, Li ZJ, Shi JZ, Shinya K, et al. (2006) Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. Journal of Virology 80: 5976–5983.HL ChenYB LiZJ LiJZ ShiK. Shinya2006Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China.Journal of Virology8059765983
- 11. FAO (2007) Gridded Livestock of the World 2007. Rome: 131 p.FAO2007Gridded Livestock of the World 2007Rome131
- 12. Chen H, Smith GJD, Li KS, Wang J, Fan XH, et al. (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 of America 103: 2845–2850.H. ChenGJD SmithKS LiJ. WangXH Fan2006Establishment of multiple sublineages of H5N1 influenza virus in Asia: Implications for pandemic control.Proceedings of The National Academy of Sciences of the United States of America10328452850
- 13. Gauthier-Clerc M, Lebarbenchon C, Thomas F (2007) Recent expansion of highly pathogenic avian influenza H5N1: a critical review. Ibis 149: 202–214.M. Gauthier-ClercC. LebarbenchonF. Thomas2007Recent expansion of highly pathogenic avian influenza H5N1: a critical review.Ibis149202214
- 14. Fouchier RAM, Munster VJ, Keawcharoen J, Osterhaus A, Kuiken T (2007) Virology of avian influenza in relation to wild birds. Journal of Wildlife Diseases 43: S7–S14.RAM FouchierVJ MunsterJ. KeawcharoenA. OsterhausT. Kuiken2007Virology of avian influenza in relation to wild birds.Journal of Wildlife Diseases43S7S14
- 15. Normile D (2006) Avian influenza. Evidence points to migratory birds in H5N1 spread. Science 311: 1225.D. Normile2006Avian influenza. Evidence points to migratory birds in H5N1 spread.Science3111225
- 16. Weber TP, Stilianakis NI (2008) Migratory birds, the H5N1 influenza virus and the scientific method. Virology Journal 5: TP WeberNI Stilianakis2008Migratory birds, the H5N1 influenza virus and the scientific method.Virology Journal5
- 17. Li L (2001) Survey results and new records of waterfowl at Qinghai Lake. China Crane News 5: 36–37.L. Li2001Survey results and new records of waterfowl at Qinghai Lake.China Crane News53637
- 18. Miyabayashi Y, Mundkur T (1999) Atlas of key sites for Anatidae in the East Asian flyway. 148 p.Y. MiyabayashiT. Mundkur1999Atlas of key sites for Anatidae in the East Asian flyway.Wetlands International - Japan, Tokyo, and Wetlands International - Asia Pacific, Kuala Lumpur. Japanese Association for Wild Geese Protection website. Available: http://www.jawgp.org/anet/aaa1999. Accessed 2010 Aug 16. 148 Japanese Association for Wild Geese Protection website. Available: http://www.jawgp.org/anet/aaa1999. Accessed 2010 Aug 16.
- 19. Boere GC, Galbraith CA, Stroud DA (2006) Waterbirds Around the World. Edinburgh, UK: The Stationary Office. 960 p.GC BoereCA GalbraithDA Stroud2006Waterbirds Around the WorldEdinburgh, UKThe Stationary Office960
- 20. BirdLife International (2009) Directory of Important Bird Areas in China (Mainland): Key Sites for Conservation;. In: Chan S, Crosby M, So S, Wang D, Cheung F, et al., editors. Cambridge, United Kingdom: 231 p.BirdLife International2009Directory of Important Bird Areas in China (Mainland): Key Sites for Conservation;S. ChanM. CrosbyS. SoD. WangF. CheungCambridge, United Kingdom231
- 21. Ramsar Convention Secretariat (2007) Designating Ramsar sites: The strategic framework and guidelines for the future development of the List of Wetlands of International Importance. Gland, Switzerland: Ramsar Convention Secretariat. 114 p.Ramsar Convention Secretariat2007Designating Ramsar sites: The strategic framework and guidelines for the future development of the List of Wetlands of International ImportanceGland, SwitzerlandRamsar Convention Secretariat114
- 22. Muzaffar SB, Ydenberg RC, Jones IL (2006) Avian influenza: An ecological and evolutionary perspective for waterbird scientists. Waterbirds 29: 243–257.SB MuzaffarRC YdenbergIL Jones2006Avian influenza: An ecological and evolutionary perspective for waterbird scientists.Waterbirds29243257
- 23. Yasue M, Feare CJ, Bennun L, Fiedler W (2006) The epidemiology of H5N1 avian influenza in wild birds: Why we need better ecological data. Bioscience 56: 923–929.M. YasueCJ FeareL. BennunW. Fiedler2006The epidemiology of H5N1 avian influenza in wild birds: Why we need better ecological data.Bioscience56923929
- 24. Xu H, Hou ZH, Ai L, Tan LC (2007) Precipitation at Lake Qinghai, NE Qinghai-Tibet Plateau, and its relation to Asian summer monsoons on decadal/interdecadal scales during the past 500 years. Palaeogeography Palaeoclimatology Palaeoecology 254: 541–549.H. XuZH HouL. AiLC Tan2007Precipitation at Lake Qinghai, NE Qinghai-Tibet Plateau, and its relation to Asian summer monsoons on decadal/interdecadal scales during the past 500 years.Palaeogeography Palaeoclimatology Palaeoecology254541549
- 25. Rhode D, Haizhou M, Madsen DB, Brantingham PJ, Forman SL, et al. (2010) Paleoenvironmental and archaeological investigations at Qinghai Lake, western China: Geomorphic and chronometric evidence of lake level history. Quaternary International 218: 29–44.D. RhodeM. HaizhouDB MadsenPJ BrantinghamSL Forman2010Paleoenvironmental and archaeological investigations at Qinghai Lake, western China: Geomorphic and chronometric evidence of lake level history.Quaternary International2182944
- 26. Dzubin A, Cooch EG (1992) Measurements of geese: General field methods. Sacramento: California Waterfowl Association. A. DzubinEG Cooch1992Measurements of geese: General field methodsSacramentoCalifornia Waterfowl Association
- 27. FAO (2007) Wild Birds and Avian Influenza: an introduction to applied field research and disease sampling techniques. FAO2007Wild Birds and Avian Influenza: an introduction to applied field research and disease sampling techniques.Rome. Food and Agriculture Organization website. Available: www.fao.org/avianflu). Accessed 2010 Aug 16. Rome. Food and Agriculture Organization website. Available: www.fao.org/avianflu). Accessed 2010 Aug 16.
- 28. Fouchier RAM, Bestebroer TM, Herfst S, Van der Kemp L, Rimmelzwaan GF, et al. (2000) Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. Journal of Clinical Microbiology 38: 4096–4101.RAM FouchierTM BestebroerS. HerfstL. Van der KempGF Rimmelzwaan2000Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene.Journal of Clinical Microbiology3840964101
- 29. OIEWorld Organization of Animal Health, editor (2004) Manual of Diagnostics Tests and Vaccines for Terrestrial Animals, 5th Edition. Chapter 2.7.12. Avian Influenza. Paris, France: Updated May 2005. pp. 465–481.OIEWorld Organization of Animal Health, editor2004Manual of Diagnostics Tests and Vaccines for Terrestrial Animals, 5th Edition. Chapter 2.7.12.Avian InfluenzaParis, FranceUpdated May 2005465481
- 30. Beyer HL (2004) Hawth's analysis tools for ArcGIS. HL Beyer2004Hawth's analysis tools for ArcGIS.Spatial Ecology website. Available: http://wwwspatialecologycom/htools. Accessed 2010 Feb 10. Spatial Ecology website. Available: http://wwwspatialecologycom/htools. Accessed 2010 Feb 10.
- 31. Liu J, Liu M, Deng X, Zhuang D, Zhang Z, et al. (2002) The land use and land cover change database and its relative studies in China. Journal of Geographical Sciences 12: 275–282.J. LiuM. LiuX. DengD. ZhuangZ. Zhang2002The land use and land cover change database and its relative studies in China.Journal of Geographical Sciences12275282
- 32. Liu JY, Zhuang DF, Luo D, Xiao X (2003) Land-cover classification of China: integrated analysis of AVHRR imagery and geophysical data. International Journal Of Remote Sensing 24: 2485–2500.JY LiuDF ZhuangD. LuoX. Xiao2003Land-cover classification of China: integrated analysis of AVHRR imagery and geophysical data.International Journal Of Remote Sensing2424852500
- 33. Horne JS, Garton EO, Krone SM, Lewis JS (2007) Analyzing animal movements using Brownian bridges. Ecology 88: 2354–2363.JS HorneEO GartonSM KroneJS Lewis2007Analyzing animal movements using Brownian bridges.Ecology8823542363
- 34. Horne JS, Garton EO (2009) Animal Space Use Version 1.3. JS HorneEO Garton2009Animal Space Use Version 1.3.College of Natural Resources University of Idaho website. Available: http://wwwcnruidahoedu/population_ecology/animal_space_use. Accessed 2010 April 13. College of Natural Resources University of Idaho website. Available: http://wwwcnruidahoedu/population_ecology/animal_space_use. Accessed 2010 April 13.
- 35. Horne JS, Garton EO (2006) Selecting the best home range model: An information-theoretic approach. Ecology 87: 1146–1152.JS HorneEO Garton2006Selecting the best home range model: An information-theoretic approach.Ecology8711461152
- 36. Ackerman JT, Takekawa JY, Orthmeyer DL, Fleskes JP, Yee JL, et al. (2006) Spatial use by wintering greater white-fronted geese relative to a decade of habitat change in California's Central Valley. Journal Of Wildlife Management 70: 965–976.JT AckermanJY TakekawaDL OrthmeyerJP FleskesJL Yee2006Spatial use by wintering greater white-fronted geese relative to a decade of habitat change in California's Central Valley.Journal Of Wildlife Management70965976
- 37. MOA (2009) Ministry of Agriculture of the People's Republic of China, Prevention and control of avian influenza. MOA2009Ministry of Agriculture of the People's Republic of China, Prevention and control of avian influenza.Ministry of Agriculture website. Available (in Chinese): http://wwwagrigovcn/ztzl/fkqlg/yqfb/. Accessed 2010 Feb 8. Ministry of Agriculture website. Available (in Chinese): http://wwwagrigovcn/ztzl/fkqlg/yqfb/. Accessed 2010 Feb 8.
- 38. Burnham KP, Anderson DR (2002) Information and likelihood theory: A basis for model selection and inference. Model selection and multimodel inference: A practical approach. New York, New York: Springer-Verlag. pp. 49–79.KP BurnhamDR Anderson2002Information and likelihood theory: A basis for model selection and inference. Model selection and multimodel inference: A practical approachNew York, New YorkSpringer-Verlag4979
- 39. Manly BFJ, McDonald LL, Thomas DL, McDonald TL, Erickson WE (2002) Resource selection by animals: Sample design and analysis for field studies. Dordrecht: Kluwer Academic Publishers. BFJ ManlyLL McDonaldDL ThomasTL McDonaldWE Erickson2002Resource selection by animals: Sample design and analysis for field studiesDordrechtKluwer Academic Publishers
- 40. R (2010) The R Project for Statistical Computing.R2010The R Project for Statistical Computing.R Project website. Available: http://www.R-project.org. Accessed 2010 July 12. R Project website. Available: http://www.R-project.org. Accessed 2010 July 12.
- 41. Therneau TM, Atkinson EJ (1997) An introduction to recursive partitioning using the RPART routines. Mayo Clinic, Section of Statistics. TM TherneauEJ Atkinson1997An introduction to recursive partitioning using the RPART routinesMayo Clinic, Section of Statistics
- 42. Breiman L, Friedman JH, Olshen RA, Stone CJ (1983) Classification and regression trees. Belmont, CA: Wadsworth. L. BreimanJH FriedmanRA OlshenCJ Stone1983Classification and regression treesBelmont, CAWadsworth
- 43. Agresti A (1992) A survey of exact inference for contingency tables. Statistical Science 7: 131–177.A. Agresti1992A survey of exact inference for contingency tables.Statistical Science7131177
- 44. Fisher RA (1954) Statistical methods for research workers. Edinburgh: Oliver and Boyd. 356 p.RA Fisher1954Statistical methods for research workersEdinburghOliver and Boyd356
- 45. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596–1599.K. TamuraJ. DudleyM. NeiS. Kumar2007MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.Molecular Biology and Evolution2415961599
- 46. Saitou N, Nei M (1987) The neighbour-joining method: a new method for recontructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.N. SaitouM. Nei1987The neighbour-joining method: a new method for recontructing phylogenetic trees.Molecular Biology and Evolution4406425
- 47. Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120.M. Kimura1980A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences.Journal of Molecular Evolution16111120
- 48. Lei FM, Tang S, Zhao DL, Zhang XW, Kou Z, et al. (2007) Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006. Avian Diseases 51: 568–572.FM LeiS. TangDL ZhaoXW ZhangZ. Kou2007Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006.Avian Diseases51568572
- 49. Zhang G-G, Liu D-P, Jiang H-X, Hou Y-Q, Dai M, et al. (2008) The current status of waterbirds after Avian Influenza outbreak at Qinghai Lake, China. Chinese Journal of Zoology 43: 51–56.G-G ZhangD-P LiuH-X JiangY-Q HouM. Dai2008The current status of waterbirds after Avian Influenza outbreak at Qinghai Lake, China.Chinese Journal of Zoology435156
- 50. Uttangi J (1987) Wintering status and site loyalty of bar-headed goose, Anser indicus, in areas of Dharwad District, Karnataka, India. Journal of Bombay Natural History Society 10: 22–24.J. Uttangi1987Wintering status and site loyalty of bar-headed goose, Anser indicus, in areas of Dharwad District, Karnataka, India.Journal of Bombay Natural History Society102224
- 51. National Bird Banding Center P.R. China (1987) Chinese Bird Banding Almanac 1982–1985. 197 p.National Bird Banding Center P.R. China1987Chinese Bird Banding Almanac 1982–1985.Gansu Technology Publisher, Lanzhou in Chinese. 197 Gansu Technology Publisher, Lanzhou in Chinese.
- 52. Bishop MA, Yanling S, Zhouma C, Binyuan G (1997) Bar-headed Geese Anser indicus wintering in south-central Tibet. Wildfowl 48: 118–126.MA BishopS. YanlingC. ZhoumaG. Binyuan1997Bar-headed Geese Anser indicus wintering in south-central Tibet.Wildfowl48118126
- 53. Liang L, Xu B, Chen Y, Liu Y, Cao W, et al. (2010) Combining Spatial-Temporal and Phylogenetic Analysis Approaches for Improved Understanding on Global H5N1 Transmission. PLoS ONE 5: e13575.L. LiangB. XuY. ChenY. LiuW. Cao2010Combining Spatial-Temporal and Phylogenetic Analysis Approaches for Improved Understanding on Global H5N1 Transmission.PLoS ONE5e13575
- 54. Kilpatrick AM, Chmura AA, Gibbons DW, Fleischer RC, Marra PP, et al. (2006) Predicting the global spread of H5N1 avian influenza. Proceedings of The National Academy of Sciences of the United States of America 103: 19368–19373.AM KilpatrickAA ChmuraDW GibbonsRC FleischerPP Marra2006Predicting the global spread of H5N1 avian influenza.Proceedings of The National Academy of Sciences of the United States of America1031936819373
- 55. Takekawa JY, Newman S, Xiao X, Prosser DJ, Spragens KA, et al. (2010) Migration of waterfowl in the East Asian Flyway and spatial relationship to HPAI H5N1 outbreaks. Avian Diseases 54: 466–476.JY TakekawaS. NewmanX. XiaoDJ ProsserKA Spragens2010Migration of waterfowl in the East Asian Flyway and spatial relationship to HPAI H5N1 outbreaks.Avian Diseases54466476
- 56. Butler D (2006) Blogger reveals China's migratory goose farms near site of flu outbreak. Nature 441: 263–263.D. Butler2006Blogger reveals China's migratory goose farms near site of flu outbreak.Nature441263263
- 57. Feare CJ, Kato T, Thomas R (2010) Captive Rearing and Release of Bar-headed Geese (Anser indicus) in China: A Possible HPAI H5N1 Virus Infection Route to Wild Birds. Journal of Wildlife Diseases 46: 1340–1342.CJ FeareT. KatoR. Thomas2010Captive Rearing and Release of Bar-headed Geese (Anser indicus) in China: A Possible HPAI H5N1 Virus Infection Route to Wild Birds.Journal of Wildlife Diseases4613401342
- 58. Brown JD, Stallknecht DE, Swayne DE (2008) Experimental infection of swans and geese with highly pathogenic avian influenza virus (H5N1) of Asian lineage. Emerging Infectious Diseases 14: 136–142.JD BrownDE StallknechtDE Swayne2008Experimental infection of swans and geese with highly pathogenic avian influenza virus (H5N1) of Asian lineage.Emerging Infectious Diseases14136142
- 59. Gaidet N, Cappelle J, Takekawa JY, Prosser DJ, Iverson SA, et al. (2010) Potential spread of highly pathogenic avian influenza H5N1 by wildfowl: Dispersal ranges and rates determined from large-scale satellite telemetry. Journal of Applied Ecology. N. GaidetJ. CappelleJY TakekawaDJ ProsserSA Iverson2010Potential spread of highly pathogenic avian influenza H5N1 by wildfowl: Dispersal ranges and rates determined from large-scale satellite telemetry.Journal of Applied Ecology
- 60. Alexander DJ (2007) An overview of the epidemiology of avian influenza. Vaccine 25: 5637–5644.DJ Alexander2007An overview of the epidemiology of avian influenza.Vaccine2556375644
- 61. Brown JD, Swayne DE, Cooper RJ, Burns RE, Stallknecht DE (2007) Persistence of H5 and H7 avian influenza viruses in water. Avian Diseases 51: 285–289.JD BrownDE SwayneRJ CooperRE BurnsDE Stallknecht2007Persistence of H5 and H7 avian influenza viruses in water.Avian Diseases51285289
- 62. Stallknecht DE, Kearney MT, Shane SM, Zwank PJ (1990) Effects Of Ph, Temperature, And Salinity On Persistence Of Avian Influenza-Viruses In Water. Avian Diseases 34: 412–418.DE StallknechtMT KearneySM ShanePJ Zwank1990Effects Of Ph, Temperature, And Salinity On Persistence Of Avian Influenza-Viruses In Water.Avian Diseases34412418
- 63. Stallknecht DE, Shane SM, Kearney MT, Zwank PJ (1990) Persistence of avian influenza viruses in water. Avian Diseases 34: 406–411.DE StallknechtSM ShaneMT KearneyPJ Zwank1990Persistence of avian influenza viruses in water.Avian Diseases34406411
- 64. Costa TP, Brown JD, Howerth EW, Stallknecht DE (2010) Effect of a prior exposure to a low pathogenic avian influenza virus in the Outcome of a heterosubtypic low pathogenic avian influenza infection in mallards (Anas platyhynchos). Avian Diseases 54: 1286–1291.TP CostaJD BrownEW HowerthDE Stallknecht2010Effect of a prior exposure to a low pathogenic avian influenza virus in the Outcome of a heterosubtypic low pathogenic avian influenza infection in mallards (Anas platyhynchos).Avian Diseases5412861291
- 65. Fereidouni SR, Starick E, Beer M, Wilking H, Kalthoff D, et al. (2009) Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses. PLoS ONE 4: e6706.SR FereidouniE. StarickM. BeerH. WilkingD. Kalthoff2009Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses.PLoS ONE4e6706
- 66. Li YB, Shi JZ, Zhong GX, Deng GH, Tian GB, et al. (2010) Continued Evolution of H5N1 Influenza Viruses in Wild Birds, Domestic Poultry, and Humans in China from 2004 to 2009. Journal Of Virology 84: 8389–8397.YB LiJZ ShiGX ZhongGH DengGB Tian2010Continued Evolution of H5N1 Influenza Viruses in Wild Birds, Domestic Poultry, and Humans in China from 2004 to 2009.Journal Of Virology8483898397
- 67. Pfeiffer DU, Minh PQ, Martin V, Epprecht M, Otte MJ (2007) An analysis of the spatial and temporal patterns of highly pathogenic avian influenza occurrence in Vietnam using national surveillance data. The Veterinary Journal 174: 302–309.DU PfeifferPQ MinhV. MartinM. EpprechtMJ Otte2007An analysis of the spatial and temporal patterns of highly pathogenic avian influenza occurrence in Vietnam using national surveillance data.The Veterinary Journal174302309
- 68. Bishop MA, Tsamchu D (2007) Tibet Autonomous Region January 2007 survey for black-necked crane, common crane, and bar-headed goose. China Crane News 11: 24–26.MA BishopD. Tsamchu2007Tibet Autonomous Region January 2007 survey for black-necked crane, common crane, and bar-headed goose.China Crane News112426
- 69. Lang A, Bishop MA, Le Sueur A (2007) An annotated list of birds wintering in the Lhasa river watershed and Yamzho Yumco, Tibet Autonomous Region, China. Forktail 23: 1–11.A. LangMA BishopA. Le Sueur2007An annotated list of birds wintering in the Lhasa river watershed and Yamzho Yumco, Tibet Autonomous Region, China.Forktail23111
- 70. Newman SH, Iverson SA, Takekawa JY, Gilbert M, Prosser DJ, et al. (2009) Migration of whooper swans and outbreaks of highly pathogenic avian influenza H5N1 virus in eastern Asia. PLoS One 4: e5729.SH NewmanSA IversonJY TakekawaM. GilbertDJ Prosser2009Migration of whooper swans and outbreaks of highly pathogenic avian influenza H5N1 virus in eastern Asia.PLoS One4e5729
- 71. Muzaffar SB, Takekawa JY, Prosser DJ, Newman SH, Xiao X (2010) Rice production systems and avian influenza: Interactions between rice, poultry and wild birds. Waterbirds 33: 219–230.SB MuzaffarJY TakekawaDJ ProsserSH NewmanX. Xiao2010Rice production systems and avian influenza: Interactions between rice, poultry and wild birds.Waterbirds33219230