Wild Waterfowl Research Articles

Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4.

Novel subtypes of Asian-origin (Goose/Guangdong lineage) H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have been identified in China since 2008 and have since evolved into four genetically distinct clade 2.3.4.4 groups (A–D). Since 2014, HPAI clade 2.3.4.4 viruses have spread rapidly via migratory wild aquatic birds and have evolved through reassortment with prevailing local low pathogenicity avian influenza viruses. Group A H5N8 viruses and its reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and North America) during 2014–2015. Novel reassortant Group B H5N8 viruses caused outbreaks in Asia, Europe, and Africa during 2016–2017. Novel reassortant Group C H5N6 viruses caused outbreaks in Korea and Japan during the 2016–2017 winter season. Group D H5N6 viruses caused outbreaks in China and Vietnam. A wide range of avian species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, seem to be permissive for infection by and/or transmission of clade 2.3.4.4 HPAI viruses. Further, compared to previous H5N1 HPAI viruses, these reassortant viruses show altered pathogenicity in birds. In this review, we discuss the evolution, global spread, and pathogenicity of H5 clade 2.3.4.4 HPAI viruses.

Understanding of Host Switch and Host Adaptation to the Avian Influenza Viruses

During recent decades, several times of frequent appearance of the newly emerging or re-emerging pathogens are posing a great threat to the human populations as well as many other animals. It is well known that most risky groups are viral pathogens. Among them, taking a pose a striking threat to the human is belong to the influenza A viruses (IAVs). Influenza or flu is a wide spread zoonotic disease caused by influenza A virus, which has their ultimate origin from avian reservoirs and may stably adapt, either directly or after several passages thru another mammalian species of host, to human populations. Novel human-adapted have emerged to cause pandemics several times in the last 100 years. Typical avian influenza A viruses are restricted from replicating efficiently and causing disease in humans. Mechanisms by which viruses evolve in one host, cause host switch, and adapt to a new host species from wild aquatic waterfowl to mammalian species including human, remain to be elucidated. Here, some insights into the mechanisms of IAVs host switch and their adaptation and viral virulence factors associated with a novel virus in human are briefly reviewed.

Novel variants of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in migratory waterfowl of Hongze Lake

Wild birds are known to play a major role in the evolution, maintenance, and spread of the avian influenza viruses (AIVs). More specifically, the waterfowl are thought to be the natural reservoirs of AIVs. Here, we conducted a survey in 2015 at the Hongze Lake and characterized 11 H5N1 highly pathogenic AIVs isolated from wild waterfowls which were found to belong to clade 2.3.2.1. In contrast, the 11 variants of H5N1 viruses did not align with the three previously defined monophyletic subclades. Antigenicity analysis revealed that antigenic drift occurred in these H5N1 variants. Hence, current vaccines may fail to confer protection against the H5N1 AIV variants in poultry.

H5N2 Highly Pathogenic Avian Influenza Viruses from the US 2014-2015 outbreak have an unusually long pre-clinical period in turkeys.

BackgroundFrom December 2014 through June 2015, the US experienced the most costly highly pathogenic avian influenza (HPAI) outbreak to date. Most cases in commercial poultry were caused by an H5N2 strain which was a reassortant with 5 Eurasian lineage genes, including a clade 2.3.4.4 goose/Guangdong/1996 lineage hemagglutinin, and 3 genes from North American wild waterfowl low pathogenicity avian influenza viruses. The outbreak primarily affected turkeys and table-egg layer type chickens. Three isolates were selected for characterization in turkeys: the US index isolate from December 2014 (A/northern pintail/WA/40964/2014), and two poultry isolates from April 2015 (A/chicken/IA/13388/2015 and A/turkey/MN/12528/2015).ResultsFour week old broad-breasted white turkeys were inoculated with one of three doses (102, 104 or 106 50% egg infectious doses [EID50] per bird) of each of the isolates to evaluate infectious dose and pathogenesis. The mean bird infectious dose of A/northern pintail/WA/40964/2014 and A/turkey/MN/12528/2015 was 105 EID50 per bird, but was 103 EID50 per bird for A/chicken/IA/13388/2015, suggesting the latter had greater adaptation to gallinaceous birds. All three isolates had unusually long mean death time of 5.3–5.9 days post challenge, and the primary clinical signs were severe lethargy and neurological signs which started no more than 24 h before death (the average pre-clinical period was 4 days). Infected turkeys also shed high levels of virus by both the oropharyngeal and cloacal routes.ConclusionsThe unusually long mean death times, high levels of virus in feces, and increased adaptation of the later viruses may have contributed to the rapid spread of the virus during the peak of the outbreak.

Low Pathogenic Avian Influenza Viruses in Wild Migratory Waterfowl in a Region of High Poultry Production, Delmarva, Maryland.

Migratory waterfowl are natural reservoirs for low pathogenic avian influenza viruses (AIVs) and may contribute to the long-distance dispersal of these pathogens as well as spillover into domestic bird populations. Surveillance for AIVs is critical to assessing risks for potential spread of these viruses among wild and domestic bird populations. The Delmarva Peninsula on the east coast of the United States is both a key convergence point for migratory Atlantic waterfowl populations and a region with high poultry production (>4,700 poultry meat facilities). Sampling of key migratory waterfowl species occurred at 20 locations throughout the Delmarva Peninsula in fall and winter of 2013-14. Samples were collected from 400 hunter-harvested or live-caught birds via cloacal and oropharyngeal swabs. Fourteen of the 400 (3.5%) birds sampled tested positive for the AIV matrix gene using real-time reverse transcriptase PCR, all from five dabbling duck species. Further characterization of the 14 viral isolates identified two hemagglutinin (H3 and H4) and four neuraminidase (N2, N6, N8, and N9) subtypes, which were consistent with isolates reported in the Influenza Research Database for this region. Three of 14 isolates contained multiple HA or NA subtypes. This study adds to the limited baseline information available for AIVs in migratory waterfowl populations on the Delmarva Peninsula, particularly prior to the highly pathogenic AIV A(H5N8) and A(H5N2) introductions to the United States in late 2014.

Neospora caninum in Wild Waterfowl: Occurrence of Parasite DNA and Low Antibody Titers.

Thirty-five adult waterfowl (14 males and 21 females) representing various orders and species were sampled during the hunting season from 2015 to 2016. Antibodies to Neospora caninum were detected by IFAT on blood samples, while heart and brain were subjected to molecular analysis for the detection of Neospora caninum DNA. Twelve birds (34.3%) (6 Anas crecca , 3 Anas platyrhynchos , 2 Anas penelope , 1 Anas acuta ) showed antibodies versus N. caninum , while 10 animals out of 35 (4 A. crecca , 2 A. platyrhynchos , 2 A. penelope , 1 A. acuta , and 1 Vanellus vanellus ) scored positive for at least 1 DNA sample, with an overall prevalence of 28.6%. The present report shows for the first time the occurrence of antibodies and N. caninum DNA in waterfowl. The avian species investigated in the present report, which feed directly from the soil and/or water, would be able to ingest oocysts excreted by final canid hosts and could contribute to parasite transmission in the sylvatic cycle. To achieve a definitive result about the role of these avian species in the epidemiology of this protozoan, the presence of viable parasites should be demonstrated by bioassay and/or culture, as well as histological evidence of N. caninum cysts in avian tissues.

Detection and Molecular Identification of Sarcocystis rileyi (Apicomplexa: Sarcocystidae) From a Northern Shoveler (Anas clypeata) in Mexico.

We detected macroscopic Sarcocystis cysts in a Northern Shoveler ( Anas clypeata ) in the Lerma Marshes, State of Mexico, Mexico in February 2014. The 5.0×2.0-mm macrocysts in the breast muscle of the duck were ovoid and yellow. Using an optical microscope, we saw parasitic forms of a Sarcocystis sp. among muscular fibers; the cysts measured 3.5×1.1 mm. The external wall had a smooth surface and the internal wall had a spongy texture. We identified the macrocysts as Sarcocystis rileyi according to sequences of the 18S rRNA gene, 28S rRNA gene, and ITS-1 region. Sarcocystosis should be considered in similar assessments in wild waterfowl in Mexico. Awareness of S. rileyi among anatids in the Lerma Marshes will contribute to more-effective conservation and management actions.

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

ABSTRACTConstant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148 h. The isolate possessed a motif of -112E-R-Q-E-R-L117- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.

Evidence for wild waterfowl origin of H7N3 influenza A virus detected in captive-reared New Jersey pheasants.

In August 2014, a low-pathogenic H7N3 influenza A virus was isolated from pheasants at a New Jersey gamebird farm and hunting preserve. In this study, we use phylogenetic analyses and calculations of genetic similarity to gain inference into the genetic ancestry of this virus and to identify potential routes of transmission. Results of maximum-likelihood (ML) and maximum-clade-credibility (MCC) phylogenetic analyses provide evidence that A/pheasant/New Jersey/26996-2/2014 (H7N3) had closely related H7 hemagglutinin (HA) and N3 neuraminidase (NA) gene segments as compared to influenza A viruses circulating among wild waterfowl in the central and eastern USA. The estimated time of the most recent common ancestry (TMRCA) between the pheasant virus and those most closely related from wild waterfowl was early 2013 for both the H7 HA and N3 NA gene segments. None of the viruses from waterfowl identified as being most closely related to A/pheasant/New Jersey/26996-2/2014 at the HA and NA gene segments in ML and MCC phylogenetic analyses shared ≥99% nucleotide sequence identity for internal gene segment sequences. This result indicates that specific viral strains identified in this study as being closely related to the HA and NA gene segments of A/pheasant/New Jersey/26996-2/2014 were not the direct predecessors of the etiological agent identified during the New Jersey outbreak. However, the recent common ancestry of the H7 and N3 gene segments of waterfowl-origin viruses and the virus isolated from pheasants suggests that viral diversity maintained in wild waterfowl likely played an important role in the emergence of A/pheasant/New Jersey/26996-2/2014.

Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States.

A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

Complete Coding Sequences of One H9 and Three H7 Low-Pathogenic Influenza Viruses Circulating in Wild Birds in Belgium, 2009 to 2012.

The complete coding sequences of four avian influenza A viruses (two H7N7, one H7N1, and one H9N2) circulating in wild waterfowl in Belgium from 2009 to 2012 were determined using Illumina sequencing. All viral genome segments represent viruses circulating in the Eurasian wild bird population.

Case Series of Turkey Farms from the H5N2 Highly Pathogenic Avian Influenza Outbreak in the United States During 2015.

Between December 2014 and June 2015, an outbreak of H5N2 HPAI caused the largest and most expensive agriculture emergency in U.S. Department of Agriculture-Animal and Plant Health Inspection Service history. The outbreak affected 21 states; 232 poultry farms (211 commercial and 21 backyard) were affected, and approximately 49.6 million birds were depopulated on poultry farms. The majority of affected farms were commercial turkey operations (n = 160). This report is a case series describing 104 H5N2 HPAI-affected turkey farms in Iowa, Minnesota, Missouri, North Dakota, South Dakota, and Wisconsin that had H5N2 HPAI virus detected between March 5 and June 1, 2015. The farm manager or farm personnel voluntarily completed an epidemiologic questionnaire administered by state and federal animal health officials. Equipment and vehicle sharing with other farms was common, particularly for feed trucks (77% of farms shared feed trucks with other farms), live haul loaders (90.4%), poult trailers (72.0%), and preloaders (80.7%). Many farms had water bodies in proximity to the farm, such as a pond (42.6%) or stream (21.8%). About one-third of farms (33.7%) reported seeing wild birds inside the turkey barns. Only 44.2% of farms reported that third-party biosecurity audits or assessments had been conducted. Because the newly introduced Asian H5N8 HPAI and two new HPAI viruses, H5N2 and H5N1, are now circulating in U.S. wild birds, primarily migratory waterfowl, a greater potential for reoccurrence exists with the spring and fall migratory seasons, representing higher risk periods for outbreaks of HPAI in commercial poultry farms in the future. Eliminating exposure to wild birds, especially waterfowl or environments contaminated by wild waterfowl, will reduce risk of reintroduction of H5N2 HPAI virus, and ensuring good on-farm biosecurity will help the poultry industry avoid introduction of influenza and lateral spread between farms.

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins.

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a α2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a α2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with α2-3 or α2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Historical and Recent Cases of H3 Influenza A Virus in Turkeys in Minnesota

Subtype H3 influenza A viruses (IAVs) are abundant in wild waterfowl and also infect humans, pigs, horses, dogs, and seals. In Minnesota, turkeys are important and frequent hosts of IAV from wild waterfowl and from pigs. Over 48 yr of surveillance history, 11 hemagglutinin (HA) subtypes of IAV from waterfowl, as well as two HA subtypes from swine, H1 and H3, have infected turkeys in Minnesota. However, there have only been two cases of avian-origin H3 IAV infections in turkeys during this 48-yr period. The first avian-origin IAV infection was detected in seven breeder and commercial flocks in 1982 and was caused by a mixed H3H4/N2 infection. In 2013, an avian-origin H3H9/N2 outbreak occurred in five flocks of turkeys between 15 and 56 wk of age. Phylogenetic analysis of the HA gene segment from the 2013 isolate indicated that the virus was related to a wild bird lineage H3 IAV. A meta-analysis of historical H3 infections in domesticated poultry demonstrated that avian-origin H3 infections have occurred in chickens and ducks but were rare in turkeys. H9N2 virus was subsequently selected during the egg cultivation of the 2013 H3H9/N2 mixed virus. A growth curve analysis suggested that passage 3 of A/Turkey/Minnesota/13-20710-2/2013(mixed) had a slightly lower replication rate than a similar avian-origin H3N2. The challenge studies indicated that the infectious dose of avian-origin H3N2 for turkey poults was greater than 106 50% egg infective dose. Considered together, these data suggest that avian-origin H3 introductions to turkeys are rare events.

Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos)

Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007–2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo-or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions.

Antibodies to Influenza A Viruses in Wintering Snow Geese (Chen caerulescens) in Texas.

Wild waterfowl in the order Anseriformes are recognized reservoirs for influenza A viruses (IAVs); however, prevalence of infection can vary greatly by species. Few isolates of IAVs have been reported from snow geese (Chen caerulescens), and generally they have not been regarded as an important component of this reservoir. In February 2013, 151 combined cloacal and oropharangeal swabs and 147 serum samples were collected from snow geese wintering on the Gulf coast of Texas. None of the swab samples tested positive by virus isolation, but antibodies to IAVs were detected in 87 (59%) birds tested by competitive blocking ELISA (bELISA). To further characterize these detected antibodies, positive samples were tested by virus microneutralization (MN) for antibodies to viruses representing 14 hemagglutinin subtypes (HA1-HA12, H14, and H15). By MN, antibodies to H1 (n = 41; 47%), H5 (n = 32; 37%), H6 (n = 49; 56%), H9 (n = 50; 57%), and H12 (n = 24; 28%) were detected. Snow goose populations have increased in North America since the 1960s, and their association with agricultural lands provides a potential indirect source of IAV infection for domestic poultry. This potential, as well as the detection of antibodies to HA subtypes H5, H9, and H12 that are not well represented in other waterfowl species, suggests that further snow geese surveillance is indicated.

Evaluation of unmanned aerial vehicle shape, flight path and camera type for waterfowl surveys: disturbance effects and species recognition.

The use of unmanned aerial vehicles (UAVs) for ecological research has grown rapidly in recent years, but few studies have assessed the disturbance impacts of these tools on focal subjects, particularly when observing easily disturbed species such as waterfowl. In this study we assessed the level of disturbance that a range of UAV shapes and sizes had on free-living, non-breeding waterfowl surveyed in two sites in eastern Australia between March and May 2015, as well as the capability of airborne digital imaging systems to provide adequate resolution for unambiguous species identification of these taxa. We found little or no obvious disturbance effects on wild, mixed-species flocks of waterfowl when UAVs were flown at least 60m above the water level (fixed wing models) or 40m above individuals (multirotor models). Disturbance in the form of swimming away from the UAV through to leaving the water surface and flying away from the UAV was visible at lower altitudes and when fixed-wing UAVs either approached subjects directly or rapidly changed altitude and/or direction near animals. Using tangential approach flight paths that did not cause disturbance, commercially available onboard optical equipment was able to capture images of sufficient quality to identify waterfowl and even much smaller taxa such as swallows. Our results show that with proper planning of take-off and landing sites, flight paths and careful UAV model selection, UAVs can provide an excellent tool for accurately surveying wild waterfowl populations and provide archival data with fewer logistical issues than traditional methods such as manned aerial surveys.

Spatiotemporal Analysis of the Genetic Diversity of Seal Influenza A(H10N7) Virus, Northwestern Europe.

Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals (Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host. Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.

Spatial Modeling of Wild Bird Risk Factors for Highly Pathogenic A(H5N1) Avian Influenza Virus Transmission.

One of the longest-persisting avian influenza viruses in history, highly pathogenic avian influenza virus (HPAIV) A(H5N1), continues to evolve after 18 yr, advancing the threat of a global pandemic. Wild waterfowl (family Anatidae) are reported as secondary transmitters of HPAIV and primary reservoirs for low-pathogenic avian influenza viruses, yet spatial inputs for disease risk modeling for this group have been lacking. Using geographic information software and Monte Carlo simulations, we developed geospatial indices of waterfowl abundance at 1 and 30 km resolutions and for the breeding and wintering seasons for China, the epicenter of H5N1. Two spatial layers were developed: cumulative waterfowl abundance (WAB), a measure of predicted abundance across species, and cumulative abundance weighted by H5N1 prevalence (WPR), whereby abundance for each species was adjusted based on prevalence values and then totaled across species. Spatial patterns of the model output differed between seasons, with higher WAB and WPR in the northern and western regions of China for the breeding season and in the southeast for the wintering season. Uncertainty measures indicated highest error in southeastern China for both WAB and WPR. We also explored the effect of resampling waterfowl layers from 1 to 30 km resolution for multiscale risk modeling. Results indicated low average difference (less than 0.16 and 0.01 standard deviations for WAB and WPR, respectively), with greatest differences in the north for the breeding season and southeast for the wintering season. This work provides the first geospatial models of waterfowl abundance available for China. The indices provide important inputs for modeling disease transmission risk at the interface of poultry and wild birds. These models are easily adaptable, have broad utility to both disease and conservation needs, and will be available to the scientific community for advanced modeling applications.

Experimental infection and pathology of clade 2.2 H5N1 virus in gulls.

During 2006, H5N1 HPAI caused an epizootic in wild birds, resulting in a die-off of Laridae in the Novosibirsk region at Chany Lake. In the present study, we infected common gulls (Larus canus) with a high dose of the H5N1 HPAI virus isolated from a common gull to determine if severe disease could be induced over the 28 day experimental period. Moderate clinical signs including diarrhea, conjunctivitis, respiratory distress and neurological signs were observed in virus-inoculated birds, and 50% died. The most common microscopic lesions observed were necrosis of the pancreas, mild encephalitis, mild myocarditis, liver parenchymal hemorrhages, lymphocytic hepatitis, parabronchi lumen hemorrhages and interstitial pneumonia. High viral titers were shed from the oropharyngeal route and virus was still detected in one bird at 25 days after infection. In the cloaca, the virus was detected sporadically in lower titers. The virus was transmitted to direct contact gulls. Thus, infected gulls can pose a significant risk of H5N1 HPAIV transmission to other wild migratory waterfowl and pose a risk to more susceptible poultry species. These findings have important implications regarding the mode of transmission and potential risks of H5N1 HPAI spread by gulls.