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Emerging Infectious Diseases

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Materials and=20 Methods=20
Results=20
Discussion=20
Acknowledgments<= /A>=20
References=20
Figure 1=20
Figure 2=20
Figure 3=20
Appendix Figure 1=20
Appendix Figure 2=20
Appendix Figure 3=20
Appendix Figure 4=20
Table=20
Appendix Table 1=20
Appendix Table 2=20
Appendix Table 3=20
Appendix Table 4=20
Suggested=20 Citation

EID Journal=20 Home > Volume = 14,=20 Number 4=96April 2008

Volume 14, Number 4=96April 2008

Research

Wild Ducks as Long-Distance Vectors of Highly Pathogenic Avian = Influenza=20 Virus (H5N1)

Juthatip Keawcharoen,* Debby van Riel,* Geert van Amerongen,* = Theo=20 Bestebroer,* Walter E. Beyer,* Rob van Lavieren,* Albert D.M.E. = Osterhaus,* Ron=20 A.M. Fouchier,* and Thijs Kuiken* 3D"Comments
*Erasmus Medical Center, Rotterdam, the=20 Netherlands

Abstract
Wild birds have been implicated in the = expansion=20 of highly pathogenic avian influenza virus (H5N1) outbreaks across Asia, = the=20 Middle East, Europe, and Africa (in addition to traditional transmission = by=20 infected poultry, contaminated equipment, and people). Such a role would = require=20 wild birds to excrete virus in the absence of debilitating disease. By=20 experimentally infecting wild ducks, we found that tufted ducks, = Eurasian=20 pochards, and mallards excreted significantly more virus than common = teals,=20 Eurasian wigeons, and gadwalls; yet only tufted ducks and, to a lesser = degree,=20 pochards became ill or died. These findings suggest that some wild duck = species,=20 particularly mallards, can potentially be long-distance vectors of = highly=20 pathogenic avian influenza virus (H5N1) and that others, particularly = tufted=20 ducks, are more likely to act as sentinels.

The currently ongoing outbreaks caused by highly pathogenic avian = influenza=20 virus (HPAIV) of the subtype H5N1 are of concern not only to the poultry = industry but also to public health (1,2). = This virus,=20 which causes a high fatality rate among infected patients, may adapt to=20 efficient human-to-human transmission and thus initiate a new human = influenza=20 pandemic (3). = Since 1996,=20 when the ancestor virus was identified in domestic geese from China = (4), = outbreaks have=20 spread and now encompass countries in Asia, the Middle East, Europe, and = Africa=20 (5). This = spread of HPAIV among poultry flocks is traditionally thought to occur = by=20 transport of infected poultry, contaminated equipment, and persons = associated=20 with the poultry industry (6). = HPAIV has=20 occasionally been detected in wild birds near affected poultry flocks, = but these=20 birds have had limited or no role in virus dissemination (7). In = the current=20 outbreaks, however, wild birds are suspected of playing a major role as=20 long-distance virus vectors.

During the expansion of HPAI (H5N1) outbreaks from Asia to Europe, 2 = events=20 implicated wild birds, particularly waterbirds, as long-distance virus = vectors=20 (8). = First,=20 virus outbreaks in 2005 rapidly spread westward from Russia and = Kazakhstan in=20 July and August to Turkey, Romania, and Ukraine in October. Wild = waterbirds were=20 suggested as a vector because the virus spread through areas that had no = record=20 of any virus presence and coincided with the fall migration of wild = waterbirds=20 between these areas. Second, at the beginning of 2006, HPAIV (H5N1) was = detected=20 in many wild waterbirds in western Europe, often in areas where no = outbreaks had=20 been detected among intensively surveyed poultry (9=9612); = this event=20 overlapped with unusual waterbird movements associated with cold weather = in the=20 Black Sea area. Quantitative analysis of the global spread of HPAIV = (H5N1) also=20 supports the potential role of migratory wild birds in virus spread = (13). In = June and=20 July of 2007, Germany, France, and the Czech Republic again reported = HPAIV=20 (H5N1) in wild waterbirds (14), = which=20 illustrates their ongoing involvement in the epidemiology of this viral=20 infection.

The main argument against the view that wild waterbirds are = long-distance=20 vectors of HPAIV (H5N1) is that most wild waterbirds in which this virus = was=20 identified were either sick or dead, which suggests that they were too = severely=20 affected to spread the virus over any substantial distance (15). = This=20 argument is supported by experimental evidence that over time HPAIV = (H5N1) has=20 become more pathogenic for domestic ducks. Although domestic ducks did = not show=20 clinical disease or death from HPAI V (H5N1) isolates from 2001 or = before,=20 experimental infection of domestic ducks with an HPAIV (H5N1) isolate = from 2002=20 caused neurologic disease and death (16). = This high=20 pathogenicity has been shown to be associated with molecular changes in = the=20 polymerase genes PA and PB1 (17). = However,=20 little is known about the pathogenicity and excretion pattern of recent = HPAIV=20 (H5N1) isolates in wild waterbird species whose migration patterns = correspond=20 with the observed westward expansion of HPAI (H5N1) outbreaks.

Figure=20 1

Figure 1. Central = nervous system=20 changes in wild ducks experimentally infected with highly = pathogenic avian=20 influenza virus (H5N1)...

Figure=20 2

Figure 2. Mean = pharyngeal=20 excretion of highly pathogenic avian influenza virus (H5N1) of = wild ducks=20 by A) virus isolation and C) reverse transcription=96PCR=20 (RT-PCR)...

Figure=20 3

Figure 3. Mean cloacal = excretion=20 of highly pathogenic avian influenza virus (H5N1) by wild ducks by = A)=20 virus isolation and C) reverse transcription=96PCR = (RT-PCR)...

Appendix Figure=20 1

Appendix Figure 1. = Distribution=20 in the eastern hemisphere of the 6 wild duck species used in this=20 study...

Appendix Figure=20 2

Appendix Figure 2. = Individual=20 pharyngeal (A) and cloacal (B) excretion of highly pathogenic = avian=20 influenza virus (H5N1) in wild duck species, by virus = isolation.

Appendix Figure=20 3

Appendix Figure 3. = Individual=20 pharyngeal (A) and cloacal (B) excretion of highly pathogenic = avian=20 influenza virus (H5N1) in wild duck species, by reverse=20 transcription=96PCR.

Appendix Figure=20 4

Appendix Figure 4. = Highly=20 pathogenic avian influenza virus (H5N1) infection in tufted=20 ducks

To test the hypothesis that wild waterbirds can excrete HPAIV (H5N1) = in the=20 absence of debilitating disease and so potentially act as long-distance = virus=20 vectors, we experimentally infected 6 species of wild ducks with an = avian=20 isolate of HPAIV (H5N1) from Europe, obtained in 2005 = (A/turkey/Turkey/1/2005).=20 We chose ducks because they are an important group in the epidemiology = of avian=20 influenza in the wild, although other waterbird species, such as geese, = swans,=20 and gulls, also play a role (15). We = chose=20 these particular duck species because of their abundance, preference for = freshwater habitats, and migratory pattern spanning Asia, Europe, and = Africa (Table; Appendix = Figure=20 1). All 6 species are listed by the European Union as carrying a = higher risk=20 for avian influenza (18).

Materials and Methods

Virus Preparation

A virus stock of influenza virus A/turkey/Turkey/1/2005 (H5N1) was = prepared=20 by 2 passages in 10-day-old embryonated chicken eggs. The harvested = allantoic=20 fluid had a titer (19) of = 1.3 =D7=20 10⁸ median tissue culture infectious dose = (TCID₅₀)/mL and=20 was diluted with phosphate-buffered saline (PBS) to obtain a final titer = of 3.3=20 =D7 10³ TCID₅₀/mL. All experiments with HPAIV = (H5N1) were=20 performed under Biosafety Level 3+ conditions.

Animals

We experimentally infected 6 species of ducks: 2 species of diving = ducks=20 belonging to the genus Aythya (tufted duck [A. = fuligula] and=20 Eurasian pochard [A. ferina]) and 4 species of dabbling ducks = belonging=20 to the genus Anas (mallard [A. platyrhynchos], common = teal=20 [A. crecca], Eurasian wigeon [A. penelope], and = gadwall=20 [A. strepera]). The Anas species represent 3 clades: = the=20 mallard represents the mallard clade, the common teal represents the=20 green-winged teal clade, and the Eurasian wigeon and gadwall represent = the=20 wigeon clade, previously belonging to the genus Strepera = (20). = For each=20 species, males and females were equally represented. All ducks used for = the=20 infection experiments were captive-bred (Dierenhandel Hoogendoorn, = Stolwijk, the=20 Netherlands) and housed indoors since hatching to minimize the risk for=20 inadvertent avian influenza virus infection. Birds were 8=9611 months of = age at=20 time of inoculation. Serum samples, cloacal swabs, and pharyngeal swabs = were=20 collected from all ducks 1 week before inoculation. Serum was analyzed = by using=20 a commercially available influenza A virus antibody ELISA kit for the = detection=20 of antibodies against nucleoprotein (European Veterinary Laboratory, = Woerden,=20 the Netherlands) according to the manufacturer's instructions. Swabs = were tested=20 by reverse transcription=96PCR (RT-PCR). No duck had antinucleoprotein = antibody,=20 except 1 pochard. Its serologic status did not protect it from HPAIV = (H5N1)=20 infection; it had the most severe clinical signs of all inoculated = pochards and=20 died at 4 days postinoculation (dpi). For 1 teal and 2 pochards, titers = were=20 suspected positive. No duck used for the infection experiments was = positive for=20 avian influenza virus by RT-PCR. We used 8 specific-pathogen=96free = White Leghorn=20 chickens, 4=966 weeks old, as controls for the pathogenicity of the = virus=20 stock.

Experimental Design

For each species, 8 birds were housed together in negatively = pressurized=20 isolator units. Each bird was inoculated with 1 =D7 10⁴=20 TCID₅₀ HPAIV (H5N1), 1.5 mL intratracheally and 1.5 mL=20 intraesophageally. We used this low dose to increase the chance of = inducing a=20 subclinical infection and to simulate field circumstances. In addition, = 4 birds=20 per duck species, which served as negative controls, were sham = inoculated in the=20 same manner with PBS-diluted sterile allantoic fluid. Each day, a = qualified=20 veterinarian scored clinical signs of disease in all birds according to = a=20 standardized list. Cloacal and pharyngeal swabs were collected in 1 mL = transport=20 medium (21) = daily for the=20 first 14 days and every 2 days thereafter.

We randomly divided each group of 8 birds into 2 groups of 4. One = group was=20 euthanized by exsanguination under isoflurane anesthesia for pathologic=20 examination at 4 dpi; the other group was monitored for virus excretion = until=20 18=9621 dpi. Two ducks were removed after inoculation: 1 tufted duck = because of=20 concurrent aspergillosis and 1 pochard because of concurrent = staphylococcosis.=20 Also, 1 pochard and 3 tufted ducks were were chosen for pathologic = examination=20 at 4 dpi because they were dead or moribund. Although this was not = random=20 sampling, it does reflect the field situation because dead ducks no = longer=20 actively excrete virus. As expected, by 2 dpi 100% of the = positive-control=20 chickens were sick or dead, whereas the negative-control ducks showed no = clinical signs and were euthanized at 4 dpi. Animal studies were = approved by an=20 independent animal ethics committee and performed under Biosafety Level = 3+=20 conditions.

Pathologic Examination and Immunohistochemical Testing

Necropsies and tissue sampling were performed according to a standard = protocol. After fixation in 10% neutral-buffered formalin and embedding = in=20 paraffin, tissue sections were examined by 1 of 2 methods: hematoxylin = and eosin=20 staining for histologic evaluation or an immunohistologic method that = used a=20 monoclonal antibody against nucleoprotein of influenza A virus as a = primary=20 antibody for detection of influenza viral antigen (22). = The positive=20 control was lung tissue of an HPAIV (H5N1)=96infected domestic cat; = negative=20 controls were omission of primary antibody, substitution of primary = antibody by=20 an irrelevant monoclonal antibody of the same isotype, and testing of = tissues=20 from sham-inoculated ducks. The following tissues were examined: brain=20 (cerebrum, cerebellum, brainstem), trachea, bronchus, lung, = caudothoracic or=20 abdominal air sac, esophagus, proventriculus, duodenum, pancreas, liver, = jejunum, ileum, cecum, colon, bursa of Fabricius, spleen, kidney, gonad = (testis=20 or ovary), heart, pectoral muscle, and adrenal gland.

RT-PCR and Virus Titration

Tissue samples were weighed and homogenized in 3 mL of transport = medium by=20 use of a homogenizer (Kinematica Polytron, Lucerne, Switzerland). RNA = isolation=20 and RT-PCR were performed as described (23). = Briefly, RNA=20 from swabs and tissue suspensions was isolated by using a MagNaPure LC = system=20 with the MagNaPure LC Total Nucleic Acid Isolation Kit (Roche = Diagnostics,=20 Almere, the Netherlands). Real-time RT-PCR assays were performed on an = ABI Prism=20 7000 Sequence Detection System machine (Applied Biosystems, Foster City, = CA,=20 USA) by using the TaqMan EZ RT-PCR Core Reagents Kit (Applied = Biosystems,=20 Nieuwerkerk a/d IJssel, the Netherlands) according to the manufacturer's = instructions. The test used a hybridization probe=20 (5=B4-6-FAM-TTT-ATT-CAA-CAG-TGG-CGA-GTT-CCC-TAG-CAC-T-TAMRA-3=B4) and = specified=20 primers (forward: 5=B4-GAG-AGG-AAA-TAA-GTG-GAG-TAA-AAT-TGG-A-3=B4 and = reverse:=20 5=B4-AAG-ATA-GAC-CAG-CTA-CCA-TGA-TTG-C-3=B4) to detect the hemagglutinin = gene of=20 HPAIV (H5N1). For each run the samples were prepared and processed in = parallel=20 with several negative and positive control samples. Virus titers were = determined=20 by serial 10-fold dilution of the homogenized tissue samples and swabs = on MDCK=20 cells, as described (19). = Virus=20 titrations were performed in quadruplicate.

Results

Despite the low doses of virus used to inoculate the ducks, rates of=20 productive infection in the 6 species were high: 76% according to virus=20 isolation and 93% according to RT-PCR (Table). HPAIV (H5N1) infection caused = clinical signs of=20 disease in only tufted ducks and pochards, both of which are diving = ducks in the=20 genus Aythya (Table). In contrast, the remaining 4 = species=97all=20 dabbling ducks belonging to the genus Anas=97were clinically = unaffected.=20 Clinical signs, which were more severe in tufted ducks than in pochards, = developed at 3 to 4 dpi and consisted of labored breathing, increased=20 recumbency, and neurologic signs (torticollis [Figure = 1, panel=20 A], circling, loss of balance, and head tremors). Severely affected = birds died=20 or were euthanized in a moribund state at 4 dpi. Mildly affected birds = recovered=20 by 7 or 8 dpi.

Severe neurologic signs in tufted ducks and pochards were associated = with=20 multifocal viral encephalitis. Although no gross brain lesions were = noted (Appendix Table 1), according to laboratory = analysis=20 these birds had multiple foci of influenza virus antigen expression (Figure = 1, panel=20 B; Appendix Table 2) associated with severe = necrosis and=20 inflammation (Figure = 1, panel=20 C) and high virus titers (10^3.5 to 10^5.2 = TCID₅₀=20 per g tissue) in the brain (Appendix Table 3). The only other ducks with = evidence=20 of HPAIV (H5N1) infection of the brain were gadwalls, none of which = showed=20 neurologic signs. Gadwalls had only focal influenza virus antigen = expression (Figure = 1, panel=20 D; Appendix Table 2), mild encephalitis (Figure = 1, panel=20 E), and low virus titers (10^1.5 TCID₅₀ per g = tissue) in=20 the brain (Appendix Table 3). No other species had = evidence of=20 HPAIV (H5N1) infection in the brain according to immunohistochemical = testing,=20 histologic examination, or virus isolation (Figure = 1, panels=20 F and G; Appendix Tables 2 and 3), although individual animals did have = positive=20 RT-PCR results for the brain (Appendix Table 4).

Pharyngeal excretion of HPAIV (H5N1) varied significantly among the 6 = duck=20 species (1-way analysis of variance of area under pharyngeal excretion = curve up=20 to 4 dpi, p<0.001), by virus isolation (Figure = 2, panel=20 A) and by RT-PCR (Figure = 2, panel=20 C). The ducks could be divided into a high-excretion group consisting of = tufted=20 ducks, pochards, and mallards, and a low-excretion group consisting of = teals,=20 wigeons, and gadwalls (Figure = 2, panels=20 B and D). Pharyngeal excretion also varied substantially among = individuals=20 within species (Appendix = Figures=20 2 and 3).=20 This finding was most extreme in tufted ducks and pochards, the species = in which=20 the individuals with the highest excretion level were also those showing = severe=20 clinical signs (Figure = 2, panels=20 B and D).

Pharyngeally excreted HPAIV (H5N1) likely originated from lung, air = sac, or=20 both, because these were the only tissues in the respiratory tract that = had=20 immunohistochemical evidence of virus replication (Figure = 2, panels=20 E and G; Appendix Table 2) and because virus was = frequently=20 detected in these tissues by virus isolation (Appendix Table 3) and RT-PCR (Appendix Table 4). The histologic lesions = corresponding=20 to influenza virus antigen expression in these tissues were = bronchointerstitial=20 pneumonia (Figure=20 2, panel F) and lymphocytic airsacculitis (Figure = 2, panel=20 H). Despite frequent isolation of HPAIV (H5N1) from trachea and = extrapulmonary=20 bronchus (Appendix Table 3), these tissues had neither=20 histopathologic nor immunohistochemical evidence of HPAIV (H5N1) = replication (Appendix Table 2), which suggests that virus = isolated=20 from these sites at 4 dpi originated from elsewhere in the respiratory=20 tract.

Cloacal excretion of HPAIV (H5N1) was uncommon; virus was detected in = cloacal=20 swabs of only 7% of ducks by virus isolation and 59% by RT-PCR (Table). Cloacal excretion was exceeded by = pharyngeal=20 excretion in all 6 duck species, according to virus isolation (Figure = 3, panels=20 A and B; Appendix=20 Figure 2) and RT-PCR (Figure = 3, panels=20 C and D; Appendix=20 Figure 3).

Cloacally excreted virus likely originated from pancreas, liver, or = both, on=20 the basis of the significant association between virus antigen = expression in=20 these tissues (Appendix Table 2) and virus detection in = cloacal swabs=20 by RT-PCR (Appendix Table 4); 7 of 8 birds with virus = antigen=20 expression in liver, pancreas, or both had PCR-positive cloacal swabs = between 1=20 and 4 dpi, in contrast to 0 of 15 birds without virus antigen expression = in=20 these tissues (Fisher exact test, p<0.00001). In the pancreas, tufted = ducks=20 and pochards had multifocal necrosis (Figure = 3, panel=20 E), which was the most prominent gross lesion associated with HPAIV = (H5N1)=20 infection in this study (Appendix Table 1). Virus antigen expression = (Figure = 3, panel=20 F) occurred at the transition between normal parenchyma and these = necrotic foci=20 (Figure = 3,=20 panel G) and corresponded with high virus titers (10^1.5 to=20 10^6.2 TCID₅₀ per g tissue) in the pancreas (Appendix Table 3). In the liver, widespread = virus=20 antigen expression (Figure = 3, panel=20 H) was associated with necrotizing hepatitis (Figure = 3, panel=20 I) and variable virus titers (no virus isolated to 10^6.2=20 TCID₅₀ per g tissue) in the liver (Appendix Table 3). Virus produced in pancreas = and liver=20 could potentially have reached the intestinal lumen through pancreatic = and bile=20 ducts, respectively. Although virus antigen expression was detected in = several=20 other tissues, virus originating from these sites likely did not = contribute to=20 virus excretion.

It is unlikely that cloacally excreted virus originated from the = intestinal,=20 urinary, or genital tracts, although all 3 tracts empty into the cloaca. = In the=20 intestine, no virus antigen expression was found in the intestinal = epithelium of=20 any of the 23 ducks examined. Virus antigen expression was found in = neurons and=20 satellite cells in the peripheral nervous system (submucosal and = myenteric=20 plexi, mesenteric ganglia) of the small intestine (Appendix = Figure=20 4, panel A), in association with necrotizing ganglioneuritis (Appendix = Figure=20 4, panel B), and in myocytes in the lamina muscularis mucosae of the = colon,=20 without associated histologic lesions. However, these tissues do not = empty into=20 the intestinal lumen. No virus antigen expression was found in tissues = of=20 urinary tract (kidney) or genital tract (testis or ovary) (Appendix Table 2). The occasional isolation = of HPAIV=20 (H5N1) from kidney samples (Appendix Table 3) may be explained by = inadvertent=20 sampling of overlying air sac wall, which did express influenza virus=20 antigen.

Evidence of HPAIV (H5N1) replication was found sporadically in = tissues other=20 than those of the respiratory, digestive, and nervous systems (Appendix Table 2). Virus antigen expression = was=20 detected in multiple foci of medullary and cortical cells of the adrenal = gland=20 (Appendix = Figure=20 4, panel C) and was associated with necrotizing adrenalitis (Appendix = Figure=20 4, panel D). Virus antigen expression was also detected in multiple = foci of=20 myocytes of the heart and was associated with myocardial necrosis.

Discussion

Our study shows that of the 6 wild duck species studied, the mallard = is the=20 prime candidate for being a long-distance vector of HPAIV (H5N1) because = it was=20 the only species to show abundant virus excretion without clinical or = pathologic=20 evidence of debilitating disease (Table; Figure = 2, panels=20 B and D). These findings fit with the absence of dead mallards in wild = bird=20 die-offs from HPAIV (H5N1) in Europe and Asia in 2005 and 2006 (14,24,25), = although=20 HPAIV (H5N1) was detected in 1 dead mallard during the recent 2007 HPAI = (H5N1)=20 outbreak in wild birds in Germany (26). = Other=20 characteristics of the mallard support its potential role as a vector = ([27]; Appendix = Figure=20 1): it is the most abundant anatid species in Western Eurasia = (≈9 million=20 birds); part of the population migrates long distances northeast to = southwest=20 between breeding and wintering areas; and it is found on nearly every = type of=20 wetland and is very tolerant of human presence, thus forming a potential = link=20 between wild waterfowl, domestic animals, and humans.

Pochards and tufted ducks are less likely candidates as long-distance = virus=20 vectors because those individuals that excreted the most virus also = developed=20 severe neurologic disease (Figure = 2, panels=20 B and D) and therefore would not have been able to fly far before = succumbing.=20 Instead, they are more likely to act as sentinels for HPAIV (H5N1) in = wild bird=20 populations, as do mute swans (Cygnus olor) (9). = However,=20 pochards cannot be ruled out as potential vectors because some birds = excreted=20 abundant virus in absence of severe clinical signs (Figure = 2, panels=20 B and D). Our results correspond with field observations of pochards and = tufted=20 ducks involved in wild bird die-offs from HPAIV (H5N1) infection in = France,=20 Germany, and Sweden early in 2006 (14). = Some of=20 these birds showed clinical signs of neurologic disease, e.g., = compulsively=20 swimming around in circles (28). = Therefore,=20 close surveillance of these 2 Aythya species for unusual = illness,=20 particularly neurologic disease, or death should provide early warning = for HPAIV=20 (H5N1) infection in an area. Redheads (Aythya americana), which = are=20 diving ducks indigenous to North America, experimentally infected with a = 2005=20 isolate of HPAIV (H5N1) neither showed clinical signs nor died (29). Of = the 6=20 species tested, the 3 remaining Anas species=97gadwall, teal, = and=20 wigeon=97are the least likely candidates as long-distance virus vectors = because=20 they had limited virus excretion (Figure = 2, panels=20 B and D).

HPAIV (H5N1) infection in these wild ducks contrasts in pattern of = excretion=20 with that of low-pathogenicity avian influenza virus infection in wild = ducks and=20 contrasts in pattern of disease with that of HPAIV infection in = chickens. Both=20 contrasts can be explained by the specific tissue tropism of HPAIV = (H5N1) in=20 wild ducks. With regard to pattern of excretion, low cloacal excretion = was=20 associated with lack of evidence for HPAIV (H5N1) replication in = intestinal=20 epithelium of any of the 23 ducks examined (Appendix Table 2), in contrast to most=20 low-pathogenicity avian influenza viruses for which intestine is the = main=20 replication site (30). = Instead,=20 HPAIV (H5N1) replicated preferentially in the respiratory tract (Appendix Tables 2 and 4), which corresponds with high pharyngeal = excretion.=20 How this preferential pharyngeal excretion of HPAIV (H5N1) affects its = spread=20 and persistence in a wild duck population remains to be determined.

Severe clinical disease in the HPAIV (H5N1)=96infected tufted ducks = and=20 pochards manifested itself mainly as neurologic signs at about 4 dpi, = although=20 pathologic examination also showed virus-induced lesions in organs other = than=20 the brain. These findings differ substantially from those of HPAIV=20 (H5N1)=96infected chickens, which are characterized mainly by widespread = hemorrhage and edema and death by about 2 dpi (31). = Again, this=20 contrast can be explained by differences in tissue tropism. Whereas the=20 cardiovascular lesions in poultry are associated with widespread = replication of=20 HPAIV (H5N1) in endothelium lining the blood vessels (31), no = such=20 endotheliotropism was detected in any of 23 ducks examined.

The knowledge gained from this study has several implications for=20 surveillance in wild ducks. Active surveillance (sampling of apparently = healthy=20 wild birds) should give priority to mallards and, to a lesser degree, = pochards.=20 Sampling should not be limited to cloacal swabs, as is the custom in=20 surveillance for low-pathogenicity avian influenza virus, but should = include=20 pharyngeal swabs. Passive surveillance (sampling of diseased or dead = birds),=20 should pay extra attention to tufted ducks and pochards, particularly = those=20 exhibiting neurologic disease. Sampling of wild duck carcasses should = not be=20 limited to cloacal, pharyngeal, and tracheal swabs and should include = internal=20 organs such as brain, trachea, lung, pancreas, liver, kidney, and spleen = (Appendix Tables 3 and 4).

Acknowledgments

We are grateful to C. Baas, R. Dias d'Ullois, R. van Herwijnen, K.=20 Hoogendoorn, P. Lexmond, V. Munster, J. Philippa, F. Read, M. van de = Bildt,=20 and F. Velkers for advice and technical assistance.

This research was funded by European Commission grant no. 044490=20 "New-FluBird" and Dutch Ministry of Economic Affairs grant "Impuls = Veterinair=20 Aviaire Influenza Onderzoek Nederland."

Dr Keawcharoen is a veterinarian at the Erasmus Medical Center in=20 Rotterdam. Her research interests include the role of animal = reservoirs in=20 emerging zoonoses and the molecular biology of influenza = virus.

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Figures

Figure 1. Central nervous system = changes=20 in wild ducks experimentally infected with highly pathogenic avian = influenza=20 virus (H5N1)...
Figure 2. Mean pharyngeal = excretion of=20 highly pathogenic avian influenza virus (H5N1) of wild ducks by A) virus = isolation and C) reverse transcription=96PCR (RT-PCR)...
Figure 3. Mean cloacal excretion = of highly=20 pathogenic avian influenza virus (H5N1) by wild ducks by A) virus = isolation and=20 C) reverse transcription=96PCR (RT-PCR)...
Appendix Figure 1. Distribution = in the=20 eastern hemisphere of the 6 wild duck species used in this = study...
Appendix Figure 2. Individual = pharyngeal=20 (A) and cloacal (B) excretion of highly pathogenic avian influenza virus = (H5N1)=20 in wild duck species, by virus isolation.
Appendix Figure 3. Individual = pharyngeal=20 (A) and cloacal (B) excretion of highly pathogenic avian influenza virus = (H5N1)=20 in wild duck species, by reverse transcription=96PCR.
Appendix Figure 4. Highly = pathogenic avian=20 influenza virus (H5N1) infection in tufted ducks...

Tables

Table. Health status and virus = excretion=20 of 46 wild ducks experimentally infected with highly pathogenic avian = influenza=20 virus (H5N1)
Appendix Table 1. Gross lesions = in wild=20 ducks at 4 days postinoculation with highly pathogenic avian influenza = virus=20 (H5N1)
Appendix Table 2. Expression of = antigen in=20 organs of wild ducks at 4 days postinoculation with highly pathogenic = avian=20 influenza virus (H5N1)
Appendix Table 3. Isolation of = highly=20 pathogenic avian influenza virus (H5N1) from organs of wild ducks at 4 = days=20 after inoculation and from swabs at 1=964 days postinoculation
Appendix Table 4. Detection of = highly=20 pathogenic avian influenza virus (H5N1) by reverse transcription=96PCR = from organs=20 of wild ducks at 4 days postinoculation and from swabs at 1=964 days=20 postinoculation