Emerg Infect DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention19861060286640609-043910.3201/eid1510.090439DispatchDucks as Sentinels for Avian Influenza in Wild BirdsDucks as Sentinels for Avian InfluenzaGlobigAnjaBaumerAnetteRevilla-FernándezSandraBeerMartinWodakEvelineFinkMariaGreberNorbertHarderTimm C.WilkingHendrikBrunhartIrisMatthesDorisKraatzUlfStrunkPeterFiedlerWolfgangFereidouniSasan R.StaubachChristophConrathsFranz J.GriotChrisMettenleiterThomas C.StärkKatharina D.C.Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany (A. Globig, M. Beer, T.C. Harder, H. Wilking, U. Kraatz, P. Strunk, S.R. Fereidouni, C. Staubach, F.J. Conraths, T.C. Mettenleiter)Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland (A. Baumer, C. Griot)Austrian Agency for Health and Food Safety, Mödling, Austria (S. Revilla-Fernández, E. Wodak, M. Fink)State of Vorarlberg Veterinary Directorate, Vorarlberg, Austria (N. Greber)Bird Ringing Centre, Radolfzell, Germany (D. Matthes, W. Fiedler)Federal Veterinary Office, Bern, Switzerland (I. Brunhart)Royal Veterinary College, London, UK (K.D.C. Stärk)Address for correspondence: Anja Globig, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany; email: anja.globig@fli.bund.de102009151016331636

To determine the effectiveness of ducks as sentinels for avian influenza virus (AIV) infection, we placed mallards in contact with wild birds at resting sites in Germany, Austria, and Switzerland. Infections of sentinel birds with different AIV subtypes confirmed the value of such surveillance for AIV monitoring.

Keywords: sentinelavian influenzaactive surveillancewild birdsinfluenzavirusesdispatch

As a consequence of infections of wild birds and poultry with highly pathogenic avian influenza virus (HPAIV) subtype H5N1, surveillance of wild birds for avian influenza viruses (AIVs) has intensified in Europe since 2005. Reporting of results is compulsory in the European Union (1,2). HPAIV surveillance includes investigation of dead or sick wild birds (3) with the aim of early detection of HPAIV (H5N1) complemented by sampling of healthy wild birds to monitor for low pathogenicity (LP) AIV strains (4). Previously, sentinel birds were used successfully to obtain information about AIV subtypes circulating in wild birds (5), but results of those studies are now outdated. Also, the effectiveness of sentinel birds has not yet been documented for AIV strains that emerged during the past decade.

We evaluated a sentinel approach to monitor the prevalence of HPAIV and LPAIV within an ecosystem, obtain information about seroconversion and duration of immunity after infection with AIV, and serve as an early warning system for the introduction of HPAIV (H5N1) and other notifiable AIVs (subtypes H5 and H7) to wild bird populations. Here we summarize results from a 2-year period of 3 international sentinel projects ongoing since 2006.

The Study

In 2006, multiple introductions and spread of HPAIV (H5N1) occurred in Europe, including the wetlands in Austria, Germany, and Switzerland (3,4,6,7). For our study, we selected 5 locations with substantial and heterogeneous wild bird populations on the basis of HPAIV (H5N1) subtype detected during 2006. Sentinel stations were located around Lake Constance and in 2 other wetlands in Germany. The sentinel flocks at Lake Constance were situated in 1) Radolfzell (Möggingen), Germany (47°45′58′′N, 8°59′45′′E); 2) Altenrhein, Switzerland (47°29′25′′N, 9°32′45′′E); and 3) Bregenz-Thal (Rheindelta), Austria (47°30′60′′N, 9°38′55′′E). The 2 other stations were situated 4) on the Isle of Koos close to the Island of Rügen in Mecklenburg–Western Pomerania, Germany (54°10′13′′N, 13°24′11′′E) and 5) near the Oder Valley at Lake Felchow (Brandenburg), Germany, (53°03′09′′N, 14°08′06′′E) (Figure 1). After their wing feathers were clipped, 10–20 hand-bred adult mallard ducks (Anas plathyrhynchos) <1 year of age were placed in pens in natural water bodies, allowing continuous direct contact with wild water birds as previously described (5). Cloacal and oropharyngeal swabs and blood were taken from the mallards and tested negatively by using 1-step TaqMan real-time reverse transcription–PCR (RT-PCR) or competitive nucleoprotein antibody ELISA (cNP ELISA) (ID-Vet, Montpellier, France; Anigen, MegaCorGmbH, Hörbanz, Austria) before their use as sentinels.

Locations of sentinel duck flocks at 5 locations in Germany, Switzerland, and Austria. A–C) Sites at Lake Constance: Radolfzell, Germany (A); Bregenz-Thal, Austria (B); and Altenrhein, Switzerland (C). D–E) Additional sentinel stations at Lake Felchow, Brandenburg, Germany (D), and Isle of Koos, Mecklenburg–Western Pomerania, Germany (E). Yellow dots mark the location of sentinel stations. Red dots mark detections of highly pathogenic avian influenza virus (HPAIV) (H5N1) in dead wild birds in 2006, and blue dots in 2007. In 2008, HPAIV (H5N1) was not found in dead wild birds in any of the 3 countries but was found in a live pochard (Aythya ferina) from Switzerland/Lake Sempach (blue dot in Switzerland).

At all sentinel stations in Germany, we collected oropharyngeal and cloacal swab samples from the sentinels every 14 days starting in autumn 2006. Sampling at the station in Austria started in February 2007 and at the station in Switzerland in October 2007. Laboratory tests were conducted in accordance with the Diagnostic Manual for Avian Influenza of the European Union (8). RNA was isolated from swabs by using viral RNA kits (QIAGEN, Hilden, Germany; Macherey-Nagel, Oensingen, Switzerland) and analyzed by real-time RT-PCR for influenza virus matrix (M) or nucleocapsid protein (NP) gene fragments. In positive samples, H5-, N1- and H7-specific real-time RT-PCRs were used to identify or exclude respective subtypes (9,10). H5 and H7 isolates were pathotyped following the European Union directive (8). Direct hemagglutinin (HA) typing or sequencing of positive samples was carried out as previously described (7,1113). The neuraminidase (NA) subtype was identified molecularly, by following the method of Fereidouni et al. (14). Simultaneously, we attempted virus isolation in embryonated chicken eggs from positive samples (8).

From October 2006 through September 2008, at least 23 specifiable AIV infections were detected at the sentinel stations by the fortnightly swabbing. After initial AIV introduction, virus was excreted during the following 1–3 sampling dates (Figure 2). All ducks at all sites tested positive at least once. Infections caused by AIV of 8 HA subtypes, including H5 and H7, and 6 NA subtypes, were found in clinically healthy sentinel birds (Tables 1, 2). Viral RNA and, in 44% of AIV cases, infectious virus also were recovered both from cloacal and oropharyngeal swabs. AIVs were subtyped as H1N1, H1Nx, H2N2, H2N5, H3N2, H3Nx, H3N8, H4N6, H5Nx, H6N5, H6N8, H7N3, H7Nx, and H9N2. Cycle threshold values ranged from 24 to 40. Pathotyping of H5 and H7 subtypes showed the exclusive presence of LP viruses. Additional AIVs were detected but could neither be isolated nor sequenced for subtype identification because of low loads of viral RNA. However, we did not detect H5, N1, and H7 subtypes by using real-time RT-PCR. Infections occurred most frequently from August through January (Figure 2; Table 1). Reinfection of the sentinels with the same subtype occurred in 2 of the sentinel flocks in Germany (Table 1).

Months with positive results for sentinel birds over a 2-year period at 5 locations in Germany, Switzerland, and Austria. Sites at Lake Constance: Radolfzell, Germany (yellow); Bregenz-Thal, Austria (gray); and Altenrhein, Switzerland (green). Additional sentinel stations at Lake Felchow, Brandenburg, Germany (red), and Isle of Koos, Mecklenburg–Western Pomerania, Germany (blue). Bars indicate the cumulative percentage of sentinel birds tested positive at each of the 5 locations at the time of sampling (maximum 500% at all 5 stations). For example, in December 2006, all sentinel ducks at station 4, but only 30% of sentinels at station 5, were positive at the date of sampling. 1, days 1–15 of month; 2, day 16 through end of month.

Detection of AIV by sampling of sentinel mallard ducks (<italic>Anas plathyrhynchos</italic>) at 5 locations in Germany, Switzerland, and Austria*†
Sentinel locationDate sampledCt range‡Duration virus excretion, dHA subtypeNA subtypeIsolateSequence of HA-cleavage site (H5, H7)
Radolfzell, Germany
2007 Aug29–37Min 28, max 42H6N8Yes
2007 Oct31–39Min 14, max 28H2N5Yes
H3N2Yes
2008 Jan33–38PunctualH3N2Yes
2008 Jun33–39PunctualH3N2No
2008 Sep
33–39
Min 14, max 28
H3
N8
Yes

Altenrhein, Switzerland
2007 Oct27–39Min 15, max 39ND§N2No
2007 Dec24–37PunctualH2N2No
2008 Aug
29–34
Punctual
H9
N2
No

Bregenz-Thal, Austria
2007 Apr28–38Min 23, max 38H3ND¶No
2007 Oct22–40Min 12, max 26H9N2Yes
2008 Jan27–38Min 14, max 42H1N1No
2008 Mar
25–35
Min 14, max 42
H7
ND¶
No
PEIPKGR GLF
Lake Felchow, Brandenburg, Germany
2006 Dec27–35Min 22, max 36H6N2Yes
2007 Jan29–38Min 14, max 34H5N3?NoPQRETR GLF
2007 Mar35–38PunctualH5ND¶No
2007 Sep31–39PunctualH6N5Yes
2007 Dec27–37Min 42, max 56H9ND¶No
H1?ND¶No
H11?ND¶No
2008 Feb/Mar
30–38
Min 56, max 70
H9
ND¶
No

Isle of Koos, Mecklenburg–Western Pomerania, Germany2006 Dec29–35Min 14, max 28H4N6Yes
2007 Aug32–38Min 35, max 49H7N3YesPEIPKGR GLF

*Radolfzell, Altenrhein, and Bregenz-Thal are located along Lake Constance. AIV, avian influenza virus; Ct, cycle threshold value; HA, hemagglutinin; NA, neuraminidase; min, minimum; max, maximum; ND, not determined.
†Each sentinel flock comprised 10–20 birds. Only the initial detection of each AIV introduction and determined HA or NA subtypes are presented.
‡Results of matrix and nucleocapsid protein gene fragments by real-time reverse transcription–PCR.
§AIV-positive, HA subtype not determined, non-H5, non-H7.
¶AIV-positive, NA subtype not determined, non-N1.

Frequency of sentinel duck sampling and frequency of AIV detection at 5 locations in Germany, Switzerland, and Austria*
Sentinel locationInvestigation periodNo. samplings of sentinel flock†No. AIV detectionsAIV subtypes
Radolfzell, Germany2006 Oct–2008 Sep5311H6N8, H2N5, H3N2, H3N8
Altenrhein, Switzerland2007 Oct–2008 Sep247H2N2, H9N2, HxN2
Bregenz-Thal, Austria2007 Feb–2008 Sep449H9N2, H3Nx, H1N1, H1Nx, LP H7Nx
Brandenburg, Germany2006 Oct–2008 May4120H1?, LP H5Nx, H6N2, H6N5, H9Nx, H11?
Mecklenburg–Western Pomerania, Germany2006 Oct– 2008 Jun406H4N6, LP H7N3

*Radolfzell, Altenrhein, and Bregenz-Thal are located along Lake Constance. AIV, avian influenza virus; LP, low pathogenicity.
†Each flock consisted of 10–20 birds.

Blood samples were collected from the ducks once a month, and serum was tested in a cNP-ELISA after heat inactivation at 56°C for 30 min. After each natural infection, sentinel animals seroconverted, and serum scored positive in the cNP-ELISA within 2–4 weeks. By hemagglutination inhibition test using homosubtypic but not autologous antigen, HA-specific antibodies were detected only rarely and at low titers.

Detection rates of AIV in sentinel ducks were compared with data from monitoring of healthy, trapped wild birds. From October 2006 through September 2008, a total of 1,953 wild birds were investigated for AIV within a radius of 30 km of Lake Constance, resulting in 47 (2.4%) AIV detections of subtypes H3Nx, LP H5N2, H6N8, LP H7Nx, H1N1, HxN1, H1Nx, and H9N2. During January 2007–May 2008, a total of 8 (0.4%) of 2,028 investigated wild bird samples from Brandenburg tested positive (subtypes H3N6, H6). In Mecklenburg–Western Pomerania, 8,066 birds were tested; 23 (0.3%) AIV infections (subtypes H1Nx, LP H5N2, H6Nx, H12Nx, H16Nx) were found.

Conclusions

In practice, AIV surveillance of live wild birds is difficult and involves substantial labor and costs, particularly for purchase and maintenance of trapping equipment, salary of trapping staff, and laboratory analysis. Trapping of wild birds also can be biased by season and by bird species that are easier to catch. Low proportions of AIV-positive results (<3%) indicate the low cost:benefit ratio of surveillance based on trapping wild birds (2). In contrast, our findings demonstrate that the use of sentinel birds in regions with substantial wild bird populations achieves a high rate of AIV detection and, therefore, is an efficient supplement to active AIV monitoring. The detection of different AIVs among the sentinel ducks reflects the natural ecology of AIV at discrete locations. Recently, all duck species, especially dabbling ducks, have been assessed as high-risk species for possibly contributing to the transmission of HPAIV (H5N1) (15). Therefore, mallards as sentinel species ensure a high probability of detecting AIV if kept in direct contact with wild water birds. In addition, sites for sentinel stations need to be selected carefully to achieve spatial representation.

Although our study was conducted in areas where HPAIV (H5N1) had circulated in wild birds in 2006, this subtype was not found by screening live wild birds or by using sentinel birds during the study period. Therefore, persistent circulation of HPAIV (H5N1) in the wild bird populations is unlikely for the area of Lake Constance, the coastal area of Mecklenburg–Western Pomerania, and the region of the Oder Valley in Brandenburg. However, because of the limited sample sizes, a low prevalence cannot be excluded. Although HPAIV (H5N1) was found only rarely in apparently healthy birds, e.g., in a pochard (Aythya ferina) in Switzerland in 2008 (Figure 1), regular testing of sentinel birds could increase the probability of detecting sporadic transmission of HPAIV in healthy wild water birds even in the absence of detectable deaths.

Suggested citation for this article: Globig A, Baumer A, Revilla-Fernández S, Beer M, Wodak E, Fink M, et al. Ducks as sentinels for avian influenza in wild birds. Emerg Infect Dis [serial on the internet] 2009 Oct [date cited]. Available from http://www.cdc.gov/EID/content/15/10/1633.htm

Acknowledgments

We thank the Ministry of Food and Rural Areas in Baden-Württemberg; nature conservation and veterinary authorities in Baden-Württemberg, Brandenburg, and Mecklenburg–Western Pomerania; and veterinary authorities of the Canton Thurgau and St. Gallen in Switzerland for authorization of sentinel stations. We also thank the Nature Conservation Society Rheindelta, Austria. We appreciate the excellent assistance of J. Anklam and M. Calderana in sample processing and virus testing.

This work was supported by the project Constanze, grant no. 1.07.01 from the Swiss Federal Veterinary Office; the Federal Ministry of Food, Agriculture and Consumer Protection, Germany (FSI, project no. 1-3.5); and the Federal Ministry of Health in Austria (BMGF-74600/0360-IV/6/2006).

Dr Globig is a veterinarian working with the International Animal Health team of the Friedrich-Loeffler-Institute, Federal Research institute for Animal Health. Her main research interests are emerging infectious diseases and veterinary epidemiology.

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