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 . the sentinel flocks at lake constance were situated in 1 ) radolfzell ( mggingen ) , germany ( 474558n , 85945e ) ; 2 ) altenrhein , switzerland ( 472925n , 93245e ) ; and 3 ) bregenz - thal ( rheindelta ) , austria ( 473060n , 93855e ) . the 2 other stations were situated 4 ) on the isle of koos close to the island of rgen in mecklenburg western pomerania , germany ( 541013n , 132411e ) and 5 ) near the oder valley at lake felchow ( brandenburg ) , germany , ( 530309n , 140806e ) ( figure 1 ) . after their wing feathers were clipped , 1020 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 , hrbanz , 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 ) . brandenburg , germany ( d ) , and  isle of koos , mecklenburg  western pomerania , germany ( e ) . 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 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 . 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 13 sampling dates ( figure 2 ) . 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 . 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 115 of month ; 2 , day 16 through end of month . aiv , avian influenza virus ; ct , cycle threshold value ; ha , hemagglutinin ; na , neuraminidase ; min , minimum ; max , maximum ; nd , not determined .  each sentinel flock comprised 1020 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 , avian influenza virus ; lp , low pathogenicity .  each flock consisted of 1020 birds . blood samples were collected from the ducks once a month , and serum was tested in a cnp - elisa after heat inactivation at 56c for 30 min . after each natural infection , sentinel animals seroconverted , and serum scored positive in the cnp - elisa within 24 weeks . by hemagglutination inhibition test using homosubtypic but not autologous antigen , 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 2007may 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 . 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 can not 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 .
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 .