raccoons are considered an invasive alien species in japan . recently , the growing population of feral raccoons has resulted in significant agricultural damage and prompted the initiation of eradication programs in several areas . we used a total of 1,088 serum samples collected from animals captured under this official eradication program over 3 periods in the western region of japan and 1 period in eastern japan during 20052009 for a serologic survey of avian influenza virus ( h5n1 ) infection ( table 1 ) . to detect antibodies specific to the h5 hemagglutinin ( ha ) in the serum samples , we performed a virus neutralization ( vn ) test ( 6 ) with 2 subtype h5n1 viruses , a / indonesia/3006/2005 ( clade 2.1.3 ) and a / whooper swan / mongolia/4/2005 ( clade 2.2 ) . as an initial screening step , we used the serum specimens ( 1:5 dilution ) after receptor - destroying enzyme treatment of the serum to remove nonspecific inhibitors . the vn antibody - positive serum samples were then further tested for their reactivity by using a panel of influenza viruses of multiple subtypes ( table 2 ) as well as western blot analysis ( figure 1 ) . in these assays , we found a total of 10 serum specimens that were positive for vn antibody to subtype h5n1 viruses , representing 0.9% positivity . the a-1 to a-6 serum specimens , which were collected from animals captured within a 10 km area , strongly reacted to a / whooper swan / mongolia/4/2005 ( clade 2.2 ) and more weakly to other clades of subtype h5n1 , h5n2 , and h5n3 viruses . these serum specimens did not react to viruses of other ha subtypes , including h1 , h3 , h7 , and h9 . of note , the a-2 , a-3 , and a-4 animals were from the same litter captured at a lair , which suggests that the detected vn antibodies in these samples might be maternal antibodies from their uncaptured mother , who may have been infected with a subtype h5n1 virus . it is possible that 2 viruses of clade 2.2 , which had slightly different antigenicities , may have infected raccoons in this area , as indicated by the different patterns of cross - reactive vn titers to subtype h5n1 clade 1 and h5n3 viruses . the b-1 and b-2 samples from animals captured at a 25-km distance strongly reacted to both subtype h5n1 clades 2.2 and 2.5 viruses . given that the subtype h5n1 clade 2.5 virus has not circulated since 2004 and that the clade 2.2 virus was more highly reactive than the clade 2.5 virus , these raccoons were likely infected with clade 2.2 viruses , as supported by timing with poultry outbreaks . by contrast , the c-1 and c-2 samples from raccoons captured in eastern japan reacted strongly to a / whooper swan / akita/1/2008 ( clade 2.3.2 ) , unlike the samples from western japan , indicating that the c-1 and c-2 animals were infected with a virus of this clade . together , these data suggest that feral raccoons in japan have been infected with subtype h5n1 viruses of different clades . * we used a / pr8/34 ( h1n1 ) , a / aichi/2/68 ( h3n2 ) , a / quail / aichi/1/09 ( h7n6 ) , a / seal / ma/1/80 ( h7n7 ) , a / hong kong /1073/99 ( h9n2 ) , a / vietnam/1194/04 ( h5n1 ; clade 1 ) , a / indonesia/3006/05 ( h5n1 ; clade 2.1.3 ) , a / whooper swan / mongolia/4/05 ( h5n1 ; clade 2.2a ) , a / chicken / miyazaki / k11/07 ( h5n1 ; clade 2.2b ) , a / whooper swan / akita/1/08 ( h5n2 ; clade 2.3.2 ) , a / hanoi/30850/05 ( h5n1 ; clade 2.3.4 ) , a / chicken / yamaguchi/8/04 ( h5n1 ; clade 2.5 ) , a / chicken / ibaraki/1/05 ( h5n2 ) , and a / whooper swan / shimane/499/83 ( h5n3 ) as antigens for the virus neutralization test . a / whooper swan / mongolia/4/05 ( h5n1 ) virus ( clade 2.2 ) was purified through a 25% sucrose cushion and used as an antigen under nonreducing conditions in the western blot assay . after blocking with 5% skim milk , each raccoon serum specimen ( 1:100 dilution ) was incubated for 1 h and then reacted with horseradish peroxidase ( hrp)labeled protein a / g ( pierce chemical co. , rockford , il , usa ) and subjected to chemiluminescence detection ( ecl plus , ge healthcare uk , ltd , chalfont st . serum from a mouse infected with a / whooper swan / mongolia/4/05 was used as a marker . the negative control reaction when only hrp - protein a / g is used is also shown . lane 1 , anti - h5n1 polyclonal ab ; lanes 211 , vn - positive serum ( 2 , a-1 ; 3 , a-2 ; 4 , a-3 ; 5 , a-4 ; 6 , a-5 ; 7 , a-6 ; 8 , b-1 ; 9 , b-2 ; 10 , c-1 ; 11 , c-2 ) ; lane 12 , vn - negative serum ; lane 13 , negative control . to assess the presence of anti - neuraminidase ( na ) antibodies in the serum samples , we used an na - inhibition ( ni ) assay for the vn - positive samples and found marked inhibition of the na activity of the n1 subtype ( figure 2 ) . we also performed the standard ni assay using another n1 virus , a / swine / iowa/15/30 ( h1n1 ) , to avoid nonspecific na inhibition by h5 antibodies , for 2 vn - positive serum specimens ( a-6 and c-2 ) and found that a-6 and c-2 had positive ni titers of 20 and 80 , respectively . these data demonstrate that vn - positive raccoon serum specimens contain anti - h5n1 antibodies , indicating that raccoons have been infected with subtype h5n1 viruses . each of the n1 to n9 viruses , consisting of a / whooper swan / mongolia/4/05 ( h5n1 ) , a / hong kong/1073/99 ( h9n2 ) , a / whooper swan / shimane/499/83 ( h5n3 ) , a / turkey / ontario/6188/68 ( h8n4 ) , a / duck / alberta/60/76 ( h12n5 ) , a / duck / england/56 ( h11n6 ) , a / seal / massachusetts/1/80 ( h7n7 ) , a / duck / ukraine/1/63 ( h3n8 ) , and a / duck / memphis/546/74 ( h11n9 ) , was incubated with a vn - positive serum sample ( a-6 , b-2 , or c-1 ) for 1 h at 37c , and viral na activity was then measured ( 6 ) . data are shown as percentage of activities compared with incubation with vn - negative serum samples ( 100% ) . three independent tests were performed , and significant reduction of na activity ( p<0.05 , t test with 2-tailed analysis ) was observed only for n1 virus ( * ) . japan has experienced 3 outbreaks of highly pathogenic subtype h5n1 viruses . in the first in early 2004 , the second , in early 2007 , involved the isolation of clade 2.2 subtype h5n1 viruses from poultry in western japan . the third occurred in mid-2008 , when clade 2.3.2 viruses were isolated from diseased swans in the lakes in the northern area of eastern japan . subtype h5n1 viruses have not been reported in any poultry or wild migratory birds under the government surveillance program . our data indicate that raccoons in western japan were likely infected with the clade 2.2 viruses , whereas those in eastern japan were infected with the clade 2.3.2 virus . notably , some antibody - positive raccoons in western japan were captured 6 months before the poultry outbreak with clade 2.2 virus , suggesting that a clade 2.2 subtype h5n1 virus had invaded japan by 2006 . we can not determine by seropositive text results the exact date when the viruses infected the raccoons , because the duration of naturally acquired antibody to subtype h5n1 virus in this species is unknown . recent data indicate that this animal maintains a detectable serum antibody response for at least 9 months after natural exposure to influenza viruses of other ha subtypes such as h1 , h3 , and h4 ( 7 ) . in humans , a detectable antibody response to seasonal viruses can last > 5 years ( 8) and in swine antibodies to the virus have been detected 28 months postinfection ( 9 ) . because wild raccoons are omnivores and highly opportunistic at exploiting foods they prefer , whenever available they could eat diseased or dead migratory birds from areas where subtype h5n1 viruses are enzootic . they also sometimes attack poultry farms for food , creating the potential to transmit virus to domestic poultry . in addition , the increasing likelihood for contact between wild raccoons and humans elevates the possibility of human infection with these viruses , posing risks to public health and increasing the possibility of the emergence of mammalian - adapted mutant viruses with pandemic potential . further investigation and surveillance of influenza virus infections in peridomestic animal species are needed to better understand influenza ecology .
although raccoons ( procyon lotor ) are susceptible to influenza viruses , highly pathogenic avian influenza virus ( h5n1 ) infection in these animals has not been reported . 
 we performed a serosurvey of apparently healthy feral raccoons in japan and found specific antibodies to subtype h5n1 viruses . 
 feral raccoons may pose a risk to farms and public health .