8,592 samples were collected from healthy ducks , chickens , quail , and pigs at live markets in vientiane , champasak , and savannakht provinces , near the borders of thailand , vietnam , and cambodia ( figure 1 ) . the samples were injected into chicken or duck eggs , and allantoic fluid was harvested after 72 h and analyzed as described ( 9 ) . as of january 2006 , no evidence of any influenza virus subtypes had been detected in any species . map of lao people 's democratic republic indicates regions ( black areas ) of influenza virus surveillance . outbreaks of h5n1 occurred in vientiane ( location of isolation of a / duck / laos/3295/2006 ) ( a ) , savannakht ( b ) , and champasak ( c ) provinces during late 2003 and early 2004 . in january 2006 , serologic surveillance was initiated to clarify whether poultry in lao pdr had been exposed to influenza viruses and recovered . vaccination of poultry is not practiced in lao pdr , which made serologic surveillance possible . serologic analysis for antibodies to influenza viruses was performed with elisa and hemagglutination inhibition ( hi ) . the idexx flockchek aiv ( idexx laboratories , inc . , westbrook , me , usa ) was used to initially screen for antibodies to influenza . sera were then tested by hi with antigens to h5 , h6 , h7 , and h9 influenza subtypes as described ( 10 ) . initial results from serologic surveillance confirmed virus isolation results and absence of vaccine use ; however , in early february 2006 , in apparently healthy ducks in a live market in vientiane , we detected antibodies to the viral hemagglutinins h5 ( hi titers 40 to < 640 ) and h9 ( hi titer 80 ) . only 21 ( 3.5% ) of 604 birds had positive test results at that time ; 1 duck had antibodies to h5 and h9 . in late february 2006 , h5n1 was isolated from healthy ducks at a layer farm in vientiane capital . the source of the isolated h5n1 is unknown , but our 8-month prospective surveillance program showed that it was recently introduced into this infected flock . cloacal samples from 40 ducks at a duck farm in vientiane tested negative for h5n1 virus in november 2005 , but upon retesting in late february 2006 , results for the same flock were positive for h5n1 . to identify the source of the new isolate , we compared the sequence of its hemagglutinin gene with those of h5n1 viruses isolated since 2003 in china , thailand , vietnam , and cambodia ( figure 2 ) . phylogenetic relationship of the hemagglutinin ( ha ) gene of representative h5n1 influenza virus strains isolated in southeast asia from 2003 through 2006 . sequence analysis indicated that the new h5n1 isolate ( a / duck / laos/3295/2006 ) ( genbank accession no . dq845348 ) was not closely related to the other viruses circulating in southeast asia during 2003 and 2004 . it clustered phylogenetically with h5n1 viruses isolated in china in 2005 and was most closely related to a 2005 human isolate from anhui , china ( a / anhui/1/2005 ) ( 11 ) . furthermore , the 2004 laotian h5n1 virus strains clustered with 2004 isolates from malaysia , thailand , and vietnam . this evidence suggests that the h5n1 virus had recently been introduced into lao pdr ; however , its direct source is unknown . despite its origin in china , it clustered with a 2005 vietnamese virus isolate ( a / duck / vietnam/568/2005 ) and therefore may have crossed into lao pdr from vietnam . a malaysian h5n1 isolate ( a / chicken / malaysia/935/2006 ) also clustered with this group rather than with malaysian isolates from 2004 , which indicates that it was likely a new introduction into malaysia . two months after the isolation of h5n1 , 40 cloacal swabs and 28 serum samples were collected from ducks at the infected and surrounding farms . all cloacal swabs had negative results for influenza viruses , and serum samples had negative results for antibodies to h5n1 . as of june 2006 , no reports of infected poultry have been made since the isolation , which indicates that h5n1 did not spread and its introduction may have been an isolated event ; however , surveillance is ongoing . as highly pathogenic h5n1 influenza viruses continue to spread into eurasia , whether they will become endemic in eurasian poultry or whether the highly pathogenic h5n1 will burn out and disappear with the possibility of reintroduction is unknown . we describe results from the first active surveillance study in lao pdr since the 2004 outbreak . we extensively sampled live markets and local farms in vientiane capital and additionally sampled districts bordering cambodia , china , thailand , and vietnam . a genetically distinct h5n1 was isolated from apparently healthy ducks , which indicates reintroduction of h5n1 in lao pdr . serologic data , antibodies to h5 in serum samples from apparently healthy ducks found in live markets , provide additional evidence of h5n1 in lao pdr ; however , we can not rule out the illegal use of vaccines . evidence of an h9 subtype was also detected in a duck that was seropositive for h9 antibodies ; however , no h9 influenza subtypes were isolated . whether h5n1 influenza viruses are endemic in wild migratory birds is unresolved ; however , our study supports the notion that highly pathogenic h5n1 viruses are not endemic in lao pdr . our findings suggest that the h5n1 virus did not become established after the 2004 outbreak in lao pdr , which is less densely populated than neighboring thailand , vietnam , and china ( high summer temperatures in lao pdr may have also been a factor ) . instead , the virus disappeared and was later replaced by a newly introduced virus . although no measures were taken to eradicate the virus , the newly introduced h5n1 has since vanished from the area . the area has been identified as high risk and has become the focus of ongoing surveillance . this virus appears to have disappeared on its own in lao pdr in the absence of vaccines and with limited surveillance , unlike what happened in the more densely populated thailand , vietnam , and china . in china , h5n1 continues to circulate in poultry despite ongoing surveillance and use of vaccines ; heroic measures were required to eradicate h5n1 in thailand , and since the use of vaccines in vietnam in 2005 , no more human cases have been reported . these findings are encouraging for the less densely populated , developing countries of africa and asia because they suggest that h5n1 viruses are not being perpetuated through wild migratory birds . to date , wild birds have not been found to carry and spread highly pathogenic h5 and h7 influenza viruses ; instead , each outbreak has been caused by a phylogenetically new virus , whose parent viruses were not pathogenic ( 12,13 ) .
a prospective surveillance program for influenza viruses was established in lao people 's democratic republic ( pdr ) in july of 2005 . 
 we report isolation of h5n1 virus genetically distinct from h5n1 circulating in 2004 , which indicates reintroduction of h5n1 into lao pdr after its disappearance ( i.e. , no virologic or serologic evidence ) for 2 years .