during 20042007 , as part of a broader biodiversity survey and inventory program , we sampled birds from mostly forested sites in guangxi and guizhou provinces in the southern part of the people s republic of china ( figure ) .
sampling was conducted by mist netting and selective harvesting with shotguns ; all birds in the study were apparently healthy and behaving normally at the time of collection . because initial sampling was focused on endoparasite communities , samples from 20042005 consisted of complete gastrointestinal tracts frozen in liquid nitrogen . in 20062007
southeastern asia , showing 5 sites in the people s republic of china where land birds were collected and tested for influenza a virus .
prevalence values were 4% ( n = 103 ) in dashahe in 2006 ; 6% ( n = 194 ) in kuan kuoshui in 2006 ; 0.3% ( n = 328 ) in shuipu in 2007 ; 3% ( n = 184 ) in jing xi , in 2004 ; and 0% ( n = 130 ) in shiwandashan in 2005 .
a total of 184 samples were collected from jing xi municipality in guangxi ( 21.122n , 105.964e ) in 2004 , 130 from shiwandashan nature reserve in guangxi ( 21.840n , 107.880e ) in 2005 , 103 from dashahe nature reserve in guizhou ( 29.167n , 107.575e ) in 2006 , 194 from kuan kuoshui nature reserve in guizhou ( 28.226n , 107.160e ) in 2006 , and 328 from shuipu village , guizhou province ( 25.485n , 107.882e ) in 2007 ( figure ) .
samples were tested for influenza a virus by real - time reverse transcription  pcr ( 6 ) in 2 diagnostic laboratories ( southeast poultry research laboratory , us department of agriculture , athens , ga , usa , and national wildlife health center , us geological survey , madison , wi , usa ) .
of 939 samples tested , 24 were positive for influenza a viruses ( prevalence 2.3% , table ; complete summary in appendix table ) .
if migratory behavior ( species classified as migratory or nonmigratory on the basis of descriptions by mackinnon and phillipps ) , was considered , 11 ( 4.8% ) of 231 samples from species showing marked seasonal migrations were influenza positive .
however , only 13 ( 1.8% ) of 708 samples from nonmigratory species were positive .
the cumulative binomial probability that such a high number ( 11 ) of positive samples would result among the 231 migratory - species samples , were the true prevalence to be 1.8% , is low ( p = 0.0013 ) .
thus , migratory species appear to have higher influenza infection rates . in terms of general habitat use ( 7 ) , open - country species were slightly more prone to be influenza positive ( 8 [ 2.9% ] of 274 samples ) than forest species ( 16 [ 2.4% ] of 665 samples ) , but the difference was not significant ( cumulative binomial probability , p>0.05 ) .
interactions between migratory behavior and habitat use were not significant ( contingency test , p>0.05 ) .
although all infections detected were among songbirds ( passeriformes ) , the sampling also concentrated on songbirds ( 94.3% ) .
thus , we could not test adequately the hypothesis that influenza prevalence was equivalent between songbirds and other birds .
an obvious question is whether the influenza a viruses we detected belong to the highly pathogenic subtype h5n1 strain currently circulating across much of asia .
pcr ( 6 ) , although this result does not exclude the possibility that h5 viruses were among the positive samples .
the preservation status of samples we tested prevented virus isolation or full , strain - level characterization of influenza viruses .
the subtype h5n1 strain of influenza virus has spread rapidly and has been detected across much of central and southern eurasia . although movements of wild birds have been implicated in this spread ( 8) , other studies question ( 9,10 ) or contradict ( 11 ) this idea .
an important part of the argument centers on the question of the occurrence of the virus in wild birds without obvious illness , which can be difficult to interpret given the low prevalence of influenza .
for instance , a recent study based on sampling > 13,000 migratory birds in china detected the subtype h5n1 strain of influenza virus only 8 times ( 12 ) , and similar results have been obtained elsewhere ( 2 ) .
our study , although not successful in characterizing influenza viruses to specific strains , nonetheless shows that influenza a virus infection occurs in more bird species than previously assumed and that influenza a infections can be found in birds that behave normally and show no sign of illness .
although a review of avian influenza virus ecology ( 1 ) discussed the occurrence of influenza viruses across all groups of birds ( and other vertebrates ) , subsequent studies have assumed that waterbirds are the primary reservoir ( 8,13,14 ) . in this study ,
although waterbirds could have higher prevalences , we have demonstrated broad occurrence of influenza viruses in diverse taxa of passeriformes ( songbirds ) in southeast asia .
this result suggests that land birds may also be a major reservoir of influenza viruses .
we have taken a step toward a more complete understanding of influenza virus ecology among wild birds .
our partial survey of influenza virus distributions across the rich avifaunas of the southern region of china demonstrated frequent infections .
we suggest that to be effective future surveillance efforts will need to include the full diversity of wild birds .
 water birds are considered the reservoir for avian influenza viruses . 
 we examined this assumption by sampling and real - time reverse transcription  
 pcr testing of 939 asian land birds of 153 species . 
 influenza a infection was found , particularly among migratory species . 
 surveillance programs for monitoring spread of these viruses need to be redesigned . 