| Qinghai Lake and the Brahmaputra rivers basin in Tibet are critical breeding grounds and wintering areas for migratory birds,which supporting thousands of migrants each year.In 2005,an unprecedented epizootic at Qinghai Lake led to an anomalously high cumulative mortality of more than 6000 migratory birds including over 3000 bar-headed geese(Anser indicus).H5N1 subsequently spread to Europe and Africa,and has re-emerged in wild birds along the Central Asia flyway several times in following years.This reminds us the role of wild birds in circulation of highly pathogenic avian influenza virus H5N1.Apart from avian influenza virus,bar-headed geese would also be the carriers of pathogenic bacteria,which poses potential menace to the health of wildlife,domestic animals and even human.Besides,every year,there are thousands of bar-headed geese migrate towards Qinghai Lake to breed and hatch.Mixing of birds would increase the likelihood of pathogen exchange among populations.In this study,1792 samples of tissues and fresh feces of bar-headed geese from Qinghai and Tibet regions were collected during 2013 ~ 2015.The nucleic acid of avian influenza virus contained in the samples were detected,and viruses were isolated by embryonated eggs afterwards.The viral genomes of each isolate were sequenced to assess the molecular and phylogenetic characteristics.In the meantime,bacteria in the feces of bar-headed geese were also isolated and identified.The major conclusions are as follows:1.AIVs detection and isolation in bar-headed geese during 2013 ~ 2015There were 124,348 and 860 samples were collected respectively from Qinghai Lake in 2013,2014 and 2015,and 460 fecal samples were also collected from Tibet region in 2015.Morever,a dead juvenile bar-headed goose was dissected and sampled.The results of AIV detection reflected that the corresponding positive rate in 2013,2014 and 2015 were 0.72%(9/124),5.7%(20/348)and 18%(157/860).And the positive rate of AIV of bar-headed geese from Tibet region was 22%(101/460).AIVs isolated from dead juvenile bird and three feces in 2015 were identified and named as A/bar-headed goose/Qinghai/F/2015(H5N1),A/bar-headed goose/47/133/2015(H5N1),A/ bar-headed goose/Qinghai/70/2015(H5N1)and A/bar-headed goose/Qinghai/133/2015(H5N1),which were known as QHL-F,QHL-47,QHL-70,QHL-133 for short.2.Molecular characteristics and phylogenetic analysisThe viral genomes of four isolated AIVs were sequenced and subsequently analysis the molecular and phylogenetic features.The results indicated that isolated AIVs of QHL-F?QHL-47 ? QHL-70 ? QHL-133 deived from the H5N1 clade 2.3.2.1c HA lineage,which arranged from 99% ~ 100%.Collectively,the gene segments of isolated AIVs were most closely related to and possess high nucleotide sequence identity to the H5N1 influenza isolate named A/whooper swan/Henan/SMX1/ 2015(H5N1).The HA cleavage site of isolated AIVs all contained a series of continuity basic amino acids which meet the characteristic of highly pathogenic AIV.The HA segment of these AIVs possess 138A?160A amino acid substitutins,indicating the potential binding preferential to mammalian receptor.The stalk of NA had a 20-amino-acid deletion,which would contribute to the pathogenicity of these AIVs in mammals.While other mutations associated with virulence and drug resistance of AIVs were not observed.3.Isolation and identification of bacteria in feces of bar-headed geese30 feces of bar-headed geese were collected from samples collected during 2015 in Qinghai Lake.A total of 123 bacteria which belong to 10 genus were isolated from 30 fecal samples.One strain from each of the 10 genus were further cultured and identified.The ten bacteria were identified by colonial morphology observation,biochemical properties tests,PCR amplification of 16 S rRNA genes,which indicated that 10 isolated strains were Escherichia coli,Rahnella aquatilis,Enterococcus mundtii,Bacillus sublitis,Arthrobacter citreus,Shewanella pulrefaciens,Enterobacter amnigenus,Pantoea agglomerans,Aeromonas salmonicida,and Klebsiella oxytoca respectively.Among these bacteria,R.aquatilis,A.salmonicida and P.agglomerans were multidrug resistant,while other bacteria were resistant to ampicillin and tetracycline and showed varying degrees of sensitivity to the rest drugs.The results obtained from this study indicated that Anser indicus would take amounts of pathogen which exhibit antibiotic resistance. |
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