Avian influenza surveillance in migration of ducks and shorebirds in Taiwan M. C. Cheng1,2, M.S. Lee1, Y. H. Ho3, W.L. Chyi3, S.H. Lee1, C. H. Wang2

1

Animal Health Research Institute, Concil of Agriculture Executive Yuan, Tamsui, Taiwan. 2 Graduate School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan. 3 Wild Bird Society of Taipei, Taiwan Abstract. Monitoring of avian influenza in migratory ducks and shorebirds has begun from spring of 1998 (after Hong Kong bird flu, 1997). The surveillance was still continue after 2003, the outbreak of H5N1 bird flu in Southeast Asia. More then twenty thousands of fecal samples and cloaca swabs were collected from ducks and shorebirds in wetlands and riverbanks distributed in Taiwan and KingMen islet. 286 of avian influenza viruses were isolated by inoculated the specemens into the eggs of special pathogen free (SPF) and these viruses were identified as influenza A by polymerase chain reaction (PCR) in NP gene and then subtyped the hemagglutinin and neunaminidase genes by one set of 15 H and 9 N primer for PCR method. Totally 27 different subtypes of viruses; H1N1, H1N3, H2N3, H2N7, H3N6, H3N8, H3N9, H4N2, H4N3, H4N6, H4N7, H4N8, H5N2, H5N6, H6N1, H6N2, H6N5, H7N1, H7N3, H8N4, H10N3, H10N4, H10N6, H10N7, H10N8, H11N9, and H14N7, were isolated. Anyway, there were no H5N1 virus be detected during eight years from 199t8 to 2005. All of the avian influenza viruses are nonpathogenic according to their intravenous pathogenicity index tests. Compare the nucleotide of some strains of H5 gene sequences and H7 gene sequences indicated that the H5 and H7 genes are static lineage in Taiwan migratory ducks. Introduction Highly pathogenic avian influenza may cause extremely high mortality and resulting in large economic impact on poultry industry and international trade, and thus, it has been in List A of Office International des Epizooties (OIE). H5N1 HPAI had been outbreak in Hong-Kong in 1997 and recently, in Southeast Asia from 2004 till now, that caused more then 90 people died. Waterfowl are AIVs reservoirs, especially in migratory duck and shorebird; there are many researches mention about. They may carry and shed viruses along the migratory pathways. Taiwan is surrounded with H5N1 outbreak regions, and there are

thousands of ducks and shorebirds may carry flu viruses migrating to here through winter season. This surveillance has done in migratory season since the year on 1998. Around the 8 years, twenty seven different subtypes of AI viruses were detected, no H5N1 but H5N2 and H5N6 viruses was find in near this two years. We will making the phylogenies of the H5 and H7 as a refer data for prevention policy. Materials and Methods Samples collection: Surveillance sites included some wetlands or riverbanks in the north (Taipei), the centra (ChanWha), the south (Tainan), the east (I-Lan) of Taiwan, and a separated islet (KingMen) near to Mainland China. Twenty fresh dropping samples were collected from the ground at each location where large numbers of birds congregated during the annually waterfowl migration season, from August to next April. Fresh drooping samples were collected using cotton swabs and subsequently stored in 2 ml transport media in cool boxes. All samples were collected with help from the Taipei Wild Bird Society. Virus isolation and Identification: Virus was isolated by specimen inoculated into the allantoic cavities of 10-day-old embryonated specific pathogen free (SPF) chicken eggs and then inoculated at 35℃ for 48 hrs and examined individually for haemagglutininating activity. Sero subtypes were determining by hemagglutinin and neuraminidase. Hemagglutination (HA) titers in virus stocks were determined with chicken erythrocytes using standard procedures. Virus isolates were characterized by hemagglutination and neuraminidase inhibition assays using reference virus strains and antisera kindly provided by Dr. H. Kida, Hokkaido University, Sapporo, Japan. The avian influenza virus is also identified by RT-PCR using a set of primers created by M.S. Lee, Animal Health Research Institute, Concil of Agriculture Executive Yuan, Tamsui, Taiwan, specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes and NA-subtypes of avian influenza virus were determined by running simultaneously 15 and 9 RT-PCR reactions, each using a set of primers specific to HA-subtype and NA-subtypes. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined, also the 9 RT-PCR for NA-subtypes. Gene sequencing and Phylogenetic analysis Representative viruses of the same H5 subtype and H7 subtype were randomly chosen for further genetic analyses. Purified PCR products were sequenced with synthetic oligonucleotides by using Rhodamine Dye- Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase FS and an ABI model 377 DNA Sequencer

(Perkin-Elmer/Applied Biosystems). Sequence data were edited by the computer packages of DNAStar and GeneDoc. The WISCONSIN Sequence Analysis Package, Version 10.0 (GCG) was used for further analyses and comparison. Phylogenetic analysis was performed with Maximum Parsimony method of PAUP (Phylogenetic Analysis Using Parsimony, Version 4.0, Swafford, Illinois Natural History Survey,Champaign, IL, USA). Results and Discussions Totally 286 avian influenza viruses isolated from the fecal samples of ducks and shorebirds in migrating winter season of 8 years of 1998 to 2005. They are distributed into 27 different subtypes H1N1, H1N3, H2N3, H2N7, H3N6, H3N8, H3N9, H4N2, H4N3, H4N6, H4N7, H4N8, H5N2, H5N6, H6N1, H6N2, H6N5, H7N1, H7N3, H8N4, H10N3, H10N4, H10N6, H10N7, H10N8, H11N9, and H14N7. All of H and N subtypes in avian influenza except H9, H12, H13, H15 and newly H16 were accepted. Most of these viruses were isolated from ducks, only two virus (H6N5 and H5N2) was isolated from shorebird. Anyway, there were no H5N1 virus be detected during eight years from 1998 to 2005. All of the avian influenza viruses are nonpathogenic according to their intravenous pathogenicity index tests and no multiple basic amino acids on the sequence of cleavage site of heamagllutinin(fig.5). Compare the nucleotide of some strains of H5 gene sequences and H7 gene sequences indicated that the H5 and H7 genes are static lineage in Taiwan migratory ducks (fig. Is preparing). NS gene of AIVs isolated from these birds dived into A and B group, all were avian Eurasia gene.

1. 2. 3. 4.

5.

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Main population of wild ducks in monitored wetlands Garganey Northern pintail 白眉鴨

尖尾鴨 Common shoveller 3% 琵嘴鴨 3%

4%

赤頸鴨European wigeon 15%

花嘴鴨 2%

Spotbill duck

其他鴨科 other 4%

Green-winged teal

小水鴨 69%

fig.1 Main population of wild ducks in monitored metlands.

Fig.2 Winter migratory birds monitoring area.

Fig 3 Fecal sample collection.

Banding and collect the cloaca swabs

Fig.4 Banding shorebird. Comparison of HA cleavage site’s amino acid sequences Strains

Cleavage site

A/Ck/Pakistan/447/95

TP-KRKRKR*GLF

HPAI

A/Ck/Victoria/1/92

IP—KKKKR*GLF

HPAI

A/Tk/England/63

TP—KRRRR*GLF

HPAI

A/FPV/Rostock/34

EPSKKRKKR*GLF

HPAI

A/Ck/Victoria/75

LP-KKRRKR*GLF

HPAI

A/Tern/Potsdam/342/6/79

IP----KGR*GLF

LPAI

A/WDK/TW/37/98(H7N1)

IP----KGR*GLF

LPAI

0.0

A/WDK/TW/54/99(H7N1)

IP----KGR*GLF

LPAI

0.0

A/WDK/TW/65/99(H7N1)

IP----KGR*GLF

LPAI

0.0

A/WDK/TW/42/00(H7N1)

IP----KGR*GLF

LPAI

0.0

A/WDK/TW/117/01(H7N1)

IP----KGR*GLF

LPAI

0.0

A/Shorebird/TW/519/04(H5N2) PQRETR*GLF

LPAI

0.0

A/WCK/TW/522/04(H5N6)

LPAI

0.0

PQRETR*GLF

IVPI

Fig. 5 Cleavage sits of H5 and H7 isloated strains