Low Pathogenic Avian Influenza (H7N1) Transmission Between Wild Ducks and Domestic Ducks

SHORT COMMUNICATION

Low Pathogenic Avian Influenza (H7N1) TransmissionBetween Wild Ducks and Domestic DucksO. R. Therkildsen1, T. H. Jensen2*, K. J. Handberg2, K. Bragstad3 and P. H. Jørgensen2

1 National Environmental Research Institute, University of Aarhus, Aarhus, Denmark2 National Veterinary Institute, Technical University of Denmark, Aarhus, Denmark3 Department of Virology, Statens Serum Institut, Copenhagen, Denmark

Impacts

• The paper describes a virological investigation in a mixed flock of domestic

ducks and geese and mallards reared for shooting following detection of

avian influenza virus antibodies subtype H5 in domestic geese.

• Low pathogenic avian influenza virus subtype H7N1 was found in both

domestic ducks and wild mallards, indicating that transmission of H7N1

virus was likely to have taken place between these.

• The importance of implementing and maintaining appropriate biosecurity

measures is re-emphasized.

Introduction

Wild waterbirds are considered the natural reservoir for

avian influenza viruses (AIV) (Olsen et al., 2006). North-

ern European outbreaks of highly pathogenic AI have

originated from low pathogenic avian influenza viruses

(LPAIV) previously found in waterbirds (Munster et al.,

2005). So far, only H5 and H7 AIV subtypes are known

to potentially become highly pathogenic after introduc-

tion to domestic birds (Webster et al., 1992; Banks et al.,

2001). Cross-species transmission of LPAIV seems to

occur regularly in wild bird populations (Ferro et al.,

2008; Siembieda et al., 2010) and LPAIV subtypes in

poultry partially reflects those found in wild birds (Olsen

et al., 2006). However, direct evidence of transmission of

LPAIV between wild and domestic birds has been difficult

to establish. Here, we present a case of concomitant isola-

tion of LPAIV H7N1 viruses in domestic ducks (Anas

platyrhynchos domesticus) and wild mallards (Anas platy-

rhynchos) indicating that disease transfer between these

had taken place.

In April 2008, the Danish national surveillance of AI

detected antibodies against AIV subtype H5 in a flock of

domestic geese (Anser anser domesticus). Accordingly, a

virological investigation was carried out on the premises,

which also held domestic ducks and mallards reared for

shooting. A flock of up to 200 wild mallards at a pond

about 50 m away from the sheds were included in the

Keywords:

Avian influenza; wild ducks; domestic ducks;

mallards; transmission

Correspondence:

O. R. Therkildsen. Department of Wildlife

Ecology & Biodiversity, National Environmental

Research Institute, Aarhus University, Kalø,

Grenavej 14, DK-8410 Rønde, Denmark.

Tel.: +45 89201700; Fax: +45 89201514;

E-mail: [email protected]

*First two authors contributed equally to this

work.

Received for publication January 23, 2010

doi: 10.1111/j.1863-2378.2010.01375.x

Summary

This article describes a virological investigation in a mixed flock of ducks and

geese following detection of avian influenza virus antibodies in domestic geese.

Low pathogenic H7N1 was found in both domestic and wild birds, indicating

that transmission of virus was likely to have taken place between these. The

importance of implementing and maintaining appropriate biosecurity measures

is re-emphasized.

Zoonoses and Public Health

312 ª 2010 Blackwell Verlag GmbH • Zoonoses Public Health. 58 (2011) 312–317

investigation. The wild mallards were considered the most

plausible source of infection as they had access to spilled

food on the premises and were seen to mix with the

domestic geese. Subsequently, isolates of LPAIV H7N1,

but none of subtype H5, were obtained from both

domestic ducks and wild mallards at the pond on the

premises. The infected flocks, but not the wild mallards,

were stamped out immediately.

To investigate the extent to which AIV was present in

local wild birds, samples from wild mallards, a common

LPAIV host (Munster et al., 2005), and shelducks (Tadorna

tadorna) were subsequently collected at nearby sites.

Mallards and shelducks are abundant breeding species in

Danish farmland occupying smaller wetlands.

Materials and Methods

The flock consisted of 300 domestic geese, 250 domestic

ducks and 1500 mallards reared for shooting (Mellerg-

aard, 2008). The domestic geese and domestic ducks were

kept in the same shed in separate sections; only the geese

had access to outside areas. The mallards reared for

shooting were kept in outside pens covered with nets.

None of these had access to the nearby pond, but the

wild mallards present at the pond were seen to mix with

the geese in the outside areas and occasionally entered the

shed to feed.

On 21 April, sera from domestic ducks (n = 23),

domestic geese (n = 22) and mallards reared for shooting

(n = 45) were collected as part of the national surveil-

lance programme. On 24 April, following the detection of

antibodies against AIV subtype H5 in the domestic geese,

swabs were collected from the domestic geese (n = 10)

and domestic ducks (n = 10) to confirm the serological

analysis. On 29 April, tracheal and cloacal swabs from

domestic ducks (n = 20) in sheds and wild mallards at a

pond in the premises (n = 24) and sera from domestic

ducks (n = 50) in sheds were collected for further diag-

nostic investigation. For animal welfare reasons, tracheal

swabs were taken only from dead birds.

The survey was extended to include 33 ponds and

flooded fields up to approximately 10 km from the farm

(Table 1, Fig. 1); here, samples of fresh droppings from

wild mallards (n = 46) and shelducks (n = 10) were col-

lected from 8 to 18 May.

Antibody titres were determined by haemagglutination

inhibition test (HI) as described by Commission of the

European Communities, Council Directive (CEC 2006).

A/Ostrich/Denmark/72420/96 (H5N2) and A/Duck/Den-

mark/64650/03 (H5N7) were used as antigens for detec-

tion of antibodies against AIV H5 in domestic geese,

domestic ducks and mallards reared for shooting (21

April). A/Turkey/England/647/77 (H7N7) and A/African

Starling/983/79 (H7N1) were used for detection of anti-

Table 1. Samples from domestic geese, domestic ducks, mallards reared for shooting, wild mallards and shelducks tested for avian influenza

Sampling

date

No. samples

(n · pools of) Species Origin of birds

Type of

sample

Results RT-PCR*Virus

isolation

Sequence

analysis

Serology

(HI)

M H5 H7 H5 H7

21 April 23 D. duck Premises (shed) Serum – – – – – 0/23 0/23

21 April 22 D. goose Premises (shed) Serum – – – – – 3/22 0/22

21 April 45 Mallard r.s. Premises (shed) Serum – – – – – 0/45 0/45

24 April 10 (2 · 5) D. goose Premises (shed) Oropharyngeal 0/2 0/2 0/2 Nd Nd – –

24 April 10 (2 · 5) D. goose Premises (shed) Cloacal 0/2 0/2 0/2 Nd Nd – –

24 April 10 (2 · 5) D. duck Premises (shed) Oropharyngeal 0/2 0/2 0/2 Nd Nd – –

24 April 10 (2 · 5) D. duck Premises (shed) Cloacal 2/2 0/2 2/2 2/2 (H7N1) LP H7 – –

29 April 20 (not pooled) D. duck Premises (shed) Tracheal 0/20 0/20 0/20 Nd Nd – –

29 April 20 (not pooled) D. duck Premises (shed) Cloacal 1/20 0/20 1/20 Nd� LP H7 – –

29 April 50 D. duck Premises (shed) Serum – – – – – 0/50 5/50

29 April 24 (4 · 5, 1 · 4) W. mallard Premises (pond) Tracheal 0/5 0/5 0/5 Nd Nd – –

29 April 24 (4 · 5, 1 · 4) W. mallard Premises (pond) Cloacal 3/5 0/5 2/5 2/5 (H7N1)� LP H7 – –

8–18 May 46 (16 · 1, 8 · 2,

2 · 3, 2 · 4)§W. mallard Nearby sites Faeces 0/28 0/28 0/28 Nd Nd – –

8–18 May 10 (1 · 1, 3 · 2, 1 · 3)§ Shelduck Nearby sites Faeces 0/5 0/5 0/5 Nd Nd – –

HI, haemagglutination inhibition test; D, domestic; W, wild; r.s.: reared for shooting; Nd, not done.

*Number of positive samples or pools of the total number of samples or pools tested. The H7 sequence analyses were performed on these sam-

ples.�The sample became unavailable for virus isolation.�One of the sample positive for the M gene by RT-PCR did not yield a virus isolate.§Pools represent different sampling sites.

O. R. Therkildsen et al. Transmission of LPAIV H7N1

ª 2010 Blackwell Verlag GmbH • Zoonoses Public Health. 58 (2011) 312–317 313

bodies against AIV H7 as recommended by CEC (2007).

Two AIV subtype antigens were used to elucidate

potential cross reactivity with the N protein (CEC, 2007).

The sera collected from the ducks on 29 April were

tested against A/Ostrich/Denmark/72420/96 (H5N2) and

A/African Starling/983/79 (H7N1) antigens.

Swab samples (Table 1) were transported in phosphate

buffered saline with antibiotics and tested by reverse

transcriptase (RT)-PCR and virus isolation (CEC, 2006).

Total RNA was extracted from swabs using the RNeasy

Mini kit (Qiagen, Sollentuna, Sweden) following the

manufacturer’s protocol. RT-PCR was performed using

the FB-AI-M52C and -M253 primers for M-gene detec-

tion (Fouchier et al., 2000); GK7-3 and -4 primers for

H7 detection and H5-KHA-1 and -3 primers for H5

identification (Slomka et al., 2007). RT-PCR amplicons

were sequenced by DNA Technology (Aarhus, Denmark)

using FB-AI-M52C and -M253 primers for M gene and

GK7-3 and -4 primers for H7 identification.

Virus isolation was achieved by inoculating swab super-

natant in the allantoic cavity of 8- to 10-day-old specific

pathogen free chicken embryos (Lohmann Tierzucht,

Cuxhaven, Germany). The allantoic fluid from eggs with

dead embryos and eggs harvested after 6 days of incuba-

tion were examined for haemagglutinating activity (CEC,

2006). Neuraminidase inhibition assay for N typing of the

isolates was performed according to Alexander (1974).

RT-PCR and sequencing of the internal segments PB2,

PB1, PA, NP, M and NS of the two isolates from the wild

mallards were performed as described by Brown et al.

(1998). Partial sequence analyses were performed as

described by Handberg et al. (2009).

Full length sequencing was performed on the virus iso-

late from the domestic ducks. Viral RNA was extracted

from the allantoic fluid by an automated MagNA Pure

LC Instrument applying the MagNa Pure LC Total

Nucleic Acid Isolation Kit (Roche Diagnostics, Basel,

Switzerland). The different gene segments were amplified

by OneStep RT-PCR Kit (Qiagen) as previously described

(Bragstad et al., 2007) including a 2-min elongation step

for all genes. The primers for RT-PCR were subtype uni-

versal targeting the highly conserved non-coding RNA

regions at the 5¢- and 3¢-end of each segment (Hoffmann

et al., 2001). PCR products were purified using the

GFX� PCR DNA and Gel Band Purification Kit (Amer-

sham Biosciences, Freiburg, Germany) prior to sequenc-

ing. Purified PCR products were sequenced directly. The

sequencing reaction was performed by ABI PRISM Big-

Dye Terminators v3.1 Cycle Sequencing Kit (Applied Bio-

systems, Foster City, CA, USA) as described previously

(Bragstad et al., 2005). The development of the sequences

was performed on an automatic ABI PRISM 3130 genetic

analyzer (Applied Biosystems) with 50 cm capillaries.

Consensus sequences were generated in seqscape Soft-

Fig. 1. Map illustrating the location of the premises, where LP H7N1 was isolated from wild mallards and domestic ducks, and nearby sites,

where samples from wild mallards and shelducks were collected.

Transmission of LPAIV H7N1 O. R. Therkildsen et al.


ware v2.5 (Applied Biosystems). Sequence assembly, mul-

tiple alignment and alignment trimming were performed

using the bioedit software v.7.0.5 (Hall, 1999).

Genbank accession numbers

Full-genome sequences of A/duck/Denmark/53-147-8/08

are available in GenBank with the following accession

numbers: HA GQ401157, NA GQ401158, NP GQ401159,

M GQ401160, NS GQ401161, PB2 GQ401162, PB1

GQ401163 and PA GQ401164.

Results and Discussion

Initially, the serological surveillance programme revealed

AIV subtype H5 antibodies in three domestic geese

(Table 1). This finding triggered further investigations. By

this, LPAIV H7 was subsequently identified by PCR and

A/duck/Denmark/ 53-147-8/08 (H7N1) was isolated from

domestic ducks housed in sheds adjacent to the domestic

geese (Fig. 2). On the basis of the partial sequence analy-

ses of the H7 gene and the internal fragments PB2, PB1,

PA, NP, M and NS, we conclude that this isolate was

A/turkey/Italy/8535/2002 (H7N3)




A/chicken/Italy/270638/02 (H7N3)

A/chicken/England/4054/2006 (H7N3)

A/chicken/England/4266/2006 (H7N3)

A/mallard/Sweden/S90597/2005 (H7N7)

A/mallard/Netherlands/33/2006 (H7N8)

A/duck/Denmark/53-147-8/08 (H7N1)





A/chicken/Italy/322/2001 (H7N1)




A/duck/Nanchang/1904/1992 (H7N1)

A/Pekin robin/California/30412/1994 (H7N1)

A/parakeet/Netherlands/267497/94 (H7N1)

A/duck/Hongkong/301/72 (H7N1)

A/chicken/Germany/1934 (H7N1)

A/FPV/Rostock/1934 (H7N1)

A/mallard/Alberta/34/2001 (H7N1)

A/rhea/North Carolina/39482/1993 (H7N1)

0.02

100

100

100100

10099

72

77

93100

100

100

97

97

7389

9997

98

79

95

100

100

A/guinea fowl/Italy/155/2000 (H7N1)

A/mallard/Italy/43/01 (H7N3)




A/mute swan/Hungary/5973/2007 (H7N7)

Fig. 2. Evolutionary relationship of the HA nucleotide sequence (1685 bp) of A/duck/Denmark/53-147-8/08(H7N1) compared to the twenty most

closely related H7 viruses and other representative H7N1 viruses public available. The Danish isolate is indicated in bold typeface in the mid-point

rooted neighbour joining tree. Bootstrap values of 1000 resamplings in percent (>70%) are indicated.



identical to the LPAIV H7N1 isolates obtained a few days

later from wild mallards at a pond in the premises. The

sequence analysis also showed that this virus has high

similarity with other European virus sequences (Fig. 2).

One sample from the domestic ducks was positive by

PCR, but unfortunately, the sample was not available for

virus isolation.

The detection of both antibodies against AIV H5 and

LPAIV H7N1 showed that at least two strains of AIV had

been introduced to the flock. Most likely, the domestic

geese had experienced AIV H5 earlier and cleared the

virus, as no AIV subtype H5 could be detected by viro-

logical examination. Transmission of AIV between the

domestic geese and domestic ducks was not evident since

the geese had H5 antibodies and no detectable virus,

whereas LPAIV H7N1 and H7 antibodies were found in

the domestic ducks (Table 1). The initial failure to detect

antibodies against AIV subtype H7 during routine surveil-

lance could reflect a recent introduction of this subtype

into the flock as only five of 50 domestic ducks had anti-

bodies against AIV subtype H7 1 week later (Table 1).

Remarkably, although the results of the serological sur-

veillance suggested an infection with AIV of subtype H5,

only subtype H7N1 was detected in swabs collected

3 days after the sera had been collected.

The concomitant isolation of LPAIV H7N1 viruses in

the wild mallards at the pond next to the premises and

domestic ducks indicates a viral transmission between

these. However, it is not possible to determine whether

wild mallards had been the source of infection or if this

disease transfer had occurred from domestic ducks to the

wild mallards. The origin of the wild mallards at the

pond on the premises remains unknown, but they were

probably non-breeders attracted to the farm by spilled

food, since the spring migration had finished at the time

of the investigation. Unfortunately, no information about

turn-over in the flock during spring migration or local

movements between the premises and neighbouring areas

could be obtained. We are therefore unable to assess the

potential for transmission between infected wild mallards

at the nearby pond and the local wild bird reservoir.

Whilst the wild mallards may have introduced LPAIV

H7N1, the possibility of virus introduction by other feral

birds, virus-contaminated material or infected domestic

birds cannot be excluded. Previous studies indicate that

most introductions of highly pathogenic H5N1 in Europe

have been through migrating birds (Kilpatrick et al.,

2006). Likewise, wild mallards were reported to be the

source of an H7N3 outbreak in turkeys in Italy (Campi-

telli et al., 2004), although it should be noted that indi-

rect transmission from environmental virus reservoirs

may be an underestimated source of infection (Rohani

et al., 2009).

As AIV was not detected in our sampling of wild mal-

lards and shelducks at nearby sites outside the premises

(Table 1, Fig. 1), we suggest that until the flocks were

stamped out, the spread of LP H7N1 from the infected

population at the premises either had not taken place or

had been limited. This suggests little or no movement of

individuals between the pond and nearby sites or low

rates of disease transmission between individuals.

Real-time PCR was not performed on the samples and

therefore data on relative virus loads are not available.

This case re-emphasizes the importance of implementing

and maintaining appropriate biosecurity measures at all

times, to minimize wild bird contact with domestic birds

at existing and new production facilities, especially through

the removal of food, and avoidance of waterbodies and

ponds, which may attract wild birds. This case also con-

firms that inappropriate biosecurity measures may facili-

tate direct transfer of AIV from infected poultry to the

avian reservoir of wild birds, which potentially could fur-

ther spread disease agents. During highly pathogenic avian

influenza outbreaks in poultry, this may even severely

threaten wild bird populations of conservation concern.

Acknowledgements

The collection of samples from wild birds was funded by

the Danish Food and Veterinary Agency. We wish to

thank Lars Hansen for collecting the samples. Tony Fox

kindly commented on the manuscript.

References

Alexander, D. J., 1974: Comparison of the neuraminidases of

three avian paramyxoviruses. Arch. Gesamte Virusforsch. 44,

28–34.

Banks, J., E. S. Speidel, E. Moore, L. Plowright, A. Piccirillo, I.

Capua, P. Cordioli, A. Fioretti, and D. J. Alexander, 2001:

Changes in the haemagglutinin and the neuraminidase genes

prior to the emergence of highly pathogenic H7N1 avian

influenza viruses in Italy. Arch. Virol. 146, 963–973.

Bragstad, K., P. H. Jørgensen, K. J. Handberg, S. Mellergaard,

S. Corbet, and A. Fomsgaard, 2005: New avian influenza A

virus subtype combination H5N7 identified in Danish mal-

lard ducks. Virus Res. 109, 181–190.

Bragstad, K., P. H. Jørgensen, K. J. Handberg, and A. Fomsg-

aard, 2007: Genome characterisation of the newly discovered

avian influenza A H5N7 virus subtype combination. Arch.

Virol. 152, 585–593.

Brown, I. H., P. A. Harris, J. W. McCauley, and D. J. Alexan-

der, 1998: Multiple genetic reassortment of avian and

human influenza A viruses in European pigs, resulting in

the emergence of an H1N2 virus of novel genotype. J. Gen.

Virol. 79, 2947–2955.

Transmission of LPAIV H7N1 O. R. Therkildsen et al.


Campitelli, L., E. Mogavero, M. A. De Marco, M. Delogu, S.

Puzelli, F. Frezza, M. Facchini, C. Chiapponi, E. Foni, P.

Cordioli, R. Webby, G. Barigazzi, R. G. Webster, and I.

Donatelli, 2004: Interspecies transmission of an H7N3 influ-

enza virus from wild birds to intensively reared domestic

poultry in Italy. Virology 323, 24–36.

CEC, 2006: Council Directive 2006/437/EF. Virological tests

and evaluation of results. The Commission of the European

Communities. In: Official Journal of the European Union –

Approving a Diagnostic Manual for Avian Influenza as Pro-

vided for in Council Directive 2005/94/EC, pp. 16–17.

CEC, 2007: Council Decision 2007/268/EF. Surveillance pro-

grams in the member states concerning avian influenza in

poultry and wild birds. In: Official Journal of the European

Union May 3, 2007. L 115/3.

Ferro, P. J., J. El-Attrache, X. Fang, S. N. Rollo, A. Jester, T.

Merendino, M. J. Peterson, and B. Lupiani, 2008: Avian

Influenza Surveillance in Hunter-harvested Waterfowl from

the Gulf Coast of Texas (November 2005–January 2006).

J. Wildl. Dis. 44, 434–439.

Fouchier, R. A., T. M. Bestebroer, S. Herfst, L. Van Der Kemp,

G. F. Rimmelzwaan, and A. D. Osterhaus, 2000: Detection

of influenza A viruses from different species by PCR ampli-

fication of conserved sequences in the matrix gene. J. Clin.

Microbiol. 38, 4096–4101.

Hall, T. A., 1999: BioEdit: a user-friendly biological sequence

alignment editor and analysis program for Windows 95/98/

NT. Nucleic Acids Symp. Ser. 41, 95–98.

Handberg, K. J., O. R. Therkildsen, and P. H. Jørgensen, 2009:

Genetic analysis of avian influenza virus form wild birds

and mallards reared for shooting in Denmark. Avian Dis.

54(Suppl. 1), 420–425.

Hoffmann, E., J. Stech, Y. Guan, R. G. Webster, and D. R.

Perez, 2001: Universal primer set for the full-length

amplification of all influenza A viruses. Arch. Virol. 146,

2275–2289.

Kilpatrick, A. M., A. A. Chmura, D. W. Gibbons, R. C.

Fleischer, P. P. Marra, and P. Daszak, 2006: Predicting the

global spread of H5N1 avian influenza. Proc. Natl. Acad. Sci.

U.S.A. 103, 19368–19373.

Mellergaard, S., 2008: Alvorligt smitsomme husdyrsygdomme i

Europa. DVT 20, 18–19 In Danish.

Munster, V. J., A. Wallensten, C. Baas, G. F. Rimmelzwaan, M.

Schutten, B. Olsen, A. D. Osterhaus, and R. A. Fouchier, 2005:

Mallards and highly pathogenic avian influenza ancestral

viruses, northern Europe. Emerg. Infect. Dis. 11, 1545–1551.

Olsen, B., V. J. Munster, A. Wallensten, J. Waldenstrom, A. D.

Osterhaus, and R. A. Fouchier, 2006: Global patterns of

influenza a virus in wild birds. Science 312, 384–388.

Rohani, P., R. Breban, D. E. Stallknecht, and J. M. Drake,

2009: Environmental transmission of low pathogenicity

avian influenza viruses and its implications for pathogen

invasion. Proc. Natl. Acad. Sci. U.S.A. 106, 10365–10369.

Siembieda, J. L., C. K. Johnson, C. Cardona, N. Anchell, N.

Dao, W. Reisen, and W. Boyce, 2010: Influenza A Viruses in

Wild Birds of the Pacific Flyway, 2005–2008. Vector Borne

Zoonotic Dis. 10. doi: 10.1089=vbz.2009.0095.

Slomka, M. J., V. J. Coward, J. Banks, B. Z. Londt, I. H.

Brown, J. Voermans, G. Koch, K. J. Handberg, P. H. Jørgen-

sen, M. Cherbonnel-Pansart, V. Jestin, G. Cattoli, I. Capua,

A. Ejdersund, P. Thoren, and G. Czifra, 2007: Identification

of sensitive and specific avian influenza polymerase chain

reaction methods through blind ring trials organized in the

European Union. Avian Dis. 51, 227–234.

Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers,

and Y. Kawaoka, 1992: Evolution and ecology of influenza

A viruses. Microbiol. Rev. 56, 152–179.



Documents

Low Pathogenic Avian Influenza (H7N1) Transmission Between Wild Ducks and Domestic Ducks