Announcements
Eurosurveillance remains in the updated list of the Directory of Open Access Journals (DOAJ). It was first added to the DOAJ on 9 September 2004. Eurosurveillance is also listed in the Securing a Hybrid Environment for Research Preservation and Access / Rights MEtadata for Open archiving (SHERPA/RoMEO) [2], a database which uses a colour‐coding scheme to classify publishers according to their self‐archiving policy and to show the copyright and open access self-archiving policies of academic journals. Eurosurveillance is listed there as a ‘green’ journal, which means that authors can archive pre-print (i.e. pre-refereeing), post-print (i.e. final draft post-refereeing) and archive the publisher's version/PDF.

Follow Eurosurveillance on Twitter: @Eurosurveillanc

Read our articles on Zika virus infection

Note of concern published for 'Epidemiological investigation of MERS-CoV spread in a single hospital in South Korea, May to June 2015', http://bit.ly/29QFXPp


In this issue


Home Eurosurveillance Edition  2016: Volume 21/ Issue 38 Article 7
Back to Table of Contents
Previous Download (pdf)
Next

Eurosurveillance, Volume 21, Issue 38, 22 September 2016
Research article
Poen, Verhagen, Manvell, Brown, Bestebroer, van der Vliet, Vuong, Scheuer, van der Jeugd, Nolet, Kleyheeg, Müskens, Majoor, Grund, and Fouchier: Lack of virological and serological evidence for continued circulation of highly pathogenic avian influenza H5N8 virus in wild birds in the Netherlands, 14 November 2014 to 31 January 2016

+ Author affiliations


Citation style for this article: Poen MJ, Verhagen JH, Manvell RJ, Brown I, Bestebroer TM, van der Vliet S, Vuong O, Scheuer RD, van der Jeugd HP, Nolet BA, Kleyheeg E, Müskens GJDM, Majoor FA, Grund C, Fouchier RAM. Lack of virological and serological evidence for continued circulation of highly pathogenic avian influenza H5N8 virus in wild birds in the Netherlands, 14 November 2014 to 31 January 2016. Euro Surveill. 2016;21(38):pii=30349. DOI: http://dx.doi.org/10.2807/1560-7917.ES.2016.21.38.30349

Received:05 April 2016; Accepted:21 July 2016


Introduction

Wild birds are the natural hosts of low pathogenic avian influenza (LPAI) viruses, which generally do not cause clinical signs of disease in these host species [1]. So far, virus subtypes H1 to H16 and N1 to N9 have been detected in wild birds, of which viruses of subtypes H5 and H7 have shown the ability to evolve to highly pathogenic avian influenza (HPAI) viruses in poultry, causing severe disease with high mortality in such animals. These HPAI viruses were historically mainly detected in rapidly contained sporadic outbreaks in poultry, until H5N1 viruses of the A/Goose/Guangdong/1/1996 (GsGd) lineage emerged in Asia in 1997. Subsequently, these viruses have continuously circulated in poultry with frequent detections in wild birds [2] and with significant expansion in global range.

HPAI H5N8 viruses of the GsGd lineage of clade 2.3.4.4 emerged in poultry and wild birds on multiple continents in 2014. The ancestral influenza H5N8 virus to the strains causing outbreaks from 2014 onwards was first detected in China in 2010 in a captive-held mallard (Anas platyrhynchos) [3]. In early 2014, HPAI H5N8 GsGd virus of clade 2.3.4.4 occurred for the first time in poultry in South Korea, soon after causing outbreaks also in Japan [4]. From late 2014 onwards, this virus spread to other areas of the world including Europe, North America, Russia and Taiwan [5-8]. The HPAI H5N8 virus detections in Europe were limited to sporadic cases in wild birds and a relatively small number of unrelated outbreaks in poultry. However in North America HPAI H5N8 viruses reassorted with co-circulating LPAI viruses, giving rise to new HPAI H5N1 and H5N2 virus subtypes that caused a large number of outbreaks in poultry with numerous detections in wild birds [9]. Despite mild clinical symptoms caused by infection with HPAI H5N8 viruses of clade 2.3.4.4 in experimentally infected mammals [10-12] and ducks [11], the widespread detection and rapid global spread of HPAI H5 clade 2.3.4.4 viruses pose a potential threat to domestic and wild animals and should be studied further.

The major challenges in understanding the epidemiology of emerging influenza viruses in wild birds are the large numbers of potential host species and the usually short period of viral shedding, combined with the difficulty of catching and sampling representative numbers per species. For instance, mallards that were experimentally infected with HPAI H5N8 virus shed infectious virus in tracheal swabs for only up to 5 days post infection [11]. These impediments result in a low probability of detecting newly emerging avian influenza viruses in wild birds through active virological surveillance and result in a delay of implementation of effective control measures. Nevertheless, to date HPAI H5N8 virus has been detected in 30 wild bird species. In addition to the host species previously described [13,14], HPAI H5N8 viruses have been detected in wild bird species belonging to the orders Anseriformes in Asia (Aythya spp.) and North America (Branta spp.) [6]. In Europe, HPAI H5N8 viruses have been detected in bird species of the orders Anseriformes (Anas spp. and Cygnus spp.) and Charadriiformes (Larus spp.) [5,6,14].

To estimate the likelihood of the involvement of live wild birds in local and long distance movement of HPAI H5 viruses, information on recent exposure of wild bird populations to HPAI H5N8 viruses using serology, in addition to virology, would add substantial power to surveillance programmes. Studies with ferret sera have shown serological tests to have substantial discriminative power between antibodies directed to HPAI H5 viruses of different clades and LPAI H5 viruses using haemagglutination inhibition (HI) assays [12,15]. Although less is known about serology in wild birds, a study on wild birds sampled in Europe and Mongolia showed that antigenic differences between the haemagglutinin (HA) of classical Eurasian LPAI H5 viruses and GsGd lineage HPAI H5 viruses can be used to define bird populations in which HPAI viruses have previously been circulating [16]. With regard to HPAI H5N8 viruses specifically, a 2014 South Korean serology study showed evidence of a rise of H5 virus antibodies occurring in long distance migratory duck species after the onset of the HPAI H5N8 virus emergence in South Korea [4].

In this study, in response to the emergence of HPAI H5N8 virus in Europe, we present data on wild bird surveillance activities in the Netherlands, including results of virological and serological assays.

Methods

Ethical statement

The capture of free-living birds was approved by the Dutch Ministry of Economic Affairs based on the Flora and Fauna Act (permit number FF/75A/2009/067 and FF/75A/2014/054). Handling and sampling of free-living birds was approved by the Animal Experiment Committee of the Erasmus Medical Centre (permit number 122–11–31). Free-living birds were released into the wild after sampling and all efforts were made to minimise animal suffering throughout the studies.

Study population

Immediately after the first detection of HPAI H5N8 virus in poultry in Europe, ongoing influenza surveillance activities in migrating and overwintering wild birds in the Netherlands were intensified (14 November 2014–13 May 2015). Hereafter, this period will be referred to as ‘during the outbreak’. Surveillance activities in wild birds in the Netherlands were again intensified from the onset of the arrival of wild migrating birds a year after the initial HPAI H5N8 virus detection in Europe (1 September–31 December 2015). This period will be referred to as ‘after the outbreak’. Sampled populations consisted of resident birds, partial migrants and long distance migrants. During both periods of intensified surveillance, blood samples were obtained in addition to samples for virus detection. A matching historical set of serum samples was compiled based on similarity in species and family, hereafter referred to as ‘before the outbreak’ (2007–2013).

Sample collection

Wild birds were captured using duck decoys, clap nets, cannon nets, mist nets, leg-nooses, swan hooks, or manually. Birds were sampled routinely for virus detection using cloacal and/or oropharyngeal swabs as described elsewhere [14]. In addition, faecal samples were collected from a limited number of species for virus detection. Blood samples were collected for antibody detection. Blood samples were collected from the brachial or metatarsal vein and centrifuged at 3,000 rpm for 10 min in 0.8 mL gel separation tubes (MiniCollect tubes, Roche). Serum samples were stored below -20 °C until analysis.

Virus detection, isolation and characterisation

Samples for virus detection were analysed for the presence of HPAI H5(N8) virus using matrix- and H5-specific real-time polymerase chain reaction (RT-PCR) assays followed by H5 and neuraminidase sequencing as previously described [14]. Samples testing positive in matrix specific RT-PCR were inoculated in embryonated chicken eggs as described previously [17].

Antibody detection

Serum samples were first tested for the presence of H5-specific antibodies in an HI assay according to standard procedures [18]. Briefly, serum samples were incubated for 16 hours at 37 °C with Vibrio cholerae filtrate containing receptor-destroying enzyme to remove non-specific inhibitors of haemagglutination activity, followed by incubation for 1 hour at 56 °C. Twofold serial dilutions of serum samples with a start dilution of 1:20 were prepared using phosphate-buffered saline (PBS) in U-bottomed 96 well microtitre plates. Serum dilutions were incubated with four haemagglutinating units (HAU) of Madin–Darby canine kidney (MDCK) (all HPAI H5 clade viruses) or egg (A/Mallard/Netherlands/3/1999) cultured virus for 30 min at 37 °C. A suspension of 1% turkey red blood cells (TRBC) was added to the serum-virus dilutions. After incubation for 1 hour at 4 °C, haemagglutination patterns were read. Negative controls, based on serum incubation without virus, were used to measure non-specific haemagglutination of each serum sample. Sera showing high background (i.e. high non-specific haemagglutination) were pre-treated with 10% TRBC for 1 hour at 4 °C and retested for the presence of H5-specific antibodies as described above. Serum samples from experimentally inoculated ferrets [12,15], a domestic duck, and a domestic goose were used as positive controls.

All serum samples were initially screened for antibodies specific for classical Eurasian LPAI H5N2 virus A/Mallard/Netherlands/3/1999 and clade 2.3.4.4 HPAI H5N8 virus A/Chicken/Netherlands/EMC-3/2014. Serum samples that tested positive for HPAI H5 clade 2.3.4.4-specific antibodies were further tested against HPAI viruses of the H5 clades 1 (A/Viet Nam/1194/2004), 2.1 (A/Indonesia/5/2005), 2.2 (A/Turkey/Turkey/1/2005), and 2.3 (A/Anhui/1/2005), and retested against the clade 2.3.4.4 virus. Samples showing more than threefold differences in titre or testing negative in the second assay after showing initial titres were tested a third time. The viruses used were recombinant viruses based on an A/PR/8/34 virus backbone, containing the HA and neuraminidase (NA) of the representative H5 strains. The sequences of the HA genes were modified to remove the multi-basic cleavage site to enable this study within biosafety level 2 laboratories. HPAI H5 virus of clade 0 was excluded from the analyses due to high overall reactivity with all avian positive control sera as previously described [16] and thus of limited discriminative value.

A representative selection (based on titre and serum availability) of serum samples that tested positive for HPAI H5 clade 2.3.4.4 antibodies were sent to the Animal and Plant Health Agency (APHA) (Weybridge, UK) for confirmation of HPAI H5 clade 2.3.4.4-specific antibodies using an HI assay. The HI assay procedure used by the APHA differed from the HI assay described above and was carried out in accordance to the World Organisation for Animal Health (OIE) [19]. In short, twofold serial dilutions of serum samples with a start dilution of 1:12 were made using phosphate-buffered saline (PBS) and prepared in V-bottomed microtitre plates. Serum dilutions were incubated with four HAU of egg cultured virus for 30 min at room temperature. A solution of 1% chicken red blood cells (CRBC) was added to the serum–virus dilutions. After incubation for 30 min at room temperature, haemagglutination patterns/streaming of red cells were read. Polyclonal chicken sera raised against the same clade 2.1, 2.2, 2.3, and 2.3.4.4 viruses as mentioned above were used as positive controls, supplemented with LPAI H5N3 virus A/Teal/England/7394–2805/2006 and clade 2.3.4.4 HPAI H5N8 virus A/Duck/England/36254/2014.

All samples that tested positive for HPAI H5 clade 2.3.4.4-specific antibodies in the initial HI assay were tested in a virus neutralisation (VN) assay if sufficient amounts of serum were available. The VN assay was performed as described previously [20], using titrated virus stocks of clade 2.1, 2.3, and 2.3.4.4. Briefly, serum was heat inactivated for 30 min at 56 °C and twofold serial dilutions of the sera starting at a 1:20 dilution were prepared and 100 median tissue culture infectious dose (TCID50) was added. After incubating antigen and serum for 1 hour at 37 °C with 5% CO2, the mixtures were transferred to 96 well flat bottom plates containing MDCK cells, which were washed once with infection medium before inoculation. The plates were incubated for 1 hour at 37 °C with 5% CO2, after which the cells were washed once with 100 μL infection medium and the medium was replaced by 200 μL infection medium. Three days later, a haemagglutination assay was performed with the supernatant to determine the antibody titres.

Results

Study population

A total of 11,355 birds were sampled for virus detection during and after the first detection of HPAI H5N8 viruses in poultry and wild birds in Europe. Of those, 5,387 birds were sampled during the outbreak and 5,968 after the outbreak. This report describes the results on 7,337 samples obtained from 21 February 2015 onwards in addition to the previously reported 4,018 samples obtained until 20 February 2015 [14]. Sampled species mainly belonged to the orders Anseriformes, Charadriiformes and Gruiformes (Table 1).

Table 1

Wild bird species sampled for virus detection during and after the emergence of highly pathogenic avian influenza H5N8 virus in Europe, the Netherlands, 21 February 2015–31 January 2016 (n = 7,337 animals)

Order Family Species During outbreak: 21 Feb 2015–13 May 2015 After outbreak: 14 May 2015–31 Jan 2016
Birds sampled
N
AIV-positive birds
N
H5-positive birds
N
Pathotype Birds sampled
N
AIV-positive birds
N
H5-positive birds
N
Pathotype
Anseriformes Ducks Common pochard (Aythya ferina) 0 0 0 NA 1 0 0 NA
Common teal (Anas crecca) 8 0 0 NA 221 39 4 LPAI
Egyptian goose (Alopochen aegyptiaca) 58 0 0 NA 136 0 0 NA
Eurasian wigeon (Anas penelope) 175 1 1 HPAI 1,034 101 2 LPAI
Gadwall (Anas strepera) 1 0 0 NA 175 15 0 NA
Mallard (Anas platyrhynchos) 748 50 0 NA 2,464 354 15 LPAI
Mandarin duck (Aix galericulata) 2 0 0 NA 0 0 0 NA
Northern pintail (Anas acuta) 0 0 0 NA 7 3 0 NA
Northern shoveler (Anas clypeata) 0 0 0 NA 17 2 0 NA
Tufted duck (Aythya fuligula) 0 0 0 NA 1 0 0 NA
Geese Barnacle goose (Branta leucopsis) 96 5 4 LPAI 926 3 0 NA
Bean goose (Anser fabalis) 0 0 0 NA 8 0 0 NA
Brent goose (Branta bernicla) 54 0 0 NA 0 0 0 NA
Canada goose (Branta canadensis) 3 0 0 NA 72 0 0 NA
Greylag goose (Anser anser) 59 0 0 NA 239 0 0 NA
Pink-footed goose (Anser brachyrhynchus) 0 0 0 NA 1 0 0 NA
Greater white-fronted goose (Anser albifrons) 0 0 0 NA 55 0 0 NA
Swans Mute swan (Cygnus olor) 3 0 0 NA 31 1 0 NA
Charadriiformes Gulls Black-headed gull (Chroicocephalus ridibundus) 84 0 0 NA 392 53 0 NA
Caspian gull (Larus cachinnans) 4 0 0 NA 4 0 0 NA
Common gull (Larus canus) 1 0 0 NA 18 0 0 NA
Great black-backed gull (Larus marinus) 1 0 0 NA 0 0 0 NA
Herring gull (Larus argentatus) 15 0 0 NA 32 2 0 NA
Lesser black-backed gull (Larus fuscus) 0 0 0 NA 33 2 0 NA
Mediterranean gull (Larus melanocephalus) 1 0 0 NA 3 1 0 NA
Yellow-legged gull (Larus michahellis) 0 0 0 NA 1 0 0 NA
Lapwings Northern lapwing (Vanellus vanellus) 6 0 0 NA 0 0 0 NA
Terns Black tern (Chlidonias niger) 0 0 0 NA 0 0 0 NA
Common tern (Sterna hirundo) 0 0 0 NA 0 0 0 NA
Columbiformes Pigeons Common wood-pigeon (Columba palumbus) 1 0 0 NA 0 0 0 NA
Gruiformes Coots Common coot (Fulica atra) 46 0 0 NA 92 0 0 NA
Rails Little crake (Porzana parva) 0 0 0 NA 1 0 0 NA
Common moorhen (Gallinula chloropus) 3 0 0 NA 4 0 0 NA
Total 1,369 56 5 NA 5,968 576 21 NA

AIV: avian influenza virus; HPAI: highly pathogenic avian influenza; LPAI: low pathogenic avian influenza; N: number; NA: not applicable.

Surveillance activities were intensified from 21 February to 13 May 2015 (n = 1,369) and 1 September to 31 December 2015 (n = 3,736).

For antibody detection, 1,443 serum samples were analysed. Among these, 945 samples from 25 avian species were obtained during the outbreak, while 349 samples from 15 species originated from after the outbreak. A total of 149 serum samples from 15 species sampled before the HPAI H5N8 virus emergence, obtained between 2007 and 2013, served as controls (Table 2). The majority of these samples were collected from birds wintering in Dutch wetlands.

Table 2

Wild bird species sampled for H5-specific antibody detection before, during and after the emergence of highly pathogenic avian influenza H5N8 virus in Europe, the Netherlands, 2007–2015 (n = 1,443)

Order Family Species Number of individuals sampled
Before outbreak
(before 2014)
During outbreak
(14 Nov 2014–13 May 2015)
After outbreak
(1 Sep 2015–31 Dec 2015)
Anseriformes Ducks Common teal (Anas crecca) 0 15 111
Egyptian goose (Alopochen aegyptiaca) 9 62 28
Eurasian wigeon (Anas penelope) 0 78 46
Gadwall (Anas strepera) 1 3 1
Mallard (Anas platyrhynchos) 21 93 18
Mandarin duck (Aix galericulata) 1 2 0
Northern pintail (Anas acuta) 0 0 1
Northern shoveler (Anas clypeata) 0 2 3
Ruddy shelduck (Tadorna ferruginea) 1 0 0
Geese Barnacle goose (Branta leucopsis) 20 19 0
Bean goose (Anser fabalis) 5 0 0
Brent goose (Branta bernicla) 0 19 0
Greylag goose (Anser anser) 0 2 0
Lesser white-fronted goose (Anser erythropus) 0 3 0
Pink-footed goose (Anser brachyrhynchus) 0 1 0
Greater white-fronted goose (Anser albifrons) 20 77 0
Swans Bewick's swan (Cygnus columbianus bewickii) 0 20 0
Mute swan (Cygnus olor) 10 90 29
Whooper swan (Cygnus cygnus) 0 1 0
Charadriiformes Gulls Black-headed gull (Chroicocephalus ridibundus) 20 262 31
Caspian gull (Larus cachinnans) 0 6 3
Common gull (Larus canus) 12 34 17
Great black-backed gull (Larus marinus) 0 1 0
Herring gull (Larus argentatus) 7 61 28
Lesser black-backed gull (Larus fuscus) 1 3 8
Mediterranean gull (Ichthyaetus melanocephalus) 2 1 0
Yellow-legged gull (Larus michahellis) 0 0 1
Gruiformes Rails Common coot (Fulica atra) 19 84 24
Moorhen (Gallinula chloropus) 0 6 0
Total 149 945 349

Virus detection, isolation and characterisation

In addition to the two previously reported HPAI H5N8 virus-infected Eurasian wigeons detected in the Netherlands in November 2014 [14], the virus was detected in a third Eurasian wigeon faecal sample obtained on 25 February 2015 (1/1,369 birds sampled in 21 February–13 May 2015), near Ilpendam (52°28′N 4°57′E) (GenBank accession numbers: AKH14448–AKH14459). Since then, no HPAI H5N8 virus has been detected in any of the samples tested (0/5,968 birds sampled in 14 May 2015–31 January 2016) (Table 1).

Influenza A H5 virus clade-specific antibody detection

As shown previously, ferret antisera raised against prototype strains representing LPAI and HPAI H5 viruses of various clades showed almost exclusive reactivity with homologous viruses in HI assays [12] (Table 3). Importantly, a ferret antiserum raised against the clade 2.3.4.4 virus did not react with other H5 viruses, and antisera raised against other prototype H5 strains did not react with the clade 2.3.4.4 virus A/Chicken/Netherlands/EMC-3/2014. Sera obtained upon inoculation of a domestic duck and a domestic goose with the clade 2.3.4.4 virus A/Turkey/Germany/AR2487/2014 reacted similar to the ferret clade 2.3.4.4 antiserum; no cross-reactivity was seen with other prototype H5 strains (Table 3). These data indicate that the antigenic differences between clade 2.3.4.4 HA and HA of LPAI and HPAI viruses belonging to other clades were sufficiently large to allow serological discrimination by HI assay.

Table 3

Details of positive control sera titres from experimentally infected ferrets, a domestic duck, and a domestic goose with one low pathogenic (LPAI) H5 and different highly pathogenic avian influenza (HPAI) H5 clades (n = 8 antisera)

Antiserum raised against Characteristics Species Haemagglutination inhibition assay Virus neutralisation assay
Viruses Viruses
LPAI HPAI clade HPAI clade
1a 2.1b 2.2c 2.3d 2.3.4.4e 2.1b 2.3d 2.3.4.4e
A/Mallard/Netherlands/3/1999 LPAI H5N2 Ferret 160 < 10 < 10 < 10 < 10 < 10 ND ND ND
A/Viet Nam/1194/2004 HPAI H5N1 clade 1 Ferret < 10 80 < 10 < 10 < 10 < 10 ND ND ND
A/Indonesia/5/2005 HPAI H5N1 clade 2.1 Ferret < 10 < 10 120 < 10 60 < 10 80 < 10 < 10
A/Turkey/Turkey/1/2005 HPAI H5N1 clade 2.2 Ferret < 10 < 10 < 10 1,280 60 < 10 ND ND ND
A/Anhui/1/2005 HPAI H5N1 clade 2.3 Ferret < 10 < 10 < 10 20 320 < 10 < 10 160 < 10
A/Chicken/Netherlands/EMC-3/2014 HPAI H5N8 clade 2.3.4.4 Ferret < 10 < 10 < 10 < 10 < 10 160 < 10 < 10 40
Turkey/Germany/AR2487/2014 HPAI H5N8 clade 2.3.4.4 Domestic duck < 10 < 10 < 10 < 10 < 10 160 ND ND ND
Turkey/Germany/AR2487/2014 HPAI H5N8 clade 2.3.4.4 Domestic goose < 10 < 10 < 10 < 10 < 10 80 ND ND ND

HPAI: highly pathogenic avian influenza; LPAI: low pathogenic avian influenza; ND: not determined.

Lowest serum dilution tested was 10. Titres indicating the reactivity of sera to viruses homologous to the viruses, which the sera were raised against are in bold.

a A/Viet Nam/1194/2004.

b A/Indonesia/5/2005.

c A/Turkey/Turkey/1/2005.

d A/Anhui/1/2005.

e A/Chicken/Netherlands/EMC-3/2014.

Influenza A virus H5-specific antibody detection in wild birds

Haemagglutination inhibition assays

Of the serum samples initially tested in the HI assay with LPAI H5N2 (A/Mallard/Netherlands/3/1999) and HPAI H5 clade 2.3.4.4 H5N8 (A/Chicken/Netherlands/EMC-3/2014) virus, LPAI H5-specific antibodies were detected in 31 of 1,443 serum samples and HPAI H5 clade 2.3.4.4-specific antibodies in 53 of 1,443 serum samples (Table 4). Among these, seven samples tested positive for both LPAI H5- and HPAI H5 clade 2.3.4.4-specific antibodies. The incidence of LPAI H5-specific antibodies was similar before, during and after the HPAI H5N8 virus emergence in Europe (Fisher exact test, p = 0.76 before vs during the outbreak; p = 0.39 during vs after the outbreak), while HPAI H5 clade 2.3.4.4-specific antibodies were detected exclusively in sera from five bird species, obtained during and after the HPAI H5N8 virus emergence in Europe (Table 4, Table 5). The incidence of HPAI H5 clade 2.3.4.4-specific antibodies a year after the outbreak (10/329 (20 samples with high background excluded), 3.0%) was lower than during the outbreak (43/940 (5 samples with high background excluded), 4.6%) (Fisher exact test, p = 0.27).

Table 4

Detected haemagglutination inhibition antibody titres to low pathogenic avian influenza H5 virusa and to highly pathogenic avian influenza H5 clade 2.3.4.4 H5N8 virusb in birds, before, during, and after detection of the highly pathogenic avian influenza H5N8 virus in Europe, the Netherlands, 2007–2015 (n = 1,443 birds)

Strain Period relative to the outbreakc Haemagglutination inhibition titre High background Total tested Total positives
BLD 10–40 40–80 80–160 160–320 320–640 ≥ 640
LPAI H5N2a Before 121 1 0 1 0 0 0 26 149 2
During 903 16 5 2 1 0 0 18 945 24
After 324 2 1 0 2 0 0 20 349 5
HPAI H5N8b Before 123 0 0 0 0 0 0 26 149 0
During 897 7 20 6 4 5 1 5 945 43
After 319 4 3 2 1 0 0 20 349 10

BLD: below limit of detection; LPAI: low pathogenic avian influenza; HPAI: highly pathogenic avian influenza.

Lowest serum dilution tested was 10.

a A/Mallard/Netherlands/3/1999.

b A/Chicken/Netherlands/EMC-3/2014.

c The ‘outbreak’ refers to the six months following the detection of the highly pathogenic avian influenza H5N8 virus in Europe and this extends from 14 November 2014 to 13 May 2015. The period before the ‘outbreak’ is from 2007 to 2013, while the period after the ‘outbreak’ is from 1 September to 31 December 2015.

Table 5

Birds species with antibodies to highly pathogenic avian influenza H5 clade 2.3.4.4 H5N8 virusa, and number of respective animals, according to their haemagglutination inhibition antibody titres to the virus, during and after detection of highly pathogenic avian influenza H5N8 virus in Europe, the Netherlands, 14 November 2014–31 December 2015 (n = 382 birds)

Species Period relative to the outbreakb HI titre to HPAI H5 clade 2.3.4.4 (H5N8) virus High background Total tested
BLD 10–40 40–80 80–160 160–320 320–640 ≥ 640
Eurasian wigeon (Anas penelope) During 66 6 4 2 0 0 0 0 78
Lesser white-fronted goose (Anser erythropus) During 2 0 1 0 0 0 0 0 3
Mute swan (Cygnus olor) During 59 1 14 4 4 5 1 2 90
Common coot (Fulica atra) During 83 0 1 0 0 0 0 0 84
Eurasian wigeon (Anas penelope) After 42 2 1 0 0 0 0 1 46
Egyptian goose (Alopochen aegyptiaca) After 27 1 0 0 0 0 0 0 28
Mute swan (Cygnus olor) After 19 1 2 2 0 0 0 5 29
Common coot (Fulica atra) After 21 0 0 0 1 0 0 2 24

BLD: below limit of detection; HI: haemagglutination inhibition; HPAI: highly pathogenic avian influenza.

Lowest serum dilution tested was 10.

a A/Chicken/Netherlands/EMC-3/2014.

b The ‘outbreak’ refers to the six months following the detection of the highly pathogenic avian influenza H5N8 virus in Europe and this extends from 14 November 2014 to 13 May 2015. The period after the ‘outbreak’ is from 1 September to 31 December 2015.

Serum samples obtained during (43/940 (5 samples with high background excluded), 4.6%) and after (10/329 (20 samples with high background excluded), 3.0%) the outbreak that tested positive for HPAI H5 clade 2.3.4.4-specific antibodies were subsequently tested in an HI assay against prototype viruses of clades 1, 2.1, 2.2, 2.3, and 2.3.4.4. Of the sera collected during the outbreak, 29/90 mute swans (Cygnus olor), 12/78 Eurasian wigeons, 1/3 lesser white-fronted geese (Anser erythropus) and 1/84 common coots (Fulica atra) tested positive for HPAI H5 clade 2.3.4.4-specific antibodies (Table 5). In these HPAI H5 clade 2.3.4.4-specific antibody positive sera, no cross-reactivity was observed in sera of Eurasian wigeons (12/12) and the lesser white-fronted goose (1/1). In contrast, the common coot (1/1) serum showed an additional titre to the clade 2.3 virus and sera of mute swans showed cross-reactivity to clade 2.3 (27/29), 2.1 (23/29), 1 (9/29) and 2.2 (4/29) viruses. In the majority of samples (22/29), titres to clade 2.1 and 2.3 exceeded those detected to clade 2.3.4.4 (Table 6).

Table 6

Titres of confirmatory haemagglutination inhibition and virus neutralisation assays for sera positive for highly pathogenic avian influenza H5 clade 2.3.4.4-specific antibodies in the initial screening, the Netherlands, 14 November 2014–31 December 2015 (n = 53 serum samples)

Period Speciesa Haemagglutination inhibition assay Virus neutralisation assay
Initial Confirmatory
LPAI
H5
HPAI clade LPAI
H5
HPAI clade HPAI clade
1 2.1 2.2 2.3 2.3.4.4b 2.1 2.2 2.3 2.3.4.4 2.1 2.3 2.3.4.4
During the outbreak:2014/15 Eurasian wigeon < 10 < 10 < 10 < 10 < 10 50 ND ND ND ND ND ND ND ND
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 100 ND ND ND ND ND ND ND ND
Eurasian wigeon 20 < 10 < 10 < 10 < 10 15 ND ND ND ND ND ND ND ND
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 60 ND ND ND ND ND ND ND 80
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 20 < 6 < 6 < 6 < 6 < 6 ND ND 20
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 40 < 6 < 6 < 6 < 6 < 6 ND ND 20
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 25 ND ND ND ND ND ND ND 40
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 15 ND ND ND ND ND ND ND 20
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 15 ND ND ND ND ND ND ND 10
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 20 ND ND ND ND ND ND ND 20
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 40 < 6 < 6 < 6 < 6 < 6 ND ND 40
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 120 ND ND ND ND ND ND ND 160
Common coot < 10 40 < 10 < 10 30 40 ND ND ND ND ND ND ND < 10
Lesser white-fronted goose 20 < 10 < 10 < 10 < 10 70 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 120 320 < 30 640 40 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 160 160 < 30 640 200 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 30 < 180 320 < 180 960 60 ND ND ND ND ND ND ND ND
Mute swan < 120 < 120 120 < 120 320 240 ND ND ND ND ND < 10 < 10 80
Mute swan < 30 30 160 40 640 480 ND ND ND ND ND < 10 < 10 60
Mute swan < 60 < 60 < 40 < 40 60 480 ND ND ND ND ND < 10 < 10 240
Mute swan < 60 < 40 240 < 30 640 70 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 120 < 60 160 < 60 640 960 12 < 6 < 6 < 6 192 < 10 10 240
Mute swan < 10 < 40 320 < 40 1,280 70 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 60 480 30 2,560 60 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 30 < 120 240 < 120 480 70 ND ND ND ND ND ND ND < 10
Mute swan < 60 < 120 320 < 120 640 50 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 60 < 120 320 < 120 640 80 ND ND ND ND ND < 10 < 10 20
Mute swan < 10 < 60 320 < 60 960 70 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 120 160 640 30 2,560 240 < 6 < 6 < 6 < 6 < 6 < 10 < 10 < 10
Mute swan < 60 40 320 30 1,280 120 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 30 30 160 < 30 640 50 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 ND ND ND ND 50 ND ND ND ND ND ND ND 20
Mute swan < 120 < 120 160 < 120 640 70 ND ND ND ND ND 20 < 10 < 10
Mute swan < 10 160 320 < 120 1,280 70 ND ND ND ND ND ND ND ND
Mute swan < 60 < 120 160 < 120 640 50 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 30 40 160 < 60 640 50 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 30 < 30 160 < 30 320 35 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 < 180 320 < 180 640 100 ND ND ND ND ND < 10 < 10 < 10
Mute swan 40 < 60 160 < 60 640 80 ND ND ND ND ND ND ND ND
Mute swan < 60 < 60 < 60 < 60 160 240 ND ND ND ND ND < 10 < 10 10
Mute swan < 60 < 240 < 240 < 240 < 240 480 12 < 6 < 6 < 6 96 < 10 < 10 60
Mute swan < 60 < 30 < 30 < 30 60 480 < 6 < 6 < 6 < 6 96 < 10 < 10 240
Mute swan < 120 < 120 < 120 < 120 320 480 ND ND ND ND ND < 10 < 10 60
After the outbreak: 2015 Eurasian wigeon < 10 < 10 < 10 < 10 < 10 20 ND ND ND ND ND ND ND 160
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 10 ND ND ND ND ND ND ND 20
Eurasian wigeon < 10 < 10 < 10 < 10 < 10 40 ND ND ND ND ND ND ND 80
Common coot < 10 80 60 60 320 160 ND ND ND ND ND ND ND 20
Egyptian goose < 10 < 10 < 10 < 10 80 25 ND ND ND ND ND ND ND < 10
Mute swan 160 80 60 < 30 160 120 ND ND ND ND ND < 10 < 10 40
Mute swan 40 80 80 80 320 45 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 < 10 < 10 < 10 30 15 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 80 80 80 320 60 ND ND ND ND ND < 10 < 10 < 10
Mute swan < 10 160 160 240 320 80 ND ND ND ND ND 80 20 < 10

HPAI: highly pathogenic avian influenza; LPAI: low pathogenic avian influenza; ND: not determined.

Lowest serum dilution tested was 10 for the initial haemagglutination inhibition (HI) and virus neutralisation assay and 6 for the confirmatory HI assay.

a Species included common coot (Fulica atra), Egyptian goose (Alopochen aegyptiaca), Eurasian wigeon (Anas penelope), lesser white-fronted goose (Anser erythropus), mute swan (Cygnus olor).

b Mean titre of in duplo tested samples.

Of the sera collected after the outbreak, 5/29 mute swans, 3/46 Eurasian wigeons, 1/28 Egyptian geese (Alopochen aegyptiaca) and 1/24 common coots tested positive for HPAI H5 clade 2.3.4.4-specific antibodies (Table 5). The sera of the Eurasian wigeons reacted with HPAI H5N8 virus exclusively. However, the common coot as well as 1/5 mute swans showed HI titres to all five H5 clades. The other 3/5 mute swans showed HI titres to multiple but not all H5 clades, while 1/5 mute swans and 1/1 Egyptian goose only showed an additional titre to clade 2.3 (Table 6).

Seven of the HPAI H5 clade 2.3.4.4-seropositive bird sera obtained during the outbreak, from four mute swans and three Eurasian wigeons, were retested in an HI assay at the APHA. Here, 3/4 mute swan samples with high initial HI antibody titres against HPAI H5 clade 2.3.4.4 (H5N8) virus were confirmed. However, 1/4 mute swan sera could not be confirmed, and HPAI H5 clade 2.3.4.4-specific antibodies were also not detected in 3/3 sera of the Eurasian wigeons that had low antibody titres in the initial tests (Table 6).

Virus neutralisation assays

For 37/43 HPAI H5 clade 2.3.4.4-positive sera collected during and 10/10 sera collected after the outbreak, sufficient serum volumes were available for retesting in a VN assay. In this assay, HPAI H5 clade 2.3.4.4-specific antibodies were detected in sera of 9/9 Eurasian wigeons and of 10/26 mute swans obtained during the outbreak. Sera of the mute swans did not react with viruses of other H5 clades. HPAI H5 clade 2.3.4.4-specific antibodies were not detected in the sera of the common coot and the lesser white-fronted goose by VN assay. HPAI H5 clade 2.3.4.4-specific antibodies were confirmed by VN assay in sera from 3/3 Eurasian wigeons, 1/5 mute swans, 1/1 common coot and 0/1 Egyptian goose collected after the outbreak (Table 6).

Discussion

In this report surveillance data for HPAI H5N8 in birds in the Netherlands are presented. In addition to bird samples previously investigated for the virus from 14 November 2014 to 20 February 2015, a new set of 7,337 samples obtained between 21 February 2015 and 31 January 2016 is analysed. One faecal sample obtained from a Eurasian wigeon (Anas penelope) on 25 February 2015 tested positive for the HPAI H5N8 virus, adding to the previous finding of the virus in two Eurasian wigeons in the country in late 2014 [14]. Virological surveillance moreover suggests that only very limited numbers of wild bird species were identified as potential hosts in Europe. Importantly, to the best of our knowledge, there are no reports of additional findings of HPAI H5N8 viruses in wild birds and poultry in Europe, since the last detection of the virus in February 2015 in the Netherlands.

Given the difficulty of detecting newly emerging HPAI virus strains in wild birds however, the application of a more sensitive and cost-effective method to detect potential host species is warranted. For this purpose, we performed serological assays specifically aimed to detect antibodies specific to HPAI H5 clade 2.3.4.4 viruses in a substantial number of sera obtained before, during, and after HPAI H5N8 emergence in the Netherlands. Three potential HPAI H5N8 host species were identified by HI assays and confirmed by VN assays; Eurasian wigeons, mute swans and common coots. Considering the results of virological studies performed worldwide since the onset of the HPAI H5N8 virus emergence in early 2014, the detection of HPAI H5 clade 2.3.4.4-specific antibodies in these species is not surprising. HPAI H5N8 virus was isolated from Eurasian wigeons in Russia [8] and the Netherlands [14], from mute swans in Sweden [6], and from a common coot in South Korea [21].

The serological results reported here were not entirely consistent between HI and VN assays and between HI assays performed in two different laboratories. Although low HI titres (e.g. in Eurasian wigeons) were reproducible within a laboratory with the same HI assay and a VN assay, they were not detected by HI assay in a second laboratory, potentially due to differences in the methods used and hence differences in sensitivity and specificity. High antibody titres in mute swan sera were reproduced by HI assay in a second laboratory and by VN assay, but low antibody titres in mute swans were not always reproduced. While it is thus clear that individual HI titres in avian sera obtained from a single test cannot be used reliably for diagnosis, use of serum panels from cohorts of birds, use of multiple tests to cross-validate results, a panel of relevant viruses and use of collections of control antisera may still enable the use of serological tests in support of HPAI H5 surveillance studies.

Previously, HI assays were shown to be discriminative enough to detect antibodies in serum samples collected from free-living wild birds in Europe and Asia to be directed to either HPAI or LPAI H5 viruses. However, widely varying results were obtained as far as HPAI H5 clade-specific antibodies were concerned [16]. In this study, most birds that tested positive for HPAI H5 clade 2.3.4.4-specific serum antibodies showed relative low HI titres. This is in accordance with findings based on experimental HPAI H5N8 virus infections of ferrets [10-12], possibly indicating low immunogenicity upon infection. In addition, there is limited knowledge about the longevity of avian antibodies after naturally occurring infection with avian influenza viruses. Antibodies specific to LPAI viruses were detected up to several months after experimental or natural infection [22-24], whereas little is known about the duration of detection of antibodies specific to HPAI viruses with a reported maximum of detection of 28 days after experimental infection in domestic ducks [25]. To date, there is no knowledge on the effect of a prior exposure to an unrelated subtype or on the phenomena of antigenic sin in avian species. Hypothetically, low immunogenicity in combination with decreasing titres in time could be an explanation for the low incidence and relative low titres of antibodies detected in wild bird sera in this study.

In conclusion, our results provide evidence that clinically unaffected long distance migratory and local wild birds sampled in the Netherlands during the H5N8 outbreak late 2014 and early 2015, and again late 2015, have been exposed to HPAI H5N8 or closely related HPAI H5 clade 2.3.4.4 viruses and seroconverted upon exposure. Since HPAI H5N8 virus has not been detected in Europe since early 2015 and because HPAI H5 clade 2.3.4.4-specific antibody incidence decreased in time, we conclude that the virus has not circulated extensively at the breeding grounds in summer and upon the return of the birds to their wintering areas in the 2015/16 winter. As a consequence, the newly emerging HPAI H5N8 clade 2.3.4.4 virus subtype appears to have already disappeared from European wild birds indicating that sustained transmission and independent maintenance may be less likely. This is an important consideration in the ongoing evolution and ecology of these viruses in wild birds and the potential risks they pose for introduction to poultry and the pathways through which they might spread. Finally we recommend that serological tools be further optimised, harmonised, and validated for avian influenza surveillance studies in wild birds.


Acknowledgements

The authors thank Stefan van Nieuwkoop, Pascal Lexmond, Anne van der Linden, Judith Guldemeester, Manon de Briedé, Suzan Pas, David van de Vijver, Teunis en Joke de Vaal, Bert Pellegrom, Jan Berkouwer, Arie Keijzer, Jan en Lilian Slijkerman, Hans Zantinge, Roy Slaterus, Peter de Vries, Manon Kaandorp, Cynthia Lange, Joanne Malotaux, Jan Beekman, Alwin Hut, Peter Volten, Evert-Jan Epping, Harma Scholten, Ton Eggenhuizen, Henk Koffijberg, Gerben Tijsma, Cor van Aart, Henk ten Klooster, Sjoerd Dirksen, Jan van der Winden and the Kreupel tern research group for providing technical assistance and samples. We thank Ger van der Water for excellent logistical assistance. We gratefully acknowledge Timm Harder and Martin Beer (Friedlich Loeffler Institute, Germany) for providing positive control avian sera. This work was supported by The Dutch Ministry of Economic Affairs, European Research Council project FLUPLAN (250136), NIAID/NIH contract HHSN272201400008C, and Horizon 2020 project COMPARE.

Conflict of interest

None declared.

Authors’ contributions

MP and JV: performing serological assays, compiling the data and drafting the manuscript; RM and IB: confirmatory serological assays; TB and SV: excellent technical assistance in preparing, performing and interpreting serological assays; OV and RS: analysing samples for virus detection; HJ, BN and EK: initiation of the study, providing field data; FM and GM: collecting field data; CG; providing positive control sera; RF: initiation of study, critically revised the manuscript.


References

  1. Kuiken T. Is low pathogenic avian influenza virus virulent for wild waterbirds? Proc. Biol. Sci. 2013;280(1763):20130990. DOI: http://dx.doi.org/10.1098/rspb.2013.0990 .PMID: 23740783

  2. Liu J, Xiao H, Lei F, Zhu Q, Qin K, Zhang XW,  et al.  Highly pathogenic H5N1 influenza virus infection in migratory birds. Science. 2005;309(5738):1206. DOI: 10.1126/science.1115273 PMID: 16000410

  3. Zhao K, Gu M, Zhong L, Duan Z, Zhang Y, Zhu Y,  et al.  Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China. Vet Microbiol. 2013;163(3-4):351-7. DOI: 10.1016/j.vetmic.2012.12.025 PMID: 23375651

  4. Jeong J, Kang HM, Lee EK, Song BM, Kwon YK, Kim HR,  et al.  Highly pathogenic avian influenza virus (H5N8) in domestic poultry and its relationship with migratory birds in South Korea during 2014. Vet Microbiol. 2014;173(3-4):249-57. DOI: 10.1016/j.vetmic.2014.08.002 PMID: 25192767

  5. World Organisation for Animal Health (OIE). Update on highly pathogenic avian influenza in animals (typeH5 and H7). Paris: OIE; 2014. [Accessed 02 Feb 2016]. Available from: http://www.oie.int/animal-health-in-the-world/update-on-avian-influenza/2014/

  6. World Organisation for Animal Health (OIE). Update on highly pathogenic avian influenza in animals (typeH5 and H7). Paris: OIE; 2015. [Accessed 02 Feb 2016]. Available from: http://www.oie.int/animal-health-in-the-world/update-on-avian-influenza/2015/

  7. Verhagen JH, Herfst S, Fouchier RAM. How a virus travels the world.Science. 2015;347(6222):616-7. DOI: 10.1126/science.aaa6724 PMID: 25657235

  8. Marchenko VY, Susloparov IM, Kolosova NP, Goncharova NI, Shipovalov AV, Durymanov AG,  et al.  Influenza A(H5N8) virus isolation in Russia, 2014. Arch Virol. 2015;160(11):2857-60. DOI: 10.1007/s00705-015-2570-4 PMID: 26306756

  9. Hall JS, Dusek RJ, Spackman E. Rapidly Expanding Range of Highly Pathogenic Avian Influenza Viruses.Emerg Infect Dis. 2015;21(7):1251-2. DOI: 10.3201/eid2107.150403 PMID: 26079209

  10. Kim HM, Kim CK, Lee NJ, Chu H, Kang C, Kim K,  et al.  Pathogenesis of novel reassortant avian influenza virus A (H5N8) Isolates in the ferret. Virology. 2015;481:136-41. DOI: 10.1016/j.virol.2015.02.042 PMID: 25776760

  11. Kim YI, Pascua PN, Kwon HI, Lim GJ, Kim EH, Yoon SW,  et al.  Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus. Emerg Microbes Infect. 2014;3(10):e75. DOI: 10.1038/emi.2014.75 PMID: 26038499

  12. Richard M, Herfst S, van den Brand JMA, Lexmond P, Bestebroer TM, Rimmelzwaan GF,  et al.  Low Virulence and Lack of Airborne Transmission of the Dutch Highly Pathogenic Avian Influenza Virus H5N8 in Ferrets. PLoS One. 2015;10(6):e0129827. DOI: 10.1371/journal.pone.0129827 PMID: 26090682

  13. European Food Safety Authority (EFSA). Highly pathogenic avian influenza A subtype H5N8.EFSA Journal.2014;12(12):3941. DOI: 10.2903/j.efsa.2014.3941

  14. Verhagen JH, van der Jeugd HP, Nolet BA, Slaterus R, Kharitonov SP, de Vries PP,  et al.  Wild bird surveillance around outbreaks of highly pathogenic avian influenza A(H5N8) virus in the Netherlands, 2014, within the context of global flyways. Euro Surveill. 2015;20(12):21069. DOI: 10.2807/1560-7917.ES2015.20.12.21069 PMID: 25846491

  15. Koel BF, van der Vliet S, Burke DF, Bestebroer TM, Bharoto EE, Yasa IWW,  et al.  Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site. MBio. 2014;5(3):e01070-14. DOI: 10.1128/mBio.01070-14 PMID: 24917596

  16. Gilbert M, Koel BF, Bestebroer TM, Lewis NS, Smith DJ, Fouchier RAM. Serological evidence for non-lethal exposures of Mongolian wild birds to highly pathogenic avian influenza H5N1 virus.PLoS One. 2014;9(12):e113569. DOI: 10.1371/journal.pone.0113569 PMID: 25502318

  17. Munster VJ, Baas C, Lexmond P, Bestebroer TM, Guldemeester J, Beyer WE,  et al.  Practical considerations for high-throughput influenza A virus surveillance studies of wild birds by use of molecular diagnostic tests. J Clin Microbiol. 2009;47(3):666-73. DOI: 10.1128/JCM.01625-08 PMID: 19109483

  18. Hirst GK. Studies of Antigenic Differences among Strains of Influenza a by Means of Red Cell Agglutination.J Exp Med. 1943;78(5):407-23. DOI: 10.1084/jem.78.5.407 PMID: 19871338

  19. World Organisation for Animal Health (OIE). Ch. 2.3.4. Avian Influenza (infection with avian influenza viruses). OIE Terrestrial Manual 2015. Paris: OIE; 2015. [Accessed 02 Feb 2016]. Available from: http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.03.04_AI.pdf

  20. Rimmelzwaan GF, Baars M, van Beek R, de Lijster P, de Jong JC, Claas EC,  et al.  Influenza virus subtype cross-reactivities of haemagglutination inhibiting and virus neutralising serum antibodies induced by infection or vaccination with an ISCOM-based vaccine. Vaccine. 1999;17(20-21):2512-6. DOI: 10.1016/S0264-410X(99)00063-8 PMID: 10418897

  21. Kim HR, Kwon YK, Jang I, Lee YJ, Kang HM, Lee EK,  et al.  Pathologic changes in wild birds infected with highly pathogenic avian influenza A(H5N8) viruses, South-Korea, 2014. Emerg Infect Dis. 2015;21(5):775-80. DOI: 10.3201/eid2105.141967 PMID: 25897841

  22. Brown J, Poulson R, Carter D, Lebarbenchon C, Pantin-Jackwood M, Spackman E,  et al.  Susceptibility of avian species to North American H13 low pathogenic avian influenza viruses. Avian Dis. 2012;56(4) Suppl;969-75. DOI: 10.1637/10158-040912-Reg.1 PMID: 23402121

  23. Tolf C, Latorre-Margalef N, Wille M, Bengtsson D, Gunnarsson G, Grosbois V,  et al.  Individual variation in influenza A virus infection histories and long-term immune responses in Mallards. PLoS One. 2013;8(4):e61201. DOI: 10.1371/journal.pone.0061201 PMID: 23637797

  24. Verhagen JH, Höfle U, van Amerongen G, van de Bildt M, Majoor F, Fouchier RAM,  et al.  Long-Term Effect of Serial Infections with H13 and H16 Low-Pathogenic Avian Influenza Viruses in Black-Headed Gulls. J Virol. 2015;89(22):11507-22. DOI: 10.1128/JVI.01765-15 PMID: 26339062

  25. Wibawa H, Henning J, Waluyati DE, Usman TB, Lowther S, Bingham J,  et al.  Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus. BMC Vet Res. 2012;8:117. DOI: 10.1186/1746-6148-8-117 PMID: 22823985



Back to Table of Contents
Previous Download (pdf)
Next

The publisher’s policy on data collection and use of cookies.

Disclaimer: The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the editorial team or the institutions with which the authors are affiliated. Neither ECDC nor any person acting on behalf of ECDC is responsible for the use that might be made of the information in this journal. The information provided on the Eurosurveillance site is designed to support, not replace, the relationship that exists between a patient/site visitor and his/her physician. Our website does not host any form of commercial advertisement. Except where otherwise stated, all manuscripts published after 1 January 2016 will be published under the Creative Commons Attribution (CC BY) licence. You are free to share and adapt the material, but you must give appropriate credit, provide a link to the licence, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.

Eurosurveillance [ISSN] - ©2007-2016. All rights reserved
 

This website is certified by Health On the Net Foundation. Click to verify. This site complies with the HONcode standard for trustworthy health information:
verify here.