Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007

  • Sasan R Fereidouni1, 2Email author,

    Affiliated with

    • Ortrud Werner1,

      Affiliated with

      • Elke Starick1,

        Affiliated with

        • Martin Beer1,

          Affiliated with

          • Timm C Harder1,

            Affiliated with

            • Mehdi Aghakhan2,

              Affiliated with

              • Hossein Modirrousta2,

                Affiliated with

                • Hamid Amini3,

                  Affiliated with

                  • Majid Kharrazian Moghaddam3,

                    Affiliated with

                    • Mohammad H Bozorghmehrifard4,

                      Affiliated with

                      • Mohammad A Akhavizadegan2,

                        Affiliated with

                        • Nicolas Gaidet5,

                          Affiliated with

                          • Scott H Newman6,

                            Affiliated with

                            • Saliha Hammoumi5,

                              Affiliated with

                              • Giovanni Cattoli7,

                                Affiliated with

                                • Anja Globig1,

                                  Affiliated with

                                  • Bernd Hoffmann1,

                                    Affiliated with

                                    • Mohammad E Sehati3,

                                      Affiliated with

                                      • Siamak Masoodi3,

                                        Affiliated with

                                        • Tim Dodman8,

                                          Affiliated with

                                          • Ward Hagemeijer8,

                                            Affiliated with

                                            • Shirin Mousakhani9 and

                                              Affiliated with

                                              • Thomas C Mettenleiter1

                                                Affiliated with

                                                Virology Journal20107:43

                                                DOI: 10.1186/1743-422X-7-43

                                                Received: 7 October 2009

                                                Accepted: 19 February 2010

                                                Published: 19 February 2010

                                                Abstract

                                                Background

                                                Virological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran.

                                                Results

                                                Overall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals.

                                                Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%.

                                                Conclusion

                                                Results of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected.

                                                Background

                                                Wild waterbirds are considered the main reservoir of all subtypes of avian influenza viruses (AIV). Low pathogenic AIV (LPAIV) are widely distributed in wild avian species around the world. They have been most frequently identified in waterbirds of the orders Anseriformes (including ducks, geese and swans) and Charadriiformes (particularly gulls and terns). These viruses replicate in epithelial cells of the respiratory and intestinal tracts of birds, and are excreted in high concentrations in their faeces [1]. It is now well recognized that global influenza virus surveillance in wild birds is important in understanding the role of wild birds in the epidemiology and ecology of these viruses.

                                                After expansion of HPAIV H5N1 from Southeast Asia into many Eurasian and African countries, the frequency and intensity of avian influenza surveys in the world increased dramatically. In particular North American and European countries gathered massive epidemiological information regarding circulation of AIV in wild birds. Yet, little is known about the prevalence of AIV in wild birds in West & Central Asian countries and the Middle East. Many countries in this region were severely affected by HPAI H5N1 in late 2005 and early 2006, with recurrent outbreaks since 2007 [2]. In Iran, two outbreaks of HPAI H5N1 have been officially reported in wild birds and domestic poultry during 2006 and 2008, respectively.

                                                The wetlands located in the southern part of the Caspian Sea represent major wintering and stopover sites during migration for many wild waterbirds from Siberia and northern Russia. Several million migratory birds usually arrive in October and either remain until February/March or migrate further south.

                                                Here, we describe the results of four years of AIV surveillance in wild birds by using different virological, molecular and serological methods. This study provides the first extensive survey of AIV in wild birds in West and Central Asia and the Middle East.

                                                Methods

                                                Sampling plan

                                                Samples were collected from 1146 waterbirds belonging to 45 species (11 families, Table 1). The samples were mainly obtained from captured or hunted birds, or during ringing activities. Mist nets with mesh sizes of 20 × 20 and 50 × 50 mm were used to capture the birds for sampling. Samples were collected between October and March from 2003 to 2007 at 18 sites located in six provinces of Iran including Mazandaran, Gilan, West Azerbaijan, Tehran, Fars and Khuzestan (Figure 1). The sampling sites comprise the most important wetlands of Iran, serving as wintering sites for migratory waterbirds. The majority of samples (83%) were collected from birds staging in the wetlands along the southern shores of the Caspian Sea which form an important ecological site for wild migratory birds along the Central Asia flyway.
                                                Table 1

                                                Wild birds sampled in Iran during different years of study, and AIV positives by rRT-PCR.

                                                Family

                                                Bird name

                                                Scientific name

                                                2003 & 2004

                                                2005

                                                2007

                                                Total Species No.

                                                Species Pos. No.

                                                No. Bird/Family

                                                Pos./Family

                                                Podicipedidae

                                                Black-necked Grebe

                                                Podiceps nigricollis

                                                7

                                                -

                                                -

                                                7

                                                0

                                                19

                                                0

                                                 

                                                Great-crested Grebe

                                                Podiceps cristatus

                                                3

                                                1

                                                -

                                                4

                                                0

                                                  
                                                 

                                                Little Grebe

                                                Podiceps ruficollis

                                                8

                                                -

                                                -

                                                8

                                                0

                                                  

                                                Phalacrocoracidae

                                                Lesser Cormorant

                                                Phalacrocorax pygmaeus

                                                -

                                                1

                                                1

                                                2

                                                0

                                                14

                                                0

                                                 

                                                Great Cormorant

                                                Phalacrocorax carbo

                                                11

                                                -

                                                1

                                                12

                                                0

                                                  

                                                Ardeidae

                                                Great White Egret

                                                Egretta alba

                                                3

                                                2

                                                -

                                                5

                                                0

                                                22

                                                0

                                                 

                                                Grey Heron

                                                Ardea cinerea

                                                6

                                                4

                                                -

                                                10

                                                0

                                                  
                                                 

                                                Little Egret

                                                Egretta garzetta

                                                3

                                                4

                                                -

                                                7

                                                0

                                                  

                                                Phoenicopteridae

                                                Greater Flamingo

                                                Phoenicopterus roseus

                                                8

                                                4

                                                -

                                                12

                                                0

                                                12

                                                0

                                                Anatidae

                                                Gadwall

                                                Anas strepera

                                                10

                                                1

                                                15

                                                26

                                                0

                                                745

                                                31

                                                 

                                                Garganey

                                                Anas querquedula

                                                3

                                                -

                                                -

                                                3

                                                1

                                                  
                                                 

                                                Greylag Goose

                                                Anser anser

                                                3

                                                -

                                                1

                                                4

                                                1

                                                  
                                                 

                                                Mallard

                                                Anas platyrhynchos

                                                78

                                                34

                                                68

                                                180

                                                15

                                                  
                                                 

                                                Northern Pintail

                                                Anas acuta

                                                17

                                                1

                                                18

                                                36

                                                1

                                                  
                                                 

                                                Common Pochard

                                                Aythya ferina

                                                5

                                                5

                                                8

                                                18

                                                1

                                                  
                                                 

                                                Red-crested Pochard

                                                Netta rufina

                                                1

                                                -

                                                -

                                                1

                                                0

                                                  
                                                 

                                                Ruddy Shelduck

                                                Tadorna ferruginea

                                                1

                                                5

                                                -

                                                6

                                                0

                                                  
                                                 

                                                Greater Scuap

                                                Aythya marila

                                                -

                                                1

                                                -

                                                1

                                                0

                                                  
                                                 

                                                Common Shelduck

                                                Tadorna tadorna

                                                10

                                                -

                                                1

                                                11

                                                0

                                                  
                                                 

                                                Northern Shoveler

                                                Anas clypeata

                                                10

                                                1

                                                4

                                                15

                                                2

                                                  
                                                 

                                                Common Teal

                                                Anas crecca

                                                79

                                                36

                                                243

                                                358

                                                9

                                                  
                                                 

                                                Tufted Duck

                                                Aythya fuligula

                                                2

                                                2

                                                1

                                                5

                                                1

                                                  
                                                 

                                                White-headed Duck

                                                Oxyura leucocephala

                                                2

                                                -

                                                -

                                                2

                                                0

                                                  
                                                 

                                                Lesser White-fronted Goose

                                                Anser erythropus

                                                -

                                                -

                                                1

                                                1

                                                0

                                                  
                                                 

                                                Greater White-fronted Goose

                                                Anser albifrons

                                                -

                                                -

                                                1

                                                1

                                                0

                                                  
                                                 

                                                Eurasian Wigeon

                                                Anas penelope

                                                4

                                                4

                                                69

                                                77

                                                0

                                                  

                                                Rallidae

                                                Common Coot

                                                Fulica atra

                                                117

                                                39

                                                77

                                                233

                                                4

                                                234

                                                4

                                                 

                                                Water Rail

                                                Rallus aquaticus

                                                1

                                                -

                                                -

                                                1

                                                0

                                                  

                                                Recurvirostridae

                                                Pied Avocet

                                                Recurvirostra avosetta

                                                4

                                                -

                                                -

                                                4

                                                0

                                                6

                                                0

                                                 

                                                Black-winged Stilt

                                                Himantopus himantopus

                                                2

                                                -

                                                -

                                                2

                                                0

                                                  

                                                Charadriidae

                                                Northern Lapwing

                                                Vanellus vanellus

                                                10

                                                1

                                                4

                                                15

                                                0

                                                17

                                                0

                                                 

                                                White-tailed Lapwing

                                                Vanellus leucurus

                                                2

                                                -

                                                -

                                                2

                                                0

                                                  

                                                Scolopacidae

                                                Black-tailed Godwit

                                                Limosa limosa

                                                3

                                                2

                                                -

                                                5

                                                0

                                                51

                                                0

                                                 

                                                Dunlin

                                                Calidris alpina

                                                5

                                                -

                                                -

                                                5

                                                0

                                                  
                                                 

                                                Jack Snipe

                                                Lymnocryptes minimus

                                                3

                                                -

                                                1

                                                4

                                                0

                                                  
                                                 

                                                Marsh Sandpiper

                                                Tringa stagnatilis

                                                13

                                                -

                                                -

                                                13

                                                0

                                                  
                                                 

                                                Common Redshank

                                                Tringa totanus

                                                12

                                                1

                                                1

                                                14

                                                0

                                                  
                                                 

                                                Common Greenshank

                                                Tringa nebularia

                                                -

                                                5

                                                -

                                                5

                                                0

                                                  
                                                 

                                                Kentish Plover

                                                Charadrius alexandrinus

                                                -

                                                2

                                                -

                                                2

                                                0

                                                  
                                                 

                                                Ruff

                                                Philomachus pugnax

                                                1

                                                2

                                                -

                                                3

                                                0

                                                  

                                                Laridae

                                                Black-headed Gull

                                                Larus ridibundus

                                                16

                                                1

                                                -

                                                17

                                                0

                                                25

                                                0

                                                 

                                                Little Gull

                                                Larus minutus

                                                3

                                                -

                                                -

                                                3

                                                0

                                                  
                                                 

                                                Slender-billed Gull

                                                Larus genei

                                                2

                                                -

                                                -

                                                2

                                                0

                                                  
                                                 

                                                Yellow-legged Gull

                                                Larus cachinnans

                                                3

                                                -

                                                -

                                                3

                                                0

                                                  

                                                Sternidae

                                                Whiskered Tern

                                                Chlidonias hybridus

                                                1

                                                -

                                                -

                                                1

                                                0

                                                1

                                                0

                                                   

                                                472

                                                159

                                                515

                                                1146

                                                35

                                                1146

                                                35

                                                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-7-43/MediaObjects/12985_2009_Article_788_Fig1_HTML.jpg
                                                Figure 1

                                                The geographical distribution of sampling sites in Iran (blue spots; capital letters in the spots indicate the province: A: West Azerbaijan, F: Fars, G: Gilan, K: Khuzestan, M: Mazandaran, T: Tehran).

                                                During 2003-2005 only cloacal samples (n = 631) and in 2007 cloacal and oropharyngeal samples were collected. In addition, 711 serum samples were collected from 27 different species (Tables 2 &3). In 2006, sampling was not permitted due to an HPAI H5N1 outbreak in the wild bird population in Iran.
                                                Table 2

                                                Time and location of sampling and prevalence of virological and serological AIV positive wild birds during 2003-2007.

                                                Year

                                                Month

                                                Province

                                                Swab Samples

                                                Pos.

                                                Serum Samples

                                                Pos.

                                                2003

                                                Feb.

                                                Khuzestan (K)

                                                30

                                                1

                                                0

                                                0

                                                 

                                                Nov.

                                                W. Azerbaijan (A)

                                                58

                                                0

                                                4

                                                1

                                                 

                                                Nov.

                                                Tehran (T)

                                                12

                                                1

                                                0

                                                0

                                                 

                                                Nov.

                                                Gilan (G)

                                                118

                                                3

                                                82

                                                22

                                                 

                                                Dec.

                                                Mazandaran (M)

                                                49

                                                0

                                                40

                                                23

                                                 

                                                Dec.

                                                Gilan

                                                52

                                                0

                                                47

                                                20

                                                 

                                                Dec.

                                                Fars (F)

                                                63

                                                0

                                                36

                                                8

                                                2004

                                                Jan.

                                                Tehran

                                                13

                                                0

                                                0

                                                0

                                                 

                                                Feb.

                                                Khuzestan

                                                26

                                                0

                                                5

                                                2

                                                 

                                                Feb.

                                                Tehran

                                                51

                                                7

                                                3

                                                1

                                                2005

                                                Jan

                                                Mazandaran

                                                22

                                                0

                                                0

                                                0

                                                 

                                                Feb.

                                                Tehran

                                                59

                                                1

                                                11

                                                5

                                                 

                                                Mar.

                                                Mazandaran

                                                78

                                                1

                                                32

                                                4

                                                2007

                                                Feb.

                                                Gilan

                                                27

                                                0

                                                23

                                                7

                                                 

                                                Feb.

                                                Mazandaran

                                                488

                                                21

                                                428

                                                252

                                                   

                                                1146

                                                35

                                                711

                                                345

                                                Table 3

                                                AIV serological results of different bird species at different sampling times.

                                                Family

                                                Bird species

                                                2003/2004

                                                2005

                                                2007

                                                Total

                                                Number/family

                                                Tested

                                                Pos.

                                                Tested

                                                Pos.

                                                Tested

                                                Pos.

                                                Tested

                                                Pos.

                                                Tested

                                                Pos.

                                                Podicipedidae

                                                Black-necked Grebe

                                                1

                                                0

                                                -

                                                -

                                                -

                                                -

                                                1

                                                0

                                                8

                                                0

                                                 

                                                Great-crested Grebe

                                                3

                                                0

                                                -

                                                -

                                                -

                                                -

                                                3

                                                0

                                                  
                                                 

                                                Little Grebe

                                                4

                                                0

                                                -

                                                -

                                                -

                                                -

                                                4

                                                0

                                                  

                                                Phalacrocoracidae

                                                Great Cormorant

                                                7

                                                1

                                                -

                                                -

                                                -

                                                -

                                                7

                                                1

                                                7

                                                1

                                                Ardeidae

                                                Great White Egret

                                                2

                                                1

                                                2

                                                0

                                                -

                                                -

                                                4

                                                1

                                                14

                                                2

                                                 

                                                Little Egret

                                                2

                                                1

                                                1

                                                0

                                                -

                                                -

                                                3

                                                1

                                                  
                                                 

                                                Grey Heron

                                                4

                                                0

                                                3

                                                0

                                                -

                                                -

                                                7

                                                0

                                                  

                                                Phoenicopteridae

                                                Greater Flamingo

                                                3

                                                3

                                                2

                                                1

                                                -

                                                -

                                                5

                                                4

                                                5

                                                4

                                                Anatidae

                                                Gadwall

                                                1

                                                0

                                                  

                                                13

                                                4

                                                14

                                                4

                                                521

                                                313

                                                 

                                                Greylag Goose

                                                2

                                                2

                                                -

                                                -

                                                -

                                                -

                                                2

                                                2

                                                  
                                                 

                                                Mallard

                                                32

                                                28

                                                10

                                                3

                                                66

                                                50

                                                108

                                                81

                                                  
                                                 

                                                Northern Pintail

                                                11

                                                5

                                                1

                                                0

                                                17

                                                16

                                                29

                                                21

                                                  
                                                 

                                                Common Pochard

                                                4

                                                1

                                                1

                                                0

                                                6

                                                6

                                                11

                                                7

                                                  
                                                 

                                                Ruddy Shelduck

                                                -

                                                -

                                                3

                                                2

                                                -

                                                -

                                                3

                                                2

                                                  
                                                 

                                                Northern Shoveler

                                                6

                                                5

                                                -

                                                -

                                                4

                                                3

                                                10

                                                8

                                                  
                                                 

                                                Common Teal

                                                41

                                                22

                                                6

                                                2

                                                225

                                                133

                                                272

                                                157

                                                  
                                                 

                                                Eurasian Wigeon

                                                2

                                                0

                                                2

                                                0

                                                68

                                                31

                                                72

                                                31

                                                  

                                                Rallidae

                                                Common Coot

                                                66

                                                1

                                                11

                                                0

                                                49

                                                16

                                                126

                                                17

                                                126

                                                17

                                                Recurvirostridae

                                                Pied Avocet

                                                1

                                                1

                                                -

                                                -

                                                -

                                                -

                                                1

                                                1

                                                1

                                                1

                                                Charadriidae

                                                Northern Lapwing

                                                5

                                                0

                                                -

                                                -

                                                3

                                                0

                                                8

                                                0

                                                8

                                                0

                                                Scolopacidae

                                                Black-tailed Godwit

                                                2

                                                0

                                                -

                                                -

                                                -

                                                -

                                                2

                                                0

                                                7

                                                3

                                                 

                                                Common Redshank

                                                4

                                                2

                                                -

                                                -

                                                -

                                                -

                                                4

                                                2

                                                  
                                                 

                                                Common Greenshank

                                                -

                                                -

                                                1

                                                1

                                                -

                                                -

                                                1

                                                1

                                                  

                                                Laridae

                                                Black-headed Gull

                                                10

                                                4

                                                -

                                                -

                                                -

                                                -

                                                10

                                                4

                                                14

                                                4

                                                 

                                                Little Gull

                                                1

                                                0

                                                -

                                                -

                                                -

                                                -

                                                1

                                                0

                                                  
                                                 

                                                Slender-billed Gull

                                                1

                                                0

                                                -

                                                -

                                                -

                                                -

                                                1

                                                0

                                                  
                                                 

                                                Yellow-legged Gull

                                                2

                                                0

                                                -

                                                -

                                                -

                                                -

                                                2

                                                0

                                                  
                                                  

                                                217

                                                77

                                                43

                                                9

                                                451

                                                259

                                                711

                                                345

                                                711

                                                345

                                                In 2007, in the framework of an international collaboration, birds were sampled in duplicate and tested independently by reference laboratories of the World Organisation for Animal Health (OIE) at the Friedrich-Loeffler-Institut (FLI), Germany and the Istituto Zooprofilattico Sperimentale delle Venezie (ISZ-Ve), Italy, as well as at the Agricultural Research Centre for International Development (CIRAD), France. Cloacal and oropharyngeal samples were collected with cotton swabs, stored in viral transport medium (Hank's medium or PBS) containing antibiotics and antimycotics (plus 5% calf serum during 2003-2005) and maintained at -70°C after arrival at the laboratory. Serum samples were stored at -20°C until tested.

                                                Diagnostic procedures

                                                The samples from 2003-2004 were analyzed by virus isolation (VI) at the Razi Institute, Iran, and real-time reverse transcription PCR (rRT-PCR) at the Friedrich-Loeffler-Institut (FLI), Germany, whilst positive samples were further characterized at the FLI. The samples from 2005 were analysed only by virus isolation (at the Razi Institute) and two positive samples were further characterized at the FLI.

                                                In 2007, rRT-PCR was performed for screening and only PCR-positive samples were processed for virus isolation. Samples duplicated in the field were analysed at the FLI (rRT-PCR and VI) and at CIRAD (France) (rRT-PCR) and the IZS-Ve (Italy) (VI).

                                                Isolates were characterized by conventional hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays, and subsequently confirmed by subtype specific RT-PCR assays and sequencing. The subtypes of PCR-positive but isolation-negative samples, were determined by subtype specific RT-PCR, DNA microarray and sequencing.

                                                Virus isolation and characterization

                                                Virus isolation was carried out in specific pathogen free (SPF) embryonated chicken eggs based on standard procedures [3].

                                                RNA extraction, RT-PCR and Real-time RT-PCR

                                                RNA was extracted either using the QIAamp Viral RNA kit (Qiagen) for swab materials (field samples), or the High Pure Viral RNA kit (Roche) for virus isolates (allantoic fluids), according to the manufacturer's instructions. The 2007 samples were processed by automated RNA extraction (Freedom Evo 3000, Tecan) using the NucleoSpin 96 Virus Core kit (Macherey & Nagel).

                                                Reverse transcription-PCR (RT-PCR) assays were performed on the basis of one-step protocols using appropriate RT-PCR Kits (Qiagen or Invitrogen) according to the manufacturers' instructions. Subtype specific RT-PCR assays using specific primers for different HA [4] and NA [5] were used for subtype identification or confirmation. Degenerate consensus primers were used for full length amplification and further sequencing of different viral segments [6].

                                                The samples were tested by a modified TaqMan one-step real-time RT-PCR assay targeting the influenza A virus M gene [7], an H5 subtype gene fragment and an H7 subtype gene fragment [3]. Brilliant QRT-PCR kit (Stratagene), SuperScript III One-step RT-PCR kit with Platinum Taq DNA polymerase (Invitrogen) and one-step RT-PCR kit (Qiagen) were used on a MX3000P Real-Time PCR System (Stratagene). In all tests, negative RNA preparation controls and negative and positive rRT-PCR controls as well as an internal transcription and amplification control (IC-2) were included [8].

                                                Sequencing and phylogenetic analyses

                                                PCR products of the anticipated size range were purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Purified DNA fragments were cycle-sequenced in both directions using the same primers as for RT-PCR. The Prism Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems) was used and amplicons were analysed on an automatic sequencer (ABI-377, Applied Biosystems). Assembled nucleotide sequences were then used in BlastN2 database searches for subtype specification. Phylogenetic analyses were carried out for complete open reading frame of HA gene of selected H9N2 AIV using the neighbour-joining (NJ) method, with 1000 bootstrap replicates implemented in the MEGA 4 programme [9].

                                                Hemagglutination (HA) and Neuraminidase inhibition (NI) assay

                                                HA assay was performed based on standard protocols [3] using reference antisera prepared from 30 different viruses representing all 16 avian influenza HA subtypes and 9 different avian paramyxoviruses. The previously described NI assay was used for determination of the NA subtype of virus isolates from 2003-2004 [5].

                                                Pathogenicity assessment

                                                Two isolates from 2003 and 2004 of subtypes H7N3 and H9N2 were selected for IVPI testing [3] due to the potential pathogenicity of these subtypes for domestic poultry. Pathogenicity of two H5 isolates from 2007 was determined by sequencing of the HA cleavage site and restriction enzyme cleavage pattern (RECP) assay [10].

                                                Competitive ELISA

                                                An in-house competitive ELISA method was used for testing of 217 serum samples from 2003-2004 [11]. For the investigation of 494 serum samples collected during 2005-2007 (Table 4), a cELISA kit based on the same assay principle was used (ID Screen, Influenza A NP Antibody Competition, ID.VET). The cut-off value of this test was used as recommended by the suppliers.
                                                Table 4

                                                AIV characterized from different species of wild birds during 2003-2007 in Iran.

                                                Year/Month

                                                ID-code

                                                Sample type

                                                subtype

                                                Bird species

                                                Virus isolation

                                                Province

                                                2003/Feb

                                                K8

                                                C

                                                H9

                                                N2

                                                Garganey

                                                Yes

                                                Khuzestan

                                                2003/Nov

                                                T9

                                                C

                                                ?

                                                N7

                                                Common Coot

                                                 

                                                Tehran

                                                2003/Nov

                                                G52

                                                C

                                                ?

                                                ?

                                                Northern Shoveler

                                                 

                                                Gilan

                                                2003/Nov

                                                G54

                                                C

                                                H9

                                                N2

                                                Mallard

                                                Yes

                                                Gilan

                                                2003/Nov

                                                G94

                                                C

                                                H9

                                                N2

                                                Northern Shoveler

                                                Yes

                                                Gilan

                                                2004/Feb

                                                V4

                                                C

                                                ?

                                                ?

                                                Common Teal

                                                 

                                                Tehran

                                                2004/Feb

                                                V10

                                                C

                                                H3

                                                N8

                                                Mallard

                                                Yes

                                                Tehran

                                                2004/Feb

                                                V15

                                                C

                                                H10

                                                N7

                                                Mallard

                                                Yes

                                                Tehran

                                                2004/Feb

                                                V16

                                                C

                                                H8

                                                N4

                                                Mallard

                                                Yes

                                                Tehran

                                                2004/Feb

                                                V17

                                                C

                                                ?

                                                ?

                                                Mallard

                                                 

                                                Tehran

                                                2004/Feb

                                                V31

                                                C

                                                H7

                                                N3

                                                Mallard

                                                Yes

                                                Tehran

                                                2004/Feb

                                                V40

                                                C

                                                H10

                                                N7

                                                Mallard

                                                Yes

                                                Tehran

                                                2005/Feb

                                                V41

                                                C

                                                H10

                                                N7

                                                Mallard

                                                Yes

                                                Tehran

                                                2005/Mar

                                                M72

                                                C

                                                H7

                                                N7

                                                Mallard

                                                Yes

                                                Mazandaran

                                                2007/Feb

                                                T31

                                                T

                                                ?

                                                ?

                                                Common Coot

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T41

                                                T

                                                ?

                                                ?

                                                Mallard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C54

                                                C

                                                H8

                                                N4

                                                Greylag Goose

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C64

                                                C

                                                H1

                                                N1

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C108

                                                C

                                                H5

                                                H11

                                                N3

                                                N9

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C136

                                                C

                                                H1

                                                N1

                                                Common Teal

                                                Yes

                                                Mazandaran

                                                2007/Feb

                                                T149

                                                T

                                                H6

                                                N2

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C154

                                                C

                                                H11

                                                N1

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T183

                                                T

                                                H11

                                                ?

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T223

                                                T

                                                H10

                                                N8

                                                Common coot

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T292

                                                T

                                                H5

                                                ?

                                                Common Pochard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C303

                                                C

                                                H3

                                                ?

                                                Tufted Duck

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C309

                                                C

                                                H10

                                                N8

                                                Common Coot

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C364

                                                C

                                                H9

                                                N2

                                                Mallard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T366

                                                T

                                                H9

                                                N2

                                                Mallard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T367

                                                T

                                                H9

                                                N2

                                                Mallard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                T370/C370

                                                C & T

                                                H9

                                                N2

                                                Mallard

                                                Yes

                                                Mazandaran

                                                2007/Feb

                                                T371

                                                T

                                                H9

                                                N2

                                                Mallard

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C381

                                                C

                                                H11

                                                N9

                                                Northern Pintail

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C388

                                                C

                                                H3

                                                ?

                                                Common Teal

                                                 

                                                Mazandaran

                                                2007/Feb

                                                C415

                                                C

                                                H11

                                                N2

                                                Common Teal

                                                 

                                                Mazandaran

                                                C: cloacal. T: oropharyngeal

                                                Microarray

                                                Microarray was used for HA and NA subtyping of samples with low viral load and which therefore did not yield enough PCR amplificates for PCR subtyping or sequencing. Sample RNA was amplified by RT-PCR assays using biotinylated generic pan HA and pan NA primers. An in vitro transcript 'LPC-pan HA' and a 'no template control' were included in every run. Microarray detection of the biotinylated PCR products was done as described by Gall et al. [12].

                                                Western blot analysis

                                                Western blot analysis was carried out according to Kothlow et al. [13], to support the cELISA results of selected serum samples. Briefly, after separation of purified virus by sodium dodecyl sulphate-polyacrylamide gel electrophoresis proteins were blotted onto nitrocellulose membrane, and strips were incubated with 1:250 diluted test and control sera overnight. After the respective washing steps, bound duck immunoglobulin (Ig) was detected by mAb (1:2000 dilution) recognising Ig of several species followed by incubation with horseradish peroxidase-conjugated goat-anti-mouse Ig-specific polyclonal antibody (Sigma). Finally, strips were allowed to react with a horseradish peroxidase substrate (ECL plus Western Blotting Detection System, Amersham Biosciences) and the reaction was visualized by autoradiography on X-ray film. Sera which reacted at least with either the NP or the M protein were considered positive. Sera from 8 mallards hatched and kept under quarantine, were used as Western blot negative controls.

                                                Results

                                                Detection of LPAIV in Iran

                                                In total 3% of all sampled birds were AIV positive by rRT-PCR of M gene (Table 1). Out of 11 bird families examined, two were positive, Anatidae and Rallidae (Tables 1 &2). The highest number of AIV (31 out of 35 rRT-PCR positive samples) were detected in dabbling ducks (genus Anas) including Mallard Anas platyrhynchos (n = 15; 8.3%), Common Teal Anas crecca (n = 9; 2.5%) and Northern Shoveler Anas clypeata (n = 2). Individual Greylag Goose Anser anser, Garganey Anas querquedula, Northern Pintail Anas acuta, Common Pochard Aythya ferina and Tufted Duck Aythya fuligula also yielded positive results. Out of 45 sampled bird species, eight were positive. In total, Anatidae made up 65% of the sample volume, and 4.3% of them were AIV positive. Four Common Coots Fulica atra of the Rallidae family were also AIV positive in this study (1.7%).

                                                Temporal and geographical distribution of AIV

                                                During five months of sampling in different years and different provinces, the highest numbers of positive samples were found in February and November (Figure 2). The number of samples collected in this study was not distributed evenly over different months. Approximately 66% of samples (n = 759) were collected during February and early March (2004, 2005 and 2007). 88% of positive samples were found in this portion of samples, though 78% of samples collected during this period originated from ducks.
                                                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-7-43/MediaObjects/12985_2009_Article_788_Fig2_HTML.jpg
                                                Figure 2

                                                Prevalence (%) of AIV in total sampled birds and in Anatidae in different months of sampling in Iran.

                                                Out of the 35 AIV-positive samples detected during the four year study, 34.3% (n = 12) were found in 2003/2004, 5.7% (n = 2) in 2005 and 60% (n = 21) in 2007. 84% of samples in 2007 belonged to Anatidae (compared to 57% in 2005 and 48% during 2003/2004); and only in 2007 both oropharyngeal and cloacal samples were collected from each bird. Prevalence of AIV was 2.54% in 2003/2004, 1.26% in 2005 and 4.08% in 2007.

                                                Out of 21 AIV-positive samples in 2007 (the year in which paired sampling from 515 birds was carried out), nine samples were positive only in the oropharyngeal swab, eleven samples positive only in the cloacal swab, and only one bird positive in both oropharyngeal and cloacal swab samples. The highest number of positive samples originated from the wetlands south of the Caspian Sea (n = 25) and in a seasonal wetland in the south-east of Tehran province (n = 9).

                                                Detection of specific AIV antibodies

                                                The results of serological investigation of 711 serum samples are shown in Tables 2 and 3. The seroprevalence rates against AIV were 35.5%, 21% and 57.4% respectively for 2003/2004, 2005 and 2007. However, the composition of bird species in these four sampling periods was different. During 2003-2005 only 45.6% of serum samples belonged to Anatidae, which increased to 88.5% in 2007. However, the proportion of positive Anatidae in 2004/2005 and 2007 samples was almost similar (64% and 61% respectively). Anatidae contributed a high proportion of positive results: 81 out of 108 Mallard, 157 out of 272 Common Teal, 8 out of 10 Northern Shoveler, 7 out of 10 Common Pochard, 21 out of 28 Northern Pintail, 31 out of 70 Eurasian Wigeon and 2 out of 2 Greylag Goose were antibody-positive. In total, birds from 17 of 25 species carried antibodies against AIV (Table 3). A randomly selected batch of 31 cELISA positive and negative serum samples belonging to different species was tested by Western blotting with a high correlation between the two tests (data not shown).

                                                Virus isolation and characterisation

                                                In total, 35 LPAIV were molecularly identified from 1601 oropharyngeal and cloacal samples originating from 1146 different wild waterbirds wintering/staging in Iran. No highly pathogenic strains, including H5N1, were detected in this survey. A total of twelve AIV were isolated and subtyped mainly during the 2003/2004 investigation; virus isolation for the additional 23 molecularly positive samples (mainly during the 2007 investigation) failed even after 2-3 passages in SPF chicken eggs (Table 3). HA and/or NA subtypes of 17 samples from this group were characterized by subtype-specific RT-PCR, sequencing and microarray (Table 4).

                                                Characterized AIV were categorised into 14 subtypes using HI, NI, subtype-specific RT-PCR, microarray and sequencing: H1N1, H3N8, H5N9, H6N2, H7N3, H7N7, H8N4, H9N2, H10N7, H10N8, H11N1, H11N2, H11N3, and H11N9. The most common HA subtypes were H9, H10 and H11, and the most common NA subtypes were N2, N7 and N1 (Figure 3). Two LPAIV H5 and two LPAIV H7 strains were identified. One cloacal sample taken from a Common Teal was positive for two different AIV subtypes: H5N3 (or N9) and H11N9 (or N3).
                                                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-7-43/MediaObjects/12985_2009_Article_788_Fig3_HTML.jpg
                                                Figure 3

                                                Frequency of different hemagglutinin and neuraminidase subtypes identified in wild bird samples during 2003-2007.

                                                One H7N3 and one H9N2 isolate (from 2003/2004) were assessed as LPAIV using IVPI test, as no evidence of disease or pathogenicity in SPF chickens was observed (IVPI = 0). Two H5 subtype viruses, from 2007, were considered as LPAIV in RECP assay. Sequencing of the HA cleavage site for the H5 and H7 viruses confirmed their status as LPAI viruses. The amino acid patterns of the HA cleavage site for H5 and H7 subtype viruses were RETR*G and PKGR*G, respectively.

                                                Inter-laboratory real-time PCR results

                                                All 515 samples from 2007 were tested independently by two AI reference laboratories (FLI tested one set of samples, ISZ-Ve and CIRAD together another set) using real-time RT-PCR (Tables 1 &3). Results for most of the samples with moderate and strongly positive results (Ct-values ≤ 33) were identical, and only 4 weakly positive samples (33.3 ≤ Ct-values ≤ 35.5) were found positive by only one laboratory. Positive and negative controls included in all tests ensured the validity of rRT-PCR results. We showed that double sampling from the wild waterbirds is possible and the rRT-PCR results revealed a very high degree of agreement.

                                                Phylogenetic analyses

                                                In total, nine H9N2 AIV were identified in samples collected during our monitoring studies, and the HA genes of five of them were sequenced (GenBank: FN600116 - FN600119). Due to widespread infection of poultry farms in Iran with H9N2 subtype virus, phylogenetic analysis was preferentially carried out on these viruses. A/Garganey/Iran/G8/2003 (H9N2) was isolated from a Garganey in the southern part of Iran which was suspected to have been kept together with backyard poultry for a short time before sampling. The four 2007 H9N2 viruses were identified in a small group of Mallards in the northern part of Iran, without any known contact with domestic poultry. The 2003 virus clustered closely with H9N2 viruses isolated in poultry in Iran during 1999-2003, while the 2007 viruses clustered together with wild bird H9N2 viruses from Russia and Hokkaido (Figure 4).
                                                http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-7-43/MediaObjects/12985_2009_Article_788_Fig4_HTML.jpg
                                                Figure 4

                                                Phylogenetic tree for the full length hemagglutinin gene of H9N2 influenza viruses. The tree constructed by Neighbour-joining method. Sequences obtained in this study were labelled by red (2003 virus) and green dots (2007 viruses). The other sequences were selected from GenBank. The numbers represent bootstrap values which were determined using 1000 replications.

                                                Discussion

                                                The important role of waterbirds, especially waterfowl, as a reservoir for avian influenza viruses of all subtypes is well known from intensive investigations from many regions of the world [1417]. Avian influenza monitoring of wild birds in natural habitats and in areas at risk of transmission between domestic poultry and wild birds will increase the knowledge of epidemiology, ecology and genetic relationships of AIV infections. This knowledge will facilitate risk assessments concerning poultry and wild bird populations and provides information on currently circulating AIV which might also have the potential to become important for human health. However, little information is available about the circulation of influenza viruses in waterbirds in West and Central Asia and in the Middle East. This is the first study of AIV-investigations in wild birds in Iran and this geographic region.

                                                In a survey during 1999-2000 in Northern Europe, 2.6% of wild ducks and 1.4% of wild geese were positive in rRT-PCR [18]. In more recent monitoring studies of wild birds during 2003-2005 in Italy, 5.1% of Anseriformes were positive in rRT-PCR [19]. Also, in an AIV screening in 2005 in Norway, 13.2% of Anseriformes were positive in rRT-PCR [20]. The prevalence of LPAIV in wild birds in Alaska and Canada seems to be more variable [2123]. The results from these studies have shown that the prevalence of AIV in wild birds, and especially ducks, depends on various factors, including geographic altitude of sampling area, bird species, seasonal parameters and different sample processing approach. In our investigation, the number of positive birds varied based on species, sampling month and sites. In total 3.4% of all sampled birds were positive, but different families and species had different number of positives. Only two out of 11 investigated families (Anatidae and Rallidae) were AIV positive, and among 17 sampled species of Anatidae, 8 species revealed positive results. Prevalence rates for Mallard, Common Teal and Common Coot, with high sample sizes, were 8.3%, 2.5% and 1.7% respectively (Table 1). No positive samples were found in shorebirds (Recurvirostridae, Charadriidae & Scolpacidae).

                                                Previous studies revealed high virus prevalence during the autumn season in the Northern Hemisphere [16], whereas the lowest prevalence rates have been measured in early spring. In contrast, 88% of positive samples in our study were found during February and early March. Interestingly, 78% of samples which were collected in this period came from Anatidae.

                                                The geographical distribution of positive samples reveals further significant differences. In Mazandaran (one of the northern provinces), 21 out of 637 samples tested positive (3.3%), while in a small wetland in the southeast of Tehran province, 9 out of 135 sampled birds were positive (6.7%, Table 2). Sample numbers per species largely reflect the proportion of the wintering populations in different geographical regions of Iran. However, with respect to statistical inferences, a bias regarding different species, seasons and geographical regions cannot be excluded.

                                                The results of this study regarding the dominant AIV infected wild bird species are consistent with several other investigations from Europe and the Americas. Dabbling ducks were found infected with LPAIV at higher prevalence rates than other taxonomic groups [2325]. Similarly, the highest number of positive AIV samples has been reported from Mallard and Common Teal, relative to any other species [20, 23]. Mallards are the most frequently sampled species in most wild birds surveillance studies in the northern hemisphere, though in our study Common Teal and Common Coot occupied a higher ranking (Table 1).

                                                No evidence was found for HPAIV H5N1 circulating in wild birds before (2003-2005) and after introduction of this virus into wild bird and poultry populations in countries around the Caspian Sea in 2006. Nevertheless, the small sample sizes of the species investigated here in comparison to their total populations make it very hard to exclude a low or very low prevalence of HPAIV in wild bird populations. The largest sample sizes in our study belonged to Common Teal, Common Coot and Mallard with 358, 233 and 180 birds, respectively.

                                                The results of recent wild bird surveillance in Sweden indicate that the proportion of positive cloacal samples exceeds positive oropharyngeal samples [26]. Our results show a similar proportion of positive oropharyngeal and cloacal samples. Among 21 positive samples from 2007, nine were only positive in oropharyngeal swabs, 11 were only positive in cloacal swabs, and one was positive in both (Table 3). Only in February 2007 oropharyngeal and cloacal samples were collected, when nearly half of the positive samples were retrieved from oropharyngeal swab samples. In previous years only cloacal swabs had been taken. This may indicate that the number of LPAIV positive birds may be significantly underestimated when relying exclusively on cloacal samples. In addition, it cannot be excluded that different AIV subtypes may exhibit different tissue tropism. For example, out of five H11 subtype viruses detected during 2007, four originated from cloacal samples.

                                                The real-time RT-PCR results of inter-laboratory analyses of 2007 samples were highly concordant, when two identical sets of the samples were tested by laboratories in Germany and France/Italy. This finding shows the robustness of results when using the standard protocols in different laboratories. In addition, it shows sampling of cloaca or oropharyngeal area of the same bird twice, does not cause false negative results of the second sample.

                                                The potentially higher sensitivity of rRT-PCR compared to virus isolation has been mentioned before in other investigations [18]. Although some investigators could demonstrate that avian viruses originating from wild bird samples require several passages in embryonated eggs for adaptation, our investigation during 2003-2005 showed acceptable results even in the first egg passage (Table 3). During the 2007 investigation, only two out of 21 positive samples were also positive in virus isolation. The efficacy of virus isolation was dramatically reduced compared to 2003-2005. This might be due to many factors including: improper sample transfer in 2007 with extended international transport and interruption of the cold chain, quality and preservation of VTM and viral load of the samples.

                                                Validation of a serological assay for detection of AIV-specific antibodies in wild birds is still a matter of debate [15, 18, 2732]. Only limited information is available about the sensitivity of the HI assay in these species. In a recent study [33], only 16.9% of wild duck sera which tested positive in a double antibody sandwich blocking ELISA were positive in HI assay. Previous studies showed that competitive ELISAs on the basis of recombinant AIV-NP antigen appear to be a valid and reliable method for testing different bird species for avian influenza infection [15, 32, 34].

                                                In this study, the overall sero-prevalence based on 711 serum samples from different species was 48.5%, and Anseriformes provided 61.45% positive c-ELISA results. Therefore, the overall sero-prevalence could be misleading due to the species composition of samples. Two duck species with high numbers of samples were Mallards with 75% positive of 108, and Common Teal with 57.7% positive of 272 tested birds. These results, together with the virological data, again emphasize the importance of these species for the epidemiology of AIV in the nature, but the high percentage of AIV positive Mallard and Common Teal may just reflect an over-representation of these species in the sample composition due to their large wintering population in Iran. Thus, although the sample size for other members of this order was small, the detected prevalence rates are impressive (Table 3). Interestingly, only one serum sample from 66 Common Coots sampled during 2003-2005 was weakly seropositive, whereas sixteen from 49 Common Coot samples collected in 2007 were seropositive (32%). In addition, in 2003-2005 we could not find any AIV positive samples from 117 Common Coots taken from six different provinces [35], whereas in 2007 three out of 77 samples from Common Coots were positive in rRT-PCR. The relevance of these findings is not clear. Although seropositive samples were found among only 12% of Non-Anseriformes in 5 different families, some species, such as Greater Flamingo (3/3 positive) and Black-headed Gull (4/10 positive), demonstrated high prevalence rates.

                                                Conclusions

                                                In summary, the results of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected. Proper sample handling and maintenance of cold chains are important to ensure sample quality which is essential at least for virus isolation. Results obtained by rRT-PCR are less dependent on sample quality as shown by higher number of positives and by the high degree of correlation of results from different laboratories. The obvious problems with virus isolation highlight the ongoing demand of molecular methods for the sensitive detection and characterization of AIV.

                                                Abbreviations

                                                AIV: 

                                                avian influenza virus

                                                cELISA: 

                                                competitive enzyme linked immunosorbent assay

                                                HA: 

                                                hemagglutinin

                                                HI: 

                                                hemagglutination inhibition

                                                HPAIV: 

                                                highly pathogenic avian influenza virus

                                                Ig: 

                                                immunoglobulin

                                                IVPI: 

                                                intravenous pathogenicity index

                                                LPAIV: 

                                                low pathogenic avian influenza virus

                                                mAb: 

                                                monoclonal antibody

                                                NP-cELISA: 

                                                nucleoprotein-specific competitive ELISA

                                                NA: 

                                                neuraminidase

                                                NI: 

                                                neuraminidase inhibition

                                                NJ: 

                                                neighbour-joining

                                                PBS: 

                                                phosphate buffered saline

                                                RECP: 

                                                restriction enzyme cleavage pattern

                                                rRT-PCR: 

                                                real time reverse-transcription polymerase chain reaction

                                                RT-PCR: 

                                                reverse-transcription polymerase chain reaction

                                                SPF: 

                                                specific pathogen free

                                                VI: 

                                                virus isolation.

                                                Declarations

                                                Acknowledgements

                                                We would like to thank the involved personnel of Department of Environment of Iran who have contributed to field sampling and the technicians in Friedrich-Loeffler-Institut (FLI), Razi Research Institute, CIRAD and ISZ-Ve who contributed to laboratory analyses. This study was supported by Federal Ministry of Food, Agriculture and Consumer Protection of Germany (Forschungs-Sofortprogramms Influenza), Food and Agriculture Organization of the United Nations (FAO), Iran Department of the Environment, Razi Research Institute and Wetland International.

                                                Authors’ Affiliations

                                                (1)
                                                Friedrich-Loeffler-Institut (FLI)
                                                (2)
                                                Razi Research Institute
                                                (3)
                                                Wildlife Bureau, Department of Environment
                                                (4)
                                                Clinical Sciences Department, Faculty of Veterinary Medicine, University of Tehran
                                                (5)
                                                Centre de Cooperation Internationale en Recherche Agronomique pour le Développement
                                                (6)
                                                EMPRES Wildlife Unit, Food and Agriculture Organization of the United Nations
                                                (7)
                                                Research and development Deptartment, Istituto Zooprofilattico Sperimentale delle Venezie
                                                (8)
                                                Biodiversity and Ecological Networks, Wetlands International
                                                (9)
                                                Bird Conservation Society of Iran

                                                References

                                                1. Stallknecht DE, Brown JD: Ecology of Avian Influenza in Wild Birds. In Avian Influenza. 1st edition. Edited by: Swayne DE. Blackwell Publishing, Ames, Iowa; 2008:43–58.View Article
                                                2. FAO EMPRES-I database: Emergency Centre for Transboundary Animal Diseases (ECTAD), Animal Health Service. Rome, Italy: Food and Agriculture Organization of the United Nations; 2007.
                                                3. Commission Decision 2006/437/EC approving a diagnostic manual for avian influenza as provided for in Council Directive 2005/94/ECOff J Eur Commun 2006, L237:1–27.
                                                4. Lee MH, Chang PC, Shien JH, Cheng MC, Shieh HK: Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J Virol Methods 2001, 97:13–22.PubMedView Article
                                                5. Fereidouni SR, Starick E, Grund C, Globig A, Mettenleiter TC, Beer M, Harder T: Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses. Vet Microbiol 2009, 135:253–260.PubMedView Article
                                                6. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR: Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 2001, 146:2275–2289.PubMedView Article
                                                7. Spackman E, Senne DA, Bulaga LL, Myers TJ, Perdue ML, Garber LP, Lohman K, Daum LT, Suarez DL: Development of real-time RT-PCR for the detection of avian influenza virus. Avian Dis 2003,47(3 Suppl):1079–1082.PubMedView Article
                                                8. Hoffmann B, Depner K, Schirrmeier H, Beer M: A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses. J Virol Methods 2006, 136:200–209.PubMedView Article
                                                9. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24:1596–1599.PubMedView Article
                                                10. Fereidouni SR, Harder TC, Starick E: Rapid pathotyping of recent H5N1 highly pathogenic avian influenza viruses and of H5 viruses with low pathogenicity by RT-PCR and restriction enzyme cleavage pattern (RECP). J Virol Methods 2008, 154:14–19.PubMedView Article
                                                11. Fereidouni SR, Bozorghmehrifard MH, Starick E, Werner O, Amini H, Aghakhan M: Serological Monitoring of Avian Influenza in Migratory birds of Iran. Arch Razi Inst 2005, 60:11–19.
                                                12. Gall A, Hoffmann B, Harder T, Grund C, Höper D, Beer M: Design and validation of a microarray for detection, hemagglutinin subtyping, and pathotyping of avian influenza viruses. J Clin Microbiol 2009, 47:327–334.PubMedView Article
                                                13. Kothlow S, Haüslaigner R, Kaspers B, Grund C: Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain. Avian Pathol 2008, 37:323–328.PubMedView Article
                                                14. Alexander DJ: Report on Avian Influenza in the Eastern Hemisphere during 1997–2002. Avian Dis 2003, 47:792–797.PubMedView Article
                                                15. De Marco MA, Fonic E, Campitelli L, Raffini E, Di Trani L, Delogu M, Guberti V, Barigazzi G, Donatelli I: Circulation of Influenza viruses in wild waterfowl wintering in Italy during the 1993–1999 period: evidence of virus shedding and seroconservation in wild ducks. Avian Dis 2003, 47:861–866.PubMedView Article
                                                16. Munster VJ, Baas C, Lexmond P, Waldenström J, Wallensten A, Fransson T, et al.: Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds. PLoS Pathog 2007, 3:e61.PubMedView Article
                                                17. Gaidet N, Dodman T, Caron A, Balança G, Desvaux S, Goutard F, et al.: Avian influenza viruses in water birds, Africa. Emerg Infect Dis 2007, 13:626–629.PubMedView Article
                                                18. Fouchier RAM, Olsen B, Bestebroer TM, Herfst S, Kemp L, Rimmelzwaan GF, Osterhaus ADME: Influenza A Virus Surveillance in Wild Birds in Northern Europe in 1999 and 2000. Avian Dis 2003, 47:857–860.PubMedView Article
                                                19. Cattoli G, Terregino C, Guberti V, De Nardi R, Drago A, Salviato A, Fassina S, Capua I: Influenza virus surveillance in wild birds in Italy: results of laboratory investigations in 2003–2005. Avian Dis 2007, 51:414–416.PubMedView Article
                                                20. Jonassen CM, Handeland K: Avian influenza virus screening in wild waterfowl in Norway, 2005. Avian Dis 2007, 51:425–428.PubMedView Article
                                                21. Ip HS, Flint PL, Franson JC, Dusek RJ, Derksen DV, et al.: Prevalence of Influenza A viruses in wild migratory birds in Alaska: patterns of variation in detection at a crossroads of intercontinental flyways. Virol J 2008, 5:71.PubMedView Article
                                                22. Krauss S, Obert CA, Franks J, Walker D, Jones K, Seiler P, et al.: Influenza in migratory birds and evidence of limited intercontinental virus exchange. PLoS Pathog 2007, 3:e167.PubMedView Article
                                                23. Parmley EJ, Bastien N, Booth TF, Bowes V, Buck PA, Breault A, et al.: Wild bird influenza survey, Canada, 2005. Emerg Infect Dis 2008, 14:84–87.PubMedView Article
                                                24. Globig A, Starick E, Werner O: Influenzavirusinfektionen bei migrierenden Wasservögeln: Ergebnisse einer zweijährigen Studie in Deutschland. Berl Münch Tierärztl Wochenschr 2006, 119:132–139.PubMed
                                                25. Gaidet N, Dodman T, Caron A, Balança G, Desvaux S, Goutard F, et al.: Influenza Surveillance in Wild Birds in Eastern Europe, the Middle East, and Africa: Preliminary Results from an Ongoing FAO-led Survey. J wildlife Dis 2007, 43:22–28.
                                                26. Ellström P, Latorre-Margalef N, Griekspoor P, Waldenström J, Olofsson J, Wahlgren J, Olsen B: Sampling for low-pathogenic avian influenza A virus in wild Mallard ducks: oropharyngeal versus cloacal swabbing. Vaccine 2008, 26:4414–4416.PubMedView Article
                                                27. Arenas A, Carranza J, Perea A, Miranda A, Maldonado A, Hermoso M: Type A influenza viruses in birds in southern Spain: serological survey by enzyme-linked immunosorbent assay and haemagglutination inhibition tests. Avian Pathol 1990, 19:539–546.PubMedView Article
                                                28. Astorga RJ, Leon L, Cubero MJ, Arenas A, Maldonado A, Tarradas MC, Perea A: Avian Influenza in wild waterfowl and shorebirds in Donana Park: serological survey using the enzyme-linked immunosorbent assay. Avian Pathol 1994, 23:339–344.PubMedView Article
                                                29. Graves IL: Influenza viruses in birds of the Atlantic flyway. Avian Dis 1992, 36:1–10.PubMedView Article
                                                30. Pfitzer S, Verwoerd DJ, Gerdes GH, Labuschagne AE, Erasmus A, Manvell RJ, Grund C: Newcastle disease and avian influenza A virus in wild waterfowl in South Africa. Avian Dis 2000, 44:655–660.PubMedView Article
                                                31. Sala G, Cordioli P, Moreno-Martin A, Tollis M, Brocchi E, Piccirillo A, Lavazza A: ELISA Test for the Detection of Influenza H7 Antibodies in Avian Sera. Avian Dis 2003, 47:1057–1059.PubMedView Article
                                                32. Shafer AL, Katz JB, Eernisse KA: Development and validation of a competitive Enzyme-linked immunosorbent assay for detection of type A influenza antibodies in avian sera. Avian Dis 1998, 42:28–34.PubMedView Article
                                                33. De Marco MA, Campitelli L, Fonic E, Raffini E, Barigazzi G, Delogu M, Guberti V, Di Trani L, Tollis M, Donatelli I: Influenza surveillance in birds in Italian wetlands (1992–1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots? Vet Microbiol 2004, 98:197–208.PubMedView Article
                                                34. Zhou EM, Chan M, Heckert JR, Cantin MF: Evaluation of a competitive ELISA for detection of antibodies against avian influenza virus nucleoprotein. Avian Dis 1998, 42:517–522.PubMedView Article
                                                35. Fereidouni SR, Aghakhan M, Werner O, Starick E, Bozorghmehrifard MH: Isolation and identification of avian influenza viruses from migratory birds in Iran. Vet Rec 2005, 157:526.PubMed

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                                                © Fereidouni et al. 2010

                                                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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