Abstract
Black-headed gulls (Chroicocephalus ridibundus) are a suitable host species to study the epidemiology of low-pathogenic avian influenza virus (LPAIV) infection in wild waterbirds because they are a common colony-breeding species in which LPAIV infection is detected frequently, limited mainly to the H13 and H16 subtypes. However, the sites of virus replication and associated lesions are poorly understood. We therefore performed virological and pathological analyses on tissues of black-headed gulls naturally infected with LPAIV. We found that 24 of 111 black-headed gulls collected from breeding colonies were infected with LPAIV (10 birds with H16N3, one bird with H13N8, 13 birds undetermined), based on virus and viral genome detection in pharyngeal and cloacal swabs. Of these 24 gulls, 15 expressed virus antigen in their tissues. Virus antigen expression was limited to epithelial cells of intestine and cloacal bursa. No histological lesions were detected in association with virus antigen expression. Our findings show that LPAIV replication in the intestinal tract of black-headed gulls is mainly a superficial infection in absence of detectable lesions, as determined recently for natural LPAIV infection in free-living mallards (Anas platyrhynchos). These findings imply that LPAIV in black-headed gulls has adapted to minimal pathogenicity to its host and that potentially the primary transmission route is faecal–oral.
Introduction
Influenza virus infection is an important problem for the health of both humans and domestic animals (Gibbs, 2010). The ultimate reservoir of the 16 haemagglutinin (H1 to H16) and nine neuraminidase (N1 to N9) subtypes are wild birds, mainly waterbirds of the orders Anseriformes (including ducks, geese, and swans) and Charadriiformes (including gulls, waders and terns). In these wild birds, influenza virus nearly always occurs as low pathogenic avian influenza virus (LPAIV). Among waterbird species, the number of H subtypes that are found varies considerably. While the mallard (Anas platyrhynchos) has been found to be infected with nearly all H subtypes of LPAIV, gulls (Laridae) are primarily infected with subtypes H13 or H16. H1, H2, H4, H6, H9, and H11 also have been reported sporadically (Hinshaw et al., 1982; Sinnecker et al., 1983; Kawaoka et al., 1988; Graves, 1992; Fouchier et al., 2005; Olsen et al., 2006). Because of the large number of both host species and virus subtypes, the epidemiology of LPAIV infection in wild birds is poorly understood. One step to increase this understanding is to determine the tissue tropism and associated pathology of LPAIV infection in wild birds.
The tissue tropism and associated pathology of LPAIV infection in domestic birds is well studied. LPAIV in domestic birds has been shown to replicate in epithelial cells of the respiratory tract, intestinal tract, or both (Slemons & Easterday, 1978; Kida et al., 1980; Pantin-Jackwood & Swayne, 2009). In chickens, associated lesions are epithelial necrosis and inflammation of the upper respiratory tract, and—more rarely—pneumonia (Mo et al., 1997; Swayne, 1997; Jones & Swayne, 2004; Spackman et al., 2010). In turkeys, associated lesions include inflammation of the reproductive tract, intestine, and pancreas (Capua et al., 2000). In domestic ducks and ratites, associated lesions occur in the upper and lower respiratory tract (Pantin-Jackwood & Swayne, 2009; Spackman et al., 2010).
Little is known about the tissue tropism and associated pathology of LPAIV infection in wild birds. A recent study of natural LPAIV infection in free-living mallards showed that virus replication was limited to epithelial cells of the intestine and cloacal bursa in the absence of microscopic lesions (Daoust et al., 2011). For other wild bird species, including black-headed gulls, this information is unknown.
The objectives of our study were therefore to determine the sites and cell types of virus replication and to determine whether virus replication was associated with microscopically detectable lesions in black-headed gulls with natural LPAIV infection. We did this by performing virological and pathological analyses on tissues of juvenile black-headed gulls that were naturally infected with LPAIV. We chose black-headed gulls for three reasons: first, the fact that, although other virus subtypes have been reported in this species, the virus subtypes that occur most frequently are H13 and H16; second, because black-headed gulls breed in colonies and therefore are relatively easy to sample; and third, because the black-headed gull is the most common gull species in Western Europe (BirdLife International, 2004). This research was undertaken as part of a larger study, in which we are using the black-headed gull as a host species to study the epidemiology of LPAIV infection.
Materials and Methods
Birds and sampling
The black-headed gulls for this study came from two sources. First, 61 recently dead juvenile (15 to 20 days old) black-headed gulls were collected from the island Griend (53.251966, 5.253568), the Netherlands, during the 2009 and 2010 breeding seasons in the course of active influenza surveillance and were necropsied on site. Second, 50 live juvenile (5 to 10 days old) black-headed gulls were collected from a colony site at Blauwestad (53.171627, 7.011681), the Netherlands on 6 June 2011, to be used as naïve birds for an infection experiment with influenza virus. They were kept in captivity and supplied with water and appropriate food ad libitum. Although they had been collected earlier in the season than when influenza virus was recorded to be circulating in Dutch black-headed gull breeding colonies in previous years (unpublished data), the birds were found to be infected with influenza virus and could not be used for the infection experiment. Fifteen days after capture, they were euthanized using an overdose of isofluorane (Upjohn, Ede, the Netherlands) and necropsied. The study was performed under appropriate permits for capture and transport from the Dutch Ministry of Economic Affairs, Agriculture, and Innovation, and all methods employed were previously approved by the institutional Animal Ethics Committee (dierexperimentencommissie DEC) of Erasmus Medical Center.
Samples for influenza virus detection by reverse transcriptase (RT)-polymerase chain reaction (PCR; see below) were oropharyngeal swabs and cloacal swabs placed separately (2009, 2011) or together (2010) into sterile containers containing 1 ml virus transport medium (Fouchier et al., 2000). Based on this test, 24 positive gulls (principal) and six negative gulls (control) were selected for further analysis (Table 1). Additional tissue samples for influenza virus RT-PCR were collected in 2009 (trachea, duodenum, jejunum, colon, cloacal bursa) and 2011 (lung, jejunum) into sterile containers containing no transport medium (2009) or 1 ml transport medium (2011). All samples were analysed directly upon arrival at the laboratory or stored at −80°C until analysis.
Table 1. Evidence of influenza virus infection in black-headed gulls naturally infected with LPAIV (P1 to P24) compared with those not infected (C1 to C6).
Samples for virus antigen expression by immunohistochemistry (IHC) and for detection of influenza-associated microscopic lesions by routine histopathology were digestive tract samples from the level of the proventriculus to the level of the cloaca. Specific sample sites were: proventriculus, ventriculus, duodenum, jejunum at 5, 15, 25 and 35 cm along its length, ileocaecal junction, colon and cloacal bursa. Additional samples for IHC and histopathology were collected in 2010 (trachea) and 2011 (nasal conchae, trachea, syrinx, bronchus, lung, air sac, cerebrum, cerebellum, brainstem, kidney, liver, spleen). Tissue samples were fixed in 10% neutral-buffered formalin and processed within 48 h for paraffin embedding.
Influenza virus detection, isolation and subtyping
Oropharyngeal and cloacal swabs were extracted using the Magnapure extraction system (Magna Pure LC; Roche Diagnostics GMBH, Penzberg, Germany) and analysed individually for the presence of the avian influenza virus (AIV) matrix gene by Taqman RT-PCR as described previously (Munster et al., 2007). In birds in which a positive result with a cycle threshold value below 40 was obtained from a swab, tissue samples were homogenized and extracted and analysed using the same methodology as the swabs. Also, an aliquot of 100 µl transport medium from positive swabs and tissue samples was inoculated into the allantoic cavity of embryonated 11-day-old specific pathogen-free chicken eggs for virus isolation. Eggs were harvested on day 3 post inoculation and tested by the haemagglutination test for the presence of haemagglutinating virus. If negative, a second passage on embryonated chicken eggs was performed. The allantoic fluid of both passages was tested by haemagglutination test using turkey red blood cells. The H subtype of isolated viruses was determined by haemagglutination inhibition test against all known H subtypes of AIV using rabbit (H1 to H12, H14, H15) and ferret (H13, H16) hyperimmune sera and turkey red blood cells. The N subtype was determined by specific PCR for all nine known N subtypes and subsequent sequencing (Fouchier et al., 2005).
Histopathology and immunohistochemistry
After paraffin embedding, tissue samples were sectioned at 3 µm intervals, fixed on silane-coated slides (Starfrost adhesive; Knittel Glass, Braunschweig, Germany), and stained with haematoxylin and eosin (HE) for histopathological examination. Duplicate slides were made for virus antigen expression by IHC. The primary antibody used was directed against the AIV nucleoprotein (HB65; American Type Culture Collection, Manassas, Virginia, USA) and IHC procedures were carried out as previously described (van Riel et al., 2010; Daoust et al., 2011). Briefly, after deparaffinization and hydration, tissue sections were incubated for 10 min at 37°C with 0.1% protease in phosphate-buffered saline (PBS). Endogenous peroxidase was blocked using 3% H2O2 for 10 min, followed by washes with PBS and PBS–Tween. After 1-h incubation with the primary antibody (mouse anti influenza A nucleoprotein Clone Hb65, mouse IgG2a 1 mg/ml, diluted 1/400 in PBS–Tween) at room temperature, and washes with PBS–Tween, a peroxidase-labelled goat antimouse IgG2a (Southern Biotech, Birmingham, Alabama, USA) was used to detect binding of the primary antibody. Peroxidase was revealed using 3-amino-9-ethyl-carbazol (Sigma Chemicals, Zwijndrecht, the Netherlands), giving bright-red labelling of positive nuclei and cytoplasm. Sections were counterstained with haematoxylin. As a positive control, lung tissue of a cat infected experimentally with Influenza A/Vietnam/1194/2004 (H5N1) was included. As negative controls, RT-PCR-negative gull tissues, matched isotype controls and omission controls were included.
Tissue sections were examined under a light microscope for the detection of virus antigen expression and microscopic lesions. Tissues were considered virus antigen-positive even if single nuclei were clearly labelled. In positive intestinal sections, virus antigen expression was scored according to the number of cells stained by IHC into three categories: less than 10 cells per tissue section positive, 10 to 50 cells per tissue section positive; and more than 50 cells per tissue section positive. Tissues expressing virus antigen were examined in detail by light microscopy for the presence of any associated microscopic lesions, in accordance with previously described LPAIV-associated lesions, specifically epithelial cell necrosis and infiltration of inflammatory cells.
Circovirus detection
Circoviruses have been described as able to induce immune compromise in infected birds (Todd, 2000). Black-headed gulls, including individuals from the Netherlands, have been shown to be susceptible to infection by circovirus (Kuiken et al., 2002; Todd et al., 2007). In order to detect infection by circovirus in the gulls included in the study, HE-stained tissue sections containing lymphoid tissue (2009 to 2011, cloacal bursa, caecal tonsil; 2011, spleen) were also screened for the presence of characteristic circovirus-associated inclusions. In addition, cloacal swabs taken in 2011 were extracted using the Magnapure system and analysed by nested PCR as described previously for the presence of circovirus genome (Todd et al., 2007).
Results
Influenza virus detection, isolation and subtyping
Of 111 black-headed gulls tested, 24 gulls (principal) were positive for influenza virus by RT-PCR on individual pharyngeal or cloacal swabs or on a pool of pharyngeal and cloacal swabs (Table 1). From swabs of these 24 RT-PCR-positive gulls, influenza virus was isolated and subtyped from 11 gulls: 10 birds with H16N3, one bird with H13N8. All six RT-PCR-negative gulls (control) were also negative by virus isolation.
Immunohistochemistry
Expression of influenza virus nucleoprotein was found in 15 of 24 principal gulls (Table 1). In these 15 gulls, virus antigen expression was detected only in the small intestine, large intestine, and cloacal bursa, and was most frequent and intense in the lower jejunum (Figure 1). None of the nine principal gulls for which respiratory tract tissue was available showed antigen expression in any of these tissues.
Published online:
14 December 2012Figure 1. Distribution and intensity of virus antigen expression in black-headed gulls naturally infected with LPAIV.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i06/03079457.2012.744447/20121214/images/medium/cavp_a_744447_o_f0001g.gif"})
Figure 1. Distribution and intensity of virus antigen expression in black-headed gulls naturally infected with LPAIV.
In the small and large intestines, cells expressing virus antigen in the nucleus—thus proving intracellular production of virus protein—were limited to enterocytes lining intestinal villi (Figures 2 and 3). Most of these positive enterocytes were located in the upper one-third of the villi, and occurred as individual cells among enterocytes not expressing virus antigen. In some intestinal villi with abundant virus antigen expression in enterocytes, there also was virus antigen expression in the subjacent lamina propria (Figure 4). However, this was present as loose granular material and was not located within cell nuclei.
Published online:
14 December 2012Figure 2. Small intestine of a black-headed gull infected naturally with LPAIV. Influenza virus antigen expression of enterocytes lining the intestinal villi (2a to 2c) and absence of associated microscopic lesions (2d). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (2a to 2c). HE, sequential section of 2c (2d). Bar=50 μm.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i06/03079457.2012.744447/20121214/images/medium/cavp_a_744447_o_f0002g.jpg"})
Figure 2. Small intestine of a black-headed gull infected naturally with LPAIV. Influenza virus antigen expression of enterocytes lining the intestinal villi (2a to 2c) and absence of associated microscopic lesions (2d). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (2a to 2c). HE, sequential section of 2c (2d). Bar=50 μm.
Published online:
14 December 2012Figure 3. Large intestine of a black-headed gull infected naturally with LPAIV. Influenza virus antigen expression of enterocytes lining the intestinal villi (3a and 3b) and absence of associated microscopic lesions (3c). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (3a and 3b). HE, sequential section of 3b (3c). Bar=50 μm.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i06/03079457.2012.744447/20121214/images/medium/cavp_a_744447_o_f0003g.jpg"})
Figure 3. Large intestine of a black-headed gull infected naturally with LPAIV. Influenza virus antigen expression of enterocytes lining the intestinal villi (3a and 3b) and absence of associated microscopic lesions (3c). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (3a and 3b). HE, sequential section of 3b (3c). Bar=50 μm.
Published online:
14 December 2012Figure 4. Large intestine of a black-headed gull naturally infected with LPAIV. Virus antigen both in epithelium (arrowhead) and lamina propria (between arrows) (4a), and absence of microscopic lesions (4b). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (4a). HE, sequential section of 4a (4b). Bar=50 μm.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i06/03079457.2012.744447/20121214/images/medium/cavp_a_744447_o_f0004g.jpg"})
Figure 4. Large intestine of a black-headed gull naturally infected with LPAIV. Virus antigen both in epithelium (arrowhead) and lamina propria (between arrows) (4a), and absence of microscopic lesions (4b). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (4a). HE, sequential section of 4a (4b). Bar=50 μm.
In the cloacal bursa, cells expressing virus antigen were limited to the surface epithelium lining the central lumen of the bursa (Figure 5). Similarly to some of the H16-infected gulls (P2, only jejunum in P3 and P4), virus antigen expression in the gull infected with H13N8 subtype (P9) was detected only in the lower part of the jejunum and the ileum. No virus antigen expression was found in any respiratory tract tissues or any other tissues outside the intestinal tract of principal gulls. No virus antigen expression was found in any tissues of the six control gulls (Table 1).
Published online:
14 December 2012Figure 5. Cloacal bursa of a black-headed gull naturally infected with LPAIV. Virus antigen in surface epithelium of central lumen (5a), and absence of microscopic lesions (5b). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (5a). HE, sequential section of 5a (5b). Bar=50 μm.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i06/03079457.2012.744447/20121214/images/medium/cavp_a_744447_o_f0005g.jpg"})
Figure 5. Cloacal bursa of a black-headed gull naturally infected with LPAIV. Virus antigen in surface epithelium of central lumen (5a), and absence of microscopic lesions (5b). Immunohistochemical method to detect nucleoprotein of influenza virus counterstained with haematoxylin (5a). HE, sequential section of 5a (5b). Bar=50 μm.
Histopathology
No microscopic lesions, such as epithelial necrosis or infiltration with inflammatory cells, were observed to co-localize with virus antigen expression. However, several incidental lesions were observed. All gulls had evidence of trematode infection of the intestinal tract, based on the presence of adult trematodes or trematode metacercarial cysts in the lumen or mucosa of the intestinal tract. All gulls had diffuse infiltration with mononuclear cells and granulocytes in the lamina propria of small and large intestines, but this did not co-localize with virus antigen expression, and was also present in the six non-AIV-infected gulls. Four gulls from 2011 had mononuclear cell infiltration in the nasal mucosa, which co-localized in one gull with a helminth parasite. Five gulls from 2011 had one or more granulomas in the lung parenchyma. One gull from 2011 had a cloacal abscess with several arthropods in the centre. None of these lesions was associated with the degree of AIV antigen expression in the examined gulls.
Circovirus detection
None of the gulls were infected by circovirus, based on the absence of typical inclusions in lymphoid tissues upon histological examination (2009 to 2011), and the negative results by PCR on cloacal swabs (2011 only).
Discussion
Our results show for the first time the replication of LPAIV in naturally infected black-headed gulls in the epithelial cells of the intestine and cloacal bursa, based on virus antigen expression. They also show that there are no detectable microscopic lesions associated with LPAIV replication in these gulls.
Overall, the results of our study in black-headed gulls correspond to those reported recently for natural LPAIV infection in free-living mallards (Daoust et al., 2011), where virus replication was restricted to intestinal and bursal epithelium in the absence of lesions. However, the site of virus replication in the two studies showed differences both in the level of the intestinal tract and in the height of the intestinal mucosa. First, while virus replication in our gulls was found more commonly in the small intestine (13 of 24 birds) than in the large intestine (seven of 24) or bursal cloaca (one of 24) (Table 1, Figure 1), Daoust et al. (2011) found virus replication more commonly in bursal cloaca (eight of 19 birds) and large intestine (three of 19) than in the small intestine (one of 19) of naturally infected free-living mallards. Possible explanations are differences in virus, host species, and temporal stage of infection between these two studies. The findings of Daoust et al. (2011) correspond with those for experimental LPAIV infection in domestic ducks by Slemons & Easterday (1978) and Kida et al. (1980), who found virus replication primarily in the epithelium of cloacal bursa and the large intestine. However, they do not correspond with the results of the study of Volmer et al. (2010), who found virus replication more frequently in the small intestine than in the large intestine of domestic ducks experimentally infected with a genetically engineered virus possessing both human-type and avian-type amino acid motifs in the NS1 protein.
Second, the height of the intestinal mucosa where virus replication occurred differed slightly between these two studies. While virus replication in our gulls was found exclusively in epithelial cells of intestinal villi (15 of 15), Daoust et al. (2011) found virus replication in epithelial cells of both intestinal villi (four of four) and submucosal glands (one of four). Furthermore, Kida et al. (1980) reported virus replication only in epithelial cells of mucosal crypts in the colon of experimentally infected domestic ducks. A possible explanation is that LPAIV in black-headed gulls has a strong tropism for the differentiated epithelial cells lining the intestinal villi, and not for the less differentiated epithelial cells lining the intestinal crypts or the submucosal gland epithelial cells.
The absence of microscopic lesions associated with LPAIV replication in our gulls matches the absence of lesions in natural LPAIV infection of free-living mallards (Daoust et al., 2011) and absence of clinical signs in experimental LPAIV infection of domestic ducks (Slemons & Easterday, 1978; Kida et al., 1980). In contrast, Volmer et al. (2010) observed moderate enteritis in domestic ducks infected experimentally with a genetically engineered LPAIV, and Jourdain et al. (2010) measured a transient increase in body temperature of mallards infected experimentally with LPAIV. While we did not detect LPAIV-associated lesions in our gulls, we cannot rule out the possibility that LPAIV infection has more subtle costs for black-headed gulls due to, for example, increased turnover of intestinal epithelial cells or host immune response.
Our results showed no association of circovirus and H13/16 AIV infection in black-headed gulls. Despite the known ability of circovirus to induce immune compromise in infected birds (Todd, 2000), and previous results (Kuiken et al., 2002) that indicate circulation of circovirus in black-headed gulls in the Netherlands, none of our AIV-positive gulls showed any evidence for circovirus infection. However, if co-infections with both viruses were possible this could have an effect on the pathogenicity and virulence of the co-infecting LPAIV in black-headed gulls, which may deserve some attention in future studies.
We could not prove respiratory tract infection in our gulls, because none of the respiratory tract tissues tested expressed virus antigen. However, we did detect virus by RT-PCR in the lung of one of nine gulls and in the trachea of one of four gulls (Table 1). Also, Velarde et al. (2010) isolated LPAIV from lung tissue of ring-billed gulls (Larus delawarensis) and Costa et al. (2011) isolated LPAIV more often from oropharyngeal swabs than from cloacal swabs of laughing gulls (Leucophaeus atricilla) inoculated intrachoanally with different subtypes of mallard-origin strains. These findings suggest that LPAIV may also infect the respiratory tract of gulls, depending on, amongst other factors, virus strain…. virus strain, gull species, and route of entry.
In conclusion, our study shows that LPAIV replicates primarily in the intestinal epithelium of black-headed gulls, without causing microscopic lesions. These results imply that LPAIV in black-headed gulls has minimal—if any—pathogenicity, that LPAIV is excreted mainly via faeces, and that the most important route of transmission is faecal–oral. This does not rule out the possibility that LPAIV in gulls also replicates in the respiratory tract and is spread via respiratory secretions. This requires further investigation. Together, this knowledge helps to build a more complete picture of the epidemiology of LPAIV infection in black-headed gulls.
| RT-PCR (Ct value) | Virus antigen expression in: | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Swabs | Tissues | Jejunum | ||||||||||||||||||
| Bird | Necropsy date | Colony site | Pharynx | Cloaca | Pharynx and cloaca | Trachea | Lung | Duodenum | Jejunum | Colon | Virus subtype | Duodenum | 5 cm | 15 cm | 25 cm | 35 cm | Ileum | Caeca | Colon | Bursa |
| P1 | 9 Jul 2009 | G | − | 26 | − | 24 | 24 | 27 | H16N3 | − | − | + | − | − | + | + | + | − | ||
| P2 | 9 Jul 2009 | G | − | 25 | 33 | 33 | 33 | 19 | H16N3 | − | − | − | + | + | + | − | + | − | ||
| C1 | 9 Jul 2009 | G | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | ||
| C2 | 9 Jul 2009 | G | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | ||
| P3 | 21 Jul 2010 | G | 22 | H16N3 | − | + | − | + | − | − | − | − | − | |||||||
| P4 | 21 Jul 2010 | G | 29 | − | + | − | + | + | + | − | − | − | − | |||||||
| P5 | 21 Jul 2010 | G | 22 | − | + | + | + | + | + | − | − | − | − | |||||||
| P6 | 21 Jul 2010 | G | 31 | − | − | − | − | − | − | − | − | − | − | |||||||
| P7 | 21 Jul 2010 | G | 23 | − | + | + | + | + | + | + | + | + | + | |||||||
| P8 | 21 Jul 2010 | G | 26 | − | + | + | + | + | − | − | − | − | − | |||||||
| P9 | 21 Jul 2010 | G | 34 | H13N8 | − | − | − | + | + | + | − | − | − | |||||||
| P10 | 21 Jul 2010 | G | 29 | − | − | − | − | − | − | + | − | + | − | |||||||
| P11 | 21 Jul 2010 | G | 36 | − | − | − | − | − | − | − | − | + | − | |||||||
| P12 | 21 Jul 2010 | G | 28 | − | − | − | − | + | + | + | − | + | − | |||||||
| P13 | 21 Jul 2010 | G | 34 | − | − | − | − | − | − | − | − | − | − | |||||||
| P14 | 21 Jul 2010 | G | 34 | − | − | − | − | − | − | − | − | − | − | |||||||
| P15 | 21 Jul 2010 | G | 32 | H16N3 | − | − | − | − | − | − | + | − | − | |||||||
| C3 | 21 Jul 2010 | G | − | − | − | − | − | − | − | − | − | − | − | |||||||
| C4 | 21 Jul 2010 | G | − | − | − | − | − | − | − | − | − | − | − | |||||||
| P16 | 22 Jun 2011 | BS | 18 | − | − | − | H16N3 | − | − | − | − | − | − | − | − | − | ||||
| P17 | 22 Jun 2011 | BS | 32 | − | − | − | H16N3 | − | − | − | − | − | − | − | − | − | ||||
| P18 | 22 Jun 2011 | BS | − | 30 | − | − | − | − | − | − | − | − | − | − | − | − | ||||
| P19 | 22 Jun 2011 | BS | 31 | − | 27 | − | − | − | − | − | − | − | − | − | − | − | ||||
| P20 | 22 Jun 2011 | BS | − | 30 | − | 19 | H16N3 | − | − | − | − | + | − | − | − | − | ||||
| P21 | 22 Jun 2011 | BS | − | 31 | − | − | H16N3 | − | − | − | − | − | − | − | − | − | ||||
| P22 | 22 Jun 2011 | BS | − | 23 | − | 17 | H16N3 | − | − | − | − | + | − | − | − | − | ||||
| P23 | 22 Jun 2011 | BS | 31 | − | − | − | − | − | − | − | − | − | − | − | − | − | ||||
| P24 | 22 Jun 2011 | BS | − | 30 | − | 17 | H16N3 | − | − | − | − | + | − | − | − | − | ||||
| C5 | 22 Jun 2011 | BS | − | − | − | − | − | − | − | − | − | − | − | − | − | − | ||||
| C6 | 22 Jun 2011 | BS | − | − | − | − | − | − | − | − | − | − | − | − | − | − | ||||
| P, principal; C, control; G, Griend; BS, Blauwe Stad; Ct, cycle threshold; −, negative; +, positive. | ||||||||||||||||||||
Acknowledgements
The authors would like to acknowledge assistance with handling of the black-headed gulls from F. Majoor, D. Lutterop, and G. Kasemir, and excellent assistance in the laboratory by P. van Run. J.H.V. and R.A.M.F. are supported by contracts with the Dutch Ministry of Agriculture and NIAID/NIH (number HHSN266200700010C). U.H. had a short-term visiting scientist “José Castillejo” fellowship from the Spanish Ministry of Innovation and Research (Ministerio de Innovación e Investigación MICINN, JC2010-0257) at Erasmus Medical Center. T. Kuiken is supported by contracts with the European Commission through the ANTIGONE project (number 278976) and with the Dutch Ministry of Economic Affairs through the Impulse Veterinary Avian Influenza Research.
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