This study was carried out in strict accordance with the recommendations of the Ethical Commission of Animal Experimentation of the Autonomous Government of Catalonia (Comissió d'Experimentació Animal de la Generalitat (CEA), Permit Number: 5567). The protocol was approved by the Ethics Committee of Animal and Human Experimentation of the Universitat Autònoma de Barcelona (Comissió d'Ètica en l'Experimentació Animal i Humana (CEEAH), Permit Number: 1066). All manipulations were performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering. Welfare information and end point criteria are included in Text S1.

Two strains of AI virus were used: the HPAI isolate A/Great crested grebe/Basque Country/06.03249/2006 (H5N1) (H5N1 HPAI) and the LPAI isolate A/Anas plathyrhynchos/Spain/1877/2009 (H7N2) (H7N2 LPAI). The deduced amino acid sequence of the region coding for the cleavage site of the haemagglutinin molecule were PEIPKGSRVRR*GLF for the isolate H5N1 HPAI and PEIPKGR*GLF for the isolate H7N2 LPAI, being typical of HPAI and LPAI viruses, respectively. In addition, the H5N1 HPAI subtype demonstrated an intravenous pathogenicity index of 3.0 [28].

Stocks of the H5N1 HPAI and the H7N2 LPAI viruses were produced in 9-day-old embryonated specific pathogen free (SPF) chicken eggs. In both cases, the allantoic fluid was harvested at 48 hours post inoculation, aliquoted and stored at −80° C until use. Virus was diluted tenfold in phosphate buffer saline (PBS) for titration in 9-day-old embryonated SPF chicken eggs. The 50% egg lethal dose (ELD₅₀) for H5N1 HPAI, and the 50% egg infective dose (EID₅₀) for H7N2 LPAI, were determined using the Reed and Muench method [29].

Juvenile (5–10 weeks old) male captive-reared ¾ gyr-saker (Falco rusticolus × Falco cherrug) hybrid falcons were obtained from a breeder (Roc Falcon S.L). From 3 to 10 weeks of age, falcons were imprinted by a person from the research group in order to minimize further stress. In order to assess that optimal health conditions existed, a complete blood cell count (CBC) was performed and a peripheral blood smear evaluated to rule out the presence of hemoparasites in all birds. Results of the CBC and blood smears were unremarkable. Also, an anti-parasite treatment (Baycox®) was carried out. In addition, non-diluted serum samples were collected to ensure that birds were serologically negative for AI antibodies by a competitive enzyme-linked immunosorbent assay (C-ELISA) (ID-VET, Montpellier, France). Furthermore, oropharyngeal and cloacal swabs were collected in order to make certain that falcons were negative for AI virus by real time RT-PCR (RRT-PCR). Birds were weighed and tagged with numbered aluminum leg bands, and brought to biosafety level 3 (BSL-3) in CReSA facility, where they were randomly distributed within the experimental groups and separately housed in negative-pressured isolators with HEPA-filtered air. It is worth highlighting that isolators were environmentally adapted for falcons' welfare (Text S1). Whole chick-preys, supplemented with vitamins were provided twice a day. Birds were adapted to their new environment for 5 days before experimental infection.

The experimental design is summarized in Table 1. Seventeen falcons were distributed into five experimental groups (A to E). Groups A (n = 4) and B (n = 4) were challenged with 10⁶ EID₅₀ of the H7N2 LPAI virus, whereas groups C (n = 3) and D (n = 4) were challenged with 10⁶ ELD₅₀ of the H5N1 HPAI virus. Two falcons from group E were inoculated nasochoanally with PBS solution and served as negative controls.

Groups A and C were inoculated nasochoanally; in group A, one of the 4 falcons was not-inoculated and was referred to as the contact animal. Groups B and D were challenged via the natural feeding route with previously infected SPF chicks. Briefly, one-day-old SPF chicks, confirmed negative for AI virus by RRT-PCR, were inoculated via oculonasal route with either 10⁶ EID₅₀ of the H7N2 LPAI virus or 10⁶ ELD₅₀ of the H5N1 HPAI virus. At 3 days post-infection (dpi), chicks were confirmed positive for AI virus by performing a RRT-PCR on oropharyngeal and cloacal swabs. Two whole H7N2 LPAI-infected chicks and five whole H5N1 HPAI-infected chicks were used to infect each falcon from groups B and D, respectively. In order to estimate the viral load ingested by the falcons, viral titration in Madin-Darby Canine Kidney (MDCK) cells was performed on tissue homogenates from LPAI-infected and HPAI-infected chicks. Two out of three homogenates of trachea, lung, kidney and small intestine from H7N2 LPAI-infected chicks contained from 10^3.2 to 10^7.2 TCID₅₀/g of tissue, whereas the viral load from homogenates of liver, lung, kidney and brain of all H5N1 HPAI-infected chicks reached titers from 10^6.5 to 10^7.4 TCID₅₀/g of tissue. Inocula titers of all the experimental groups were verified by performing a RRT-PCR of both the original non-diluted viruses and the diluted inocula.

All falcons were monitored daily for the development of clinical signs, and oropharyngeal and cloacal swabs were obtained to measure viral shedding by RRT-PCR. Besides, blood samples were collected before euthanasia to detect AI antibodies by C-ELISA testing. As it was terminal, bleeding was done from the heart after previous anesthesia with intramuscular injection of ketamine/xylazine (10 g/kg body weight, Imalgene® 1000 and 1 g/kg body weight, Xilagesic® 2%). Mortality and mean death time (MDT) were calculated. Ethically euthanized and naturally dead falcons were necropsied to evaluate gross lesions and obtain samples for pathological and molecular studies. Negative control falcons and surviving infected falcons were euthanized at the end of the experiment (11 dpi for LPAI groups and 10 dpi for HPAI groups). Falcons were euthanized using intravenous sodium pentobarbital (100 mg/kg, Dolethal®,Vétoquinol, Cedex, France). Oropharyngeal and cloacal swabs, blood samples and tissue samples for molecular studies were stored at −80° C until further use.

Necropsies and tissue sampling were performed according to a standard protocol. After fixation in 10% neutral buffered formalin and paraffin embedding, tissue sections were processed routinely for haematoxylin/eosin (H/E) staining. The following tissues were examined: esophagus, crop, proventriculus-ventriculus, duodenum, jejunum-ileum, cecum/cecal tonsil, rectum, pancreas, liver, kidney, adrenal gland, gonad, nasal turbinates, trachea, lung, heart, breast muscle, skin, bone marrow, spleen, bursa of Fabricius, thymus, brain, spinal cord and sciatic nerve.

An immunohistochemical technique based on the Avidin-biotin complex immunoperoxidase system was performed as previously described [30], [31]. The primary antibody was a mouse-derived monoclonal commercial antibody against nucleoprotein (NP) of influenza A virus (IgG2a, Hb65, ATCC). As a secondary antibody, a biotinylated goat anti-mouse IgG antibody (GaMb, Dako E0433, Glostrup, Denmark) was used. Tissues previously demonstrated to be positive against nucleoprotein of influenza A virus by IHC were used as a positive control. Tissues from sham-inoculated animals were incubated without the primary antibody and served as a negative control. The following score was used in order to grade the staining in tissues: no positive cells (−), single positive cells (+), scattered groups of positive cells (++), widespread positivity (+++).

Oropharyngeal and cloacal swabs were placed in 0.5 mL of Dulbecco's Modified Eagle's Medium (DMEM) with antibiotics. Additionally, tissue samples from trachea, lung, kidney and small intestine of H7N2 LPAI infected falcons, and from lung, kidney, duodenum/pancreas, liver and brain of H5N1 HPAI infected falcons were placed in 0.5 mL of PBS. Viral RNA was extracted with NucleoSpin® RNA Virus kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. A RRT-PCR assay was used to detect the viral M gene in Fast7500 equipment (Applied Biosystems, Foster City, CA, USA), using the primers and probe previously described [32], at a concentration of 400 nM for each primer and 300 nM for the TaqMan probe, the AgPath-ID one-step RT-PCR reagents (Applied Biosystems, Foster City, CA, USA) and 3 µL of eluted RNA in a total volume of 20 µL. The amplification conditions were as follows: reverse transcription at 48° C 10 min; initial denaturation at 95° C for 10 min and 40 PCR-cycles of 97° C for 2 sec and 61° C for 30 sec.

A C-ELISA test was carried out in order to detect antibodies against the A nucleoprotein of AI virus using the commercially available kit ID Screen® Influenza A Antibody Competition (ID-VET, Montpellier, France), performed according to the manufacturer's instructions.

Lectin histochemistry was carried out in respiratory (nasal turbinates, trachea and lung) and digestive (proventriculus, duodenum, ileum, cecum and rectum) tracts of control falcons using the lectins Maackia amurensis agglutinin II (MAAII) and Sambucus nigrans agglutinin (SNA), which show affinity for α-2,3 (avian type) and α-2,6 (human type) receptors, respectively.

Lectin histochemistry was performed using previously described procedures [33] with minor modifications. Briefly, 3 µm-thick sections were dewaxed and treated with 3% H₂O₂ in methanol to eliminate endogenous peroxidase activity, washed with TNT (0.1 M Tris HCl, 0.15 M NaCl, pH 7.5) and blocked with TNB (TNT plus blocking reagent) (Perkin Elmer, US) for 30 minutes at room temperature (RT). Tissue sections were then incubated with biotynilated SNA (10 µg/ml) and MAAII (15 µg/ml) (Vector Laboratories Inc, CA, US) in TNB at 4° C, overnight. After washing with TNT, sections were incubated with streptavidin-horse radish peroxidase (SA-HRP) 1∶100 for 1 hour, and again incubated with SA-HRP for 30 min at RT. The reaction was developed with diaminobenzidine (Sigma-Aldrich, MO, US) at RT for 30 seconds followed by counterstaining with Mayer's haematoxylin. To rule out the non-specific binding of lectins, two sequential slides were used as negative controls. One slide was pretreated with neuraminidase, which cleaves both α-2,3 and α-2,6 residues, as previously described [33], and the other was incubated with PBS instead of the lectins. Negative controls consisted of the substitution of the lectin with a TNB buffer. Human, pig and mice tissue samples were used as positive controls because of previous publications reporting the lectin pattern of staining of these species [33]–[36]. For each slide, and in order to compare receptor expression patterns among the tissues included in this study, the relative intensity of receptor expression was scored based on the percentage of cells in a section showing positivity, and was graded as: no positive cells (−), single positive cells (+), scattered groups of positive cells (++), widespread positivity (+++).

Oral viral shedding obtained from RRT-PCR was analyzed by Kruskal-Wallis test for significant differences (p<0.05) between H7N2 and H5N1 groups and between routes of infection. The statistical tests were performed using the Statistical Package for the Social Sciences for Windows Version 17.0.

There were no relevant differences between nasochoanally inoculated animals and animals challenged via the natural feeding route in both H5N1 HPAI and H7N2 LPAI groups. Falcons from both H5N1 HPAI challenged groups (groups C and D) showed first clinical signs at 5 dpi which consisted of depression, apathy, impaired respiration and, above all, slight neurological signs that in a matter of hours turned into moderate or severe, and included torticollis, head tilt, ataxia, circling, incoordination, leg/wing paralysis, opisthotonus and tremors (Movie S1). Some birds were also found recumbent and unresponsive. Following the endpoint criteria established in the ethical protocol, one falcon per group was ethically euthanized at 5 dpi. At 6 dpi, one falcon infected by feeding and showing severe neurological signs was also ethically euthanized, while at 7 dpi one falcon per group was found dead. Remaining falcons (one per group) survived until the end of the experiment at 10 dpi and did not develop clinical signs. To sum up mortality data, two out of three nasochoanally inoculated falcons, and three out of four falcons challenged by feeding died between 5 and 7 dpi, and the MDT was 6 dpi. Neither morbidity nor mortality was observed in the negative control group (group E) or in the H7N2 LPAI groups (groups A and B).

Findings associated with AI virus infection were only observed in some of the H5N1 HPAI infected falcons (groups C and D). Lesions were very similar regardless of the infection group and day of necropsy, being pancreas, proventriculus, ventriculus and brain the most affected organs. Regarding the nasochoanal group (group C), multifocal hemorrhagic necrosis in the pancreas was found in the falcon necropsied at 5 dpi (Figure 1), while the falcon found dead at 7 dpi showed multifocal petechia on the proventriculus-ventriculus junction mucosa, as well as brain and eyelid congestion. The surviving falcon from this group also showed multifocal petechia in the proventriculus-ventriculus junction mucosa at time of necropsy (10 dpi). Concerning the prey ingestion group (group D), the falcon necropsied at 5 dpi did not show significant lesions, whereas multifocal petechia on the proventriculus-ventriculus junction mucosa and brain and eyelid congestion were found in the falcon euthanized at 6 dpi. In this group, the falcon found dead at 7 dpi also showed multifocal petechia on the proventriculus-ventriculus junction mucosa and multifocal hemorrhagic necrosis in the pancreas. No gross lesions were observed in the H5N1 HPAI infected falcon of the prey ingestion group euthanized at 10 dpi.

Histological lesions and influenza A viral antigen (NP) were only observed in H5N1 HPAI challenged falcons (groups C and D) (Tables 2 and 3). Neither significant lesions nor positive IHC staining were observed in H7N2 LPAI challenged animals (groups A and B), or in the control birds. Tissues not present in Tables 2 or 3 appeared overtly normal on histopathological analysis and did not show positive IHC staining.

The most severely affected organ was the brain (Tables 2 and 3). The main findings consisted in moderate (5–6 dpi) to severe (7 dpi) multifocal areas of malacia in the cortex, present in all hemispheres of the brain, associated with spongiosis of the neuropil, chromatolysis, gliosis and caryolysis (Figure 2A). Vascular endothelial swelling was also observed, especially at 5–6 dpi. Evident severe necrosis of ependymal cells of the ventricles and epithelial cells of the choroid plexus was present in almost all falcons. The cerebellum frequently showed multifocal areas of moderate to severe chromatolysis of Purkinje neurons, sometimes associated with necrosis of the Purkinje cell layer and non-suppurative inflammatory infiltrate. Severe non-suppurative choroiditis was observed in two falcons of the prey ingestion group, particularly in the falcon found dead at 7 dpi and in the falcon euthanized at 10 dpi, as well as in the falcon found dead at 5 dpi of the nasochoanal group. Moderate multifocal areas of perivascular cuffing were present, being severe in the falcon from the prey ingestion group necropsied at 10 dpi. This falcon had the same histopathological findings as the neurologically affected falcons, whereas the falcon from the nasochoanal group necropsied at 10 dpi did not show any significant lesions in the brain. Significant microscopic lesions were seen in the spinal cord of the falcon from the nasochoanal group necropsied at 5 dpi, where severe malacia in the grey matter, and severe gliosis and necrosis of the ependym and neuropil surrounding the medullary canal were observed. Interestingly, almost all lung tissues showed positive IHC staining in bronchi, in particular bronchial epithelial cells and goblet cells. In general, antigenic staining was mainly nuclear and also often cytoplasmic in distribution and correlated well with histopathological findings (Figure 2B).

Real time RT-PCR was performed on oropharyngeal and cloacal swabs, and on various tissues obtained at necropsy. In both H7N2 LPAI-infected groups (groups A and B) (Figure 3A), viral RNA was only detected on oropharyngeal swabs. Regarding H7N2 LPAI prey-infected falcons (group B) the amount of viral RNA detected orally was consistent and reached minimum cycle of threshold (Ct) values of 22.29 at 2 dpi. Besides, viral RNA was already detected at 1 dpi in one animal, with a Ct value of 37.31. H7N2 LPAI viral RNA was detected in all falcons until the end of the experiment (11 dpi), although amounts of viral RNA were evidently declining. Regarding falcons inoculated via the nasochoanal route (group A), detection was similar to that observed in the feeding group, although quantities of viral RNA declined more rapidly. No viral RNA was detected in swabs from the contact animal during the whole experiment.

For H5N1 HPAI infected falcons (groups C and D) (Figure 3B), viral RNA detection had a different profile than for that of H7N2 LPAI infected animals. Regarding oropharyngeal swabs, both nasochoanally and feeding infected falcons showed viral RNA from 1 dpi until prior to death, peaking at 2 dpi (Ct 24.08). Falcons that survived until the end of the experiment (10 dpi) stopped shedding at 7 dpi. In contrast with H7N2 LPAI falcons, H5N1 HPAI infected animals showed some viral RNA detection in cloacal swabs. In particular, the falcon from the feeding group that was euthanized at 5 dpi showed low amounts of virus (Ct 33.15) at the time of necropsy, as well as two falcons from the nasochoanal group: the one that was euthanized at 5 dpi showed virus from 3 to 5 dpi (Ct 33.77, Ct 37.96 and Ct 29.21), and the one found dead at 7 dpi had a Ct value of 31.51 at 5 dpi.

Results of viral detection by RRT-PCR from the tissues are shown in Table 4. In general, all falcons that showed clinical signs and died, or were ethically euthanized had positive results for almost all the selected tissues. The highest amounts of viral RNA were detected in the brain. The surviving falcon's brain from the feeding group was positive for RRT-PCR.

The statistical analysis performed on the results of the oropharyngeal swabs until 7 dpi (both individual and mean Ct) showed no significant differences between routes of infection for both H5N1 HPAI and H7N2 LPAI groups (p>0.05), whereas amounts of viral RNA of H7N2 LPAI groups were significantly higher than in H5N1 HPAI groups (p<0.05).

With the exception of the contact bird, all H7N2 LPAI infected falcons were seropositive at the end of the experiment (11 dpi). Concerning H5N1 HPAI infected falcons, only serum samples from 10 dpi were seropositive. No seroconversion was observed in the negative control falcons (group E).

Lectin immunohistochemistry was carried out in the respiratory and digestive tracts of control falcons in order to assess the distribution pattern of SNA (α-2,6) and MAAII (α-2,3). Regarding respiratory tract (Figure 4), moderate expression of α-2,6 was observed in ciliated epithelial cells and mucous gland cells of the respiratory tract of nasal turbinates and in salivary gland epithelium. Mild expression of α-2,6 receptors was observed in ciliated epithelial cells, mucous gland epithelium and goblet cells of the trachea. However, expression of receptors was predominantly α-2,3 (avian type) on the respiratory tract, being as follows: strong in bronchial epithelial cells; moderate in ciliated epithelial cells and goblet cells of the trachea; and mild in ciliated epithelial cells of the respiratory tract of nasal turbinates and in nasal gland epithelium, and in mucous gland epithelium of the trachea. Regarding digestive tract (Figure 5), strong α-2,3 expression was noted in enterocytes of the rectum, and mild α-2,3 expression was observed in goblet cells of the same region. Other cell types, such as endothelial cells, macrophages and lymphocytes, gave mild positive results for both α-2,3 and α-2,6 receptors in the cecum/cecal tonsil and rectum.