Adult male chickens (White Leghorns) with no vaccination history were used to produce antiserum against avian influenza virus. At our laboratory, an inactivated Indonesian vaccine strain of A/H5N1 antigen (50PD₅₀/bird) was mixed with an adjuvant and inoculated into the pectoral muscle of the chicken. Four weeks later, blood samples were collected from the chicken, and the serum was separated by centrifugation. Antibody titers of the obtained antiserum were measured by an enzyme-linked immunosorbent assay (ELISA). Furthermore, the neutralizing activity against avian influenza virus was scored by a hemagglutination inhibition (HI) test using erythrocytes [20] .

Based on our previous papers, binding ability of the chicken antisera to the viral antigen was measured by ELISA [20] . Each well of 96-well ELISA plates (Sumitomo Bakelite, Japan) was coated with antigens of pandemic influenza virus A/H1N1 (A/California/9/2009 (H1N1) pdm09) or avian A/H5N1 virus in phosphate-buffered saline (PBS), and the ELISA plate was stored overnight at 4˚C. After washing the wells twice with PBS containing 0.05% Tween 20, each of the subsequent incubation steps were performed. A commercial blocking buffer (DS Pharma Biomedical, Japan) were added into the wells and incubated at 37˚C for 2 h. Serial dilutions of preimmune or antiserum were added vertically to the wells and incubated at 37˚C for 1 h. Then, horse radish peroxidase (HRP)-conjugated secondary antibody (rabbit IgG against chicken IgY Fc fraction) (Nakarai Tesque, Japan) diluted in PBS (1:5000) was dispensed into each well, and incubated for 1 h at 37˚C. Next, a substrate buffer containing TMB (Sumitomo Bakelite, Japan) was added to each well, and samples were incubated at 37˚C for 15 min. The color development reaction was stopped by the addition of a stopping reagent (1.25 M sulfuric acid). The absorbance was measured at 450 nm using the ELISA plate reader.

Whole blood from guinea pigs was washed with PBS, and the erythrocytes were prepared after centrifugation. Serial 2-fold dilutions of pandemic influenza virus A/H1N1 or A/H5N1 viruses were mixed with erythrocyte solutions in clear 96-well micro test polystyrene assay plates (Becton Dickinson, USA). After 45 minutes at room temperature, the hemaggregation activity was observed, and the HA titers of the virus were measured. The highest dilutions (indicating clear hemaggregation) were scored as HA titers [20] .

Serial dilutions of preimmune or antiserum were mixed with 8-HA units of the influenza viruses in clear 96-well micro test polystyrene assay plates (Becton Dickinson, USA). After 30-minute incubation at room temperature, erythrocytes were added, pipetted gently, and then incubated for another 45 minutes at room temperature. The hemaggregations in each well were observed, and the HI titers were scored based on the HA titer with preimmune serum/HA titer with antiserum (a higher ratio indicates a stronger inhibitory activity of the antiserum against the pandemic influenza virus) [20] .

Serial dilutions of antiserum were mixed with pandemic influenza viruses A/H1N1 (A/Osaka/47/2009 (H1N1) pdm) or avian A/H5N1 (A/Bogor 2/FKH-IPB/2008 (H5N1)) at 10² TCID₅₀ and incubated for 1 h at 37˚C. Thereafter, these viral fluids were adsorbed onto MDCK monolayers for 1 h at 35˚C, and then incubated in GIT media (Nakarai Tesque, Japan) containing low trypsin at 35˚C. At 5 days post-incubation, the cultures were inspected for the cytopathic effect (CPE): the neutralizing titer, expressed as the reciprocal of antiserum dilution at which virus growth is 50% inhibited, was calculated by the number of virus-negative wells and the serum dilution according to the report by Reed et al. [21] .

Specific-pathogen-free (SPF) chicks were housed under controlled conditions in a BSL3 laboratory and received food and water ad libitum. At 10 days old, the birds were inoculated intranasally with A/H5N1 (A/Bogor 2/FKH-IPB/2008 (H5N1)) virus (10⁵ TCID₅₀/ml) [22] [23] [24] . Some chicks were injected intramuscularly with antiserum (8, 400, 2000 mg/kg body weight) at 2 h prior to infection, and all birds were housed for another 3 days. A higher volume of preimmune serum (2000 mg/kg) was administered to some birds as controls. At least 10 birds were prepared for each volume of the sera. At 3 days after inoculation, the dead birds were counted, and the survival rate was scored in each group (surviving birds/all birds examined). The survivors were sacrificed with pentobarbital sodium solution, and the tracheae and lungs were removed and immersed in 10% neutral buffered formalin for further histopathology and immunohistochemistry studies.

For the histopathology studies, paraffin sections were cut at 3-μm with a microtome and stained with hematoxylin and eosin (H & E) per routine procedures and then observed under a light microscope.

All of the animal experiments were performed in accordance with the guidelines for studies with laboratory animals of the Kyoto Prefectural University Experimental Animal Committee.

Tracheae and lungs were fixed in buffered formalin and washed in PBS. The organs were soaked in 30% sucrose in PBS overnight. The organ pieces were mounted in a compound, frozen and cut into 20-μm sections with a cryostat. The frozen sections were attached to glass slides and air-dried at room temperature. After being washed in PBS, the samples were incubated with a FITC-conjugated ostrich IgY against A/H5N1 viruses (1:1000) at 4˚C overnight. Finally, they were mounted with glycerol following sufficient washing with PBS, and specific signals for viral antigens were examined under a fluorescent microscope [24] .

The immunoreactivities of antisera against A/H5N1 virus were estimated by ELISA. The titers for A/H5N1 were dramatically increased in the serum at four weeks after the initial immunization (Table 1). In contrast, the antisera bound only slightly to A/H1N1 viral antigen. These findings indicate that specific antibodies were generated and transmitted into the bloodstream.

First, the HA activities of the pandemic influenza A/H1N1 and avian A/H5N1 viruses were estimated using erythrocytes, since the viral strains in this study originated from sporadic cases of infection and their characteristics have not yet been clarified. Fortunately, both strains caused strong aggregations of erythrocytes. The highest dilutions of viral fluids showing hemaggregation were scored as a single HA unit, and both strains were used for further HI testing at 8 HAU. Hemaggregation by A/H5N1virus was dramatically inhibited by the antiserum whereas that by A/H1N1 was not inhibited (Table 2).

The antibody titer against pandemic influenza A/H1N1 (A/Osaka/47/2009 (H1N1) pdm) and avian A/H5N1 (A/Bogor 2/FKH-IPB/2008 (H5N1)) viruses was estimated by ELISA. At 4 weeks post-immunization, the ELISA titers against A/H5N1 were clearly increased in chicken antiserum. In contrast, the reactivity against A/H1N1 virus did not increase in the serum. The ELISA titers were defined as the reciprocal of the highest dilution of antiserum that produced an ELISA signal twice as intense as the signal from equivalently diluted preimmune serum.

Purified erythrocytes were reacted with a pandemic influenza virus A/H1N1 (A/Osaka/47/2009 (H1N1) pdm) or an avian influenza virus A/H5N1 (A/Bogor 2/FKH-IPB/2008 (H5N1)) after incubation with antisera. The activity of antisera against each of the viruses was represented as HI titer ratios (HA titer with preimmune serum/HA titer with antiserum), with higher HI titers indicating a strong inhibitory activity of the antiserum against aggregation by the viruses. Note that the antiserum against A/H5N1virus shows inhibitory activities on the hemaggregation by A/H5N1 virus.

The pandemic influenza virus A/H1N1 and avian influenza virus A/H5N1 were reacted with antiserum followed by inoculation into MDCK cells. As shown in Table 3, the cytopathic effects of MDCK cells infected with A/H5N1 were inhibited by the antiserum: the serum volume at 50% inhibition of A/H5N1 virus infection was much lower than that of A/H1N1 infection. These findings indicated that antiserum had neutralization activity against infection of A/H5N1 virus.

The experimental challenge in living SPF chickens (10 days old) with A/H5N1 (A/Bogor 2/FKH-IPB/2008 (H5N1)) caused high lethality among birds (100%) within three days post-infection, indicating that this virus was a highly virulent type. Of note, all birds showed only slight symptoms, including dehydration and depression, followed by sudden death. In addition, all birds injected with preimmune serum died within three days after viral infection (Figure 2). A histopathological study of the infected chickens showed typical avian flu findings of acute inflammation accompanied by heterophilic infiltration, hemorrhage, edema and severe congestion in various organs, mainly in the pulmonary tissues, including the trachea and lung (Figure 3). The respiratory sections of infected birds showed slight-to-moderate inflammation with epithelial necrosis and

The neutralization assays were performed using MDCK infected with pandemic influenza A/H1N1 virus (A/Osaka/47/2009 (H1N1) pdm) or avian A/H5N1 virus (A/Bogor 2/FKH-IPB/2008 (H5N1)). The neutralizating titers are indicated as the mean of 50% inhibition on CPE at 5-days post infection.

heterophilic infiltration in the interstitium and parabronchial cavities. Severe hemorrhaging and congestion, accompanied by edema and mucosal exudates, were predominately seen. The viral antigens were found in the tracheal epithelial cells and pulmonary tissues of infected chicks at three days post-inoculation.

The antiserum was directly administered to A/H5N1-infected chicks to confirm the therapeutic and protective effects on avian flu infections. Interestingly, the survival rate of the infected birds was dramatically increased by an injection of antiserum (Figure 2). In addition, the pathological reactions in the infected tracheae and lungs in infected chicks were decreased by the injection of antiserum (Figure 3). The edema, hemorrhaging, congestion and mucosal exudation were clearly inhibited in the lesions. Viral antigens were scarcely found in the trachea and pulmonary tissues. These findings were consistent with results showing that the mortality of A/H5N1 chicks was decreased by antiserum administration. Accordingly, antiserum injection at a high volume was able to rescue all birds from death following A/H5N1 infection.