Thirty newly-hatched chicks were collected from poultry farms selected at random in Indonesia. We confirmed that these birds had no history of vaccination with avian influenza vaccine. In addition, specific-pathogen-free (SPF) chicks were purchased and used for experiments as controls. They were reared under conditions of water and feed provided ad libitum in BSL-3 facility, and blood was sampled at 10 days of age. The serum titer of antibodies against A/H5N1 antigens was measured by an enzyme-linked immunosorbent assay (ELISA).

The antibody titer was measured by an ELISA according to our previous report [13] . A/H5N1 antigens (0.2 µg) (Protein Science, USA) in phosphate-buffered saline (PBS) were coated onto each well of 96-well ELISA plates and stored overnight at 4˚C. Each of the subsequent incubation steps was preceded by sufficient washing the wells with 0.05% Tween 20 in PBS. The wells were blocked from nonspecific binding by the addition of a commercial blocking reagent (DS Pharma Biomedical, Japan) and incubated at 37˚C for 2 h. The serial dilutions of subject serum from each chick were added vertically to the wells and incubated at 37˚C for 1 h. The serum from SPF chicks was also used as a negative control. Next, HRP-conjugated rabbit IgG against chicken IgY Fc fraction (Nakarai Tesque, Japan) diluted in PBS (1:5000) was dispensed into each well. The plate was incubated for 1 h at 37˚C. Later, a substrate buffer containing TMB (Sumitomo Bakelite, Japan) was added to each well, and samples were incubated at 37˚C for 15 min. The reaction was terminated by the addition of a stopping reagent (1.25 M sulfuric acid). The absorbance was recorded at 450 nm using the ELISA plate reader. The ELISA titers were defined as the reciprocal of the highest dilution of subject serum that produced an ELISA signal twice as intense as that from equivalently diluted SPF chick serum.

At 10 days of age, seventy-two chicks from farm and ten SPF chicks were inoculated intranasally with a field-isolated strain of A/H5N1 virus (A/LAWANG/FKH-IPB/H5N1) at a dose of 10⁵TCID₅₀ per bird. This virus strain was isolated from a chicken spontaneously infected with H5N1 in a local poultry farm in the Bogor area of Indonesia. Five days after the viral inoculation, the lethality of infected birds was scored as the percentage of dead birds. The surviving chickens were sacrificed by administration of overdose pentobarbital sodium solution. The tracheae and lungs of subjects were removed and immersed in 10% neutral buffered formalin for further histopathology and immunostaining studies. 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. After completion of the experiment, all samples were completely sterilized by autoclave.

The formalin fixed samples were dehydrated with serial concentrations of ethanol and embedded with xylene and paraffin using an automatic embedding processor. The sections were then cut using a microtome and attached to slide-glasses. The sections were dried and de-paraffinized inxylene and ethanol and then stained with hematoxylin and eosin. They were next serial-dehydrated with ethanol and immersed in xylene before finally being mounted with cover-glass using Canada balsam and examined under a light microscope.

The chick organs fixed in buffered formalin were washed in PBS, cut into 5-mm cubes, and soaked in 30% sucrose in PBS overnight. The 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 an FITC conjugated ostrich IgY against A/H5N1 viruses (1:1000) at 4˚C overnight [13] [14] [15] . Finally, they were mounted with glycerol following sufficient washing with PBS, and specific signals for viral antigens were examined under a fluorescent microscope.

The immunoreactivity of subject sera against A/H5N1 virus was estimated by ELISA. The titers for A/H5N1 varied among the chicks, as shown in Figure 1. The antibody titer in chicks’ blood was 102,400 at the maximum and 1600 at the minimum, and there were individual differences. This suggested that the amount and activity of transitional antibody from the mother differed among individual chicks.

The experimental challenge in SPF chickens (10 days old) with A/H5N1 caused high lethality among the birds (100%) within 5 days after infection, indicating that this viral strain isolated from poultry farm was a highly virulent type (Figure 2). Of note, all SPF birds showed only slight symptoms, including dehydration and depression, followed by sudden death. In contrast, a large individual

difference was observed in the fatality rate of the infected chicks purchased from the poultry farm. The mortality rate due to virus infection was quite high in chicks with low antibody titers in blood, whereas the birds with high serum antibody titers showed low mortality. Furthermore, 100% of birds with an ELISA value of 102,400 survived despite virus infection. We therefore concluded that viral infection could be suppressed in individuals with high transitional antibody titers.

The histopathological study of the infected SPF chickens showed the typical avian flu findings of acute inflammation accompanied by heterophilic infiltration, hemorrhaging, edema and severe congestion in various organs, mainly in the pulmonary tissues, including the trachea and lung (Figure 3). Pathological findings similar to SPF chickens were observed in dead chickens or infected chickens with low ELISA values (≤25,600). The pulmonary sections of infected birds showed slight to moderate inflammation with epithelial necrosis and heterophilic infiltrations in the interstitium and parabronchial cavities. Severe hemorrhaging and congestion, accompanied by edema and mucosal exudates, were predominately seen. In contrast, the pathological reactions were decreased in the tracheae and lungs in survival chicks with an ELISA value exceeding 51,200. 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 the results showing that the mortality of A/H5N1 chicks was decreased by higher titer of migratory antibodies.

Viral antigens were found in the tracheal epithelial cells and pulmonary tissues of dead chickens or infected chickens with low ELISA values (≤25,600). In contrast, viral antigens were scarcely seen in the pulmonary tissues of in surviving chicks showing ELISA values exceeding 51,200 (Figure 4).