Virus.
Clade 2.3.4.4e HPAIV Tottori/1 was used for experimental infection of ducks. The strain was isolated from a fecal sample of teal in Tottori City located in the Midwestern region of Japan [44]. The virus was categorized into the C2 group of the clade 2.3.4.4e, which has the most popular gene constellation in the 2016–2017 outbreak in Japan [9]. The accession numbers of the gene sequences were LC199865–199872. Virus was propagated in 10-day-old chicken embryos (Aoki Breeder Farm, Tochigi, Japan) for 48 h at 35°C. After the incubation period, eggs were chilled at 4°C for 12 h. The allantoic fluid was harvested and stored as virus stock at −80°C.
Birds.
Ten each of three Anatidae species (Eurasian wigeon, Mallard, and Northern pintail) were captured at Togo and Koyama Ponds in Tottori Prefecture in Japan, with the approval of Tottori Prefecture (permission numbers: 201800217771 and 201900216410) in two consecutive winter seasons, January and December 2019 (see Additional file 1). Their ages (juvenile or adult) were identified by feather growth and molt. The pharyngolaryngeal, cloacal, and conjuntival swabs were collected and examined for influenza A virus antigen via rapid diagnostic kits (ESPLINE INFLUENZA A & B-N, Fujirebio Inc., Tokyo, Japan) and/or for virus isolation by egg inoculation. Blood was also collected, and subsequently checked for specific serum antibody against the challenge virus, Tottori/1, by HI testing [45]. These sera were also assayed for influenza A subtype viruses using a cELISA kit (IDEXX Influenza A Ab Test, IDEXX laboratories, ME, USA). The ducks were house at Tottori University for a maximum of 5 weeks. The ducks were grouped according to the following criteria: serum HI titers under the detection limit (<2 HI) were preferentially applied to 106 EID50 inoculation group as stated below; cELISA-positive ducks were impartially distributed to each group as possible; their age (adult/juvenile) and sex were also taken into consideration for grouping to reduce biases.
Experimental design.
Seven of each duck were intranasally inoculated with 200 µL of allantoic fluid containing the Tottori/1 at 106, 104, or 102 EID50, then observed for clinical signs at 24-hour intervals for 10 days. Pharyngolaryngeal and cloacal swabs were collected at 1, 2, 3, 5, 7, and 10 dpi to assess viral shedding. The swabs were collected in 2 mL of nutrient broth medium (Nissui Pharmaceutical, Tokyo, Japan) with 10 mg of streptomycin sulfate (Meiji Seika Pharma, Tokyo, Japan) and 1 × 104 units of penicillin G (Meiji Seika Pharma). At the end of the 10-day period, the ducks were also checked for specific antibodies against the challenge virus in serum by HI testing. The surviving birds were euthanized using isoflurane (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan) after collection of conjunctival swabs and blood at 10 dpi, and their tissues (brain, trachea, breast muscle, lung, liver, pancreas, spleen, heart, kidney, colon, eyeball, and wing shaft) were sampled for virus isolation and histopathological study, as described below. The remaining three of each duck species were intranasally inoculated with 200 µL of allantoic fluid containing the virus at 106 EID50, then euthanized at 3 dpi. The samples were collected in the same manner as above.
Portions of the tissue samples were homogenized using a Multi-Bead Shocker (Micro SmashTM MS-100R, Tomy Seiko, Tokyo, Japan) at 3,000 rpm for 30 s to create a 10% (weight/volume) organ emulsion in nutrient broth medium with antibiotics. Samples serially 10-fold diluted in phosphate buffered saline with streptomycin sulfate and penicillin G were inoculated into 10-day-old chicken embryos. Eggs were incubated at 35°C for 48 hours. Hemagglutination (HA) testing [46] was then performed using allantoic fluid, and the EID50 was calculated using the Reed and Müench method [47]. The sampled tissues were also subjected to histopathological analysis. Tissues fixed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Corporation) were processed according to routine methods, then embedded in paraffin wax. Sections were stained with haematoxylin and eosin for histopathological examination. Immunohistochemical staining was also performed using antigen retrieval solution, 0.05% citraconic anhydride, pH 7.4 (Immunosaver; Nissin EM, Tokyo, Japan), mouse anti-influenza A virus matrix protein monoclonal antibody (clone GA2B; Serotec Ltd., Oxford, UK), and the Simple Stain MAX-PO (M) kit (Nichirei Bioscience Inc., Tokyo, Japan), in accordance with the manufacturers’ instructions.
Ethics statement.
All animal experiments were conducted in self-contained isolator units (CLEA Japan, Tokyo) in a biosafety level 3 laboratory at the Avian Zoonosis Research Center, Tottori University, Japan. The experiments were approved by the Ethics Committee of Tottori University and performed in accordance with the guidelines of the institutional animal care and use committee of Tottori University (approval number 19-T-03). The study protocol stipulated that throughout the study any birds that became unable to eat or drink were to be euthanized, and recorded as dead at the time of the following day’s observation.