During February and March in 2019, an epidemic of pneumoenteritis in cattle occurred on a dairy farm in Kyoto, Japan. In this farm, a 20-month-old Holstein-Friesian calf (#1) exhibited typical symptoms, including loss of appetite, slight fever (40.1oC), dyspnea with cough, and conjunctival hyperemia. This and other affected animals were treated with antibiotics but recovered very slowly, therefore, viral agents were suspected to be involved in the disease. Clinical samples, including sera, nasal swabs, and stools were collected from the animals, and referred to the Chutan Livestock Hygiene Service Center for viral and bacterial examination. The clinical condition of calf #1 had improved two weeks after the onset.
Viral isolation and bacterial culture
Approximately 1 g of fecal samples was homogenized in 9 mL of maintenance medium (MM), minimal essential medium (MEM) containing 10 mg/ml gentamicin (Takata Pharmaceutical, Saitama, Japan), and centrifuged at 3,000 rpm for 20 min. The supernatant was filtrated through a 0.45 mm cellulose acetate filter membrane (Advantec, Tokyo, Japan) and stored at -80oC until use. Madin-Darby bovine kidney (MDBK) cells were used for virus isolation. Cells and all chemicals used in this study were checked beforehand by RT-PCR  and all were pestivirus-free. Fecal homogenates were inoculated into MDBK cells. The inoculated cells were incubated at 37oC, 5% CO2 for 1 h, then MM was added and the cells were maintained in the same culture conditions. The cells were checked daily using an inverted microscope (Nikon, Tokyo, Japan) and monitored for the appearance of cytopathic effects (CPE). When 80% of the cells showed CPE, the cell culture was harvested, vortexed vigorously, and centrifuged at 3,000 rpm for 10 min to remove cellular debris. The supernatants were stored at -80oC.
The fecal homogenates were also inoculated onto blood agar (supplemented with 5% horse blood) and deoxycholate-hydrogen sulfide-lactose agar and incubated aerobically at 37oC for 1-2 days.
Transmission electron microscopic analysis
For transmission electron microscopy, infected MDBK cells were detached from the petri dish using a rubber policeman. The cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4), pelleted by centrifugation, post fixed with 1% osmium tetroxide in 0.1 M PB, dehydrated in graded ethanol, and embedded in LUVEAK-812 resin (Nacarai Tesque Inc., Kyoto, Japan). Ultrathin sections were stained with uranyl acetate and lead solutions and observed using a JEM-1400Flash transmission electron microscope (Jeol Ltd., Tokyo, Japan).
Total DNA and RNA were extracted simultaneously from the fecal homogenates and cell culture supernatants using a MagDEA Dx SV magtraction reagents (Precision System Science Co. Ltd., Matsudo, Japan) according to the manufacturer’s instructions. A nested PCR assay was conducted to detect the BAdV hexon gene, as described previously . The amplicons were gel purified using a QIAquick Gel Extraction kit (Qiagen) and direct sequencing was performed with forward and reverse primers using an ABI PRISM Big Dye Terminator cycle sequencing kit (Applied Biosystems) and an ABI 3130 genetic analyzer (Applied Biosystems). The nucleotide sequences of both strands were obtained for verification and the finalized sequences were used for a BLAST search. Other respiratory viruses, including bovine viral diarrhea virus (BVDV), bovine infectious rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza 3 virus (BPI3V), and bovine coronavirus (BCoV), were also investigated by PCR or RT-PCR, as described previously [16-19].
Complete genome sequencing and phylogenetic analysis
The infected cell culture supernatants were collected six days after virus inoculation and centrifuged to remove cellular debris. The stocks were further semi-purified by discontinuous sucrose density gradient ultra-centrifugation and dissolved in phosphate-buffered saline (pH 7.4). DNA was extracted using a QIAamp MinElute Virus Spin kit (Qiagen) and submitted to Macrogen Japan Co. Ltd (Tokyo, Japan) for whole-genome sequencing. Briefly, sequence libraries were constructed using a Nextera XT DNA library preparation kit (Illumina, Tokyo, Japan). DNA sequencing was performed with a deep sequencing protocol using Novaseq 6000 (Illumina). De novo assembly of the resultant reads and determination of 5’ and 3’ terminal sequences were performed as described previously . The sequences were aligned using a Muscle program available in MEGA X (https://www.megasoftware.net/) with reference adenovirus strains retrieved from GenBank. Phylogenetic trees were constructed using MEGA X with maximum-likelihood methods and 1,000 bootstrap replicates.
Virus neutralization tests
Calves and cows from the 36 dairy farms were selected randomly for sampling. A total of 1,325 serum samples and the isolated virus were used for virus neutralization (VN) tests. Serial two-fold dilutions of sera, heat-inactivated at 56oC for 30 min, were incubated in 96-well tissue culture plates with an equal volume of a viral suspension containing 200 median tissue culture infective doses (TCID50)/0.05 ml of the isolate at 37°C for 1 h. An MDBK cell suspension containing 1.2 × 105 cells/ml was added with 0.1 ml of each serum-virus mixture, and the mixtures were incubated at 37°C for 7 days. VN antibody titers are expressed as the reciprocal of the highest dilution of serum that inhibited CPE completely.