Advance Publication The Journal of Veterinary Medical Science Accepted Date: 7 July 2020 J-STAGE Advance Published Date: 17 July 2020 ©2020 The Japanese Society of Veterinary Science Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Pathology 2 NOTE 3 4 Inclusion body hepatitis caused by Aviadenovirus in a tropical 5 screech owl (Megascops choliba) 6 7 Tetsuya KOMATSU1), Takashi KUBO1), Rena KITOU1), Naomi 8 KAWAMOTO1), Masaji MASE2), Yu YAMAMOTO3) and 9 Tomoyuki SHIBAHARA3, 4)* 10 11 1) Aichi Prefectural Chuo Livestock Hygiene Service Center, 1-306 12 Jizono, Miaicho, Okazaki, Aichi 444-0805, Japan 13 2) Division of Viral Disease and Epidemiology Research, National 14 Institute of Animal Health, National Agriculture and Food 15 Research Organization (NARO), 3-1-5 Kannondai, Tsukuba, 16 Ibaraki 305-0856, Japan 17 3) Division of Pathology and Pathophysiology, National Institute of 18 Animal Health, National Agriculture and Food Research 19 Organization (NARO), 3-1-5 Kannondai, Tsukuba, Ibaraki 20 305-0856, Japan 21 4) Department of Veterinary Science, Graduate School of Life and 22 Environmental Sciences, Osaka Prefecture University, 1-58 23 Rinku-oraikita, Izumisano, Osaka 598-8531, Japan 24 1 25 *Correspondence to: SHIBAHARA, T., Division of Pathology and 26 Pathophysiology, National Institute of Animal Health, National 27 Agriculture and Food Research Organization (NARO), 3-1-5 28 Kannondai, Tsukuba, Ibaraki 305-0856, Japan 29 Fax: 029-838-7774 30 E-mail: [email protected] 31 Running head: AVIADENOVIRUS INFECTION IN AN OWL 32 33 2 34 ABSTRACT 35 In 2016, tropical screech owl (Megascops choliba) (Tso) chicks were 36 suddenly found dead in a Japanese breeding facility. We autopsied a 37 9-day-old Tso and discovered white spots scattered on the liver 38 surface. Multifocal necrosis was diffused, and macrophages had 39 infiltrated the necrotic hepatic lesions. Hepatocytes contained 40 numerous intranuclear inclusion bodies. Immunohistochemical 41 staining detected Adenovirus antigen only in the liver. Next, PCR and 42 sequencing (LC536616) identified Tso Adenovirus (TsoAd). Basic 43 Local Alignment Search Tool (BLAST) and phylogenic analyzes 44 suggested TsoAd is an owl Aviadenovirus. Our study contributes to an 45 improved understanding of infectious disease among captive raptors. 46 47 KEY WORDS: Aviadenovirus, inclusion body hepatitis, Japan, 48 tropical screech owl 49 3 50 Adenoviruses infect numerous species of mammals, birds, 51 reptiles, and amphibians [6]. Adenoviridae is divided into five 52 groups, with Aviadenovirus and Atadenovirus being 53 infectious-disease agents in birds [6]. 54 Aviadenovirus is a major cause of inclusion body hepatitis 55 [12], gizzard erosion, and ulceration [4] in chickens. Inclusion body 56 hepatitis has high mortality [9], and outbreaks have been reported 57 worldwide [19], including in Japan [17]. Although inclusion body 58 hepatitis caused by Aviadenovirus infection has been observed in 59 birds other than chicken, including turkey, duck [26], racing pigeon 60 [8], and Gambel's quail [1], it is limited in raptors, except in falcon 61 [2, 15, 20, 21, 23]. 62 The tropical screech owl (Megascops choliba) is a common 63 raptor occasionally kept as a pet. Endemic to South America, pet 64 screech owls are typically imported to Japan, although some are bred 65 domestically. Although this species is widespread, we know little 66 regarding infectious diseases that affect them, especially in breeding 67 facilities. 68 In this study, we report the detection of inclusion body 69 hepatitis in the tropical screech owl, caused by an owl Aviadenovirus. 70 Our research aims to improve current understanding of owl infectious 71 disease in captive conditions, using pathological and phylogenetic 72 data. 73 From March to May 2016, numerous tropical screech owls (age 4 74 range 1–20 days) in a breeding facility were presented anorexia, 75 vomiting, diarrhea, and sudden mortality. Owls were treated with 76 toltrazuril as an anticoccidial agent and manually fed. Most birds 77 died within one week. The facility houses other raptors, such as 78 Eurasian eagle owl (Bubo bubo), rock eagle owl (Bubo bengalensis), 79 barn owl (Tyto alba), and several falcon species. Most were imported, 80 with some individuals used for breeding. Juvenile screech owls were 81 housed in independent cages that caretakers disinfected with chlorine 82 once a day. Their diet was a commercial feed comprising minced 83 chicks of chicken and Japanese quail. 84 A 9-day-old tropical screech owl that died suddenly without 85 external symptoms was moved to Aichi Prefectural Chuo Livestock 86 Hygiene Service Center by a veterinary hospital and was autopsied 87 (Fig. 1a). We observed white spots scattered across the liver surface 88 (Fig. 1b) and hemorrhaging in the gizzard. No other gross lesions 89 were detected. Intestinal contents did not contain coccidian oocysts. 90 At autopsy, the liver, kidney, heart, lung, pancreas, trachea, 91 stomach, intestine, and brain tissue samples were fixed in 10% 92 neutral-buffered formalin. Fixed tissues were then embedded in 93 paraffin wax, sectioned (~3 µm), then stained with hematoxylin and 94 eosin (H&E) for histological examination. Gram staining was also 95 performed on the liver, kidney, and gizzard sections. For 96 immunohistochemical examination, formalin-fixed 97 paraffin-embedded (FFPE) sections of the liver, gizzard, pancreas, 5 98 and intestines were deparaffinized and incubated with 3% hydrogen 99 peroxide in methanol solution to suppress endogenous peroxidase 100 activity. Antigen retrieval was conducted using 0.1% actinase E 101 solution in phosphate buffered saline at 37°C for 20 min. After 102 adding 10% normal goat serum to block non-specific reactions, 103 sections were incubated with rabbit anti-Aviadenovirus antibody [18] 104 for 1 hr. Sections were incubated with secondary antibody (Histofine 105 Simple Stain MAX-PO Multi; Nichirei Bioscience Inc., Tokyo, Japan) 106 for 30 min at room temperature, then treated with aminoethyl 107 carbazole (AEC) substrate solution (Histofine Simple Stain AEC 108 solution; Nichirei Bioscience Inc.), also at room temperature. Finally, 109 sections were counterstained with hematoxylin. 110 To identify the virus associated with intranuclear inclusion 111 bodies, formalin-fixed liver tissue was observed under transmission 112 electron microscopy (TEM) (JEM-1400 Flash; JOEL, Tokyo, Japan), 113 as described previously [14]. 114 Histopathological examination revealed that multifocal 115 necrosis was randomly diffused in the liver (Fig. 1c). Macrophages 116 had infiltrated into necrotic lesions, and we also observed a few 117 granulomatous necrotizing lesions. Hepatocytes in the surrounding 118 necrotic areas contained numerous full, basophilic, and Cowdry type 119 A, eosinophilic intranuclear inclusion bodies (Fig. 1d). Kidney 120 interstitium exhibited infiltration of lymphocytes and a few 121 macrophages. Gizzard surface displayed hemorrhaging and heterophil 6 122 infiltration. Gram staining revealed a few gram-positive bacteria on 123 the surface of the gizzard although no bacteria were detected in the 124 liver and kidney. 125 Immunohistochemical staining revealed positive reactions in 126 hepatocyte nuclei and infiltrated macrophage cytoplasm (Fig. 1e). We 127 did not observe reactions in other tissues. 128 TEM observation revealed that hepatocyte nuclei had 129 deformed membranes and contained nonenveloped isometric viral 130 particles of approximately 90 nm in size (Fig. 1f). 131 Next, we conducted PCR on FFPE sections to investigate 132 viruses associated with inclusion body hepatitis. Genomic DNA was 133 extracted from liver sections using the QIAamp DNA FFPE Tissue kit 134 (Qiagen GmbH, Hilden, Germany), following manufacturer protocol. 135 Primers used targeted those encoding hexon [11], DNA-dependent 136 Adenovirus DNA polymerase [25], and Herpesvirus DNA polymerase 137 [24]; only Adenovirus DNA polymerase was amplified (516 bp). 138 Amplicons of the first degenerate PCR were sequenced. Basic Local 139 Alignment Search Tool (BLAST) searches of the National Center for 140 Biotechnology Information (NCBI) revealed that partial nucleotide 141 and amino acid sequences exhibited the highest similarity with those 142 of fowl Aviadenovirus E (accession number: MK572855) (74.61%) 143 and fowl Aviadenovirus A (accession number: APP94056) (86.63%). 144 This result implies that the tropical screech owl Adenovirus (TsoAd) 145 that we detected possibly represents a novel Aviadenovirus because 7 146 the novelty threshold for an Aviadenovirus is a 5%–15% sequence gap 147 in the DNA-dependent DNA polymerase amino acid sequence [5]. 148 However, the full-length sequence of DNA polymerase, genome 149 organization, cross-neutralization, and other characteristics should 150 be investigated for greater certainty [5]. The TsoAd sequence was 151 deposited to the DNA Data Bank of Japan (accession number: 152 LC536616). 153 We performed a phylogenetic analysis of the TsoAd amino acid 154 sequence with the other aviadenoviruses. Sequences included were 155 those of fowl, goose, duck, psittacine, pigeon, and owl adenoviruses, 156 with egg drop syndrome virus set as an outgroup. Multiple alignments 157 were performed in MEGA version 7.0, using Clustal W. All candidate 158 sequences were retrieved from the NCBI nucleotide database. A 159 neighbor-joining phylogenic tree was constructed in MEGA 7. The 160 TsoAd amino