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NO56 - 4

Development of New Real-time PCR Assays for Detecting Megalocytivirus Across Multiple Genotypes

Yasuhiko Kawato1*, David M. Cummins2, Stacey Valdeter2, Peter G. Mohr2, Takafumi Ito1, Kaori Mizuno3, Hidemasa Kawakami3, Lynette M. Williams2, Mark St. J. Crane2 and Nicholas J. G. Moody2

1Pathology Division, Nansei Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 519-0193, Japan
2CSIRO Australian Centre for Disease Preparedness, Geelong, VIC 3220, Australia
3Ehime Fisheries Research Center, Ehime 798-0087, Japan

(Received February 18, 2021)

ABSTRACT―In the present study, two real-time PCR assays (Cummins and Kawato-1) were newly developed for the detection of broad range of Megalocytivirus (MCV) genotypes. The analytical sensitivity (ASe) and analytical specificity (ASp) of four real-time PCR assays, including two previously reported assays (Kawato-2 and Mohr), targeting the major capsid protein gene were compared across the four MCV genotypes including infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and threespine stickleback iridovirus (TSIV). The four assays were tested using artificially synthesized plasmids containing seven different representative nucleotide sequences of the target region from the genotypes. The ASe and ASp for the detection of each MCV genotype differed between assays while the three assays other than Kawato-2 detected all ISKNV, RSIV, and TRBIV targets to a detection limit of approximately 2 plasmid copies per reaction. Diagnostic capability of each assay was validated using spontaneously RSIV-infected Japanese amberjack Seriola quinqueradiata. Diagnostic sensitivity in the dead fish of RSIV infection was equivalent among the four assays. However, in the asymptomatic fish, the detection rate of Cummins assay was higher than that of Kawato-1 assay and had equivalent sensitivity to previously reported assays. Thus, in addition to screening with synthesized plasmids, diagnostic capability of the assays should be tested using fish infected with the other MCV genotypes depending on target of host and the viral genotypes.

Key words: Megalocytivirus, real-time PCR, ISKNV, RSIV, TRBIV, analytical sensitivity and specificity

Outbreak of Nontuberculous Mycobacteriosis with Dematiaceous Fungus Co-infection in Aquarium-reared Sharphead Flyingfish (Hirundichthys oxycephalus)

Takeshi Komine1, Saralee Srivorakul2, Kentaro Tomaru1, Yoshiaki Tanaka3, Mari Inohana1, Hanako Fukano4, Osamu Kurata1 and Shinpei Wada1*

1Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyohnan-cho, Musashino, Tokyo 180-8602, Japan
2Veterinary Diagnostic Center, Faculty of Veterinary Medicine, Chiang Mai University, Mae Hia Muang, Chiang Mai 50100, Thailand
3Shimane Aquarium (AQUAS), 1117-2 Kushiro-cho, Hamada, Shimane 697-0004, Japan
4Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aoba-cho, Higashi-Murayama, Tokyo 189-0002, Japan

(Received May 17, 2021)

ABSTRACT―An episode of serial mortality occurred in a population of wild-caught, aquarium-reared sharphead flyingfish Hirundichthys oxycephalus. Most diseased fish had ulcerative hemorrhagic lesions on the skin, especially on the head, and displayed multiple, fine, whitish nodules scattered on the internal organs, including the kidney, spleen and liver. Histopathological examination revealed the nodules to be granulomatous lesions consisting of epithelioid cells with a thin outermost rim of fibroblasts and connective tissue, and the central caseous core of the granulomas was associated with acid-fast bacilli. Though the granulomatous lesions were systemically present in the major internal organs, the most severe lesions were in the kidney and spleen. In addition, melanized fungal hyphae were also found in the kidney and spleen. Acid-fast bacterial strains were isolated from affected fish and most were identified as Mycobacterium marinum by molecular examination. A few dematiaceous fungal strains were also isolated from some fish and identified as Exophiala aquamarina by morphological and molecular examination. This is a novel description of a case of co-infection of a nontuberculous mycobacterium (M. marinum) and a dematiaceous fungus (E. aquamarina) in a flyingfish species.

Key words: nontuberculous mycobacteriosis, dematiaceous fungus, sharphead flyingfish, co-infection

Infection of a Species of Digenean in Common Octopus Octopus sinensis in Japan

Shotaro Izumi1*, Nobuhiko Akiyama1, Yuta Suzumura1 and Kazuo Ogawa2

1School of Marine Science and Technology, Tokai University, Shizuoka 424-8610, Japan
2Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received June 17, 2021)

ABSTRACT―During a post-mortem inspection of common octopus Octopus sinensis, digenean parasites were found on the visceral surface. Parasitological examination revealed that 77 of 131 octopuses were infested, with maximum intensity of 975 digeneans. In all the retrieved digeneans, 92.3% were found to be attached to the renal sac. Species identification based on morphology was not possible because no fully mature digenean worms were obtained. Based on molecular analysis of 18S and 28S rDNA, the digenean was identified as Proctoeces cf. major. This is the first report of Proctoeces infection in common octopus in Japan.

Key words: Proctoeces cf. major, Octopus sinensis, Fellodistomidae

Efficacy of Febantel Against Gill Monogeneans of Amberjacks I: Laboratory Assessment for Effective Dose and Administration Period

Sho Shirakashi1*, Satoshi Nishikawa2, Yuji Ishii2, Hidemasa Kawakami2, Jun Satoh3, Chihaya Nakayasu3, Mamoru Ishihara4, Tomoko Fukui4 and Kazuhiko Hirose4

1Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
2Ehime Fisheries Research Center, Ehime 798-0087, Japan
3Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
4Animal Health Department, Agricultural & Veterinary Division, Meiji Seika Pharma Co., Ltd., Tokyo 104-8002, Japan

(Received May 14, 2021)

ABSTRACT―Three experimental trials were conducted to determine the effective dose and administration period of a pro-benzimidazole anthelmintic drug febantel (FBT) against two gill monogenean species on amberjacks; Zeuxapta japonica on Seriola dumerili and Heteraxine heterocerca on Seriola quinqueradiata. The mean Z. japonica intensities were checked at 3 days after given FBT-coated feeds at the doses of 1, 5, 10, or 20 mg/kg body weight (BW)/day (d) for 5 consecutive days (trial 1), and 10 mg/kg BW/d for 1, 3, or 5 days (trial 2). Based on the results of these trials, the efficacy of oral FBT treatment was assessed for H. heterocerca infections at the treatment regime of 10 mg/kg BW/d for 3 or 5 days (trial 3). Over 95% FBT efficacies were achieved for both monogenean species when the fish received the dose of 10 mg/kg/d or higher for 5 consecutive days. Amberjacks showed no apparent avoidance to the FBT-coated feed and no apparent signs of the drug toxicity were detected. Our results demonstrated that the in-feed FBT treatment at the dose of 10 mg/kg BW/d for 5 days is an effective, easy, and practical control measure against gill monogenean infections in aquaculture of Seriola fish.

Key words: Heteraxine heterocerca, Zeuxapta, Seriola, Gill fluke, Fenbendazole, Benzimidazole

Efficacy of Febantel against Gill Monogeneans of Amberjacks II: Field Trial Assessment

Sho Shirakashi1*, Satoshi Nishikawa2, Yuji Ishii2, Hidemasa Kawakami2, Yoshiaki Imaoka3, Keisuke Yoshii4, Jun Satoh5, Chihaya Nakayasu5, Mamoru Ishihara6, Tomoko Fukui6 and Kazuhiko Hirose6

1Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
2Ehime Fisheries Research Center, Ehime 798-0087, Japan
3Kagoshima Prefecture Fisheries Technology and Development Center, Kagoshima 891-0315, Japan
4Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602, Japan
5Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
6Animal Health Department, Agricultural & Veterinary Division, Meiji Seika Pharma Co., Ltd., Tokyo 104-8002, Japan

(Received May 14, 2021)

ABSTRACT―Field trials were conducted to assess the efficacy of oral febantel (FBT) treatment at the dose of 10 mg/kg for 5 days against Heteraxine heterocerca and Zeuxapta japonica in Seriola fish. Numbers of gill flukes were checked 1 day before and 3 days after the treatment. In comparison to the control groups, fish given FBT-coated feed showed deworming efficacies of 99.9% to 100%. No mortality and no signs of drug toxicity were observed. These results indicate that in-feed administration of FBT is an effective and practical treatment for gill fluke infections in Seriola aquaculture.

Key words: Benzimidazole, Seriola, Heteraxine heterocerca, Zeuxapta

Histological Distribution of MHC Class II‒expressing Cells Responsible for Ingestion of Blood‒borne Exogenous Particles in Japanese Flounder Paralichthys olivaceus

Osamu Kurata*, Suyu Miyashita and Shinpei Wada

Laboratory of Aquatic Medicine, School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan

(Received May 24, 2021)

ABSTRACT―This study investigated the ability of MHC class II‒expressing cells to ingest blood-borne exogenous particles and their tissue distribution in Japanese flounder Paralichthys olivaceus. Carbon particles were injected into a blood vessel in the caudal peduncle. After 3 h, carbon-laden MHC class II‒expressing cells were found in the spleen and the kidney, and localised in ellipsoids, splenic cords, peritubular sinuses and interstitial veins of the kidney. Additionally, the percentage of these cells was significantly higher in the spleen than the kidney. These results suggest that the spleen is the main organ that plays a role in the ingestion of blood‒borne exogenous particles for antigen presentation.

Key words: antigen‒presenting cells, lymphoid organs, immunohistochemistry, spleen, ellipsoid