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NO56 - 2

Temporal Change in Genetical Lineages of Infectious Hematopoietic Necrosis Virus (IHNV) in the North Kanto Region of Japan from 1981 to 2015

Aki Namba1, Kai Minakami1, Taro Takee1, Kaori Shiibashi1, Miyu Sugino1, Shuta Yasuda1, Hisato Takeuchi1,4, Takanori Ishikawa1,2, Toshimitsu Matsubara3, Hajime Arai3, Hajime Arai1 and Nobuhiro Mano1*

1 College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
2 Tochigi Prefectural Fisheries Experimental Station, Tochigi 324-0404, Japan
3 Gunma Prefectural Fisheries Experimental Station, Gunma 371-0036, Japan
4 South Ehime Fisheries Research Center, Ehime University, Ehime 798-4292, Japan

(Received March 17, 2021)

ABSTRACT―We analyzed the full-length glycoprotein gene sequences of 72 infectious hematopoietic necrosis virus (IHNV) isolates that had been obtained from diseased salmonid fish in the North Kanto region of Japan from 1981 through 2015. The analysis identified three genetical lineages in the genogroup J of IHNV; two known Shizuoka (JS) and Nagano (JN) lineages and a new lineage named as North Kanto lineage (Jnk). Most of the isolates from the 1980s were classified into JS lineage, whereas most collected after 2000 belonged to JN lineage. Jnk lineage was classified into North Kanto A clade [Jnk-A] and B clade [Jnk-B], and the number of isolates belonging to Jnk-B in the 2010s was higher than in the 2000s. These results suggest that the diversity of IHNV found in salmonid farms in the North Kanto region have been increasing with genetical lineage shifting from 1980 to the present, probably due to change of the fish trade.

Key words: infectious hematopoietic necrosis virus, IHNV, temporal change, lineage, glycoprotein, salmonid fish, North Kanto region

Ceratothoa verrucosa (Isopoda: Cymothoidae) Infection in the Buccal Cavity of Red Seabream Caught in Iyo-Nada, Western Japan, with Some Notes on Its Co-infection with Choricotyle elongata (Monogenea: Diclidophoridae)

Tomomichi Ohtani1, Izumi Kawamoto2, Masamitsu Chiba2, Noriyuki Kurono2, Satoru Matsuoka2 and Kazuo Ogawa3*

1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2 Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Ehime 798-0104, Japan
3 Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received March 29, 2021)

ABSTRACT―Long-term parasitological surveys were conducted on the isopod Ceratothoa verrucosa infecting red seabream Pagrus major caught in Iyo-Nada, Ehime Prefecture during the period of 2003-2007. The isopods were almost always in a pair of a male and a female, attached to the buccal cavity wall. The earliest infection occurred when seabream was 0+ year-old and the isopods grew as host fish grew. Prevalence of infection ranged from 12.2% to 21.2% when the host was 0+ to 5-year-old and decreased to 3.4% at 6-year-old and no infection among 7-year-old and older fish. These results suggest that infection was established when the host was 0+ year-old, and their life span of C. verrucose was estimated to be up to 6 years. Infected fish sometimes showed deformation in the upper jaw and growth retardation, but there was no evidence of infection-related mortality. The main reproduction season of the isopod was in summer, estimated by the presence of manca larvae in the marsupium of the female. Sometimes the monogenean Choricotyle elongata attached to the pleotelson of C. verrucosa. Prevalence and abundance of the monogenean were higher in the hyperparasitized seabream than in the fish without isopod infection, suggesting that the isopod favored the monogenean infection.

Key words: Ceratothoa verrucosa, parasitic Isopoda, Pagrus major, red seabream, Choricotyle elongata, hyperparasitism

Development of Quantitative Real-time PCR and Loop-mediated Isothermal Amplification (LAMP) Assays for Detection of Microsporidium seriolae

Tohru Mekatai1*, Jun Satoh1, Chihaya Nakayasu1, Yuji Ishii2, Shogo Harakawa2, Hidemasa Kawakami2 and Soetsu Yanagi3

1 Aquaculture Research Department, Nansei Branch, FisheriesTechnology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
2 Ehime Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Ehime 798-0087, Japan
3 Kagoshima Prefecture Fisheries Promotion Section, Kagoshima 890-8577, Japan

(Received October 3, 2020)

ABSTRACT―Beko disease, caused by the Microsporidium seriolae infection, has been a problem in yellowtail aquaculture in western Japan. In recent years, severe cases of this disease have been confirmed, resulting in a significant decrease in product value due to mass mortality and residual cysts. Only polymerase chain reaction (PCR) assays have been reported so far as a detection method for the disease. In this study, quantitative real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP) methods have been optimized for M. seriolae detection in order to establish a more sensitive and rapid diagnosis. Target regions for each detection method were selected based on the nucleotide sequences obtained by the gene analysis of cysts in diseased fish. Primer sets for the qPCR and LAMP methods were designed, and the gene amplification efficiency of each method was evaluated. The results showed that the newly developed qPCR method could detect 1.4 copies of the target gene, and the LAMP method detected 100 copies within 15 minutes. In this study, the newly developed qPCR and LAMP assays were shown to be rapid and highly sensitive methods for quantitative detection of M. seriolae.

Key words: Microsporidium seriolae, real-time PCR, LAMP

Chemical Treatment of Beko Disease in Seriola Fishes: Laboratory Trials of Commercially Available Fishery and Veterinary Medicines

Soetsu Yanagi1*, Jun Satoh2, Tohru Mekata2, Takamitsu Sakai3, Hidemasa Kawakami4, Shogo Harakawa4, Sho Shirakashi5, Shogo Hamano5, Kei Fukudome6, Kazuhiko Wada1, Masatoshi Yamasaki2†, Toyohiro Nishioka7, Chihaya Nakayasu2 and Koh-ichiro Mori3

1 Kagoshima Prefecture Fisheries Promotion Section, Kagoshima 890-8577, Japan
2 Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
3 Nagasaki Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Nagasaki 851-2213, Japan
4 Ehime Fisheries Research Center, Ehime 798-0087, Japan
5 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
6 Osumi Regional Promotion Bureau, Kagoshima 893-0011, Japan
7 Production Engineering Division, Yashima Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Kagawa 761-0001, Japan
Present address: Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan

(Received February 17, 2021)

ABSTRACT―Beko disease, caused by infection of the microsporidian Microsporidium seriolae, results in significant damage to farmed amberjack, Seriola spp., in Japan. Little is known about the disease, including the general biology of the causative agent, and no therapeutic method has been established to treat it. We determine the therapeutic efficacies of various drugs by administering them in-feed to juvenile Seriola spp. naturally infected with M. seriolae. Candidate drugs include commercially available fishery anthelminthic, febantel, and eight other compounds. Fish were given various doses of drugs, and M. seriolae infection in their trunk muscle was then checked by quantitative PCR (qPCR) and macroscopic cyst detection. Fish treated with febantel at an early stage of infection had significantly lower rates of cyst formation and qPCR-positive. However, febantel administered to fish after cyst formation did not reduce cyst numbers. Other drugs showed no apparent efficacy to prevent cyst formation or to promote cyst reduction. Results indicate febantel is a promising therapeutic agent for beko disease when applied at an early stage of infection.

Key words: Febantel, Fenbendazole, Microsporidia, Seriola dumerili, Seriola quinqueradiata

Field Trials of Febantel for the Treatment of the Beko Disease in Seriola spp.

Hidemasa Kawakami1*, Yuji Ishii1, Kentaro Nakajima1, Soetsu Yanagi2 and Jun Satoh3

1 Ehime Fisheries Research Center, Ehime 798-0087, Japan
2 Kagoshima Prefecture Fisheries Promotion Section, Kagoshima 890-8577, Japan
3 Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan

(Received January 13, 2021)

ABSTRACT―Outbreaks of beko disease caused by the microsporidian Microsporidium seriolae have been a serious problem in Seriola aquaculture in Japan. Recent laboratory experiments demonstrated that the oral administration of a probenzimidazole febantel (FBT) at the early stage of infection effectively suppresses the formation of the microsporidian cysts in the trunk muscle. This opens the possibility of drug therapy for the beko disease. Here, we conduct a series of field trials to assess the efficacy and practicality of FBT treatment for beko disease in farmed Seriola quinqueradiata and Seriola dumerili. The trial tested FBT doses of 0, 1, 3, 5, and 10 mg/kg bw for 5 days resulted in significantly lower beko disease-related mortality, cyst detection rate, and progression of cyst formation in the 10 mg/kg group of S. quinqueradiata. This 10 mg/kg bw for 5 days FBT administration was also effective to suppress cyst formation in S. dumerili. Another trial used fifteen repeated administrations of 10 mg/kg FBT for 5 days with 2 days interval in between completely prevented the cyst formation in juvenile S. quinqueradiata, in comparison to the cyst detection rate of as high as 83.3% in non-drug treated fish. No adverse effects of FBT on the fish were observed in any of the trials. Our field trials indicate that the oral administration of FBT at the dose of 10 mg/kg bw is highly effective and practical chemotherapy for beko disease in Seriola fish.

Key words: Microsporidium seriolae, Benzimidazole, Chemotherapy, Seriola quinqueradiata, Seriola dumerili

Evaluations of Lethal and Sub-lethal Toxicity of Febantel in the Juvenile Japanese Amberjack Seriola quinqueradiata

Sho Shirakashi1*, Satoshi Miwa2, Takuya Katsuki1, Shogo Harakawa3, Hidemasa Kawakami3, Chihaya Nakayasu2 and Koh-ichiro Mori4

1 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
2 Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
3 Ehime Fisheries Research Center, Ehime 798-0087, Japan
4 Nagasaki Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Nagasaki 851-2213, Japan

(Received February 25, 2021)

ABSTRACT―Recent studies have shown efficacy of febantel (FBT) against Microsporidium seriolae in Seriola fish. This study evaluated the toxicity of FBT in Seriola quinqueradiata. Fish were gavage fed with feed containing various doses of FBT (50 to 1,000 mg/kg/day (d)) for 14 or 28 consecutive days and monitored for growth and survival. Major mortality started at 7 days in fish given 200 and 1,000 mg/kg/d FBT, while the onset of mortality in the 50 and 100 mg/kg/d groups was delayed until 15 days. Doses of 50 mg/kg/d or higher affected fish growth. Histological analyses revealed changes in the distribution and density of Nissl bodies in the brain, and a reduction of vacuolies in hepatocytes. These histological changes were rarely observed after a 9-day drug washout period, suggesting that such changes were reversible. There was no drug-related mortality in fish that received 15 mg/kg/d for 20 days or 20 mg/kg/d for 14 days. These results suggest that, although FBT can have lethal toxicity in S. quinqueradiata when administrated at doses over 50 mg/kg/d for more than 14 days or 200 mg/kg/d for more than 7 days, the drug is considered safe at the normal antiparasitic dose, including the effective dose for M. seriolae treatment (10 mg/kg/d for 5 days).

Key words: Benzimidazole, Histopathology, Nissl body, Microsporidia, Yellowtail

Early Infection Dynamics of the Beko Disease in Seriola spp.

Soetsu Yanagi1*, Jun Satoh2, Yoshiaki Imaoka3, Hidemasa Kawakami4, Shogo Harakawa4, Tohru Mekata2, Chihaya Nakayasu2 and Koh-ichiro Mori5

1 Kagoshima Prefecture Fisheries Promotion Section, Kagoshima 890-8577, Japan
2 Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
3 Kagoshima Prefecture Fisheries Technology and Development Center, Kagoshima 891-0315, Japan
4 Ehime Fisheries Research Center, Ehime 798-0087, Japan
5 Nagasaki Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Nagasaki 851-2213, Japan

(Received April 28, 2021)

ABSTRACT―The early infection dynamics of Microsporidium seriolae, the causative agent of beko disease were investigated in wild and hatchery-reared juvenile Seriola quinqueradiata and S. dumerili at several grow-out and farming locations in western Japan. The progression of M. seriolae infection in the somatic muscle of the fish and its DNA in seawater were monitored by quantitative PCR and macroscopic detection of the cysts. In sea areas where outbreaks of beko disease have been confirmed in the past, fish became infected by M. seriolae after placing in sea cages when 102-103 copies/L of M. seriolae gene was continuously detected from the seawater. The parasite DNA was detected from the somatic muscle within 2 wk after exposure to the contaminated water and their cysts became visible within next 1 wk. M. seriolae infections were confirmed in S. quinqueradiata and S. dumerili even during the winter months (November to January), indicating that its infection season is longer than previously thought. Although direct comparison was not made, S. dumerili appeared to be more susceptible to this disease than S. quinqueradiata.

Key words: Microsporidium seriolae, Seriola quinqueradiata, Seriola dumerili, disease prediction, environmental DNA

Persistence Assessment of Febantel and its Effects on Blood Profile in Juvenile Japanese Amberjack Seriola quinqueradiata

Sho Shirakashi1*, Nanami Asai1, Jun Satoh2 and Chihaya Nakayasu2

1 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
2 Aquaculture Research Department, Nansei Branch, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan

(Received January 19, 2021)

ABSTRACT―Persistence of Febantel (FBT) and its metabolites (FBZ) were assessed in Seriola quinqueradiata after 5 days gavage administration of 20 mg/kg body weight. FBT was not detected in any organs at 1 day post administration (dpa), but traces of FBZ were detected in the liver and kidney at 7 dpa. Neither compound was detected at 14 dpa. While S. quinqueradiata received 20 mg/kg of FBT had higher bilirubin and lower triglyceride values in the blood at 1 dpa, and lower plasma creatinine level at 7 dpa, these were not likely associated with severe damages of organs and physiological functions.

Key words: Fenbendazole, Oxfendazole, Bilirubin, Triglyceride