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NO52 - 2

Pseudotuberculosis

Kinya Kanai*

Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan

ABSTRACT― Pseudotuberculosis caused by Photobacterium damselae subsp. piscicida (= Pasteurella piscicida) is a bacterial disease of cultured fish belonged to genus Seriola. The disease frequently occurs among juveniles at a water temperature ranging from 20 to 25°C with a high mortality rate. A characteristic disease sign is occurrence of a lot of small, white spots in the spleen and kidney, which consist of bacterial colonies and focal tissue necrosis. The disease can be cured by administrating antibiotics, but P. damselae subsp. piscicida possessing multiple drug resistance has been found. In Japan, several adjuvant vaccines are commercially available.

Red Sea Bream Iridoviral Disease

Yasuhiko Kawato1*, Jun Kurita2 and Kazuhiro Nakajima1,3

1National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
2Tamaki Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 519-0423, Japan
3Headquarters, Japan Fisheries Research and Education Agency, Kanagawa 220-6115, Japan

ABSTRACT― Red sea bream iridoviral disease (RSIVD) has caused severe economic damages to mariculture in Japan since it was first found in 1990 in the western part of Japan. The causative agent (red sea bream iridovirus: RSIV) was successfully isolated and identified in few years and then a useful diagnosis method by indirect fluorescent antibody test using anti-RSIV monoclonal antibody was developed. In addition, effectiveness of formalin-inactivated virus as a vaccine was demonstrated in 1997 and the vaccine started to be sold in 1999 in Japan as the first commercially available vaccine against a viral disease in marine fish. In this review, we summarized the history of RSIVD research in the past few decades.

Myxosporean Emaciation Disease

Tetsuya Yanagida*

Laboratory of Veterinary Parasitology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan

ABSTRACT― Myxosporean emaciation disease is caused by the enteric myxosporeans Enteromyxum leei and Sphaerospora fugu (syn. Leptotheca fugu). It emerged in the aquaculture of tiger puffer since mid-1990s in Japan. Thus far, more than 50 marine fishes have been reported as susceptible to E. leei in Europe and East Asia. As it has a wide host range and is feasible to transmit directly from fish to fish, E. leei is considered to be one of the most devastating parasites in warm water seawater aquacultures. Regardless of its economic impact, neither effective treatment nor preventive measure for the disease has been established until now. To control this disease, further studies are needed for better understanding of the biology of the parasites and of the host-parasite interaction.

Aeromonas veronii Infection Associated with High Morbidity and Mortality in Farmed European Seabass Dicentrarchus labrax in the Aegean Sea, Greece

Maria Smyrli1,2, Athanasios Prapas3, George Rigos1, Constantina Kokkari1, Michail Pavlidis2 and Pantelis Katharios1*

1Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Heraklion 71003, Greece
2Department of Biology, University of Crete, Heraklion 70013, Greece
3Department of Pathology of Aquatic Organisms, Veterinary Center of Athens, Ag. Paraskevi 15310, Greece

ABSTRACT― In the present study, we examined a disease caused by Aeromonas veronii bv. sobria in sea cage-farmed European seabass Dicentrarchus labrax, in the Aegean Sea, Greece. Commercial sized fish were affected by A. veronii bv. sobria and exhibited high morbidity and mortality. Gross pathologic features and histology revealed a systemic infection characterized by the presence of abscesses and chronic granulomatous inflammation. Two clinical bacterial isolates (Aero NS and Aero PDB) were identified as A. veronii bv. sobria based on bacteriological characteristics and sequence analysis for 16S rRNA and gyrB genes. Infectivity tests in the form of intraperitoneal injection administration (Aero NS) and immersion in a bacterial suspension (Aero NS and Aero PDB) revealed that both isolates could cause clinical signs similar to those observed in the field and high mortality rate. To our knowledge, this is the first report of A. veronii bv. sobria isolated from farmed European seabass in the Mediterranean Sea accompanied by supporting data of its pathogenicity.

Key words: Dicentrarchus labrax, European seabass, Aeromonas veronii bv. sobria, pathogenicity, Aegean Sea, Mediterranean Sea, Greece

A Novel Plasmid Carrying Capsule Gene Cluster Found in Lactococcus garvieae Isolated from Filefish

Kinya Kanai1*, Masakazu Tsujikura2, Kimihiro Shutou2, Toshio Honma2, Fumihiro Matsumoto1, Koushirou Suga1, Shusaku Takagi3, Yutaka Fukuda4 and Yukitaka Sugihara5

1Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki 852-8521, Japan
2Bio Science Co. Ltd., Tokushima 779-1292, Japan
3Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Ehime 798-0087, Japan
4Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602, Japan
5Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan

ABSTRACTLactococcus garvieae is recognized as a crucial bacterial pathogen of freshwater and marine fish species. It has been divided into two serological phenotypes, namely KG- and KG+. Difference of the two phenotypes is owing to the presence or absence of polysaccharide capsule, and a phenotypic change from KG- to KG+ occurs during stocking of isolates for a long period or by repeated subculturing. We found that the phenotypic change occurred more readily in L. garvieae isolates from cultured filefish, thread-sail filefish Stephanolepis cirrhifer and black scraper Thamnaconus modestus, than those from other fish species. Thus we studied the gene cluster for capsular polysaccharide biosynthesis (capsule gene cluster) of a filefish isolate, strain BSLG13015, and revealed that the strain possessed the same capsule gene cluster as those from other fish species, but that it was integrated in a newly identified plasmid. The plasmid, a size of 31,654 bp and circular, was named pBSLG13015. It was detected in all of KG- filefish isolates but not in KG+ filefish isolates or L. garvieae from other fish species. It is highly probable that the easier change from KG- to KG+ in L. garvieae filefish isolates is attributed to the loss of the plasmid.

Key words: Lactococcus garvieae, capsule gene cluster, plasmid, filefish, KG, KG+, phenotypic change

Subclinical Infection of <em>Renibacterium salmoninarum</em> in Fry and Juveniles Chum Salmon Oncorhynchus keta in Hokkaido, Japan

Kunio Suzuki*, Naoyuki Misaka, Shinya Mizuno and Yoshitaka Sasaki

Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan

ABSTRACT― Genomic and mRNA sequences of Renibacterium salmoninarum the causative agent of bacterial kidney disease (BKD), were detected in the kidney tissue and surface mucus of asymptomatic fry and juveniles chum salmon Oncorhynchus keta, using nested PCR, quantitative real-time PCR and RT-nested PCR. Fry were obtained from a hatchery belonging to the Hokkaido Salmon Resources Association from March 25, through May 10, 2016. In addition, the fry were transferred from the hatchery into a wet laboratory at the Salmon and Freshwater Fisheries Research Institute, reared in running well water at 8°C-10°C, and monitored for the bacterium from April 19, 2016 through January 4, 2017. The fish showed no mortality from BKD, and live bacteria were not detected in the kidney tissue by culture. Yet prevalence of R. salmoninarum genome and positive rate of the mRNA in the kidney tissue samples were 0%-100% and 0%-70%, respectively, with number of genome of 1.0 × 101-1.7 × 104 copies/mg. Surface mucus samples, which were obtained in November 21, 2016 and January 4, 2017, showed similar detection levels to the kidney tissue based on prevalence and intensity of the genome. The results verify subclinical infection with R. salmoninarum in chum salmon fry in Hokkaido, Japan.

Key words: subclinical infection, Renibacterium salmoninarum Oncorhynchus keta, prevalence, surface mucus, kidney tissue, BKD

Application of Tissue Clearing Techniques to 3D Study of Infectious Disease Pathology in Fish

Shimon Ohnuma, Takayuki Katagiri, Masashi Maita, Makoto Endo and Kunihiko Futami*

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan

ABSTRACT― Conventional fish pathological diagnoses are limited in ability to detect pathogens in infected fish due to limitations of partial or 2 dimensional images. Here, we tested three tissue clearing techniques (ClearT, SeeDB and CUBIC), which make the fish body transparent, allowing visualization of pathogens. CUBIC was most effective in clearing the fish body, and was applicable to immunohistochemistry and GFP (green fluorescent protein) imaging. Furthermore, DAPI-labeled Aeromonas hydrophila was visible under a fluorescence microscope. Thus, CUBIC is the most suitable tissue clearing technique allowing 3 dimensional analysis of infectious diseases in fish.

Key words: CUBIC, tissue clearing techniques, infectious disease, 3D pathology

PCR- Based Detection and Quantification of Renibacterium salmoninarum in Ovarian Fluid of Returning Chum Salmon Oncorhynchus keta and Masu Salmon O. masou in Hokkaido, Japan

Kunio Suzuki*, Naoyuki Misaka, Shinya Mizuno and Yoshitaka Sasaki

Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan

ABSTRACTRenibacterium salmoninarum was detected in ovarian fluid of returning salmonids using nested PCR; chum salmon from seven rivers (925 samples) and masu salmon from three rivers (309 samples) in Hokkaido in 2015 and 2016. Real-time PCR was also performed for the samples in 2016. In chum salmon prevalence of R. salmoninarum genome was 0%-90% in 2015 and 12%-98% in 2016. In masu salmon the prevalence was 0% in 2015 and 50%-84% in 2016. In 2016, the prevalence had risen in the samples in eight of ten rivers. The numbers of R. salmoninarum genome were estimated to be 1.0 × 101-8.0 × 102 copies/μL of ovarian fluid.

Key words: Renibacterium salmoninarum, prevalence, Oncorhynchus keta, Oncorhynchus masou, ovarian fluid

Development of Loop-Mediated Isothermal Amplification Methods for Detection of the Enteric Myxosporeans Causing Myxosporean Emaciation Disease

Masahide Katata* and Yoshio Okuyama

Wakayama Prefectural Fishery Experiment Station, Wakayama 649-3503, Japan

ABSTRACT― In this study, we developed loop-mediated isothermal amplification (LAMP) methods for detecting the enteric myxosporeans Enteromyxum leei and Sphaerospora fugu which cause myxosporean emaciation disease. The LAMP primer sets were designed based on the sequences of the small subunit ribosomal RNA gene (SSU rDNA) of E. leei and S. fugu. Reaction time and temperature were optimized for 60 minutes at 62°C, respectively. Cross-reactivity with other myxosporeans was not observed. These LAMP methods showed detection sensitivities of 100-1,000 times higher than those of the PCR methods applied so far. These LAMP methods established in this study are the specific and sensitive methods, allowing for the rapid and reliable diagnosis of this disease.

Key words: LAMP method, myxosporean emaciation disease, Enteromyxum leei, Sphaerospora fugu