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NO56 - 3

A Preliminary Study on Sinking Disease in Koi Carp

Shinya Yasumoto*, Masato Okada, Hideaki Moroi, Hiroki Shimizu and Masakazu Kondo

Department of Applied Aquabiology, National Fisheries University, Japan Fisheries Research and Education Agency, Yamaguchi 759-6595, Japan

(Received November 26, 2020)

ABSTRACT―"Sinking disease" (Sensui-byo in Japanese) has become a major issue in the field of koi carp breeding. Although many diseased fish did not exhibit any significant changes in their appearance, some had ulcers and erosions on the abdomen and at the base of the fins due to physical scratching caused by being at the bottom of a pond for a prolonged period. At necropsy, the swim bladder of the fish was filled with fluid (swim bladder fluid, SBF) and very little gas. Histopathologically, the fish had hyperplastic connective tissue in the tunica intima of the swim bladder, and some were accompanied by inflammation. Furthermore, one to three species of bacteria were isolated from six out of fourteen SBF samples of diseased fish. To reproduce this disease, two experiments were performed using the three major isolates (live or formalin-killed bacteria) from SBF as well as SBF itself. Immobility and the accumulation of SBF occurred in the live bacteria-injected groups. Based on these results, we concluded that the major cause of sinking disease is the accumulation of SBF due to bacterial invasion in the swim bladder and resulting loss of buoyancy.

Key words: koi carp, swim bladder, sinking disease, sensui-byo, Streptococcus, Pseudomonas, Mycobacterium

Morphological and Molecular Phylogenetic Analyses of an Ectoparasitic Trichodinid Ciliate, Trichodina hokkaidoensis n. sp., Infecting Artificially Reared Barfin Flounder Verasper moseri

Shinya Mizuno1*, Taihei Matsuda2, Shotaro Nishikawa1 and Shingo Ito1

1 Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan
2 Mariculture Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 051-0013, Japan

(Received January 13, 2021)

ABSTRACT―Infections by ectoparasitic trichodinid ciliates cause epidermal damages to artificially reared juvenile barfin flounder Verasper moseri of mariculture fisheries in Hokkaido, northern Japan. In the present study, morphological observation and molecular phylogenetic analysis using the small subunit ribosomal RNA gene (SSU rDNA) were performed to identify the trichodinid ciliates. Morphological analysis of denticles and adoral ciliary spiral demonstrated that the trichodinid ciliate belongs to the genus Trichodina within ten genera of Trichodinidae. Denticles of the trichodinid ciliate were clearly distinguishable from those of other Trichodina species by small denticle span, and extensive elongation of the central conical part and anterior ray apophysis. All the isolated ciliate had the same 1,699 base pair SSU rDNA nucleotide sequences, which revealed that the trichodinid ciliates comprised a single species. The SSU rDNA of the trichodinid ciliate showed the highest homology with that of T. pectenis (98.5%) among 30 species of trichodinid ciliates, and the two species formed a single clade in the phylogenetic tree. However, no identical sequence with the present trichodinid ciliate was found in the database. We propose Trichodina hokkaidoensis n. sp. for the ciliate infecting artificially reared juvenile barfin flounder, based on both morphological and molecular phylogenetic analyses.

Key words: morphological analysis, denticle, molecular phylogenetic analysis, SSU rRNA, central conical part, anterior ray apophysis

Biology and Pathogenicity of Benedenia akajin Infecting Hatchery-reared Plectropomus leopardus Juveniles

Hirotaka Mizuochi1,Yutaro Fujiwara2, Sho Shirakashi2*, Yuji Fujikura3 and Kazuo Ogawa4

1 Stock Enhancement Center of Lake Biwa, Shiga 525-0005, Japan
2 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
3 Yaeyama Field Station, Fisheries Technology Institute, National Research and Development Agency, Japan Fisheries Research and Education Agency, Okinawa 907-0451, Japan
4 Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received February 10, 2021)

ABSTRACTBenedenia akajin has recently been described as a new species from cultured coral grouper Plectropomus leopardus. We conducted annual monitoring for the skin monogenean among hatchery-reared P. leopardus juveniles to understand the seasonality of the infections. In addition, growth, development, reproduction, site specificity, and pathogenicity of B. akajin were investigated. The infections were most prominent at a temperature above 25°C and B. akajin was the only capsalid species found in the hatchery-reared P. leopardus juveniles. The parasite took approximately 9 days to reach maturity and had an estimated longevity of approximately 1 month at 26°C. At this temperature, the shortest egg-laying frequency was 131 s, but the oviposition rate was strongly affected by the size of the worm. Most B. akajin individuals were found on the fins of the host. Wounds and lesions were observed on the fins of infected P. leopardus and the host condition factor was negatively correlated with the parasite intensity. These results demonstrated that B. akajin caused decreased growth in cultured P. leopardus juveniles and indicated the needs to control this skin fluke in P. leopardus rearing facilities.

Key words: Capsalidae, skin fluke, grouper, reproduction, site-specificity

A New Microsporidian Disease of Rainbow Trout Oncorhynchus mykiss Farmed in Shiga Prefecture, Japan, and Possible Involvement of Common Prawn Palaemon paucidens for Transmission of the Pathogen

Michitaka Yamamoto1a,Sho Shirakashi2*, Nanami Asai2, Tetsuya Yanagida3, Kazuhiro Sugahara1 and Hiroshi Yokoyama4

1 Shiga Prefectural Fisheries Experiment Station, Shiga 522-0057, Japan
2 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
3 Laboratory of Parasitology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8511, Japan
4 Faculty of Veterinary Medicine, Okayama University of Science, Ehime 794-8555, Japan
a Present address: Shiga Prefecture Fisheries Management Division, Shiga 520-8577, Japan

(Received March 15, 2021)

ABSTRACT―A microsporidian disease showing abnormal petechiae-like red spots on the trunk muscle, beneath the skin, occurred among rainbow trout Oncorhynchus mykiss farmed in Shiga Prefecture. Numerous microsporidian spores were detected from the affected area and the causative agent was identified as a microsporidian closely related to Kabatana takedai based on morphology and molecular analyses. Anecdotal information indicated the possible involvement of common prawn Palaemon paucidens for the transmission of the pathogen. Three prawn feeding trials using O. mykiss and Biwa trout Oncorhynchus masou subsp. successfully reproduced the disease in both fish species. The microsporidian infections were confirmed in the fish given fresh and chilled prawns and mortalities occurred in these groups. No infection occurred in the groups fed with frozen prawn. These results strongly suggest that the microsporidian infections can be lethal to Oncorhynchus spp. and that P. paucidens plays important role in the transmission of the pathogen. These findings may lead to the elucidation of the multi-host lifecycle of fish microsporidians.

Key words: Microsporidia, Kabatana, petechiae, rainbow trout, Palaemon, SSU rDNA, Lake Biwa

Infections of Kudoa septempunctata (Myxozoa: Multivalvulida) in Wild Grass Puffer Takifugu alboplumbeus and Japanese Whiting Sillago japonica

Sho Shirakashi1*,Sang Phil Shin2,Tohru Mekata3 and Ikunari Kiryu3

1 Aquaculture Research Institute, Kindai University, Wakayama 649-2211, Japan
2 Department of Marine Life Science, Jeju National University, Jeju 63333, Republic of Korea
3 Pathology Division, Nansei Field Station, Fisheries Technology Institute, Japan Fisheries Research and Education Agency, Mie 519-0193, Japan

(Received March 16, 2021)

ABSTRACTKudoa septempunctata is an important myxozoan parasite in culture and capture fisheries as it causes food poisoning in humans. This species is generally considered a parasite of olive flounder Paralichthys olivaceus because K. septempunctata-related food poisoning cases are associated with consumption of raw infected P. olivaceus. However, the host range of K. septempunctata remains unclear. Moreover, there is scarce information on the lifecycle of the parasite, and this hinders the development of a fundamental prophylactic measure in fish farms. During the survey of wild fishes at a K. septempunctata endemic area, kudoid myxospores were detected in the somatic muscle of grass puffer Takifugu alboplumbeus and Japanese whiting, Sillago japonica. Our morphological, molecular, and histological analyses identified them as K. septempunctata. While the infection levels in T. alboplumbeus were higher (up to 92.3% prevalence) than those in S. japonica (23.8%), the maximum spore density in the muscle tissue was about 7×104/g in both fishes, which is far less than the food poisoning risk value of 1 × 106/g. These findings indicate a wide host range of K. septempunctata and further attention is required for its possible infections in fishes other than P. olivaceus.

Key words: Kudoidae, host specificity, pseudocyst, myxospore, food poisoning

Effects of Peptidoglycan and Polyinosinic: Polycytidylic Acid on the Recombinant Subunit Vaccine Efficacy Against Edwardsiella tarda in Japanese Flounder Paralichthys olivaceus

Hidehiro Kondo1*,Tatsuharu Kikumoto1,Keisuke Yoshii2,Naoya Murase2,Hidetoshi Yamada2,Yutaka Fukuda2 and Ikuo Hirono1

1 Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2 Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita 879-2602, Japan

(Received April 5, 2021)

ABSTRACT―The protective effects of a subunit vaccine, a recombinant protein of Edwardsiella tarda glyceraldehyde 3-phosphate dehydrogenase (Rec) combined with peptidoglycan (PGN) and polyinosinic:polycytidylic acid (polyIC), against edwardsiellosis in Japanese flounder Paralichthys olivaceus were investigated to evaluate an adjuvant property of pathogen-associated molecular patterns. By injection of the mixture of the recombinant protein plus peptidoglycan and polyIC (Rec + PGN and Rec + IC, respectively), the interleukin-1β and interferon (IFN)-γ mRNA levels were upregulated in comparison to injection of phosphate-buffered saline (PBS). In addition, type I IFN mRNA was induced in the Rec + IC group. Although antibody titers were increased not only in the recombinant injected Rec group but also in the Rec + PGN and Rec + IC groups, the Rec + PGN and Rec + IC values were slightly higher and lower than those of the Rec group, respectively. After the challenge test with E. tarda, the Rec + IC group showed a higher survival rate than the PBS group, whereas the rates of the Rec and Rec + PGN groups were lower than the PBS group. Furthermore, bacteria were reisolated from only 1 out of 16 survivors in the Rec + IC group. These results suggested that polyIC might be a candidate for a vaccine adjuvant for intracellular bacterial infection.

Key words: Edwardsiella tarda, glyceraldehyde 3-phosphate dehydrogenase, Paralichthys olivaceus, peptidoglycan, polyinosinic:polycytidylic acid, recombinant protein, subunit vaccine