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NO55 - 3

Heavy Metacercarial Infection in the Abdominal Cavity of Hatchery-reared Japanese Dace Tribolodon hakonensis

Norihiko Komatsu1, Naoki Itoh2 and Kazuo Ogawa3*

1 Horticulture & Livestock Division, Agricultural Administration Department, Nagano Prefectural Government, Nagano 380-8570, Japan
2 Laboratory of Fish Diseases, The University of Tokyo, Tokyo 113-8657, Japan
3 Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received January 7, 2020)

ABSTRACT―In August 2013, metacercarial infection in the abdominal cavity of Japanese dace Tribolodon hakonensis fingerlings occurred at a hatchery in Nagano Prefecture, Japan. All fish examined in this study (n = 45) were infected, and heavily infected fish showed an abnormally distended abdomen and ascites, with the number of metacercariae reaching 66. The metacercariae were distinctly bipartite, representing a Neascus type and classified as a member of Diplostomidae, and phylogenetical analyses based on ITS2 and cox1 sequences tentatively classified them in the genus Posthodiplostomum. When they were excysted, only a few were alive and intact, suggesting that dace is not a suitable host. Histopathologically, cysts containing dead worms and empty and vestigial cysts were in the abdominal cavity, detached from the host visceral organs and surrounded by host inflammatory cells. Diplostomid cercarial larvae were not found in the three species of snails (n = 613) sampled from ponds in the station. At the end of rearing in September, the survival rate was 0.96% in a pond with a high infection rate, whereas it was 12.5% in another pond where fingerlings showed no clear disease signs. Oral administration of praziquantel at 150 mg/kg fish body weight for 3 consecutive days effectively killed metacercariae in the abdominal cavity.

Key words: metacercaria, Tribolodon hakonensis, Japanese dace, seed production

Quantitative Analysis of Diplomonad Flagellate Spironucleus salmonis Infection in Intestines of Hatchery and Wild Salmonid Fishes in Hokkaido

Shinya Mizuno1*, Shigehiko Urawa2, Yoshitomo Katsumata1, Takumi Morishita2, Yui Minowa2 and Masatoshi Ban2

1 Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan
2 Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Hokkaido 062-0922, Japan

(Received April 21, 2020)

ABSTRACT―Diplomonad flagellate Spironucleus salmonis infection in hatchery-reared juvenile chum salmon Oncorhynchus keta and masu salmon O. masou has been previously reported in Hokkaido, northern Japan. We established a quantitative real-time PCR (qPCR) assay for the S. salmonis ribosomal RNA gene (rDNA) using a fish intestine nucleic acid template, which we then use to investigate the infection intensity of S. salmonis in hatchery-reared, farmed, and wild salmonids throughout Hokkaido. Our new qPCR assay enabled the measurement of rDNA from 1.0-1.0 × 108 copies/μL. Our study reported a significantly positive correlation between parasite trophozoite cell number and its rDNA copy number, and presented this as a simple regression (parasite rDNA copy number = -768 + 338 × parasite cell number). By reporting S. salmonis infection in juvenile and/or adult chum and masu salmon collected from three hatcheries, one fish farm, and one river in Hokkaido, we demonstrate the utility of our qPCR assay for the surveillance of S. salmonis infection in hatchery-reared, farmed and wild fish.

Key words: Spironucleus salmonis, Oncorhynchus keta, Oncorhynchus masou, hatchery, fish farm, wild fish, Hokkaido, salmon

Different Virulence of Flavobacterium psychrophilum Isolates in Two Stocks of Ayu Plecoglossus altivelis

1 Fisheries and Marine Technology Center, Hiroshima Prefectural Technology Research Institute, Hiroshima 737-1207, Japan
2 Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan

(Received May 12, 2020)

ABSTRACT―The susceptibility of ayu Plecoglossus altivelis to infection by Flavobacterium psychrophilum differs among hatchery stocks. We challenged two ayu stocks, domesticated stock (DS) and amphidromous stock (AS), with 18 F. psychrophilum isolates derived from diseased ayu using intraperitoneal (IP) injection and immersion methods. In both methods, 6 isolates were more virulent to DS than to AS (designated as the Do pathotype), whereas the other 12 isolates were more virulent to AS than to DS (designated as the Am pathotype). The LD50s (CFU/fish) of the representative isolates, PH-1034 (Do pathotype) and PH-1037 (Am pathotype) by IP injection in DS and AS were 1.4 × 102 and 5.0 × 105, and 5.9 × 105 and 4.6 × 102, respectively. The difference in virulence between pathotypes was supported by the in vivo bacterial counts in the blood and organs of IP-challenged fish, but not by the in vitro growth ability in the serum of ayu, which is a virulence factor of F. psychrophilum. Additionally, the 18 F. psychrophilum isolates could be classified into any one of the previously reported PCR-RFLP-based genotypes. However, no clear correlation was found between these genotypes and pathotypes.

Key words: Flavobacterium psychrophilum, virulence, pathotype, genotype, Plecoglossus altivelis, bacterial cold-water disease, ayu

Effects of Hydrogen Peroxide on Egg Laying and Hatching of the Skin Monogenean Neobenedenia girellae

Noritaka Hirazawa*, Naoko Umeda and Kousuke Akiyama

Oita Marine Biological Technology Center, Nippon Suisan Kaisha, Ltd., Oita 876-1204, Japan

(Received October 17, 2019)

ABSTRACT―The effects of concentrations and exposure durations of H2O2 on the egg laying and egg hatching of Neobenedenia girellae were investigated. Four groups of eight adults or approximately 250 eggs were treated with H2O2 as follows: 0 ppm for 30 min (control), 300 ppm for 3 min; 75 ppm for 30 min; 100 ppm for 30 min. The adults in the H2O2 exposed groups produced significantly fewer number of normal eggs. Additionally, the H2O2 exposures to the eggs significantly reduced the hatching success of the eggs at < 24 h after laying, but the eggs at 48-71 h after laying were not affected.

Key words: Capsalid monogenean, Neobenedenia girellae, hydrogen peroxide, egg laying, egg hatching

Development of New PCR and Quantitative PCR Protocols for the Detection of Plecoglossus altivelis Poxvirus-like Virus in Atypical Cellular Gill Disease of Ayu

Tomoki Koyama1,#, Daiki Komatsu1,#, Tsubasa Uchino1,2, Yuki Midorikawa1, Goshi Kato1, Takanori Ishikawa3, Tomohiro Nishimura3, Korenori Takeda3, Hideo Fukuda1, Shinpei Wada4 and Motohiko Sano1*

1 Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2 Aquaculture Research Department, Fisheries Technology Institute , Japan Fisheries Research and Education Agency, Mie 519-0423, Japan
3 Tochigi Prefectural Fisheries Experimental Station, Tochigi 324-0404, Japan
4 Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan

(Received April 22, 2020)

ABSTRACTPlecoglossus altivelis poxvirus-like virus (PaPV) is the presumptive causative agent of atypical cellular gill disease (ACGD), which has caused significant losses in aquaculture of ayu P. altivelis in Japan. In this study, we investigated a new PCR protocol and a quantitative PCR (qPCR) protocol with primers designed from the vaccinia virus A16L homologous gene sequence of PaPV. The new PCR showed a positive reaction for affected fish in all eight ACGD cases tested and no cross-reaction with carp edema virus, and was more sensitive than a PCR with BOKE30 primers. The qPCR quantified around 108-109 virus DNA copies/mg in the diseased fish gills.

Key words: Plecoglossus altivelis poxvirus-like virus, PCR, qPCR, atypical cellular gill disease, ayu