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NO44 - 4

Virulence Change of Infectious Hematopoietic Necrosis Virus against Rainbow Trout Oncorhynchus mykiss with Viral Molecular Evolution

<>Mamiko Mochizuki1, Hyoung Jun Kim2, Hisae Kasai2, Toyohiko Nishizawa2 and Mamoru Yoshimizu2*<>1Fuji Trout Hatchery, Shizuoka Prefectural Research Institute of Fishery, Shizuoka 418-0108, Japan
2Faculty of Fisheries Sciences, Hokkaido University, Hokkaido 041-8611, Japan
(Received February 9, 2009)
ABSTRACT―Infectious hematopoietic necrosis virus (IHNV) is the causative agent of IHN, one of the most serious viral diseases of salmonid fish. A total of five major genogroups including JRt for Asian isolates were confirmed among worldwide isolates based on glycoprotein (G) gene nucleotide (nt) sequences. The present study revealed existence of new two lineages, JRt Shizuoka and JRt Nagano, in the genogroup JRt by addition of new isolates obtained in 2006. The maximum nt diversity of G gene within JRt Shizuoka or JRt Nagano lineage was 6.3% or 3.5%, while that between JRt Shizuoka and JRt Nagano lineages was 7.0%. To evaluate influence of the evolutional divergence to virulence of IHNV, experimental challenges to rainbow trout Oncorhynchus mykiss were conducted by bath exposure at 104 TCID50/mL of RtShiz06s and RtShiz06a (JRt Shizuoka lineage), RtNag96 and RtNag06a (JRt Nagano lineage), and ChAb76 (a representative of the genogroup U). Distinct difference was observed in IHNV virulence to rainbow trout, i.e. the highest virulence was in RtShiz06s and RtShiz06a (≥ 76% of mortalities), and subsequently in RtNag96 and RtNag06a (20-40%), but scarcely any virulence in ChAb76 (≤ 10%). Thus it was suggested that nt diversity of Japanese IHNV continued rapidly with changing its virulence in rainbow trout farm environments.

Key words: infectious hematopoietic necrosis virus, IHNV, virulence, rainbow trout, phylogeny, nucleotide diversity

Antifungal Effect of Potassium Chloride (KCl) on Water Mold Infection in Ayu Plecoglossus altivelis Eggs

<>Masayuki Miura1*, Kishio Hatai2, Heisuke Oono2, Nana Kaji3 and Jun Nagura3<>1Yamanashi Prefectural Fisheries Technology Center, Yamanashi 400-0121, Japan
2Laboratory of Fish Diseases, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan
3Oshino Trout Hatchery, Yamanashi Prefectural Fisheries Technology Center, Yamanashi 401-0511, Japan
(Received June 22, 2009)
ABSTRACT―The antifungal effects of potassium chloride (KCl) against Saprolenia diclina, S. parasitica and Achlya sp. and fungal infection in ayu Plecogrossus altivelis eggs were evaluated. Zoospore motility of the fungi was greatly inhibited within 1 min in 0.03% KCl, although hyphal growth and zoospore germination of the fungi were not inhibited at a concentration of 0.24% KCl. When fertilized ayu eggs were continuously kept in 0.03%, 0.06% and 0.12% KCl until the eyed-egg stage, treatment at concentrations of 0.06% and 0.12% apparently decreased the rate of fungal infection in three separate experiments. These concentrations showed no effect on the egg-eyed ratio, the subsequent hatching and the larval deformity rates. It was confirmed that KCl was a useful chemical to prevent mold infection in ayu eggs.

Key words: potassium chloride, KCl, antifungal effect, water mold, fungal infection, zoospore, motility, Plecoglossus altivelis

Seawater Survival of Enteromyxum leei (Myxozoa) Evaluated by In Vitro Viability and In Vivo Infectivity Assays

<>Hiroshi Yokoyama1*, Masahiro Kageyama1, Tetsuya Yanagida2 and Kazuo Ogawa1<>1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2Department of Parasitology, Asahikawa Medical College, Hokkaido 078-8510, Japan
(Received July 6, 2009)
ABSTRACT―A vital staining assay using Hoechst 33342 and propidium iodide was developed for the determination of viability of Enteromyxum leei (Myxozoa), a causative agent of myxosporean emaciation disease. Using this in vitro viability assay combined with in vivo infectivity test, the survival of developmental stages of E. leei in seawater was evaluated. Developmental stages of E. leei were freshly isolated from infected tiger puffer Takifugu rubripes and incubated in filtered natural seawater at 20コC for 0, 6, 12 and 24 h. The in vitro staining assay showed a time-dependent decrease in viability, and no viable stages were detected at 24 h. In the first trial of in vivo infectivity, naive tiger puffer were exposed to E. leei suspensions which were incubated in seawater for 0-48 h. In the second trial, naive grass puffer T. niphobles were fed E. leei incubated for 0-24 h. After rearing for 3-4 weeks, infection with E. leei was detected in fish challenged with the parasite incubated for 24 and 6 h in the first and second trials, respectively. Results of the in vitro and in vivo assays showed that the survival time of developmental stages of E. leei in seawater was variable but estimated less than 24 h.

Key words: Enteromyxum leei, Myxozoa, Hoechst 33342, propidium iodide, myxosporean emaciation disease, Takifugu rubripes, tiger puffer

A Bath Challenge Method with Tenacibaculum maritimum for Japanese Flounder Paralichthys olivaceus

<>Toyohiro Nishioka1*, Ken-ichi Watanabe2 and Motohiko Sano1<>1National Research Institute of Aquaculture, Mie 516-0193, Japan
2National Research Institute of Aquaculture, Oita 879-2602, Japan
(Received May 8, 2009)
ABSTRACT―A bath challenge method with Tenacibaculum maritimum was examined using juvenile Japanese flounder Paralichthys olivaceus. After immersed in the bacterial suspensions at different concentrations for 1 h, fish were maintained at 15コC, 20コC and 25コC. At any temperatures, mortalities were 80-100% and 20-40% in fish challenged at 107.9 and 106.9 CFU/mL, respectively. No disease signs were observed in fish challenged at 105.9 CFU/mL. The affected fish exhibited mouth reddening and skin erosion, and T. maritimum were re-isolated from the hemorrhagic skin lesions. The seed lot method used in the present study probably provided reproducible results with high mortality.

Key words: Tenacibaculum maritimum, gliding bacterial disease, bath challenge, Paralichthys olivaceus, Japanese flounder

Surveillance of Salmonid Viruses Especially Targeting Infectious Salmon Anemia Virus in Japan

<>Hisae Kasai*, Shuhei Iwawaki and Mamoru Yoshimizu<>Faculty of Fisheries Sciences, Hokkaido University, Hokkaido 041-8611, Japan
(Received June 24, 2009)
ABSTRACT― Infectious salmon anemia (ISA) is a virus disease of Atlantic salmon Salmo salar in Europe and the Americas, but it has not been isolated in Far East Asia. In this study, we conducted virus isolation with ASK and ASE cells targeting ISA virus (ISAV) from a total of 5,967 fish belonging to eight salmonid species in Japan from 2005 to 2007. ISAV was not isolated from any fish examined but infectious hematopoietic necrosis virus was isolated from 116 fish belonging to three species, while infectious pancreatic necrosis virus was found in 14 fish from three species. It was considered that Japan is still free from ISAV.

Key words: surveillance, salmonid virus, infectious salmon anemia, ISAV, IHNV, IPNV

Development of a PCR-RFLP Method for Differentiation of Perkinsus olseni and P. honshuensis in the Manila Clam Ruditapes philippinarum

<>Miki Takahashi, Tomoyoshi Yoshinaga*, Tsukasa Waki, Jun Shimokawa and Kazuo Ogawa<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
(Received August 24, 2009)
ABSTRACT―We developed a method using PCR-RFLP to differentiate Perkinsus olseni and P. honshuensis, the latter of which was recently discovered as a new species in Mie, Japan, in the Manila clam Ruditapes philippinarum. In normal gill samples spiked with cultured trophozoites of the parasites, the minimum infection levels that could be detected by this method were 100 cells per 10 mg sample for P. olseni and one cell for P. honshuensis. Using this method, we found that clams from the western Seto Inland Sea were infected with both species of the parasites. This is the first report of P. honshuensis from areas outside the type locality of this species.

Key words: Perkinsus olseni, Perkinsus honshuensis, Ruditapes philippinarum, PCR-RFLP