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NO48 - 4

Efficacy of High-concentration Ascorbic Acid Supplementation against Infectious Hematopoietic Necrosis in Salmonid Fish Influenced by Viral Strain and Fish Size

Takanori Ishikawa1, Nobuhiro Mano2*, Kai Minakami2, Aki Namba3,4, Takahito Kojima2, Hitomi Hirose2
and Teruyuki Nakanishi3
1Tochigi Prefectural Fisheries Experimental Station, Tochigi 324-0404, Japan
2Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
3Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
4Research Fellow of Japan Society for the Promotion of Science

(Received April 16, 2013)

ABSTRACT―Although dietary supplementation with high-concentration ascorbic acid (AsA) can be a promising approach for reducing fish losses due to infectious hematopoietic necrosis (IHN), ineffective cases of AsA supplementation have occasionally been reported in fish farming operations. In the present study, we investigated the influence of viral strains and fish sizes on the efficacy of AsA supplementation in rainbow trout Oncorhynchus mykiss and kokanee O. nerka. Fish with different body weights were fed commercial diets supplemented with 5,000 mg of AsA per kg of diet for 7 days, and then challenged by bath exposure with two IHNV isolates (TK8901 and TV0026) belonging to different genogroups. AsA supplementation in the diet did not significantly reduce the mortality of 1-g rainbow trout as a result of infection with either isolate. On the other hand, the mortality of 2-g kokanee and 6-g rainbow trout fed AsA decreased significantly against TV0026 infection. The efficacy of AsA against TK8901 infection was, however, not confirmed with these fish sizes. These results revealed that differences in IHNV strain or fish size can affect the efficacy of high-concentration AsA administration in IHN of farmed salmonid fish.
Key words: ascorbic acid, vitamin C, prophylactic effect, infectious hematopoietic necrosis, IHN, Oncorhynchus mykiss, Oncorhynchus nerka

Microsporidian Encephalomyelitis in Cultured Yellowtail Seriola quinqueradiata

Hiroshi Yokoyama*, Yohei Miyazaki and Tomoyoshi Yoshinaga
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

(Received July 16, 2013)

ABSTRACT―Microsporidian encephalomyelitis of the greater amberjack Seriola dumerili is characterized by a spiral swimming movement in diseased fish, and has been reported in fish imported into Japan from China. In 2010, a similar disease was found in cultured yellowtail Seriola quinqueradiata, which were raised in Japan using wild-captured fingerlings. In the present study, a microsporidian was consistently observed in the nervous tissue of all diseased yellowtail specimens, supporting the hypothesis that the microsporidian is the etiologic agent of the disease. Morphological and molecular similarities between the yellowtail and amberjack microsporidia indicate that they belong to the same species. Phylogenetic analysis showed that the present microsporidian was closely related to the genus Spraguea. Histopathological examination of affected yellowtail revealed encephalomyelitis with severe gliosis, degenerate axons and necrotic nerve cells with cytoplasmic vacuoles, together with aggregates of microsporidian spores in the lesions. Field investigations of cultured yellowtail at the same farm where the disease occurred in 2010 showed that disease incidence and infection prevalence decreased in 2011 and 2012. The present microsporidian was probably transferred to Japan from China through importation of juvenile greater amberjack, followed by transmission to cultured yellowtail.
Key words: microsporidian, Seriola quinqueradiata, yellowtail, encephalomyelitis, Spraguea, greater amberjack

Establishment of a Cell Line from Chinese Soft-shelled Turtle Pelodiscus sinensis with the Practicability of Transfection and Viral Replication

Jian Ping Fu1,2, Zhang Luo1, Yang Yan3, Peng Fei Zou1, Shu Huan Zhang1, Qi Wei Qin3 and Pin Nie1,4*
1State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, China
2University of Chinese Academy of Sciences, Beijing 100049, China
3CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangdong Province 510301, China
4College of Fisheries, Jimei University, Xiamen, Fujian Province 361021, China

(Received July 16, 2013)

ABSTRACT―A continuous cell line derived from extracranial carotid artery of the Chinese soft-shelled turtle Pelodiscus sinensis was established and characterized. It was named as STA (soft-shelled turtle artery) cell line. The cells were muscle-cell like, and were stained with antibody against smooth muscle myofilament marker, a-smooth muscle actin. These cells had a normal diploid chromosome number of 2n = 66, and their 12S rRNA and 16S rRNA sequences shared 99% identity with ones of the turtle in database. The cell line can be cultured well in media DMEM/F12 or M199 supplemented with 10% FBS at 28°C. It was further demonstrated that the cells were transfected successfully with pTurbo plasmid, with a transfection efficiency being over 30%. The soft-shelled turtle iridovirus (STIV) propagated in the cell line, causing typical CPE with the formation of inclusion bodies. It is suggested that the established STA cell line provides a convenient platform for studying the pathogenesis of STIV and biological aspects of the turtle.
Key words: cell line, Pelodiscus sinensis, Chinese soft-shelled turtle, transfection, soft-shelled turtle iridovirus, STIV

Genotyping of Flavobacterium psychrophilum Isolated from Chum Salmon Oncorhynchus keta in Hokkaido, Japan

Makoto Hatakeyama*, Naoyuki Misaka, Shinya Mizuno and Nobuhisa Koide
Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan

(Received September 10, 2013)

ABSTRACT―We examined matured female chum salmon Oncorhynchus keta returning to a river, at a hatchery three times a year between 2006 and 2008, and chum salmon fry suffering from bacterial coldwater disease at the same hatchery in 2008 for Flavobacterium psychrophilum isolation. The isolates were genotyped by plasmid and RAPD analyses. In adults, the frequency distribution of genotypes varied among different years but was seemingly similar among different sampling opportunities within the same years. The RAPD genotype of the isolates from fry was one of major genotypes isolated from their parental group, which suggests vertical transmission of the bacterium.
Key words: Flavobacterium psychrophilum, Oncorhynchus keta, chum salmon, plasmid profiling, RAPD