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NO52 - 1

Infectious Hematopoietic Necrosis

Toyohiko Nishizawa1* and Mamoru Yoshimizu2

1Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea
2Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan

ABSTRACT― Infectious hematopoietic necrosis (IHN) is a disease of salmonid fishes, mainly Oncorhynchus spp., including rainbow trout O. mykiss and Atrantic salmon Salmo salar. IHN causes serious economic losses. It was initially known as endemic in sockeye salmon fry O. nerka and chinook salmon fry O. tschawytscha in West Coast of North America since the 1950s. However, it was spread to Japan, Korea and Taiwan in the 1970s. It was spread to Italy and France in the 1990s. Currently, IHN can be found in many parts of the world, including Russia and South America. Mortality due to IHN in fish with ≤ 0.5 g of body weight can reach 60% to 100%. Mortality caused by IHN will reduces when fish grow bigger. However, the onset of IHN has also increased recently in fish with larger. Here, we describe the molecular epidemiology and virulence changes of IHN virus (IHNV) in Japan, and the detection and identification methods for IHNV. Furthermore, we will discuss the future prospects of IHNV researches.

Neoheterobothrium hirame Infection of Olive Founder

Tomoyoshi Yoshinaga*

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

ABSTRACT― In the mid-1990s, wild olive flounder showing heavy anemia characterized by deformed erythrocytes with low-stained cytoplasm appeared in Japanese waters. Parasitological, histopathological and hematological studies and surveys revealed that the anemia was caused by the infection with the monogenean Neoheterobothrium hirame on the gill and buccal cavity wall of host. The parasite was later revealed to originally infect southern flounder Paralichthys lethostigma in north America and invade Japanese waters. Bathing in NaCl-supplemented seawater is effective to remove worms from fish. The parasite has been prevailing in wild stocks of olive flounder and may threat the stocks especially in warmer waters.

Akoya Oyster Disease

Hirofumi Yamashita* and Kazushi Odawara

Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries Shitaba, Ehime 798-0104, Japan

ABSTRACT― Since 1996, mass mortalities of cultured Japanese pearl oyster Pinctada fucata martensii have been occurring in western Japan. Mortalities are accompanied with reddish-brown discoloration of the soft body, especially the adductor muscle of oysters. This disease was named "akoya oyster disease (AOD)". However, the cause of the disease is still unidentified. As a control strategy of this disease, selective breeding of pearl oysters resistant to AOD has been conducted in several public institutes and private groups. The recent decline in outbreaks of AOD can be attributed, in part, to the introduction of selectively bred oysters that are resistant to this disease.

Molecular Characterization and Expression Analysis of Tumor Necrosis Factor Alpha-induced Protein 3 (TNFAIP3/A20)
Gene from Japanese Pufferfish Takifugu rubripes

Jun-ichi Hikima1*, Misaki Morita1, Shunsuke Kinoshita1, Madhubanti Basu2, Gouranga Biswas3, Tomoya Kono1 and Masahiro Sakai1

1Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
2Fish Health Management Division, ICAR-Central Institute of Freshwater Aquaculture, Odisha 751002, India
3Kakdwip Research Centre of ICAR-Central Institute of Brackishwater Aquaculture, West Bengal 743347, India

ABSTRACT― Tumor necrosis factor alpha (TNF-α)-induced protein 3 (TNFAIP3/A20) is an important deubiquitinating enzyme that takes part in homeostasis of immunity induced by TNF-α. During viral and bacterial infection, it plays a crucial role in negative regulation of innate immune responses in mammals. However, this molecule in fish is still poorly understood. In this study, we identified full-length A20 gene of Japanese pufferfish (Fugu) Takifugu rubripes and performed its expression analyses in various tissues and stimulated cells. Total length of the determined Fugu A20 gene spaned 6,229 bp and consisted of 9 exons and 8 introns. The Fugu A20 gene contained a 2,394 bp open reading frame (ORF) that encoded a 797-amino acid residue. In the exon-intron structure of Fugu A20 gene, the intron-boundary positions in the region having 7 zinc finger (Zf) domains were different from those of human. Expression analysis of Fugu A20 gene exhibited a higher transcription in thymus, head kidney (HK) and spleen tissues than the others. In HK and spleen cells stimulated with LPS, polyI:C and imiquimod, A20 gene was expressed after up-regulation of TNF-α gene expression. These results together suggest that expression of Fugu A20 gene is associated with bacterial and viral infections.

Key words:Tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20), TNF-α, Zinc finger domain, Takifugu rubripes, Japanese pufferfish, Fugu

Evaluation of an RSIVD-resistant Trait of Red Sea Bream
Pagrus major Broodstock Using DNA-based
Pedigree Tracings: A Field Study

Eitaro Sawayama1* and Motohiro Takagi2

1R&D Division, Marua Suisan Co., Ltd., Ehime 794-2410, Japan
2South Ehime Fisheries Research Center, Tarumi Branch, Ehime University, Ehime 790-8566, Japan

ABSTRACT― In this study, we evaluated the red sea bream iridoviral disease-resistant (RSIVD-resistant) trait of broodstock of red sea bream used in a commercial production based on DNA parentage analysis. We compared family structures of two groups from the same production lot and cultured at two different farms: a population without any outbreak of diseases (farm A) and a high mortality population after an RSIVD outbreak (farm B). The survival percentage of the fish at the farm A and farm B were 86.4% and 20.5%, respectively. Pedigree tracing was conducted on 200 individuals from each farm using eight microsatellite DNA markers among 22 potential breeders (20 dams and 2 sires), and parental pairs at farm A and B were successfully assigned with 93.0 and 90.5%, respectively. After allocating parentage information to each specimen collected from both farms, the estimated survival percentages of each family were calculated. The estimated survival of offspring from a male was 82.3%, and this survival was higher than that from the other male (estimated survival was 2.5%). Also, offspring from a female showed a high survival. These results suggest that some broodstock have resistant traits against RSIVD and shows the potential for developing an RSIVD-resistant strain of red sea bream.

Key words:Pagrus major, red sea bream, RSIVD, pedigree tracing, resistant, family structure, microsatellite, field study

Haliphthoros sabahensis sp. nov. Isolated from Mud Crab Scylla tranquebarica Eggs and Larvae in Malaysia

Yih Nin Lee1, Kishio Hatai1* and Osamu Kurata2

1Microbiology and Fish Disease Laboratory, Borneo Marine Research Institute, Universiti Malaysia Sabah, Sabah 88400, Malaysia
2Laboratory of Aquatic Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan

ABSTRACT―Mass mortality of eggs and larvae of the mud crab Scylla tranquebarica occurred at a hatchery in Sabah, Malaysia in June 2014. The mortality of the larvae reached nearly 100% within 5 days post-hatching. We isolated a pathogenic Peronosporomycetes (Oomycota) from the mud crab eggs and larvae using peptone-yeast-glucose-seawater (PYGS) agar, and then randomly selected the strain IPMB 1402 from the isolates for further study. We observed the formation of fragments inside the hyphae, a characteristic of Haliphthoros species. The strain morphologically most resembled Haliphthoros milfordensis, but comparisons of DNA sequences of the ITS1 region confirmed that the strain was not identical to that species. We classified the strain into a clade with Haliphthoros sp. group 2 (comprising strains NJM 0440, NJM 0449 and NJM 0535) after determining 97-100% intraspecific similarity. Based on distinctive morphological and molecular characteristics, the strain IPMB 1402 is here assigned to the genus Haliphthoros and designated Haliphthoros sabahensis sp. nov. Like other recognized species of Haliphthoros, it is an obligate marine fungus; growth was observed in pH 4-9, and optimum growth temperature was 25-30°C. The strain was experimentally pathogenic to nauplii of the brine shrimp Artemia salina.

Key words:Haliphthoros sabahensis sp. nov., Scylla tranquebarica, Artemia salina, oomycetes, pathogenicity, Peronosporomycetes

Mortality of Pond-cultured Litopenaeus vannamei Associated with Acute Hepatopancreatic Necrosis Disease (AHPND) and White Spot Syndrome Virus (WSSV) Infection in the Philippines

Leobert D. de la Peña1*, Nikko Alvin R. Cabillon2, Edgar C. Amar1, Demy D. Catedral1, Roselyn C. Usero3, Joseph P. Faisan, Jr.1, Joey I. Arboleda1, Wilberto D. Monotilla4, Adelaida T. Calpe5, Dalisay D. G. Fernandez5 and Cynthia P. Saloma4

1Aquaculture Department, Southeast Asian Fisheries Development Center, Iloilo 5021, Philippines
2University of Highlands and Islands-Scottish Association for Marine Science, Oban PA37 1QA, United Kingdom
3Negros Prawn Producers Cooperative Analytical and Diagnostic Laboratory, Bacolod City 6100, Philippines
4Philippine Genome Center, University of the Philippines Diliman, Quezon City 1101, Philippines
5Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development, Laguna 4030, Philippines

ABSTRACT― Mortalities of up to 60% were observed in pond-cultured Litopenaeus vannamei in Bohol, Philippines. Histopathological examination revealed typical acute hepatopancreatic necrosis disease (AHPND) pathology. PCR test generated 1,269 bp and 230 bp amplicons confirmative for the toxin-producing AHPND strain of Vibrio parahaemolyticus among shrimp sampled from eight ponds. The same samples were subjected to PCR analyses for the presence of other viruses, namely WSSV, IHHNV, IMNV, and TSV. The samples were negative for the viruses except WSSV, which was detected after one-step PCR in six out of eight ponds. These results suggested that shrimp were infected dually with AHPND V. parahaemolyticus and WSSV.

Key words:AHPND, Vibrio parahaemolyticus, WSSV, Litopenaeus vannamei, dual infection

Selective Medium Design for the Japanese Eel Pathogen Edwardsiella tarda by SMART: Selective Medium- design Algorithm Restricted by Two Constraints

Atsushi Yamamoto*, Hiroshi Kitano and Kyosuke Araki

Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan

ABSTRACT― The SMART established first to selectively isolate plant pathogens was applied to develop a selective medium for isolation of fish-pathogenic Edwardsiella tarda. The SMART medium selectively allowed the growth of E. tarda only when a mixture of E. tarda and other fish pathogens was inoculated onto the medium. The medium also enabled the direct culturing of E. tarda from diseased Japanese eel Anguilla japonica. However, two other strains from eel intestinal contents and diseased tilapia did not grow on the medium. Most of E. tarda strains from diseased marine fishes also did not grow. Therefore, the medium made by SMART was applicable to selectively isolate E. tarda from diseased Japanese eel.

Key words:SMART, selective medium, Edwardsiella tarda, Anguilla Japonica, Japanese eel

PCR-mediated Identification of the Newly Emerging Pathogen Lactococcus garvieae Serotype II from Seriola quinqueradiata and S. dumerili

Kazuya Ohbayashi1, Daisaku Oinaka1, Truong Dinh Hoai1, Terutoyo Yoshida1 and Issei Nishiki2*

1Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
2Research Center for Bioinformatics and Biosciences, National Research Institute of Fisheries Science, Fisheries Research and Education Agency, Yokohama 236-8648, Japan

ABSTRACT― The newly emerging pathogen Lactococcus garvieae serotype II, which appears to be morphologically and biochemically indistinguishable from L. garvieae serotype I, has been isolated from farmed Seriola quinqueradiata and S. dumerili. To distinguish the two serotypes, a primer set was designed to target the glxR-argS intergenic region. PCR with the primer set amplified different-sized fragments: 1,285 bp for serotype II and 285 bp for serotype I. Therefore, the primer set enables the PCR-mediated identification of serotype II strains isolated from marine fish species in Japan.

Key words: Lactococcus garvieae, serotypes II, PCR, Seriola quinqueradiata, S. dumerili