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NO37 - 2

Changes in Variance Value of Immunological and Hematological Parameters Observed in a Population of Japanese Flounder Suffering from Edwardsiellosis

<>Tougo Miyazaki<>Toyama Prefecture Sea Farming Center, 15-1, Sugata, Himi, Toyama, 935-0411, Japan.

ABSTRACT輸 group consisting of 20 individuals of Japanese flounder Paralichthys olivaceus was reared for 178 days, during which edwardsiellosis occurred. Monitoring of immunological (phagocytic rate, NBT reduction and potential killing activity of blood phagocytes and lysozyme activity in the plasma) and hematological (protein concentration and glucose concentration in the plasma) parameters were carried out throughout the period, using the blood periodically collected from each fish. Relative standard deviation (SD/average) values of most of the parameters were expanded several weeks before mortality started. The expansion of these values is thought to be caused by increased physiological differences between normal and infected fish in the group. The variance value of these parameters is suggested to be useful to recognize a sign of outbreaks of infectious diseases.

Preparation of an Inoculum of White Spot Syndrome Virus for Challenge Tests in Penaeus japonicus

<>Jin Lu Wu1, Kazunori Suzuki1, Misao Arimoto2, Toyohiko Nishizawa1 and Kiyokuni Muroga1*<>1Laboratory of Fish Pathology, Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan
2Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan

ABSTRACT輸n inoculum of white spot syndrome virus (WSSV), called penaeid rod-shaped DNA virus (PRDV) in Japan, was prepared for challenge tests in kuruma shrimp Penaeus japonicus. The hemolymph was drawn with PBS from moribund shrimp, which were intramuscularly (IM) inoculated three days before with a virus suspension prepared from naturally affected shrimp. The virus concentration in the hemolymph was quantified as 1.5x107 genome copies /mL hemolymph by competitive PCR. The LD50 of the inoculum prepared from the hemolymph stored at ・0ーC for 40 days was calculated by IM challenges as 10・.2 mL hemolymph /g shrimp or 9.5x102 genome copies/g shrimp. Virulence testes were also conducted through IM injections with the virus preparation stocked for 5 and 16 months. As a result, there was no significant difference in cumulative mortalities (p > 0.05) among the 3 challenge tests made after preservation of the virus in the hemolymph at ・0ーC for 40 days, 5 or 16 months. 

Genomic Similarity of Taura Syndrome Virus (TSV) between Taiwan and Western Hemisphere Isolates

<>Tzu-Wen Lien, Hann-Chang Hsiung, Chih-Cheng Huang and Yen-Ling Song*<>Department of Zoology, National Taiwan University, Taipei, Taiwan ROC

ABSTRACT柚ortalities of white shrimp Litopenaeus vannamei juveniles exceeding 80% have occurred in Taiwan since late 1998. It has been determined that Taura syndrome virus (TSV) is responsible for the epizootic outbreaks.In order to clarify the origin of these epizootics, a 3288 base pair fragment that encodes a TSV coat protein was amplified by RT-PCR. Analysis showed that the Taiwan isolate had 97% and 98% identity to the sequences in Genebank originated from isolates obtained from Mexico and Hawaii, respectively, both in cDNA and deduced amino acid sequences.These results suggest that TSV that infected the white shrimp in Taiwan in late 1998 and early 1999 is similar to the Western hemisphere isolates. We conclude that the Taiwan isolate originated in the Western hemisphere. Urgent and strict quarantine is advised to prevent Taura syndrome dissemination to other areas in Asia.

Occurrence of Bacterial Kidney Disease in Cultured Ayu

<>Takahiro Nagai* and Yoshisuke Iida<>Hiroshima Prefectural Fisheries Experiment Station, Ondo, Hiroshima 737-1207, Japan

ABSTRACT輸 disease characterized by white nodules in the kidney, swollen abdomen and ascitic fluid occurred in cultured ayu Plecoglossus altivelis from April to July, 2001. The bacterial isolate on KDM-2 from the affected ayu was identified to Renibacteirum salmoninarum using IFAT and PCR, and experimental infection resulted in the same clinical signs and re-isolation of the bacterium. From clinical signs, bacteriological examinations and challenge test, this disease in ayu was diagnosed as bacterial kidney disease (BKD). In experimental infection, cumulative mortalities of ayu and masu salmon Oncorhynchus masou were 100% and 60% when injected with 107 cells/fish, and 100% and 0% when injected with 103 cells/fish, respectively. These results indicate that ayu was more susceptible to the isolate than masu salmon, although there is no reported case of BDK in ayu. The affected ayu were originally introduced from another farm in April 2001, where salmonid fishes were reared in neighboring ponds, and it was revealed that masu salmon cultured in the latter farm was infected with R. salmoninarum. This result indicates that R. salmoninarum was horizontally transmitted from masu salmon to ayu.

Renomegaly Associated with a Mycobacterial Infection in Summer Flounder Paralichthys dentatus

<>Kathleen P. Hughes, Robert B. Duncan, Jr. and Stephen A. Smith*<>Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 USA

ABSTRACT輸 population of juvenile (28 cm, 180 g) summer flounder Paralichthys dentatus developed severe coelomic distention, anorexia and lethargy over a period of 4 to 6 months. Gross internal lesions included a large coelomic mass that replaced the posterior kidney and yellow foci on the heart, spleen and liver. Histopathologically, the posterior kidney was obliterated by extensive granulomatous inflammation and rare calcified granulomas. Special stains revealed extracellular acid-fast bacilli in the areas of inflammation. An isolated bacterium belonging to the genus Mycobacterium was the presumptive causative agent of these lesions.

Nodavirus Infection in Hatchery-Reared Orange-Spotted Grouper Epinephelus coioides : First Record of Viral Nervous Necrosis in the Philippines

<>Yukio Maeno1*, Leobert D. de la Pena2 and Erlinda R. Cruz-Lacierda2<>1Japan International Research Center for Agricultural Sciences, Ohwashi 1-1, Tsukuba, Ibaraki 305-8686, Japan
2Southeast Asian Fisheries Development Center, Tigbauan 5021, Iloilo, Philippines

ABSTRACT柚ass mortality occurred in 34-day old larval orange-spotted grouper Epinephelus coioides reared at a hatchery in the Philippines with clinical signs such as anorexia and abnormal swimming behavior. Histopathology of moribund fish demonstrated marked vacuolation of the brain, spinal cord and retina. Cytopathic effects were observed in SSN-1 cells inoculated with the tissue filtrate of affected grouper. Electron microscopy revealed non-enveloped virus particles measuring 20 to 25 nm in diameter in the cytoplasm of degenerated SSN-1 cells. Piscine nodavirus (betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues and SSN-1 cells inoculated with the tissue filtrate of affected fish by RT-PCR. This is the first record of VNN in the Philippines.

Effectiveness of a Vaccine against Red Sea Bream Iridoviral Disease in Various Cultured Marine Fish under Laboratory Conditions

<>Kazuhiro Nakajima1*, Takafumi Ito1, Jun Kurita1, Hidemasa Kawakami2, Tomokazu Itano2, Yutaka Fukuda3, Yumiko Aoi4, Tetsurou Tooriyama4 and Sadao Manabe4<>1Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan
2Ehime Prefectural Fish Disease Control Center, Sakashizu, Uwajima, Ehime 798-0087, Japan
3Oita Institute of Marine and Fisheries Science, Kamiura, Minami-Amabe, Oita 879-2602, Japan
4Research Foundation for Microbial Diseases of Osaka University, Yahata, Kanonji, Kagawa 768-0061, Japan

ABSTRACT裕he effectiveness of a vaccine made of the cell culture supernatant of RSIV (red sea bream iridovirus)-infected cells inactivated with formalin (0.1% v/v) in yellowtail Seriola quinqueradiata, amberjack S. dumerili, kelp grouper Epinephlelus moara, striped jack Pseudocaranx dentex and spotted parrot fish Oplegnathus punctatus was evaluated under experimental conditions. Juvenile or young fish were intraperitoneally injected with the vaccine, and challenged with RSIV by intraperitoneal injection at 10th day post-vaccination. Statistical analysis showed significantly higher survival rates in the vaccinated groups of all tested fish species.

Age-dependent Humoral Immune Response of Barfin Flounder against Recombinant Coat Protein of Barfin Flounder Nervous Necrosis Virus

<>Ken-ichi Watanabe1* and Mamoru Yoshimizu2<>1Akkeshi Station of Japan Sea-Farming Association, Chikushikoi, Akkeshi, Hokkaido, 088-1108, Japan.
2Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, 041-8611, Japan.

ABSTRACT輸ge-dependent humoral immune response of barfin flounder Verasper moseri was investigated. One hundred mL of recombinant coat protein of barfin flounder nervous necrosis virus(BFNNV)adjusted to 250 mg/mL in Tris-EDTA buffer(pH 8.0)was injected intravascularly to 4, 5, 6 and 7 month-old fish. One month after injection, an enzyme-linked immunosorbent assay(ELISA)antibody titer was measured. Although no ELISA antibody titers were detected in injected or non-injected fish of 4 and 5 month-old, the antibody titers were remarkably increased(p<0.025)in 6 and 7 month-old fish. High ELISA antibody titers were kept for at least 2 months.

Effect of Water Temperature on Pathological States of Japanese Flounder Experimentally Infected with Viral Hemorrhagic Septicemia Virus, an Flounder Isolate KRRV-9601

<>Tadashi Isshiki 1*, Taizou Nagano1 and Teruo Miyazaki2<>1Kagawa Prefectural Fisheries Experiment Station, Takamatsu, Kagawa 761-0111, Japan
2Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan

ABSTRACT裕he effect of water temperature on virus propagation, mortality and histopathological changes of Japanese flounder Paralichthys olivaceus intraperitoneally injected with an isolate (KRRV-9601) of VHSV from flounder were examined. Mortalities for 15 days at 10, 15 and 20ーC were 100, 60 and 0%, respectively. At 10ーC, all of the infected fish showed high virus titers in both the heart and kidney, and histopathologically displayed necrotizing myocarditis and necrotic lesions in the spleen, hematopoietic tissue and liver. At 15ーC, all fish showed higher virus titers in the heart than in the kidney, and myocardial necrosis. No histopathological changes were observed and no VHSV was recovered from fish at 20ーC.

Iron-acquisition Ability of Edwardsiella tarda with Involvement in Its Virulence

<>Arisa Igarashi1, Takaji Iida2* and Jorge H. Crosa3<>1The United Graduate School of Agricultural Sciences, Kagoshima University,Korimoto 1-21-24, Kagoshima 890-0065, Japan
2Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-nishi 1-1, Miyazaki, Miyazaki 889-2192, Japan.
3Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 93702, USA

ABSTRACT友orty-one strains of Edwardsiella tarda including both virulent and avirulent strains were examined in their iron-acquisition ability. The strains were divided into two groups by growth under an iron-limiting condition (LB broth containing casamino acid in the presence of 6.25 mm EDDHA); one group was able to grow more than 102 times the initial bacterial density, but the other was not. The bioassay and the modified CAS assay for detecting bacterial siderophore production showed that the strains of the former group produced the siderophore with a higher ability for iron-acquisition than those of the latter group. The siderophore-producing strains include all virulent ones. In addition, a natural mutant that showed lowered siderophore production had a remarkably reduced virulence. All of strains produced two outer membrane proteins, one of which is considered to be a receptor of the siderophore under the iron-limiting condition, indicating that these proteins are hardly related to the virulence of E. tarda. Our data in the present study suggest that the iron-acquisition ability of E. tarda by the siderophore is involved in its virulence.
Fish Pathology, 37:53-57, 2002.