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NO38 - 3

Detection of White Spot Syndrome Virus from Small Penaeid Shrimp Species Caught in the Western Seto Inland Sea

<>Kazuo Momoyama<>Inland Sea Division, Yamaguchi Prefectural Fisheries Research Center,
Aio-Futashima, Yamaguchi 754-0893, Japan
(Received February 26, 2003)
ABSTRACT悠nfection rates with white spot syndrome virus (WSSV) in 4 small penaeid shrimp species, Metapenaeopsis acclivis, M. barbata, M. dalei and Trachypenaeus curvirostris collected from the western Seto Inland Sea of Japan, was examined by PCR, and transmission of the virus to juvenile Penaeus japonicus was tried by oral or intramuscular inoculation of infected samples. No shrimp collected from the Aki-Nada and Iyo-Nada regions were shown to carry WSSV, except 2 out of 274 individuals of M. dalei from Iyo-Nada. Infection rates of M. acclivis, M. barbata and T. curvirostris collected from the Suo-Nada region were 29, 40 and 23%, respectively. Oral administration of small pieces of PCR positive shrimp muscle did not kill any challenged shrimp during the test period, but could establish infection with WSSV. Half of the challenged shrimp injected with a filtered homogenate of PCR positive shrimp muscle died with typical signs of WSS within 11 days.

Key words: shrimp virus, WSS, WSSV, PAV, PRDV, infection trial, penaeid shrimp, carrier

Fate of Viral Hemorrhagic Septicemia Virus in Japanese Flounder Paralichthys olivaceus Challenged by Immersion<>Hideki Iida1, Koh-ichiro Mori2, Toyohiko Nishizawa1,3, Misao Arimoto2 and Kiyokuni Muroga1,4*

<>1Graduate School of Biosphere Sciences, Hiroshima University, Higashihiroshima 739-8528, Japan
2Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan
3Present address: Graduate School of Fisheries Sciences, Hokkaido University,
Hakodate 041-8611, Japan
4Present address: Graduate School of Agricultural Science,
Tohoku University, Sendai 981-8555, Japan
(Received March 6, 2003)
ABSTRACT友ate of viral hemorrhagic septicemia virus (VHSV) in tissues of Japanese flounder Paralichthys olivaceus (20.7 g in average body weight) challenged by immersion for 1 h with a flounder isolate (Obama 25, an American genotype) of VHSV at 104.0 TCID50/mL was monitored for 7 weeks by virus isolation method with FHM cell line. The mortality of Japanese flounder challenged by the same method in a companion experiment reached 68% at 13°C at week 6 post-immersion challenge (PIC). VHSV was detected in the gills just after the immersion, in the kidney, spleen and skin at day 1 PIC, and in all the tissues tested at day 3 PIC. The infective titers of VHSV higher than 109.0 TCID50/g were measured in the heart and blood as well as the foregoing tissues except skin at week 1or 2 PIC. VHSV was not detected from most tissues at week 4 PIC except from the gills, heart, brain and blood, and at week 6 it was isolated only from the heart and brain. When the virus was not isolated from any tissues at week 7 PIC, the surviving fish were given stresses by 3 different methods, i.e. shaking of the fish container, or abrupt raise (13°C to 18°C) or fall (13°C to 8°C) in water temperature. As a result, VHSV was detected from the heart of some fish after the temperature stresses, indicating that the heart is the most probable hiding site of the host for VHSV.

Key words: viral hemorrhagic septicemia virus, Paralichthys olivaceus, carrier, exprimental infection, viral fate, temperature stress, Japanese flounder, VHSV

A PCR Method to Detect Nocardia seriolae in Fish Samples

<>Yasuyuki Miyoshi and Satoru Suzuki*<>Center for Marine Environmental Studies (CMES), Ehime University,
Matsuyama 790-8577, Japan
(Received March 7, 2003)
ABSTRACT祐pecies-specific polymerase chain reaction (PCR) targeting the 16S rRNA gene of Nocardia seriolae was developed. The PCR targeted nucleotide #609 to 1038 (Escherichia coli numbering), which gave a 432 bp-length product. This method could detect N. seriolae type strain (JCM3360) and eight clinical isolates of this species from yellowtail Seriola quinqueradiata and Japanese flounder Paralichthys olivaceus, but not those of other bacterial species including 5 other Nocardia spp. and 4 yellowtail pathogens. The detection limit of the PCR was 102 CFU. Eight diseased yellowtail were employed for detection of the bacterium by the PCR. Positive results were obtained from all fish.

Key words: Nocardia seriolae, PCR, 16S rDNA, Seriola quinqueradiata

Influence of Hemoglobin Concentration on Tolerance to Bacterial Gill Disease in Rainbow Trout

<>Atsushi Yamamoto1*, Jun Nagura2 and Takaji Iida3<>1Department of Aquatic Resource Science, Faculty of Fisheries, Kagoshima University,
Kagoshima 860-0056, Japan
2Oshino Trout Hatchery, Yamanashi Prefectural Fisheries Technology Center, Oshino,
Yamanashi 401-0511, Japan
3Aquatic Animal Health Division, National Research Institute of Aquaculture,
Fisheries Research Agency, Nansei, Mie 516-0193, Japan
(Received March 12, 2003)
ABSTRACT邑e induced three types of triploid rainbow trout by retention of the second polar body (RSP) and cross fertilization between tetraploids and diploids (4♀× 2♂ and 2♀× 4♂) and found that hemoglobin concentration of triploid (4♀× 2♂) was higher than those of the others. No difference was observed in their hypoxic tolerance between normal two triploids (4♀× 2♂ and 2♀× 4♂). However, significant differences were observed in the mortalities in bacterial gill disease (BGD)-infected triploids. The tolerance to BGD in triploid (4♀× 2♂) was higher than those in triploid (RSP) and triploid (2♀× 4♂). Since it is known that fish with BGD die from asphyxiation and its mortality is affected by the environmental oxygen level, hemoglobin concentration of fish would be closely associated with the tolerance against BGD.

Key words: hemoglobin, bacterial gill disease, triploid, anemia, Oncorhynchus mykiss, rainbow trout, hypoxic tolerance

Effect of Water Temperature and Flow Rate on the Transmission of Microsporidial Gill Disease Caused by Loma salmonae in Rainbow Trout Oncorhynchus mykiss

<>Joy A. Becker*, David J. Speare and Ian R. Dohoo<>Atlantic Veterinary College, University of Prince Edward Island Charlottetown,
Prince Edward Island Canada, C1A 4P5
(Received May 21, 2003)
ABSTRACT裕wo studies were designed to quantify the effect of water temperature and flow rate on the transmission potential of the important salmonid gill pathogen, Loma salmonae. Using survival analysis, increased water temperature and low flow rates were determined as risk factors for the transmission of microsporidial gill disease caused by L. salmonae in rainbow trout Oncorhynchus mykiss. Fish were experimentally infected with L. salmonae via a cohabitation exposure model and monitored for the development of branchial xenomas. On any given day, fish held at 11°C and 15°C had a hazard ratio equal to 0.80 and 0.68, respectively, for the development of branchial xenomas compared with fish held at 19°C. From the flow rate trial, fish housed in a low flow tank (0.83 L/min) had an increased chance of developing branchial xenomas when compared to fish in tanks at normal (1.67 L/min) and high (2.5 L/min) flow rates with hazard ratios reported as 0.69.

Key words: survival analysis, Loma salmonae, temperature, flow rate, disease management, hazard function, Oncorhynchus mykiss, rainbow trout

Susceptibility of Various Marine Fish Species to Viral Hemorrhagic Septicemia Virus Isolated from Japanese Flounder

<>Tadashi Isshiki1*, Taizou Nagano1 and Teruo Miyazaki2<>1Kagawa Prefectural Fisheries Experiment Station, Takamatsu, Kagawa 761-0111, Japan
2Faculty of Bioresources, Mie University,
Tsu, Mie 514-8507, Japan

(Received March 26, 2003)

ABSTRACT・Susceptibilities of six species of marine fish to a flounder isolate KRRV-9601 (a European genotype) of VHSV were examined by intraperitoneal injection. Mortalities were induced in Japanese flounder Paralichthys olivaceus (100%), black sea bream Acanthopagrus schlegeli (90%), red spotted grouper Epinephelus akaara (70%) and rockfish Sebastes schlegeli (10%), but not in marbled sole Pleuronectes yokohamae and red sea bream Pagrus major. VHSV was recovered from dead and moribund fish and the surviving red spotted grouper at 60th day after the virus injection. The neutralizing antibody against VHSV was detected in the sera of surviving rockfish, but not in other surviving fishes. Histopathological features in the
affected fish for each species were generally similar to each other, although the severity of hemorrhage varied among fish species.

Key words: viral hemorrhagic septicemia virus, host range, experimental infection, susceptibility, Paralichthys olivaceus, VHSV

Rapid In Situ Hybridization Technique for the Detection of Fish Microsporidian Parasites<>Sun-Joung Lee, Hiroshi Yokoyama* and Kazuo Ogawa

<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Tokyo 113-8657, Japan

(Received April 23, 2003)

ABSTRACT由apid in situ hybridization (ISH) technique was developed for the detection of fish microsporidian parasites. All developmental stages of Glugea plecoglossi including the initial stage immediately after entry into the fish tissue could be detected by rapid ISH using DIG-labelled oligonucleotide probes. However, ISH signals were only faintly visible from mature spores of Microsporidium seriolae, while the pre-sporogonic stages were clearly positive. Thus, it is recommended to use both rapid ISH and Uvitex 2B stain for the detection of all stages of M. seriolae. The rapid ISH allows the whole process to be completed within 2 h, and thus this technique is both convenient and practical as a diagnostic and research tool for studying the life cycle of microsporidians.

Key words: in situ hybridization, microsporidia, diagnosis, detection, Glugea plecoglossi, Microsporidium seriolae