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NO38 - 1 (2003)

Development of a Real-time PCR Assay for the Detection and Quantification of Red Seabream Iridovirus (RSIV)

<>Christopher Marlowe Caipang, Ikuo Hirono and Takashi Aoki<>Laboratory of Genetics and Biochemistry, Department of Aquatic Biosciences,
Tokyo University of Fisheries, Konan 4-5-7, Minato-ku,
Tokyo 108-8477, Japan
(Received June 7, 2002)

ABSTRACT輸novel real-time PCR detection and quantification method for the red seabream iridovirus (RSIV) was developed using the major capsid protein (MCP) gene. A linear relationship was observed between the amount of input DNA and the threshold cycle (CT) values over a range of 1 to 109 viral copies. The real-time PCR was 2 orders of magnitude more sensitive than the conventional PCR. It was also shown to be specific to RSIV, i.e., it did not react with either hirame rhabdovirus (HIRRV), viral hemorrhagic septicemia virus (VHSV) or a DNA sample from apparently healthy red seabream Pagrus major. The viral load of dead juvenile red seabream (1 g average body weight) experimentally challenged with RSIV was in the range of 2.4 エ 105 ミ 5.4 エ 107 MCP gene copy number. To determine the target tissues of RSIV in the infected fish, 2 red seabream with average body weight of 50 g were experimentally challenged with RSIV and DNA was
extracted from the various tissues and organs at 3rd day post-infection. Highest viral load was obtained in the heart with a mean MCP gene copy number of 2.7 エ 105, while the blood had the lowest viral load at 8.8 エ 103 mean MCP gene copy number. After the viral challenge, the real-time PCR assay was still able to detect RSIV from vaccinated survivors, which otherwise tested negative for RSIV by conventional PCR.

Key words: real-time PCR, red seabream iridovirus, RSIV, Pagrus major

Identification of Japanese Flounder Leucocytes Involved in the Host Response to Neoheterobothrium hirame

<>Chihaya Nakayasu1*, Nobuyuki Tsutsumi2, Tomoyasu Yoshitomi3,
Tomoyoshi Yoshinaga1,2 and Akira Kumagai1,4<>1Inland Station, National Research Institute of Aquaculture, Fisheries Research Agency,
Tamaki, Mie 519-0423, Japan
2Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan
3Aqua Restoration Research Center, Independent Administrative Institution
Public Works Research Institute Government Land Reserve,
Kasada-machi, Kawashima-cho, Gifu, 501-6021 Japan
4Miyagi Prefectural Freshwater Fisheries Experimental Station, Taiwa,
Miyagi 981-3625, Japan
(Received October, 21, 2002)

ABSTRACT有eucocyte types involved in the cellular response of Japanese flounder Paralichthys olivaceus against the monogenean Neoheterobothrium hirame were identified. With electron microscopy, peripheral blood leucocytes (PBL) of Japanese flounder could be classified into four types: lymphocytes, thrombocytes, monocytes and granulocytes. Granulocytes were positive for acid phosphatase and peroxidase activities, and stained with Sudan black B. In fish infected with N. hirame, the ratios of granulocytes and monocytes significantly increased in the PBL composition. Infiltration of monocytes/macrophages and granulocytes, and cells having large electron-dense granules (DGC) was observed in the infection sites. These cells were often observed adhering to the tegument of N. hirame. Monocytes/macrophages showed particularly higher adherent ability to N. hirame than the other leucocytes under in vitro and in vivo conditions. We conclude that monocytes/macrophages, granulocytes and DGC are major effector cells in the defense of the flounder against N. hirame.

Key words: Paralichthys olivaceus, defense system, Neoheterobothrium hirame, Monogenea, granulocyte, monocyte, macrophage, leucocyte

Experimental Coinfection with Aquabirnavirus and Viral Hemorrhagic Septicemia Virus (VHSV),Edwardsiella tarda or Streptococcus iniae in Japanese Flounder Paralichthys olivaceus

<>Rolando Pakingking Jr.1, Ryoko Takano1, Toyohiko Nishizawa2, Koh-ichiro Mori3, Yoshisuke Iida4, Misao Arimoto3 and Kiyokuni Muroga1*<>1Graduate School of Biosphere Sciences, Hiroshima University, Higashi-hiroshima 739-8528, Japan
2Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
3Kamiura Station of Japan Sea-Farming Association, Kamiura, Oita 879-2602, Japan
4Hiroshima Prefectural Fisheries Experiment Station, Ondo,
Hiroshima 737-1207, Japan
(Received October 24, 2002)

ABSTRACT憂apanese flounder Paralichthys olivaceus, farmed or wild, are often infected with aquabirnavirus (ABV) but the virus does not usually exhibit pathogenicity to the fish under usual experimental conditions. The present study investigated the experimental coinfection in flounder with ABV and three flounder pathogens, i.e. viral hemorrhagic septicemia virus (VHSV), Edwardsiella tarda and Streptococcus iniae. When young flounder were injected with ABV (106.5 TCID50/ fish) and subsequently (1 week later) injected with VHSV (106.7 or 104.7 TCID50/ fish), higher survival rates were obtained in these flounder compared with those in fish infected with VHSV alone. In contrast, higher mortalities occurred in the flounder dually injected with ABV (106.8 TCID50/fish) and E. tarda (1.4 エ 101 CFU/fish) or S. iniae (1.7 エ 102 CFU/fish) at a 1 week-interval than those in fish infected with bacteria alone. These results indicate that the primary ABV infection in flounder suppresses the secondary viral infection but facilitates the secondary bacterial infections.

Key words: coinfection, Paralichthys olivaceus, aquabirnavirus, viral hemorrhagic septicemia virus, Edwardsiella tarda, Streptococcus iniae, VHSV

Outbreak of Salmonid Herpesviral Disease in Cultured Rainbow Trout

<>Mitsuru Furihata1*, Akira Hosoe1, Kaoru Takei1, Masakazu Kohara1,3, Jun Nakamura1, Akira Motonishi1 and Mamoru Yoshimizu2<>Mitsuru Furihata1*, Akira Hosoe1, Kaoru Takei1, Masakazu Kohara1,3, Jun Nakamura1,
Akira Motonishi1 and Mamoru Yoshimizu2
1Nagano Prefectural Fisheries Experimental Station, Nakagawate 2871, Akashina,
Nagano 399-7102, Japan
2Graduate School of Fisheries Sciences, Hokkaido
University, Minato 3-1-1, Hakodate,
Hokkaido 041-8611, Japan
3Horticulture & Special Products Division, Nagano
Prefectural Government, Minaminagano
Habashita 692-2, Nagano, Nagano
380-8570, Japan

(Received July 15, 2002)

ABSTRACT友rom February 2000 to January 2001, epizootics occurred in cultured rainbow trout Oncorhynchus mykiss weighing 12 ミ 1,503 g at 18 fish farms in Nagano Prefecture, Japan. A virus was isolated from diseased fish in RTG-2 cells with a CPE characterized by syncytia.
High infectivity titers were demonstrated in the main internal organs and multiple necrotic foci were observed in the liver. The virus was identified as salmonid herpesvirus 2 by serological tests and PCR. Based on these results, the epizootic was diagnosed as salmonid herpesviral disease. In more than 80% cases, the outbreaks were linked with introductions of live fish.

Key words: salmonid herpesviral disease, Oncorhynchus mykiss, salmonid herpesvirus 2, rainbow trout, SaHV-2

Failure of Vaccination in Brook Trout Salvelinus fontinalis against Loma salmonae (Microspora)

<>David J. Speare* and Joanne Daley<>Project Loma, Department of Pathology & Microbiology,
Atlantic Veterinary College, University of Prince
Edward Island, Prince Edward Island,
Canada C1A 4P3

(Received October, 7 2002)

ABSTRACT裕he effectiveness of vaccines made of viable spores of two strains of Loma salmonae (Microspora) delivered orally to rainbow trout Oncorhynchus mykiss and brook trout Salvelinus fontinalis was evaluated under experimental conditions of re-challenge. Whereas prior exposure to
either of two strains of L. salmonae led to marked resistance of rainbow trout to a second challenge, brook trout failed to mount an effective protective response.

Key words: Loma salmonae, vaccination, Salvelinus fontinalis, Oncorhynchus mykiss, brook trout, rainbow trout