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NO42 - 1 (2007)

Review: Overview on the Studies of Humoral Defense Factors in Bivalve Molluscs

<>Kiyokuni Muroga* and Keisuke G. Takahashi<>Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
(Received September 29, 2006)
ABSTRACT涌wing to the global development of shellfish culture and increasing awareness of the risks associated with infectious diseases in bivalve molluscs, the importance of studies on defense mechanisms of bivalves has become widely recognized. Japan has a long history of oyster culture, however, there have been meager investigations on diseases and defense mechanisms of bivalves in the country. In foreign countries, especially those in the West, the immunological responses of bivalves, being composed of cellular and humoral factors, have been approarched from various standpoints such as comparative immunology, pathology/health management, environmental monitoring, and exploitation of novel antimicrobial agents. In this paper, studies on humoral defense factors in bivalves represented by oysters, mussels and clams, made mainly with the interest in interaction between hosts and pathogens, are reviewed. For each humoral defense factor, history of researches, chemical and molecular characteristics, and functions of the following factors are presented here with some critical comments: lectins (agglutinins), lysozymes, antimicrobial peptides, pro-phenoloxidase (PO) activating system, and other humoral foctors (protease inhibitors, lysosomal enzymes, heat-shock proteins).

Key words: bivalve mollusc, humoral defense factor, lectin, lysozyme, antimicrobial peptide, proPO system, protease inhibitor

Purification of Two Different Immunoglobulins (Igs) from Olive Flounder Paralichthys olivaceus and Analysis of Lactococcus garvieae Antigens by the Igs

<>Gee Wook Shin1, Young Rim Kim1, Yong Seung Shin1, Eung Goo Lee1, Myung Joo Oh2, Terutoyo Yoshida3 and Tae Sung Jung1*<>1Laboratory of Fish & Shellfish Diseases, College of Veterinary Medicine,
Gyeongsang National University, Chinju 660-701, Korea
2Department of Fish Pathology, Yosu National University,
Yosu 550-747, Korea
3Department of Biological Production and Environmental Science,
Faculty of Agriculture, Miyazaki University,
Miyazaki 889-2192, Japan
(Received February 24, 2006)
ABSTRACT悠mmunoglobulins (Igs) were purified from sera obtained from olive flounder Paralichthys olivaceus immunized with goat IgG using immunoaffinity and mannan-binding protein (MBP) affinity columns and designated IMMIg and MBPIg respectively. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) analyses demonstrated that the olive flounder serum contained at least two different types of Ig in terms of molecular weight and pI of heavy chain. 2-DE separation of the Igs followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) revealed that these two Igs were IgM and IgM precursor of olive flounder. Monoclonal antibodies (MAbs) were raised against the IMMIg and MBPIg and proteome analysis of Lactococcus garvieae using these MAbs and olive flounder immune sera demonstrated that IMMIg and MBPIg recognized different antigens of L. garvieae. This suggested that different Igs are possibly involved in protecting olive flounder against L. garvieae infection.

Keywords: Paralichthys olivaceus, Lactococcus garvieae, immunoglobulin, affinity chromatography, immunoproteomics, monoclonal antibodies, olive flounder

Detection of Kuchijirosho Associated Proteins by Western Blot with Sera of Tiger Puffer which Survived Kuchijirosho

<>Ikuo Takami1,2, Yuki Kokawa1, Toyohiko Nishizawa1 and Mamoru Yoshimizu1*<>1Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
2Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan
(Received August 21, 2006)
ABSTRACT揖uchijirosho (snout ulcer disease) is an infectious disease of tiger puffer Takifugu rubripes in Japan and Korea. Little is known about detailed characteristics on the causative agent of kuchijirosho, except it is a filterable agent. In the present study, kuchijirosho associated proteins (KAPs) with molecular weight of 100・20 kDa were detected in brain tissues of tiger puffer by western blot analysis with sera of tiger puffer which survived kuchijirosho, but not in the kidney or spleen tissues. Interestingly, KAPs existed in brain tissues of tiger puffer with no history of kuchijirosho, however, the KAPs were not recognized by those healthy fish sera. KAPs were fractionated in supernatant of brain tissue homogenate containing the kuchijirosho agent by ultra-centrifugation, while pathogenicity of the kuchijirosho agent was observed in the precipitated fraction rather than the supernatant. These results suggest that KAPs are not structural proteins of the kuchijirosho agent, and could be brain tissue proteins antigenically altered by infection of the kuchijirosho agent. It is also considered that the causative agent could be low in antigenicity to host fish because structural proteins of the causative agent was never recognized by host fish sera.

Key words: kuchijirosho, Takifugu rubripes, tiger puffer, kuchijirosho associated protein, snout ulcer disease

Induction of Kuchijirosho in Yellowtail by Injection with the Brain Homogenate of Affected Tiger Puffer

<>Ikuo Takami1,2, Yuki Kokawa1, Toyohiko Nishizawa1 and Mamoru Yoshimizu1*<>1Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
2Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan
(Received August 21, 2006)

ABSTRACT裕he causative agent of kuchijirosho (snout ulcer disease), an infectious disease of tiger puffer Takifugu rubripes, is filterable, detailed characteristics of the agent have not been clear. Here, susceptibility of eight fish species against kuchijirosho was studied by injection with the kuchijirosho agent. A hundred percent mortality with typical signs of kuchijirosho was observed in tiger puffer, grass puffer T. niphobles, panther puffer T. pardalis and blue-spotted boxfish Ostracion immaculatus, while no mortality occurred in Japanese parrotfish Oplegnathus fasciatus, largescale blackfish Girella punctata and red seabream Pagrus major. Moreover, yellowtail Seriola quinqueradiata was demonstrated to be susceptible to this disease, because cumulative mortalities of 100% and 60% were recorded in young (average 12 g in body weight) and adult (average 530 g in body weight) yellowtail, respectively. Affected young yellowtail exhibited convulsion with the curvature of the spine (scoliosis), although no typical disease sign was observed in the dead adult yellowtail. In experimental infection with the brain homogenate of the dead adult yellowtail, scoliosis was produced in young yellowtail.

Key words: kuchijirosho, Takifugu rubripes, Seriola quinqueradiata, kuchijirosho associated protein, snout ulcer disease, tiger puffer

Virulence Attenuation of Capsular Polysaccharide-deleted Mutants of Streptococcus iniae in Japanese Flounder Paralichthys olivaceus

<>Kimihiro Shutou1, Kinya Kanai2* and Kazuma Yoshikoshi2<>1Graduate School of Science and Technology,
2Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan
(Received December 6, 2006)
ABSTRACT祐treptococcus iniae is known as an important bacterial pathogen causing serious damage in fish culture industry worldwide. Although the capsule of this pathogen is suggested to play a role in the disease process, it has not been verified yet. In this study we created isogenic uncapsulated mutants of S. iniae NUF631 by Tn916 transposon mutagenesis and investigated the role of capsule in the virulence and resistance to phagocytic activities of peritoneal macrophages of Japanese flounder Paralichthys olivaceus. Mutants transformed from K+ phenotype to K・/SUP> phenotype were all uncapsulated. They exhibited neither K+ type-specific precipitation line in an immunodiffusion test nor ladder-like banding pattern characteristic for bacterial acidic polysaccharides in polyacrylamide gel electrophoresis. Their virulence to Japanese flounder was highly attenuated. In contrast to the mutant strains, the parent capsulated strain resisted phagocytic and bactericidal activities of the macrophages and prevented chemiluminescence reaction of the macrophages upon phagocytosis. From these results, it is verified that the S. iniae capsule is an important factor for intramacrophage survival of the pathogen.

Key words: Streptococcus iniae, capsule, transposon mutagenesis, virulence, Paralichthys olivaceus, Tn916, macrophage, chemiluminescence

Endoparasitic Dinoflagellate of the Genus Ichthyodinium Infecting Fertilized Eggs and Hatched Larvae Observed in the Seed Production of Leopard Coral Grouper Plectropomus leopardus

<>Koh-ichiro Mori1*, Kazuhisa Yamamoto1, Kazuhisa Teruya1, Satoshi Shiozawa2,Kenzo Yoseda3, Takuma Sugaya1, Sho Shirakashi4,Naoki Itoh4 and Kazuo Ogawa4<>1National Research Institute of Aquaculture, Fisheries Research Agency, Saiki,
Oita 879-2602, Japan
2National Center for Stock Enhancement, Fisheries Research Agency, Setouchi,
Kagoshima 894-2414, Japan
3Seikai National Fisheries Research Institute, Fisheries Research Agency,
Ishigaki, Okinawa 907-0451, Japan
4Department of Aquatic Bioscience, Graduate School of Agricultural
and Life Sciences, The University of Tokyo,
Tokyo 113-8657, Japan
(Received October 6, 2006)
ABSTRACT祐ince 1990, mass mortality of fertilized eggs and hatched larvae of the leopard coral grouper Plectropomus leopardus has occurred repeatedly during its seed production in Okinawa Prefecture. A hitherto unknown protozoan parasite multiplied in the yolk sac in great numbers. The yolk sac of fertilized eggs and hatched larvae eventually burst and parasites were released to sea water. The development of the parasite was briefly described, which is very similar to that of the dinoflagellate Ichthyodinium chabelardi, known to infect yolk of several marine fish larvae in Europe. Based on the similarities of, and minor differences in, the mode of infection, development and SSU rDNA sequence, the parasite is tentatively designated as Ichthyodinium sp. PL (= PL for the abbreviated form of the scientific name for the host). Although the life cycle of the parasite is unknown, the disease outbreak was controlled by rearing the broodstock and incubating fertilized eggs in oxidant-treated seawater. The PCR diagnosis using specific primers designed from part of SSU rDNA sequence of the parasite revealed that disease outbreaks in different years were caused by a single species of parasite. This is the first report of Ichthyodinium infection in tropical fish from the Asia-Pacific region.

Key words: Plectropomus leopardus, endoparasite, seed production, mass mortality, leopard coral grouper, dinoflagellate, Ichthyodinium chabelardi


Infection by a Protozoan Endoparasite of the genus Ichthyodinium in Embryos and Yolk-sac Larvae of Yellowfin Tuna Thunnus albacares

<>Kei Yuasa1*, Takashi Kamaishi1, Koh-ichiro Mori2, John Harianto Hutapea3,Gusti Ngurah Permana3 and Akio Nakazawa4<>1National Research Institute of Aquaculture, Fisheries Research Agency,
Minami-Ise, Mie 516-0193, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency,
Saiki, Oita 879-2602, Japan
3Gondol Research Institute for Mariculture, Indonesia,
P.O. Box 140 Singaraja Bali-Indonesia
4Overseas Fishery Cooperation Foundation,
Tokyo 107-0052, Japan
(Received August 2, 2006)
ABSTRACT悠n October 2005, a mass mortality of yolk-sac larvae of yellowfin tuna Thunnus albacares due to an endoparasite infection occurred during the seed production in a semi-closed system tank in Bali, Indonesia. Prevalence of the infection reached 100% maximally. The affected embryo or yolk sac of hatched larvae was filled with numerous parasites by 24 h post spawning. The larvae died due to a burst of the yolk sac. Phylogenetic analysis with 18S rRNA sequence and morphological characteristics of the parasite indicated that the present parasite was identified as a protozoan endoparasite Ichthyodinium chabelardi infecting the yolk-sac larvae of Atlantic sardine Sardina pilchardus or as its closely related species. PCR with a primer set designed from the 18S rRNA sequence detected the genome of the parasite from infected fertilized eggs, yolk-sac larvae and rearing water but not from the gonads of broodstock or feed fish for broodstock. The infection was not observed when the spawned eggs were immediately transferred into sterilized seawater, but the eggs kept in the rearing water for more than 1 h were infected by the parasite. These results suggest that the parasite infects fertilized eggs horizontally through the rearing water.

Key words: Ichthyodinium chabelardi, endoparasite, Thunnus albacares, yellowfin tuna, embryo, PCR, phylogenetic analysis, prevention

Experiments on Induction of Protection against Kuchijirosho of Tiger Puffer

<>Ikuo Takami1,2, Toyohiko Nishizawa1 and Mamoru Yoshimizu1*<>1Faculty of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Japan
2Nagasaki Prefectural Institute of Fisheries,
Nagasaki 851-2213, Japan

(Received August 21, 2006)

ABSTRACT揖uchijirosho (snout ulcer disease) is an infectious disease of tiger puffer Takifugu rubripes, but little is known about detailed characteristics of its causative agent. Homogenate of brain from naturally diseased fish containing the kuchijirosho agent was treated with 0.3% formalin for 7 days. Injection of the homogenate induced 79% mortality in tiger puffer rearing at 25゚C, indicating that the kuchijirosho agent was not inactivated by the formalin treatment However, no mortality was observed in fish injected with the formalin-treated homogenate rearing at 18・0゚C. A challenge test at 25゚C with the untreated homogenate showed that the formalin-treated homogenate did not induce protection against kuchijirosho in the survivors.

Key Words: kuchijirosho, Takifugu rubripes, tiger puffer, formalin inactivation, snout ulcer disease

Occurrence of a Monogenean Gill Parasite Pseudorhabdosynochus epinepheli on Red Spotted Grouper Epinephelus akaara and Its Experimental Treatment by Hydrogen Peroxide Bathing

<>Tadashi Isshiki1*, Taizou Nagano2 and Katsuhiro Miki2<>1Graduate School of Bioresouces, Mie University,
Tsu, Mie 514-8507, Japan
2Kagawa Prefectural Fisheries Experiment Station, Takamatsu, Kagawa 761-0111, Japan

(Received Octorber 6, 2006)

ABSTRACT祐ince 1997, infection of a monogenean gill parasite Pseudorhabdosynochus epinepheli has been occurring in red spotted grouper Epinephelus akaara at the Kagawa Prefectural Sea-Farming Center, Japan. The efficacy of hydrogen peroxide (H2O2) bathing was examined in vivo. Bathing in a 700 ppm H2O2 solution for 45 min at 10 or 15゚C, or for 15 min at 20 or 25゚C, with infected fish of 230・100 g in body weight, resulted in 100% mortality of P. epinepheli on the gills. Mortality occurred in fish (550・730 g) bathed for 30 min or longer at 25゚C, but not in fish bathed for at least 60 min at 10, 15 or 20゚C. Results indicate that chemotherapy with H2O2 has a high potential as an effective treatment for P. epinepheli infection in red spotted grouper.

Key words: Pseudorhabdosynochus epinepheli, Monoャgenea, hydrogen peroxide, Epinephelus akaara, red spotted grouper