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NO41 - 3

Histopathological Features of Ayu Plecoglossus altivelis Experimentally Infected with Pseudomonas plecoglossicida

<>Tatsuya Kobayashi* and Makoto Imai<>Kyowa Yakuhin Co., Ltd., Wakayama, 641-0036, Japan
(Received November 21, 2005)
ABSTRACT幽istopathology of experimentally infected ayu Plecoglossus altivelis with bacterial hemorrhagic ascites (BHA) was studied in order to determine its histopathological features. Fish (2.3 g in average weight, n=108) were infected with Pseudomonas plecoglossicida by immersion (IM) (ca. 107 CFU/mL), intraperitoneal injection (IPI) (ca. 104 and 107 CFU/fish) and intramuscular injection (IMI) (ca. 104 and 107 CFU/fish). The cumulative mortalities ranged from 92.9 to 100%. In the IM challenge groups, necrotic lesions accompanied by edema, fibrin deposition and hemorrhage were observed in the splenic and hematopoietic tissues. In some fish, the liver had necrotic lesions and microabscesses.In both the IPI and IMI challenge groups, lesions of the spleen and kidney were similar to those of the IM challenge groups. In addition, necrotic lesions and hemorrhage in the abdominal adipose tissue and pancreas were often observed in the IPI challenge groups. In the IMI challenge groups, the infected fish had definite lesions of the musculature at the injected site. The results revealed that the lesion formation in the spleen and kidney was the main pathological changes of BHA in ayu.

Keywords: Pseudomonas plecoglossicida, bacterial hemorrhagic ascites, Plecoglossus altivelis, ayu, experimental infection, histopathology

Susceptibility and Immune Response to Flavobacterium psychrophilum between Different Stocks of Ayu Plecoglossus altivelis

<>Takahiro Nagai1* and Takashi Sakamoto2<>1Hiroshima Prefectural Fisheries and Marine Technology Center, Kure,
Hiroshima 737-1207, Japan
2Faculty of Marine Science, Tokyo University of Marine Science
and Technology, Minato, Tokyo 108-8477, Japan
(Received March 10, 2006)
ABSTRACT裕hree stocks of ayu Plecoglossus altivelis, viz. an amphidromous stock, domesticated stock and hybridized stock between amphidromous and domesticated stocks, were challenged by intraperitoneal injection and by field spontaneous infection with Flavobacterium psychrophilum to compare susceptibility to bacterial coldwater disease (BCWD). Cumulative mortalities of the hybrid stock were medium between those of the amphidromous (low) and domesticated stock (high). It is possible that susceptibility to BCWD was inherited to the hybrid stock. The efficacy of immersion vaccine with the formalin-killed bacterin of F. psychrophilum and the serum aggulutinin titer of fish immunized with the formalin-killed cells of F. psychrophilum or Vibrio anguillarum were compared between these stocks. Although the mortality of the vaccinated fish was significantly lower than that of unvaccinated fish for each stock, no obvious difference was observed in the efficacy of immunization between these stocks. However, the serum aggulutinin titer of the amphidromous stock against both antigens was highest among the three stocks, possibly explaining the low susceptibility of the stock to BCWD.

Key wards: bacterial coldwater disease, Plecoglossus altivelis, resistance stock, immune
response, Flavobacterium psychrophilum, ayu

Rapid Discrimination of Fish Pathogenic Vibrio and Photobacterium Species by Oligonucleotide DNA Array

<>Tomomasa Matsuyama*, Takashi Kamaishi and Norihisa Oseko<>Aquatic Animal Health Division, National Research Institute of Aquaculture,
Fisheries Research Agency, Minami-Ise, Mie 516-0193 Japan
(Received April 17, 2006)
ABSTRACT邑e developed a low-density oligonucleotide DNA array for the discrimination of 17 Vibrio and 1 Photobacterium species that are pathogenic to aquatic animals. We designed oligonucleotide probes targeting non-coding regions of the intergenic transcribed spacers (ITS) between 16S and 23S ribosomal DNA. Based on the alignment of ITS sequences obtained from the database and the results in this study, three oligonucleotide probes from a bacterial species were designed and immobilized on a nylon membrane. The ITS regions were PCR-amplified using a pair of universal primers, followed by hybridization of the digoxigenin (DIG)-labeled PCR products to the membrane. Specific signals were produced with alkaline phosphatase-conjugated anti-DIG antibody and chemiluminescent substrate. Athough cross hybridization was observed, the patterns from each species were different among the species. This DNA array is useful for a rapid and easy discrimination of Vibrio species. The total process, including PCR amplification, can be carried out within a day. Our method enables a global screening of fish pathogenic Vibrio and Photobacterium species

Key words: DNA array, discrimination, Vibrio, Photobacterium

Fate of Oncomiracidia of Heterobothrium okamotoi (Monogenea) Attaching to the Body Surface of Tiger Puffer Takifugu rubripes

<>Tomoyoshi Yoshinaga*, Masayoshi Yasuzaki and Kazuo Ogawa<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

(Received April 10, 2006)

ABSTRACT涌ncomiracidia of the gill monogenean Heterobothrium okamotoi experimentally attached to both the body surface and gills of tiger puffer Takifugu rubripes. While worms disappeared from the body surface in 3 days, those on the gills showed an increase in number in 12 days. When worms were removed from the body surface with a formalin treatment, such an increase was not observed in worms on the gills. Na・e fish became infected, when cohabiting with fish just after challenge, although they were separated with a mesh screen. These results demonstrate that H. okamotoi can reinfect new hosts after detachment from the body surface of previous hosts.

Key words: Monogenea; Heterobothrium okamotoi; Takifugu rubripes; oncomiracidium; transmission

Disinfection Method for Fertilized Eggs of Kuruma Prawn Penaeus japonicus Using Povidone-iodine

<>Jun Satoh1*, Koh-ichiro Mori1, Toyohiro Nishioka2, Keita Hattori3, Masakazu Oka4 and Ken-ichi Watanabe5<>1Aquatic Animal Health Division, Kamiura Station, National Research Institute of Aquaculture, Fisheries Research Agency, Kamiura, Saiki, Ooita 879-2602, Japan
2Diagnosis and Training Center for Fish Diseases, National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan
3Goto Station, National Center for Stock Enhancement, Fisheries Research Agency, Tamanoura,
Nagasaki 853-0508, Japan
4Headquarters, Fisheries Research Agency, Yokohama, Kanagawa 220-6115, Japan
5Aquatic Animal Health Division, Komame Station, National Research Institute of Aquaculture, Fisheries Research Agency, Ootsuki, Kouchi 788-0315, Japan

(Received May 15, 2006)

ABSTRACT佑onditions suitable for disinfection of fertilized eggs of Penaeus japonicus using povidone-iodine were investigated. Eggs 10 h after fertilization were exposed to 0, 2.5, 5.0 and 10.0 mg/L of active iodine for 5, 10, 15 and 20 min. Hatching rate showed no significant difference between control and test groups in concentration of 2.5 mg/L of active iodine for 5 to 20 min and 5.0 mg/L for 5 to 15 min. Viable bacterial counts in these conditions decreased by more than 90% when compared with those of control groups. There was no significant difference in the hatching rates of eggs with eight different developmental stages between control and tested groups exposed to 5.0 mg/L of active iodine for 5 min.

Key words: Penaeus japonicus, kuruma prawn, fertilized egg, disinfection, povidone-iodine

Vaccination with Acetone-dry Bacterin against Bacterial Hemorragic Ascites of Ayu Plecoglossus altivelis

<>Hiroaki Kintsuji* Koji Ninomiya and Michitaka Yamamoto<>Shiga Prefectural Fisheries Experimental Station, Hikone, Shiga 522-0057, Japan

(Received May 15, 2006)

ABSTRACT邑e developed an acetone-dry oral bacterin against bacterial hemorragic ascites of ayu Plecoglossus altivelis. The bacterin was prepared from the cultured cells of Pseudomonas plecoglossicida, which were killed in aceton at 37゚C for 2 h and dehydrated. The experiment was performed twice in 2002. The bacterin was orally administered to ayu (average body weight: 4.0 g and 3.6 g) with food twice at 2-week-interval. The fish were challenged by immersion or injection with P. plecoglossicida 2 weeks after the second vaccination. The bacterin showed significant efficacy with RPS of 40・9 %.

Keywords: bacterial hemorragic ascites, Pseudomonas plecoglossicida, oral vaccination, Plecoglossus altivelis, ayu

A Preliminary Study on the Infection of Anisakid Larvae in Juvenile Greater Amberjack Seriola dumerili Imported from China to Japan as Mariculture Seedlings

<>Tomoyoshi Yoshinaga*, Ryuhei Kinami, Kathryn A. Hall and Kazuo Ogawa<>Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

(Received May 18, 2006)

ABSTRACT憂uvenile greater amberjack Seriola dumerili (fork length: 39.5・3.0 cm) imported from China to Japan as mariculture seedlings were found infected with larval anisakid nematodes in the spring of 2005. The parasite was morphologically identified as Anisakis type I larva causing human anisakiasis. Based on the nucleotide sequence of ITS1-5.8S rRNA-ITS2 region, the parasite was tentatively identified as A. pegreffii, one of the species comprising A. simplex sensu lato. The main infection site was the wall and serous membrane of the stomach. No worms were found in the ventral side of the body muscle of fish. This is the first documented case of Anisakis infection in cultured marine fishes.

Key words: Anisakis simplex sensu lato, Anisakis pegreffii, Seriola dumerili, greater amberjack

Rapid and Simple Method for Detection of Yersinia ruckeri, the Causal Agent of Enteric Redmouth Disease

<>Takamitsu Sakai*, Norihisa Oseko and Takaji Iida<>Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan
(Received May 29, 2006)
ABSTRACT輸vailability of known selective-differential culture media and PCR methods for the detection of Yersinia ruckeri, the causal agent of enteric redmouth disease, was investigated. The attempted three selective-differential culture media had some difficulties in the selective growth of Y. ruckeri and the differentiation of the bacterium from other fish-pathogenic bacteria. Three PCR methods targeting 16S rDNA were evaluated to be effective in sensitivity and specificity for the detection of Y. ruckeri. The rapid and accurate diagnosis of redmouth disease will be performed by PCR following Gram stain and cytochrome-oxidase test.

Key words: Yersinia ruckeri, enteric redmouth disease, PCR