HOME > Volume and Number > NO43 - 3

NO43 - 3

Electron Microscopy on the Heart of Kuruma Prawn Penaeus japonicus Artificially Infected with Penaeid Rod-shaped DNA Virus

<>Teruo Miyazaki1*, Kana Yamaguchi1, Shinya Yasumoto1 and Yukinori Takahashi2<>1Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
2National Fisheries University, Shimonoseki, Yamaguchi 759-6595, Japan
(Received December 6, 2007)
ABSTRACT猶enaeid acute viremia (PAV) or white spot disease (WSD) causes mass mortality in cultured penaeid shrimp. Despite many histopathological studies, the principal lesion in the diseased shrimp remained unclear. Here we reevaluated the histopathology and ultrastructure of moribund kuruma prawn Penaeus japonicus artificially infected with penaeid rod-shaped DNA virus (PRDV) as a pathogen of PAV. We showed that PRDV infected cardiac satellite cells, primary myocardial cells and myocardial cells of the heart, where the nuclei of these cells displayed virus propagation that resulted in severe myocardial necrosis. Myocardial necrosis was suggested to be a primary cause of the viremia of PAV.

Key words: penaeid acute viremia, PAV, WSSV, cardiac satellite cell, myocardial cell, myocardial necrosis, Penaeus japonicus

In Vitro and In Vivo Activities of Drugs against Mycobacterium marinum in Yellowtail Seriola quinqueradiata

<>Sompoth Weerakhun1, Kishio Hatai1*, Takuya Murase2 and Tatsumu Hirae2<>1Laboratory of Fish Diseases, Nippon Veterinary and Life Science University, Musashino, Tokyo 180-8602, Japan
2Kagoshima Prefecture Fisheries Technology and Development Center, Ibusuki, Kagoshima 891-0315, Japan
(Received February 18, 2008)
ABSTRACT柚ycobacterium marinum (NJB 0419, MY 0401) isolated from yellowtail Seriola quinqueradiata showed drug susceptibility to rifampin, streptomycin (SM), ethambutol, kanamycin, sulfamethoxazole, amikacin, ciprofloxacin and ofloxacin by the broth dilution method. Furthermore, SM, kanamycin and sulfamonomethoxine activities were tested using the proportion method, and M. marinum was most susceptible to SM. Therefore, SM was used for the treatment of yellowtail (mean body weight 236 g) intramuscularly injected with NJB 0419 at a dose of 5.6 ∞ 104 CFU/fish. The diet medicated with SM was orally administered at doses of 25 and 50 mg/kg body weight/day for 7 consecutive days after 4 h or 10 days following injection. Fish receiving 50 mg SM had lower mortality than the controls when medication started 4 h after injection. Therefore, SM may be an effective drug to control M. marinum infection. The timing for initiating oral medication with SM requires further study.

Key words: Mycobacterium marinum, Seriola quinqueradiata, yellowtail, drug susceptibility, treatment, mycobacteriosis

Detection of Specific Antibodies against Infectious Hematopoietic Necrosis Virus from Rainbow Trout Sera by ELISA using Two Novirhabdoviruses<>Wi-Sik Kim1, Mamiko Mochizuki2, Toyohiko Nishizawa1* and Mamoru Yoshimizu1

<>1Faculty of Fisheries Sciences, Hokkaido University, Hakodate, 041-8611, Japan
2Fuji Trout Hatchery, Shizuoka Prefectural Research Institute of Fishery, Fujinomiya, Shizuoka 418-0108, Japan
(Received February 21, 2008)
ABSTRACT輸n enzyme-linked immunosorbent assay (ELISA) with viral culture fluids as capture antigens was performed to detect specific antibodies against infectious hematopoietic necrosis virus (IHNV) from rainbow trout Oncorhynchus mykiss sera. When using IHNV antigen (IHNV-Ag), ELISA OD values for IHN-survived (IHN-Surv) rainbow trout sera were relatively higher than those for specific pathogen free (SPF) fish sera. However, some of the SPF sera were diagnosed as positive due to high OD values (> 0.2). To clarify reasons for these high OD values, each of five IHN-Surv and SPF sera was subjected to ELISA with viral hemorrhagic septicemia virus (VHSV) and hirame rhabdovirus (HIRRV) antigens (VHSV-Ag and HIRRV-Ag). Some of the IHN-Surv and SPF sera showed high OD values in both VHSV-Ag and HIRRV-Ag plates even though there was no possibility that those sera contained antibodies against VHSV or HIRRV antigen. These results suggest that some of the sera contained antibodies against impurities in viral culture fluids such as FBS and cell debris, and caused the false-positive reactions. The corrected OD values, subtracted the OD values in VHSV-Ag plates from those in IHNV-Ag plates, were all < 0.2, in SPF sera (n = 148) while those in IHN-Surv sera (n = 238) were randomly distributed from 0 to 0.7. It was considered that the corrected OD values may represent true values recognized by IHNV-specific antibodies.

Key words: antibody detection, ELISA, Oncorhynchus mykiss, IHNV, background

Quantitative Detection of Viable Flavobacterium psychrophilum in Chum Salmon Oncorhynchus keta by Colony Blotting and Immunostaining

<>Naoyuki Misaka1*, Toyohiko Nishizawa2 and Mamoru Yoshimizu2<>1The Hokkaido Fish Hatchery, Eniwa, Hokkaido 061- 1433, Japan.
2Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
(Received March 4, 2008)
ABSTRACT猷uantitative detection of viable Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease, was evaluated by colony blotting and immunostaining. Bacterial colonies isolated from chum salmon Oncorhynchus keta ovarian fluids on a modified Cytophaga agar plate were blotted onto a nitrocellulose membrane and immunostained with antiserum against F. psychrophilum. Although the blotted colonies were strongly or weakly stained with the antiserum, blots from colonies of F. psychrophilum were distinguishable from those of other yellowish colonies by digital processing of the colony-blotted membrane photograph with an image-analyzing software. It was also confirmed that 12 strains of F. psychrophilum were all positive by the present method, while the subjected six isolates, which formed yellowish colonies but were not identified as F. psychrophilum by PCR targeting gyrB gene, and other reference six strains, F. branchiophilum, F. limicola, F. granuli, Pseudomonas flavescens, P. fluorescens, Chryseobacterium daecheongense, were all negative. From these results, the present procedure using colony blotting and immunostaining is useful for quantitative detection of viable F. psychrophilum from ovarian fluids and kidneys of chum salmon.

Key words: Flavobacterium psychrophilum, bacterial coldwater disease, BCWD, colony blotting, immunostaining, digital processing

Reliability of PCR Methods for the Detection of Flavobacterium psychrophilum

<>Kyuma Suzuki1,2*, Hajime Arai1, Toshihiro Kuge1, Takayuki Katagiri2 and Shotaro Izumi1<>1Gunma Prefectural Fisheries Experiment Station, Maebashi, Gunma 371-0036, Japan
2Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Minato, Tokyo 108-8477, Japan

(Received July 11, 2005)

ABSTRACT邑e compared the sensitivity and specificity of PCR methods targeting 16S rDNA, DNA gyrase subunit genes (gyrA, gyrB) and peptidyl-prolyl cis-trans isomerase C gene (ppiC) for the detection of Flavobacterium psychrophilum using 82 bacterial isolates and 55 washings of fish gills. To identify F. psychrophilum among the bacterial isolates, the PCRs targeting 16S rDNA, gyrB and ppiC exhibited the same sensitivity and specificity but some false-positive results were found in the PCR targeting gyrA. The PCR targeting 16S rDNA was more sensitive than the other PCRs in detecting the bacterium from gill washings but occasionally gave false-positives. To avoid the false-positive result in gill washings, the PCRs targeting gyrB and ppiC seem to be preferable.

Key words: Flavobacterium psychrophilum, PCR, detection, sensitivity, specificity

Spontaneous Intestinal Adenocarcinoma in a Blue Gularis Fundulopanchax sjostedti: an Immunohistochemical Study

<>Gian Enrico Magi*, Emiliano Di Cicco and Giacomo Rossi<>Department of Veterinary Science, University of Camerino, Via Circonvallazione 93/95, 62024 Matelica, Italy

(Received February 14, 2008)

ABSTRACT輸 spontaneous intestinal adenocarcinoma in a 3-year-old blue gularis Fundulopanchax sjostedti with a history of swelling of the trunk and anorexia is described. At necropsy, there was a large, firm, irregular mass originated from the intestine and distending the abdominal cavity. Histologically the intestinal mass showed proliferation of neoplastic epithelial cells arranged in irregularly shaped islands and dense sheets with papillotubular ingrowths. Immunohistochemically the neoplastic cells expressed cytokeratin AE1/AE3, PCNA and interestingly the mutant p53 protein. On the basis of the histopathological and immunohistochemical features, an intestinal adenocarcinoma was diagnosed. The expression of the mutant p53 protein in tumour cells may demonstrate its role in neoplastic transformation of enterocytes as observed in human cancers.

Key words: Fundulopanchax sjostedti, p53, intestinal adenocarcinoma, immunohistochemistry, killifish