In a study of immune mechanisms in fish, specific cell-mediated cytotoxic activity against trinitrophenyl (TNP) -modified autologous peripheral blood leucocytes (PBL) was successfully induced in in vivo primed carpCyprinus carpio. The cytotoxic activity increased in proportion to the number of immunizations. The cytotoxic activity was shown to be gradually reduced with time afterin vivosensitization, and was finally lost. In addition, cytotoxic activities against TNP-modified allogeneic PBLs (T-ALLO-PBL) in carp immunized with TNP-modified autologous PBLs (T-AUTO-PBL) were not detected and effector cells from carp immunized with T-ALLO-PBL were not significantly cytotoxic against T-AUTO-PBL. Thus, the occurrence of a genetic restriction in carp was shown using thein vivopriming model. These results suggest that specific cell-mediated cytotoxicity ofin vivoprimed carp in some respects resembles mammalian cytotoxic T cells.
Histopathological studies were carried out to investigate a previously unknown disease characterized by severe anemia in both wild and cultured Japanese flounder, Paralichthys olivaceus. Light microscopy revealed necrotic cells in the hematopoietic tissues of affected fish. No other tissues showed any notable pathological changes except for atrophy of the hepatopancreas. Virus-like particles of about 27 nm in diameter were observed with electron microscopy in cells in the hematopoietic tissue of the kidney and spleen, including granulocytes and red blood cells (RBCs) which exhibited abnormal features of cytoplasmic organelles. However, the relationship of these particles and the disease is uncertain. RBCs in the blood smears from affected fish were often deformed and staining of the cytoplasm was very weak. These histopathological and hematological characteristics were common among affected flounder from various places in Japan, suggesting that the anemic fish observed in this study were affected by the same disease.
Thirteen wild molluscan shellfish species collected from 8 Prefectures in Japan were surveyed for the presence of marine birnavirus (MABV) by the polymerase chain reaction (PCR) technique and culture method. Approximately 60% of bivalves and 35% of gastropods tested had detectable MABV genome, although the prevalence of positive specimens varied among species. The PCR-positive shellfish were submitted to virus isolation. The isolation rate was low, suggesting that the MABV was in a state of persistent infection in these shellfish. Seventy four % of the examined virus strains were different in a position of the nucleotide sequences of the PCR products from that of MABV strains obtained from fish.
In April 1998, unidentified parasites were found in Manila clam Tapes philippinarumin an inner bay of the western part of Japan. Ray's fluid thioglycollate medium (RFTM) culture of tissues from infected Manila clams yielded prezoosporangia of the parasite. Many free motile zoospores were released from the prezoosporangia when they were transferred to seawater. Histologically two types of trophozoites were observed : cells in a cluster and free single cells. They were found in the digestive gland, gill, mantle and foot of the clam. In immunohistochemical assays using an antiserum against Perkinsus marinus, the antibody reacted to both types of trophozoites. These results indicated that these parasites in Manila clam were species belonging to the genus Perkinsus. However, they differed from previously described species in the morphology of trophozoites and prezoosporangia.
To understand the kinetics of the defense activities of inflammatory neutrophils during inflammation, their chemotactic, phagocytic and respiratory burst activities were examined in inflamed carp (Cyprinus carpio) and red sea bream (Pagrus major) . Formalin-killed Escherichia coli cells were injected into the swim bladder of fish. Inflammatory neutrophils were withdrawn from the swim bladder of the animals at various times (12, 24, 36, 48, 60 and 70 h) after injection. The results obtained for the two species were similar. Neutrophils and macrophages were collected from the swim bladder, while neither lymphocytes nor thrombocytes were found in this region. Neutrophils accounted for the great part of cells exuded into the swim bladder. The number of exudate cells peaked 48 h after injection. The neutrophils isolated at the first sampling time had the highest chemotactic activity. Phagocytic activity of carp neutrophils had the greatest value 48 h after injection, whereas no significant difference in phagocytic activity was observed in red sea bream neutrophils during the experiment. Respiratory burst activities in carp and red sea bream were highest at 48 and 36 h post-injection, respectively. These observations suggest the following course of events : An invasion of irritants induces inflammation. In the early stage of inflammation, the neutrophils with higher migration activity first arrive at the inflammatory site to prevent the spread of irritants. As the inflammation progresses, the neutrophils with higher killing activity follow to eliminate the irritants.
To apply the biocontrol technique for vibriosis of larvae of the Pacific oyster, Crassostrea gigas, we isolated marine bacterial strains from the rearing seawater of oyster brood stock and screened the strains having suppressive activity for the growth of pathogenic vibrios. Twelve strains of the 51 isolated strains clearly showed inhibitory effects on growth of three vibrios (Vibrio alginolyticus, V. tubiashii and Vibrio sp.) by a smear method with agar plates. One strain (S21) demonstrated the highest vibriostatic activity among the 12 tested strains, while S21 did not have adverse effect on the survival of larval oysters. The addition of strain S21 suppressed the growth of V. alginolyticus in a value of 1.0 × 105 CFU/mL, whereas mono-culture of V.alginolyticus augmented to 3.7 × 108 CFU/mL in seawater. The larval survival rate at 24 h after challenge with V. alginolyticus was 78% in the group added with strain S21 at 105 CFU/mL, while it was 8.4% in the control group. These results indicate that strain S21 protects larvae from V. alginolyticus and that strain S21 is a potential biocontrol agent for vibriosis in the larval oyster rearing system.
Nucleotide sequences of an internal fragment of iron-cofactored superoxide dismutase gene (sodB) from members of the genus Edwardsiella were determined directly from amplified DNA fragments. Sequences obtained were all 454 bp in length, and there was no insertion or deletion against that of Escherichia coli. Identical sequences were obtained for the three Edwardsiella ictaluri strains, the four atypical Edwardsiella tarda strains isolated from red sea bream Chrysophrus major, and the five strains of Edwardsiella sp. from Japanese eel Anguilla japonica, respectively. In contrast, five classes of sodB sequences were found in 26 biochemically typical E. tarda strains and were assigned to sodB types 1 to 5. A phylogenetic tree based on the sodB sequences reveals that members of the genus Edwardsiella can be divided into two clusters, I and II, which differed from each other in their pathogenicity to fish. Cluster I is composed of the pathogenic E. tarda strains isolated from Japanese eel, Japanese flounder Paralichtys olivaceus, nile tilapia Oreochromis niloticus, and ayu Plecoglossus altivelis, atypical E. tarda from red sea bream, Edwardsiella sp. from Japanese eel, and E. ictaluri, whereas cluster II is composed of the non-pathogenic E. tarda strains and Edwardsiella hoshinae. These data indicated that sequence analysis of sodB is an effective tool for identifying fish-pathogenic strains belonging to the genus Edwardsiella.
In order to clarify the effect of a prolonged exposure period on vaccine effectiveness, juvenile rainbow trout were immersed in 100, 1, 000 and 10, 000 times dilutions of a commercial vibrio bacterin for 3 min (brief immersion, BI) and for 24 h (prolonged immersion, PI). Two weeks and 2, 4 and 6 mo after the vaccination, fish were challenged by waterborne exposure to Vibrio ordalii. Prolonged exposure to dilute bacterin resulted in better protection than the standard BI method. Two weeks after vaccination, the cumulative mortality was 0% in the group vaccinated by PI with the 100, 1, 000 and 10, 000 times diluted vaccine. In contrast, mortality was 58 and 77% in the groups vaccinated by BI with the 100 and 10, 000 times diluted vaccine respectively, and was 100% in the unimmunized control group. Furthermore, 6 mo after vaccination, the mortality was 20% (100 times dilution) to 77% (10, 000 times dilution) in the PI group, while it was 64% (100 times dilution) to 93% (10, 000 times dilution) in the BI group. Mortality in the corresponding control group was 97%. These results suggest that prolonged exposure period improves vaccine effectiveness.
Since 1996, an epizootic ulcerative disease has affected both coloured-and common carp in Japan. We isolated atypical Aeromonas salmonicida from ulcerative lesions in the skin of diseased fish. It took more than 5 days for the isolates to make visible colonies on heart infusion agar at 18deg;C. All isolates were Gram-negative, facultatively anaerobic, non-motile, short rods, which reacted positively to rabbit anti-A. salmonicida subsp. salmonicida ATCC14174 serum by fluorescent antibody technique. However, the isolates differed from typical A. salmonicida in the following characters : coloured carp isolates were negative in the catalase test, and common carp isolates were negative in the oxidase test. Sequence analysis of 16S rRNA genes indicated that they could be identified as atypical A. salmonicida, and that coloured carp isolates differed in only one base of the sequences from common carp isolates. Subcutaneous injections of common carp with a representative strain of each type confirmed its virulence in producing ulcerative lesions on the skin.