Seven species of fish pathogens, Vibrio alginolyticus, V. anguillarum, V. damsela, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus were collected. Genomic DNA were extracted and cleaved by restriction enzymes, such as BclI, ClaI, DpnI, EcoRI, HindIII, HpaII, MboI, MspI, SacI, Sau3A, SpeI and XhoI. Best results were obtained from ClaI, EcoRI, HindIII, and SacI. The resulting DNA fragments, ranging from 2 to 23 kb, were separated electrophoretically on horizontal agarose gel to form specific restriction patterns. The restriction patterns of each Vibrio species were characteristic as fingerprint. The fingerprint of genomic DNA also reflects intraspecific differences as shown by serovars and biogroups. Restriction endonuclease analysis (REA) were highly identical for strains of V. anguillarum (Ita172, LS174 and NIE275) which were categorized as serovar C. Nevertheless, among serovars A, B and C of V. anguillarum, two patterns, HindIII and SacI, were similar; the other two patterns, ClaI and EcoRI, were not closely similar. Genomic DNA obtained from V. vulnificus strains (ATCC27562, SG716, TG617 and UE516) were also examined in this study. It showed that the restriction patterns of three formers, which belong to biogroup 1, were almost identical. However, these fingerprint patterns were different from those of strain UE516, which was isolated from eel and categorized as biogroup 2. We therefore conclude that REA has potential for studying the epidemiology of vibriosis in fish, shrimp and shellfish.
Recently a fatal disease with muscular atrophy has often occurred in cultured juvenile abalone Nordotis discus at various hatcheries in Japan. The disease is characterized by the formation of tumors in the pleuro-pedal neuro-trunk. Nothing is known about the cause of the disease. In order to investigate the cause of the disease, an infection experiment was made by exposing uninfected juvenile abalones to a 220 nm filtrate of diseased abalone homogenates and to the drain of an aquarium containing diseased abalones. As a result, the disease identical grossly and histopathologically to naturally diseased animals was produced by either method. This indicated that the disease was infectious and the causative agent might be a virus. The progress of the disease was suppressed when the ambient water temperature rose to 23°C or higher.
The fate of Edwardsiella tarda antigens, crude lipopolysaccharide (LPS) and formalin-killed cells (FKC) which was injected intramuscularly in eel, Anguilla japonica, was investigated. Location of the antigens was observed using fluorescent antibody technique. After injection, crude LPS was detected in the liver, kidney and spleen at 30 min and 1 h, in the heart and liver at 3 and 6 h, but not detected in any tissues at 12 h. FKC were detected in the gill, liver and kidney at 30 min, in the gill, stomach, intestine, liver and kidney at 1 h, in the intestine, heart, liver, kidney and spleen at 3 h, in the liver, kidney and spleen at 6 h, and in the kidney at 12 h. FKC disappeared from all tissues except the kidney until 12 h. Intracellular specific fluorescence was observed in the cells of the heart and liver of both fish groups injected with crude LPS or FKC. These findings suggest that after injection, the antigens are rapidly accumulated mainly in the liver and kidney, then decomposed or excluded.
Orally administered fumagillin DCH failed to protect rainbow trout (Oncorhynchus mykiss) from natural or experimentally-induced ceratomyxosis. No differences in mortality were observed between groups of trout exposed to Ceratomyxa shasta and fed fumagillin at 0.25 or 0.50 g/kg of diet at 2.0% fish body weight per day. Treatments with the drug however, did extend the mean time to death of trout due to C. shasta when compared to exposed but unmedicated controls. Extended feeding of the drug resulted in mortality in both infected and uninfected control groups. Malachite green was also ineffective in controlling ceratomyxosis when applied at 7 days intervals at 1.6 ppm for 40 min over a 64 day period.
Changes in the number of antibody producing cells were studied by measuring plaqueforming cells (PFC) of the spleen and kidney in carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), ayu (Plecoglossus altivelis), Japanese eel (Anguilla japonica), Japanese flounder (Paralichthys olivaceus), and red seabream (Pagrus major) after an intraperitoneal injection with Vibrio anguillarum 0-antigen. It was confirmed in carp and ayu that the injected antigen was distributed at the highest levels in the spleen and kidney 3 to 6 h after the injection. PFCs appeared in the spleen and kidney of all the fish species tested. The number of PFC reached maximum value after 8-17 days. The number of PFC in the spleen was always higher than that in the kidney in carp, rainbow trout, ayu and eel, whereas in red seabream the former was lower than the latter. There was no difference in PFC number between the two organs in the Japanese flounder. In an additional experiment, ayu was immunized by immersing in the 0-antigen suspension (3g/l) for 30 min. As a result, no PFCs were detected in the spleen or kidney and no aggulutinating antibody was measured in the serum.
Glycogen content was determined in the neutrophils of eel, Anguilla japonica. Prior to collecting neutrophils, the eel was intraperitoneally injected with formalin-killed Edwardsiella tarda, 2% casein or physiological saline as an irritant. Samples were taken from head kidney, peripheral blood and peritonel cavity exudate. Separation of neutrophils from the samples was carried out by centrifuging cell suspension loaded on a gradient Ficoll-Metrizoate solution. Neutrophils were washed with MEM supplemented with 1mg/ml of glucose, because glycogen depletion was demonstrated in the neutrophils washed with glucose-free Krebs-Ringer phosphate. Glycogen content of neutrophils in the fish injected with 2% casein or formalin-killed E. tarda was significantly higher than that in the fish injected with physilological saline. Both the peripheral and peritoneal neutrophils contained more glycogen than head kidney neutrophils.
Whether Edwardsiella tarda produces siderophore or not was investigated. CAS assay was used for detecting siderophore and Eagle's MEM was employed as a basal medium with a slight modification. Iron was eliminated from the basal medium by chloroform-8-hydroxyquinoline extraction. On this iron deficient agar medium, all the strains of E. tarda showed siderophore positive reactions. The siderophores were prepared from two virulent and two avirulent strains and examined their electrophoretic mobilities at pH 5.6. The siderophore mobilities against H2PO4-were all-0.05 and no differece was found among these 4 strains.
Large and small plaque-producing populations of infectious hematopoietic necrosis virus were separated in a salmon embryo cell line, CHSE-214. Plaques produced by the two viral populations increased in size at different rates, indicating that the two plaque types were genetically determined. The small plaque type was numerically dominant in low-passage virus stocks from four locations and in unpassed virus from naturally and experimentally infected fish, suggesting that it is the wild type. The pH of the overlay medium appeared to influence the size of the plaque.