Replicability and yield of 2 strains of IHNV in 9 salmonid cell lines (CHSE-214, KO-6, RTE, RTG-2, RTH, RTT, SEH, STE, RTE-2) and 3 non-salmonid cell lines (FHM, EPC, EPG) were compared at a low M.O.I.of 0.001.Yield of IHNV in RTE-2 derived from rainbow trout embryo was as high as those of RTG-2, EPG, FHM and EPC cell lines. Although EPC and FHM cell lines showed high sensitivity to toxic substances in the ovarian fluid from masu salmon Oncorhychus masou, RTE-2 and RTG-2 showed low susceptibility to these substances.These results indicate that the RTE-2 and RTG-2 cell lines are suitable for the isolation of IHNV from the ovarian fluid.
An etiological study was conducted to identify the causative agent of contagious edema of cultured juvenile colorcarp, Cyprinus carpio, in Japan. The disease outbreaks were often observed during the rainy season, extending from late June to late July, and the mortality frequently reached 80-100%. High mortality with edema was demonstrated among carp fry (0.10-0.26g) exposed to the filtered homogenates of diseased fish. Repeated infection trials demonstrated that the disease was transmissible by using filtered homogenates even after 12 times passage.Pox-like virus particles, 335 (300-400) nm in length and 265 (245-290) nm in width, were detected in the gilllamellar epithelial cells from both spontaneously affected and experimentally infected fish. Pancreas, kidney, spleen, and intestine of experimentally infected fish showed histopathological changes, but pox-like virus or other pathogenic organisms were not found in these organs. In the case of experimentally infected fish, more than 80%of infectivity was found in gills when the titers were compared by quantitative infection trials. Although the virus has not been successfully isolated, the facts mentioned above indicate that the pox-like virus observed in gill epithelial cells is the pathogen of this disease. We suggest that this infectious disease be termed “viral edema of carp”.
A disease showing erosive and ulcerative cutaneous lesions occurred in Japanese eels Anguilla japinica, reared in warm-water ponds in the spring of 1992. In this study, virological investigation, histological and electron microscopic observations were performed in both naturally and experimentally infected eels. A herpesvirus was isolated from the skin lesions and EK-1 cells inoculated with the isolated virus formed polynulcear giant cells at 25°C within 2 days after inoculation. The isolate was neutralized with an anti-Herpesvirus anguillae serum. Electron microscopy revealed enveloped virions with a diameter of 200nm in the cytoplasmic vesicle and nucleocapsids with a diameter ranging 114-115nm in nuclei of infected EK-1 cells. In experimental infections, the isolate did not have lethal effect on eels during rearing period of 14 days at 25°C. However cutaneous lesions were produced at the inoculated sites. Histologically, the cutaneous lesions of naturally as well as experimentally infected fish, showed similar signs : fibrocytes in the dermis were infected and necrotized, accompanied with necrotic changes of melanocytes and subsequent infiltration of inflammatory cells. The fibrocytic necrosis and the infiltration of inflammatory cells extended into the subcutaneous adipose tissue and lateral musculature.
Outbreaks of bacterial gill disease (BGD) occurred among cultured rainbow trout (Oncorhynchus mykiss) in Chungbuk province of Korea. Gram-negative, filamentous and yellow-pigmented bacteria were isolated from the diseased fish. The morphological, physiological, biochemical, and antigenic properties of the Korean isolates were consisted with those of Flavobacterium branchiophilum (ATCC 35035T and ATCC 35036) isolated from salmonid fish in Japan and Oregon, USA. It was concluded that the Korean isolates were identified as F. branchiophilum.
Epizootiological surveys on salmonid herpesvirus-2 (SaHV-2) infection in coho salmon (Oncorhynchus kisutch) culture were undertaken at freshwater hatcheries and seawater farms in order to clarify the infection source. At seawater farms, the outbreaks were observed only in the fish populations derived from particular hatcheries, and in some cases, the disease was observed during the seawater acclimatization period. Outbreaks during mariculture period tended to occur repeatedly year after year when the seedlings were introduced from hatcheries where coho salmon was reared together with other salmonids. On the other hand, in fish derived from hatcheries where only coho salmon was reared, the outbreak occurred only when the hatchery replenished the juveniles from virally contaminated hatcheries. In the surveillance of virus carrier fish, SaHV-2 highly virulent to coho salmon was isolated from apparently normal rainbow trout collected at a hatchery, where coho salmon seedlings supplied every year had been found to be contaminated. These results suggest that the SaHV-2 infection to coho salmon occurred at freshwater hatcheries and that the transportation of infected juveniles spread the virus among the hatcheries. It was also suggested that the infection source might be asymptomatically infected other salmonid species which were reared together with coho salmon.
The randomly amplified polymorphic DNA (RAPD) method of PCR amplification was used to analyze a group of non-motile aeromonads. Genomic DNA fingerprints were generated, using 12-mer random primers, for 29 strains of atypical Aeromonas salmonicida isolated from eight different species of fish in Japan and for references strains of A. salmonicida subsp. salmonicida, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia. All RAPD profiles, using primers A22 and A32, of atypical A. salmonicida strains were identical. The RAPD profile amplified by primer A31 was similar among isolates of the same species of fish. Some common fragments were found in atypical A. salmonicida strains amplified by primers A24 and A29. This indicates genomic homogeneity among atypical A. salmonicida strains isolated from the eight species of fish in Japan. However, RAPD patterns of the 4 reference strains differed from those of atypical A. salmonicida strains examined, suggesting genomic differences between atypical A. salmonicida and the others.
An invesigation on optimal temperature for the growth and protease production of a mesophilic bacterium, Aeromonas hydrophila was carried out. The studied 12 strains of fish origin were collected and stored at -80deg;C. Optimal growth temperature was determined by a temperature gradient incubator using TYE medium, a suitable medium for protease production. The protease production was estimated by the Bio-Rad DC protein assay with broth culture supernatant at around stationary phase. Casein (Hammarsten quality) was used as a protein substrate. There was no considerable amount of protease within log phase culture. Although the optimum temperatures for growth and protease production were variable with the strain, optimal temperatures for protease production were always lower than those for the growth. Peak growth was favoured at 34.5±1.0deg;C, while the protease production at 27.6±4.9deg;C. The differences among the peaks of protease production and growth were 1 to 15deg;C.
The bactericidal activity of Japanese eel neutrophils in the presence of oxygen radical scavengers was investigated. Catalase inhibited bacterial killing, while superoxide dismutase and hydroxyl radical scavengers (D-mannitol, sodium benzoate) did not. Superoxide dismutase had no synergetic effect with catalase. These observations indicate that hydrogen peroxide acts as a potent factor in the bactericidal process by eel neutrophils. The phagocytic index of cytochalasin B-treated neutrophils was less than one third of that of control neutrophils, although their phagocytic rate was more than 90%. Cytochalasin B, which inhibits phagosome formation without suppressing radical production, decreased the bactericidal activity of the neutrophils, and catalase did not affect the bacterial killing of cytochalasin B-treated neutrophils. Exposure to hydrogen peroxide at concentrations, which are expected within eel neutrophils during the respiratory burst, effectively killed the bacteria tested. Thus, it is suggested that the hydrogen peroxide-dependent bactericidal activity in eel neutrophils is expressed only within phagosomes but not extracellularly and that phagosome formation is essential for this activity.
Prior to the challenge with Enterococcus seriolicida, 50 yellowtail Seriola quinqueradiata were reared with the high dissolved oxygen (HDO), and another fifty with the low dissolved oxygen (LDO), for 10 days. After the fish were exposed to the pathogen, each group was separated into 2 subgroups and each subgroup was kept either in LDO or in HDO water. Low DO level during the pre-infection period shortened the length of the incubation period, and the low DO level during post-infection period increased the cumulative mortality of the disease.