In this study, Japanese eels were injected with bacterial sonicate and then challenged with viable cells of Vibrio anguillicida. Bacterial sonicate was injected intramuscularly at a rate of 2.0 mg per 100 g body weight. After 24 hours, the treated fishes were challenged with an intramuscular injection of the viable bacteria at rates of 0.5 and 1.0 mg per 100 g body weight. Sixty percent of challenged fishes survived for three to seven days. The fishes produced protrudent lesions at the injected areas within three days and ulcerative lesions on the fifth day. In the protrudent lesions, many neutrophils infiltrated and phagocytized bacteria extensively in the affected tissues which exhibited necrosis, edema and hemorrhage. In the viscera, reactions of neutrophils and cells belonging to the reticulo-endothelial system were extensive, which prevented bacterial dissemination. Forty percent of challenged fishes became moribund within two days. The moribund fishes had protrudent lesions at the injected areas and exhibited the congested liver, splenomegaly and enteritis. They underwent systemic infection and the infected tissues exhibited extensive necrosis, circulatory disturbances and slight reactions of neutrophils. Based on the results, it was clarified that the fishes which were previously treated with the bacterial sonicate, manifested extensive reactions of neutrophils as defensive responses against challenge with the viable bacteria.
From June to October in 1980, mortalities occurred among the cultured Japanese horse mackerel (Trachurus japonicus) in Tachibana Bay, Nagasaki Prefecture. The typical external symptom of the diseased fish was corneal opacity and reddening of the eye-ball which finally progressed to evulsion of the orbital contents. A bacterium was isolated from the eye, liver, kidney, etc. of all the diseased fish. By inoculation experiments the bacterium was proved to be pathogenic to Japanese horse mackerel. The bacterium was placed in the genus Vibrio from its biological and biochemical characteristics. Although the biochemical and biological characters of the isolates resembled closely to those of Vibrio parahaemolyticus, they could be distinguished from V. parahaemolyticus by its lower NaCl and temperature tolerance. As above mentioned this bacterium may be regarded as a new fish pathogenic vibrio in the future.
From June through October 1980, an epizootic occured among the Ayu (Plecoglossus altivelis) at fresh water farms in Tochigi Prefecture, Japan. All the diseased fish examined exhibited clinical signs as follows; Externally, numerous petechial hemorrhages in the anal region and ventral body surface, and secretion of abnormal slime on the gill were common. Internally, excessive reddish ascitic fluid accumulation in body cavity and numerous hemorrhages in the intestine were common. The cultural and biochemical characteristics of five isolates from diseased Ayu as causative organism were quite uniform and resembled those of Streptococcus sp. which ONISHI et al. (1978) and KITAO et al. (1981) reported previously. The characteristics of all five isolates have been considered to justify classification in genus Streptococcus, and resemble S. pyogenes, S. equisimilis, S. zoopidemicus, S. equi, or S. dysgalactiae especially S. pyogenes mentioned in Bergy's manual 8ed. However, from the isolates exhibited, certain characteristics including serological character served to differentiate them from S. pyogenes. Under these circumstances it was not possible to classify isolates as members of any the species of genus Streptococcus described previously.
A continuous cell line, designated EO-2, was established from the ovary of Japanese eel, Anguilla japonica. The cells have been passed for 110 times during 19 months in Leibovitz's L-15 plus 10% foetal calf serum under a temperature of 32±1°C. The cell line consists of fibroblastic cells with large or small nuclei. Multinucleated cells were present in most of the cultures. The number of chromosome counted from 100 EO-2 metaphase cells was ranging from 22 to 74 with a modal number of 38. A low plating efficiency of approximately 8% was recorded for EO-2 cell at 32±1°C. The temperature ranged from 20 to 37°C could support the growth of EO-2 cell line. EO-2 cells were also demonstrated to be susceptible to Eel Virus Europe (EVE), Eel Virus Europe X (EVEX) and Eel Virus America (EVA). In comparison, EO-2 cells are more sensitive to these three viruses than RTG-2 or FHM cells. The cell line was found to be free of bacterial, fungal and mycoplasma contamination.
From August in 1978 to August in 1980, a continuous field investigation was made on Alella macrotrachelus infestation in black sea-bream (Acanthopagrus schlegeli) cultured in a private farm in the Inland Sea of Japan. 1) A. macrotrachelus was observed on the gills of the fish from the farm throughout the year.The parasite was also common in some other farms, but few in which were situated near the mouth of rivers. 2) The average number of the female parasites on a fish began to increase immediately after the start of rearing (August), and reached the first peak (ca. 15 worms/fish) in winter (January or February). It attained the second peak in the next spring, then decreased considerablly in late summer, when some of the cultured population began to be harvested. 3) The most dangerous stage of this parasitic disease seemed to be in the first winter. 4) From histopathological observations, it was found that the female parasite took the epithelial cells of gill lamellae and erythrocytes. The hyperplasia of the epithelial cell layer, edema and hemorrhage in the gill lamellae were also caused by the parasite.
Procedures for isolating of the leucocytes from fish blood were studied. Blood samples were obtained by heart puncture from carp, Cyprinus carpio and gold fish, Carassius auratus. The following two methods were adopted. Method 1. To one volume of the blood collected into a heparinized plastic syringe, added 2/3 volumes of physiological saline for freshwater fish(PSF). Mixed well by inversion. Vertically kept the syringe at room temperature for 3 hours. Collected the leucocytes from the buffy coat. Method 2. To heparinized blood, added an equal volume of PSF. Layered the blood-saline mixture onto the ficoll and sodium diatrizoate mixture(Histopaque®). Centrifuged at 400×g for 30 minutes at room temperature. Collected the. leucocytes from the opaque interphase. About 106 cells of leucocytes were isolated from 1 ml of the blood by these methods. The viability of isolated leucocytes was shown by their non-staining with trypan blue and phagocytic ability on bacterial cells. The peroxidase negative leucocytes had the adhesive property to the glass surface. These results indicate that the leucocytes isolated by these two methods are useful for studying the functions of fish leucocytes in vitro.