In the course of diagnostic works of diseased ayu, Plecoglossus altivelis, and amago, Oncorhynchus rhodurus var. macrostomus, cultured in freshwater ponds in Tokushima Prefecture from 1977 to 1978, a new bacterial disease was found among them. The characteristic sign of the diseased fishes was hemorrhagic lesions of body surface, especially operculum, abdomen, and anus. A β-hemolytic streptococcus was isolated from these diseased fishes, and it proved to be able to kill ayu and amago with typical symptom of this disease by intraperitoneal injection. From the results of biochemical and biological tests, it became evident that the etiological agent of this disease was identical with the isolate from cultured yellowtail, Seriola quinqueradiata, by MINAMI et al. (1979). Yet, the species name could not be determined for the organism, because the antigen prepared from the isolate did not react with commercial antisera to Streptococcus groups A, B, C or G.
The incidence of Aeromonas salmonicida as the causative agent of furunculosis in the kindney of anadromous salmonids in Hokkaido were examined. During September to November in 1979 a total of 2, 028 fish of four species of salmonids, chum salmon (Oncorhynchus keta), pink salmon (O. gorbuscha), masu salmon (O. masou), and kokanee salmon (O. nerka) were collected for this study at the salmon capture stations in 18 rivers and one lake. The obtained results are summarized as follow. 1) In chum salmon, 1, 290 fish from 12 rivers were examined and A. salmonicida was isolated from 148 fish. Incidence in each river varied from 0 to 60.5 percent and was greater in the fish reared for maturation than the others. 2) A. salmonicida was isolated from 66 pink salmon of the 562 fish at 9 rivers. The highest incidence was 36.6 percent at Ichani river. 3) No sign of furunculosis were observed in any chum salmon or pink salmon examined. 4) Other than the salmonids examined, in 116 masu salmon, 60 kokanee salmon and 266 fresh water fish, no A. salmonicida were isolated.
The relative efficiencies of various methods of concentration and purification of the eel virus of Europe (EVE) were examined. Whilst Freon treatment had no obvious effect on yields, EVE was effectively concentrated 10 times using polyethylene glycol (PEG), and could be concentrated 100 times using a 2 step PEG technique, or by use of high speed centrifugation as alternative to the 2nd PEG step. High speed centrifugation had the advantage of excluding UV absorptive substances other than virus, which could not be excluded by the 2 step PEG method. EVE was partially purified by sucrose linear 15-45% density gradient centrifugation at 80, 000g for 2 hrs. Well purified EVE was obtained by CsCl isopycnic centrifugation at 160, 000g for 24 hrs at a bouyant density of 1.33g/cm3. IPNV-VR299 was concentrated and purified in a similar manner to EVE and was also shown to have a density of 1.33g/cm3. The mean diameter of uranyl-acetate stained EVE was 65.5nm and that of IPNV 66.6nm. CsCl isopycnic centrifugation of EVE, but not of IPNV, revealed a faint band at a density of 1.30g/cm3 composed of many particles of average diameter 20nm apparently morphological subunits. This feature, together with the preserve of empty and collapsed particles in the main EVE band, suggested a major difference between EVE and IPNV in virus stability.
In this study, the possibility of using passive immunization with rabbit immunoglobulin (RIg) as a preventive measure and a medical treatment for pseudotuberculosis in cultured yellowtail, Seriola quinqueradiata, was investigated. Yellowtail were passively immunized with RIg prepared from anti-Pasteurella piscicida rabbit serum by intraperitoneal injection. The absorption of RIg into serum and skin mucus after immunization were examined. The artificial challenges with virulent P. piscicida were carried out 6, 12, 24 hours before and 1, 12, 24 hours after immunization. The efficacy of passive immunization from the results was studied. In immunized fish, agglutinating antibody titers in sera increased rapidly and the highest mean titer of 1: 42 was obtained 3 hours after immunization. Maximum concentration of RIg in serum was detected 12 hours after immunization. However, no RIg was detected in serum after 24 hours post-immunization, and neither in all of skin mucus samples. Survival ratios for 8 days after challenge of fish immunized passively were not less than 70% in comparison with 0% survival in the control. On the contrary, survival ratios for 8 days after passive immunization of challenged fish were not more than 30%. It is concluded that passive immunization of yellowtail against pseudotuberculosis with RIg is highly effective for at least 24 hours after immunization.
Little work has been done on phagocytosis of fish leucocytes and on the role of phagocytes in the host-parasite relations in fish. The purpose of the work reported in this paper was to classify the leucocytes of goldfish(Carassius auratus) by peroxidase reaction, and to examine which of the leucocytes could phagocytize bacteria. Inoko & Itoga's method using o-tolidine as a hydrogen donator was applied to blood smears to detect the peroxidase reaction of leucocytes. Phagocytosis of leucocytes on bacteria was examined in vitro. Heparinized blood was obtained from goldfish by heart puncture. Eighteen hour broth cultures of Staphylococcus aureus and Escherichia coli were used as bacterial cell suspensions. Zero point zero one ml of the bacterial cell suspension was added to 0.09ml of the blood in a small test tube. The mixture was incubated for several hours at 25°C with shaking. After the last incubation, the mixture was spread on slides.The peroxidase staining was applied to these slides. The peroxidase reaction and the structure of the leucocytes phagocytized bacteria were examined. The peroxidase positive granules in the leucocyte of goldfish stained yellow brown by Inoko & Itoga's method and the nucleus of the peroxidase positive leucocyte could be clearly observed. The positive reaction could be observed on the blood smears preserved for a period of 47 days without fixation. The average number of the peroxidase positive leucocytes per 5, 000 erythrocytes was 17 in the peripheral blood of goldfish. Many peroxidase positive cells were observed on touch preparations from kidney and head kidney. Three types of leucocytes, peroxidase positive leucocyte, peroxidase negative large leucocyte and peroxidase negative small leucocyte, could phagocytize bacteria. The appearance of peroxidase positive leucocyte phagocytized bacteria was similar to that of granulocyte in mammals.