Three cell lines derived from spleen and kidney of black porgy (Acanthopagrus schlegeli), designated as BPS-1, BPS-4, and BPK, were established and their characteristics were determined. The BPS-1cell line and BPS-4 cell line consist of epithelial-like and fibroblast-like cells, respectively. The BPK cell line contains both fibroblast-like and epithelial-like cells. Using Leibovitz's-15 medium supplemented with 10%fetal calf serum and 0.15M NaCl at 28°C, BPS-1, BPS-4 and BPK cells have been passaged 105, 104, and 87 times, respectively. The chromosome numbers of the BPS-4 cell ranged from 10 to 58 with a modal number of 30 at passage 77, while the chromosome numbers for the BPS-1 cells and the BPK cells varied even more widely from 25 to 98 at passage 76 and 9 to 94 at passage 28, respectively.With low seeding densities, all the three cell lines showed a low plating efficiency.All three cell lines were susceptible to a variety of fish birnaviruses and herpesviruses from eels in Taiwan.
HB-1 virus, a strain of infectious pancreatic necrosis virus (IPNV), was isolated from hard clam, Meretrix lusoria, with TO-2 cell line derived from tilapia ovary. The defective interfering (DI) particles of HB-1 virus could be generated by serial undiluted passaging. The polypeptides and RNA in virus particles of diluted and undiluted virus preparations were compared in order to investigate the nature of defective interfering particles involved in viral interference of HB-1 virus. Cesium chloride density gradient centrifugation was applied to purify virus particles of diluted and undiluted virus preparations. By comparing the polypeptides and RNA in virus particles of diluted and undiluted virus preparations, it was revealed that (I) the virus particles of undiluted virus preparation had smaller polypeptides than normal ; (II) subgenomic RNA of large segment was found in virus particles of undiluted virus preparation. These two findings suggested that DI particles of HB-1 virus was a deletion mutant of standard virus. This is the first report indicating the polypeptides and RNA possibly related to IPN virus DI particles.
Histopathological studies were made on cultured ayu with systemic, multiple granuloma. Examined fish were collected from a farm in Tochigi Prefecture in 1989 and 1990. Macroscopically the disease was characterized by discoloration of the body, severely protruded eyes, and numerous small nodules on the surface of many organs as follows; liver, kidney, spleen, heart and intestine. Histology showed that these nodules were granulomas composed of mononuclear cells and surrounded by the thin connective tissue. Granulomas were also found in gill filaments and trunk muscles. Gram-positive, selener, long rods were abundantly detected in the granulomas. Some of these long rods were also stained positively with Ziehl-Neelsen method for acid-fast bacteria and methenamine silver-nitrate stain (Grocott's variation). From the results, the case of these fish was diagnosed as “systemic, multiple granuloma caused by acid-fast bacteria”.
We tried several transmission experiments of naturally infected actinosporeans in the tubificid oligochaete, Branchiura sowerbyi, to myxosporean-free goldfish, Carassius auratus. Almost all actinosporean spores collected by squashing tubificids or filtering effluent from a container with tubificids were physically damaged and/or unclean, and all infection experiments using such spores failed. However, coexistence of uninfected goldfish with a stock of B. sowerbyi shedding spores of several actinosporeans in the same container resulted in infections with three species of myxosporeans : Zschokkella, Myxobolus, and Thelohanellus spores in 15-20% of goldfish after 2.5-4 months. We improved the collection method of actinosporeans; tubificids were individually placed in the cell-well plates with 24 2 ml-wells and waterborne actinosporeans were detected by inverted phase-contrast microscopy. Actinosporeans harvested by this method were intact and purely concentrated. Eventually, in goldfish kept with tubificids harbouring the actinosporean Raabeia sp. in the same tank for 3 weeks, and in goldfish exposed to Raabeia suspension for 1 hour, spores of an unidentified Myxobolus were found 3-4 months after the exposure. Unexposed goldfish and ones exposed to actinosporean-free tubificids were both negative for the infection. We developed the collection method of actinosporeans, and confirmed that the actinosporean Raabeia infecting the oligochaete B. sowerbyi was transformed into Myxobolus in goldfish.
Prevalence of Aeromonas salmonicida was studied in mature chum (Oncorhynchus keta), pink (O. gorbuscha), and masu salmon (O. masou) without signs of furunculosis. From September 1979 to November 1990, a total of 15, 264 chum, pink and masu salmon were collected from 33 rivers. We isolated A. salmonicida from chum salmon in 18 of 28 rivers and the prevalence ranged from 1.8 to 34.4%. For pink salmon populations, A. salmonicida was isolated in 9 of 14 rivers with prevalence ranging from 1.7 to 14.6%. In masu salmon, A. salmonicida was isolated in 6 of 10 rivers and the prevalence ranged from 0.5 to 3.7%. The above results indicated that A. salmonicida distributed widely in the populations of mature salmonids, except those in the rivers on the coast from Tsugaru Strait to Uchiura Bay. The annual change in prevalence of A. salmonicida in salmon was closely related to the fish density in the holding ponds for maturation. The viable numbers of A. salmonicida in the kidney were found to be from 103 to 105 cfu/g.
In the previous paper, we reported that Aeromonas salmonicida was isolated from mature chum salmon (Oncorhynchus keta), pink salmon (O. gorbuscha) and masu salmon (O. masou) without signs of furunculosis. In the present study, we determined the prevalence of A. salmonicida in immature chum salmon without signs of furunculosis captured in rivers and on the coast of Hokkaido. A. salmonicida was not isolated from 480 immature chum salmon were examined from September 1979 to October 1990. We compared the prevalence of A. salmonicida in the kidney of immature and mature chum salmon which ascended the Shibetsu, Nishibetsu and Ishikari rivers. The prevalence of A. salmonicida in the immature fish was lower than that in the mature fish. A. salmonicida was neither detected from the kidneys of chum and masu salmon fry, nor isolated from the surface of chum salmon eggs in the hatcheries. From these results, we considered that these salmonids were not infected with A. salmonicida until they entered the rivers.