• Korean Journal of Veterinary Pathology
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• v.3 no.1
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• pp.1-5
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• 1999

This experiment was carried out to establish the immunohistochemical diagnostic method for infectious pancreatic necrosis in the monolayers of CHSE-214 cell cultures and paraffin-embedded tissue sections from rainbow trout infected with infectious pancreatic necrosis virus(IPNV). Specific identification of IPNV antigens was often demonstrated in the pancreatic exocrine cells, and less in the intestinal mucous epithelia and the renal hemopoietic tissues by the use of monoclonal antibodies against capsid protein VP2. The specific reaction was seen as a distinct red cytoplasmic color, often as small granules of various sizes. The result showed that streptavidin alkaline phosphatase immunohistochemisry specifically identified IPNV antigens in both infected cell cultures and tissue sections.

• Journal of fish pathology
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• v.30 no.2
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• pp.71-78
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• 2017

The glycoprotein of novirhabdoviruses is known to play a critical role in the determination of host specificity. Viral hemorrhagic septicemia viruses (VHSVs) in different genotypes have different glycoprotein sequences and show different preferences for specific cell lines. In this study, to know whether the glycoprotein is solely responsible for the host cell preference of VHSV, a recombinant VHSV expressing vesicular stomatitis virus (VSV) glycoprotein instead of VHSV IVa glycoprotein (rVHSV-VSV-G) was generated by reverse genetics and inoculated into several fish cell lines, then, cytopathic effect (CPE) and viral growth caused by rVHSV-VSV-G infection were compared with those caused by rVHSV-wild that was previously generated and has the same genomic sequence with wild-type VHSV except a few nucleotides. The plaque numbers of rVHSV-VSV-G were significantly higher in EPC, BF-2 and GF cells than those of rVHSV-wild. However, in HINAE cells (originated from olive flounder), rVHSV-VSV-G titer was significantly lower than rVHSV-wild titer, and both recombinant VHSVs were not grown well in CHSE-214 cells. Although statistical significances were detected in the titers between rVHSV-wild and rVHSV-VSV-G in several cell lines, the cell line-preference order of rVHSV-VSV-G was not different from that of rVHSV-wild. These results suggest that the replacement of VHSV glycoprotein may not completely change host cell preference, and other regions of VHSV might also involve in the determination of host cell preference.

• Journal of Microbiology
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• v.34 no.2
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• pp.124-130
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• 1996

A survey was conducted to determine the prevalance of infectious pancreatic necrosis virus (IPNV) on fish farms in Korea and the epidemiology of IPNV infection in the farmed rainbow trout. In total, 43 pools of rainbow trout with apparent signs of viral infection from five provinces were obtained and analyzed. Evident cytopathic effects, including karyopycnosis and cell destruction, were observed in CHSE (chinook samlmon embyro)-214 cells infected with the virus isolates. Of these, ten viral isolates were assumed to be IPNV based on biophysical properties. RNA analysis revealed that the isolates contained two-segmented RNA genomes, further indicating that the viral isolates are IPNV. Antigenic comparison of the IPNV isolates identified three distinct serological groups separable by the cross-neutralization test. Of the ten IPNV isolates, six could be classified as strain DRT, two as strain Ab, and two as strain VR299. We were not able to isolate new strain of IPNV or any isolate serologically similar to the standard strain Sp.poraceae and families of the Agaricales, they are genetically more related to the Polyporaceae. These results are consistent with morphological characters observed in those mushrooms. However, it is premature to conclude taxonomic status Ganoderma species in the present study employing small sample size.

• Journal of fish pathology
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• v.4 no.2
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• pp.87-94
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• 1991

In the late summer of 1990 and 1991, mass mortality occured among cage-cultured grouper, Epinephelus septemfasciatus in south cost of Korea. The moribund fish didn't feed and became pale or dark chestnut colour and irregularly swimmed due to the loss of equilibrium, finally the diseased fish fell down side away on the bottom or the surface of cage showing the bent of body and died. The diseased fish showed the extensive hemorrahge in brain, the swelling of spleen and bile duct as the specific syptoms of internal organs. So the gill, skin and other organs of the diseased fish were examined for the presence of pathogenic parasites and bacteria. The parasitic Trichodina sp. were detected only from the gill lamella of the diseased fish, but these parasites seemed to be not a direct causative agents that induced the gross mortality of the cultured grouper. because these parasites were also observed in normal grouper, yellowtail, red seabream and rock bream co-cultured with the diseased grouper in same or near cages. In the viral examination, although isolation of the causative agent by the use of estabilshed cell Lines, RTG-2 and CHSE-214, was not succeed, the normal grouper inoculated intramuscularly with the filtered homogenate of the organs of the diseased fish showed the same external and internal signs with the naturally infected grouper. They died within a week. By using the naturally and the artificially infected fishes, electron microscopic observation revealed numerous hexagonal or polygonal particles in the cytoplasm of liver cells. Based on the these results, we suggest that the mass mortality of the cultured grouper would be occurred by the infection of a viral agent.

• Journal of fish pathology
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• v.6 no.2
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• pp.205-218
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• 1993

On the development of hirame(Paratichtys olivaceus) culture, outbreak of scuticociliata infection was reported to cause severe damage in Japan. To establish effective measures for isolation and cultivation of this ciliate, we tried to culture this pathogenic ciliate using medium for bacteria and fish cell lines in vitro. Scuticociliata from the brain tissues of infected fish was aseptically inoculated to CHSE-214 cells cultured in MEM-10 without antibiotic. Scuticociliata grew well and the number of ciliate reached $10^6\;cells/ml$ at temperatures of $15^{\circ}C$ to $20^{\circ}C$ for 10d. The number of ciliate cultured in the cell lines is 10 times higher than the numbers cultured in the liquid medium alone. This ciliata could be cloned by dilution method. Scuticociliata isolated could grow well on 42 different cell lines that were established from marine fish, warm freshwater fish, and salmonids. This ciliate could be preserved in liquid nitrogen for more than 6 months. Subsequently, we observed the optimal temperature and salinity for growth, and tested the sensitivities of this organism to formaldehyde, flagyl(Metronidazole), Ekuteshin(Combination compound of sulfamonometoxin and ormethoprim), and ozonixation. Optimal temperature for growth was $25^{\circ}C$ and salinity was 1.0 to 1.5%. Washed scuticociliata was killed by formaldehyde at the concentration of 50ppm for 10min, but was not completely killed even at a high concentration of 400ppm for 20min in MEM-5. Flagyl and Ekuteshin can inhibit the growth of scuticociliata at the concentration of 1,000 and $100{\mu}g/ml$ in MEM-10, respectively. More than 99% of this scuticociliata could be killed by ozonization at a dose equivalent to $1.0mg/\ell$ oxidant for 30sec in sea water. Isolated scuticociliata showed the pathogenicity to the cultured hirame by artificial infection(I. P. injection, $10^5\;cells$/fish). The number of scuticociliata in the water could be counted by most probable number(MPN) method using tissue culture, and the minimum detectable number was $1.8\;cells/\ell$. The number in the reservoir tank for water supply to the culture tank was 110 cells/l. After cleaning by elimination of the sediments from of the reservoir tank and disinfected with formaldehyde, number of scuticociliata decreased and was counted less than $1.8\;cells/\ell$ and infection rate of cultured hirame was decreased.

• Journal of fish pathology
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• v.24 no.3
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• pp.213-223
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• 2011

Among wild marbled sole, Pleuronectes yokohamae caught in west coastal area, Chungnam, tumor-bearing fish were found. Parasites and pathogenic bacteria were not detected and CPE was not observed. Using the light microscope, the epidermal layer of wild P. yokohamae was significantly thickened compared to the normal skin. The lesion was formed papillary folds. Hypertropical epithelial cells revealed karyolysis, marked nucleolus and cloud swelled cytoplasm. In the epidermal layer of the lesion, X-cell that is characterized by oval and small pale nucleus and prominent nucleolus was observed. Dermal layer had newly formed vessels. The size of mucous cell in the papilloma lesion was significantly increased compared to the normal. In this study, no pathogens were found, so future works for finding cause of the papilloma in the P. yokohamae are needed.

• Journal of fish pathology
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• v.11 no.2
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• pp.97-104
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• 1998

Since 1989, mass mortality has repeatly occurred in cage-cultured sevenband grouper, Epinephelus septemfasciatus along the southern coast of Korea in the summer season and usually reached over 80% within a few months. Diseased fish showed the clinical signs of anorexia, dark coloration, loss of eqilibrium, spinal swimming behaviour, vertebral deformity and inflation of swim bladder. Histopathologically, necrosis and/or vacuolation of the nerve cells in the brain and retina were observed. We previously reported that the causative agent was filtrable. The causative agent was not culturable in various fish cells; RTG-2, CHSE-214, BF-2, EPC and FHM. However, electron microscopic observation revealed unenveloped icosahedral viral particles with about 30 nm in diameter in the cytoplasm of nerve cells of the brain. The characteristics of the virus was tested by an artificial infection with the filtrate of the homogenate of diseased fish. The pathogenicity of the virus was retained after treatment with ether or heat ($50^{\circ}C$, 30 min) but partly lost by pH 3 or 11 treatment. These results suggest that the causative agent are similar to the fish nodavirus. In order to compare the causative agent with a fish nodavirus, Striped Jack Nervous Necrosis Virus (SJNNV), a polymerase chain reaction (PCR) was performed with primers specific to SJNNV. As a result, about 430 by PCR products were detected from the brain and the eye of both naturally and artificially infected sevenband grouper. All these results represent that the mass mortality in the cultured sevenband grouper is caused by the infection of a nodavirus similar to SJNNV and this is the first report of a fish nodavirus from the cultured sevenband grouper in Korea.

• Journal of fish pathology
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• v.18 no.2
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• pp.167-178
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• 2005

Streptococcus iniae is one of the major bacterial pathogens of cultured olive flounder, Paralichthys olivaceus in Korea. This study was carried out to investigate the interaction between morphological characteristics and immune response such as phagocytosis and serum killing activity. They were differentiated phenotypically into two groups, the viscous colonies and the non-viscous colonies, but the strains in both groups were similar in physiological and biochemical characteristics. Electron microscopic examination of the viscous form revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the exopolysaccharide material was absent around the non-viscous form of S. iniae. The viscous type strains were disclosed more virulent than those of non-viscous type for olive flounder. The viscous strains were resistant to normal serum killing activity, while the non-viscous strains were susceptible to bactericidal activity of normal serum from olive flounder. The viscous strains were more resistant to opsonophagocytosis and decreased the chemiluminescence response of head kidney macrophages of olive flounder. All of the tested non-viscous S. iniae strains were efficiently destroyed by the macrophage after phagocytosis. Both the viscous and the non-viscous strains invaded and replicated in cultured fish cell line (CHSE-2l4). This cellular invasion may contribute to the pathogenesis of invasive S. iniae infection.