• Journal of fish pathology
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• v.9 no.2
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• pp.169-176
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• 1996

An indirect double antibody enzyme-linked immunosorbent assay (ELISA) was developed for rapid detection of a new virus isolated from abnormally swimming salmonid fish, RVS (Retrovirus of salmonid). Results using brain tissue homogenates, and infected cell cultures are described. The sensitivity of the methods is $10^{2.6}$ $TCID_{50}/100{\mu}l$ of the examined cell culture fluid. The specificity was confirmed by the ELISA inhibition test and virological examinations. Viral antigen could be detected in artificially infected fish tissue homogenates. The assay will allow the diagnosis of RVS-infected fish within a day.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.2
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• pp.136-140
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• 1986

The change of cell counts of Vibrio parahaemolyticus in fish muscle by the storage time and temperature was examined to get basic informations for precautionary steps against food poisoning of slices of raw fish (sashimi). There fore, we inoculated fish homogenate of oceanic bonito (Katsuwonus pelamis), yellow tail (Seriola quinqueradiata) with Kanagawa positive Vibrio parahaemolyticus and stored it at $30^{\circ}C,\;18^{\circ}C,\;4^{\circ}C\;and\;-20^{\circ}C$ for 24 hours. The number of the Vibrio parahaemolyticus upon fish homogenate stored at $30^{\circ}C\;and\;18^{\circ}C$ decreased for the first two hours and increased thereafter. When the fish homogenates inoculated with Vibrio parahaemolyticus at about $10^3$ per gram were stored at $18^{\circ}C\;and\;30^{\circ}C$ for 10 hours, the cell numbers increased about 10 times and 1,000 times initial cell numbers, respectively. The survival rate of Vibrio parahaemolyticus was about $20\%$, when the inoculated fish homogenates were stored at $-20^{\circ}C$ for 24 hours. Vibrio parahaemolyticus inoculated in fish homogenates was decreased by about $10\%$ of initial cell numbers by the storage at $4^{\circ}C$ for 4 hours and it was decreased by about $50\%$ after 24 hours storage of the samples at the same temperature. The decreasing rate of inoculated Vibrio parahaemolyticus in fresh fish muscle homogenate was higher than that in frozen fish muscle homogenate during the storage time at a refrigerator.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.21 no.2
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• pp.80-84
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• 1988

The change of cell counts of Vibrio vulnificus in meat homogenates of fish and shellfish by the storage time and temperature was examined to get basic information for precautionary steps against septicemia from slices of raw fish (sashimi). Therefore, we inoculated raw and cooked meat homogenates of fish and shellfish with Vibrio vulificus M-8 (isolated from shellfish ) and stored them at $-20^{\circ}C,\;4^{\circ}C\;and\;30^{\circ}C$ for 72 hours. Vibrio vulnificus M-8 was not detected in 32 hours when it was frozen and stored at $-20^{\circ}C$ after inoculating them into phosphate buffer solution at concentration of $10^5\;cell/ml$, while the existance of Vibrio vulnificus was identified after 72 hours of storage at the same temperature in case of inoculation into the meat homogenate of yellow tail. The cell count of Vibrio vulnificus was decreased as about $20\%$ of initial count after 2 hours storage at $4^{\circ}C$ in phosphate buffer solution with fish and shellfish homgenates. From the experimental results it was recognized that Vibrio vulnificus was labile to the cold stress. In comparison to the growth of growth of Vibrio M-8 at $30^{\circ}C$ in the raw and cooked meat of the yellow tail(Seriola guingueradita), snapper(Chrysophrys major), ark shell(Anadra brouhgtonii), and oyster(Crassostrea gigas), the raw meat homogenates were more excellent than the cooked ones though all fish and shellfish meat homogenates were proves to be good for the growth of the microbe.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1457-1463
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• 2016

Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3-C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10³ CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10³ CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10² CFU/ml, confirming the reliability of the method.

• Journal of fish pathology
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• v.32 no.1
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• pp.45-48
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• 2019

We developed and subsequently characterized mouse monoclonal antibodies (MAbs) against recombinant VP28 structural protein (rVP28) of white spot syndrome virus (WSSV). We established six hybridoma clones secreting MAbs against rVP28: 15A11, 20G6, 31H2, 34H6, 38D1 and 43A1. All six MAbs recognized the 25 kDa of protein in gill homogenates of WSSV-infected shrimp by western blot analysis, while no reactivity was observed in gill homogenates of normal shrimp. Moreover, high enzyme-linked immunosorbent assay (ELISA) optical density (OD) values (0.8-2.68) were observed in the hemolymphs from WSSV-infected shrimp, while low OD values (less than 0.24) were recorded in the hemolymphs from normal shrimp, by using these six MAbs produced in this study. These results suggest that these six MAbs are useful for the detection of WSSV.

• Korean journal of food and cookery science
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• v.12 no.1
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• pp.6-12
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• 1996

The survival and growth of Vibrio parahaemolyticus in tryptic soy broth(TSB), flounder homogenate and oyster homogenate with 0 or 5% of ethanol was tested at -20, 5, 35, 45 and 50$^{\circ}C$. Growth pattern of V. parahaemolyticus was similar in TSB and flounder homogenate but slightly poor in oyster homogenate at 35$^{\circ}C$. Growth occured at 5% ethanol, in TSB and flounder homogenate after a prolonged lag period but decreased in oyster homogenate during incubation at 35$^{\circ}C$. TSB and fish homogenates containing 0 or 5% of ethanol were inoculated with 10$\^$6/-10$\^$7/ cells/ml of V. parahaemolyticus and cold or heat resistance of the cells were determined at -20, 5, 45 and 50$^{\circ}C$. At 5$^{\circ}C$, the viability in culture broth with 5% of ethanol or without ethanol was not vary with the culture broth. In the presence of 5% of ethanol at -20$^{\circ}C$, cells of V. parahaemolyticus in flounder homogenate and oyster homogenate were more significantly inhibited than in TSB. The D-valves for V. parahaemolyticu at 45 and 50$^{\circ}C$ was significantly lower in oyster homogenate than in TSB and flounder homogenate with 5% of ethanol or without ethanol. The D-values in each culture broth without ethanol were 1.9-3.5 times of that value in each culture broth containing 5% of ethanol at 45 and 50$^{\circ}C$.

• Korean journal of food and cookery science
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• v.11 no.3
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• pp.261-266
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• 1995

The survival and growth of Staphylococcus aureus and Listeria monocytogenes in fish homogenates (flounder, shrimp and oyster homogenate) and tryptic soy broth (TSB) were tested during storage at simulated ambient (35$^{\circ}C$), refrigerated (5$^{\circ}C$) and frozen (-20$^{\circ}C$) temperature. A similar growth pattern of S. aureus at 35$^{\circ}C$ was observed in fish homogenates and TSB. Survival of S. aureus decreased at refrigerated or frozen temperature and that was greater at -20$^{\circ}C$ (0.3-1.2 log reduction/6 weeks) than at 5$^{\circ}C$ (1-1.6 log reduction/3 weeks). Viable cells of L. monocytogenes increased rapidly at 35$^{\circ}C$ in flounder homogenate, shrimp homogenate and TSB but after a prolonged lag period in oyster homogenate. During 3 weeks of storage at 5$^{\circ}C$, the levels of L. monocytogenes increased 3.8-5.0 log cycles in flounder homogenate, shrimp homogenate and TSB whereas levels increased 2.2 log cycles in oyster homogenate. Viable cells of L. monocytogenes during 6 weeks of frozen storage decreased 1.5-1.8 log cycles in flounder homogenate, shrimp homogenate and TSB while decreased 2.8 log cycles in oyster homogenate.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.4
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• pp.471-476
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• 1998

Microbiological spoilage of marine fish is complex process occurring by bacteria, yeasts and molds. There have been rare study for saprophytic yeasts although having enormous numbers of bacteriological studies on the spoilage of marine fish. The 14 genera of yeasts isolated from mackerel (Scomber japonicus) with high frequency of occurrence were Candida sp., Rhodotorula sp., Torulopsis sp., Cryptotoccus sp. and Tricosporon sp. Among these ones Candida lipolytica was identified as the strongest proteolytic yeast, then named Candida lipolytica FM5 (C. lipolytica FM5). C. lipolytica FM5 showed optimum growth at $25^{\circ}C$, pH 7.0 and could grow at $5^{\circ}C$ and in medium containing $10\%$ sodium chloride, To evaluate the saprophytic activity of the selected strain, C, lipolytica FM5 and Pseudomonas fluorescens ATCC 17571 which is one of representative spoilage bacteria were individually inoculated into the sterilized fish muscle homogenates, and then pH changes and volatile basic nitrogen (VBN) values were checked during the storage at various temperatures. According to the experimental results, the productions of VBN by C. lipolytica FM5 in the fish muscle homogenates were 50 mg-N/100 g at $5^{\circ}C$, 152 mg-N/100 g at $15^{\circ}C$ and 379 mg-N/100 g at $25^{\circ}C$ for 1 week storage, respectively. Above results were nearly same as in case of Ps. fluorescens ATCC 17571 inoculation. It suggest that sapyophytic yeasts also have important role in spoilage of marine fish.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.4
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• pp.356-362
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• 1986

This experiment was designed to evaluate effects of freezing and frozen storage on survival of sanitary indicative bacteria in seafoods. Culture of bacteria such as Escherichia coli type I, Citrobacter freundii type I, Klebsiella aerogenes type I and Streptococcus faecalis was inoculated into homogenates of pollack, shrimp, and sardine frozen in a contact plate freezer at $-40^{\circ}C$ and chest freezer at $-20^{\circ}C$, stored at $-20^{\circ}C$, and then survival of the inoculated bacteria was determined over a period of 95 days. Coliform group was highly sensitive to freezing and frozen storage showing survival of about $2\%$ after 95 days of frozen storage at $-20^{\circ}C$, whereas Streptococcus faecalis was relatively resistant with $20\%$ survival rate. The sanitary indicative bacteria count was rapidly decreased in the early stage of frozen storage revealing 90 to $95\%$ loss of coliform group and 40 to $70\%$ loss in case of Streptococcus faecalis after 10 days storage. In determining recovery rate, most probable number (MPN) method gave more reproducible recovery of the tested strain than did the selected agar plate method.

• Journal of fish pathology
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• v.32 no.2
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• pp.59-67
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• 2019

We developed and subsequently characterized mouse antibodies (MAbs) against viral hemorrhagic septicemia virus (VHSV, genotype IVa), the causative agent of VHS. Five hybridoma clones secreting MAbs against VHSV were established. The MAbs recognized the glycoprotein (MAbs 2C10, 18H4, 23H6, and 30B7) and nucleocapsid protein (15E10) of VHSV by western blot analysis. All five MAbs reacted with VHSV-infected cells and tissue homogenates of VHSV-infected olive flounder (Paralichthys olivaceus) by western blot analysis. Whereas, no reactivity was observed in normal cells and tissue homogenates of normal olive flounder. Moreover, these MAbs reacted with VHSV, but did not react with other fish viruses (infectious hematopoietic necrosis virus, hirame rhabdovirus, spring viraemia of carp virus, infectious pancreatic necrosis virus, marine birnavirus, and nervous necrosis virus) by enzyme linked immunosorbent assay (ELISA). These results indicate that the MAbs are specific to VHSV and can be of value in VHSV detection.