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Duwoon Kim,
Byeong-Mun Jeong,
Wi-Sik Kim,
Jong-Oh Kim,
Chae Hong Lim,
Jeong-Young Cho,
Jong-Bang Eun,
Jae-Hak Moon, Seok-Ryel Kim,
Myoung-Ae Park,
Myung-Joo Oh
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ABSTRACT: Efficiency of extraction and concentration methods for the detection of the major capsid protein gene of lymphocystis disease
virus from different tissues of olive flounders (Paralichthys olivaceus) was tested. Tris elution buffer showed a 100 fold higher polymerase chain reaction (PCR) detection limit than TE elution
buffer in the virus extraction step from skin tissues. Using the TRPD (Tris elution buffer, polyethylene, and DNA extraction
kit) procedure, we confirmed that skin tissues and lymphocystis cells of olive flounders had a detection limit of 10−6 and 10−7 PCR-U/μL, meaning that 106 and 107 fold dilutions of lymphocystis disease virus (LCDV) were PCR positive, respectively.
Keywordslymphocystis disease virus–lymphocystis disease virus (LCDV)–
Paralichthys olivaceus
–polymerase chain reaction (PCR)–fish
Food science and biotechnology 04/2012; 19(6):1693-1696. · 0.49 Impact Factor
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ABSTRACT: The viral diseases have been the serious problem in salmonid farming, and rainbow trout is not an exception. In this study, routine surveys were conducted for detecting of viruses in farmed rainbow trout (Oncorhynchus mykiss) in Korea during 2009-2010. Head kidneys from individual fish were employed for virus detection by using a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) were the target viruses in this study. 53.5% (46/86) were found to be IPNV-positive, while IHNV and VHSV showed RT-LAMP negative during examination for 2 years. Ten IPNV-positive samples were randomly selected for viral isolation and the cells showing CPEs were subjected to RT-LAMP, RT-PCR, and direct sequencing. Phylogenetic analysis showed that the rainbow trout isolate has high similarity homologies with the VR-299 strain, as previously described.
The Journal of Microbiology 10/2011; 49(5):741-6. · 1.10 Impact Factor
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ABSTRACT: Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activity-guided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC(50)) and each viral replication (EC(50)) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihy-droxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC(50)/EC(50)) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activités against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.
The Journal of Microbiology 02/2011; 49(1):102-6. · 1.10 Impact Factor
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ABSTRACT: The results described the structure of Longicollum pagrosomi and histopathological characters of the intestine of the red sea bream, Pagrus major, infected with acanthocephalans, using the light and electron microscopes. Among the six samples of P. major, L. pagrosomi was identified in the posterior intestine of five fish samples. Adult L. pagrosomi (total length, 8-27 mm) is divided into the presoma (proboscis, anterior neck, and posterior neck) and metasoma (trunk). The proboscis had vertically arranged hooks (40 μm in length), with ten hooks per row, and the septum was observed between the posterior neck and trunk. The tegument thickness of the proboscis was approximately 15 μm, and it was composed of thin, circular muscle fibers. The outer fibrous membrane was approximately 1 μm, and the connective tissue layer was approximately 35 μm in thickness in the anterior neck. The tegument of the posterior neck enclosed the cephalic ganglion and had longitudinal and vertical muscle fibers, and the tegument thickness was approximately 45 μm. The tegument of the body, which was approximately 1 mm in thickness, was composed primarily of muscle and collagen fibers, and the structure of the tegument was different, depending on the body region. The acanthocephalans had ovaries and oval-shaped eggs with an eggshell (77.5 × 17.1 μm), floating within the body cavity of the trunk. In the infected posterior intestine of P. major, the presoma and the anterior part of the metasoma of L. pagrosomi passed through the intestinal wall and infected the intestinal tissue, perforating the loose connective tissue. In the inflammatory connective tissue, collagen and muscle fibers were fragmented and revealed partial necrosis. Lipid drops and eosinophilic granular cells aggregated in the connective tissue of the tissue capsule. In the vicinity of the acanthocephalan, the mucosal epithelia contained hypertrophied nuclei, and the epithelial layer was collapsed. In an extreme case, the mucosal fold was degenerated because of pressure from the acanthocephalan.
Parasitology Research 01/2011; 109(1):175-84. · 2.15 Impact Factor
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ABSTRACT: A marine bacterial strain, designated A71(T), was isolated from marine algae collected from the South Sea, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A71(T) belonged to the family Flavobacteriaceae and was closely related to Aequorivita antarctica SW49(T) (96.5 % sequence similarity). Cells of strain A71(T) were Gram-negative, aerobic, oxidase-negative, catalase-positive, yellow/orange-pigmented and non-motile. The major fatty acids were iso-C(15 : 0) (20.6 %), iso-C(17 : 1)omega9c (13.3 %), anteiso-C(15 : 0) (13.1 %), iso-C(17 : 0) 3-OH (12.7 %) and summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c; 6.6 %). The DNA G+C content was 36.9 mol%. Several phenotypic characteristics served to differentiate the isolate from recognized members of the genus Aequorivita. Data from this polyphasic study clearly demonstrated that strain A71(T) represents a novel species of the genus Aequorivita. The name Aequorivita capsosiphonis sp. nov. is proposed, with strain A71(T) (=KCTC 22183(T) =JCM 15070(T)) as the type strain. In addition, an emended description of the genus Aequorivita is presented.
International journal of systematic and evolutionary microbiology 05/2009; 59(Pt 4):724-8. · 2.27 Impact Factor
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ABSTRACT: The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 10(5) fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.
The Journal of Microbiology 09/2008; 46(4):436-40. · 1.10 Impact Factor
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ABSTRACT: Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.
The Journal of Microbiology 07/2008; 46(3):265-73. · 1.10 Impact Factor
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ABSTRACT: Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.
The Journal of Microbiology 03/2008; 46(1):29-33. · 1.10 Impact Factor
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ABSTRACT: Sephadextrade bead agarose electrophoresis (SBAE) was used to enrich the higher dextran binding phages. Phage collection obtained after the 2<sup>nd</sup> round of SBAE was 3.5 fold higher color intensity than that of phage collections obtained after the 1<sup>st</sup> round. DNA sequencing of phage collection (SBAE-2R) confirmed that human origin antibody was present. The PCR products of lambda, k light chains and heavy chain from phage collection (SBAE-2R) were approximately 420 bp, 550 bp and 600 bp.
Frontiers in the Convergence of Bioscience and Information Technologies, 2007. FBIT 2007; 11/2007
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Frontiers in the Convergence of Bioscience and Information Technologies 2007, FBIT 2007, Jeju Island, Korea, October 11-13, 2007; 01/2007
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ABSTRACT: Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.
FEMS Microbiology Letters 09/2004; 237(1):147-56. · 2.04 Impact Factor
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Wi-Sik Kim, Seok-Ryel Kim,
Duwoon Kim,
Jong-Oh Kim,
Myoung-Ae Park,
Shin-Ichi Kitamura,
Heung-Yun Kim,
Do-Hyung Kim,
Hyun-Ja Han,
Sung-Ju Jung,
Myung-Joo Oh
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ABSTRACT: We examined the cause of a disease outbreak in juvenile and market-sized olive flounder Paralichthys olivaceus from aquaculture farms in the south sea of Korea in 2005. Principal signs included expanded abdomen, congested liver and enlarged spleen and kidney, with fish suffering heavy mortality (40 to 60%). Although no parasites or bacteria were isolated from diseased fish, tissue filtrates still produced cytopathic effects (CPE) in FHM, CHSE-214 and FSP cells. PCR reactions of tissue filtrates from diseased fish and supernatants of cell cultures showing CPE indicated specific 587 bp fragments from the glycoprotein (G) gene of the viral hemorrhagic septicemia virus (VHSV). Supporting this, the nucleotide sequences among three isolates had shown 100% homology, and 99.3% and 99.8% homology with the VHSV JY-0112 isolate from flounder in Korea and VHSV obama25 isolate from flounder in Japan, respectively. Experimental infection trials using supernatants of cell cultures showing CPE gave cumulative mortalities of 100% and 60% for virus-injected and virus-immersed flounder, respectively. The pathological signs shown were generally similar to those of naturally diseased fish. Results of this investigation indicate that VHSV was the causative agent of the natural epizootic.
Aquaculture. 296:165-168.
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ABSTRACT: Japanese flounder, Paralichthys olivaceus, is the most important commercial fish in Korea, and viral diseases in this fish cause important economic losses. To control fish viral diseases, an understanding of the viral dynamics in the host fish and also in viral vectors and/or reservoirs is needed. In this study, polymerase chain reaction (PCR) was used to investigate seasonal changes of lymphocystis disease virus (LCDV), aquatic birnavirus (ABV), viral haemorrhagic septicaemia virus (VHSV) and megalocytivirus in Japanese flounder. Level of antibody titers in flounder sera against ABV was also determined by a neutralization test. In addition, because wild blue mussel Mytilus galloprovincialis is considered a candidate for the vector and/or reservoir of the viruses, PCR was performed to determine if viruses were present in the shellfish collected near the aquaculture facilities. During the course of fish development from juvenile size to commercial size, ten fish and ten shellfish were collected monthly from a Korean aquaculture farm. They were used for virus detection by PCR. Sera from the fish collected at the end of the study were examined for antibody titers. Neither megalocytivirus nor VHSV was detected by PCR in any of the fish or shellfish collected throughout the experimental period. LCDV was detected in the fish collected in August and November, and the detection rates of the virus were 50% and 10%, respectively. Regarding the two peaks of virus detection, LCDV most probably extensively replicates in Japanese flounder in summer, when the water temperature is around 20 °C because the virus can grow from 20 to 25 °C in cell lines such as HINAE and GCO. In winter, the virus would have been able to replicate in the fish that had not previously acquired antibodies against the virus. ABV was detected in 10% of the fish collected in July and August. Although the ABV detection rate was low in the fish during the observation period, almost all the fish collected in November had neutralization antibody titers (> 1:128). The fish may acquire neutralization antibodies against the virus during seeding production, because the disease primarily occurs in hatcheries in Korea, and/or in aquatic environments, such as seawater, where the virus is quite common. ABV was also detected in blue mussels around the same time that the virus was detected in the fish, suggesting that the shellfish may be a vector and/or reservoir for the virus.
Aquaculture.
