• Korean Journal of Veterinary Research
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• v.61 no.2
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• pp.19.1-19.7
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• 2021

Feline panleukopenia virus (FPV) causes leukopenia and severe hemorrhagic diarrhea, killing 50% of naturally infected cats. Although intact FPV can serve as an antigen in the hemagglutination inhibition (HI) test, an accidental laboratory-mediated infection is concern. A non-infectious diagnostic reagent is required for the HI test. Here, we expressed the viral protein 2 (VP2) gene of the FPV strain currently prevalent in South Korea in a baculovirus expression system; VP2 protein was identified by an indirect immunofluorescence assay, electron microscopy (EM), Western blotting (WB), and a hemagglutination assay (HA). EM showed that the recombinant VP2 protein self-assembled to form virus-like particles. WB revealed that the recombinant VP2 was 65 kDa in size. The HA activity of the recombinant VP2 protein was very high at 1:2¹⁵. A total of 143 cat serum samples were tested using FPV (HI-FPV test) and the recombinant VP2 protein (HI-VP2 test) as HI antigens. The sensitivity, specificity, and accuracy of the HI-VP2 test were 99.3%, 88.9%, and 99.3%, respectively, compared to the HI-FPV test. The HI-VP2 and HI-FPV results correlated significantly (r = 0.978). Thus, recombinant VP2 can substitute for intact FPV as the serological diagnostic reagent of the HI test for FPV.

• Journal of Microbiology and Biotechnology
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• v.32 no.6
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• pp.749-760
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• 2022

α-Galactosidase is a debranching enzyme widely used in the food, feed, paper, and pharmaceuticals industries and plays an important role in hemicellulose degradation. Here, T26, an aerobic bacterial strain with thermostable α-galactosidase activity, was isolated from laboratory-preserved lignocellulolytic microbial consortium TMC7, and identified as Parageobacillus thermoglucosidasius. The α-galactosidase, called T26GAL and derived from the T26 culture supernatant, exhibited a maximum enzyme activity of 0.4976 IU/ml when cultured at 60℃ and 180 rpm for 2 days. Bioinformatics analysis revealed that the α-galactosidase T26GAL belongs to the GH36 family. Subsequently, the pET-26 vector was used for the heterologous expression of the T26 α-galactosidase gene in Escherichia coli BL21 (DE3). The optimum pH for α-galactosidase T26GAL was determined to be 8.0, while the optimum temperature was 60℃. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90℃. Furthermore, Ca²⁺ and Mg²⁺ promoted the activity of T26GAL while Zn²⁺ and Cu²⁺ inhibited it. The substrate specificity studies revealed that T26GAL efficiently degraded raffinose, stachyose, and guar gum, but not locust bean gum. This study thus facilitated the discovery of an effective heat-resistant α-galactosidase with potent industrial application. Meanwhile, as part of our research on lignocellulose degradation by a microbial consortium, the present work provides an important basis for encouraging further investigation into this enzyme complex.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1011-1016
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• 2022

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

• Journal of Veterinary Science
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• v.21 no.4
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• pp.63.1-63.9
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• 2020

Background: Canine adenovirus type 2 (CAV-2) induces infectious laryngotracheitis in members of the family Canidae, including dogs. To date, no ELISA kits specific for CAV-2 antibody have been commercialized for dogs in Korea. Objectives: We aimed to develop new indirect enzyme-linked immunosorbent assay (I-ELISA) to perform rapid, accurate serological surveys of CAV-2 in dog serum samples. Methods: In total, 165 serum samples were collected from dogs residing in Chungbuk and Gyeongbuk provinces between 2016 and 2018. The Korean CAV-2, named the APQA1701-40P strain, was propagated in Madin-Darby canine kidney cells and purified in an anion-exchange chromatography column for use as an antigen for I-ELISA. The virus-neutralizing antibody titers of CAV-2 in the dog sera were measured by virus neutralization (VN) test. Results: We compared the results obtained between the VN and new I-ELISA tests. The sensitivity, specificity, and accuracy of new I-ELISA were 98.6%, 86.4% and 97.0% compared with VN test, respectively. New I-ELISA was significantly correlated with VN (r = 0.91). Conclusions: These results indicate that new I-ELISA is useful for sero-surveillance of CAV-2 in dog serum.

• Journal of Veterinary Science
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• v.23 no.5
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• pp.32.1-32.11
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• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• Research in Plant Disease
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• v.29 no.4
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• pp.420-424
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• 2023

In 2020, within the Dongbaekdongsan area in Jeju Island, a Septobasidium sp. associated with a felt disease in Castanopsis sieboldii (Makino) Hatus. ex T. Yamaz. & Mashiba was identified. The symptom included the presence of brown, thin, and silk-like mycelial mats attached to the tree's bark, displaying variations in size from large to small. To induce hyphal growth, the samples collected were incubated in a moist chamber, and the newly formed hyphae were subjected to genomic DNA extractions. The nucleotide sequences of the internal transcribed spacer and small subunit rDNA genes were determined, and molecular characteristics among the isolates were investigated through polymerase chain reaction-based restriction fragment length polymorphism analysis. This Septobasidium sp. exhibited distinct morphological and phylogenetic features compared to those that were previously reported in South Korea. Consequently, this strain is taxonomically classified as a provisionally novel species of Septobasidium. Furthermore, the observed felt disease exhibited a high degree of host specificity, as it was exclusively identified in C. sieboldii without occurrence in other tree species at the time of observation.

• Korean Journal of Veterinary Research
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• v.64 no.3
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• pp.26.1-26.9
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• 2024

Japanese encephalitis virus (JEV) is a mosquito-borne virus that can infect pigs, horses, and other mammals, including humans. Sero-epidemiological investigations of JEV have been performed using hemagglutination inhibition (HI), virus neutralization (VN) tests and enzyme-linked immunosorbent assay (ELISA). A need exists for a new ELISA that can detect JEV antibodies in the sera of several animal species. We aimed to develop a blocking ELISA (B-ELISA) for detecting JEV antibodies in pig and horse serum samples. JEV antibodies in 218 pig and 315 horse serum samples were measured using HI and VN tests. The purified KV1899-306 strain was used as an antigen for B-ELISA. The purified antibody (7A13) was conjugated with horseradish peroxidase and used as a detector antibody. The sera of pigs and horses to measure antibody against JEV were subjected to B-ELISA and analyzed. The B-ELISA had a diagnostic sensitivity of 94.6% to 100%, a specificity of 91.2 to 100%, and an accuracy of 94.9 to 98.6% compared with those of the HI and VN tests in pig and horse sera. The B-ELISA had a higher correlation with pig sera (r = 0.89 and 0.90 for VN and HI) than with horse sera (r = 0.75 and to 0.79). The new B-ELISA could be useful in the sero-surveillance of JEV in pig and horse sera and replace indirect ELISA.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.91-95
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• 2004

For the detection of the oil-degrading bacterium, Nocardia sp. Hl7-1, inoculated during the bioremediation of oil-contaminated soil, a species-specific primer was constructed based on the 16S rDNA sequence of this strain. Two forward primers and two reverse primers were designed and tested against both closely and distantly related bacterial strains. All the primers designed were specific to the Nocardia sp. H17-1. Particularly, primer sets NH169F-NH972R and NH575F-NH972R could be used to detect 50 fg of template DNA and TEX>$1.2${\times}$10^4$ CFU/g of sandy soil. These two PCR primer sets successfully detected the H 17-1 strain in the oil-con-laminated soil samples containing heterogeneous DNA. We also conformed the primer specificity by restriction-enzyme cleavage of the PCR products and denaturing gradient gel electrophoresis.

• Journal of Life Science
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• v.19 no.10
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• pp.1424-1431
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• 2009

Shrimp Jeot-Gal is a popular traditional Korean fermented seafood and has been used for seasoning. We isolated a bacterium showing strong extra-cellular fibrinolysis and chitinase activity from shrimp Jeot-Gal and the strain was designated SC082. SC082 was identified as Bacillus licheniformis by 16S rRNA sequence homology search. B. licheniformis SC082 exhibited optimum temperature, pH, and salt concentration at $37^{\circ}C$, pH 7.0, and 6%, respectively. Substrate specificity of the culture supernatant from B. licheniformis SC082 was detected in fibrin, skim milk, and chitin plate. The fibrinolytic activity was highly maintained up to $50^{\circ}C$ at a pH of 7.0 for 3 hr and was stable up to pH 9.0 at $37^{\circ}C$ for 3 hr. The chitinase activity was remarkably induced by addition of 1.0% colloidal chitin and the pH and temperature optima of the enzyme were 5.0 and $45^{\circ}C$, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis, this strain produced three fibrinolytic isozymes and two chitinase isozymes. The approximate molecular weights of the putative fibrinolytic enzymes were 23.0, 62.0, and 72.0 kDa and those of the chitinases were 62.0 and 55.0 kDa, respectively. The antioxidant activity of SC082 was also measured by using 2,2-diphenyl-l-picryl-hydrazyl (DPPH) free radical. The DPPH radical scavenging was slightly increased in a dose-dependent manner.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.6
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• pp.852-858
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• 1998

We have isolated a bacterial strain that tends to kill P. micans from the mixed culture of p. minns plus seawater filtrate (poresize, 0.8 $\mu$m) collected at Masan bay in July 1996, in which the mixed culture grown in the f/2 medium. According to the experimental results of the isolated bacterium such as fatty acids analysis, morphological and biochemical characteristic tests, the strain was supposed to be a Pseudomonas and then it was named as Pseudomonas sp. LG-2. The killing effect of Pseudomonas sp. LG-2 against P. micans was proportionally increased with the concentrations of culture filtrate (pore size, 0.8 $\mu$m) is well as with the number of bacterium inoculated. In the mixed culture inoculated with $1.3\times10^6$ cells/ml of Pseudomonas sp. LG-2, the number of P. micans (2,000 cells/ml) was gradually decreased and then killed below 100 cells/ml within 7 days. In addition, the culture filtrate with $30\%$ of final concentration revealed a significant killing effect against P. micans around 3 days after culture. In the relationship between killing effects and growth stage of Pseudomonas sp. LG-2, the culture filtrate at lag phase has little effects on P. micans. In constant, the culture filtrate at mid-log phase showed the killing effect by decreasing P. micans to 112 in number within 5 days. In particular, the culture filtrate at stationary phase showed a significant killing effect against P. micans in which the majority of it was killed after 3 day culture. The species specificity of killing effects of Pseudomonas sp. LG-2 against 5 species of dinoflagellate was only found in P. micans and Scrippsiella trochoidea.