• Asian-Australasian Journal of Animal Sciences
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• v.30 no.3
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• pp.439-445
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• 2017

Objective: Production of alpha-1,3-galactosyltransferase (${\alpha}GT$)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous ${\alpha}GT$ knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce ${\alpha}GT$-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods: Miniature pig fibroblasts were transfected with ${\alpha}GT$ gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous ${\alpha}GT$ gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-${\alpha}$-1,3-galactose, an epitope produced by ${\alpha}GT$. Using magnetic activated cell sorting, cells with monoallelic disruption of ${\alpha}GT$ were removed. Remaining cells with LOH carrying biallelic disruption of ${\alpha}GT$ were used for the second round NT to produce homozygous ${\alpha}GT$ gene-targeted piglets. Results: Monoallelic mutation of ${\alpha}GT$ gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous ${\alpha}GT$ gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous ${\alpha}GT$ knockout piglets. Conclusion: The present study demonstrates that the time required for the production of ${\alpha}GT$-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a substantial length of time for breeding.

• Research in Plant Disease
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• v.17 no.1
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• pp.66-74
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• 2011

In 2005, a survey was conducted to identify virus diseases on victory onion, Allium victorialis var. platyphyllum grown in Ulleung island located in the East Sea. A total of 61 samples were collected from victory onion in the neighborhood of Seonginbong. The identification of viruses from the samples were carried out by electron microscopy and RT-PCR using primers species specific to GCLV, LYSV, SLV, OYDV and genus specific to Allexivirus, respectively. From sixty-one samples, filamentous rod particles (600-900 nm) were detected from four victory onion samples in EM, three samples containing SLV and one sample containing both SLV and Allexivirus in RT-PCR analysis, respectively. Victory onions naturally infected by the viruses were asymptomatic apparently. The viruses detected by RT-PCR were further characterized by the nucleotide sequence analysis of the coat protein region. Three isolates of SLV showed approximately 99% identities in the nucleotide and amino acid sequences, suggesting that they were likely to be the same strain. On the other hand, they showed approximately 75.7~83.7% identities in the nucleotide and 89.2~97.0% in amino acid sequences compared with the previously reported SLV isolates in Allium. The CP gene of the Allexivirus showed approximately 99.2% nucleotide identities and 98.8% amino acid identities with Garlic virus A. However, there was relatively low homology ranging from 60.6% to 81.5% compared with other Allexiviruses (GarV-C, GarV-E, GarV-X, GMbMV, and Shal-X). These data suggested that two viruses, SLV and GarV-A identified from victory onion, are named SLV-Ulleungdo and GarV-A-Ulleungdo, respectively. This is the first report of viruses infecting victory onion.

• Korean Journal of Food Science and Technology
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• v.39 no.4
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• pp.452-457
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• 2007

In this study, Lilium sp. were separated into bulbs, leaves, and flowers. Then, total polyphenol contents, electron donating ability (EDA), superoxide dismutase (SOD)-like activity, hydroxyl radical scavenging activity, and antimicrobial activity were measured from the extracts of each of the three aforementioned parts. The examination of physiologically active substances in the three parts revealed that Lilium davidii leaves had high total polyphenol contents, SOD-like activity, hydroxyl radical scavenging activity, and EDA, while the flowers of L. lancifolium showed high SOD-like activity, hydroxyl radical scavenging activity, and EDA, as well as a high level of total polyphenols in the bulb. Measurements of the antimicrobial activities of the extracts against Gram positive bacteria revealed that the leaves and flowers of L. davidii and L. lancifolium caused Bacillus subtilis and Salmonella enteritidis to form clear zones greater than 10 mm. Furthermore, the flowers of L. lancifolium showed particularly high antimicrobial activity against B. subtilis, and the flowers of L. davidii had high activity against S. enteritidis. For the Gram negative bacteria, the leaves and flowers of L. davidii and L. lancifolium caused Listeria monocytogenes and Escherichia coli to form clear zones greater than 10 mm, and finally, the flowers of L. davidii and L. lancifolium showed high antibacterial activity, with inhibition exceeding 12 mm.

• Food Science of Animal Resources
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• v.33 no.3
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• pp.403-410
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• 2013

This study was performed to evaluate the microbiological contamination level of raw beef from retail markets in Seoul, Korea. The sampling and laboratory test were performed according to the procedure of "Standard for processing and ingredients specification of livestock product" and "Korean food code". Enterotoxin of Staphylococcus aureus isolates were detected using VIDAS$^{(R)}$ and PCR-based methods. Listeria monocytogenes serotyping and genotyping were carried out using Listeria antisera and L. monocytogenes Fingerprinting kit, respectively. A total of 48 samples were collected from 16 retail markets (butcher's shop: 5, department store: 6, supermarket: 5) in 2011. The level of total bacteria counts in the butcher's shop, department store and supermarket were $4.4{\times}10^3$ CFU/g, $3.9{\times}10^5$ CFU/g and $1.0{\times}10^4$ CFU/g, respectively. The concentrations of Escherichia coli of these three retail markets were $6.4{\times}10$ CFU/g, 7.6 CFU/g and $2.0{\times}10$ CFU/g, respectively. Salmonella species was not detected on all samples. However, S. aureus was isolated in the 3 samples (6.25%) from each type of three retail markets. L. monocytogenes was isolated in the 4 samples (8.3%) from department stores. The level of contamination of these foodborne bacteria was less than 100 CFU/g. The enterotoxin-encoding genes of S. aureus isolates were sea, seh, sei and sep gene. The gene similarity of L. monocytogenes isolated from two retail markets by Rep-PCR showed 57.8-98.1% and 68.1-98.1%, respectively. These results suggest that the HACCP guideline for environmental control in slaughterhouse and retail markets should be provided to prevent cross contamination and manage foodborne pathogens such as L. monocytogenes and S. aureus.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.277-285
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• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.

• Applied Microscopy
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• v.41 no.3
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• pp.205-213
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• 2011

Numerous anisakids were parasitic near the digestive tract of the black rockfish, Sebastes schlegeli and some anisakids observed on the liver of the host. Anisakids in the host were identified three species (Hysterothylacium sp., Anisakis simplex, A. pegreffii) and Hysterothylacium sp. was the high occurrence of anisakid worms in the host. Hysterothylacium sp. was shorter and thinner than A. simplex. Both of anisakids observed lip, mouth, nerve ring, excretory pore and excretory duct in the anterior portion. In the mid portion, anisakids had esophagus, ventriculus and intestine and especially, Hysterothylacium sp. had intestinal ceacum and ventricular appendage. There was conical nodulose apex at the end of the posterior portion in Hysterothylacium sp. and spine in A. simplex. SEM examination revealed that there was three lip near the mouth of Hysterothylacium sp. Dorsal lip was approximately 65 ${\mu}m$ and paried lateroventral lip were approximately 60 ${\mu}m$ in the width. All lips were found double papilla (approximately 8 ${\mu}m$ in the width). The body width of the Hysterothylacium sp. and A. simplex was approximately 480 ${\mu}m$ and 900 ${\mu}m$ respectively. The hight of the lateral alae was about 7 ${\mu}m$ and width of papilla on the cornical nodulose apex was about 3.3 ${\mu}m$ in Hysterothylacium sp. The hight of spine was approximately 20 ${\mu}m$ in A. simplex. There was mainly Hysterothylacium sp. in the intestinal lumen of the host. The nematod worms were parasitic near the mucosal fold and in the submucosal. In the mucosal epidermal layer, it was increased mucous cells by the infection of the parasites.

• Journal of fish pathology
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• v.18 no.1
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• pp.39-48
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• 2005

Pathogenicity of Vibrio tapetis, the causative bacterium of 'brown ring disease (BRD)' was evaluated in Manila clams (Ruditapes philippinarumi by artificially 0.1 $m\ell$ infection of $1.0\times10^5$cells and $1.0\times10^8$ cells at 20 $^{\circ}C$. A PCR assay based on 16S rRNA to detect the bacteria in clam tissues was established. Accumulative mortality of clams infected with $1.0\times10^7$cells and $1.0\times10^4$ cells per an individual of the bacteria was 67.5% and 7.5%, respectively. However, the deposit of brown pigment in the inner shells by accumulation of chonchiolin was not found. The bacteria were not be able to re-isolate from the infected clams by the conventional agar plate method but were easily detected by PCR assay established in this experiment. In clams artificially infected with 10 species of Vibrio, a 414bp for V. tapetis was detected in PCR assay. The specific band in the clams infected with $1.0\times10^4$cells per an individual of V. tapetis was detected only in gills one day after the infection but never be found in any tissues including gills three days after the infection. In the case of clams infected with $1.0\times10^8$cells per an individual of V. tapetis the specific band was detected in gills and intestine one day after the infection, in all tissues three days after the infection, and then in gills and adductor muscle nine days after the infection. The PCR assay was applied to detect V. tapetis in manila clam, surf clam (Mactra veneriformis), oyster (Crassostrea gigas) and Thomas' rapa whelk (Rapana venosa) taken from Taean and Gochang from April to July 2004. The infection rates were detected to 23.1% and 9.4% in the oyster and surf clam, while manila clam and Thomas' rapa whelk were not found.

• Korean Journal of Ecology and Environment
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• v.45 no.2
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• pp.218-231
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• 2012

The objectives of this study were to evaluate the physico-chemical water quality, trophic and tolerance guilds in the control ($C_o$) and impacted streams of the abandoned mine, along with the ecological health, using a multimetric health model and physical habitat conditions of Qualitative Habitat Evaluation Index (QHEI), during the period of three years, 2005~2007. Also, eco-toxicity ($EE_t$) enclosure tests were conducted to examine the toxic effects on the outflows from the mine wastewater, using the sentinel species of Rhynchocypris oxycephalus, and we compared the biological responses of the control ($C_o$) and treatment (T) to the effluents through a Necropybased Health Assessment Index ($N_b$-HAI). Tissue impact analysis of the spleen, kidney, gill, liver, eyes, and fins were conducted in the controlled enclosure experiments (10 individuals). According to the comparisons of the control ($C_o$) vs. the treatment (T) in physicochemical water quality, outflows from the abandoned mine resulted in low pH of 3.2, strong acid wastewater, high ionic concentrations, based on an electrical conductivity, and high total dissolved solid (TDS). Physical habitat assessments, based on Qualitative Habitat Evaluation Index (QHEI) did not show any statistical differences (p>0.05) in the sampling sites, whereas, the $M_m$-EH model values in a multimetric ecological health ($M_m$-EH) model of the Index of Biological Integrity (IBI), using fish assemblages, were 16~20 (fair condition) in the control and all zero (0, poor condition) in the impacted sites of mine wastewater. In addition, in enclosure eco-toxicity ($EE_t$) tests, the model values of $N_b$-HAI ranged between 0 and 3 in the controls during the three years, indicating an excellent~good condition (Ex~G), and were >100 (range: 100~137) in the impacted sites, which indicates a poor condition (P). Under the circumstances, organ tissues, such as the liver, kidney, and gills were largely impaired, so that efficient water quality managements are required in the outflow area of the abandoned mine watershed.

• Korean Journal of Ecology and Environment
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• v.44 no.1
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• pp.31-41
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• 2011

To optimize the natural chemical agents against nuisance phytoplankton, we examined algal removal activity (ABA) of Plant-Mineral Composite (PMC), which already developed by our teams (Kim et al., 2010), on various conditions. The PMC are consisted of extracted-mixtures with indigenous plants (Camellia sinensis, Quercusacutissima and Castanea crenata) and minerals (Loess, Quartz porphyry, and natural zeolite), and characterized by coagulation and floating of low-density suspended solids. A simple extraction process was adopted, such as drying and grinding of raw material, water-extraction by high temperature-sonication and filtering. All tests were performed in 3 L plastic chambers varying conditions; six different concentrations ($0{\sim}1.0\;mL\;L^{-1}$), six light intensities ($8{\sim}1,400\;{\mu}mol\;m^{-2}s^{-1}$), three temperatures ($10{\sim}30^{\circ}C$), four pHs (7~10), five water depths (10~50 cm), and three different waters dominated by cyanobacteria, diatom, and green algae, respectively. Results indicate that the highest ABA of PMC was seen at $0.05\;mL\;L^{-1}$ in treatment concentrations, where showed a reduction of more than 80% of control phytoplankton biomass, while $1,400\;{\mu}mol\;m^{-2}s^{-1}$ in light intensity (>90%), $20{\sim}30^{\circ}C$ temperature (>60%), 7~9 in pH (>90%), below 50 cm in water depth (>90%), and cyanobacterial dominating waters (>80%), respectively. Over the test, ABA of PMC were more obvious on the algal biomass (chlorophyll-${\alpha}$) than suspended solids, suggesting a selectivity of PMC to particle size or natures. These results suggest that PMC agents can play an important role as natural agents to remove the nuisant algal aggregates or seston of eutrophic lake, where occur cyanobacterial bloom in a shallow shore of lake during warm season.

• Journal of Food Hygiene and Safety
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• v.33 no.5
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• pp.404-411
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• 2018

To evaluate bacteriological and toxicological safety hygienic indicator bacterium and paralytic and diarrhetic shellfish toxins in the shellfish produced in Hansan Geojeman 2013-2017 were investigated. Fecal coliforms were < 18~330 MPN/100 g in 404 oyster samples. But all samples tested, did not exceed 230 E. coli MPN/100 g. Geometric mean of E. coli for oyster samples collected during major shellfish production period was 24.3 MPN/100 g, considerde stable results. Bacteriological quality of oysters collected from Hansan Geojeman meets the standard value based on shellfish hygiene of the Food Sanitation Act of Korea and also meets Grade A, according to classification of shellfish harvesting areas of the European Union. For toxicological evaluation of Hansan Geojeman, 532 oyster samples and 268 mussel samples as an indicator, were analyzed. Paralytic shellfish toxins were detected in the range of 0.42~2.29 mg/kg in eight mussel samples, and exceeded criteria in three samples from early to late April 2013. Diarrhetic shellfish toxin was detected in three of 120 samples, but it was revealed to be under regulation value (0.16 mg Okadaic Acid equ./kg). As a result of toxicological evaluation, paralytic and diarrhetic shellfish toxins were not detected in oyster samples, but it was found that mussel as an indicator species, exceeded the threshold value of paralytic shellfish toxin. Accordingly, sanitary surveys were continuously requested for food safety management of shellfish.