• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1701-1710
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• 2017

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that $RecCpp40_{+10}$ failed to induce fever in rabbits, whereas $RecCpp80_{+10}$ caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

• Journal of Veterinary Clinics
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• v.32 no.4
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• pp.324-329
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• 2015

The primary aim of this study was to investigate biosecurity practices in pig farms and to determine the major risk factors associated with PCV2 infection for a sampled swine population in Korea. To this end, we analyzed data from a cross-sectional study of 296 farrow-to-finish farms, which was conducted between March and September 2014 to explore the prevalence of swine disease at farm level. Face-to-face interviews by on-site visit of trained veterinarians were conducted with the farm owners or managers using a standardized questionnaires with information about basic demographical data and management practices. Farms were classified as negative or positive through the use of infection profiles that combined data on serological testing including PCR antigen test result, antibody titer and sero-conversion pattern at each age category taking into account vaccination status. Data were analyzed using multivariate ordinal logistic regression. Results from this study indicated that biosecurity level of the farms was considered not good given low compliance of the biosecurity programs and facilities in the farm: off-site removal of dead stocks (7%), off-site location of storage facility for incoming feeds (12.6%), off-site pick-up location for finishers (19.3%), restrictions on feed supplier vehicles for farm entrance (19.6%), restriction of finisher trucks entering the farm (22.4%), and restriction on manure disposal trucks entering the farm (26.4%). In the final model (n = 255), allowance of finisher truck driver to the pig unit had increased risk of infection (OR = 2.4, 95% CI 1.22-4.67) whereas farms with a sign forbidding the entrance had decreased risk of infection (OR = 0.19, 95% CI 0.10-0.58). Further comprehensive research with larger sample size is required to better understand the multifactorial characteristic that some predisposing risk factors that were not available in this study. To the best knowledge of the authors, this was the first study to use empirical data to report risk factors associated with PCV2 infection in the Korean pig farms. Results from the current study could be used to decide optimal biosecurity measures to reduce the impact of PCV2 infection to farmers and policy makers.

• Journal of Veterinary Clinics
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• v.30 no.2
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• pp.115-118
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• 2013

An 8-year-old spayed female domestic shorthair cat was presented with a chief complaint of chronic nasal discharge and dyspnea. Physical examination revealed pyohemorrhagic nasal discharge, inspiratory dyspnea and stertor, and an enlarged right mandibular lymph node. Abnormalities of blood works and serum chemistry included mildly increased hematocrit, and globulin concentration. Serologic tests for FeLV and FIV, and a panel of polymerase chain reaction tests for Chlamydophila felis, Feline Calicivirus, Herpesvirus, Bordetella, Mycoplasma felis, and H1N1 influenza was all negative. Only radiographic finding showed increasing soft tissue density in the right nasal cavity and computed tomography disclosed soft tissue/fluid opacification in the right nasal cavity, paranasal sinus, and pharyinx along with slight deviation to the right of the osseous nasal septum. Focal lysis of ventral nasal septum was also suspected in CT scan. Cytological evaluation of fine needle aspirate smears of the enlarged mandibular lymph nodes revealed numerous fungal yeasts having variably thick capsule both extracellularly and intracellularly with low numbers of macrophages. Some yeasts showed narrow based budding, which was a consistent finding with Cryptococcus organisms. Serum protein electrophoresis was a polyclonal consistent with chronic infection and serum was submitted for a fungal serology panel test. In serologic tests Cryptococcus antigen titer was 1 : 32,768. In vitro culture was unsuccessful. Treatment was initiated with administration of fluconazole, clindamycin, and tocopherol. Clinical signs resolved within 3 days after the initial treatment. The cat was discharged and scheduled for periodic evaluation and continued therapy, but was lost to follow-up thereafter.

• Korean Journal of Veterinary Service
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• v.25 no.3
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• pp.245-257
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• 2002

The gene encoding the HN protein from the CBP-1 strain, a heat stable Newcastle disease virus (NDV) isolated from diseased pheasants in Korea, was characterized by reverse transcriptase- polymerase chain reaction(RT-PCR) and the nucleotide and amino acid sequences were analyzed following cloning of the HN gene. In all of the NDV strains studied, a 1.75 kb size cDNA fragment for the HN gene was generated by RT-PCR and smaller specific band sizes harboring the internal portions of the HN gene were also detected by using four pairs of primers. The RT-PCR was sensitive enough to detect viral transcripts when the virus titer was above 25 hemagglutination units. The amplified 1.75 kb cDNA was cloned into a BamHI site of the pVL1393 Baculo transfer vector. The nucleotide sequences of the 1,758 bp HN gene from the CBP-1 strain were determined by the dye terminator cyclic sequencing method. The gene sequences were compared among the strains of CBP-1, Texas GB, Beaudette C, LaSota, B1 and Ulster. The homology of the CBP-1 HN gene to other HN variants was 97.8% to Texas GB, 98.4% to Beaudette C, 95.4% to LaSota, 95.6% to B1 and 90.2% to Ulster. As the deduced 577 amino acid sequences were compared among the strains, the homology for CBP-1 HN appeared to be 96.7% to Texas GB, 97.9% to Beaudette C, 95.5% to LaSota, 95.5% to B1 and 92.7% to Ulster. It was evident that the amino acid sequences included 5 sites for N-asparagine linked glycosylation and 12 cysteine residues. The three conserved leucine residues within the predicted transmembrane domain of the HN protein are amino acid 30, 37 and 44. The three antigenic sites on the HN protein of NDV are amino acids 347(Glu), 481(Asn) and 495(Glu). These data indicate that the genotype of the CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than it is for the LaSota, B1 and Ulster strains.

• Korean Journal of Veterinary Research
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• v.23 no.1
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• pp.81-89
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• 1983

Epizootics and sporadic outbreaks of the cattle disease characterized by fever, stomatitis and dysphagia were observed in Korea in September through November, 1982. The number of cattle showed typical symptoms of the disease was estimated to 73 and these animals were concentrated in Gyongido province, the central district of Korea. In other districts around Gyongido province the disease tended to be sporadic occurrence. The main lesions of the disease were defined as edema, hemorrhage, degeneration and necrosis of the oral mucosa and musculatures of the esophagus and laryngopharyngeal area, and abomasum. Of these lesions hyaline degeneration and hemorrhage of the striated muscle of the esophagus and laryngopharyngeal area were the most striking changes of the disease and rated to be the lesion of diagnostic significance. The serum samples of the cattle with typical symptoms showed significant positive titer of the antibodies against Ibaraki virus. On these clinical, epidemiological, pathological and serological findings of the disease it was diagnosed as Ibaraki disease presenting the only report on the epizootics in the countries other than Japan.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.623-632
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• 1993

This study was conducted to develop a sensitized latex-antigen for serodiagnosis of toxoplasmosis in animals. Tachyzoites of T gondii(RH-strain) harvested from mouse peritoneal cavity were purified through the filtraton of polycarbonate membrane(pore size, $3.0{{\mu}m}$, Costar Co.) and disrupted by ultrasonicator. The tachyzoite suspension was ultracentrifuged for 30 min at $60,000{\times}g(4{^{\circ}C})$ and the supernatant was used as a water-lysate antigen. Polystyrene latex particles of $0.8{{\mu}m}$ in diameter(Sigma) were used for the preparation of sensitized latex-antigen suspension. The several parameters including the preparation conditions, incubation buffer. serum dilution buffer and stability of agglutination reactions were evaluated and the results obtained were summarized as follows : 1. The antigen consisting of a water-lysate of T gondii tachyzoites was adsorbed onto polystyrene latex particles of $0.8{{\mu}m}$ in diameter by adding a latex suspension to an equal volume of diluted antigen solution and by incubating the mixture at $37{^{\circ}C}$ under different conditions. 2. The optimum incubation buffer used for the antigen sensitization was 0.1M Tris-HCl buffer(pH 8.0). 3. The optimum serum dilution buffer used for the latex agglutination test was 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300 mM NaCl. But 0.1M Tris-HCl-NaCl buffer(pH 7.4) containing 300-600 mM NaCl, 0.5% BSA and 0.01% Tween-20 improved the agglutination pattems and cleared the background of microplate well without the effects on L.A titer. 4. The time required for antigen sensitization was 40 and 60 min in incubation buffer(pH 8.0) at $37{^{\circ}C}$. But the optimun time for antigen sensitization was min at $37{^{\circ}C}$. 5. The optimun quantity of antigen absorbed on latex particles for proper agglutination was the range of 20 to $32{\mu}g$ of latex particles. 6. The optimun concentration of the latex-antigen suspension for the proper agglutination reaction was determined as 0.2%(w/v). 7. The specificity, rapidity and simplicity of the latex-particle agglutination test suggested that it might be adaptable to large scale serum screening.

• Parasites, Hosts and Diseases
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• v.25 no.1
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• pp.1-6
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• 1987

This study was to observe the changes of blastogenic responses of splenic Iymphocytes to T-cell mitogens, N. fcwleri Iysate and concanaualin A, and serum antibody titer during the course of experimental PAM in mice. Naegleria fcwleri, strain 0359, was cultured in the CGVS medium axenically and inoculated intranasally with $7{\times}10^4$ trophozoites for the development of experimental PAM in mice. The amoebae were subjected to ultrasonication and centrifuged at 20,000g for 60 minutes, and filtered through $0.2{\mu\textrm{m}}$ filter membrane. The supernatant, N. fcwleri Iysate, was used as T-cell mitogen, and antigen for ELISA. The serum antibody was examined by ELISA using peroxidase conjugate. Two hundred ${\mi}l$ of $10^6$ splenocytes in RPMI 1640 containing 0% fetal calf serum were added to each well of a microtiter plate. To each well was added T-cell mitogens, $100{\mu}g/ml$ of N. fowleri Iysate or $4{\mu}g/ml$ of con. A, and the plates were incubated for 42 hours at $37^{\circ}C$ in 5% $CO_2$ incubator. Cultures were pulsed with of $methyl-(^3H)-thymidine$ 6 hour before harvesting. The mean blastogenic response of the splenocytes to N. fewleri Iysate was reduced, whereas that to con. A was also reduced up to on day 11 after infection. Both of these results were statistically significant compared with those of uninfected control group. The serum antibody titers were increased gradually up to day 15. The results indicated that there was an impairment of the blastogenic response of splenocytes to N. fowleri Iysate during the acute course of experimental PAM in mice.

• Journal of Food Hygiene and Safety
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• v.17 no.3
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• pp.137-145
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• 2002

Tributyltin compounds have been increasingly used in the form of plastic stabilizers, catalytic agents, industrial agricultural biocides, antifouling paint, and pesticides. Among these organotin compounds, large amounts of tributyltin(TBT) and triphenyltin(TPT) have been used as antifouling agents because they have a superior ability to prevent marine organism from being encrusted on ship bottoms and in culturing nets. Environmental pollution by these organotin compounds in the aquatic environment were undertaken. The international maritime Organization's established a provisional tolerable daily intake(TDI) of 1.6[micro]g TBTO/kg/ B.W. The Food and Agiculture Organization (of the United Nations)/world Health Organization's (FAO/WHO) proposed a TDI of 0.5ug TPT/kg BW/d. This study is conducted monitoring of TBT on seafoods in Korea and risk assessment for exposure on TBT in seafoods. Total hazard index(using Reference Dose : 0.3 ug TBTO/kg B.W/day) of intake exposure on seafoods is 0.04 as the 50th percentile, 0.08 as the 95th percentile. This value is estimated by Monte-Carlo simulation using Crystal Ball(Decisioneering Co., 2001).

• The Journal of the Korean Society for Microbiology
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• v.22 no.3
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• pp.309-321
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• 1987

For the investigation of microbiological and immunological specificity of Bacteroides gingivalis, Bacteroides gingivalis were isolated, enumerated and characterized from 13 Korean rapidly progressive periodontitis and 7 healthy control by anaerobic culture technique. The total proportion of black-pigmented Bacteroides from Korean R.P.P. patients and healthy control were 8.78% and 0.92%, respectively, among total isolated black-pigmented Bacteroides. In antibiotic susceptibility test, Bacteroides gingivalis isolated from R.P.P. patients were sensitive to Ampicillin and Tetracycline, and resistant to Gentamicin and Erythromycin in disc diffusion method. In antibiotic broth dilution method, the minimum inhibitory concentration(MIC) to Bacteroides gingivalis was 2 unit/ml of Penicillin and $0.25{\sim}1{\mu}g/ml$ of Tetracycline, respectively. The concentration of serum IgG in rapidly progressive periodontitis patients were sigificantly higher than that of healthy control, and concentration of diluted gingival crevicular IgG has not any significant differences between two groups. Serum and gingival crevicular IgG antibody to Bacteroides gingivalis were significantly higher titer in rapidly progressive periodontitis patients to compare with healthy control. The lipopolysaccharide profiles of 2 Korean B. gingivalis in silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis were similar to type strains of B. gingivalis and typical LPS band were appeared around the 24-Kd molecular weight. Immunodiffusion test and immunoelectrophoresis of the L.P.S. extracted from 2 Korean B. gingivalis and 2 kinds of type strains of B. gingivalis showed that B. gingivalis Korean-1 was reacted identically to B. gingivalis ATCC 33277. In trypsin and ${\alpha}$-glucosidase activity test of 2 Korean B. gingivalis, both of them revealed positive trypsin and negative ${\alpha}$-glucosidase activity, respectively. These investigation suggested that B. gingivalis is important pathogenic plaque bacteria for the pathogenesis of periodontitis and further study is needed to purify and characterize of the species-specific antigens of this organisms to develop monoclonal antibody and potential diagnostic reagents.