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하재이,손상규,허민도,정현도,HA Jae Yi,SOHN Sang Gyu,HUH Min-Do,JEONG Hyun Do 한국수산과학회 1996 한국수산과학회지 Vol.29 No.5
여러 다른 S. aureus strains및 토끼항체를 사용하여 항체감작을 시킬 때 나타나는 S. aureus의 자체응집을 방지키 위한 분석과 함께 cogglutination의 최적 조건을 확립하였다. 적정화된 coagglutination기법을 실험실과 현장에서의 edwardsiellosis의 진단에 응용하였을 때 약 $10\;{\mu}g/m1$의 E. tarda까지 검출 할 수 있었다. 더구나 이 방법은 E. tarda의 FKC, EDTA또는 열탕추출 항원에 대해서 까지 좋은 진단결과를 보여주었다. 현장에서 edwardsiellosis에 감염된 어류로부터 직접 분리된 E. tarda 균주들은 토끼 항체생성을 위해 사용된 E. tarda 219와 응집항체법및 cogglutination법에서 모두 교차반응을 보여 주었다. 이러한 교차반응의 정도는 현장에서 나타나는 여러다른 E. tarda 균주에 감염 될 수 있는 어류의 질병진단에 사용하기에 충분한 정도로 나타났으며 감염어의 조직마쇄물을 1000배 이상 희석하여도 토끼 항 E. tarde 항체로 감작시킨 S. aureus와 coagglutination 될수 있는 양의 E. tarda를 함유하고 있었다. 자연감염 또는 인위감염된 넙치, 틸라피아의 조직마쇄물, 열탕추출항원에 대한 이 방법의 적용 결과는 본방법이 특별한 장비없이 현장에서 질병진단 기법으로 사용할 수 있다는 것을 보여 준다. To avoid the self-agglutination of Staphylococcus aureus sensitized with rabbit antibody in the absence of antigen, we determined the optimum concentration of rabbit antibody for sensitization. It was analyzed by using three different kinds of S. aureus strains at various concentraions of antibody. The optimized coagglutination test using the S. aureus sensitized with rabbit antibody was applied to the diagnosis of edwardsiellosis in field and in laboratory. The presence of E. tarda as low as $10\;{\mu}g/ml$ was detected by this method. Moreover, it showed good coagglutination results against several different forms of antigens such as FKC, EDTA or heat extracted antigen of E. tarda. E. tarda strains, isolated from the flounders suffering from edwardsiellosis in fields, showed some cross-reactions to the E. tarda 219 analyzed by both agglutination and coagglutination test with rabbit anti-E, tarda 219 antibody. The degree of cross-reactions analyzed was enough to apply the coagglutination test for the diagnosis of edwardsiellosis in field. Thus, even 1,000 fold diluted tissue homogenate of infected flounder naturally contained enough E. tarda as an antigen to show good coagglutination with S. aueus sensitized with rabbit anti-E, tarda 219 antibody. The successful application of this method to the homogenate and heat extract of tissues from naturally or artificially infected flounder or tilapia preyed that coagglutination test was a simple and rapid reliable dignostic technique suitable for using in laboratory and field without any special equipments.
수산생물의 생산과 관리에 관한 기초연구 : ELISPOT 기법을 이용한 넙치의 항체생성 세포분석
하재이,박준효,김명석,정준기,정현도,HA Jai Yi,PARK Jun-Hyo,KIM Myoung Sug,CHUNG Joon-Ki,JEONG Hyun Do 한국수산과학회 1999 한국수산과학회지 Vol.32 No.4
한국 양식산업에서 중요한 어종인 넙치 (Paralichthys olivaceus) 포르말린으로 처리한 E. tarda를 항원으로 하였을 때의 면역반응 분석을 위하여 ELISPOT 기법을 적정화시킨 후 넙치의 각 장기에 있는 총 항체생성세포와 특이 항체생성세포를 계수하는데 응용하고자 하였다. 전신과 비장의 항체생성세포를 2.5시간 이상 96 well plate에 배양하면 충분히 분석이 가능하였다. 그러나 총 또는 특이 항체생성세포 분석을 위하여 과량의 토끼 항 넙치 면역글로불린 또는 E. tarda 항원을 plate에 coating하는 것은 오히려 ELISPOT법의 감도를 감소시키는 것으로 나타났다. ELISPOT법의 특이성은 단백질 합성 억제제인 cycloheximide를 처리한 임파세포에서 총 항체생성세포가 발견되지 않는 것으로서 입증할 수 있었다. 특이 항체생성세포 수의 최대치는 면역 3주째에 나타났으며 이후 계속 빠르게 감소하여 7주째는 거의 발견되지 않았다. 이러한 반응은 신장과 비장에서 유사하게 나타나 임파장기에 따른 차이점은 발견할 수 없었다. 면역 후 2주와 3주 사이에 혈청내 특이 항체량 또한 빠르게 증가하여 ELISPOT법으로 분석된 특이 항체생성세포 수의 변화와 일치함을 발견할 수 있었다. 그러나 증가된 혈청내 특이 항체량이 면역 5주부터 실험 종료 시점까지 계속 높은 수준으로 유지되고 있는 것은 급격한 감소를 보이는 특이 항체생성세포의 동력학적 변화와는 명확히 구별되는 점이었다. We examined the immune response in flounder, Paralichthys olivaceus, with immunization of formalin killed Edwardsiella tarda as an antigen. The ELISPOT-assay (enzyme-linked immunospot assay) was optimized technically and applied to count the number of total and specific antibody secreting cells (TASC and SASC) in lymphocytes of different lymphatic organs. Incubation of lymphocytes on 96 well plate for more than 2.5hrs came out enough time in ELISPOT-assay for counting the antibody secreting cells in the anterior kidney and spleen. However, too much of plate-coated antigen or rabbit anti-flounder immunoglobulin for SASC or TASC counting, respectively, was appeared to decrease the sensitivity of the assay system. Specificity of the system was also confirmed by the absence of TASC in lymphocytes treated with cycloheximide to prevent protein synthesis. The peak numbers of SASC appeared at wk 3 post immunization after that there was a sharp decrease and reached to almost zero at wk 7. In the spleen and kidney, the timing and numbers of SASC on peak response were concurrent without preferential organ distribution. The specific antibody level in the sera increased rapidly between wk 2 and 3 after immunization, i.e. like the specific cellular response found with ELISPOT-assay on that period, However, the remained high level of specific serum antibody from wk 5 after immunization until the end of experiment was clearly distinguishable from the kinetics of SASC response decreased sharply.
나일 틸라피아에 대한 질병예방제 및 면역보조제로서의 PS-K 효과 분석
허성회(Sung Hoi Huh),박경현(Kyun Hyun Park),정현도(Hyun Do Jeong),하재이(Jai Yi Ha),허민도(Min Do Huh) 한국어병학회 1997 한국어병학회지 Vol.10 No.1
A protein-bound polysaccharide preparation, PS-K, isolated from Coriolus versicolor was evaluated for its ability to enhance resistance against Edwardsiella tarda, causal agent of classical edwardsiellosis in tilapia, Oreochromis niloticus. Fish treated with a dose rate of 0.1 ㎎ PS-K g^(-1) body weight were challenged by intraperitoneal injection of E. tarda with different concentrations. Against low burdens of injections(1.2×10^7 and 12 ×10^8 cfu/fish), PS-K treated fish did not show any mortality compared to 50% and 100% mortality of control groups, respectively. However there was no increase of resistance against challenge with high concentrations of E. tarda (1.2×10^9 and 1.2×10^(10)cfu /fish) except few days delaying of death. Tilapia injected with PS-K one day after intraperitoneal inoculation of E tarda(1.2×10^7 cfu/fish) showed 100% survival rate compared to control group of 50% survival rate. The result indicates the potential of PS-K as a prophylactic agent in aquaculture. The increased antibody response in fish received PS-K one week prior to FKC administration suggested the influence of PS-K on the specific humoral immunity and increased resistance against bacterial infection.
정준기(Joon Ki Chung),정현도(Hyun Do Jeong),하재이(Jai Yi Ha),허민도(Min Do Huh) 한국어병학회 1992 한국어병학회지 Vol.5 No.2
The absorption and excretion times of oxolinic acid(OX) used in farms as new aquatic antibiotics commonly were evaluated with determination of the effects of water temperature and feeding to parameters by using the bioassay technique. On the same time, antibacterial activity and the complex formation of oxolinic acid with serum proteins of two different fishes were compared to those oxytetracycline(OTC). With more than 10 times lower MIC values than those of OTC in the strains among 13 analyzed fish pathogens. OX did not show the decresed antibacterial activity by the binding of serum proteins in carp and tilapia. It implies more powerful potential of OX as aquatic medicine OTC. The serum concentration of OX after different administrations the oral, i.m., i.v and dipping methods were compared. The higher beginning concentration in serum and faster excretion times were obserbed in i.m. and dipping methods respectively. In the oral and i.m. administration, peak serum concentration after 24-48 hrs and slow excretion times demonstrated in both methods. These pharmacokinetic characteristics similar at 30℃ and 20℃ water temperature conditions, however, beginning serum concentration of OX in fish dipped in 50㎎/ℓsol after starvation for 2 wks was appeared lower than those of fed fish. It suggests the importance of biological condition of the gill or skin for absorption of antibiotics after dipping administration.
하동 지역에 서식하는 바지락의 미생물총 분포에 관한 정량 및 정성적 분석
허성회(Sung Hoi Huh),김명석(Myoung Sug Kim),박준효(Jun Hyu Park),정현도(Hyun Do Jeong),하재이(Jai Yi Ha),허민도(Min Do Huh) 한국어병학회 1998 한국어병학회지 Vol.11 No.2
Characteristics and distribution of the natural commensal flora in the surrounding environment and tissues of clam in Hadong area were studied under varying conditions of growth media and incubation temperatures. Total numbers of bacteria present in intestinal tract, gill, body fluid and surrounding mud were found to be not influenced by the used BHIA, STA and SNA media. Although the growth rate of bacteria at the condition of 15℃ incubation temperature was slower than that of 25℃ and 35℃, it showed the highest number of total bacteria compared with other two different conditions of incubation temperature. Interestingly, the proportion of bacteria able to form colony on several selective media was higher in replica analysis from nutrient media to selective media than that in direct smearing from samples. The generic diversity of bacteria isolated from the tissues and analyzed by API 20E and API 20NE kit showed similar pattern with each other and distinct from that of environment. The distribution of bacteria in the surrounding mud or mantle fluid of clam indicated a high diversity comparable to that found for the gill or intestinal tract microflora, with Pseudomonas being the prevalent group. It implies that the tissues of clam may probide a selective habitat for a commensal microflora.