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Characteristics of Two Serotypes of Streptococcus parauberis Isolated from the Farmed Olive Flounder and Protective Efficacy of Combined Vaccine in Farmed Olive Flounder : 넙치에서 분리한 두가지 혈청형의 Streptococcus parauberis의 특성 및 넙치의 세균성질병 예방을 위한 3종 혼합백신의 효과에 관한 연구

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Authors

한상윤

Advisor
박세창
Major
수의학과
Issue Date
2012-02
Publisher
서울대학교 대학원
Description
학위논문 (석사)-- 서울대학교 대학원 : 수의학과, 2012. 2. 박세창.
Abstract
Olive flounder, Paralichthys olivaceus has become a major mariculture species of aquaculture industry in Korea. According to the statistics issued by the Statistics Korea, the production of olive flounder in Korean hatcheries was more than 40,923 tons in 2010. And there are many bacterial diseases threatening olive flounder aquaculture production in Korea. This research was focused on characteristics of Streptococcus parauberis, which is major causative agent of Streptococcosis in olive flounder. Furthermore, we evaluated the efficacy of a combined vaccine to prevent Streptococcosis and Edwardsiellosis in olive flounder.
This thesis is composed of two chapters. Some new findings obtained on the causative pathogen S. parauberis were described in Chapter Ⅰ; Prevalence and Different Characteristics of Two Serotypes of Streptococcus parauberis Isolated from the Farmed Olive flounder in Korea. In Chapter Ⅱ, we evaluated the protective efficacy of a combined vaccine containing formalin-inactivated Edwardsiella tarda, Streptococcus iniae, and S. parauberis using olive flounder.

Chapter Ⅰ. Prevalence and Different Characteristics of Two Serotypes of Streptococcus parauberis Isolated from the Farmed Olive flounder in Korea.
Streptococcs parauberis is known as important causative agent of streptococcosis in cultured olive flounder. The diverse characteristics and pathogenicity of S. parauberis were already reported. However, there are no report about serotype of this pathogen in Korea. In this study, we identified two serotypes of S. parauberis isolated from cultured olive flounders using anti-serum and compared presence of capsule and pathogenicity of two serotypes.
Twenty nine strains of S. parauberis were isolated from cultured olive flounder between 2003 and 2010 in Korea. Anti-S. parauberis rabbit serum were produced using S1735 (Serotype 1) and S53 (Serotype 2) strains mixed with Freund's adjuvant. Agglutination test was performed by serial twofold dilution method with 96-well microplates using each serotyope of rabbit antisera. In order to identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S-23S rRNA intergenic spacer region (ISR) and experimental infection test in olive flounder.
Agglutination assay using anti serum indicated two distinct serotype of S. parauberis isolated from Korean fish farm except for one strain. Twenty four strains were agglutinated with only anti-S1735 (Serotype 1) and four strains were agglutinated with only anti-S53 (Serotype 2). Mean agglutination titers of Serotype 1 strains (32 to 128) against anti-S1735 were higher than those of Serotype 2 strains (16 to 32) against anti-S53, relatively, which may reflect different antigenicities between two serotypes. S1091 strain was agglutinated with both type of anti-serum, with different level of agglutination titer against each anti-serum. In TEM, both serotype of S. parauberis strains were observed capsule layer around the cell surface. Serotype 1 strain was revealed thicker polycationic ferritin labelled capsule layer comparing with Serotype 2 strain. Cumulative mortalities of Serotype 1 strains were higher than those of Serotype 2 strains. Cumulative mortalities of S1735 strain were 80%, 50%, 30% and 30% for 109, 108, 107 and 106 CFU/fish. And those of S2018 strain were 100%, 100%, 60% and 40%. In contrast, cumulative mortalities of Serotype 2 strains (S53 and S2018) were below than 30% at all concentrations. However, no distinguishable biochemical reactions and sequence variations of 16S-23S rRNA ISR were observed between the serotypes. Our results suggested that S. parauberis isolated from fish farms in Korea were divided into two serologically distinct type and Serotype 1 strains were dominant and more virulent type in olive flounders.

Chapter Ⅱ. Protective Efficacy of a Combined Vaccine against Edwardsiella tarda, Streptococcus iniae and Streptococcus parauberis in Farmed Olive Flounder, Paralichthys olivaceus
We evaluated the protective efficacy of a combined vaccine containing formalin-inactivated E. tarda, S. iniae, and S. parauberis using olive flounder Paralichthys olivaceus. The fish were injected intraperitoneally with 0.1 mL of the combined vaccine. The fish were then challenged by intraperitoneal injection of the three bacterial strains. Mortality was significantly lower in the vaccinated groups compared with the control groups 3 weeks post vaccination. Furthermore, there was a significant increase in antibody titer up to 16 weeks after vaccination. Our results suggest that the combined vaccine successfully induced humoral and protective immunity against the three pathogens in olive flounder.
넙치는 한국 해수어 양식산업에서 가장 큰 비중을 차지하는 중요한 어종이다. 그러한 현재 연쇄구균증, 에드와드병 등의 세균성질병에 의해 넙치양식산업이 큰 경제적 피해를 입고있다.
이에 첫 번째 연구에서는 넙치 연쇄구균증의 주요 원인체인 Streptococcus parauberis의 특성에 대해 연구하였다. 2003년부터 2010년까지 양식넙치에서 분리한 29주의 Streptococcus parauberis를 사용하여 혈청형을 구분하고 각각의 혈청형에 따른 특성을 조사하였다. 29주의 S. parauberis는 PCR법을 사용하여 동정하였으며 토끼항혈청을 이용한 응집반응법으로 각각의 혈청형을 조사하였다. 그 결과 S1901주를 제외한 28개의 S. parauberis는 두가지의 혈청형(1형, 2형)으로 구분되었고, 1형이 29주 중 24주로 우점종으로 확인되었다. 각 혈청형의 특성을 조사하기 위하여 API20 Strep kit 를 이용한 생화학적 특성 검사 및 16S-23S rRNA ISR의 염기서열분석을 통한 유전학적 특성검사를 실시하였으나 혈청형에 따른 차이점은 확인되지 않았다. 그러나 전자투과현미경 촬영결과 1형에서 2형에 비해 두꺼운 협막이 관찰되었으며, 넙치에서의 감염시험을 통한 병원성 시험에서도 1형이 2형보다 높은 병원성을 나타내었다. 본 연구의 목적은 양식넙치에서 연쇄구균증을 일으키는 주요 세균인 S. parauberis의 혈청형을 조사하고 각각의 형청형별 유병율 및 특성을 조사하는 것이다.
두 번째 연구에서는 Edwardsiella tarda, S. iniae, S. parauberis 포르말린 불활화 혼합백신의 방어효과시험을 실시하였다. 넙치에 0.1mL씩 혼합백신을 복강 접종하고 3주후에 각 항원별로 공격접종을 실시하였다. 그 결과 백신군이 대조군보다 모든 항원에 대해 낮은 누적폐사율을 보였고 백신접종 2주후부터 항체가가 상승하여 16주까지 유지 또는 상승하였다. 이 결과를 통하여 E. tarda, S. iniae, S. parauberis 포르말린 불활화 혼합백신이 넙치에서 각 항원에 대한 특이항체뿐만 아니라 방어능을 유도한다는 것을 증명하였다.
Language
eng
URI
https://hdl.handle.net/10371/155098

http://dcollection.snu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000001015
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