Study on vaccine Development for Streptococcosis of Olive Flounder (Paralichthys olivaceus) : Olive Flounder (Paralichthys olivaceus) 의 연쇄구균증에 대한 백신 개발에 관한 연구
Graduate School, Gyeongsang National University
Department of veterinary medicine
연쇄구균증 ECP Olive flounder 수의학;
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넙치연쇄구균증은 국내 제주도 양식어장에서 많이 발생하는 질병으로서 이 질병으로 인한 경제적 피해는 막대할 것으로 사료되어진다. 현 연구에서, 제주도 및 남해 일대에서 분리된 양성구균에 대한 역학조사를 실시한 바 제주균주에서 S. iniae 가 가장 많이 분리되었으며 그리고, 남해 일대에서는 L. garvieae 가 많이 동정되었다. 제주도에서 분리된 S. iniae 에 대한 넙치에 대한 균력을 수행한 바 제주-13 그리고 -45 분리주에서 50%이상의 높은 폐사율을 보였고 L. garvieae 로 동정된 제주-21 분리주에서도 높은 폐사율을 확인할 수 있었다. S. iniae 와 L. garvieae의 2-DE map을 건설하여 균력에 차이를 보이는 제주-13분리주와 제주-45분리주를 비교한 결과 GAPDH사이의 한 단백질 spot에서 차이를 보였다. 또한, 제주-21 과 제주-45 분리주에 대한 항혈청을 이용하여 제주-13, 제주-45 그리고 제주-21 분리주에 대해 2-DE immunobloting을 수행한 바 각 분리주간에 공통항원이 다수 존재는 하나 서로 다른 양상을 보였다. 제주-13, 제주-45 그리고 제주-21 분리주의 formalin killed 백신을 투여 한 후 각 분리주로 공격하였던바 S. iniae의 백신은 효율적으로 L. garvieae 를 방어하였으나 동종의 분리주에 대한 방어력은 약한 것을 알 수 있었다. 이러한 차이는 extracellular protein(ECP) 그리고, capsule에 의한 것으로 사료되어진다. 그래서 ECP를 2-DE에 의해 조사 하였던 바 많은 차이가 있는 것으로 확인 되었고 이러한 ECP는 백신재료로서 유용하게 사용 될 수 있을 것이라 생각되어지며 또한 넙치 백신 실험에서 확인되었다.
Olive-Olive-flounder (Paralichthys olivaceus) is a highly popular mariculture fish in Korea. However, a number of epidemiological studies reported the frequent occurrence of streptococcosis led to heavy economic losses to olive-flounder farmers. The aims of present studies are selection of vaccine strain and development of vaccine against olive-flounders streptococcosis. In chapter 1, bacteriological studies using bacteria from streptococcosis of olive-flounder were performed for identification of causative agents and investigation the virulence between the isolates. All Jeju isolates, except Jeju 21, and isolate of Yosu 10 showed β-heamolysis on blood agar, negative reaction for voges-proskauers VP test, and positive reaction for alkaline phosphatase. However, Jeju 21 and isolates from Namhae showed not β-heamolysis but α-haemolysis on blood agar, positive for VP test, and negative for alkaline phosphatase. PCR assays for the isolates were showed that β-haemolytic isolates were identified as S. iniae by its specific primers, α-haemolytic isolates were recognized as L. garvieae by it's specific primers. Comparison of proteins and antigens with the bacterial lysates were also demonstrated apparent differences between S. iniae and L. garvieae similar to biochemical test and PCR assay. Six of selected isolates were injected IP to olive-flounder in order to examine their virulence. Jeju 21 and 45 isolates were showed as high virulent, Jeju 13 isolate was moderate, and Jeju 24, 33, and 39 isolates were nearly avirulent. This result was matched with biochemical test, ADH negative strains of S. iniae (Jeju 13 and Jeju 45 isolates) were more virulent compared to ADH positive strains (Jeju 24, 33, and 39 isolates). Consequently, Jeju 13 and Jeju 45 isolates of S. iniae might be vaccine candidate strains for olive-flounder streptococcosis. In chapter 2, two-dimensional gel electrophoresis (2-DE) was performed not only to construct protein maps of S. iniae and L. garvieae but also to identify proteins associated with virulence by comparison of virulent and moderately virulent strains of S. iniae. In addition, 2-DE immunoblot assay was employed to explore antigenic proteins using olive-flounder sera raised against live S. iniae and L. garvieae isolates. Peptides mass fingerprinting (PMF) with (full name) MALDI-MS was able to identify 17 and 26 from 2-DE map of S. iniae ATCC29178 and L. garvieae KG (-), respectively. Both S. iniae ATCC29178 and L. garvieae KG(-) strains were revealed similar characteristics in 2-DE profiles, such as overcrowding proteins at the range of pH 4-7 and molecular weight (MW) 30 to 70 kDa, and identification of glycolytic enzymes at acidic region. On the other hand, clear difference was observed at spots of arginine deiminase (ADI) and ornithine carbamoltransferase between 2-DE profiles of L. garvieae and S. iniae. Proteins associated with virulence in two difference virulent strains (Jeju 13 and Jeju 45 isolate) of S. iniae was searched by 2-DE. The distant spot localized at between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 2-DE map of Jeju 13 isolate could not be observed in 2-DE map of Jeju 45 isolates. However, this spot can not be identified proteins database by PMF. Therefore, this spot is not known whether isoforms of GAPDH or protein associated with virulence. Nevertheless, because of more virulent Jeju 45 isolate, this spot may be protein associated with down regulation of virulence of S. iniae. In 2-DE immunoblot assay, the anti- live Jeju 21 and anti-live Jeju 45 sera of olive-flounder reacted weakly with antigenic proteins of S. iniae isolates, but were detected strongly with that of L. garvieae isolate. Therefore, Cell surface or cytosolic proteins of S. iniae may be not played an important role in production of antibody or vaccine as immunogen because of encapsulating of cell surface. However, cross reactivity of antisera to S. iniae isolate with Jeju 21 isolate suggested that S. iniae may be secreted extracellular proteins as immunogens.
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