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Identification, characterization, rapid detection technique development and vaccine production of major fish pathogen, Pseudomonas anguilliseptica isolated from farmed olive flounder, Paralichthys olivaceus

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Abstract
-
Author(s)
장영환
Issued Date
2014
Awarded Date
2014. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006672
Alternative Author(s)
Jang, Yeoung Hwan
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
허문수
Table Of Contents
INTRODUCTION 1
Part Ⅰ. Identification, phenotyping and genotyping of Pseudomonas anguilliseptica isolated from olive flounder (Paralichthys olivaceus) cultured in low rearing temperature
in Jeju Island
ABSTRACT 9
METERIALS AND METHODS 11
1. Fish 11
2. Isolation of pathogenic bacterial cells 11
3. DNA extraction of pathogenic bacterial cells 15
4. 16S rRNA gene sequence analysis of bacterial cells 16
5. Phylogenetic analysis 17
6. Susceptibility assay of antimicrobial agents 17
7. Pathogenicity assay 18
RESULTS 20
1. Phenotypic and biochemical characterization 20
2. Phylogenetic analysis 24
3. Susceptibility to antimicrobial agents 34
4. Pathogenicity by intra-peritoneal challenge 35
5. Pathogenicity by immersion challenge 43
DISCUSSION 45
Part Ⅱ. Rapid detection of P. anguilliseptica from olive flounder (Paralichthys olivaceus) using Real-Time PCR Taqman Fluorescent probes technique
ABSTRACT 48
METERIALS AND METHODS 50
1. P. anguilliseptica culture and genomic DNA extraction 50
2. Taqman probe Real-time PCR assay 53
3. Specificity, sensitivity and amplification efficiency of Taqman PCR technique 54
4. Challenge experiment 54
5. Statistical analysis 54
RESULTS 55
1. Challenge experiment 55
2. Taqman probe detection assay 57
DISCUSSION 61
Part Ⅲ. Efficacy of P. anguilliseptica formalin-killed vaccine on immune gene expression pattern and protection of farmed olive flounder (Paralichthys olivaceus)
ABSTRACT 63
METERIALS AND METHODS 65
1. Fish 65
2. Vaccine preparation 65
3. Vaccination-preparation of fish anti-Ps sera 66
4. Preparation of rabbit anti-Ps sera 66
5. Agglutination assay 67
6. Blood sampling 68
7. Blood biochemistry 68
8. Denaturing polycrylamide gel electrophoresis SDS-PAGE and immunoblotting analysis 69
9. Immune gene expression of vaccinated fishes 70
10. Primer design and quantitative PCR 71
11. Vaccinated fish challenge assay 73
12. Statistical analysis 73
RESULTS 74
1. Blood biochemical constituents 74
2. Protection of fish after vaccination 76
3. Antigenic protein recognition of vaccinated fishes 79
4. Real-time PCR expression of immune gene in vaccinated fishes 81
DISCUSSION 85
REFERENCE 87
ACKNOWLEDGEMENT 97
Degree
Doctor
Publisher
제주대학교 대학원
Citation
장영환. (2014). Identification, characterization, rapid detection technique development and vaccine production of major fish pathogen, Pseudomonas anguilliseptica isolated from farmed olive flounder, Paralichthys olivaceus
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General Graduate School > Marine Life Sciences
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