Studies on growth phenotypes and development of molecular detection of Aphanomyces invadans, and bio-control of epizootic ulcerative syndrome
- Alternative Title
- Aphanomyces invadans의 성장 특성과 분자적 검출법 개발 및 유행성궤양성증후군의 생물학적 방제에 관한 연구
- Abstract
- Pathogenic oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Despite this, little is known about the influence of environmental factors on growth phenotypes, quantitative detection methods, and control of infection caused by A. invadans. Therefore, the purpose of this study was to investigate the growth phenotypes of A. invadans under different environmental factors and develop a sensitive and specific quantitative PCR targeting A. invadans and its effective bio-control method. The results showed that A. invadans could survive and produce the zoospores in a wide range of environmental factors: salinity from 0-16 ppt, pH from 4-9, and temperature from 10-35˚C and are pathogenic to Korean native snakeheads and catfish. The pathogenicity of A. invadans on catfish varied depending on temperatures. In this study, the developed assay showed the limit of detection of 4.68 copies of A. invadans genomic DNA per reaction (95% CI, 2.75 to 19.05 copies/reaction). The assay showed 50 times higher sensitivity compared to WOAH-recommended PCR assays and is highly specific to only A. invadans. The assay also showed high repeatability and reproducibility. The assay showed diagnostic sensitivity of 100% and diagnostic specificity of 75.76 and 78.43% for defined and undefined samples, respectively. Carp mucus-derived Enterococcus malodoratus CL6 and Enterococcus hawaiiensis CL21 exhibited strong anti-fungal activity against the mycelia growth and germination of A. invadans zoospores and other Aphanomyces species. These isolates could inactivate the germination of A. invadans zoospores, thus reducing the mortality of catfish from infection with A. invadans without causing any adverse effects. This study could provide fundamental data about EUS and its causative agent, A. invadans, transboundary disease management and pathogen monitoring of the aquatic environment, and bio-control strategies for infection with A. invadans.
- Issued Date
- 2023
- Awarded Date
- 2023-02
- Type
- Dissertation
- Keyword
- library
- Publisher
- 부경대학교
- Affiliation
- Pukyong National University, Graduate School
- Department
- 대학원 수산생명의학과
- Advisor
- Kim Do-Hyung
- Table Of Contents
- I. General introduction 1
1. Epizootic ulcerative syndrome 2
1.1. Agent factors 2
1.1.1. Causative agent 2
1.1.2. The life cycle of Aphanomyces invadans 3
1.1.3. Survival outside the host 6
1.1.4. Stability of Aphanomyces invadans against environmental factors 6
1.1.5. Isolation, culturing, and maintenance of Aphanomyces invadans 7
1.2. Epizootic ulcerative syndrome 7
1.2.1. Susceptible species and life stage 8
1.2.2. Clinical signs and target organs 8
1.2.3. Geographical distribution 9
1.2.4. Transmission mechanisms 14
1.2.5. Environmental and climate factors 14
1.3. Available diagnosis methods 15
1.3.1. Microscopy-based 15
1.3.2. Immunological methods 15
1.3.3. Polymerase chain reaction assays 15
1.4. Economic and social impacts 16
2. Treatment and control 16
3. Objectives of this study 18
II. Growth phenotypes, pathogenicity, and shedding of Aphanomyces invadans 19
1. Introduction 20
2. Materials and methods 21
2.1. Fungal strains, cultivation, and sporulation 21
2.2. Effects of pH on Aphanomyces invadans 22
2.2.1. Growth of Aphanomyces invadans mycelia at different pH levels 22
2.2.2. Sporulation ability of Aphanomyces invadans at different pH levels 22
2.3. Effects of different salinities on Aphanomyces invadans 23
2.3.1. Growth of Aphanomyces invadans at different salinities 23
2.3.2. Sporulation of Aphanomyces invadans at different salinities 23
2.4. Effects of different temperatures on Aphanomyces invadans 23
2.4.1. Growth of Aphanomyces invadans at different temperatures 23
2.4.2. Sporulation of Aphanomyces invadans at different temperatures 24
2.4.3. Viability of Aphanomyces invadans at different temperatures 24
2.5. Susceptibility of Korean native fish species to Aphanomyces invadans 24
2.5.1. Susceptibility of northern snakehead and amur catfish to infected intramuscularly with Aphanomyces invadans 25
2.5.2. Susceptibility of amur catfish infected with Aphanomyces invadans by immersion 25
2.6. Effects of different temperatures on pathogenicity and shedding of Aphanomyces invadans 26
3. Results 27
3.1. Effects of different pH levels on Aphanomyces invadans 27
3.1.1. Mycelia growth 27
3.1.2. Sporulation 29
3.2. Effects of different salinities on Aphanomyces invadans 31
3.2.1. Mycelia growth 31
3.2.2. Sporulation 33
3.3. Effects of different temperatures Aphanomyces invadans 35
3.3.1. Mycelia growth 35
3.3.2. Effects of different temperatures on the sporulation of Aphanomyces invadans 37
3.4. Effects of different temperatures on the viability of Aphanomyces invadans 39
3.5. Susceptibility of Korean native fish species to Aphanomyces invadans 41
3.5.1. Susceptibility of amur catfish infected intramuscularly with Aphanomyces invadans 41
3.5.2. Susceptibility of amur catfish infected with Aphanomyces invadans by immersion 43
3.6. Effects of different temperatures on the pathogenicity and shedding of Aphanomyces invadans 45
4. Discussion 50
III. Development and characterization of the quantitative assay to detect and quantify Aphanomyces invadans 53
1. Introduction 54
2. Materials and methods 55
2.1. Development of quantitative PCR method 55
2.1.1. Aphanomyces invadans specific primer and probe design 55
2.1.2. Positive control plasmid instruction 55
2.1.3. TaqMan quantitative assay 56
2.2. Characterization of EUS quantitative assay 56
2.2.1. Analytical characteristics 56
2.2.2. Clinical characteristics of EUS qPCR assay 61
2.3. Statistical analysis 62
3. Results 63
3.1. EUS quantitative PCR assay design 63
3.2. Analytical characteristic 65
3.2.1. Analytical specificity test 65
3.2.1.1. In silico analysis 65
3.2.1.2. Cross-reaction test 65
3.2.2. Analytical specificity test 67
3.2.2.1. Limit of detection and cut-off value 67
3.2.2.2. Interfering substances 69
3.2.2.3. Sensitivity comparisons of three WOAH-recommended conventional PCR and EUS qPCR assay 69
3.2.3. Precision test 71
3.2.3.1. Reaction efficiency 71
3.2.3.2. Repeatability 71
3.2.3.3. Reproducibility test 73
3.2.3.4. The whole system failure rate 75
3.2.3.5. Instrument compatibility 75
3.3. Clinical characteristics 77
3.3.1. Diagnostic sensitivity and diagnostic specificity using defined samples 77
3.4. Diagnostic sensitivity and diagnostic specificity using undefined samples 79
4. Discussion 82
II. Antifungal activity of bacteria isolated from fish skin mucus in vitro and in vivo against Aphanomyces invadans 85
1. Introduction 86
2. Materials and methods 87
2.1. Microbial isolations 87
2.2. Oomycete strains, cultivation, and sporulation 88
2.3. Mycelia inhibition assay 88
2.4. Zoospore inhibition assay 88
2.5. Microbial identification 89
2.6. Antagonistic activity against other oomycetes 91
2.7. Determination of the optimal concentration to inhibit Aphanomyces invadans 91
2.8. Efficacy of antagonistic bacteria in in vivo test 91
2.9. Statistical analysis 92
3. Results 93
3.1. Bacterial isolation and their inhibitory activity against Aphanomyces invadans 93
3.2. Zoospore germination inhibition, identification of selected mucus-derived bacteria, and their antagonistic activity against other oomycetes 95
3.3. The optimal concentration of CL6 and CL21 to inhibit A. invadans 101
3.4. Efficacy of CL6 and CL21 in in vivo test 104
4. Discussion 110
Conclusion 113
Appendix 115
Acknowledgment 129
References 130
- Degree
- Doctor
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