Title Page
Contents
List of Abbreviations 15
요약 17
Abstract 18
Chapter 1. General introduction 19
1. Epizootic ulcerative syndrome 20
1.1. Agent factors 20
1.2. Epizootic ulcerative syndrome 25
1.3. Available diagnosis methods 33
1.4. Economic and social impacts 34
2. Treatment and control 34
3. Objectives of this study 36
Chapter 2. Growth phenotypes, pathogenicity, and shedding of Aphanomyces invadans 37
1. Introduction 38
2. Materials and methods 39
2.1. Fungal strains, cultivation, and sporulation 39
2.2. Effects of pH on Aphanomyces invadans 40
2.3. Effects of different salinities on Aphanomyces invadans 41
2.4. Effects of different temperatures on Aphanomyces invadans 41
2.5. Susceptibility of Korean native fish species to Aphanomyces invadans 42
2.6. Effects of different temperatures on pathogenicity and shedding of Aphanomyces invadans 44
3. Results 45
3.1. Effects of different pH levels on Aphanomyces invadans 45
3.2. Effects of different salinities on Aphanomyces invadans 49
3.3. Effects of different temperatures Aphanomyces invadans 53
3.4. Effects of different temperatures on the viability of Aphanomyces invadans 57
3.5. Susceptibility of Korean native fish species to Aphanomyces invadans 59
3.6. Effects of different temperatures on the pathogenicity and shedding of Aphanomyces invadans 63
4. Discussion 68
Chapter 3. Development and characterization of the quantitative assay to detect and quantify Aphanomyces invadans 71
1. Introduction 72
2. Materials and methods 73
2.1. Development of quantitative PCR method 73
2.2. Characterization of EUS quantitative assay 74
2.3. Statistical analysis 80
3. Results 81
3.1. EUS quantitative PCR assay design 81
3.2. Analytical characteristic 83
3.3. Clinical characteristics 95
3.4. Diagnostic sensitivity and diagnostic specificity using undefined samples 97
4. Discussion 100
Chapter 4. Antifungal activity of bacteria isolated from fish skin mucus in vitro and in vivo against Aphanomyces invadans 103
1. Introduction 104
2. Materials and methods 105
2.1. Microbial isolations 105
2.2. Oomycete strains, cultivation, and sporulation 106
2.3. Mycelia inhibition assay 106
2.4. Zoospore inhibition assay 106
2.5. Microbial identification 107
2.6. Antagonistic activity against other oomycetes 109
2.7. Determination of the optimal concentration to inhibit Aphanomyces invadans 109
2.8. Efficacy of antagonistic bacteria in in vivo test 109
2.9. Statistical analysis 110
3. Results 111
3.1. Bacterial isolation and their inhibitory activity against Aphanomyces invadans 111
3.2. Zoospore germination inhibition, identification of selected mucus-derived bacteria, and their antagonistic activity against other oomycetes 113
3.3. The optimal concentration of CL6 and CL21 to inhibit A. invadans 119
3.4. Efficacy of CL6 and CL21 in in vivo test 122
4. Discussion 128
Conclusion 131
Appendix 133
References 148
Table 1-1. Occurrence of epizootic ulcerative syndrome 30
Table 2-1. Summary of clinical signs exhibition and infection rate 65
Table 3-1. Primers used in this study 82
Table 3-2. Cross-reaction test results 84
Table 3-3. Sensitivity comparisons between WOAH-recommended conventional PCR and the EUS qPCR assay using different templates 88
Table 3-4. Summary of repeatability test 90
Table 3-5. EUS qPCR reproducibility assay 92
Table 3-6. Compatibility of the EUS qPCR assay with commonly utilized quantitative PCR instruments 94
Table 3-7. Diagnostic performance of the EUS qPCR assay in comparison with the reference conventional PCR assay conducted on defined samples 96
Table 3-8. Diagnostic performance of the EUS qPCR assay in comparison with the reference conventional PCR assay conducted on undefined samples 98
Table 4-1. Primers used in this study 108
Table 4-2. Identification of mucus-derived bacteria obtained in this study 116
Supplementary Table 1. Glucose peptone yeast broth media 133
Supplementary Table 2. Peptone glucose 1 media 134
Supplementary Table 3. Determination of limit of detection of EUS qPCR assay at 6 different positive plasmid concentrations 135
Supplementary Table 4. Verification of limit of detection value using EUS qPCR assay 136
Supplementary Table 5. Amplification of EUS qPCR assay with and without potential interfering substances. The cycle threshold (Ct) was expressed as average ± standard deviation (SD). △Ct value is the difference between the Ct value of the positive sample and the...[이미지참조] 137
Supplementary Table 6. The whole system failure rate of EUS qPCR assay. 138
Supplementary Table 7. Compatibility of EUS qPCR assay with commonly utilized quantitative PCR instruments 139
Supplementary Table 8. Sequencing results of the PCR products were amplified using EUS qPCR assay of the discordant samples from both defined and undefined samples. 143
Figure 1-1. Life cycles of oomycetes 21
Figure 1-2. The life cycle of Aphanomyces invadans 23
Figure 1-3. Geographical map showing the current global distribution of epizootic ulcerative syndrome (1971-2020) 29
Figure 2-1. Growth of Aphanomyces invadans NJM9701 (A) and S1 (B) in PG-1 agar at different pH levels 46
Figure 2-2. Zoospore production of Aphanomyces invadans NJM9701 (A) and S1 (B) in autoclaved pond water at different pH levels. Asterisk indicates the oomycete colonies at 200 times dilution 48
Figure 2-3. Growth of Aphanomyces invadans NJM9701 (A) and S1 (B) in PG-1 agar at different salinities 50
Figure 2-4. Zoospore production of Aphanomyces invadans NJM9701 (A) and S1 (B) in autoclaved pond water at different salinities. Asterisk indicates the oomycete colonies at 200 times dilution 52
Figure 2-5. Growth of Aphanomyces invadans NJM9701 (A) and S1 (B) in PG-1 agar at different temperatures 54
Figure 2-6. Zoospore production of Aphanomyces invadans NJM9701 (A) and S1 (B) in autoclaved pond water at different temperatures. Asterisk indicates the oomycete colonies at 200 times dilution 56
Figure 2-7. Viability of mycelia and zoospore of Aphanomyces invadans NJM9701 (A) and S1 (B) in autoclaved pond water at different temperatures 58
Figure 2-8. Clinical signs (A) and accumulated mortality (B) of amur catfish (Silurus asotus) and snakehead (Channa argus) infected intramuscularly with Aphanomyces invadans 60
Figure 2-9. Accumulated mortality of amur catfish (Silurus asotus) infected with Aphanomyces invadans by immersion 62
Figure 2-10. Accumulated mortality of amur catfish (Silurus asotus) infected intramuscularly with Aphanomyces invadans NJM9701 (A) and S1 (B) at different temperatures 64
Figure 2-11. Shedding of Aphanomyces invadans from amur catfish (Silurus asotus) infected with strain NJM9701 (A) and strain S1 (B) at different temperatures 67
Figure 3-1. Detection curve for the EUS qPCR assay (A), regression analysis for determining the cut-off value (B), and the dynamic range (C). The solid black line denotes the proportion of detection means with a 95% confidence interval between the solid line in red color. 86
Figure 3-2. Cycle threshold (Ct) distribution of the positive and negative sample in defined samples (A, n=200) and undefined samples (B, n=100) at a cut-off value of 41.03 for the cycle threshold (horizontal line) 99
Figure 4-1. Inhibition of mycelial growth of Aphanomyces invadans NJM9701 (A) and S1 (B) by bacterial-free supernatant in PGY broth. The blue arrow indicates the growth of Aphanomyces invadans mycelia, and the red arrow indicates the inhibition zones 112
Figure 4-2. The optical density of Aphanomyces invadans NJM9701 (A) and S1 (B) spore suspensions in the presence of extracellular products of fish skin-derived bacteria. Asterisks indicate a significant difference (p 〈0.05) 114
Figure 4-3. Antagonistic activity of bacterial-free supernatant from mucus-derived isolates CL6 (A) and CL21 (B) against Aphanomyces species (upper) and the graphical representation of the percent suppression of growth (lower). The blue arrow indicates... 118
Figure 4-4. Antagonistic activity of different bacterial cells of Enterococcus malodoratus CL6 and Enterococcus malodoratus CL21 at different concentrations on the growth of Aphanomyces invadans NJM9701 (A) and CL21 (B) and the summary of inhibition... 120
Figure 4-5. Antagonistic activity of bacterial cells of Enterococcus malodoratus CL6 and Enterococcus malodoratus CL21 at 1E+07 CFU·ml-1 on the germination of zoospores of Aphanomyces invadans NJM9701 (A) and CL21 (B) and the summary of inhibition... 121
Figure 4-6. Observation of wounds in catfish (Silurus asotus) infected with Aphanomyces invadans S1 with/without the presence of Enterococcus malodoratus CL6 and... 123
Figure 4-7. Accumulated mortality of catfish (Silurus asotus) infected with Aphanomyces invadans S1 in the presence of Enterococcus malodoratus CL6 and Enterococcus... 125
Figure 4-8. The concentration of Enterococcus malodoratus CL6 (A) and Enterococcus hawaiiensis CL21 (B) in the water sample and mucus of catfish (Silurus asotus) after 9 days supplemented to the culture water 127
Supplementary Figure 1. The non-specific amplification of three WOAH-recommended conventional PCR assays using two Aphanomyces invadans strains, other oomycetes, four fish species, and water samples. Lane 1, Aphanomyces invadans NJM9701; Lane 2, Aphanomyces invadans... 145
Supplementary Figure 2. Positive rate for determine of limit of detection (left tables) and regression analysis for determining the cut-off value(right figures) of CFX96 Touch Real-Time PCR Detection System (A) and AriaMx Real-time PCR System System (B) 146