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Quantitative analysis of myxosporean parasites (Enteromyxum leei, E. fugu and Parvicapsula anisocaudata) detected from emaciated marine fish in Korea

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Abstract
Quantitative analysis of myxosporean (Enteromyxum leei and Parvicapsula anisocaudata) were performed using real-time PCR on the internal organs (head kidney, body kidney, intestine, spleen, brain, liver, heart, muscle, blood, and eye) of emaciated olive flounder, Paralichthys olivaceus from farm-A. The highest DNA copy number of E. leei was shown in the intestine (1.3 × 108 copies/mg tissue) of emaciatied P. olivaceus and DNA copy number in the other internal organs (1.3 × 103 - 4.6 × 105 copies/mg tissue) showed lower than intestine. From the result of real-time PCR for P. anisocaudata, it was considered mildly infected, due to the low DNA copy numbers of the head kidney (1.3 × 103 copies/mg tissue) and body kidney (9.1 × 103 copies/mg tissue). In order to investigate whether myxosporean parasites can be detected in a non-lethal methods, quantitative analysis of E. leei and P. anisocaudata isolated from rearing water of three farms were performed by real-time PCR. The DNA copy number of E. leei from rearing water of farm-A and farm-B were 8 × 104 and 5 × 105 copies/L. However, it was not detected in farm-C, for P. anisocaudata from rearing water from farm-B and farm-C were detected (2.0 × 106 and 5.1 × 106 copies/L). These results indicated that the two species of myxosprean can be diagnosed using rearing water, and it was considered that it could be used as a non-lethal diagnostic method through rearing water. Result of multiplex PCR using EM-F/R primer set (Kim et al., 2015) and EL-F/R primer set (Kang et al., 2020), It was confirmed that two clear bands were detected targeting P. anisocaudata and E. leei. As a result of performing multiplex-PCR on 9 tissues of olive flounder, one clear band of P. anisocaudata in kidney tissue and one clear band of E. leei in intestine tissue were detected. These results showed the multiplex PCR method used in this study can detect two types of myxosporeans through one PCR, and it was expected to be used as an economical, fast and accurate diagnostic method to diagnosis myxosporeans causing emaciation disease.
As a result of monitoring E. leei and P. anisocaudata from 2021 to 2022, . E. leei was detected starting from June when the water temperature increased until november, and it was confirmed that P. anisocaudata was detected throughout the year. Among 117 olive flounders suspected of emaciation disease, 73 olive flounders (62.4%) were confirmed that was single infection by P. anisocaudata and 4 olive flounders (3.4%) were confirmed that was single infection by E. leei. 28 olive flounders (23.9%) were confirmed that was co-infected by P. anisocaudata and E. leei and 12 olive flounder (10.3%) was confirmed that was non-infected, In the case of olive flounder single infected by P. anisocaudata, it was confirmed that almost all of them showed emaciation symptoms, the average relative condition factor, rCF values of individuals single-infected by P. anisocaudata was 84.6%, and it was confirmed that symptoms appeared from mild to severe. In the case of E. leei, it was confirmed that only 4 out of 22 farms were detected and the average rCF values of individuals single-infected by E. leei was 80.6%, it was confirmed them showed emaciation symptoms similarly P. anisocaudata, it was in contrast to the previously reported result that P. anisocaudata has a low correlation with emaciation disease (Shin et al., 2018).
Myxosporean parasite E. fugu was occurred for the first time in Korea. Result of sequencing, it was confirmed that E. leei and E. fugu were co-infected to tiger puffer, Takifugu rubripes. To early diagnosis E. fugu and E. leei, we developed 1-step PCR, 2-step PCR, real-time PCR and multiplex PCR for detection of E. fugu, E. leei. Myxosporeans detection was tested using 1-step PCR, 2-step PCR, real-time PCR, multiplex PCR. These results showed that real-time PCR and 2-step PCR were higher sensitive compared to PCR results and real-time PCR was higher sensitivity than 2-step PCR. Multiplex PCR has similar sensitivity to 1-step PCR. The real-time PCR method developed in this study has advantage that it is less time consuming than 1-step PCR technique because real time PCR does not require endpoint detection. 2-step PCR was consuming more time than 1-step PCR while, it has higher sensitive and exclude cross-reaction with other myxosporeans. In the case of multiplex PCR, has a similar sensitivity to 1-step PCR so it is considered helpful to less consuming time and resource. The diagnostic methods conducted in this study are expected to identify emaciation diseases in advance and reduce economic losses through rapid control.
Author(s)
이영준
Issued Date
2023
Awarded Date
2023-02
Type
Dissertation
URI
https://dcoll.jejunu.ac.kr/common/orgView/000000010977
Alternative Author(s)
Lee, Young Juhn
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
정준범
Table Of Contents
Contentes ⅰ
Summary ⅳ
List of figures ⅶ
List of tables ⅷ
Chapter 1. Development and optimization of diagnostic methods for diagnosing and monitoring emaciation disease 1
1.1 Introduction 1
1.2. Material and methods 1
1.2.1. Experimental fish 3
1.2.2. Experimental water 3
1.2.3. DNA extraction 3
1.2.4. PCR analysis 4
1.2.5. Multiplex PCR analysis 5
1.2.6 Real-time PCR analysis 5
1.3. Results 8
1.3.1. PCR anaylsis results 8
1.3.2. Multiplex PCR anaylsis results 8
1.3.3. Real-time PCR results 10
1.4. Discussion 13
Chapter 2. Monitoring of emaciation disease in 2021-2022 15
2.1. Introduction 15
2.2. Material and methods 15
2.2.1. Experimental fish 16
2.2.2. DNA extraction 16
2.2.3. Multiplex PCR analysis 17
2.2.4. Comparison of condition factor (CF) 17
2.2.5 Statistical analysis 17
2.3. Results 21
2.3.1. Multiplex PCR anaylsis results 21
2.3.2. Comparison of condition factor 24
2.3.3 Comparison of relative condition factor (rCF) 24
2.3.4 Statistical analysis 33
2.4. Discussion 35
Chapter 3. 37
3.1. Introduction 37
3.2. Material and methods 40
3.2.1. Experimental fish and water sample 40
3.2.2. DNA extraction 41
3.2.3. Primer design 42
3.2.4. 1-step PCR analysis 43
3.2.5. 2-step PCR analysis 44
3.2.6 Sequencing for identifying myxosporean (E. leei and E. fugu) 45
3.2.7. Multiplex PCR analysis 45
3.2.8. Real-time PCR analysis 46
3.2.9. Comparison sensitivity 49
3.3. Results 50
3.3.1. sequencing for identify myxosporean (E. leei and E. fugu) 50
3.3.2. 1-step PCR analysis 50
3.3.3. 2-step PCR analysis 51
3.3.3.1 2-step PCR analysis result of tiger puffers 51
3.3.3.2 2-step PCR analysis result oof olive flounders 52
3.3.4. Multiplex PCR analysis 52
3.3.5. Standard curve efficiency 52
3.3.6. Real-time PCR results 53
3.3.7. Comparison of sensitivity 53
3.4. Discussion 60
4. Reference 64
Degree
Master
Publisher
제주대학교 대학원
Citation
이영준. (2023). Quantitative analysis of myxosporean parasites (Enteromyxum leei, E. fugu and Parvicapsula anisocaudata) detected from emaciated marine fish in Korea.
Appears in Collections:
General Graduate School > Marine Life Sciences
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