Abstract
Introduction
Miamiensis avidus is the major parasitic pathogen affecting the olive flounder, Paralichthys olivaceus. Recent epidemiological studies have shown that M. avidus infections are becoming increasingly severe and frequent in the olive flounder farming industry.
Objectives
This study aimed to evaluate the infection density of M. avidus in various organs of the olive flounder including spleen, liver, kidney, stomach, esophagus, intestine, gill, muscle, heart, and brain. Olive flounders were collected from a local fish farm.
Methods
Each fish was injected subcutaneously with 2.75 × 103 CFU M. avidus/ fish. Organs infected with M. avidus were obtained after 7 and 25 days. Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene.
Results
Each organ was examined for parasitic infection using real-time PCR. The primers were designed according to the sequences of 28 s in M. avidus, which was used as a target gene. The levels of 28 s rRNA were used to calculate quantitative gene copy number. Real-time PCR of brain (60.58 ± 38.41), heart (64.03 ± 62.40), muscle (6.10 ± 3.12), gill (5.06 ± 4.56), intestine (2.38 ± 1.69), esophagus (4.22 ± 3.72), stomach (3.25 ± 2.68), kidney (0.81 ± 0.15), liver (0.63 ± 0.15), and spleen (11.18 ± 4.08) was performed at 3 days post-infection. At 7 days post-infection, heart (754.15 ± 160.85), brain (247.90 ± 62.91), spleen (38.81 ± 17.52), liver (7.47 ± 4.54), kidney (10.90 ± 3.41), stomach (19.50 ± 8.86), esophagus (39.37 ± 14.10), intestine (17.54 ± 12.63), gill (38.27 ± 20.20), and muscle (33.62 ± 15.07) were measured.
Conclusion
The present study, together with previous data, demonstrated that the gill, intestine, and brain are the major target organs of M. avidus in olive flounder. However, this does not mean that tiny amounts of DNA extracted from those tissues of fish during the early stages of infection can guarantee successful detection and/or quantification of M. avidus. Our data suggest that the brain might be the best organ for detection in the early stage.
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Acknowledgements
This research was a part of a project titled ‘Omics based on fishery disease control technology development and industrialization (20150242),’ funded by the Ministry of Oceans and Fisheries, Korea.
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Hyunsu Kim declares that he has no conflict of interest. Kyung-Wan Baek declares that he has no conflict of interest. Ahran Kim declares that he has no conflict of interest. Nguyen Thanh Luan declares that he has no conflict of interest. Yunjin Lim declares that he has no conflict of interest. Heyong Jin Roh declares that he has no conflict of interest. Nameun Kim declares that he has no conflict of interest. Do-Hyung Kim declares that he has no conflict of interest. Yung Hyun Choi declares that he has no conflict of interest. Suhkmann Kim declares that he has no conflict of interest. Heui-Soo Kim declares that he has no conflict of interest. Mee Sun Ock declares that he has no conflict of interest. Hee-Jae Cha declares that he has no conflict of interest.
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13258_2019_792_MOESM1_ESM.pptx
Figure S1. Melting curves from real-time PCR for various M. avidus-infected organs of olive flounder, including spleen, liver, head-kidney, body-kidney, muscle, esophagus, stomach, intestine, gill, and brain 25 days post-infection. Figure S2. For quantification of DNA copy number, a standard curve was generated for each of the 28s genes. Standards containing 100 ng/μl (41,700,000 copies per μl), 10 ng/μl (4,170,000 copies per μl), 1 ng/μl (417,000 copies per μl), 0.1 ng/μl (41,700 copies per μl), 0.01 ng/μl (4,170 copies per μl), 1 pg/μl (417 copies per μl), 0.1 pg/μl (41.7 copies per μl), and 0.01 pg/μl (4.17 copies per μl) were used to generate the calibration curve. (A) Results of real-time PCR for the standards monitored in serial dilution. (B) The standard curve for the real-time PCR assay. The standard curve generated from the mean of Ct values plotted against log10 of eightfold serial dilutions of M. avidus genomic DNA. The calibration curve showed a good linear correlation (r2 = 0.9991). (PPTX 627 KB)
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Kim, H., Baek, KW., Kim, A. et al. Genome based quantification of Miamiensis avidus in multiple organs of infected olive flounder (Paralichthys olivaceus) by real-time PCR. Genes Genom 41, 567–572 (2019). https://doi.org/10.1007/s13258-019-00792-z
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DOI: https://doi.org/10.1007/s13258-019-00792-z