{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T05:27:11Z","timestamp":1786512431675,"version":"build-2736575974"},"reference-count":24,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100008787","name":"National Institute of Fisheries Science","doi-asserted-by":"publisher","award":["R2026052"],"award-info":[{"award-number":["R2026052"]}],"id":[{"id":"10.13039\/501100008787","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R2026052"],"award-info":[{"award-number":["R2026052"]}],"id":[{"id":"https:\/\/ror.org\/02chzeh21","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Emaciation disease, primarily caused by the myxosporean parasite Enteromyxum leei, poses a significant economic threat to the olive flounder (Paralichthys olivaceus) aquaculture industry. Research on E. leei has been severely restricted by its status as an unculturable obligate parasite and its extreme sensitivity to cryopreservation. In this study, we developed a standardized in vitro culture system that achieved stable maintenance of E. leei for over 12 months for the first time. To overcome the challenges of primary isolation, a physical aggregation technique was implemented, which effectively concentrated the parasites while reducing bacterial contamination by 99.1%, as validated by qPCR. The established culture, maintained in minimum essential medium supplemented with 30% FBS at 25 \u00b0C, exhibited a consistent 20-day asexual proliferative cycle consisting of six distinct developmental stages. Morphological stability and genetic identity were rigorously monitored, showing no phenotypic drift or genetic divergence throughout the 12-month period. Our results demonstrate that the entire proliferative phase of E. leei can be recapitulated in vitro without host intervention. Given that cryopreservation remains non-viable, this continuous culture system provides a critical, unique platform for high-throughput drug screening and myxozoan developmental biology, offering a robust tool to mitigate emaciation syndrome in marine fish farms.<\/jats:p>","DOI":"10.3390\/pathogens15080836","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T16:35:16Z","timestamp":1786379716000},"page":"836","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Stable In Vitro Cultivation of the Myxozoan Enteromyxum leei for over 12 Months: Integration of a Physical Aggregation Technique for Long-Term Antiparasitic Interventions"],"prefix":"10.3390","volume":"15","author":[{"given":"Eun Ji","family":"Jeon","sequence":"first","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Han Gill","family":"Seo","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1911-7060","authenticated-orcid":false,"given":"Na Young","family":"Kim","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"},{"name":"Aquaculture Industry Research Division, South Sea Fisheries Research Institute, National Institute of Fisheries Science, Yeosu 59780, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahran","family":"Kim","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7247-5345","authenticated-orcid":false,"given":"Myoung Sug","family":"Kim","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeong Wan","family":"Do","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8982-9947","authenticated-orcid":false,"given":"Hyun Ja","family":"Han","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyun Sil","family":"Kang","sequence":"additional","affiliation":[{"name":"Subtropical Fisheries Research Institute, National Institute of Fisheries Science, Jeju 63068, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5337-6042","authenticated-orcid":false,"given":"Soo Ji","family":"Woo","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Lee, H., Kim, T.M., Oh, H.M., Lim, H.K., and Cho, J.H. (2025). Transcriptomic Profiling Reveals Immune-Related Pathway Alterations in Paralichthys olivaceus Infected with Enteromyxum leei. Fishes, 10.","DOI":"10.3390\/fishes10120601"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kang, G., Woo, W.S., Kang, B.J., Kang, W.C., and Park, C.I. (2024). The Emaciation Disease: The Possibility of Non-Pathogenic Occurrence in Olive Flounder (Paralichthys olivaceus). Animals, 14.","DOI":"10.3390\/ani14223176"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.aquaculture.2018.04.041","article-title":"Molecular diagnostics for verifying an etiological agent of emaciation disease in cultured olive flounder Paralichthys olivaceus in Korea","volume":"493","author":"Shin","year":"2018","journal-title":"Aquaculture"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"11","DOI":"10.7847\/jfp.2011.24.1.011","article-title":"Pathophysiology of olive flounder Paralichthys olivaceus suffering from emaciation","volume":"24","author":"Kim","year":"2011","journal-title":"J. Fish Pathol."},{"key":"ref_5","first-page":"119","article-title":"Co-infection of two myxosporean parasites-Parvicapsula anisocaudata and an unidentified myxosporean-in the kidney of cultured olive flounder, Paralichthys olivaceus","volume":"18","author":"Cho","year":"2005","journal-title":"J. Fish Pathol."},{"key":"ref_6","first-page":"337","article-title":"Characterization of the myxosporean parasite isolated from emaciated olive flounders Paralichthys olivaceus on Jeju Island","volume":"48","author":"Kim","year":"2015","journal-title":"Korean J. Fish. Aquat. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"29","DOI":"10.3147\/jsfp.37.29","article-title":"Fish-to-fish transmission of Myxidium spp. (Myxozoa) in cultured tiger puffer suffering from emaciation disease","volume":"37","author":"Yasuda","year":"2002","journal-title":"Fish Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"738641","DOI":"10.1016\/j.aquaculture.2022.738641","article-title":"Comparison of oral and anal inoculation of Enteromyxum leei into olive flounder Paralichthys olivaceus","volume":"561","author":"Shin","year":"2022","journal-title":"Aquaculture"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1111\/jfd.12912","article-title":"Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)","volume":"42","author":"Shin","year":"2019","journal-title":"J. Fish Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"145","DOI":"10.3147\/jsfp.35.145","article-title":"Myxosporeans and their hyperparasitic microsporeans in the intestine of emaciated tiger puffer","volume":"35","author":"Tun","year":"2000","journal-title":"Fish Pathol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"175","DOI":"10.3147\/jsfp.40.175","article-title":"Occurrence of the myxosporean emaciation disease caused by Enteromyxum leei in cultured Japanese flounder Paralichthys olivaceus","volume":"40","author":"Yasuda","year":"2005","journal-title":"Fish Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"234","DOI":"10.47853\/FAS.2023.e20","article-title":"Infection of Enteromyxum leei in cultured starry flounder Platichthys stellatus","volume":"26","author":"Shin","year":"2023","journal-title":"Fish. Aquat. Sci."},{"key":"ref_13","unstructured":"KOSIS (Korean Statistical Information Service) (2025, February 15). Fishery Production Survey. Available online: https:\/\/kosis.kr\/eng\/."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/s42523-025-00388-x","article-title":"Interactions between gilthead seabream intestinal transcriptome and microbiota upon Enteromyxum leei infection: A multi\u2013omic approach","volume":"7","author":"Estensoro","year":"2025","journal-title":"Anim. Microbiome"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"45","DOI":"10.3147\/jsfp.43.45","article-title":"Myxosporean emaciation disease of cultured red sea bream Pagrus major and spotted knifejaw Oplegnathus punctatus","volume":"43","author":"Yanagida","year":"2008","journal-title":"Fish Pathol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1475","DOI":"10.1111\/jfd.13449","article-title":"Infection dynamics of Enteromyxum leei (Myxozoa, Myxosporea) in culture water and its effects on cultured olive flounder, Paralichthys olivaceus (Temminck & Schlegel)","volume":"44","author":"Sohn","year":"2021","journal-title":"J. Fish Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2 \u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Clifford, R.J., Milillo, M., Prestwood, J., Quintero, R., Zurawski, D.V., Kwak, Y.I., Paige, E.W., Lesho, E.P., and Mc Gann, P. (2012). Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0048558"},{"key":"ref_19","first-page":"55","article-title":"Therapeutic study of myxosporean emaciation disease of olive flounder (Paralichthys olivaceus) in Jeju using toltrazuril","volume":"33","author":"Kang","year":"2020","journal-title":"J. Fish Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1219","DOI":"10.1017\/S0031182022000762","article-title":"Morphological and molecular study of Didymodiclinus marginati n. sp. (Trematoda: Didymozoidae) gill parasite of Epinephelus marginatus from the central and western Mediterranean Sea","volume":"149","author":"Mele","year":"2022","journal-title":"Parasitol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/j.fsi.2006.02.004","article-title":"Innate and adaptive immune responses of turbot, Scophthalmus maximus (L.), following experimental infection with Enteromyxum scophthalmi (Myxosporea: Myxozoa)","volume":"21","author":"Redondo","year":"2006","journal-title":"Fish Shellfish Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"133","DOI":"10.3354\/dao055133","article-title":"In vitro studies on viability and proliferation of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of cultured turbot Scophthalmus maximus","volume":"55","author":"Redondo","year":"2003","journal-title":"Dis. Aquat. Organ."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"339","DOI":"10.14411\/fp.2004.022","article-title":"Studies on transmission and life cycle of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of turbot Scophthalmus maximus","volume":"51","author":"Redondo","year":"2004","journal-title":"Folia Parasit."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.cryobiol.2017.11.010","article-title":"Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia)","volume":"80","author":"Folgueira","year":"2018","journal-title":"Cryobiology"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/8\/836\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:29:56Z","timestamp":1786508996000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/8\/836"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":24,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["pathogens15080836"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15080836","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,10]]}}}