{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T21:54:25Z","timestamp":1791928465794,"version":"4.3.4"},"reference-count":32,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T00:00:00Z","timestamp":1781049600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Rapid and accurate diagnosis of infectious diseases in aquaculture is essential for preventing major economic and ecological losses. Traditional culture-based methods focus on isolation of individual pathogens, and often are burdened with extended processing times, particularly during investigations of polymicrobial infections. Application of Oxford Nanopore Technologies (ONT) sequencing offers a rapid, culture-independent workflow for the identification of bacterial and fungal pathogens directly from fish tissues. Swab and organ samples from four cases (1: Salmo spp.; 2: Cyprinus carpio; 3: Salvelinus fontinalis; 4: Heniochus acuminatus) were analyzed using ONT long-read sequencing for metagenomic screening and bioinformatic classification. The results revealed case-, species-, and tissue-specific microbial profiles, with external tissues showing higher microbial diversity and internal organs enriched in pathogenic taxa. Dominant pathogens included Streptococcus iniae, Aeromonas hydrophila, Pseudomonas spp., and Saprolegnia parasitica, alongside opportunistic zoonotic bacteria such as Escherichia coli and Acinetobacter baumannii. We demonstrate the potential for diagnostic application of ONT sequencing in investigations and detection of multi-pathogen infections, including assessments of microbial community structure changes during disease outbreaks in aquatic species. The presented workflow enables rapid, cost-effective, and comprehensive pathogen profiling, supporting early disease surveillance and improved management in aquatic veterinary practice.<\/jats:p>","DOI":"10.3390\/pathogens15060622","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T02:59:31Z","timestamp":1781146771000},"page":"622","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Rapid Culture-Independent Detection of Fish Pathogens Using Oxford Nanopore Technologies: Case-Based Insights Across Multiple Species and Tissues"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1145-8263","authenticated-orcid":false,"given":"Konrad","family":"Wojnarowski","sequence":"first","affiliation":[{"name":"Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5880-6400","authenticated-orcid":false,"given":"Paulina","family":"Cholewi\u0144ska","sequence":"additional","affiliation":[{"name":"Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongqing","family":"Zhao","sequence":"additional","affiliation":[{"name":"Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshikazu","family":"Hasegawa","sequence":"additional","affiliation":[{"name":"Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7985-8047","authenticated-orcid":false,"given":"Daniela","family":"Denk","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Pathology, Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2980-821X","authenticated-orcid":false,"given":"Du\u0161an","family":"Pali\u0107","sequence":"additional","affiliation":[{"name":"Chair for Fish Diseases and Fisheries Biology, Ludwig-Maximilians-University Munich, 80539 Munich, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,10]]},"reference":[{"key":"ref_1","unstructured":"FAO (2024). The State of World Fisheries and Aquaculture\u20142024, Blue Transformation in Action, FAO."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"737050","DOI":"10.1016\/j.aquaculture.2021.737050","article-title":"Therapeutic modulation of fish gut microbiota, a feasible strategy for aquaculture?","volume":"544","author":"Cicala","year":"2021","journal-title":"Aquaculture"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1111\/raq.12745","article-title":"Fish disease prevention via microbial dysbiosis-associated biomarkers in aquaculture","volume":"15","author":"Mougin","year":"2022","journal-title":"Rev. Aquac."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1111\/raq.12862","article-title":"Signatures of Dysbiosis in Fish Microbiomes in the Context of Aquaculture","volume":"16","author":"Xavier","year":"2024","journal-title":"Rev. Aquac."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6915","DOI":"10.1128\/AEM.01826-15","article-title":"Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin","volume":"81","author":"Lowrey","year":"2015","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/j.fsi.2018.11.079","article-title":"Analysis of the Gut and Gill Microbiome of Resistant and Susceptible Lines of Rainbow Trout (Oncorhynchus mykiss)","volume":"86","author":"Brown","year":"2019","journal-title":"Fish Shellfish Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1186\/s40168-021-01113-x","article-title":"Host Habitat Is the Major Determinant of the Gut Microbiome of Fish","volume":"9","author":"Kim","year":"2021","journal-title":"Microbiome"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/0044-8486(84)90100-5","article-title":"Comparison between the Bacterial Flora Associated with Fingerling Rainbow Trout Cultured in Two Different Hatcheries in the North-West of Spain","volume":"42","author":"Nieto","year":"1984","journal-title":"Aquaculture"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1186\/s42523-022-00201-z","article-title":"Intestine and Spleen Microbiota Composition in Healthy and Diseased Tilapia","volume":"4","author":"Ofek","year":"2022","journal-title":"Anim. Microbiome"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1111\/1751-7915.13537","article-title":"Specific Pathogens and Microbial Abundance within Liver and Kidney Tissues of Wild Marine Fish from the Eastern Mediterranean Sea","volume":"13","author":"Meron","year":"2020","journal-title":"Microb. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Austin, B., and Austin, D.A. (2016). Aeromonadaceae Representative (Aeromonas salmonicida). Bacterial Fish Pathogens: Disease of Farmed and Wild Fish, Springer International Publishing.","DOI":"10.1007\/978-3-319-32674-0_5"},{"key":"ref_12","first-page":"256261","article-title":"The Main Aeromonas Pathogenic Factors","volume":"2012","year":"2012","journal-title":"Int. Sch. Res. Not."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.vetmic.2007.03.002","article-title":"Streptococcus Iniae: An Aquatic Pathogen of Global Veterinary Significance and a Challenging Candidate for Reliable Vaccination","volume":"122","author":"Agnew","year":"2007","journal-title":"Vet. Microbiol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2002","DOI":"10.1038\/ismej.2014.44","article-title":"Deciphering Microbial Landscapes of Fish Eggs to Mitigate Emerging Diseases","volume":"8","author":"Liu","year":"2014","journal-title":"ISME J."},{"key":"ref_15","unstructured":"Woo, P.T.K., and Bruno, D.W. (1999). Saprolegnia and other Oomycetes. Fish Diseases and Disorders: Vol. 3. Viral, Bacterial and Fungal Infections, CABI Publishing."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.mycol.2006.06.004","article-title":"Saprolegnia parasitica, an Oomycete Pathogen with a Fishy Appetite: New Challenges for an Old Problem","volume":"20","year":"2006","journal-title":"Mycologist"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ofek, T., Izhaki, I., and Halpern, M. (2023). Aeromonas Hydrophila Infection in Tilapia Triggers Changes in the Microbiota Composition of Fish Internal Organs. FEMS Microbiol. Ecol., 99.","DOI":"10.1093\/femsec\/fiad137"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1111\/raq.12513","article-title":"Microbiome Diversity and Dysbiosis in Aquaculture","volume":"13","author":"Huerlimann","year":"2021","journal-title":"Rev. Aquac."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.1111\/raq.12674","article-title":"Moving towards Improved Surveillance and Earlier Diagnosis of Aquatic Pathogens: From Traditional Methods to Emerging Technologies","volume":"14","author":"MacAulay","year":"2022","journal-title":"Rev. Aquac."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Aladhadh, M. (2023). A Review of Modern Methods for the Detection of Foodborne Pathogens. Microorganisms, 11.","DOI":"10.3390\/microorganisms11051111"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1007\/s00203-022-02757-5","article-title":"Microbial Adaptation to Different Environmental Conditions: Molecular Perspective of Evolved Genetic and Cellular Systems","volume":"204","author":"Wani","year":"2022","journal-title":"Arch. Microbiol."},{"key":"ref_22","first-page":"89","article-title":"MinION-A Third Generation Portable Nanopore Sequencer: Potential Applications in Fisheries and Aquaculture","volume":"3","author":"Kumar","year":"2022","journal-title":"Vigyan Varta Int. E-Mag. Sci. Enthus."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"16338","DOI":"10.1038\/s41598-025-01136-6","article-title":"Presence of Microplastic Particles Increased Abundance of Pathogens and Antimicrobial Resistance Genes in Microbial Communities from the Oder River Water and Sediment","volume":"15","author":"Wojnarowski","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1007\/s42995-019-00002-5","article-title":"Methods for the Diagnosis of Bacterial Fish Diseases","volume":"1","author":"Austin","year":"2019","journal-title":"Mar. Life Sci. Technol."},{"key":"ref_25","first-page":"131","article-title":"Molecular diagnosis of fish diseases: A review","volume":"3","author":"Altinok","year":"2013","journal-title":"Turk. J. Fish. Aquat. Sci."},{"key":"ref_26","first-page":"240","article-title":"MALDI-TOF MS: A Diagnostic Tool for Identification of Bacterial Fish Pathogens","volume":"40","author":"Jansson","year":"2020","journal-title":"Bull. Eur. Assoc. Fish Pathol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Strutt, J.P.B., Bessarab, I., Min, J.G.Y., Marican, H., and Summers, S. (2024). Rapid Nanopore Cloud-Based Monitoring and Analysis of Commercial Aquaculture Microbiomes. bioRxiv.","DOI":"10.1101\/2024.08.07.607111"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1186\/s13059-019-1727-y","article-title":"Performance of Neural Network Basecalling Tools for Oxford Nanopore Sequencing","volume":"20","author":"Wick","year":"2019","journal-title":"Genome Biol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Sehnal, L., Brammer-Robbins, E., Wormington, A.M., Blaha, L., Bisesi, J., Larkin, I., Martyniuk, C.J., Simonin, M., and Adamovsky, O. (2021). Microbiome Composition and Function in Aquatic Vertebrates: Small Organisms Making Big Impacts on Aquatic Animal Health. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.567408"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"16646","DOI":"10.1038\/s41598-022-16553-0","article-title":"Tracing the Oomycete Pathogen Saprolegnia parasitica in Aquaculture and the Environment","volume":"12","author":"Grbin","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"351","DOI":"10.2754\/avb200271030351","article-title":"Aeromonas Causes Severe Skin Lesions in Rainbow Trout (Oncorhynchus mykiss): Clinical Pathology, Haematology, and Biochemistry","volume":"71","year":"2002","journal-title":"Acta Vet. Brno"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhao, D., Wojnarowski, K., Cholewi\u0144ska, P., and Pali\u0107, D. (2025). Current Trends in Approaches to Prevent and Control Antimicrobial Resistance in Aquatic Veterinary Medicine. Pathogens, 14.","DOI":"10.3390\/pathogens14070681"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/6\/622\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T03:42:14Z","timestamp":1781149334000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/6\/622"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,10]]},"references-count":32,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["pathogens15060622"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15060622","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,10]]}}}