{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T07:40:37Z","timestamp":1789544437033,"version":"build-2803163510"},"reference-count":51,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,12,22]],"date-time":"2023-12-22T00:00:00Z","timestamp":1703203200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Thai Union Feedmill Public Company Limited (Thailand)","award":["P-19-51469"],"award-info":[{"award-number":["P-19-51469"]}]},{"name":"National Science and Technology Development Agency (NSTDA)","award":["P-19-51469"],"award-info":[{"award-number":["P-19-51469"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>In the present study, chitosan-based bivalent nanovaccines of S. iniae and F. covae were administered by immersion vaccination at 30 and 40 days after hatching (DAH), and the third vaccination was orally administered by feeding at 50 DAH. ELISA revealed that the levels of total IgM and specific IgM to S. iniae and F. covae were significantly elevated in all vaccinated groups at 10, 20, and 30 days after vaccination (DAV). A qRT-PCR analysis of immune-related genes revealed significantly higher IgT expression in the vaccinated groups compared to the control group, as revealed by 44\u2013100-fold changes in the vaccinated groups compared to the control (p &lt; 0.001) at every tested time point after vaccination. All vaccinated groups expressed IgM, MHCII\u03b1, and TCR\u03b1 at significantly higher levels than the control group at 10 and\/or 20 DAV (p &lt; 0.05). In the S. iniae challenge tests, the survival of vaccinated groups ranged from 62.15 \u00b1 2.11 to 75.70 \u00b1 3.36%, which significantly differed from that of the control group (44.44 \u00b1 1.92%). Similarly, all vaccinated groups showed higher survival rates of 68.89 \u00b1 3.85 to 77.78 \u00b1 5.09% during F. covae challenge than the control groups (50.00 \u00b1 3.33%) (p &lt; 0.05).<\/jats:p>","DOI":"10.3390\/vaccines12010017","type":"journal-article","created":{"date-parts":[[2023,12,22]],"date-time":"2023-12-22T08:53:01Z","timestamp":1703235181000},"page":"17","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Development of Immersion and Oral Bivalent Nanovaccines for Streptococcosis and Columnaris Disease Prevention in Fry and Fingerling Asian Seabass (Lates calcarifer) Nursery Farms"],"prefix":"10.3390","volume":"12","author":[{"given":"Pakapon","family":"Meachasompop","sequence":"first","affiliation":[{"name":"Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"},{"name":"Center of Excellence in Aquatic Animal Health Management, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1782-4079","authenticated-orcid":false,"given":"Anurak","family":"Bunnoy","sequence":"additional","affiliation":[{"name":"Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"},{"name":"Center of Excellence in Aquatic Animal Health Management, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1502-9514","authenticated-orcid":false,"given":"Wisawat","family":"Keaswejjareansuk","sequence":"additional","affiliation":[{"name":"National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piroonrat","family":"Dechbumroong","sequence":"additional","affiliation":[{"name":"National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3763-9388","authenticated-orcid":false,"given":"Katawut","family":"Namdee","sequence":"additional","affiliation":[{"name":"National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathumthani 12120, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0354-6538","authenticated-orcid":false,"given":"Prapansak","family":"Srisapoome","sequence":"additional","affiliation":[{"name":"Laboratory of Aquatic Animal Health Management, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"},{"name":"Center of Excellence in Aquatic Animal Health Management, Faculty of Fisheries, Kasetsart University, 50 Paholayothin Rd., Ladyao, Chatuchak, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,22]]},"reference":[{"key":"ref_1","unstructured":"Lawley, D. (2010). Repositioning Australian Farmed Barramundi: Online Consumer Survey Findings, University of Sunshine Coast."},{"key":"ref_2","first-page":"e643","article-title":"Modelling selective breeding in protandrous, batch-reared Asian sea bass (Lates calcarifer, Bloch) using walkback selection","volume":"41","author":"Robinson","year":"2010","journal-title":"Aquac. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"177","DOI":"10.3354\/dao036177","article-title":"Streptococcus iniae, a bacterial infection in barramundi, Lates calcarifer","volume":"36","author":"Bromage","year":"1999","journal-title":"Dis. Aquat. Org."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/1297-9716-44-27","article-title":"Columnaris disease in fish: A review with emphasis on bacterium-host interactions","volume":"44","author":"Declercq","year":"2013","journal-title":"Vet. Res."},{"key":"ref_5","first-page":"341","article-title":"Streptococcus iniae infection in cultured Asian sea bass (Lates calcarifer) and red tilapia (Oreochromis sp.) in southern Thailand","volume":"32","author":"Suanyuk","year":"2010","journal-title":"Songklanakarin J. Sci. Technol."},{"key":"ref_6","first-page":"3","article-title":"Experimental Streptococcus iniae infection in barramundi (Lates calcarifer) cultured in Vietnam","volume":"4","author":"Wergeland","year":"2013","journal-title":"Int. J. Aquat. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"R1321","DOI":"10.1111\/1750-3841.12222","article-title":"Considerations on the aquaculture development and on the use of veterinary drugs: Special issue for fluoroquinolones da review","volume":"78","author":"Quesada","year":"2013","journal-title":"J. Food Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1016\/S0065-3519(99)80040-8","article-title":"Fish vaccines","volume":"41","author":"Vinitnantharat","year":"1999","journal-title":"Adv. Vet. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1080\/23308249.2016.1261277","article-title":"Advances in aquaculture vaccines against fish pathogens: Global status and current trends","volume":"25","author":"Dadar","year":"2017","journal-title":"Rev. Fish. Sci. Aquac."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1256","DOI":"10.1016\/j.dci.2011.03.007","article-title":"Advances in fish vaccine delivery","volume":"35","author":"Plant","year":"2011","journal-title":"Dev. Comp. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1971","DOI":"10.1007\/s10499-022-00884-w","article-title":"A review on the recent advances and application of vaccines against fish pathogens in aquaculture","volume":"30","author":"Mondal","year":"2022","journal-title":"Aquac. Int."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"108813","DOI":"10.1016\/j.fsi.2023.108813","article-title":"Development of a bivalent mucoadhesive nanovaccine to prevent francisellosis and columnaris diseases in Nile tilapia (Oreochromis niloticus)","volume":"138","author":"Bunnoy","year":"2023","journal-title":"Fish Shellfish Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Kitiyodom, S., Yata, T., Thompson, K.D., Costa, J., Elumalai, P., Katagiri, T., Temisak, S., Namdee, K., Rodkhum, C., and Pirarat, N. (2021). Immersion vaccination by a biomimetic-mucoadhesive nanovaccine induces humoral immune response of red tilapia (Oreochromis sp.) against Flavobacterium columnare challenge. Vaccines, 9.","DOI":"10.3390\/vaccines9111253"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1051","DOI":"10.1016\/j.fsi.2022.07.056","article-title":"Novel development of cationic surfactant-based mucoadhesive nanovaccine for direct immersion vaccination against Francisella noatunensis subsp. orientalis in red tilapia (Oreochromis sp.)","volume":"127","author":"Thangsunan","year":"2022","journal-title":"Fish Shellfish Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1111\/raq.12518","article-title":"Developing oral nanovaccines for fish: A modern trend to fight infectious diseases","volume":"13","author":"Angulo","year":"2021","journal-title":"Rev. Aquac."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.cbpc.2008.08.001","article-title":"Recommendations for control of pathogens and infectious diseases in fish research facilities","volume":"149","author":"Kent","year":"2009","journal-title":"Comp. Biochem. Physiol. Part C Toxicol. Pharmacol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"109269","DOI":"10.1016\/j.fsi.2023.109269","article-title":"Efficacy of the whole-cell based monovalent and bivalent vaccines of Streptococcus iniae and Flavobacterium covae in fingering Asian seabass (Lates calcarifer)","volume":"144","author":"Tumree","year":"2023","journal-title":"Fish Shellfish Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.fsi.2019.09.064","article-title":"Enhanced efficacy of immersion vaccination in tilapia against columnaris disease by chitosan-coated \u201cpathogen-like\u201d mucoadhesive nanovaccines","volume":"95","author":"Kitiyodom","year":"2019","journal-title":"Fish Shellfish Immunol."},{"key":"ref_19","first-page":"86","article-title":"Hematoxylin and eosin staining of tissue and cell sections","volume":"49","author":"Fischer","year":"2008","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1523","DOI":"10.1248\/cpb.40.1523","article-title":"Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, remazol brilliant blue R, as a substrate","volume":"40","author":"Ito","year":"1992","journal-title":"Chem. Pharm. Bull."},{"key":"ref_21","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\u2212\u0394\u0394CT method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Bunnoy, A., Na-Nakorn, U., and Srisapoome, P. (2019). Probiotic effects of a novel strain, Acinetobacter KU011TH, on the growth performance, immune responses, and resistance against Aeromonas hydrophila of bighead catfish (Clarias macrocephalus G\u00fcnther, 1864). Microorganisms, 7.","DOI":"10.3390\/microorganisms7120613"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.fsi.2020.04.031","article-title":"Development of fish vaccine in Southeast Asia: A challenge for the sustainability of SE Asia aquaculture","volume":"103","author":"Kayansamruaj","year":"2020","journal-title":"Fish Shellfish Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"737149","DOI":"10.1016\/j.aquaculture.2021.737149","article-title":"Resistome characterization of Flavobacterium columnare isolated from freshwater cultured Asian sea bass (Lates calcarifer) revealed diversity of quinolone resistance-associated genes","volume":"544","author":"Chokmangmeepisarn","year":"2021","journal-title":"Aquaculture"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1683","DOI":"10.1016\/j.fsi.2013.09.031","article-title":"A history of fish vaccination: Science-based disease prevention in aquaculture","volume":"35","author":"Gudding","year":"2013","journal-title":"Fish Shellfish Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1007\/s10499-009-9287-x","article-title":"Oral and parenteral vaccines against Flavobacterium columnare: Evaluation of humoral immune response by ELISA and in vivo efficiency in Nile tilapia (Oreochromis niloticus)","volume":"18","author":"Leal","year":"2010","journal-title":"Aquac. Int."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"5873","DOI":"10.1016\/j.vaccine.2022.08.075","article-title":"Modern biotechnological strategies for vaccine development in aquaculture-prospects and challenges","volume":"40","author":"Priya","year":"2022","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"633","DOI":"10.1016\/j.fsi.2022.06.059","article-title":"Mucoadhesive cationic lipid-based Flavobacterium oreochromis nanoencapsulation enhanced the efficacy of mucoadhesive immersion vaccination against columnaris disease and strengthened immunity in Asian sea bass (Lates calcarifer)","volume":"127","author":"Bunnoy","year":"2022","journal-title":"Fish Shellfish Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"231","DOI":"10.4155\/tde.14.89","article-title":"Nanocarriers: A versatile approach for mucosal vaccine delivery","volume":"6","author":"Mody","year":"2015","journal-title":"Ther. Deliv."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.fsi.2022.08.035","article-title":"Chitosan-polymer based nanovaccine as promising immersion vaccine against Aeromonas veronii challenge in red tilapia (Oreochromis sp.)","volume":"129","author":"Sukkarun","year":"2022","journal-title":"Fish Shellfish Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Pumchan, A., Sae-Ueng, U., Prasittichai, C., Sirisuay, S., Areechon, N., and Unajak, S. (2022). A novel efficient piscine oral nano-vaccine delivery system: Modified halloysite nanotubes (HNTs) preventing streptococcosis disease in tilapia (Oreochromis sp.). Vaccines, 10.","DOI":"10.21203\/rs.3.rs-1564012\/v1"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/j.biomaterials.2015.11.009","article-title":"Controlled and targeted release of antigens by the intelligent shell for improving the applicability of oral vaccines","volume":"77","author":"Zhang","year":"2016","journal-title":"Biomaterials"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"738535","DOI":"10.1016\/j.aquaculture.2022.738535","article-title":"Mannose functionalized biomimetic nanovaccine enhances immune responses against tilapia lake virus","volume":"560","author":"Gong","year":"2022","journal-title":"Aquaculture"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1007\/s10126-004-3018-0","article-title":"Gene expression in tilapia following oral delivery of chitosan-encapsulated plasmid DNA incorporated into fish feeds","volume":"7","author":"Ramos","year":"2005","journal-title":"Mar. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.virol.2023.01.014","article-title":"Biomimetic nanovaccine based on erythrocyte membrane enhances immune response and protection against tilapia lake virus","volume":"580","author":"Wei","year":"2023","journal-title":"Virology"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Thompson, K.D., Rodkhum, C., Bunnoy, A., Thangsunan, P., Kitiyodom, S., Sukkarun, P., Yostawornkul, J., and Yata, T. (2023). Addressing nanovaccine strategies for tilapia. Vaccines, 11.","DOI":"10.3390\/vaccines11081356"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7959","DOI":"10.2147\/IJN.S165876","article-title":"Novel application of trimethyl chitosan as an adjuvant in vaccine delivery","volume":"13","author":"Malik","year":"2018","journal-title":"Int. J. Nanomed."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Gong, X., Gao, Y., Shu, J., Zhang, C., and Zhao, K. (2022). Chitosan-based nanomaterial as immune adjuvant and delivery carrier for vaccines. Vaccines, 10.","DOI":"10.3390\/vaccines10111906"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1111\/j.1365-2761.1993.tb00848.x","article-title":"Vaccination of channel catfish, Ictalurus punctatus (Rafinesque), by immersion and oral booster against Edwardsiella ictalurid","volume":"16","author":"Plumb","year":"1993","journal-title":"J. Fish Dis."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1016\/j.fsi.2009.08.012","article-title":"Combined immersion and oral vaccination of Vietnamese catfish (Pangasianodon hypophthalmus) confers protection against mortality caused by Edwardsiella ictalurid","volume":"27","author":"Thinh","year":"2009","journal-title":"Fish Shellfish Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1016\/j.fsi.2012.12.011","article-title":"Immune responses and immune-related gene expression profile in orange-spotted grouper after immunization with Cryptocaryon irritans vaccine","volume":"34","author":"Dan","year":"2013","journal-title":"Fish Shellfish Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1016\/j.fsi.2016.05.042","article-title":"Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment","volume":"55","author":"Gao","year":"2016","journal-title":"Fish Shellfish Immunol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.dci.2013.01.014","article-title":"Gene expression profiling in live attenuated Edwardsiella tarda vaccine immunized and challenged zebrafish: Insights into the basic mechanisms of protection seen in immunized fish","volume":"40","author":"Yang","year":"2013","journal-title":"Dev. Comp. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Beck, B.H., and Peatman, E. (2015). Mucosal Health in Aquaculture, Academic Press.","DOI":"10.1016\/B978-0-12-417186-2.00013-3"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3463","DOI":"10.4049\/jimmunol.1400797","article-title":"Transcriptional characterization of the T cell population within the salmonid interbranchial lymphoid tissue","volume":"193","author":"Aas","year":"2014","journal-title":"J. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1111\/j.1095-8649.1997.tb01951.x","article-title":"Gill anomalies of perch and roach from four lakes differing in water quality","volume":"50","author":"Haaparanta","year":"1997","journal-title":"J. Fish Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1139\/f85-083","article-title":"Fish gill structural changes induced by toxicants and other irritants: A statistical review","volume":"42","author":"Mallatt","year":"1985","journal-title":"Can. J. Fish. Aquat. Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"142869","DOI":"10.1016\/j.scitotenv.2020.142869","article-title":"Seasonal effect of land use management on gill histopathology of Barbel and Douro Nase in a Portuguese watershed","volume":"764","author":"Santos","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1016\/j.vaccine.2007.11.065","article-title":"Development of an oral vaccine for immunisation of rainbow trout (Oncorhynchus mykiss) against viral haemorrhagic septicaemia","volume":"26","author":"Adelmann","year":"2008","journal-title":"Vaccine"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e0158968","DOI":"10.1371\/journal.pone.0158968","article-title":"Antigen uptake during different life stages of zebrafish (Danio rerio) using a GFP-tagged Yersinia ruckeri","volume":"11","author":"Korbut","year":"2016","journal-title":"PLoS ONE"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"e0117263","DOI":"10.1371\/journal.pone.0117263","article-title":"Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry","volume":"10","author":"Ohtani","year":"2015","journal-title":"PLoS ONE"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/12\/1\/17\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:40:30Z","timestamp":1760132430000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/12\/1\/17"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,22]]},"references-count":51,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["vaccines12010017"],"URL":"https:\/\/doi.org\/10.3390\/vaccines12010017","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,12,22]]}}}