Evaluation of a novel potential probiotics Bacillus spp. isolated from the gut of healthy gibel carp (Carassius auratus gibelio): Effects on the growth performance, nonspecific immunity and disease resistance against CyHV-2 infection

Evaluation of a novel potential probiotics Bacillus spp. isolated from the gut of healthy gibel carp (Carassius auratus gibelio): Effects on the growth performance, nonspecific immunity and disease resistance against CyHV-2 infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of a novel potential probiotics Bacillus spp. isolated from the gut of healthy gibel carp (Carassius auratus gibelio): Effects on the growth performance, nonspecific immunity and disease resistance against CyHV-2 infection Mingming Zhang, Yaqin Yang, Ni Ma, Mingfeng Zhu, Hu Zhang, Zhigang Zhao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7766598/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Aims: For sustainable aquaculture, probiotics are viable alternatives to antibiotics. However, most commercial probiotics originate from non-aquatic environments, limiting their efficacy in fish. This study aimed to isolate and evaluate indigenous probiotics from the intestinal microbiota of wild and cultured gibel carp (Carassius auratus gibelio) to provide effective, host-specific probiotics. Methods and Results: A total of 112 bacterial isolates (47 from cultured, 65 from wild carp) were obtained; 45 showed enzyme-producing activity. Isolates SY01 and SY02 outstandingly produced amylase, protease, and cellulase, and were non-pathogenic to gibel carp. They were identified as Bacillus subtilis (SY01) and B. amyloliquefaciens (SY02) via morphological, biochemical, and 16S rRNA sequencing analyses. Dietary supplementation with 1×10⁷ CFU/g for 30 days significantly improved growth, digestive enzyme activity, and serum antioxidant capacity (CAT, T-AOC, SOD), and upregulated immune-related genes (ITLN1, IL11, IFN). After CyHV-2 challenge, survival rates were 71% (SY01), 57% (SY02) vs. 14% (control). Conclusions: The two host-derived probiotics enhanced gibel carp’s growth, digestion, antioxidant status, and disease resistance, promising as antibiotic alternatives. This study provides a scientific basis for indigenous probiotic application in sustainable, eco-friendly aquaculture. Impact Statement As a key freshwater aquaculture species in China, gibel carp (Carassius auratus gibelio) faces major threats from Cyprinid herpesvirus 2 (CyHV-2), with no effective control measures available. Probiotics are a promising alternative to antibiotics, but non-host-derived strains are often less effective. This study addresses this by isolating and identifying host-specific Bacillus strains from gibel carp intestines. These native probiotics lay a new foundation for antibiotic substitution in aquaculture and offer a novel microecological strategy to prevent CyHV-2 outbreaks. Their use is expected to enhance the sustainability and biosafety of gibel carp farming. Probiotics Bacillus sp. CyHV-2 Carassius auratus gibelio Aquaculture Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviewers agreed at journal 01 Dec, 2025 Reviews received at journal 09 Nov, 2025 Reviewers agreed at journal 02 Nov, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers invited by journal 13 Oct, 2025 Editor assigned by journal 03 Oct, 2025 Submission checks completed at journal 03 Oct, 2025 First submitted to journal 02 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7766598","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534211709,"identity":"fa82560c-b256-493c-9b79-71dd63b993b7","order_by":0,"name":"Mingming Zhang","email":"","orcid":"","institution":"Yancheng Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mingming","middleName":"","lastName":"Zhang","suffix":""},{"id":534211710,"identity":"14e3baf1-4f92-48ed-ba62-d7983116bca8","order_by":1,"name":"Yaqin Yang","email":"","orcid":"","institution":"Yancheng Institute of 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infection","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Probiotics, Bacillus sp., CyHV-2, Carassius auratus gibelio, Aquaculture","lastPublishedDoi":"10.21203/rs.3.rs-7766598/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7766598/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAims:\u003c/strong\u003e For sustainable aquaculture, probiotics are viable alternatives to antibiotics. However, most commercial probiotics originate from non-aquatic environments, limiting their efficacy in fish. This study aimed to isolate and evaluate indigenous probiotics from the intestinal microbiota of wild and cultured gibel carp (Carassius auratus gibelio) to provide effective, host-specific probiotics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e A total of 112 bacterial isolates (47 from cultured, 65 from wild carp) were obtained; 45 showed enzyme-producing activity. Isolates SY01 and SY02 outstandingly produced amylase, protease, and cellulase, and were non-pathogenic to gibel carp. They were identified as Bacillus subtilis (SY01) and B. amyloliquefaciens (SY02) via morphological, biochemical, and 16S rRNA sequencing analyses. Dietary supplementation with 1×10⁷ CFU/g for 30 days significantly improved growth, digestive enzyme activity, and serum antioxidant capacity (CAT, T-AOC, SOD), and upregulated immune-related genes (ITLN1, IL11, IFN). After CyHV-2 challenge, survival rates were 71% (SY01), 57% (SY02) vs. 14% (control).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The two host-derived probiotics enhanced gibel carp’s growth, digestion, antioxidant status, and disease resistance, promising as antibiotic alternatives. This study provides a scientific basis for indigenous probiotic application in sustainable, eco-friendly aquaculture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact Statement\u003cbr\u003e\n \u003c/strong\u003eAs a key freshwater aquaculture species in China, gibel carp (Carassius auratus gibelio) faces major threats from Cyprinid herpesvirus 2 (CyHV-2), with no effective control measures available. Probiotics are a promising alternative to antibiotics, but non-host-derived strains are often less effective. This study addresses this by isolating and identifying host-specific Bacillus strains from gibel carp intestines. These native probiotics lay a new foundation for antibiotic substitution in aquaculture and offer a novel microecological strategy to prevent CyHV-2 outbreaks. Their use is expected to enhance the sustainability and biosafety of gibel carp farming.\u003c/p\u003e","manuscriptTitle":"Evaluation of a novel potential probiotics Bacillus spp. isolated from the gut of healthy gibel carp (Carassius auratus gibelio): Effects on the growth performance, nonspecific immunity and disease resistance against CyHV-2 infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 16:19:59","doi":"10.21203/rs.3.rs-7766598/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-03T16:39:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T16:30:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98918611804427669619335240097170806171","date":"2025-12-01T08:25:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T15:11:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245786418240122207796317272778227537293","date":"2025-11-03T00:12:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297274817930854722083979448646967045057","date":"2025-10-24T01:36:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-13T12:20:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-03T11:24:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-03T08:51:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquaculture International","date":"2025-10-02T12:50:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"","identity":"aquaculture-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"10499","submissionUrl":"https://submission.nature.com/new-submission/10499/3","title":"Aquaculture International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"","reportingPortfolio":"VoR Journals","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"29eca748-4e57-4e90-801e-f2774c612393","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-04T11:41:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 16:19:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7766598","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7766598","identity":"rs-7766598","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}