Abstract

Background

Post-weaning diarrhea (PWD) in piglets, primarily caused by enterotoxigenicEscherichia coli(ETEC) K88 (F4) infection, presents a major challenge in swine production. This study aimed to isolate bacteriophages (phages) specific to ETEC K88, utilizing ETEC K88 as the host strain, and to assess the efficacy of dietary supplementation with the isolated phages in weaned piglets over a two-week period using an ETEC K88 challenge model in a pilot study.

Results

Three ETEC K88-specific phages (EC-P1, EC-P2, and EC-P3) were isolated and identified as tailed phages. These phages displayed a short latency period, broad acid–base stability, and thermal stability, effectively inhibiting ETEC K88 growth and disrupting ETEC K88 biofilms in vitro. Lyophilized phage powder was prepared and supplemented at 400, 600 or 800 mg/kg in the diets. Compared to the ETEC K88 group, piglets in the ETEC K88 + 600 or 800 mg/kg phages group exhibited markedly lower diarrhea scores and rectal temperatures at 12, 24, and 48 h post-infection. Supplementation with 600 mg/kg phages enhanced intestinal integrity of ETEC K88-infected piglets, as evidenced by an increased jejunal villus height and villus height-to-crypt depth ratio, reduced serum diamine oxidase and D-lactate levels, and upregulated jejunal ZO-1 protein expression. Concomitantly, systemic and jejunal inflammatory responses were attenuated by supplementation with 600 mg/kg of phages, as evidenced by decreased serum LPS, IL-1β, IL-10 and TNF-α levels, down-regulated jejunalIL-1βandIL-6mRNA expression, and suppressed NF-κB signalling (downregulated p-IκBα/IκBα and p-p65/p65 ratios). Supplementation with 600 mg/kg phages also shifted the faecal microbiota toward eubiosis, increasing the Shannon index, decreasing Proteobacteria and Enterobacteriaceae abundances, and elevating beneficial taxa (Patescibacteria, Muribaculaceae, andSubdoligranulum). Correlation analysis further revealed that Proteobacteria and Enterobacteriaceae abundances were positively associated with diarrhoea characteristics, whereas Muribaculaceae showed a negative correlation.

Conclusions

Three ETEC K88-targeting phages were successfully isolated, characterized, and prepared as lyophilized phage powder for dietary supplementation. Dietary supplementation with 600 mg/kg of lyophilized phage powder alleviated PWD in piglets by modulating gut microbiota and inflammatory responses.

Data Availability

The raw genome data of the three phages were deposited in the GenBank database of the National Center for Biotechnology Information (NCBI) under the following accession numbers: BankIt2899308 EC-P1 (PQ657772), BankIt2895556 EC-P2 (PQ631072), and BankIt2899079 EC-P3 (PQ657771). The 16S raw sequences of fecal microbiota were submitted to the NCBI Sequence Read Archive under accession number PRJNA1290277.

Abbreviations

  • ADFI:: Average daily feed intake
  • ADG:: Average daily gain
  • DAO:: Diamine oxidase
  • ETEC:: Enterotoxigenic Escherichia coli
  • FCR:: Feed conversion ratio
  • IL-1β:: Interleukin-1β
  • IL-6:: Interleukin-6
  • IL-10:: Interleukin-10
  • IκBα:: NF-kappa-B inhibitor alpha
  • LB:: Luria-Bertani
  • MOI:: Multiplicity of infection
  • NF-κB:: Nuclear factor kappa-B
  • PBS:: Phosphate-buffered saline
  • PFU:: Plaque forming unit
  • PWD:: Post-weaning diarrhea
  • TLR4:: Toll-like receptor 4
  • TNF-α:: Tumor necrosis factor-α
  • ZO-1:: Zonula occludens-1

References

  1. 1.Yu J, Song Y, Yu B, He J, Zheng P, Mao X, et al. Tannic acid prevents post-weaning diarrhea by improving intestinal barrier integrity and function in weaned piglets. J Anim Sci Biotechnol. 2020;11.(2020)org/10.1186/s40104-020-00496-5.: 87.
    10.1186/s40104-020-00496-5
    Article|CAS|PubMed|Google Scholar
  2. 2.Rhouma M, Fairbrother JM, Beaudry F, Letellier A. Post weaning diarrhea in pigs.(2017)risk factors and non-colistin-based control strategies.Acta Vet Scand.: 31.
    10.1186/s13028-017-0299-7
    Article|CAS|PubMed|Google Scholar
  3. 3.Wang W, Wang Y, Hao X, Duan Y, Meng Z, An X, et al. Dietary fermented soybean meal replacement alleviates diarrhea in weaned piglets challenged with enterotoxigenicEscherichia coliK88 by modulating inflammatory cytokine levels and cecal microbiota composition. BMC Vet Res. 2020;16.(2020)org/10.1186/s12917-020-02466-5.: 245.
    10.1186/s12917-020-02466-5
    Article|CAS|PubMed|Google Scholar
  4. 4.Heim G, Sweeney T, O’Shea CJ, Doyle DN, O’Doherty JV. Effect of maternal supplementation with seaweed extracts on growth performance and aspects of gastrointestinal health of newly weaned piglets after challenge with enterotoxigenicEscherichia coliK88. Br J Nutr. 2014;112(12).(2014)org/10.1017/S0007114514003171.: 1955.
    10.1017/S0007114514003171
    Article|CAS|PubMed|Google Scholar
  5. 5.Han Y, Zhan T, Tang C, Zhao Q, Dansou DM, Yu Y, et al. Effect of replacing in-feed antibiotic growth promoters with a combination of egg immunoglobulins and phytomolecules on the performance, serum immunity, and intestinal health of weaned pigs challenged withEscherichia coliK88. Animals. 2021;11(5).(2021)org/10.3390/ani11051292.: 1292.
    10.3390/ani11051292
    Article|CAS|PubMed|Google Scholar
  6. 6.Chen J, Han J, Yang Z, Zhou W, He Y, Chen X, et al. Bacteriophages as potential anti-pathogenic agents for intestinal health of weaned piglets in the post-antibiotic era.(2025)an updated review.Animals.: 1713.
    10.3390/ani15121713
    Article|CAS|PubMed|Google Scholar
  7. 7.Fang Q, Yin X, He Y, Feng Y, Zhang L, Luo H, et al. Safety and efficacy of phage application in bacterial decolonisation.(2024)a systematic review.Lancet Microbe.
    10.1016/S2666-5247(24)00002-8
    Article|CAS|PubMed|Google Scholar
  8. 8.Gencay YE, Jasinskytė D, Robert C, Semsey S, Martínez V, Petersen AØ, et al. Engineered phage with antibacterial CRISPR–Cas selectively reduceE. coliburden in mice. Nat Biotechnol. 2024;42(2).(2024)org/10.1038/s41587-023-01759-y.: 265.
    10.1038/s41587-023-01759-y
    Article|CAS|PubMed|Google Scholar
  9. 9.Choi Y, Lee W, Kwon J-G, Kang A, Kwak M-J, Eor J-Y, et al. The current state of phage therapy in livestock and companion animals. J Anim Sci Technol. 2024;66(1).(2024)57–78. https://doi. org/10.5187/jast.: 57.
    10.5187/jast.2024.e5
    Article|CAS|PubMed|Google Scholar
  10. 10.Yosef I, Manor M, Kiro R, Qimron U. Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc Natl Acad Sci USA. 2015;112(23).(2015)1073/pnas.1500107112.: 7267.
    10.1073/pnas.1500107112
    Article|CAS|PubMed|Google Scholar
  11. 11.Zeng Y, Wang Z, Zou T, Chen J, Li G, Zheng L, et al. Bacteriophage as an alternative to antibiotics promotes growth performance by regulating intestinal inflammation, intestinal barrier function and gut microbiota in weaned piglets. Front Vet Sci. 2021;8.(2021)623899. https://doi. org/10.3389/fvets.: 623899.
    10.3389/fvets.2021.623899
    Article|CAS|PubMed|Google Scholar
  12. 12.Duan Y, Young R, Schnabl B. Bacteriophages and their potential for treatment of gastrointestinal diseases. Nat Rev Gastroenterol Hepatol. 2022;19(2).(2022)org/10.1038/s41575-021-00536-z.: 135.
    10.1038/s41575-021-00536-z
    Article|CAS|PubMed|Google Scholar
  13. 13.Dong C, Chen Y, Ding M, Liu Y, Chen X, He Y, et al. Dietary bacteriophage administration alleviates enterotoxigenicEscherichia coli-induced diarrhea and intestinal impairment through regulating intestinal inflammation and gut microbiota in a newly weaned mouse model. Int J Mol Sci. 2024;25(19).(2024)org/10.3390/ijms251910736.: 10736.
    10.3390/ijms251910736
    Article|CAS|PubMed|Google Scholar
  14. 14.Li XY, Jin LJ, Uzonna JE, Li SY, Liu JJ, Li HQ, et al. Chitosan–alginate microcapsules for oral delivery of egg yolk immunoglobulin (IgY).(2009)in vivo evaluation in a pig model of enteric colibacillosis.Vet Immunol Immunopathol.: 132.
    10.1016/j.vetimm.2008.12.016
    Article|CAS|PubMed|Google Scholar
  15. 15.Nicolas M, Trotereau A, Culot A, Moodley A, Atterbury R, Wagemans J, et al. Isolation and characterization of a novel phage collection against avian-pathogenicEscherichia coli. Microbiol Spectr. 2023;11(3).(2023)1128/spectrum.04296-22.
    10.1128/spectrum.04296-22
    Article|CAS|PubMed|Google Scholar
  16. 16.Madec F, Bridoux N, Bounaix S, Cariolet R, Duval-Iflah Y, Hampson DJ, et al. Experimental models of porcine post-weaning colibacillosis and their relationship to post-weaning diarrhoea and digestive disorders as encountered in the field. Vet Microbiol. 2000;72(3–4).(2000)org/10.1016/s0378-1135(99)00202-3.: 295.
    10.1016/s0378-1135(99)00202-3
    Article|CAS|PubMed|Google Scholar
  17. 17.Zhou Y, Li L, Han K, Wang L, Cao Y, Ma D, et al. A polyvalent broad-spectrumEscherichiaphage tequatrovirus EP01 capable of controllingSalmonellaandEscherichia colicontamination in foods. Viruses. 2022;14(2).(2022)org/10.3390/v14020286.: 286.
    10.3390/v14020286
    Article|CAS|PubMed|Google Scholar
  18. 18.Shende RK, Hirpurkar SD, Sannat C, Rawat N, Pandey V. Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens. Vet World. 2017;10(8).(2017)973–8. https://doi. org/10.14202/vetworld.: 973.
    10.14202/vetworld.2017.973-978
    Article|CAS|PubMed|Google Scholar
  19. 19.Zhang L, Bao H, Wei C, Zhang H, Zhou Y, Wang R. Characterization and partial genomic analysis of a lytic Myoviridae bacteriophage againstStaphylococcus aureusisolated from dairy cows with mastitis in mid-east of China. Virus Genes. 2015;50(1).(2015)org/10.1007/s11262-014-1130-4.: 111.
    10.1007/s11262-014-1130-4
    Article|CAS|PubMed|Google Scholar
  20. 20.Kher L, Santoro D. Biofilm models.(2023)different ways of biofilm characterization and drug discovery.Curr Protoc.
    10.1002/cpz1.894
    Article|CAS|PubMed|Google Scholar
  21. 21.Rydal MP, Jørgensen CB, Gambino M, Poulsen LL, Nielsen JP. Complete association between CHCF1 genotype and enterotoxigenicEscherichia coliF4ab-associated post-weaning diarrhea in a pig challenge trial. Vet Microbiol. 2023;282.(2023)109771. https://doi. org/10. 1016/j.vetmic.: 109771.
    10.1016/j.vetmic.2023.109771
    Article|CAS|PubMed|Google Scholar
  22. 22.Garavito-Duarte Y, Duarte ME, Kim SW. Efficacy of ground herb-based and essential oil-based phytobiotics on the intestinal health and performance of nursery pigs challenged with F18+Escherichia coli. J Anim Sci. 2025;103.(2025)org/10.1093/jas/skaf018.
    10.1093/jas/skaf018
    Article|CAS|PubMed|Google Scholar
  23. 23.Garavito-Duarte Y, Bonetti A, Tugnoli B, Choi H, Piva A, Grilli E, et al. Investigation of the nutritional and functional roles of a microencapsulated blend of botanicals on intestinal health and growth of nursery pigs challenged with F18+Escherichia coli. J Anim Sci. 2025;103.(2025)org/10.1093/jas/skaf047.
    10.1093/jas/skaf047
    Article|CAS|PubMed|Google Scholar
  24. 24.National Research Council. Nutrient requirements of swine.(2012)eleventh revised edition.Washington, DC: The National Academies Press;.
    Article|CAS|PubMed|Google Scholar
  25. 25.Deng Q, Shao Y, Wang Q, Li J, Li Y, Ding X, et al. Effects and interaction of dietary electrolyte balance and citric acid on the intestinal function of weaned piglets. J Anim Sci. 2020;98(5).(2020)org/10.1093/jas/skaa106.
    10.1093/jas/skaa106
    Article|CAS|PubMed|Google Scholar
  26. 26.Wang W, Zijlstra RT, Gänzle MG. Identification and quantification of virulence factors of enterotoxigenicEscherichia coliby high-resolution melting curve quantitative PCR. BMC Microbiol. 2017;17(1).(2017)org/10.1186/s12866-017-1023-5.: 114.
    10.1186/s12866-017-1023-5
    Article|CAS|PubMed|Google Scholar
  27. 27.Youmans BP, Ajami NJ, Jiang ZD, Petrosino JF, DuPont HL, Highlander SK. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenicEscherichia coli(ETEC) heat labile and heat stable toxin genes in travelers’ diarrhea samples. Am J Trop Med Hyg. 2014;90(1).(2014)4269/ajtmh.13-0383.: 124.
    10.4269/ajtmh.13-0383
    Article|CAS|PubMed|Google Scholar
  28. 28.AOAC. Official methods of analysis. 16th ed. Washington, DC: Association of Official Analytical Chemists; 1995.
    Article|CAS|PubMed|Google Scholar
  29. 29.Zhang L, Cheng Y, Lu L, Zhong S, Zou T, Qu M, et al. Effects of dietary potassium diformate supplementation on growth performance, nutrient digestibility, gastrointestinal pH, jejunal morphology, digestive enzyme activity, and antioxidant status in weaned piglets. Animals. 2025;15(19).(2025)org/10.3390/ani15192916.: 2916.
    10.3390/ani15192916
    Article|CAS|PubMed|Google Scholar
  30. 30.Chen J, Jia X, Hu Y, Zhao X, Cheng Y, Lu L, et al. Benzoic acid as a dietary supplement mitigates inflammation and intestinal injury in acute enterotoxigenicEscherichia coli-infected mice without adverse effects in healthy mice. Food Funct. 2025;16(8).(2025)org/10.1039/d5fo00514k.: 3195.
    10.1039/d5fo00514k
    Article|CAS|PubMed|Google Scholar
  31. 31.Chen J, Xia Y, Hu Y, Zhao X, You J, Zou T. A blend of formic acid, benzoic acid, and tributyrin alleviates ETEC K88-induced intestinal barrier dysfunction by regulating intestinal inflammation and gut microbiota in a murine model. Int Immunopharmacol. 2023;114.(2023)2022.109538.: 109538.
    10.1016/j.intimp.2022.109538
    Article|CAS|PubMed|Google Scholar
  32. 32.Chen J, Chen J, Jia X, Hu Y, Zhao X, You J, et al. Dietary benzoic acid supplementation attenuates enterotoxigenicEscherichia coliK88-induced inflammation response and intestinal barrier dysfunction associated with gut microbiota modulation in newly-weaned mice. J Funct Foods. 2024;113.(2024)106044. https://doi. org/10. 1016/j.jff.: 106044.
    10.1016/j.jff.2024.106044
    Article|CAS|PubMed|Google Scholar
  33. 33.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCTmethod. Methods. 2001;25(4).(2001)402–8. https://doi. org/10.1006/meth.: 402.
    10.1006/meth.2001.1262
    Article|CAS|PubMed|Google Scholar
  34. 34.Li S, Liang P, Wang B, Chen J, You J, Zou T. Fibroblast growth factor 21/extracellular signal-regulated kinase 1/2 pathway as a key link mediates the effects of low-protein diet on skeletal muscle fiber type transformation in pigs. Anim Nutr. 2025;21.(2025)207–21. https://doi. org/10. 1016/j.aninu.: 207.
    10.1016/j.aninu.2025.03.001
    Article|CAS|PubMed|Google Scholar
  35. 35.Heo JM, Opapeju FO, Pluske JR, Kim JC, Hampson DJ, Nyachoti CM. Gastrointestinal health and function in weaned pigs.(2013)a review of feeding strategies to control post-weaning diarrhoea without using in-feed antimicrobial compounds.J Anim Physiol Anim Nutr (Berl).: 207.
    10.1111/j.1439-0396.2012.01284.x
    Article|CAS|PubMed|Google Scholar
  36. 36.Luise D, Lauridsen C, Bosi P, Trevisi P. Methodology and application ofEscherichia coliF4 and F18 encoding infection models in post-weaning pigs. J Anim Sci Biotechnol. 2019;10.(2019)org/10.1186/s40104-019-0352-7.: 53.
    Article|CAS|PubMed|Google Scholar
  37. 37.Zou T, Yang J, Guo X, He Q, Wang Z, You J. Dietary seaweed-derived polysaccharides improve growth performance of weaned pigs through maintaining intestinal barrier function and modulating gut microbial populations. J Anim Sci Biotechnol. 2021;12.(2021)org/10.1186/s40104-021-00552-8.: 28.
    Article|CAS|PubMed|Google Scholar
  38. 38.Zhang K, Shen X, Han L, Wang M, Lian S, Wang K, et al. Effects on the intestinal morphology, inflammatory response and microflora in piglets challenged with enterotoxigenicEscherichia coliK88. Res Vet Sci. 2023;157.(2023)50–61. https://doi. org/10. 1016/j.rvsc.: 50.
    10.1016/j.rvsc.2023.02.011
    Article|CAS|PubMed|Google Scholar
  39. 39.Guo X, Chen J, Yang J, He Q, Luo B, Lu Y, et al. Seaweed polysaccharide mitigates intestinal barrier dysfunction induced by enterotoxigenicEscherichia colithrough NF-κB pathway suppression in porcine intestinal epithelial cells. J Anim Physiol Anim Nutr (Berl). 2021;105(6).(2021)1111/jpn.13540.: 1063.
    10.1111/jpn.13540
    Article|CAS|PubMed|Google Scholar
  40. 40.Zhang B, Guo Y. Supplemental zinc reduced intestinal permeability by enhancing occludin and zonula occludens protein-1 (ZO-1) expression in weaning piglets. Br J Nutr. 2009;102(5).(2009)org/10.1017/S0007114509289033.: 687.
    10.1017/S0007114509289033
    Article|CAS|PubMed|Google Scholar
  41. 41.Xu J, Qiao H, Gan L, Wang P, Zhao Y, Lei Z, et al. Impacts of zinc caproate supplementation on growth performance, intestinal health, anti-inflammatory activity, and Zn homeostasis in weaned piglets challenged withEscherichia coliK88. J Anim Sci Biotechnol. 2025;16.(2025)org/10.1186/s40104-025-01172-2.: 44.
    Article|CAS|PubMed|Google Scholar
  42. 42.El-Zayat SR, Sibaii H, Mannaa FA. Toll-like receptors activation, signaling, and targeting.(2019)an overview.Bull Natl Res Cent.: 187.
    10.1186/s42269-019-0227-2
    Article|CAS|PubMed|Google Scholar
  43. 43.Tian M, Li L, Tian Z, Zhao H, Chen F, Guan W, et al. Glyceryl butyrate attenuates enterotoxigenicEscherichia coli-induced intestinal inflammation in piglets by inhibiting the NF-κB/MAPK pathways and modulating the gut microbiota. Food Funct. 2022;13(11).(2022)org/10.1039/d2fo01056a.: 6282.
    10.1039/d2fo01056a
    Article|CAS|PubMed|Google Scholar
  44. 44.Liu J, Chang G, Huang J, Wang Y, Ma N, Roy AC, et al. Sodium butyrate inhibits the inflammation of lipopolysaccharide-induced acute lung injury in mice by regulating the toll-like receptor 4/nuclear factor κB signaling pathway. J Agric Food Chem. 2019;67(6).(2019)jafc.8b06359.: 1674.
    10.1021/acs.jafc.8b06359
    Article|CAS|PubMed|Google Scholar
  45. 45.Gan Z, Guo Y, Zhao M, Ye Y, Liao Y, Liu B, et al. Excitatory amino acid transporter supports inflammatory macrophage responses. Sci Bull. 2024;69(15).(2024)2405–19. https://doi. org/10. 1016/j.scib.: 2405.
    10.1016/j.scib.2024.03.055
    Article|CAS|PubMed|Google Scholar
  46. 46.Zhang Y, Tan P, Zhao Y, Ma X. EnterotoxigenicEscherichia coli.(2022)intestinal pathogenesis mechanisms and colonization resistance by gut microbiota.Gut Microbes.: 2055943.
    10.1080/19490976.2022.2055943
    Article|CAS|PubMed|Google Scholar
  47. 47.Yan H, Xing Q, Xiao X, Yu B, He J, Mao X, et al. Effect ofSaccharomyces cerevisiaepostbiotics and essential oil on growth performance and intestinal health of weanling pigs during K88 ETEC infection. J Anim Sci. 2024;102.(2024)org/10.1093/jas/skae007.
    10.1093/jas/skae007
    Article|CAS|PubMed|Google Scholar
  48. 48.Gao Y, Han F, Huang X, Rong Y, Yi H, Wang Y. Changes in gut microbial populations, intestinal morphology, expression of tight junction proteins, and cytokine production between two pig breeds after challenge withEscherichia coliK88.(2013)a comparative study.J Anim Sci.: 5614.
    10.2527/jas.2013-6528
    Article|CAS|PubMed|Google Scholar
  49. 49.Shin NR, Whon TW, Bae JW. Proteobacteria.(2015)microbial signature of dysbiosis in gut microbiota.Trends Biotechnol.: 496.
    10.1016/j.tibtech.2015.06.011
    Article|CAS|PubMed|Google Scholar
  50. 50.Moreira de Gouveia MI, Bernalier-Donadille A, Jubelin G. Enterobacteriaceae in the human gut.(2024)dynamics and ecological roles in health and disease.Biology.: 142.
    Article|CAS|PubMed|Google Scholar
  51. 51.Li S, Guo J, Liu R, Zhang F, Wen S, Liu Y, et al. Predominance ofEscherichia-Shigellain gut microbiome and its potential correlation with elevated level of plasma tumor necrosis factor alpha in patients with tuberculous meningitis. Microbiol Spectr. 2022;10(6).(2022)1128/spectrum.01926-22.
    10.1128/spectrum.01926-22
    Article|CAS|PubMed|Google Scholar
  52. 52.Gu H, Tian Y, Xia J, Deng X, Chen J, Jian T, et al. Li-Hong Tang alleviates dextran sodium sulfate-induced colitis by regulating NRF2/HO-1 signaling pathway and gut microbiota. Front Pharmacol. 2024;15.(2024)1413666. https://doi. org/10.3389/fphar.: 1413666.
    10.3389/fphar.2024.1413666
    Article|CAS|PubMed|Google Scholar
  53. 53.O’Sullivan KM, Snelson M, Tan DS, Nguyen J, Le A, Huang U-SS, et al. Enhanced growth of gut bacteriaMuribaculaceaereduces inflammation and kidney injury in an experimental model of anti-neutrophil cytoplasmic antibody vasculitis.(2023)TH-PO557.J Am Soc Nephrol.: 245.
    10.1681/ASN.20233411S1245c
    Article|CAS|PubMed|Google Scholar
  54. 54.Van Hul M, Le Roy T, Prifti E, Dao MC, Paquot A, Zucker J-D, et al. From correlation to causality.(2020)the case ofSubdoligranulum.Gut Microbes.: 1849998.
    10.1080/19490976.2020.1849998
    Article|CAS|PubMed|Google Scholar

Acknowledgements

Not applicable.

Funding

This research was supported by the National Natural Science Foundation of China (No. 32472940 and No. 32160806), the Jiangxi Provincial Cultivation Program for Academic and Technical Leaders of Major Subjects (No. 20213BCJ22005), the Key Research and Development Program of Jiangxi Province (No. 20223BBF61018), and the Key Research and Development Program of Jiangxi Province (No. 20224BBF61029), China.

Ethics Declaration

Ethics approval and consent to participate

The animal protocol was approved by the Animal Care and Use Committee of Jiangxi Agricultural University (Ethics Approval Number: JXAULL-2024-823).

Consent for publication

All authors have read and agreed to the published version of manuscript.

Competing interests

We declare that we have no competing interests.

Rights and Permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reprints and permissions