{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,19]],"date-time":"2026-10-19T20:30:49Z","timestamp":1792441849361,"version":"4.5.0"},"reference-count":49,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2025,8,23]],"date-time":"2025-08-23T00:00:00Z","timestamp":1755907200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31972698"],"award-info":[{"award-number":["31972698"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["D18007"],"award-info":[{"award-number":["D18007"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"riority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)","award":["31972698"],"award-info":[{"award-number":["31972698"]}]},{"name":"riority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)","award":["D18007"],"award-info":[{"award-number":["D18007"]}]},{"DOI":"10.13039\/501100013314","name":"111 Project","doi-asserted-by":"publisher","award":["31972698"],"award-info":[{"award-number":["31972698"]}],"id":[{"id":"10.13039\/501100013314","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100013314","name":"111 Project","doi-asserted-by":"publisher","award":["D18007"],"award-info":[{"award-number":["D18007"]}],"id":[{"id":"10.13039\/501100013314","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>SSB proteins play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. This study investigates the transcript levels, identification, expression and purification, subcellular localization, and immune protective potential of the SSB-like proteins of Eimeria necatrix (EnSSB), exploring its role in the development of E. necatrix and its potential as a candidate antigen for a subunit vaccine against avian coccidiosis. The level of EnSSB gene transcription was highest in unsporulated oocysts (UO), followed by gametocytes (GAM) (p &lt; 0.05). The gene consisted of an open reading frame of 1488 nucleotides encoding a protein of 495 amino acid residues with a predicted molecular weight of 53.31 kDa. EnSSB contained a SSB domain with a conserved OB (oligonucleotide\/oligosaccharide binding) fold. The molecular mass of the native protein, as determined by Western blot analysis, was ~58 kDa in second-generation merozoites (MZ-2) and UO. In addition to the 58 kDa band, four other bands (~98 kDa, ~82 kDa, ~36 kDa and ~28 kDa) were detected in GAM. No bands were detected in MZ-3. Indirect immunofluorescence and immuno-electron microscopy localized EnSSB in the cytoplasm of macrogametocytes but not in wall-forming bodies and oocyst wall. Animal challenge experiments demonstrated that rEnSSB elicited robust IgY responses, increased splenic T lymphocytes and body weight gain, reduced intestinal lesion scores and oocyst shedding, and presented anticoccidial index (ACI) more than 160. These findings not only offer a foundation for understanding the role of EnSSB protein in regulating the development of E. necatrix, but also present a potential protective antigen of E. necatrix for the development of a subunit vaccine against avian coccidiosis.<\/jats:p>","DOI":"10.3390\/ani15172482","type":"journal-article","created":{"date-parts":[[2025,8,25]],"date-time":"2025-08-25T00:09:32Z","timestamp":1756080572000},"page":"2482","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Molecular Characterization and Protective Efficacy of a Novel Protein (EnSSB) Containing a Single-Stranded DNA-Binding Domain from Eimeria necatrix"],"prefix":"10.3390","volume":"15","author":[{"given":"Yu","family":"Zhu","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7135-6843","authenticated-orcid":false,"given":"Dandan","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lele","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianqian","family":"Feng","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nianyu","family":"Xue","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaofeng","family":"Hou","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinjun","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianping","family":"Tao","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/S0065-308X(05)60005-X","article-title":"The biology of avian Eimeria with an emphasis on their control by vaccination","volume":"60","author":"Shirley","year":"2005","journal-title":"Adv. Parasitol."},{"key":"ref_2","first-page":"430610","article-title":"Control of avian coccidiosis: Future and present natural alternatives","volume":"2015","year":"2015","journal-title":"Biomed. Res. Int."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1080\/03079450802050705","article-title":"A survey of the economic impact of subclinical Eimeria infections in broiler chickens in Norway","volume":"37","author":"Haug","year":"2008","journal-title":"Avian Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1186\/s13567-020-00837-2","article-title":"Re-calculating the cost of coccidiosis in chickens","volume":"51","author":"Blake","year":"2020","journal-title":"Vet. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.molbiopara.2007.02.005","article-title":"Transient transfection of Eimeria tenella using yellow or red fluorescent protein as a marker","volume":"153","author":"Hao","year":"2007","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"485","DOI":"10.2307\/1591441","article-title":"An outbreak of Eimeria necatrix coccidiosis in breeder pullets: Analysis of immediate and possible long-term effects on performance","volume":"34","author":"McDougald","year":"1990","journal-title":"Avian Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"711","DOI":"10.2307\/1593117","article-title":"Eimeria brunetti and Eimeria necatrix in chickens of Argentina and confirmation of seven species of Eimeria","volume":"44","author":"Mattiello","year":"2000","journal-title":"Avian Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"104663","DOI":"10.1016\/j.psj.2024.104663","article-title":"Coccidiosis in poultry: Disease mechanisms, control strategies, and future directions","volume":"104","author":"Mathis","year":"2025","journal-title":"Poult. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.jcpa.2022.04.004","article-title":"An experimental dermal oedema model for apx toxins of Actinobacillus pleuropneumoniae","volume":"195","author":"Soutter","year":"2022","journal-title":"J. Comp. Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.ijpddr.2014.10.002","article-title":"Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production","volume":"4","author":"Chapman","year":"2014","journal-title":"Int. J. Parasitol. Drugs Drug Resist."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1016\/j.meegid.2017.10.009","article-title":"Recombinant anticoccidial vaccines\u2014A cup half full?","volume":"55","author":"Blake","year":"2017","journal-title":"Infect. Genet. Evol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1788","DOI":"10.3382\/ps.2010-00931","article-title":"Forty years of monensin for the control of coccidiosis in poultry","volume":"89","author":"Chapman","year":"2010","journal-title":"Poult. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2657981","DOI":"10.1155\/2016\/2657981","article-title":"Herbal remedies for coccidiosis control: A review of plants, compounds, and anticoccidial actions","volume":"2016","author":"Muthamilselvan","year":"2016","journal-title":"Evid. Based Complement. Alternat. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1186\/s13071-023-06076-6","article-title":"Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix","volume":"16","author":"Wang","year":"2023","journal-title":"Parasit. Vectors"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"109480","DOI":"10.1016\/j.vetpar.2021.109480","article-title":"Full-length transcriptome sequence analysis of Eimeria necatrix unsporulated oocysts and sporozoites identifies genes involved in cellular invasion","volume":"296","author":"Gao","year":"2021","journal-title":"Vet. Parasitol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1186\/s13071-021-05015-7","article-title":"Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing","volume":"14","author":"Gao","year":"2021","journal-title":"Parasit. Vectors"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1159","DOI":"10.1007\/s00436-019-06242-9","article-title":"Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization","volume":"118","author":"Su","year":"2019","journal-title":"Parasitol. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.vetpar.2018.01.019","article-title":"Comparative transcriptome analysis of Eimeria necatrix third-generation merozoites and gametocytes reveals genes involved in sexual differentiation and gametocyte development","volume":"252","author":"Su","year":"2018","journal-title":"Vet. Parasitol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1834","DOI":"10.1073\/pnas.84.7.1834","article-title":"Replication of simian virus 40 origin-containing DNA in vitro with purified proteins","volume":"84","author":"Wobbe","year":"1987","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2523","DOI":"10.1073\/pnas.85.8.2523","article-title":"Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA","volume":"85","author":"Wold","year":"1988","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1038\/2271313a0","article-title":"T4 bacteriophage gene 32: A structural protein in the replication and recombination of DNA","volume":"227","author":"Alberts","year":"1970","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1146\/annurev.biochem.66.1.61","article-title":"Replication protein A: A heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism","volume":"66","author":"Wold","year":"1997","journal-title":"Annu. Rev. Biochem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1146\/annurev.bi.63.070194.002523","article-title":"Escherichia coli single-stranded DNA-binding protein: Multiple DNA-binding modes and cooperativities","volume":"63","author":"Lohman","year":"1994","journal-title":"Annu. Rev. Biochem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"12220","DOI":"10.1016\/S0021-9258(18)63844-X","article-title":"The bacteriophage T4 DNA replication fork. Only DNA helicase is required for leading strand DNA synthesis by the DNA polymerase holoenzyme","volume":"264","author":"Cha","year":"1989","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"10173","DOI":"10.1073\/pnas.90.21.10173","article-title":"Bacteriophage T7 gene 2.5 protein: An essential protein for DNA replication","volume":"90","author":"Kim","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","first-page":"1388","article-title":"Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism","volume":"73","author":"Pestryakov","year":"2008","journal-title":"Biochemistry"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"569","DOI":"10.18388\/abp.2005_3416","article-title":"Single-stranded DNA-binding proteins (SSBs)\u2014Sources and applications in molecular biology","volume":"52","author":"Kur","year":"2005","journal-title":"Acta Biochim. Pol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1080\/03079450500178378","article-title":"Guidelines for evaluating the efficacy and safety of live anticoccidial vaccines, and obtaining approval for their use in chickens and turkeys","volume":"34","author":"Chapman","year":"2005","journal-title":"Avian Pathol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10227","DOI":"10.1073\/pnas.89.21.10227","article-title":"Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata","volume":"89","author":"Brown","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1111\/j.1574-6968.1999.tb13685.x","article-title":"Cryptosporidium parvum possesses a short-type replication protein A large subunit that differs from its host","volume":"176","author":"Zhu","year":"1999","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1477","DOI":"10.1016\/S0020-7519(02)00135-2","article-title":"Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum","volume":"32","author":"Millership","year":"2002","journal-title":"Int. J. Parasitol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"17493","DOI":"10.1074\/jbc.M200100200","article-title":"Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript","volume":"277","author":"Voss","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/1756-3305-7-27","article-title":"Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation","volume":"7","author":"Liu","year":"2014","journal-title":"Parasit. Vectors"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1186\/s13071-023-05742-z","article-title":"Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy","volume":"16","author":"Wang","year":"2023","journal-title":"Parasit. Vectors"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Zhu, Y., Liu, D., Wang, L., Feng, Q., Wang, F., Xue, N., Hou, Z., Xu, J., Hu, J., and Tao, J. (2025). Observations of the fine structural changes associated with merogony and gametogony in Eimeria necatrix and localization of two gametocyte proteins. Microorganisms, 13.","DOI":"10.3390\/microorganisms13051135"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1186\/s13071-017-2325-z","article-title":"Comparative transcriptome analysis of second- and third-generation merozoites of Eimeria necatrix","volume":"10","author":"Su","year":"2017","journal-title":"Parasit. Vectors"},{"key":"ref_37","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(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"111318","DOI":"10.1016\/j.molbiopara.2020.111318","article-title":"Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix","volume":"240","author":"Liu","year":"2020","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/0014-4894(70)90063-9","article-title":"Anticoccidial drugs: Lesion scoring techniques in battery and floor-pen experiments with chickens","volume":"28","author":"Johnson","year":"1970","journal-title":"Exp. Parasitol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.2307\/3276989","article-title":"Development of resistance to quinoline coccidiostats under field and laboratory conditions","volume":"54","author":"McManus","year":"1968","journal-title":"J. Parasitol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1080\/10409230802341296","article-title":"SSB as an organizer\/mobilizer of genome maintenance complexes","volume":"43","author":"Shereda","year":"2008","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1002\/j.1460-2075.1993.tb05726.x","article-title":"OB(oligonucleotide\/oligosaccharide binding)-fold: Common structural and functional solution for non-homologous sequences","volume":"12","author":"Murzin","year":"1993","journal-title":"EMBO J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e15379","DOI":"10.1371\/journal.pone.0015379","article-title":"Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein","volume":"5","author":"Oliveira","year":"2010","journal-title":"PLoS ONE"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1099\/mic.0.26833-0","article-title":"Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum","volume":"150","author":"Millership","year":"2004","journal-title":"Microbiol. (Read. )"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"31460","DOI":"10.1074\/jbc.M504466200","article-title":"The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits","volume":"280","author":"Rider","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"3421","DOI":"10.1002\/j.1460-2075.1992.tb05421.x","article-title":"A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB","volume":"11","author":"Foury","year":"1992","journal-title":"EMBO J."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"7037","DOI":"10.1093\/nar\/gkq565","article-title":"Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast","volume":"38","author":"Prusty","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"16367","DOI":"10.1016\/S0021-9258(17)36246-4","article-title":"Multiple species of single-stranded nucleic acid-binding proteins in Saccharomyces cerevisiae","volume":"260","author":"Jong","year":"1985","journal-title":"J. Biol. Chem."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e70610","DOI":"10.1096\/fj.202402555RR","article-title":"The third conserved cysteine residue in the zinc-binding domain of duck plague virus ICP8 is responsible for its single-stranded DNA-binding ability, viral attenuation and protection against lethal challenge","volume":"39","author":"Wu","year":"2025","journal-title":"FASEB J."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/17\/2482\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:34:49Z","timestamp":1760034889000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/15\/17\/2482"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,23]]},"references-count":49,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2025,9]]}},"alternative-id":["ani15172482"],"URL":"https:\/\/doi.org\/10.3390\/ani15172482","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,23]]}}}