{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T22:50:58Z","timestamp":1778626258616,"version":"3.51.4"},"reference-count":41,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,5,31]],"date-time":"2024-05-31T00:00:00Z","timestamp":1717113600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100005825","name":"Arkansas Biosciences Institute","doi-asserted-by":"publisher","award":["1012366"],"award-info":[{"award-number":["1012366"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"Arkansas Biosciences Institute","doi-asserted-by":"publisher","award":["1018699"],"award-info":[{"award-number":["1018699"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"Arkansas Biosciences Institute","doi-asserted-by":"publisher","award":["2019-69012-29905"],"award-info":[{"award-number":["2019-69012-29905"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"Arkansas Biosciences Institute","doi-asserted-by":"publisher","award":["2020-67016-31346"],"award-info":[{"award-number":["2020-67016-31346"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"name":"NIFA SAS","award":["1012366"],"award-info":[{"award-number":["1012366"]}]},{"name":"NIFA SAS","award":["1018699"],"award-info":[{"award-number":["1018699"]}]},{"name":"NIFA SAS","award":["2019-69012-29905"],"award-info":[{"award-number":["2019-69012-29905"]}]},{"name":"NIFA SAS","award":["2020-67016-31346"],"award-info":[{"award-number":["2020-67016-31346"]}]},{"DOI":"10.13039\/100005825","name":"NIFA","doi-asserted-by":"publisher","award":["1012366"],"award-info":[{"award-number":["1012366"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"NIFA","doi-asserted-by":"publisher","award":["1018699"],"award-info":[{"award-number":["1018699"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"NIFA","doi-asserted-by":"publisher","award":["2019-69012-29905"],"award-info":[{"award-number":["2019-69012-29905"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005825","name":"NIFA","doi-asserted-by":"publisher","award":["2020-67016-31346"],"award-info":[{"award-number":["2020-67016-31346"]}],"id":[{"id":"10.13039\/100005825","id-type":"DOI","asserted-by":"publisher"}]},{"name":"AAES Research Incentive","award":["1012366"],"award-info":[{"award-number":["1012366"]}]},{"name":"AAES Research Incentive","award":["1018699"],"award-info":[{"award-number":["1018699"]}]},{"name":"AAES Research Incentive","award":["2019-69012-29905"],"award-info":[{"award-number":["2019-69012-29905"]}]},{"name":"AAES Research Incentive","award":["2020-67016-31346"],"award-info":[{"award-number":["2020-67016-31346"]}]},{"name":"Poultry Federation","award":["1012366"],"award-info":[{"award-number":["1012366"]}]},{"name":"Poultry Federation","award":["1018699"],"award-info":[{"award-number":["1018699"]}]},{"name":"Poultry Federation","award":["2019-69012-29905"],"award-info":[{"award-number":["2019-69012-29905"]}]},{"name":"Poultry Federation","award":["2020-67016-31346"],"award-info":[{"award-number":["2020-67016-31346"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Clostridium perfringens is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from B. subtilis was analyzed by a Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU\/mL after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU\/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and the virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in Lactobacillus johnsonii and B. longum under anaerobe but not L. johnsonii under 10% CO2 air. After the sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned bsh and amylase signal peptide sequence was inserted into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU\/mL in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against C. perfringens virulence, and recombinant secretory BSH from B-sub-BSH reduced C. perfringens growth, suggesting a new potential intervention against the pathogen-induced chicken NE.<\/jats:p>","DOI":"10.3390\/pathogens13060464","type":"journal-article","created":{"date-parts":[[2024,5,31]],"date-time":"2024-05-31T03:46:49Z","timestamp":1717127209000},"page":"464","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Recombinant Bile Salt Hydrolase Enhances the Inhibition Efficiency of Taurodeoxycholic Acid against Clostridium perfringens Virulence"],"prefix":"10.3390","volume":"13","author":[{"given":"Tahrir","family":"Alenezi","sequence":"first","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"College of Medical Applied Sciences, The Northern Border University, Arar 91431, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bilal","family":"Alrubaye","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ying","family":"Fu","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janashrit","family":"Shrestha","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samar","family":"Algehani","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hong","family":"Wang","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rohana","family":"Liyanage","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5601-0812","authenticated-orcid":false,"given":"Xiaolun","family":"Sun","sequence":"additional","affiliation":[{"name":"Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA"},{"name":"Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR 72701, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1080\/03079457.2016.1158781","article-title":"NetB and necrotic enteritis: The hole movable story","volume":"45","author":"Rood","year":"2016","journal-title":"Avian Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"14541","DOI":"10.1038\/s41598-019-51104-0","article-title":"Microbial metabolite deoxycholic acid controls Clostridium perfringens-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling","volume":"9","author":"Wang","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1080\/03079450400013162","article-title":"Clostridium perfringens in poultry: An emerging threat for animal and public health","volume":"33","author":"Pasmans","year":"2004","journal-title":"Avian Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Bansal, M., Fu, Y., Alrubaye, B., Abraha, M., Almansour, A., Gupta, A., Liyanage, R., Wang, H., Hargis, B., and Sun, X. (2020). A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens. J. Anim. Sci. Biotechnol., 11.","DOI":"10.1186\/s40104-020-00441-6"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1080\/03079450500112195","article-title":"Intercurrent coccidiosis and necrotic enteritis of chickens: Rational, integrated disease management by maintenance of gut integrity","volume":"34","author":"Williams","year":"2005","journal-title":"Avian Pathol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1080\/03079457.2016.1163521","article-title":"Epidemiologic aspects of necrotic enteritis in broiler chickens-disease occurrence and production performance","volume":"45","author":"Kaldhusdal","year":"2016","journal-title":"Avian Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"199","DOI":"10.3389\/fvets.2018.00199","article-title":"Evaluation of the Epithelial Barrier Function and Ileal Microbiome in an Established Necrotic Enteritis Challenge Model in Broiler Chickens","volume":"5","author":"Latorre","year":"2018","journal-title":"Front. Vet. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1542\/peds.75.1.151","article-title":"Lipid digestion and absorption","volume":"75","author":"Watkins","year":"1985","journal-title":"Pediatrics"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1038\/nature13828","article-title":"Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile","volume":"517","author":"Buffie","year":"2015","journal-title":"Nature"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1751","DOI":"10.1053\/j.gastro.2018.01.042","article-title":"Microbiota-Derived Metabolic Factors Reduce Campylobacteriosis in Mice","volume":"154","author":"Sun","year":"2018","journal-title":"Gastroenterology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1038\/s41586-021-03832-5","article-title":"Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians","volume":"599","author":"Sato","year":"2021","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1194\/jlr.R500013-JLR200","article-title":"Bile salt biotransformations by human intestinal bacteria","volume":"47","author":"Ridlon","year":"2006","journal-title":"J. Lipid Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1080\/19490976.2015.1127483","article-title":"Consequences of bile salt biotransformations by intestinal bacteria","volume":"7","author":"Ridlon","year":"2016","journal-title":"Gut Microbes"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1348-0421.1981.tb00001.x","article-title":"Deconjugation of bile salts by Bacteroids and Clostridium","volume":"25","author":"Masuda","year":"1981","journal-title":"Microbiol. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2329","DOI":"10.1110\/ps.9.12.2329","article-title":"Structural comparison of Ntn-hydrolases","volume":"9","author":"Oinonen","year":"2000","journal-title":"Protein Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5739","DOI":"10.1021\/bi0473206","article-title":"Conjugated bile acid hydrolase is a tetrameric N-terminal thiol hydrolase with specific recognition of its cholyl but not of its tauryl product","volume":"44","author":"Rossocha","year":"2005","journal-title":"Biochemistry"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1017\/S1466252316000153","article-title":"Bacterial bile salt hydrolase: An intestinal microbiome target for enhanced animal health","volume":"17","author":"Geng","year":"2016","journal-title":"Anim. Health Res. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1016\/j.foodres.2018.06.035","article-title":"New insights into bacterial bile resistance mechanisms: The role of bile salt hydrolase and its impact on human health","volume":"112","author":"Bustos","year":"2018","journal-title":"Food Res. Int."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1128\/AEM.72.3.1729-1738.2006","article-title":"Bile salt hydrolase activity in probiotics","volume":"72","author":"Begley","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"32516","DOI":"10.1074\/jbc.M604172200","article-title":"Structural and functional analysis of a conjugated bile salt hydrolase from Bifidobacterium longum reveals an evolutionary relationship with penicillin V acylase","volume":"281","author":"Kumar","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1107\/S2053230X16005707","article-title":"Crystal structure of bile salt hydrolase from Lactobacillus salivarius","volume":"72","author":"Xu","year":"2016","journal-title":"Acta Crystallogr. Sect. F Struct. Biol. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1016\/j.bbapap.2018.01.003","article-title":"Structure and function of a highly active Bile Salt Hydrolase (BSH) from Enterococcus faecalis and post-translational processing of BSH enzymes","volume":"1866","author":"Chand","year":"2018","journal-title":"Biochim. Biophys. Acta Proteins Proteom."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1038\/s41589-020-0467-3","article-title":"Development of a covalent inhibitor of gut bacterial bile salt hydrolases","volume":"16","author":"Adhikari","year":"2020","journal-title":"Nat. Chem. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6425","DOI":"10.1128\/AEM.68.12.6425-6428.2002","article-title":"Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens","volume":"68","author":"Knarreborg","year":"2002","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1038\/s41586-024-07017-8","article-title":"Bile salt hydrolase acyltransferase activity expands bile acid diversity","volume":"626","author":"Guzior","year":"2024","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1038\/s41586-020-2396-4","article-title":"A metabolic pathway for bile acid dehydroxylation by the gut microbiome","volume":"582","author":"Funabashi","year":"2020","journal-title":"Nature"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Kim, D.M., Liu, J., Whitmore, M.A., Tobin, I., Zhao, Z., and Zhang, G. (2024). Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis. J. Anim. Sci. Biotechnol., 15.","DOI":"10.1186\/s40104-024-00995-9"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Bansal, M., Alenezi, T., Fu, Y., Almansour, A., Wang, H., Gupta, A., Liyanage, R., Graham, D.B., Hargis, B.M., and Sun, X. (2021). Specific Secondary Bile Acids Control Chicken Necrotic Enteritis. Pathogens, 10.","DOI":"10.3390\/pathogens10081041"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Alenezi, T., Fu, Y., Alrubaye, B., Alanazi, T., Almansour, A., Wang, H., and Sun, X. (2023). Potent Bile Acid Microbial Metabolites Modulate Clostridium perfringens Virulence. Pathogens, 12.","DOI":"10.3390\/pathogens12101202"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Fu, Y., Bansal, M., Alenezi, T., Almansour, A., Wang, H., and Sun, X. (2022). Vaccines Using Clostridium perfringens Sporulation Proteins Reduce Necrotic Enteritis in Chickens. Microorganisms, 10.","DOI":"10.3390\/microorganisms10061110"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"20841","DOI":"10.1038\/s41598-020-77789-2","article-title":"Microbiota attenuates chicken transmission-exacerbated campylobacteriosis in Il10(-\/-) mice","volume":"10","author":"Fu","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2502","DOI":"10.1128\/AEM.66.6.2502-2512.2000","article-title":"Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization","volume":"66","author":"Tanaka","year":"2000","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1111\/j.1574-6968.1995.tb07780.x","article-title":"The anaerobic life of Bacillus subtilis: Cloning of the genes encoding the respiratory nitrate reductase system","volume":"131","author":"Hoffmann","year":"1995","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e03717","DOI":"10.1016\/j.heliyon.2020.e03717","article-title":"Impact of deoxycholate on Clostridioides difficile growth, toxin production, and sporulation","volume":"6","author":"Usui","year":"2020","journal-title":"Heliyon"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e2301252120","DOI":"10.1073\/pnas.2301252120","article-title":"Intestinal bile acids provide a surmountable barrier against C. difficile TcdB-induced disease pathogenesis","volume":"120","author":"Icho","year":"2023","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Liggins, M., Ramirez Ramirez, N., and Abel-Santos, E. (2023). Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1143399"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Song, Z., Cai, Y., Lao, X., Wang, X., Lin, X., Cui, Y., Kalavagunta, P.K., Liao, J., Jin, L., and Shang, J. (2019). Taxonomic profiling and populational patterns of bacterial bile salt hydrolase (BSH) genes based on worldwide human gut microbiome. Microbiome, 7.","DOI":"10.1186\/s40168-019-0628-3"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2530","DOI":"10.3168\/jds.S0022-0302(99)75506-2","article-title":"Screening of lactic acid bacteria for bile salt hydrolase activity","volume":"82","author":"Tanaka","year":"1999","journal-title":"J. Dairy. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Kusada, H., Morinaga, K., and Tamaki, H. (2021). Identification of Bile Salt Hydrolase and Bile Salt Resistance in a Probiotic Bacterium Lactobacillus gasseri JCM1131(T). Microorganisms, 9.","DOI":"10.3390\/microorganisms9051011"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1079","DOI":"10.1016\/S0022-2275(20)38464-9","article-title":"Purification and characterization of bile salt hydrolase from Clostridium perfringens","volume":"29","author":"Hylemon","year":"1988","journal-title":"J. Lipid Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/S0021-9258(18)96311-8","article-title":"The enzymatic cleavage of the carbon-nitrogen bond in 3-alpha, 7-alpha, 12-alpha-trihydroxy-5-beta-cholan-24-oylglycine","volume":"242","author":"Nair","year":"1967","journal-title":"J. Biol. Chem."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/6\/464\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:51:29Z","timestamp":1760107889000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/6\/464"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,31]]},"references-count":41,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["pathogens13060464"],"URL":"https:\/\/doi.org\/10.3390\/pathogens13060464","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,5,31]]}}}