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To address these limitations, we developed and evaluated RRbTB-E, a recombinant fusion protein comprising ESAT-6, as a defined alternative for skin testing. RRbTB-E was produced in Escherichia coli, purified by affinity chromatography, and characterized by SDS-PAGE and Western blot. In M. bovis-sensitized guinea pigs, RRbTB-E induced robust DTH reactions comparable to PPD-B, with consistent performance across six independent experiments and long-term stability after storage at 4\u20138 \u00b0C for more than 900 days. Furthermore, RRbTB-E did not induce significant reactions in non-sensitized or Mycobacterium avium\u2013sensitized animals, confirming antigenic specificity. RRbTB-E also elicited DTH responses in Mycobacterium tuberculosis\u2013sensitized guinea pigs. In naturally infected cattle, it triggered responses similar in magnitude to PPD-B, while remaining negative in non-infected animals. These findings support RRbTB-E as a stable, reproducible, and specific candidate for standardized intradermal testing in bovine tuberculosis diagnosis.<\/jats:p>","DOI":"10.3390\/ijms27052407","type":"journal-article","created":{"date-parts":[[2026,3,5]],"date-time":"2026-03-05T10:31:14Z","timestamp":1772706674000},"page":"2407","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of an ESAT-6 Recombinant Skin Test Reagent for Bovine Tuberculosis Diagnosis in Guinea Pigs and Cattle"],"prefix":"10.3390","volume":"27","author":[{"given":"Mat\u00edas Iv\u00e1n","family":"Gatto","sequence":"first","affiliation":[{"name":"Pharmaceutical Biotechnology Laboratory, Institute of Biology and Experimental Medicine (IBYME-CONICET), Ciudad Aut\u00f3noma de Buenos Aires C1428BFA, Argentina"},{"name":"Agropharma Salud Animal S.A., Moreno, Buenos Aires B1744JFN, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sol","family":"Ferrero","sequence":"additional","affiliation":[{"name":"Pharmaceutical Biotechnology Laboratory, Institute of Biology and Experimental Medicine (IBYME-CONICET), Ciudad Aut\u00f3noma de Buenos Aires C1428BFA, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Florencia","family":"Tonini","sequence":"additional","affiliation":[{"name":"A.V.I.S. Veterinary Practice, Suardi, Santa Fe S2349, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7550-6878","authenticated-orcid":false,"given":"Marcela","family":"Desio","sequence":"additional","affiliation":[{"name":"National Administration of Laboratories and Health Institutes \u201cDr. Carlos Malbr\u00e1n\u201d (ANLIS), National Institute of Biological Production (INPB), Department of Diagnostic Reagents, Mycobacterial Derivatives Service, Ciudad Aut\u00f3noma de Buenos Aires C1281AEF, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gerardo","family":"Rodr\u00edguez","sequence":"additional","affiliation":[{"name":"National Administration of Laboratories and Health Institutes \u201cDr. Carlos Malbr\u00e1n\u201d (ANLIS), National Institute of Biological Production (INPB), Department of Diagnostic Reagents, Mycobacterial Derivatives Service, Ciudad Aut\u00f3noma de Buenos Aires C1281AEF, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fernando","family":"Martino","sequence":"additional","affiliation":[{"name":"A.V.I.S. Veterinary Practice, Suardi, Santa Fe S2349, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bernardo","family":"Alonso","sequence":"additional","affiliation":[{"name":"National Food Safety and Quality Service (SENASA), General Directorate of Laboratory and Technical Control (DILAB-SENASA), Mart\u00ednez, Buenos Aires C1063ACD, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudio","family":"Paolazzi","sequence":"additional","affiliation":[{"name":"Agropharma Salud Animal S.A., Moreno, Buenos Aires B1744JFN, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"Arg\u00fcelles","sequence":"additional","affiliation":[{"name":"National Administration of Laboratories and Health Institutes \u201cDr. Carlos Malbr\u00e1n\u201d (ANLIS), National Institute of Biological Production (INPB), Department of Diagnostic Reagents, Mycobacterial Derivatives Service, Ciudad Aut\u00f3noma de Buenos Aires C1281AEF, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1509-3564","authenticated-orcid":false,"given":"Gustavo","family":"Helguera","sequence":"additional","affiliation":[{"name":"Pharmaceutical Biotechnology Laboratory, Institute of Biology and Experimental Medicine (IBYME-CONICET), Ciudad Aut\u00f3noma de Buenos Aires C1428BFA, Argentina"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e0053521","DOI":"10.1128\/mSphere.00535-21","article-title":"A Comprehensive Map of Mycobacterium tuberculosis Complex Regions of Difference","volume":"6","author":"Bespiatykh","year":"2021","journal-title":"mSphere"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2611","DOI":"10.1016\/j.vaccine.2012.02.018","article-title":"Bovine tuberculosis vaccine research: Historical perspectives and recent advances","volume":"30","author":"Waters","year":"2012","journal-title":"Vaccine"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/nature13529","article-title":"A dynamic model of bovine tuberculosis spread and control in Great Britain","volume":"511","author":"Roberts","year":"2014","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/S1473-3099(18)30013-6","article-title":"A roadmap for zoonotic tuberculosis: A One Health approach to ending tuberculosis","volume":"18","author":"Dean","year":"2018","journal-title":"Lancet Infect. 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