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Protection and diagnostic interference induced by heat-inactivated, phage-inactivated and live vaccine prototypes against animal tuberculosis Fernández Veiga, Leire Fuertes, Miguel Geijo, María V. Elguezabal, Natalia Serrano-Mestre, Jose L. Vázquez-Iniesta, Lucía Prados-Rosales, Rafael Michelet, Lorraine Boschiroli, Maria Laura Pérez de Val, Bernat Jones, Gareth J. Juste, Ramón A. Garrido, Joseba M. Sevilla, Iker A. Producció Animal Sanitat Animal 619 Introduction: Vaccination emerges as a promising cost-eective tool to reduce the impact and spread of animal tuberculosis, especially in regions where testand-slaughter eradication strategy is socioeconomically unfeasible or unfruitful for dierent reasons, provided it is safe, e cacious and compatible with diagnosis. Methods: In this study, we preliminarily evaluated the diagnostic interference (using guinea pigs) and the protective e cacy (using mice) of three heatinactivated, three phage-inactivated and one live attenuated vaccine prototypes prepared from M. bovis, M. caprae, and M. microti. Results and discussion: Phage-inactivation killed almost all (96.41–99.92%) bacteria to be included in vaccines and filtering was used to remove the remaining viable cells. All the assayed vaccines induced skin test reactions in response to bovine tuberculin, but they were smaller in the phage-inactivated vaccine groups. All the vaccines were diagnosis-compatible with defined skin test antigens based on ESAT-6, CFP-10, and Rv3615c. In contrast with the rest of prototypes, vaccination with heat- and phage-inactivated M. microti did not prompt the production of detectable anti-MPB70+MPB83 antibodies. Mean bacterial burden was lower in all vaccinated groups in comparison with the control, being significantly reduced in the lungs of the heat-inactivated M. microti and M. caprae and phage-inactivated M. caprae groups. Considering both diagnostic interference and protection collectively, the heat-inactivated M. microti vaccine showed the best performance. Further studies to evaluate these vaccines and to improve phage-driven inactivation are warranted. The author(s) declare that financial support was received for the research and/or publication of this article. This study has been carried out with funds from Grants PID2019-105155RB-C33 and PID2022-142939OR-C21 funded by MCIN/AEI/10.13039/501100011033 and by ERDF/EU, and Grant EFA115/01 INNOTUB II funded by the INTERREG POCTEFA 2021–2027 Program (co-funded by the European Union). LF-V holds a pre-doctoral fellowship from the Department of Economic Development, Sustainability and Environment of the Basque Government. RP-R thanks support from grants NIH RO1AI162821 and Spanish MICINN contracts PID2019-110240RB-I00 and PID2022-136611OB-I00. info:eu-repo/semantics/publishedVersion 2025-07-21 info:eu-repo/semantics/article Fernández-Veiga, Leire, Miguel Fuertes, María V Geijo, Natalia Elguezabal, Jose L Serrano-Mestre, Lucía Vázquez-Iniesta, Rafael Prados-Rosales, Bernat Pérez de Val, et al. 2025. “Protection and Diagnostic Interference Induced by Heat-inactivated, Phage-inactivated and Live Vaccine Prototypes Against Animal Tuberculosis.” Frontiers in Veterinary Science 12 (July). https://doi.org/10.3389/fvets.2025.1620497. 2297-1769 http://hdl.handle.net/20.500.12327/4808 https://doi.org/10.3389/fvets.2025.1620497 eng Frontiers in Veterinary Science MICINN/Programa Estatal de generación del conocimiento y fortalecimiento científico y tecnológico del sistema I+D+I/PID2019- 105155RB-C33/ES/DEVELOPMENT OF INACTIVATED VACCINE PROTOTYPES FOR THE CONTROL OF TB IN DOMESTIC ANIMALS AND ASSESSMENT OF THE INTEREFERENCE CAUSED BY NON TUBERCULOUS ORGANISMS ON ITS DIAGNOSI/ MICINN/Programa Estatal para Impulsar la Investigación Científico-Técnica y su Transferencia/PID2022-142939OR-C21/ES/DESARROLLO DE NUEVOS ABORDAJES DE VACUNACION MUCOSAL Y PARENTERAL FRENTE A LA TUBERCULOSIS ANIMAL Y EVALUACION DE SUS EFECTOS EN LOS MODELOS DE TUBERCULOSIS INTRANASAL DE RATO/ EC/INTERREG-POCTEFA/EFA115-01/EU/Red transpirenaica de investigación y desarrollo de herramientas innovadoras para el control de la tuberculosis animal/INNOTUB II FEDER/ / /EU/ / MICINN/Programa Estatal de generación del conocimiento y fortalecimiento científico y tecnológico del sistema I+D+I/PID2019-110240RB-I00/ES/IDENTIFICACION DE VULNERABILIDADES EN MYCOBACTERIUM TUBERCULOSIS A TRAVES DE LA DISECCION GENETICA Y QUIMICA DEL PROCESO DE VESICULACION/ MICINN/Programa Estatal para Impulsar la Investigación Científico-Técnica y su Transferencia/PID2022-136611OB-I00/ES/ IDENTIFICACION DE VULNERABILIDADES EN MYCOBACTERIUM TUBERCULOSIS A TRAVES DE LA DISECCION GENETICA Y QUIMICA DEL PROCESO DE VESICULACION/ Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess 14 Frontiers Media