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Live attenuated vaccines remain the most effective countermeasure, yet their application is limited by residual virulence and diagnostic interference. This study developed three rough-type attenuated Brucella melitensis mutants (G7, G8, G16) and evaluated their potential as DIVA (Differentiating Infected from Vaccinated Animals) vaccine candidates. Methods: Rough phenotypes were characterized through heat agglutination, acridine orange staining, and immunoblotting. Macrophage cytotoxicity was assessed via LDH release assays, while RT-qPCR analyzed macrophage activation capacity. Mouse infection and immunization-challenge experiments, complemented by histopathology, evaluated residual virulence and protective immunity. Antibody profiles were determined by ELISA, and DIVA capability was verified using LPS-coated ELISA. Results: G7 and G8 exhibited complete rough phenotypes, whereas G16 retained partial O-antigen (semi-rough). All rough mutants induced macrophage cytotoxicity and activation. The strains showed attenuated virulence with no viable bacteria recovered from spleens at 4 weeks post-inoculation. Histopathology revealed no liver lesions at 6 weeks post-inoculation. Immunized mice predominantly produced IgG2a-dominated Th1-type responses. The immune protection levels of G7 and G16 matched the reference vaccine M5\u201390\u039426, while G8 showed slightly lower efficacy. LPS-ELISA effectively differentiated vaccinated from infected animals via concurrent IgM\/IgG detection. Conclusions: This study demonstrates that the rough-type B. melitensis mutants G7 and G16 serve as promising DIVA vaccine candidates, offering strong protection with low residual virulence while enabling serological differentiation between vaccinated and infected animals, highlighting their potential as effective vaccines for brucellosis control.<\/jats:p>","DOI":"10.3390\/vaccines13080857","type":"journal-article","created":{"date-parts":[[2025,8,13]],"date-time":"2025-08-13T15:53:27Z","timestamp":1755100407000},"page":"857","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Biological Characterization and DIVA Potential of Three Rough Brucella melitensis Vaccine Strains"],"prefix":"10.3390","volume":"13","author":[{"given":"Jinyue","family":"Liu","sequence":"first","affiliation":[{"name":"Hebei Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao 066600, China"},{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Yin","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinmei","family":"Yang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengsi","family":"Li","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Qu","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaohui","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3259-1637","authenticated-orcid":false,"given":"Yanqing","family":"Bao","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingjing","family":"Qi","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4410-427X","authenticated-orcid":false,"given":"Tonglei","family":"Wu","sequence":"additional","affiliation":[{"name":"Hebei Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao 066600, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6794-2639","authenticated-orcid":false,"given":"Mingxing","family":"Tian","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/S1369-5274(00)00165-X","article-title":"Brucellosis: A worldwide zoonosis","volume":"4","author":"Boschiroli","year":"2001","journal-title":"Curr. 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