The commonly used live attenuated vaccine in ovine brucellosis prophylaxis is Brucella melitensis Rev. 1. This vaccine is known to induce antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in challenged animals. Brucella BP26 and Omp31 proteins have shown an interesting potential as diagnostic antigens for ovine brucellosis. Accordingly, the bp26 gene and both bp26 and omp31 genes have been deleted from the vaccine strain Rev. 1. Immunogenicity and vaccine efficacy of the parental Rev. 1 strain and of both mutants in protecting sheep against B. inelitensis strain H38 challenge was evaluated by clinical and bacteriological examination of ewes. They were conjunctivally or subcutaneously vaccinated when 4 months old and then challenged with B. melitensis H38 at the middle of the first pregnancy following vaccination. Deletion of bp26 and omp31 genes did not significantly affect the well recognised capacity of Rev. 1 to protect sheep against B. melitensis challenge. However, the protection conferred by the CGV2631 mutant was significantly lower than that conferred by the CGV26 mutant or the Rev. I strain. Vaccinated and challenged animals were detected positive in classical serological tests and in the IFN-gamma assay. A BP26-based ELISA was investigated to discriminate between ewes vaccinated by the mutants and ewes challenged with B. inelitensis H38. The cut-off which was chosen in order to have 100% specificity resulted in a moderate sensitivy for the detection of challenged ewes. The use in the field of one of the mutants as vaccine against a B. inelitensis infection, combined with classic diagnostic tests and a BP26 ELISA, could thus give an improvement in the differentiation between vaccinated and infected animals and contribute to the objective of eradication of brucellosis in small ruminants. (c) 2006 Elsevier Ltd. All rights reserved.