Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.Brucellosis is a major zoonotic disease, widely distributed in humans and domestic and wild animals, especially in developing countries. Among the different species of the Brucella genus, B. abortus and B. melitensis are the most pathogenic and virulent, not only for cattle or for sheep and goats, respectively, but also for other animal species. The occurrence of the disease in humans is largely dependent on the animal reservoir, with the highest rate of human infection in areas where rates of brucellosis in sheep and goats are high (6,35).Brucellosis vaccines are essential elements in control programs. Attenuated B. abortus strain 19 and B. melitensis strain Rev1 are proven effective vaccines; they induce good levels of protection against B. abortus in cattle and against B. melitensis in sheep and goats, preventing premature abortions (7, 30, 31). However, both vaccines have the drawback of inducing Opolysaccharide-specific antibodies that interfere with the discrimination between vaccinated and infected animals during serological screening (7, 31). In addition, they retain pathogenicity and sometimes cause abortion in vaccinated animals (10, 18, 41) and remain infectious for humans (3,5,26). The use of the conjunctival route when administering B. melit...

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Brucellosis is essentially a disease of domesticated livestock; however, humans can also be infected via the consumption of contaminated meat or dairy products, underlying the need for rapid and accurate identification methods. Procedures for microbiological identification and typing of Brucella spp. are expensive, time-consuming, and must be conducted in biohazard containment facilities to minimize operator risk. The development of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based assay has reduced the processing time while maintaining performance standards. In this study, to improve the identification accuracy and suitability of the MALDI-TOF-based assay for routine diagnosis, we developed a new protein extraction protocol and generated a custom reference database containing Brucella strains representative of the most widespread species. The reference library was then challenged with blind-coded field samples isolated from infected animals. The results indicated that the database could be used to correctly identify 99.5% and 97% of Brucella strains at the genus and species level, respectively, indicating that the performance of the assay was not affected by the different culture conditions used for microbial isolation. Moreover, the inactivated samples were stored and shipped to reference laboratories with no ill effect on protein stability, thus confirming the reliability of our method for routine diagnosis. Finally, we evaluated the epidemiological value of the protocol by comparing the clustering analysis results of Brucella melitensis strains obtained via multiple locus variable-number tandem repeat analysis or MALDI-TOF MS. The results showed that the MALDI-TOF assay could not decipher the true phylogenetic tree, suggesting that the protein profile did not correspond with the genetic evolution of Brucella.

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B.abortus RB51, B.melitensis B115 and B.melitensis B18. RB51 derived from B.abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory.The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B.melitensis 16M strain.The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain.Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.

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