dThis study aimed to evaluate the Brucella ovis ⌬abcBA strain as a vaccine candidate in the murine model. BALB/c mice were subcutaneously or intraperitoneally immunized with a single dose or three doses of the B. ovis ⌬abcBA strain and then were challenged with wild-type B. ovis. Single or multiple immunizations provided only mild protection, with significantly smaller numbers of wild-type B. ovis CFU in the livers of immunized mice but not in the spleens. Encapsulation of B. ovis ⌬abcBA significantly improved protection against experimental challenges in both BALB/c and C57BL/6 mice. Furthermore, immunization with encapsulated B. ovis ⌬abcBA markedly prevented lesions in the spleens and livers of experimentally challenged mice. These results demonstrated that the encapsulated B. ovis ⌬abcBA strain confers protection to mice; therefore, this strain has potential as a vaccine candidate for rams. Brucella ovis is a facultative, intracellular, Gram-negative coccobacillus (1) that causes economic losses due to its potential to induce reproductive failure in sheep (2, 3). The most important lesions caused by B. ovis in rams are epididymitis and seminal vesiculitis (4), and B. ovis occasionally causes abortion in ewes (5, 6).In spite of attempts to develop safe, effective vaccines (7-10), there is no available vaccine specifically against B. ovis. The ideal vaccine against B. ovis must prevent infection and clinical signs or at least reduce the risk of infection and transmission (11). Rev-1, a live attenuated Brucella melitensis strain, is the most commonly used vaccine against B. melitensis, and it also protects against B. ovis (12). However, the Rev-1 strain retains pathogenic potential, so it is capable of infecting humans. It may cause abortions in ruminants, it may interfere with serological tests, and it is resistant to streptomycin (13,14).Mice have been extensively used as a model for brucellosis, which is very important due to the limitations of experimentation with larger animals (15-18). The murine model has allowed studies for the development of safe, effective brucellosis vaccines (15,19,20).Inactivation of a predicted abcEDCBA-encoded ATP-binding cassette (ABC) transporter of B. ovis (21), which is located in B. ovis pathogenicity island 1 (BOPI-1), results in strong attenuation of this pathogen in the murine model (22). Although the substrate of this ABC transporter is not known, it is required for expression of B. ovis virulence proteins, including Omp31, Sod, and the virB-encoded type IV secretion system (23), which is required for B. ovis intracellular survival and in vivo persistence (17). Furthermore, a B. ovis ⌬abcBA strain is capable of triggering humoral and cellular immune responses like the wild-type strain in rams; importantly, however, the mutant strain is not excreted in semen and urine (24). Therefore, this mutant strain has great potential as an attenuated vaccine strain. However, considering that the ⌬abcBA mutant strain is attenuated at very early stages during infection, resulting in p...

This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams.

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Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.

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