Structures of Brucella ovis leucine-, isoleucine-, valine-, threonine- and alanine-binding protein reveal a conformationally flexible peptide-binding cavity.

Brucella ovis is an etiologic agent of ovine epididymitis and brucellosis that causes global devastation in sheep, rams, goats, small ruminants and deer. There are no cost-effective methods for the worldwide eradication of ovine brucellosis. B. ovis and other protein targets from various Brucella species are currently in the pipeline for high-throughput structural analysis at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), with the aim of identifying new therapeutic targets. Furthermore, the wealth of structures generated are effective tools for teaching scientific communication, structural science and biochemistry. One of these structures, B. ovis leucine-, isoleucine-, valine-, threonine- and alanine-binding protein (BoLBP), is a putative periplasmic amino acid-binding protein. BoLBP shares less than 29% sequence identity with any other structure in the Protein Data Bank. The production, crystallization and high-resolution structures of BoLBP are reported. BoLBP is a prototypical bacterial periplasmic amino acid-binding protein with the characteristic Venus flytrap topology of two globular domains encapsulating a large central cavity containing the peptide-binding region. The central cavity contains small molecules usurped from the crystallization milieu. The reported structures reveal the conformational flexibility of the central cavity in the absence of bound peptides. The structural similarity to other LBPs can be exploited to accelerate drug repurposing.

Influence of the co-encapsulation of different excipients on the properties of polyester microparticle-based vaccine against brucellosis.

This work evaluates the influence of different pharmaceutical auxiliaries (Pluronic F68, polyvinylpyrrolidone [PVP] or Tween 20), when mixed with an antigenic extract from Brucella ovis (hot saline; HS), on the characteristics of the resulting poly(epsilon-caprolactone) (PEC) and poly(lactide-co-glycolide) (PLGA) microparticles. In all cases, PEC microparticles were smaller than PLGA ones. Concerning the HS loading, PLGA microparticles were highly dependent on the type of the excipient used, whereas all the PEC formulations displayed similar encapsulation efficiencies. For both types of microparticles, the presence of PVP induced a burst release effect. On the contrary, the use of Tween 20 or Pluronic F68 dramatically modified this profile. For PLGA-Tween 20 and PEC-Pluronic F68 microparticles, the HS was released in a pulsatil way during the first 7 days followed by a continuous release for at least 3 weeks. The antigenicity of the HS components was kept in all cases. Phagocytosis by murine monocytes showed a clear difference based just on the hydrophobicity of the polymer, being PEC microparticles better engulfed. Cell activation quantified by the release of H2O2 did not showed major differences between batches, however, microparticles of PEC and Pluronic F68 induced the highest nitric oxide production. Together, these results confirm the advantageous qualities of the "HS-PEC-Pluronic F68 microparticles" as favorable candidate for vaccine purposes against brucellosis.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams / Nested PCR espécie-específica para o diagnóstico da infecção por Brucella ovis em carneiros

The aim of the present study was to evaluate a species-specific nested PCR based on a previously described species-specific PCR for detection of B. ovis in semen and urine samples of experimentally infected rams. The performance of the species-specific nested PCR was compared with the results of a genus-specific PCR. Fourteen rams were experimentally infected with the Brucella ovis REO 198 strain and samples of semen and urine were collected every week up to 180 days post infection. Out of 83 semen samples collected, 42 (50.6%) were positive for the species-specific nested PCR, and 23 (27.7%) were positive for the genus-specific PCR. Out of 75 urine samples, 49 (65.3%) were positive for the species-specific nested PCR, whereas 11 (14.6%) were genus-specific PCR positive. Species-specific nested PCR was significantly more sensitive (P<0.001) than the genus-specific PCR in semen and urine from experimentally infected rams. In conclusion, the species-specific nested PCR developed in this study may be used as a diagnostic tool for the detection of B. ovis in semen and urine samples from suspected rams.

O presente estudo objetivou avaliar uma técnica de nested PCR espécie-específica delineada a partir de PCR espécie-específica descrita anteriormente para detecção de B. ovis em sêmen e urina de carneiros infectados experimentalmente. O desempenho da nested PCR espécie-específica foi comparado com os resultados de uma PCR gênero-específica. Quatorze carneiros foram infectados experimentalmente com Brucella ovis REO 198 e amostras de sêmen e de urina foram colhidas semanalmente até 180 dias após a infecção. De 83 amostras de sêmen, 42 (50,6%) foram positivas pela nested PCR espécie-específica, e 23 (27,7%) foram positivas pela PCR gênero-específica. De 75 amostras de urina, 49 (65,3%) foram positivas pela nested PCR espécie-específica, enquanto 11 (14,6%) foram positivas em PCR gênero-específica. A técnica de nested PCR espécie-específica foi significativamente mais sensível (P<0,001) do que a PCR gênero-específica no sêmen e na urina de carneiros infectados experimentalmente. Em conclusão, a nested PCR espécie-específica desenvolvida neste estudo pode ser utilizada como ferramenta de diagnóstico para detecção de B. ovis em sêmen e urina de carneiros suspeitos.

A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination.

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably due to its decameric arrangement and remarkable stability. In this work we decided to develop a chimera with the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31kDa (Omp31) from Brucella spp. Vaccination of BALB/c mice with the chimera as a recombinant protein (rBLSOmp31) provided the best protection level against Brucella ovis, which was higher than the given by the co-delivery of both recombinant proteins (rBLS + rOmp31) and similar than the control vaccine Brucella melitensis strain Rev.1. Moreover rBLSOmp31 induced protection against Brucella melitensis but to a lesser degree than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific T helper 1 and cytotoxic T responses. In conclusion, our results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.

Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization assay.

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.