First study of Brucella ovis antibodies in purebred sheep flocks in the State of Parana, Brazil / [Primeiro estudo de anticorpos para Brucella ovis em rebanhos de ovinos puros de Origem, no estado do Paraná, Brasil]

Brucella ovis, a non-zoonotic species, is the etiological agent of ovine brucellosis, an infectious disease of clinical or subclinical occurrence in sheep flocks. Until then, there is no serological study of anti-Brucella ovis antibodies in purebred sheep herds. This study aimed to determine the presence of anti-Brucella ovis antibodies in purebred sheep flocks with breeding purposes from Parana State. Blood samples from 728 animals, of which 563 were females and 165 males, between 8 and 56 months of age from the six major sheep producing mesoregions of Parana, were submitted to detection of anti-Brucella ovis antibodies by the Agar Gel Immunodiffusion technique using an antigen from the bacteria Brucella ovis (Reo 198). The results indicate the presence of this disease in purebred sheep from Parana State in a low occurrence of 0.27% (2/728). The only two positive animals were rams, Santa Inês breed, from the same flock in the East Center region of Parana, without clinical disease. In conclusion, Brucella ovis is present in purebred sheep in Parana State, Brazil, and this low occurrence may have occurred due to rigorous breeding systems that may contribute to reduce the transmission of this disease.(AU)

Brucella ovis, espécie não zoonótica, é o agente etiológico da brucelose ovina, doença infecciosa de ocorrência clínica ou subclínica. Atualmente, não existe estudo sorológico de anticorpos anti-Brucella ovis em rebanhos de ovinos puros de origem. Este estudo teve como objetivo determinar a presença de anticorpos anti-Brucella ovis em rebanhos ovinos de raça pura de origem, com fins reprodutivos do estado do Paraná. Amostras de sangue de 728 animais, sendo 563 fêmeas e 165 machos, entre oito e 56 meses de idade, pertencentes a seis principais mesorregiões produtoras de ovinos no Paraná, foram submetidas à detecção de anticorpos anti-Brucella ovis pela técnica de imunodifusão em ágar gel usando-se um antígeno da bactéria Brucella ovis (Reo 198). Os resultados indicam a presença da doença em ovinos puros de origem do estado do Paraná em baixa ocorrência de 0,27% (2/728). Os dois únicos animais positivos foram reprodutores da raça Santa Inês, do mesmo rebanho da região Centro Leste do Paraná, sem manifestação clínica. Em conclusão, Brucella ovis está presente em ovinos puros de origem no estado do Paraná, e essa baixa ocorrência pode ter ocorrido devido a sistemas rigorosos de criação, que podem contribuir para a redução da transmissão dessa doença.(AU)

Brucella ovis mutant in ABC transporter protects against Brucella canis infection in mice and it is safe for dogs.

BACKGROUND/OBJECTIVES: Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. METHODS: Intracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs. RESULTS: Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. CONCLUSION: Encapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.

Pathogenic potential of Brucella ovis field isolates with different genotypic profile and protection provided by the vaccine strain B. ovis ΔabcBA against B. ovis field isolates in mice / Vacina viva atenuada Brucella ovis Δ abcBA encapsulada protege camundongos frente a desafios de B. ovis isoladas de campo

Brucella ovis causes economic and reproductive losses in sheep herds. The goal of this study was to characterize infection with B. ovis field isolates in a murine model, and to evaluate protection induced by the candidate vaccine strain B. ovis ΔabcBA in mice challenged with these field isolates. B. ovis field strains were able to colonize and cause lesions in the liver and spleen of infected mice. After an initial screening, two strains were selected for further characterization (B. ovis 94 AV and B. ovis 266 L). Both strains had in vitro growth kinetics that was similar to that of the reference strain B. ovis ATCC 25840. Vaccination with B. ovis ΔabcBA encapsulated with 1% alginate was protective against the challenge with field strains, with the following protection indexes: 0.751, 1.736, and 2.746, for mice challenged with B. ovis ATCC25840, B. ovis 94 AV, and B. ovis 266 L, respectively. In conclusion, these results demonstrated that B. ovis field strains were capable of infecting and inducing lesions in experimentally infected mice. The attenuated vaccine strain B. ovis ΔabcBA induced protection in mice challenged with different B. ovis field isolates, resulting in higher protection indexes against more pathogenic strains.(AU)

Brucella ovis é responsável por perdas econômicas e reprodutivas em rebanhos ovinos. O objetivo deste trabalho foi caracterizar a infecção com as cepas isoladas de campo de B. ovis em modelo murino e avaliar a eficiência vacinal da mutante B. ovis ΔabcAB para proteção contra desafio com as cepas isoladas de campo. Foram utilizadas sete cepas isoladas de campo foram capazes de colonizar e provocar lesões no fígado e no baço de camundongos após sete dias pós-infecção. Após triagem, duas cepas foram selecionadas para a melhor caracterização (B. ovis 94 AV and B. ovis 266L). Ambas apresentaram crescimento em placa de cultivo semelhante ao da cepa de referência B. ovis ATCC 25840. A vacinação com a cepa de Brucella ovis ΔabcBA encapsulada com alginato a 1% foi capaz de proteger camundongos desafiados com as cepas isoladas de campo, com os seguintes índices de proteção: 0,751, 1,736 e 2,746, para camundongos desafiados com B. ovis ATCC 25840, B. ovis 94 AV e B. ovis 266 L, respectivamente. Estes resultados demonstraram que as cepas isoladas de campo de B. ovis são capazes de infectar e induzir lesão em camundongos experimentalmente infectados. O uso da cepa mutante atenuada B. ovis ΔabcBA para vacinação de fêmeas C57BL/6 desafiados com diferentes cepas de B. ovis induziu proteção nos camundongos desafiados com diferentes cepas de B. ovis. Deste modo, mostrando-se eficiente na proteção das cepas de campo de B. ovis.(AU)

Prevalência de anticorpos anti-Brucella ovis e anti-Brucella abortus em ovinos do município de Colinas, Tocantins, Brasil / Prevalence of anti-Brucella ovis and anti-Brucella abortus in sheep in the city of Colinas, Tocantins, Brazil

A brucelose é uma enfermidade infecciosa, causada por bactérias do gênero Brucella spp. responsáveis por desordens reprodutivas nos animais, especialmente nos ruminantes. Este trabalho objetivou determinar a presença de anticorpos anti-Brucella ovis e anti-Brucella abortus em ovinosde 14 propriedades cadastradas na Associação de Ovinocultores do município de Colinas, Tocantins,Brasil. Para isso, amostras de soro de 450 ovinos foram analisadas por meio da aglutinação rápida em placa com Antígeno Acidificado e Tamponado (AAT) e, quando reagentes, foram realizados os testes de Aglutinação Lenta em Tubos (SAT) e 2-Mercaptoetanol (2-ME) para pesquisar cepas lisas (B. abortus). Para a pesquisa de cepas rugosas (B. ovis), foi realizada a Imunodifusão em Gel de Ágar (IDGA) e, quando reagente, foi realizada a Fixação de Complemento (FC). Das amostras analisadas,142 (31,6por cento) reagiram ao teste de IDGA, dentre estas, apenas 4 (2,8por cento) foram confirmadas na FC.Ante o AAT, apenas 20 (4,4por cento) se mostraram positivas, das quais 14 (70por cento) foram confirmadas noSAT/2-ME. Os resultados obtidos nos testes de FC e SAT/2-ME e analisados pelo Teste de Fisher e OR demonstraram significância estatística entre a positividade e a faixa etária, sendo maior a chance de um animal em reprodução ser positivo para brucelose.

Respuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación / Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy

La brucelosis ovina por Brucella ovis es una enfermedad de prevalencia alta en Argentina. Para evaluar la patogenicidad de B. ovis y la respuesta serológica durante el último mes de gestación, 6 ovejas se distribuyeron en dos grupos: G1, ovejas preñadas, n = 4 y G2, ovejas no preñadas, n = 2. Tres ovejas del G1 (15 días preparto) y una del G2 fueron inoculadas con B. ovis. Se analizaron muestras de suero mediante diferentes pruebas serológicas. Se realizó aislamiento y PCR a partir de mucus cérvico-vaginal (mcv), placenta y leche. En las muestras de placenta se realizó histopatología. Las hembras del G1 parieron corderos vivos; se detectaron anticuerpos en las ovejas desafiadas del G1 a partir de los 5 días posinoculación. El mcv de las ovejas desafiadas resultó negativo al aislamiento en ambos grupos. Las muestras de leche del G1 fueron positivas por cultivo y PCR a B. ovis. La técnica de PCR resultó positiva en las placentas de las ovejas desafiadas del G1. La histopatología reveló una placentitis necrótica supurativa en una de las ovejas desafiadas. El desafío con B. ovis preparto resultó en la invasión de la placenta y de la glándula mamaria, con la consecuente excreción de la bacteria por leche. La infección con B. ovis indujo una respuesta humoral temprana en las ovejas. La colonización de la placenta por B. ovis y la excreción de la bacteria por la leche sugieren un potencial riesgo de infección activa para los corderos y la posibilidad de que estos se comporten como portadores latentes de la infección.

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsGI (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. G1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. Sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams.

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1ß, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30 dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1ß and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1ß and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240 dpi), cytokine production was down-regulated in the epididymus (IL-1ß and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1ß and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation.

Brucella melitensis, B. neotomae and B. ovis elicit common and distinctive macrophage defense transcriptional responses.

Brucella spp. establish an intracellular replicative niche in macrophages, while macrophages attempt to eliminate the bacteria by innate defense mechanisms. Brucella spp. possess similar genomes yet exhibit different macrophage infections. Few B. melitensis and B. neotomae enter macrophages with intracellular adaptation occurring over 4-8 hr. Conversely, B. ovis are readily ingested by macrophages and exhibit a persistent plateau of infection. Evaluating early macrophage interaction with Brucella spp. allows discovery of host entry and intracellular translocation mechanisms. Microarray analysis of macrophage transcriptional response following a 4 hr infection by different Brucella spp. revealed common macrophage genes altered in expression compared to uninfected macrophages. Macrophage infection with three different Brucella spp. provokes a common innate immune theme with increased transcript levels of chemokines and defense response genes and decreased transcript levels of GTPase signaling and cytoskeletal function that may affect trafficking of Brucella containing vesicles. For example, transcript levels of genes associated with chemotaxis (IL-1beta, MIP-1alpha), cytokine regulation (Socs3) and defense (Fas, Tnf) were increased, while transcript levels of genes associated with vesicular trafficking (Rab3d) and lysosomal associated enzymes (prosaposin) were decreased. Genes with altered macrophage transcript levels among Brucella spp. infections may correlate with species specific host defenses and intracellular survival strategies. Depending on the infecting Brucella species, gene ontology categorization identified genes differentially involved in cell growth and maintenance, endopeptidase inhibitor activity and G-protein mediated signaling. Examples of decreased gene expression in B. melitensis infection but not other Brucella spp. were growth arrest (Gas2), immunoglobulin receptor (FcgammarI) and chemokine receptor (Cxcr4) genes, suggesting opposing effects on intracellular functions.

Design and influence of gamma-irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic complex from Brucella ovis.

Despite vaccination campaigns, brucellosis is still one of the most common bacterial zoonosis in the world. This work describes the development of a novel formulation strategy to the delivery of the Brucella ovis antigenic extract (HS) into ovine mucosal surfaces. Thus, HS was entrapped in conventional and mannosylated poly(anhydride) nanoparticles by the solvent displacement method, and the resulting nanosystems were gamma-irradiated to accomplish the sterilization required for the ophthalmic administration route. Sterilization, at either 10 kGy or 25 kGy, did not modify the size, morphology and antigen content of the nanoparticles. Similarly, the integrity and antigenicity of the entrapped antigen were not affected by gamma-irradiation. The 25 kGy gamma-irradiation dose seemed to influence negatively the HS release from the carriers. However, and in accordance with the Pearson's correlation, all the release patterns followed a similar tendency. Furthermore, the stability of the vaccine systems on lachrymal and nasal ovine fluids, showed that gamma-irradiation had no significant effects on the vaccine systems. Since all the vaccine systems accomplished the pharmacopoeial biological tests required for gamma-irradiation doses under 25 kGy, these results are highly suggestive for the use of HS loaded poly(anhydride) nanoparticles as an efficient vaccine delivery system for brucellosis immunoprophylaxis, especially for ophthalmic administration.

Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection.

The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis.

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.