Antibody prevalence of Leptospira spp. and Brucella abortus in domestic cats from Araguaína, Tocantins, North Region of Brazil / Prevalência de anticorpos para Leptospira spp. e Brucella Abortus em gatos domésticos de Araguaína, Tocantins, Região Norte do Brasil

Leptospira spp. and Brucella abortus are bacterial pathogens that can infect humans and animals. The present study aimed to detect anti-Leptospira and anti-B. abortus antibodies and verified the presence of factors associated with seropositivity in cats. One hundred and eighty serum samples were collected from domestic cats (Felis silvestris catus) from the urban area of the municipality of Araguaína-Tocantins by phlebocentesis of the cephalic and jugular veins. The samples were subjected to detection of anti-Leptospira and anti-B.abortus antibodies, respectively, by microscopic seroagglutination and buffered acidified antigen testing, followed by confirmation by the 2-mercaptoethanol test and slow seroagglutination in tubes. Data from the epidemiological questionnaire (the age, sex, origin, breed, and presence of clinical signs) were analyzed using Epi Info® software with seropositivity data found to search for associated factors using the chi-square test. In the present study, the prevalence of Leptospira spp. was 5.56% (10/180). However, no sample was reactive to B. abortus. None of the studied variables were associated with seropositivity for the pathogens evaluated. Therefore, there is contact between Leptospira spp. and the feline population of the municipality, indicating the possibility of the circulation of pathogenic serovars and that the presence of anti-Leptospira antibodies does not depend on the variables analyzed.(AU)

Leptospira spp. e Brucella abortus são patógenos bacterianos que podem infectar humanos e animais. O presente estudo teve como objetivo detectar anticorpos anti-Leptospira e anti-B.abortus e verificar a presença de fatores associados com a soropositividade em gatos. Foram coletadas 180 amostras de soro de gatos domésticos (Felis silvestris catus) da zona urbana do município de Araguaína-Tocantins por flebocentese das veias cefálica e jugular. As amostras foram submetidas à detecção de anticorpos anti-Leptospira e anti-B. abortus, respectivamente, por soroaglutinação microscópica e teste do antígeno acidificado tamponado, seguido de confirmação pelo teste de 2-mercaptoetanol e soroaglutinação lenta em tubos. Os dados do questionário epidemiológico (idade, sexo, procedência, raça e presença de sinais clínicos) foram analisados no software Epi Info® com os dados de soropositividade encontrados para pesquisa de fatores associados pelo teste do qui-quadrado. No presente estudo, a prevalência de Leptospira spp. foi de 5,56% (10/180). No entanto, nenhuma amostra foi reativa para B. abortus. Nenhuma das variáveis estudadas foi associada com a soropositividade para os patógenos avaliados. Portanto, há contato entre Leptospiraspp. e a população felina do município, indicando a possibilidade de circulação de sorovares patogênicos e que a presença de anticorpos anti-Leptospira independe das variáveis analisadas.(AU)

STING regulates metabolic reprogramming in macrophages via HIF-1α during Brucella infection.

Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Epitope-Based Vaccine of a Brucella abortus Putative Small RNA Target Induces Protection and Less Tissue Damage in Mice.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and animals. Currently available live attenuated vaccines against brucellosis still have drawbacks. Therefore, subunit vaccines, produced using epitope-based antigens, have the advantage of being safe, cost-effective and efficacious. Here, we identified B. abortus small RNAs expressed during early infection with bone marrow-derived macrophages (BMDMs) and an apolipoprotein N-acyltransferase (Int) was identified as the putative target of the greatest expressed small RNA. Decreased expression of Int was observed during BMDM infection and the protein sequence was evaluated to rationally select a putative immunogenic epitope by immunoinformatic, which was explored as a vaccinal candidate. C57BL/6 mice were immunized and challenged with B. abortus, showing lower recovery in the number of viable bacteria in the liver, spleen, and axillary lymph node and greater production of IgG and fractions when compared to non-vaccinated mice. The vaccinated and infected mice showed the increased expression of TNF-α, IFN-γ, and IL-6 following expression of the anti-inflammatory genes IL-10 and TGF-ß in the liver, justifying the reduction in the number and size of the observed granulomas. BMDMs stimulated with splenocyte supernatants from vaccinated and infected mice increase the CD86+ marker, as well as expressing greater amounts of iNOS and the consequent increase in NO production, suggesting an increase in the phagocytic and microbicidal capacity of these cells to eliminate the bacteria.

Factors associated with the prevalence of antibodies against Brucella abortus in water buffaloes from Santarém, Lower Amazon region, Brazil.

We evaluated the factors associated with the prevalence of antibodies against Brucella abortus in buffaloes in the municipality of Santarém, Western Pará, northern Brazil. The study was conducted on 60 farms, representing 25.8% of the total buffalo farms in the region. From those farms, a total of 426 buffaloes were sampled, males of any age and females more than 24 months of age, to avoid a false-positive reaction in the serological test due to vaccination. The Acidified Agglutination Serum Test was carried out on serum samples using B. abortus strain 1,119-3 as the antigen. Univariate and multivariate analysis were performed to investigate the association between brucellosis and potential risk factors. Of the 426 tested buffaloes, 29 were positive, resulting in an overall animal prevalence of antibodies against B. abortus at the animal level of 6.8% (4.6-9.6; 95% confidence interval). The herd level prevalence was 30% (18 of 60) and seroprevalence range within farms was from 0% to 100%. At the animal level, buffaloes raised in the floodplains tended (p = 0.06) to present a higher seroprevalence (9.70%) of antibodies against B. abortus than buffaloes raised in dry land (4.98%) and cows tended (p = 0.054) to have a higher seroprevalence than male buffaloes. Multivariate herd-level analysis revealed association between farm type and brucellosis seroprevalence (p = 0.015); dairy farms were two times more likely to have seropositive buffalo than beef farms. Our survey demonstrated a high farm seroprevalence of B. abortus in buffalo raised in an Amazonian ecosystem with positive animals found in one third of sampled farms.

Bacterial RNA Contributes to the Down-Modulation of MHC-II Expression on Monocytes/Macrophages Diminishing CD4+ T Cell Responses.

Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.

Omp19 Enables Brucella abortus to Evade the Antimicrobial Activity From Host's Proteolytic Defense System.

Pathogenic microorganisms confront several proteolytic events in the molecular interplay with their host, highlighting that proteolysis and its regulation play an important role during infection. Microbial inhibitors, along with their target endogenous/exogenous enzymes, may directly affect the host's defense mechanisms and promote infection. Omp19 is a Brucella spp. conserved lipoprotein anchored by the lipid portion in the Brucella outer membrane. Previous work demonstrated that purified unlipidated Omp19 (U-Omp19) has protease inhibitor activity against gastrointestinal and lysosomal proteases. In this work, we found that a Brucella omp19 deletion mutant is highly attenuated in mice when infecting by the oral route. This attenuation can be explained by bacterial increased susceptibility to host proteases met by the bacteria during establishment of infection. Omp19 deletion mutant has a cell division defect when exposed to pancreatic proteases that is linked to cell-cycle arrest in G1-phase, Omp25 degradation on the cell envelope and CtrA accumulation. Moreover, Omp19 deletion mutant is more susceptible to killing by macrophage derived microsomes than wt strain. Preincubation with gastrointestinal proteases led to an increased susceptibility of Omp19 deletion mutant to macrophage intracellular killing. Thus, in this work, we describe for the first time a physiological function of B. abortus Omp19. This activity enables Brucella to better thrive in the harsh gastrointestinal tract, where protection from proteolytic degradation can be a matter of life or death, and afterwards invade the host and bypass intracellular proteases to establish the chronic infection.

Recombinant Omp2b antigen-based ELISA is an efficient tool for specific serodiagnosis of animal brucellosis.

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gram-negative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni2+-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.

Serology for Neosporosis, Q fever and Brucellosis to assess the cause of abortion in two dairy cattle herds in Ecuador.

BACKGROUND: Determining the infectious cause of abortion in cattle is difficult. This case-control study was set up to investigate the infectious causes of abortion by determining the seroprevalence of three reproductive pathogens in dairy cattle in Ecuador and their association with abortion: Brucella abortus, Neospora caninum and Coxiella burnetii. RESULTS: Ninety-five blood samples were obtained from cows that had experienced a mid- or late gestation abortion of their first calf and seventy-seven samples from a control group of cows with the same age that did not experience abortion problems. No antibodies were detected for B. abortus in any of the serum samples, but a high seroprevalence for both C. burnetii (52.9%) and N. caninum infection (21.5%) was found in group of cows. The seroprevalence of N. caninum infection in cattle that had experienced abortions was significantly higher (p < 0.05) than the seroprevalence in the control cows on one of the cattle farms, but no association between abortion and seropositivity for C. burnetii was found. CONCLUSION: We conclude that Neosporosis plays an important role in the epidemiology of abortion on one cattle farm, but that Q fever is apparently not an important cause for abortion in this setting.

Neutrophils as Trojan Horse Vehicles for Brucella abortus Macrophage Infection.

Brucella abortus is a stealthy intracellular bacterial pathogen of animals and humans. This bacterium promotes the premature cell death of neutrophils (PMN) and resists the killing action of these leukocytes. B. abortus-infected PMNs presented phosphatidylserine (PS) as "eat me" signal on the cell surface. This signal promoted direct contacts between PMNs and macrophages (Mϕs) and favored the phagocytosis of the infected dying PMNs. Once inside Mϕs, B. abortus replicated within Mϕs at significantly higher numbers than when Mϕs were infected with bacteria alone. The high levels of the regulatory IL-10 and the lower levels of proinflammatory TNF-α released by the B. abortus-PMN infected Mϕs, at the initial stages of the infection, suggested a non-phlogistic phagocytosis mechanism. Thereafter, the levels of proinflammatory cytokines increased in the B. abortus-PMN-infected Mϕs. Still, the efficient bacterial replication proceeded, regardless of the cytokine levels and Mϕ type. Blockage of PS with Annexin V on the surface of B. abortus-infected PMNs hindered their contact with Mϕs and hampered the association, internalization, and replication of B. abortus within these cells. We propose that B. abortus infected PMNs serve as "Trojan horse" vehicles for the efficient dispersion and replication of the bacterium within the host.

Bayesian Evaluation of Three Serological Tests for Detecting Antibodies against Brucella spp. among Humans in the Northwestern Part of Ecuador.

Brucellosis is an important but neglected zoonosis that causes serious economic losses both in livestock and human populations. The aim of the present study was to estimate the true prevalence of brucellosis together with diagnostic sensitivity and specificity of three serological tests in humans of the northwestern part of Ecuador using a Bayesian approach adjusted for the dependencies among the multiple tests to avoid any misinterpretation. In addition, the causal agent responsible for human brucellosis was also identified. Using a total of 3,733 samples collected from humans in this area between 2006 and 2008, the prevalence of human brucellosis and the diagnostic test characteristics of the Rose Bengal fast agglutination test (RBT), Wright's slow agglutination test with ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) (SAT-EDTA), and indirect ELISA (iELISA) were estimated using a Bayesian approach. The estimated true prevalence of human brucellosis was 1% (credibility interval: 0.4-1.6). The sensitivities of iELISA and RBT were higher than and similar (95.1% and 95.0%, respectively) to those of SAT-EDTA (60.8%). Even though all tests indicated a high specificity (> 99.0%), the specificity of SAT-EDTA was highest (99.9%). The circulating strain in this study area was identified to be Brucella abortus biotype 4 based on culture and microbiological characterization. The RBT and the iELISA are recommended for estimating the true prevalence of human brucellosis and/or for surveillance programs following their high sensitivities and specificities. The proposed strategy supports evidence-based medicine for clinicians and policy-makers to ensure appropriate preventive and control program of brucellosis worldwide.

Inmunopatología de la brucelosis osteoarticular / Immunopathology of osteoarticular brucelosis

La brucelosis es una de las enfermedades zoonóticas más importantes a nivel mundial capaz de producir enfermedad crónica en los seres humanos. La localización osteoarticular es la presentación más común de la enfermedad activa en el hombre. Sin embargo, algunos de los mecanismos moleculares implicados en la enfermedad osteoarticular han comenzado a dilucidarse recientemente. Brucella abortus induce daño óseo a través de diversos mecanismos en los cuales están implicados TNF-α y RANKL. En estos procesos participan células inflamatorias que incluyen monocitos/macrófagos, neutrófilos, linfocitos T del tipo Th17 y linfocitos B. Además, B. abortus puede afectar directamente las células osteoarticulares. La bacteria inhibe la deposición de la matriz ósea por los osteoblastos y modifica el fenotipo de estas células para producir metaloproteinasas de matriz (MMPs) y la secreción de citoquinas que contribuyen a la degradación del hueso. Por otro lado, la infección por B. abortus induce un aumento en la osteoclastogénesis, lo que aumenta la resorción de la matriz ósea orgánica y mineral y contribuye al daño óseo. Dado que la patología inducida por Brucella afecta el tejido articular, se estudió el efecto de la infección sobre los sinoviocitos. Estos estudios revelaron que, además de inducir la activación de estas células para secretar quemoquinas, citoquinas proinflamatorias y MMPs, la infección inhibe la muerte por apoptosis de los sinoviocitos. Brucella es una bacteria intracelular que se replica en el retículo endoplásmico de los macrófagos. El análisis de los sinoviocitos infectados con B. abortus indicó que las bacterias también se multiplican en el retículo endoplasmático, lo que sugiere que la bacteria podría usar este tipo celular para la multiplicación intracelular durante la localización osteoarticular de la enfermedad. Los hallazgos presentados en esta revisión intentan responder a preguntas sobre los mediadores inflamatorios implicados en el daño osteoarticular causado por Brucella. (AU)

Brucellosis is one of the most important zoonotic diseases that can produce chronic disease in humans worldwide. Osteoarticular involvement is the most common presentation of human active disease. The molecular mechanisms implicated in bone damage have started to be elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and RANKL are implicated. These processes are driven by inflammatory cells, including monocytes/macrophages, neutrophils, Th17 lymphocytes and B cells. Also, Brucella abortus (B. abortus) can directly affect osteoarticular cells. The bacterium inhibits bone matrix deposition by osteoblast and modifies the phenotype of these cells to produce matrix methalloproteinases (MMPs) and cytokine secretion that contribute to bone matrix degradation. B. abortus also affects osteoclast increasing mineral and organic bone matrix resorption and contributing to bone damage. Since the pathology induced by Brucella species involves joint tissue, experiments conducted in sinoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits sinoviocyte apoptosis. Brucella is an intracellular bacterium that replicate in the endoplasmic reticulum of macrophages. The analysis of B. abortus infected sinoviocytes indicated that bacteria also replicate in their reticulum suggesting that the bacterium could use this cell type for intracellular replication during the osteoarticular localization of the disease. The findings presented in this review try to answer key questions about the inflammatory mediators involved in osteoarticular damage caused by Brucella. (AU)

Neutrophils Dampen Adaptive Immunity in Brucellosis.

Brucella organisms are intracellular stealth pathogens of animals and humans. The bacteria overcome the assault of innate immunity at early stages of an infection. Removal of polymorphonuclear neutrophils (PMNs) at the onset of adaptive immunity against Brucella abortus favored bacterial elimination in mice. This was associated with higher levels of interferon gamma (IFN-γ) and a higher proportion of cells expressing interleukin 6 (IL-6) and inducible nitric oxide synthase (iNOS), compatible with M1 macrophages, in PMN-depleted B. abortus-infected (PMNd-Br) mice. At later times in the acute infection phase, the amounts of IFN-γ fell while IL-6, IL-10, and IL-12 became the predominant cytokines in PMNd-Br mice. IL-4, IL-1ß, and tumor necrosis factor alpha (TNF-α) remained at background levels at all times of the infection. Depletion of PMNs at the acute stages of infection promoted the premature resolution of spleen inflammation. The efficient removal of bacteria in the PMNd-Br mice was not due to an increase of antibodies, since the immunoglobulin isotype responses to Brucella antigens were dampened. Anti-Brucella antibodies abrogated the production of IL-6, IL-10, and IL-12 but did not affect the levels of IFN-γ at later stages of infection in PMNd-Br mice. These results demonstrate that PMNs have an active role in modulating the course of B. abortus infection after the adaptive immune response has already developed.

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG.

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen-binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti-Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA-IgG anti Brucella biosensor was three-fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 106 CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody-coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and confocal microscopy.

AIM2 senses Brucella abortus DNA in dendritic cells to induce IL-1ß secretion, pyroptosis and resistance to bacterial infection in mice.

Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis, Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1ß as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1ß production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection.

IL-1R and Inflammasomes Mediate Early Pulmonary Protective Mechanisms in Respiratory Brucella Abortus Infection.

Brucella spp. infection is frequently acquired through contaminated aerosols. The role of interleukin-1 beta (IL-1ß) in the early pulmonary response to respiratory Brucella infection is unknown. As shown here, IL-1ß levels in lung homogenates and bronchoalveolar lavage fluid (BALF) of mice intratracheally inoculated with B. abortus were increased at 3 and 7 days p.i. At 7 days p.i., pulmonary CFU numbers were higher in IL-1 receptor (IL-1R) knockout (KO) mice than in wild type (WT) mice. At different times p.i. CFU in lungs and BALF were higher in mice lacking some inflammasome components (caspase-1, AIM2, NLRP3) than in WT mice. At 2 days p.i. pulmonary levels of IL-1ß and CXCL1 (neutrophils chemoattractant) were lower in caspase-1/11 KO mice. At day 3 p.i., neutrophils counts in BALF were lower in caspase-1/11 KO mice than in WT mice. During in vitro infections, IL-1ß secretion was lower in alveolar macrophages from caspase-1/11, NLRP3 or AIM2 KO mice than in WT controls. Similarly, IL-1ß production by B. abortus-infected alveolar epithelial cells was reduced by pretreatment with a specific caspase-1 inhibitor. This study shows that IL-1R, probably through IL-1ß action, and the NLRP3 and AIM2 inflammasomes are involved in pulmonary innate immune protective mechanisms against respiratory B. abortus infection.

Evaluation of dot-blot test for serological diagnosis of bovine brucellosis

Abstract The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests; the results were compared using the Kappa indicator. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.

Depletion of Complement Enhances the Clearance of Brucella abortus in Mice.

Brucellosis is a bacterial disease of animals and humans. Brucella abortus barely activates the innate immune system at the onset of infection, and this bacterium is resistant to the microbicidal action of complement. Since complement stands as the first line of defense during bacterial invasions, we explored the role of complement in B. abortus infections. Brucella abortus-infected mice depleted of complement with cobra venom factor (CVF) showed the same survival rate as mice in the control group. The complement-depleted mice readily eliminated B. abortus from the spleen and did so more efficiently than the infected controls after 7 days of infection. The levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6) remained within background levels in complement-depleted B. abortus-infected mice. In contrast, the levels of the immune activator cytokine gamma interferon and the regulatory cytokine IL-10 were significantly increased. No significant histopathological changes in the liver and spleen were observed between the complement-depleted B. abortus-infected mice and the corresponding controls. The action exerted by Brucella on the immune system in the absence of complement may correspond to a broader phenomenon that involves several components of innate immunity.

Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.

Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1ß, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A2, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.

Evaluation of dot-blot test for serological diagnosis of bovine brucellosis.

The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests; the results were compared using the Kappa indicator. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.

High level of B19 strain detection in Brazilian cattle semen.

Semen contaminated with microorganisms can disseminate serious diseases including brucellosis. The objectives of this study were to detect Brucella-specific antibodies and Brucella abortus DNA in samples of blood and fresh semen from 100 animals older than 20 months. The samples were collected on farms and in semen collection and processing centers (CCPS). The serum samples were evaluated by Rose Bengal test (RBT). B. abortus DNA was detected by a polymerase chain reaction (PCR) using BAB and IS771 primers. The difference between the vaccine field strain was identified using ery-1, ery-2, and ery-3 primers, using the hemi-nested PCR method. No anti-B. abortus antibodies were detected in the serum samples. Out of the total semen samples, 68% (68/100) presented amplifications of the B. abortus genes. All (68/68) were identified as B19 strain of Brucella abortus vaccine. It was concluded that even bulls that are seronegative for brucellosis can eliminate the bacteria in the semen. The presence in the DNA of the B19 vaccine strain should be investigated for a better understanding of the epidemiological importance of this strain in these animals.