Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription.

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Brucella abortus triggers the differential expression of immunomodulatory lncRNAs in infected murine macrophages.

Introduction: The lncRNAs (long non-coding RNAs) are the most diverse group of non-coding RNAs and are involved in most biological processes including the immune response. While some of them have been recognized for their influence on the regulation of inflammatory activity, little is known in the context of infection by Brucella abortus, a pathogen that presents significant challenges due to its ability to manipulate and evade the host immune system. This study focuses on characterize the expression profile of LincRNA-cox2, Lethe, lincRNA-EPS, Malat1 and Gas5 during infection of macrophages by B. abortus. Methods: Using public raw RNA-seq datasets we constructed for a lncRNA expression profile in macrophages Brucella-infected. In addition, from public RNA-seq raw datasets of RAW264.7 cells infected with B. abortus we constructed a transcriptomic profile of lncRNAs in order to know the expression of the five immunomodulating lncRNAs studied here at 8 and 24 h post-infection. Finally, we performed in vitro infection assays in RAW264.7 cells and peritoneal macrophages to detect by qPCR changes in the expression of these lncRNAs at first 12 hours post infection, a key stage in the infection cycle where Brucella modulates the immune response to survive. Results: Our results demonstrate that infection of macrophages with Brucella abortus, induces significant changes in the expression of LincRNA-Cox2, Lethe, LincRNA-EPS, Gas5, and Malat1. Discussion: The change in the expression profile of these immunomodulatory lncRNAs in response to infection, suggest a potential involvement in the immune evasion strategy employed by Brucella to facilitate its intracellular survival.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Introduction: Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. Methods and results: We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1ß production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Discussion/conclusion: Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

A spatiotemporal analysis of bovine brucellosis cases in Minas Gerais state, Brazil, from 2011 to 2018.

Our study explored the patterns of bovine brucellosis dissemination in Minas Gerais state, Brazil, by examining data on passive surveillance of bovine brucellosis cases from the Instituto Mineiro de Agropecuaria (IMA) (Animal Health Authority), as well as cattle population and bovine brucellosis testing, from 2011 to 2018 by means of a spatiotemporal analysis. We plotted cases, populations and testing distributions and performed spatial autocorrelation (Moran's I test) and local indicators of spatial autocorrelation (LISA) analyses. Moreover, we assessed the correlation of the spatial distribution and the compiled data (brucellosis cases, cattle populations, and brucellosis testing) by Lee's test. Our results showed that bovine brucellosis cases occurred mainly in the Triângulo Mineiro, Alto Paranaíba and Northwest regions, which reported cases in all analyzed years (2011 to 2018). The cattle population of Minas Gerais was concentrated in the same regions as bovine brucellosis cases, and the performed tests through the analyzed years (2011 to 2018). Moran's I test results of the case data showed significant spatial autocorrelation in 2011, 2015 and 2018 (p value < 0.05), and from 2011 to 2018, the population and testing data were also significant in Moran's I test (p value < 0.01). The results of cluster analysis (LISA) of cases showed clusters mainly in the Triângulo Mineiro, Alto Paranaíba, Northwest and South regions in 2011, 2015 and 2018. The local clusters for cattle populations and brucellosis testing were also observed in the same regions as bovine brucellosis cases in all years (2011 to 2018). The correlation results between clusters (Lee's test) were 0.22 (p value < 0.01) in 2011, 0.15 (p value < 0.01) in 2015 and 0.43 (p value <0.01) in 2018 between cases and populations, and 0.25 (p value <0.01) in 2011, 0.14 (p value <0.01) in 2015 and 0.38 (p value < 0.01) in 2018 for testing and cases. Therefore, our results showed that brucellosis cases were distributed together with cattle populations and brucellosis testing data, indicating that brucellosis in cattle in Minas Gerais state is being identified where there are more animals and where more tests are performed.

Distribution, seroprevalence and risk factors for bovine brucellosis in Brazil: Official data, systematic review and meta-analysis.

Bovine brucellosis is an endemic disease in Brazil, and evidence-based assessments of the available literature on its seroprevalence and risk factors are limited. The aim of this study was to systematically review and summarize studies related to seroprevalence and risk factors of bovine brucellosis in the entire Brazil, in addition to comparing published data with the most recent official reports. Articles available in scientific databases and published between October 2006 and October 2021 were evaluated. Forty-five publications were included in the meta-analysis on the seroprevalence of brucellosis and 29 publications in the review on risk factors. The largest number of publications was found for the State of Mato Grosso do Sul (n=4), and the highest and lowest seroprevalences were observed in Acre (11%; 95% CI: 8.0-14.0%) and in the Federal District (0.4%; 95% CI: 0.2-0.7%). The main risk factors were the purchase of animals for breeding, vaccination, the number of heifers (female ≥2 years), the presence of calving paddocks and the occurrence of abortions. The need for new official studies has been suggested to determine the true prevalence of bovine brucellosis in Brazil, supported by the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis.

Prevalence of Brucella melitensis and Brucella abortus aminoglycoside-resistant isolates: a systematic review and meta-analysis.

INTRODUCTION: Aminoglycosides are vital antibiotics for treating Brucella infections, because they interfere with bacterial protein production and are often combined with other antibiotics. They are cost-effective, have fewer side effects, and can penetrate biofilms. The prevalence of brucellosis has increased in recent years, increasing the need for effective treatments. In addition, the emergence of multidrug-resistant Brucella strains has highlighted the need for an updated and comprehensive understanding of aminoglycoside resistance. This systematic review aimed to provide a comprehensive overview of the global prevalence of aminoglycoside resistance in B. melitensis and B. abortus. METHODS: A systematic search of online databases was conducted and eligible studies met certain criteria and were published in English. Quality assessment was performed using the JBI Checklist. A random-effects model was fitted to the data, and meta-regression, subgroup, and outlier/influential analyses were performed. The analysis was performed using R and the metafor package. RESULTS: The results of this systematic review and meta-analysis suggested that the average prevalence rates of streptomycin, gentamicin, and amikacin resistance were 0.027 (95% confidence interval [CI], 0.015-0.049), 0.023 (95% CI, 0.017-0.032), and 0.008 (95% CI, 0.002-0.039), respectively. The prevalence of streptomycin resistance was higher in the unidentified Brucella group than in the B. abortus and B. melitensis groups (0.234, 0.046, and 0.017, respectively; p < 0.02). The prevalence of gentamicin resistance increased over time (r = 0.064; 95% CI, 0.018 to 0.111; p = 0.007). The prevalence of resistance did not correlate with the quality score for any antibiotic. Funnel plots showed a potential asymmetry for streptomycin and gentamicin. These results suggest a low prevalence of antibiotic resistance in the studied populations. CONCLUSION: The prevalence of aminoglycoside resistance in B. melitensis and B. abortus was low. However, gentamicin resistance has increased in recent years. This review provides a comprehensive and updated understanding of aminoglycoside resistance in B. melitensis and B. abortus.

Molecular detection of Brucella species among aborted small ruminants in southeast Iran.

Brucellosis, caused by Brucella bacteria, is a common zoonotic infectious disease with various clinical manifestations in humans and animals. The disease is endemic in human and ruminant populations in Iran, with a particular prevalence in areas where humans have close interactions with livestock. Since domestic animals serve as the primary reservoir for brucellosis, this study aimed to identify the presence of Brucella spp. among aborted small ruminants in southeast Iran. Between 2021 and 2022, aborted fetuses of small ruminants (46 sheep and 4 goats) were collected from Zarand County in the Kerman province. Swab samples from the abomasum contents of these fetuses were obtained and subjected to DNA extraction. The samples were then tested for Brucella spp. detection using the polymerase chain reaction (PCR) method. Out of the 50 aborted fetuses examined, Brucella spp. was detected in 15 (30%) specimens, comprising 13 (28%) sheep and 2 (50%) goats. Species typing revealed the presence of Brucella ovis (6 sheep and 1 goat), Brucella melitensis (6 sheep), and Brucella abortus (1 sheep) among the positive specimens. This cross-sectional study highlights the high prevalence of various Brucella species in samples from small ruminant abortions in southeast Iran. Additionally, the identified Brucella species were not limited to their primary host livestock. These indicated potential cross-species transmission among small ruminants.

Serological and Molecular Survey of Brucella Species in Owners and Their Dogs Living on Island and Mainland Seashore Areas of Brazil.

Background: Although Brucella abortus, Brucella suis, and Brucella canis may infect humans and dogs worldwide, no study to date has assessed and compared owners and their dogs between island and mainland seashore areas. Materials and Methods: Accordingly, the study herein has applied serological tests, including Microplate Agglutination Test with 2-Mercaptoethanol, immunochromatographic assay, and Rose Bengal Test, and a Brucella genus-specific PCR assay to 195 owners and their 148 dogs living on 1 mainland seashore area and three nearby oceanic islands of southern Brazil. Results: No seropositivity to B. abortus and B. suis was detected in owner or dog sera. Anti-B. canis seropositivity was observed in 3/148 (2.0%) dogs, but no owner sample was seropositive to B. canis. In addition, all blood samples from both owners and dogs were negative on Brucella genus-specific PCR assay. Conclusions: The seropositive dogs were not related and lived on the seashore mainland area of Guaraqueçaba city. The absence of seropositivity on the islands and the low seropositivity on the seashore mainland could be attributed to geographic isolation, and suggest the low impact of the disease in the region. Despite being a zoonotic disease, brucellosis by B. canis is not included in the National Program for Control and Eradication of Brucellosis, and its diagnosis and notification are not mandatory. The presence of seropositive dogs highlights the risk to human health and the importance of epidemiological surveillance actions in the region, as well as the need for the implantation of preventive measures to avoid the transmission of the pathogen.

Accuracy of serological tests for bovine brucellosis: A systematic review and meta-analysis.

The direct methods for diagnosis of bovine brucellosis have several limitations, therefore serological tests are the basis for the diagnosis of the disease. However, a meta-analysis estimating the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) on the main tests used in bovine brucellosis control programs worldwide has not been performed. This systematic review and meta-analysis aimed to estimate the DSe, DSp and thereby accuracy of serological tests individually used in the diagnosis of bovine brucellosis. The databases CABI, Cochrane Library, PubMed/MEDLINE, SciELO, Scopus and Web of Science were used to select articles. The search resulted in 5308 studies, of which 71 were selected for systematic review using quality assessment tools and 65 studies were included in the meta-analysis. For the meta-analysis, 178 assays and 11 different serological tests were considered. To estimate DSe and DSp of the tests, studies were divided according to animal selection for the studies: (1) studies that carried out a random or consecutive selection of participants (noncasecontrol studies) and (2) all studies, including casecontrol studies. Considering only the non-case-control studies to estimate the DSe, the tests that exhibited the best and worst performance were the iELISA test (indirect enzyme immunoassay - bacterial suspension as antigen - BS) (96.5%, 95% CI: 94.1-97.9%) and 2ME (2- mercaptoethanol test) (85.0%, 95% CI: 79.6-89.1%), respectively; while for DSp, the FPA (fluorescence polarization assay) (99, 7%, 95% CI: 99.5-99.8%) and PCFIA tests (protein concentration fluorescence immunoassay) (78.5%, 95% CI: 70.0-85.1%) showed better and worse performance, respectively. Overall, our results showed an overestimation in the DSe and DSp of the eleven serological tests assessed when casecontrol studies were included in the meta-analysis, which is a concern considering its impacts on the time and costs associated with populational diagnosis of the diseases, since several of these tests are routinely used in the control and eradication programs of bovine brucellosis worldwide. Furthermore, the tests that exhibited the best DSe and DSp, iELISA (BS) and FPA, respectively, are relatively easy to perform and interpret and the test which showed the best overall accuracy was FPA.

The Brucella abortus two-component system response regulator BvrR binds to three DNA regulatory boxes in the upstream region of omp25.

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and ß-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales.

Modelling the Unidentified Abortion Burden from Four Infectious Pathogenic Microorganisms (Leptospira interrogans, Brucella abortus, Brucella ovis, and Chlamydia abortus) in Ewes Based on Artificial Neural Networks Approach: The Epidemiological Basis for a Control Policy.

Unidentified abortion, of which leptospirosis, brucellosis, and ovine enzootic abortion are important factors, is the main cause of disease spread between animals and humans in all agricultural systems in most developing countries. Although there are well-defined risk factors for these diseases, these characteristics do not represent the prevalence of the disease in different regions. This study predicts the unidentified abortion burden from multi-microorganisms in ewes based on an artificial neural networks approach and the GLM. METHODS: A two-stage cluster survey design was conducted to estimate the seroprevalence of abortifacient microorganisms and to identify putative factors of infectious abortion. RESULTS: The overall seroprevalence of Brucella was 70.7%, while Leptospira spp. was 55.2%, C. abortus was 21.9%, and B. ovis was 7.4%. Serological detection with four abortion-causing microorganisms was determined only in 0.87% of sheep sampled. The best GLM is integrated via serological detection of serovar Hardjo and Brucella ovis in animals of the slopes with elevation between 2600 and 2800 meters above sea level from the municipality of Xalatlaco. Other covariates included in the GLM, such as the sheep pen built with materials of metal grids and untreated wood, dirt and concrete floors, bed of straw, and the well water supply were also remained independently associated with infectious abortion. Approximately 80% of those respondents did not wear gloves or masks to prevent the transmission of the abortifacient zoonotic microorganisms. CONCLUSIONS: Sensitizing stakeholders on good agricultural practices could improve public health surveillance. Further studies on the effect of animal-human transmission in such a setting is worthwhile to further support the One Health initiative.

A bvrR/bvrS Non-Polar Brucella abortus Mutant Confirms the Role of the Two-Component System BvrR/BvrS in Virulence and Membrane Integrity.

Brucella abortus is a bacterial pathogen causing bovine brucellosis worldwide. This facultative extracellular-intracellular pathogen can be transmitted to humans, leading to a zoonotic disease. The disease remains a public health concern, particularly in regions where livestock farming is present. The two-component regulatory system BvrR/BvrS was described by isolating the attenuated transposition mutants bvrR::Tn5 and bvrS::Tn5, whose characterization led to the understanding of the role of the system in bacterial survival. However, a phenotypic comparison with deletion mutants has not been performed because their construction has been unsuccessful in brucellae and difficult in phylogenetically related Rhizobiales with BvrR/BvrS orthologs. Here, we used an unmarked gene excision strategy to generate a B. abortus mutant strain lacking both genes, called B. abortus ∆bvrRS. The deletion was verified through PCR, Southern blot, Western blot, Sanger sequencing, and whole-genome sequencing, confirming a clean mutation without further alterations at the genome level. B. abortus ∆bvrRS shared attenuated phenotypic traits with both transposition mutants, confirming the role of BvrR/BvrS in pathogenesis and membrane integrity. This B. abortus ∆bvrRS with a non-antimicrobial marker is an excellent tool for continuing studies on the role of BvrR/BvrS in the B. abortus lifestyle.

Optimization of the first extraction protocol for metabolomic studies of Brucella abortus.

Brucellosis is a zoonosis prevalent worldwide and very recurrent in less developed or developing regions. This zoonosis affects livestock, generating high financial losses to producers, in addition to transmitting diseases to humans through meat consumption or handling contaminated products and animals. In this study, five extraction methods for Brucella abortus intracellular metabolites, using different solvent compositions and cell membrane disruption procedures, were evaluated. Derivatized extracts were analyzed by GC-HRMS. Raw data were processed in XCMS Online and the results were evaluated through multivariate statistical analysis using the MetaboAnalyst platform. The identification of the extracted metabolites was performed by the Unknowns software using the NIST 17.L library. The extraction performance of each method was evaluated for thirteen representative metabolites, comprising four different chemical classes. Most of these compounds are reported in the cell membrane composition of Gram-negative bacteria. The method based on extraction with methanol/chloroform/water presented the best performance in the evaluation of the extracted compounds and in the statistical results. Therefore, this method was selected for extracting intracellular metabolites from cultures of Brucella abortus for untargeted metabolomics analysis.

Equine Brucellosis: Current Understanding and Challenges.

Brucellosis in equines, including horses, donkeys, and mules, is characterized by abscesses in tendons, bursae, and joints. Reproductive disorders, which are common in other animals, are rare in both males and females. Joint breeding of horses, cattle, and pigs was found as the main risk factor for equine brucellosis, with the transmission from equines to cattle or among equines possible, although unlikely. Hence, evaluation of the disease in equines can be considered an indirect indicator of the effectiveness of brucellosis control measures employed for other domestic species. Generally, the disease in equines reflects disease status in the sympatric domestic species, mainly cattle. It is important to note that in equines, the disease has no validated diagnostic test, which limits the interpretation of available data. Finally, it is important to mention that equines also represent significant Brucella spp. infection sources for humans. Considering the zoonotic aspect of brucellosis, the significant losses due to infection, and the representativeness of horses, mules, and donkeys in the society, as well as the continuous efforts to control and eradicate the disease in livestock, in this review, we covered the various aspects of brucellosis in equines and compile the sparse and diffuse information on the subject.

Brucella abortus induces mast cell activation through TLR-2 and TLR-4.

The Gram-negative bacteria Brucella abortus is a major cause of brucellosis in animals and humans. The host innate immune response to B. abortus is mainly associated with phagocytic cells such as dendritic cells, neutrophils, and macrophages. However, as mast cells naturally reside in the main bacterial entry sites they may be involved in bacterial recognition. At present, little is known about the role of mast cells during B. abortus infection. The role of the innate immune receptors TLR2 and TLR4 in activation of mast cells by B. abortus (strain RB51) infection was analyzed in this study. The results showed that B. abortus did not induce mast cell degranulation, but did induce the synthesis of the cytokines IL-1ß, IL-6, TNF-α, CCL3, CCL4, and CCL5. Furthermore, B. abortus stimulated key cell signaling molecules involved in mast cell activation such as p38 and NF-κB. Blockade of the receptors TLR2 and TLR4 decreased TNF-α and IL-6 release by mast cells in response to B. abortus. Taken together, our results demonstrate that mast cells are activated by B. abortus and may play a role in inducing an inflammatory response during the initial phase of the infection.

Selection of Brucella abortus mimetic epitopes for fast diagnostic purposes in cattle.

Bovine brucellosis is a disease that significantly impacts animal production and human health. Although many sensitive diagnostic tests are used, there is still no ideal fast serological test for all epidemiological situations. In this context, we developed peptides that mimic regions of antigenic proteins of Brucella abortus and can be used in serological diagnosis. RESULTS: From phage display technology, we randomly selected nine clones of phage displaying peptide binders to B. abortus. These clones were sequenced and translated. After molecular docking analysis, two peptides (Ba4 and Ba9) were selected, chemically synthesized, and verified for their potential diagnostic value. By enzyme-linked immunoassay (ELISA), Ba9 showed a sensitivity of up to 97.5% to detect antibodies circulating in animals with brucellosis. We incorporated the peptide Ba9 onto a bioelectrode (graphite modified with poly-3-hydroxyphenylacetic acid). Then, direct serum detection was demonstrated by differential pulse voltammetry, micrographs, and topographic analyses in addition to the average roughness coefficient (Ra) and the value of the mean squared deviation of the roughness (Rms). CONCLUSION: This work shows that the mimetic epitope of B. abortus can be useful for developing new platforms for diagnosing brucellosis. In addition, we propose a fast test based on an electrochemical sensor using graphite modified with poly-3-hydroxyphenylacetic acid.

Bystander activation of microglia by Brucella abortus-infected astrocytes induces neuronal death via IL-6 trans-signaling.

Inflammation plays a key role in the pathogenesis of neurobrucellosis where glial cell interactions are at the root of this pathological condition. In this study, we present evidence indicating that soluble factors secreted by Brucella abortus-infected astrocytes activate microglia to induce neuronal death. Culture supernatants (SN) from B. abortus-infected astrocytes induce the release of pro-inflammatory mediators and the increase of the microglial phagocytic capacity, which are two key features in the execution of live neurons by primary phagocytosis, a recently described mechanism whereby B. abortus-activated microglia kills neurons by phagocytosing them. IL-6 neutralization completely abrogates neuronal loss. IL-6 is solely involved in increasing the phagocytic capacity of activated microglia as induced by SN from B. abortus-infected astrocytes and does not participate in their inflammatory activation. Both autocrine microglia-derived and paracrine astrocyte-secreted IL-6 endow microglial cells with up-regulated phagocytic capacity that allows them to phagocytose neurons. Blocking of IL-6 signaling by soluble gp130 abrogates microglial phagocytosis and concomitant neuronal death, indicating that IL-6 activates microglia via trans-signaling. Altogether, these results demonstrate that soluble factors secreted by B. abortus-infected astrocytes activate microglia to induce, via IL-6 trans-signaling, the death of neurons. IL-6 signaling inhibition may thus be considered a strategy to control inflammation and CNS damage in neurobrucellosis.

Ring test in milk samples preserved with Bronopol® / Teste do anel em amostras de leite conservadas com Bronopol ®

ABSTRACT: This study evaluated if the milk ring test (MRT) used to the brucellosis diagnostic at dairy herd level can be done in milk samples with conservative (Bronopol®) collected to the somatic cell count (SCC) that is used to mastitis monitoring. It were analyzed 38 bulk tank milk samples (BTMS) from 19 dairy herds artisanal cheese producers and 36 BTMS from a brucellosis free dairy herd, used as negative control (NC) and positive control (PC), when was added serum from vaccinated animals. The milk samples were collected in two different bottles (with or without Bronopol®). All the 38 BTMS from artisanal cheese producers (samples with and without Bronopol®) were non-reagent to MRT, as all NC. All PC were reagent to MRT, without interference of Bronopol®. Results showed that the milk sample used to SCC presented the proportion of agreement of results in all milk samples (100%), that is, false positive and false negative results were not observed. Results indicated that Bronopol® did not interfere with the color in the time of MRT reading suggesting that the same milk samples could be used to monitoring mastitis and brucellosis.

RESUMO: O objetivo deste estudo foi avaliar se o teste do anel do leite (TAL), utilizado para o diagnóstico da brucelose em nível de rebanho leiteiro, pode ser realizado em amostras de leite com conservador (Bronopol®) coletado para contagem de células somáticas (CCS) que é utilizado para monitoramento de mastite. Foram analisadas 38 amostras de leite de tanques de expansão (ALTE) de 19 rebanhos leiteiros produtores de queijo artesanal e 36 ALTE de rebanho leiteiro livre de brucelose, utilizadas como controle negativo (CN) e controle positivo (CP), quando foi adicionado soro de animais vacinados. As amostras de leite foram coletadas em dois frascos diferentes (com e sem Bronopol®). Todos as 38 ALTE de produtores artesanais de queijo (amostras com e sem Bronopol®) foram não reagentes ao TAL, como todos os CN. Todos os CP foram reagentes ao TAL, sem interferência do Bronopol®. Os resultados mostraram que a amostra de leite utilizada para CCS apresentaram a proporção de concordância dos resultados em todas as amostras de leite (100%), ou seja, não foram observados resultados falso-positivos e falso-negativos. Os resultados indicam que o Bronopol® não interferiu na cor no momento da leitura do TAL, sugerindo que as mesmas amostras de leite poderiam ser usadas para monitorar mastite e brucelose.

Phenotypic and genotypic characterization of Brucella abortus biovar 4 isolates from cattle in Brazil / Caracterização fenotípica e genotípica de isolados de Brucella abortus biovar 4 em bovinos no Brasil

ABSTRACT: The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country.

RESUMO: O objetivo do presente estudo foi caracterizar (fenotipicamente e genotipicamente) duas cepas de Brucella abortus identificadas como pertencentes ao biovar 4 isolada de bovinos no Brasil. As cepas foram isoladas de bursite cervical de bovinos dos estados do Pará e Rio Grande do Sul, respectivamente. Na identificação fenotípica, os isolados foram positivos na exigência de CO2, produziram H2S, foram resistentes à fucsina básica (20 µg / mL) e sensíveis à tionina (20 µg / mL e 40 µg / mL) e apresentaram antígeno de superfície M, mas o antígeno de superfície A foi ausente. Os isolados foram positivos na PCR para o gene bcsp31 (gênero específico) e na PCR - AMOS, ambos os isolados apresentaram perfil de banda consistente com B. abortus biovar 1, 2 ou 4. Além disso, ambos os isolados também apresentaram padrões de restrição idêntica à cepa de referência quando testada pelo omp2b PCR-RFLP. Na genotipagem usando Multiple Locus Variable Number of Tandem Repeat (VNTR) - MLVA (MLVA16), os isolados apresentaram diferenças em vários loci (Bruce42, Bruce19, Bruce04, Bruce16 e Bruce30); no Multiple Locus Sequence Typing (MLST), os isolados também exibiram diferenças na sequência tipo (ST), amostra 16/02 ST1 (2-1-1-2-1-3-1-1-1) e amostra 128/11 ST (22-1-1-8-9-3-1-1-1). A extensa tipagem de cepas de B. abortus isoladas de bovinos no Brasil por diferentes abordagens confirmou a rara ocorrência de B. abortus biovar 4 no país.