Expression of cytokine and Apoptosis-Associated genes in mice bone Marrow-Derived Macrophages stimulated with Brucella recombinant type IV secretion effectors.

BACKGROUND: Brucellosis is an economically important infectious caused by most commonly by Brucella. Detection of infected animals at the early stage is important for controlling the disease. The diagnostic antigens, usually protein antigens, have attracted much interest. However, the accurate mechanism of immune response is still unknown. The secretory effectors (BPE005, BPE275, and BPE123) of the type IV secretion system (T4SS) were involved in the intracellular circulation process of Brucella and the immune responses of the host. METHODS: Genes encoding three B. abortus effector proteins (BPE005, BPE275, and BPE123) of T4SS were cloned and the recombinant proteins were expressed and purified. The purified recombinant proteins were named rBPE005, rBPE275 and rBPE123. Then, the expressions of Th1- and Th2-related cytokine genes were analyzed in mice bone marrow-derived macrophages (BMDMs) after stimulation with rBPE005, rBPE275, and rBPE123. Furthermore, four apoptosis-associated genes (Caspase-3, Caspase-8, Bax, and Bcl-2) were also detected to explore the damage of the proteins to the cells. RESULTS: Expressions of all Th1- and Th2-related cytokine genes were induced with three proteins, and different cytokine expression patterns induced by each protein depend on the stimulation time and dose of protein. However, expressions of apoptosis-related genes did not change. CONCLUSION: These results showed that the secreted antigens of Brucella induced an immune reaction via the production of Th1- and Th2-type cytokines in BMDMs without exerting any damage on the cells.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Detection of Brucella S19 Vaccine Strain DNA in Domestic and Wild Ungulates from Brazilian Pantanal.

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome.

Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle.

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55-100) and the specificity 98.74 % (95 % CI: 97.68-99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Beyond its preferential niche: Brucella abortus RNA down-modulates the IFN-γ-induced MHC-I expression in epithelial and endothelial cells.

Brucella abortus (Ba) is a pathogen that survives inside macrophages. Despite being its preferential niche, Ba infects other cells, as shown by the multiple signs and symptoms humans present. This pathogen can evade our immune system. Ba displays a mechanism of down-modulating MHC-I on monocytes/macrophages in the presence of IFN-γ (when Th1 response is triggered) without altering the total expression of MHC-I. The retained MHC-I proteins are located within the Golgi Apparatus (GA). The RNA of Ba is one of the PAMPs that trigger this phenomenon. However, we acknowledged whether this event could be triggered in other cells relevant during Ba infection. Here, we demonstrate that Ba RNA reduced the surface expression of MHC-I induced by IFN-γ in the human bronchial epithelium (Calu-6), the human alveolar epithelium (A-549) and the endothelial microvasculature (HMEC) cell lines. In Calu-6 and HMEC cells, Ba RNA induces the retention of MHC-I in the GA. This phenomenon was not observed in A-549 cells. We then evaluated the effect of Ba RNA on the secretion of IL-8, IL-6 and MCP-1, key cytokines in Ba infection. Contrary to our expectations, HMEC, Calu-6 and A-549 cells treated with Ba RNA had higher IL-8 and IL-6 levels compared to untreated cells. In addition, we showed that Ba RNA down-modulates the MHC-I surface expression induced by IFN-γ on human monocytes/macrophages via the pathway of the Epidermal Growth Factor Receptor (EGFR). So, cells were stimulated with an EGFR ligand-blocking antibody (Cetuximab) and Ba RNA. Neutralization of the EGFR to some extent reversed the down-modulation of MHC-I mediated by Ba RNA in HMEC and A-549 cells. In conclusion, this is the first study exploring a central immune evasion strategy, such as the downregulation of MHC-I surface expression, beyond monocytes and could shed light on how it persists effectively within the host, enduring unseen and escaping CD8+ T cell surveillance.

Molecular epidemiology of Brucella abortus isolated from the environment in Ningxia Hui autonomous region, China.

Brucellosis is among the key zoonotic infectious diseases in China, and The Ningxia Hui Autonomous Region represents a major endemic area, and it is one of the main causes of poverty in the region due to illness. In Ningxia, there is substantial research on Brucella melitensis, studies on the molecular epidemiology of Brucella abortus are notably scarce. Consequently, this study aims to undertake pathogenic isolation and molecular epidemiological research on Brucella abortus isolated from the environment in Ningxia, providing insights and evidence to advance the prevention and control measures for brucellosis in the region. Building on traditional pathogenic detection methods, this research employs whole-genome sequencing(WGS) techniques and bioinformatics software to conduct a phylogenetic comparison of Ningxia strains and strains of Brucella abortus from various geographical origins. The results indicate that four Brucella abortus strains are classified as biovar 3 and MLST type ST2. It is shown that the local strains were closer phylogenetic relationships with strains from Asian and European countries. The presence of Brucella abortus in certain environmental sectors of Ningxia indicates a risk of transmission from the environment to animals and subsequently to humans. In conclusion, the Brucella abortus exists in some farming environments in Ningxia, and exists for a long time. Therefore, it is necessary to strengthen the monitoring of the disinfection effect of the farming environment to provide a basis for the forward movement of the gate of brucellosis prevention and control.

Construction of recombinant Omp25 or EipB protein loaded PLGA nanovaccines for Brucellosis protection.

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate recombinant Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed theirin vitro/in vivoimmunostimulatory activities to develop new vaccine candidates. The recombinant Omp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using FE-SEM, Zeta-sizer, and FT-IR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV-Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2 ± 5.21 nm, while E-NPs reached sizes of 274.4 ± 9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of thein vitrocytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells,in vivoimmunization experiments were conducted using concentrations of 16µg ml-1for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing producedBrucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to triggerBrucella-specific humoral and cellular immune response.

Detection and characterization of Brucella species in rodents: A threat for the persistence of brucellosis in livestock farms.

Brucellosis, caused by various Brucella species, poses a significant threat to global public health and livestock industries. This study aims to fill the knowledge gap concerning the presence of Brucella spp. in rodents on livestock farms in Iran. Both bacteriological and molecular surveys were conducted to assess the prevalence of Brucella spp. in these rodent populations. A total of 16 rodents were captured in four seropositive dairy cattle farms (n = 7) and two seropositive sheep farms (n = 9) and were then examined for the presence of the Brucella-infection. Five cow milk samples and 53 bovine lymph node samples from these farms were also tested for Brucella spp. Lymph node samples from dairy cattle farms contained 32 B. abortus biovar 3 isolates and one B. melitensis Rev1 vaccine isolate. The bacterial culture of rodents identified 12.5% of them (Mus musculus and Rattus norvegicus) harboring Brucella strains in dairy cattle farms. The rodents had B. abortus biovar 3 and B. melitensis biovar 1, suggesting a reservoir for these bacteria. A two-step molecular assay, utilizing the Omp28 sequences in tissue samples of rodents, demonstrated that 68.75% (n = 11) of the tested rodents yielded positive results. Bruce-ladder PCR and wboA typing on isolated bacteria revealed a close relationship to field strain of Brucella species. The study reveals that rodents on seropositive livestock farms in Iran harbor Brucella spp., indicating a potential reservoir for these bacteria. This highlights the importance of monitoring rodent populations through the molecular and bacterial methods to manage and control brucellosis in livestock.

Transcriptome analysis of macrophages during Brucella abortus infection clarifies the survival mechanisms of the bacteria.

Brucellosis is a critical zoonotic disease impacting humans and animals globally, causing symptoms like fever and arthritis in humans and reproductive issues in animals. The disease stems from the Brucella genus, adept at evading the immune system and proliferating within host cells. This study explores how Brucella abortus manipulates host cellular mechanisms to sustain infection, focusing on the interaction with murine macrophages over 24 h. Initial host defenses involve innate immune responses, while Brucella's survival strategies include evading lysosomal degradation and modulating host cell functions through various pathways. The research identified significant transcriptional changes in macrophages post-infection, highlighting pathways such as cytokine storm, pyroptosis signaling, Toll-like receptor pathways, and LXRs/RXRs signaling. The findings shed light on Brucella's complex mechanisms to undermine host defenses and underscore the need for further investigation into therapeutic targets to combat brucellosis.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

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.

Comparison of CcrM-dependent methylation in Caulobacter crescentus and Brucella abortus by nanopore sequencing.

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report.

BACKGROUND: It is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus. CASE PRESENTATION: A 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI-TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence. CONCLUSION: Automated identification instruments and MALDI-TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI-TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

A novel vaccine strategy against Brucellosis using Brucella abortus multi-epitope OMPs vaccine based on Lactococcus lactis live bacterial vectors.

Brucella infections typically occur in mucosal membranes, emphasizing the need for mucosal vaccinations. This study evaluated the effectiveness of orally administering Lactococcus lactis (L. lactis) for producing the Brucella abortus multi-epitope OMPs peptide. A multi-epitope plasmid was generated through a reverse vaccinology method, and mice were administered the genetically modified L. lactis orally as a vaccine. The plasmid underwent digestion, synthesizing a 39 kDa-sized protein known as OMPs by the target group. The sera of mice that were administered the pNZ8124-OMPs-L. lactis vaccine exhibited a notable presence of IgG1 antibodies specific to outer membrane proteins (OMPs), heightened levels of interferon (IFN-λ) and tumor necrosis factor alpha (TNF-α), and enhanced transcription rates of interleukin 4 (IL-4) and interleukin 10 (IL-10). The spleen sections from the pNZ8124-OMPs-L. lactis and IRIBA group had less morphological damage associated with inflammation, infiltration of lymphocytes, and lesions to the spleen. The findings present a novel approach to utilizing the food-grade, non-pathogenic L. lactis as a protein cell factory to synthesize innovative immunological candidate OMPs. This approach offers a distinctive way to evaluate experimental medicinal items' practicality, safety, affordability, and long-term sustainability.

Evaluation of the immunization of camels with Brucella abortus vaccine (RB51) in Egypt.

Background: Brucellosis is a highly contagious zoonotic disease caused by an intracellular facultative microorganism termed Brucella spp. Control of brucellosis depends on test and slaughter policy as well as vaccination programs. Aim: Estimation of the cell-mediated immunity (CMI) [total leukocytic count (TLC), phagocytic activity, phagocytic index, interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α)] in camels after vaccination with RB51 using real-time polymerase chain reaction (PCR). Methods: A total of eight camels were grouped into two groups as follows: group (A): vaccinated with RB51 vaccine [1 dose/2 ml S/C (3 × 1010 CFU)] and group (B): control group. IL-6 and TNF-α were used for estimation of the CMI using real-time PCR on serum samples that were collected at 0, 7, 14, 21, 28, and 60 days after vaccination from each group. In addition, TLC, phagocytic activity, and phagocytic index were evaluated on heparinized blood samples at 0 and 60 days post-vaccination. Results: RB51 vaccine provides a protective immune response which progressively increases from the first week to 60 days after vaccination. Moreover, the levels of TNF-α and IL-6 differed between camels in the vaccinated group. Conclusion: Vaccination of camels with RB51 vaccine (with dose 3 × 1010 CFU) could induce good protective immune responses and this immunological response will be a good indication for a safe field vaccine that can be used for the control of camel brucellosis.

The immunostimulatory roles of gold nanoparticles in immunization and vaccination against Brucella abortus antigens.

One effective antigen carrier proposed for use in immunization and vaccination is gold nanoparticles. Prior work has shown that gold nanoparticles themselves have adjuvant properties. Currently, gold nanoparticles are used to design new diagnostic tests and vaccines against viral, bacterial, and parasitic infections. We investigated the use of gold nanoparticles as immunomodulators in immunization and vaccination with an antigen isolated from Brucella abortus. Gold nanoparticles with a diameter of 15 nm were synthesized for immunization of animals and were then conjugated to the isolated antigen. The conjugates were used to immunize white BALB/c mice. As a result, high-titer (1:10240) antibodies were produced. The respiratory and proliferative activities of immune cells were increased, as were the serum interleukin concentrations. The minimum antigen amount detected with the produced antibodies was âˆ¼ 0.5 pg. The mice immunized with gold nanoparticles complexed with the B. abortus antigen were more resistant to B. abortus strain 82 than were the mice immunized through other schemes. This fact indicates that animal immunization with this conjugate enhances the effectiveness of the immune response. The results of this study are expected to be used in further work to examine the protective effect of gold nanoparticles complexed with the B. abortus antigen on immunized animals and to develop test systems for diagnosing brucellosis in the laboratory and in the field.

Getting to the point: unipolar growth of Hyphomicrobiales.

The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome. Polar incorporation of envelope components relies on homologous proteins shared by the Hyphomicrobiales, reviewed here. Ongoing and future work will reveal how unipolar growth is integrated into the alphaproteobacterial cell cycle and coordinated with other processes such as chromosome segregation and cell division.

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.