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.

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.

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.

Comparative genomics of Brucella abortus and Brucella melitensis unravels the gene sharing, virulence factors and SNP diversity among the standard, vaccine and field strains / La genómica comparada de Brucella abortus y Brucella melitensis revela el intercambio de genes, los factores de virulencia y la diversidad de SNP entre las cepas estándar, vacunales y de campo

Brucella abortus and Brucella melitensis are the primary etiological agents of brucellosis in large and small ruminants, respectively. There are limited comparative genomic studies involving Brucella strains that explore the relatedness among both species. In this study, we involved strains (n=44) representing standard, vaccine and Indian field origin for pangenome, single nucleotide polymorphism (SNP) and phylogenetic analysis. Both species shared a common gene pool representing 2884 genes out of a total 3244 genes. SNP-based phylogenetic analysis indicated higher SNP diversity among B. melitensis (3824) strains in comparison to B. abortus (540) strains, and a clear demarcation was identified between standard/vaccine and field strains. The analysis for virulence genes revealed that virB3, virB7, ricA, virB5, ipx5, wbkC, wbkB, and acpXL genes were highly conserved in most of the Brucella strains. Interestingly, virB10 gene was found to have high variability among the B. abortus strains. The cgMLST analysis revealed distinct sequence types for the standard/vaccine and field strains. B. abortus strains from north-eastern India fall within similar sequence type differing from other strains. In conclusion, the analysis revealed a highly shared core genome among two Brucella species. SNP analysis revealed B. melitensis strains exhibit high diversity as compared to B. abortus strains. Strains with absence or high polymorphism of virulence genes can be exploited for the development of novel vaccine candidates effective against both B. abortus and B. melitensis.(AU)

Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection.

Brucellosis, caused by the bacterium Brucella, poses a significant global threat to both animal and human health. Although commercial live Brucella vaccines including S19, RB51, and Rev1 are available for animals, their unsuitability for human use and incomplete efficacy in animals necessitate the further study of vaccine-mediated immunity to Brucella. In this study, we employed in vivo B-cell depletion, as well as immunodeficient and transgenic mouse models, to comprehensively investigate the roles of B cells, antigen uptake and presentation, antibody production, and class switching in the context of S19-mediated immunity against brucellosis. We found that antibody production, and in particular secretory IgM plays a protective role in S19-mediated immunity against virulent Brucella melitensis early after the challenge in a manner associated with complement activation. While T follicular helper cell deficiency dampened IgG production and vaccine efficacy at later stages of the challenge, this effect appeared to be independent of antibody production and rather was associated with altered T-cell function. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy at later timepoints after the challenge. In addition, B-cell depletion after vaccination, but before the challenge, enhanced S19-mediated protection against brucellosis, suggesting a deleterious role of B cells during the challenge phase. Collectively, our findings indicate antibody production is protective, while B-cell MHCII expression is deleterious, to live vaccine-mediated immunity against brucellosis. IMPORTANCE: Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

Structure-function analysis of the cyclic ß-1,2-glucan synthase from Agrobacterium tumefaciens.

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic ß-1,2-glucan (CßG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CßG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CßG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.

Gated nanoprobe utilizing metal-organic frameworks for identifying and distinguishing between the wild strains and the vaccine strains of brucella.

In the assay for Brucella, the identification and differentiation of wild strains and vaccine strains present a significant challenge. Currently, there aren't any commercially available product to address this issue. In this study, we have developed a novel gated nanoprobe by utilizing Metal-Organic Frameworks (MOFs) as a scaffold and hairpin DNA as a "gating switch". Specifically, Probe 1 with hairpin structure (P1h) targets a gene that is present in both wild strains Y3 (B. melitensis biovar 3) and vaccine strains A19 (Brucella abortus strains A19). We successfully applied this probe to screen positive samples of Brucella without any cross-reactivity with other substances. Additionally, we identified another specific gene exclusively found in wild strains, which serves as Probe 2 with hairpin structure (P2h) to confirm the strain type. Simultaneous detachment of both P1h and P2h from the MOFs leads to the release of Rhodamine 6G (Rho 6G) and Fluorescein (Flu), specifically indicating the presence of wild strains. If only P1h detaches and the Flu signal is detected, it suggests the presence of vaccine strains. Importantly, this method offers high accuracy, with a detection rate of 90% and a recovery rate of 94.71% to 107.65%, while avoiding cross-reactions with MO and TB. This one-step experiment provides reliable identification and differentiation of Y3 and A19, addressing concerns related to long periodicity, interference from individual variations, and the complex design of primers in existing laboratory methods. Furthermore, our approach successfully detects target 1 (T1) and target 2 (T2) at concentrations ranging from 10-6 M to 10-9 M, with a detection limit of 6.7 × 10-10 M and 6.4 × 10-10 M, respectively. Importantly, our strategy is cost-effective (around $1) and offers higher detection efficiency compared to traditional laboratory methods.

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.

Effects of the Immunocontraceptive Gonacon on Pregnancy in Brucella-Seropositive American bison (Bison bison).

The purpose of this study was to determine if the number of pregnancies in naturally infected Brucella abortus-positive bison (Bison bison) cows would be reduced over a period of 5 yr after one treatment with 3000 µg gonadotropin-releasing hormone immunocontraceptive (GonaCon) compared to a similar group of naturally infected B. abortus-positive bison cows not treated with GonaCon. In each of the 5 yr, GonaCon-treated cows produced fewer offspring in relation to number of cows than the nontreated cows. Fisher's Exact test comparing offspring produced during the first reproductive season showed a significant difference between the two groups (P=0.0028). Differences in number of calves produced in GonaCon-treated and control groups were also noted in remaining years, but statistics were not applied because of data constraints. These data indicate that one treatment with GonaCon in brucellosis-seropositive female bison reduced pregnancies over five reproductive years. Thus, immunocontraception could potentially be used to manage brucellosis in affected herds.

Effects of Pregnancy Prevention on Brucella abortus Shedding in American bison (Bison bison).

Products of parturition are the predominant source of Brucella abortus for transmission in bison (Bison bison). Our objective was to assess whether preventing pregnancy in Brucella-seropositive bison reduced B. abortus shedding. Brucella-seropositive and -seronegative bison from Yellowstone National Park, Wyoming, USA were used in a replicated experiment. Each of two replicates (rep1, rep2) included a group of seropositive females treated with a single dose of gonadotropin-releasing hormone-based immunocontraceptive (Treatment rep1, n=15; Treatment rep2, n=20) and an untreated group (Control rep1, n=14; Control rep2, n=16) housed separately. Seronegative sentinel females were placed in each group to monitor horizontal transmission. Seronegative males were co-mingled for breeding each year. Pregnant females were removed from treatment groups in the first year, but not thereafter. Each January-June we monitored for B. abortus shedding events-any parturition associated with culture-positive fluids or tissues. We analyzed probability of shedding events using a negative binomial generalized linear mixed model fit by maximum likelihood using Laplace approximation. Over 5 yr, we observed zero shedding events in Treatment rep1 vs. 12 in Control rep1. All five Control rep1 sentinels but zero (0/5) Treatment rep1 sentinels seroconverted. In the second replicate, Treatment rep2 had two shedding events over 3 yr and Control rep2 had five events over 2 yr. Sentinels in both Control rep2 (3/6) and Treatment rep2 (5/6) seroconverted by trial endpoint. Treatment rep1 showed a reduced shedding probability relative to Control rep1, Treatment rep2, and Control rep2 (log odds value -25.36 vs. -1.71, -1.39, and -0.23, respectively). Fixed effect predictor covariates, year and age, had no explanatory value. These data suggest that successful contraception of brucellosis-seropositive female bison prevents shedding of B. abortus by individual animals. However, contraceptive treatment may or may not sufficiently reduce disease transmission to reduce brucellosis prevalence in an affected herd.