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 genome-wide CRISPR screen identified host genes essential for intracellular Brucella survival.

Brucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Brucella targets the host ubiquitin-specific protease, Usp8, through the effector protein, TcpB, for facilitating infection of macrophages.

Brucella species are Gram-negative intracellular bacterial pathogens that cause the worldwide zoonotic disease brucellosis. Brucella can infect many mammals, including humans and domestic and wild animals. Brucella manipulates various host cellular processes to invade and multiply in professional and non-professional phagocytic cells. However, the host targets and their modulation by Brucella to facilitate the infection process remain obscure. Here, we report that the host ubiquitin-specific protease, USP8, negatively regulates the invasion of Brucella into macrophages through the plasma membrane receptor, CXCR4. Upon silencing or chemical inhibition of USP8, the membrane localization of the CXCR4 receptor was enriched, which augmented the invasion of Brucella into macrophages. Activation of USP8 through chemical inhibition of 14-3-3 protein affected the invasion of Brucella into macrophages. Brucella suppressed the expression of Usp8 at its early stage of infection in the infected macrophages. Furthermore, we found that only live Brucella could negatively regulate the expression of Usp8, suggesting the role of secreted effector protein of Brucella in modulating the gene expression. Subsequent studies revealed that the Brucella effector protein, TIR-domain containing protein from Brucella, TcpB, plays a significant role in downregulating the expression of Usp8 by targeting the cyclic-AMP response element-binding protein pathway. Treatment of mice with USP8 inhibitor resulted in enhanced survival of B. melitensis, whereas mice treated with CXCR4 or 14-3-3 antagonists showed a diminished bacterial load. Our experimental data demonstrate a novel role of Usp8 in the host defense against microbial intrusion. The present study provides insights into the microbial subversion of host defenses, and this information may ultimately help to develop novel therapeutic interventions for infectious diseases.

In vivo biological validation of in silico analysis: A novel approach for predicting the effects of TLR4 exon 3 polymorphisms on brucellosis.

The role of the Toll-like receptor 4 (TLR4) is of recognising intracellular and extracellular pathogens and of activating the immune response. This process can be compromised by single nucleotide polymorphisms (SNPs) which might affect the activity of several TLRs. The aim of this study is of ascertaining whether SNPs in the TLR4 of Bubalus bubalis infected by Brucella abortus, compromise the protein functionality. For this purpose, a computational analysis was performed. Next, computational predictions were confirmed by performing genotyping analysis. Finally, NMR-based metabolomics analysis was performed to identify potential biomarkers for brucellosis. The results indicate two SNPs (c. 672 A > C and c. 902 G > C) as risk factor for brucellosis in Bubalus bubalis, and three metabolites (lactate, 3-hydroxybutyrate and acetate) as biological markers for predicting the risk of developing the disease. These metabolites, together with TLR4 structural modifications in the MD2 interaction domain, are a clear signature of the immune system alteration during diverse Gram-negative bacterial infections. This suggests the possibility to extend this study to other pathogens, including Mycobacterium tuberculosis. In conclusion, this study combines multidisciplinary approaches to evaluate the biological and structural effects of SNPs on protein function.

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.

DNA Sensor for the Detection of Brucella spp. Based on Magnetic Nanoparticle Markers.

Due to the limitations of conventional Brucella detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of Brucella in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of Brucella DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from Brucella genetic material. In addition, the detection specificity was examined using gDNA from Brucella and other zoonotic bacteria that may coexist in the same niche, confirming the method's selectivity for Brucella DNA. Our proposed biosensor has the potential to be used for the early detection of Brucella bacteria in the field and can contribute to disease control measures.

Intracellular Growth Inhibition and Host Immune Modulation of 3-Amino-1,2,4-triazole in Murine Brucellosis.

Catalase, an antioxidant enzyme widely produced in mammalian cells and bacteria, is crucial to mitigating oxidative stress in hostile environments. This function enhances the intracellular survivability of various intracellular growth pathogens, including Brucella (B.) abortus. In this study, to determine whether the suppression of catalase can inhibit the intracellular growth of B. abortus, we employed 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, in both RAW 264.7 macrophage cells and an ICR mouse model during Brucella infection. The intracellular growth assay indicated that 3-AT exerts growth-inhibitory effects on B. abortus within macrophages. Moreover, it contributes to the accumulation of reactive oxygen species and the formation of nitric oxide. Notably, 3-AT diminishes the activation of the nucleus transcription factor (NF-κB) and modulates the cytokine secretion within infected cells. In our mouse model, the administration of 3-AT reduced the B. abortus proliferation within the spleens and livers of infected mice. This reduction was accompanied by a diminished immune response to infection, as indicated by the lowered levels of TNF-α, IL-6, and IL-10 and altered CD4+/CD8+ T-cell ratio. These results suggest the protective and immunomodulatory effects of 3-AT treatment against Brucella infection.

Nano and microparticle drug delivery systems for the treatment of Brucella infections.

Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients.

Micro- and nanoformulations of antibiotics against Brucella.

Brucellosis, a zoonotic intracellular bacterial infection primarily transmitted through the consumption of unpasteurized milk from infected animals, remains a challenging condition to clinically control. This is mainly because of the limited effectiveness of conventional antibiotics in targeting intracellular Brucella. Micro- and nanoformulations of antibiotics, whether used as a mono- or combination therapy, have the potential to reduce the antibiotic doses required and treatment duration. Extensive research has been conducted on various organic, semiorganic, and inorganic nanomaterials with different morphologies, such as nanoparticles (NPs), nanotubes, nanowires, and nanobelts. Metal/metal oxide, lipidic, polymeric, and carbonic NPs have been widely explored to overcome the limitations of traditional formulations. In this review, we discuss the advances and challenges of these novel formulations based on recent investigations.

New insights into the genetic predisposition of brucellosis and its effect on the gut and vaginal microbiota in goats.

Goats contribute significantly to the global food security and industry. They constitute a main supplier of meat and milk for large proportions of people in Egypt and worldwide. Brucellosis is a zoonotic infectious disease that causes a significant economic loss in animal production. A case-control genome-wide association analysis (GWAS) was conducted using the infectious status of the animal as a phenotype. The does that showed abortion during the last third period of pregnancy and which were positive to both rose bengal plate and serum tube agglutination tests, were considered as cases. Otherwise, they were considered as controls. All animals were genotyped using the Illumina 65KSNP BeadChip. Additionally, the diversity and composition of vaginal and fecal microbiota in cases and controls were investigated using PCR-amplicone sequencing of the V4 region of 16S rDNA. After applying quality control criteria, 35,818 markers and 66 does were available for the GWAS test. The GWAS revealed a significantly associated SNP (P = 5.01 × 10-7) located on Caprine chromosome 15 at 29 megabases. Four other markers surpassed the proposed threshold (P = 2.5 × 10-5). Additionally, fourteen genomic regions accounted for more than 0.1% of the variance explained by all genome windows. Corresponding markers were located within or in close vicinity to several candidate genes, such as ARRB1, RELT, ATG16L2, IGSF21, UBR4, ULK1, DCN, MAPB1, NAIP, CD26, IFIH1, NDFIP2, DOK4, MAF, IL2RB, USP18, ARID5A, ZAP70, CNTN5, PIK3AP1, DNTT, BLNK, and NHLRC3. These genes play important roles in the regulation of immune responses to the infections through several biological pathways. Similar vaginal bacterial community was observed in both cases and controls while the fecal bacterial composition and diversity differed between the groups (P < 0.05). Faeces from the control does showed a higher relative abundance of the phylum Bacteroidota compared to cases (P < 0.05), while the latter showed more Firmicutes, Spirochaetota, Planctomycetota, and Proteobacteria. On the genus level, the control does exhibited higher abundances of Rikenellaceae RC9 gut group and Christensenellaceae R-7 group (P < 0.05), while the infected does revealed higher Bacteroides, Alistipes, and Prevotellaceae UCG-003 (P < 0.05). This information increases our understanding of the genetics of the susceptibility to Brucella in goats and may be useful in breeding programs and selection schemes that aim at controlling the disease in livestock.

Combining multi-omics analysis to identify host-targeted targets for the control of Brucella infection.

Human infections caused by Brucella (called brucellosis) are among the most common zoonoses worldwide with an estimated 500,000 cases each year. Since chronic Brucella infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As a facultative intracellular bacterium, Brucella is strictly parasitic in the host cell. Here, we performed proteomic and transcriptomic and metabolomic analyses on Brucella infected patients, mice and cells that provided an extensive "map" of physiological changes in brucellosis patients and characterized the metabolic pathways essential to the response to infection, as well as the associated cellular response and molecular mechanisms. This is the first report utilizing multi-omics analysis to investigate the global response of proteins and metabolites associated with Brucella infection, and the data can provide a comprehensive insight to understand the mechanism of Brucella infection. We demonstrated that Brucella increased nucleotide synthesis in the host, consistent with increased biomass requirement. We also identified IMPDH2, a key regulatory complex that controls nucleotide synthesis during Brucella infection. Pharmacological targeting of IMPDH2, the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits B. abortus growth both in vitro and in vivo. Through screening a library of natural products, we identified oxymatrine, an alkaloid obtained primarily from Sophora roots, is a novel and selective IMPDH2 inhibitor. In further in vitro bacterial inhibition assays, oxymatrine effectively inhibited the growth of B. abortus, which was impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. This moderately potent, structurally novel compound may provide clues for further design and development of efficient IMPDH2 inhibitors and also demonstrates the potential of natural compounds from plants against Brucella.

Prevalence of Brucella species in stray cattle, dogs and cats: A systematic review.

Stray animals are unowned free roaming, homeless, abandoned, street or sheltered animals, particularly dogs, cats and cattle. They could act as carrier of several zoonotic pathogens such as rabies virus, Mycobacterium and Brucella species. However, comprehensive information on the prevalence of zoonotic pathogens in stray animals is very limited. We conducted a systematic review as per Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) 2020 guidelines to estimate the prevalence of brucellosis in stray dogs, cats and cattle. Eligibility criteria for the study were determined using the PECOS classification (population, exposure, comparison, outcome, study design) as a tool to guide the research and adjust the search strategy. Major bibliographic databases [Web of Science, Medline, Scopus, ScienceDirect, Google Scholar and PubMed] were searched using predefined keywords for published epidemiological studies on brucellosis in stray animals (dogs, cats and cattle). Systematic assessments of all the studies since 1990-2022 were conducted and those reporting the prevalence of brucellosis in stray dogs, cats and cattle using appropriate diagnostic tests (culture, molecular, serological) were included. Studies reporting prevalence of brucellosis (Brucella infection or exposure) in kennel dogs, dairy herds, livestock farms, humans or marine species were excluded. The apparent individual test- wise prevalence along with 95% confidence intervals (CI) was estimated using Epitools. Out of 2689 studies, 37 met the inclusion criteria and were included in the systematic review. Of 37 studies, 28 (75.7%) were conducted in stray dogs, 7 (18.9%) in cattle and 2 (5.4%) in cats. Furthermore, only 21.62% studies (8/37) used probabilistic random sampling approaches and 13.51% studies (5/37) explained and justified the study sample size using appropriate methods for estimation of disease prevalence in the study populations. Higher sero-prevalence in stray dogs has been reported in studies conducted in Jordan (38.0% (95% CI: 24.0-54.0) and Pakistan (38.0% (95% CI: 31.0-45.0) whereas no sero-positivity was recorded in the studies conducted in Brazil, North Colombia, Cyprus, South Korea and USA. All studies on brucellosis (n = 7) in stray cattle were from India; conducted in stray cattle reared in cow-shelters. Sero-prevalence in the range of 4.3%- 64.3% was reported in stray cattle. Differences in diagnostic tests and host species, as well as limited number and non-randomized studies and high statistical heterogeneity did not allow us to determine combined meta-analysed prevalence estimates. Stray animals are likely to pose a zoonotic and disease spillover risk to human and livestock populations.

Proteomics and bioinformatics investigations to improve serological diagnosis of canine brucellosis.

PURPOSE: Brucella canis is pathogenic for dogs and humans. Serological diagnosis is a cost-effective approach for disease surveillance, but a major drawback of current serological tests is the cross-reactivity with other bacteria that results in false positive reactions. Development of indirect tests with improved sensitivity and specificity that use selected B. canis proteins instead of the whole antigen remain a priority. EXPERIMENTAL DESIGN: A western blotting assay was developed to define the serum antibody patterns associated to infection using a panel of positive and negative dog sera. B. canis positive sera recognized immunogenic bands ranging from 7 to 30 kDa that were then submitted to ESI-LC-MS/MS and analyzed by bioinformatics tools. RESULTS: A total of 398 B. canis proteins were identified. Bioinformatics tools identified 16 non cytoplasmic immunogenic proteins predicted as non-homologous with the most important Brucella cross-reactive bacteria and nine B. canis proteins non-homologous to B. ovis; among the latter, one resulted non-homologous to B. melitensis. Data are available via ProteomeXchange with identifier PXD042682. CONCLUSIONS AND CLINICAL RELEVANCE: The western blotting test developed was able to distinguish between infected and non-infected animals and may serve as a confirmatory test for the serological diagnosis of B. canis. The mass spectrometry and in silico results lead to the identification of specific candidate antigens that pave the way for the development of more accurate indirect diagnostic tests.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade.

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species-Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration.

Intracellular growth of Brucella is mediated by Dps-dependent activation of ferritinophagy.

Bacteria of the genus Brucella cause brucellosis, one of the world's most common zoonotic diseases. A major contributor to Brucella's virulence is the ability to circumvent host immune defense mechanisms. Here, we find that the DNA-binding protein Dps from Brucella is secreted within the macrophage cytosol, modulating host iron homeostasis and mediating intracellular growth of Brucella. In addition to dampening iron-dependent production of reactive oxygen species (ROS), a key immune effector required for immediate bacterial clearance, cytosolic Dps mediates ferritinophagy activation to elevate intracellular free-iron levels, thereby promoting Brucella growth and inducing host cell necrosis. Inactivation of the ferritinophagy pathway by Ncoa4 gene knockout significantly inhibits intracellular growth of Brucella and host cell death. Our study uncovers an unconventional role of bacterial Dps, identifying a crucial virulence mechanism used by Brucella to adapt to the harsh environment inside macrophages.

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis.

BACKGROUND: Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen. METHOD: Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free-pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti-omp25-specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25-specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real-time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain. RESULTS: Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the phosphate-buffered saline control group). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups had significantly greater IL-4 and IL-10 transcription levels than the other groups. The spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. CONCLUSION: Our study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost-effective, safe, sustainable, simple pilot development of pharmaceutical products.

Diagnostic utility of LAMP PCR targeting bcsp-31 gene for human brucellosis infection.

PURPOSE: Human brucellosis is a neglected zoonotic disease of significant public health concern. Molecular diagnosis of brucella remains challenging in low resource settings, due to the high infrastructure and cost involved. Loop-mediated isothermal amplification (LAMP) is a rapid point of care polymerase chain reaction (PCR) with the utility of on-field molecular diagnosis and offers a convenient alternative to conventional PCR. In the present study, we developed and evaluated the diagnostic utility of in house LAMP PCR targeting the Brucella genus-specific bcsp-31 gene in patients having febrile illness. MATERIALS AND METHODS: The analytical sensitivity and specificity of bcsp-31 LAMP PCR was first evaluated using brucella (n â€‹= â€‹8) and non-brucella cultures (n â€‹= â€‹5), along with spiked clinical samples. The overall diagnostic utility of developed LAMP PCR was then further evaluated in 393 human samples suspected of brucellosis. RESULTS: The developed LAMP PCR could detect as low as 8 â€‹fg of DNA by visual detection within 35min. We report sensitivity and specificity of the developed LAMP PCR as 90.91% and 99.37%.The accuracy of the developed test assay was found to be 98.60%. In clinical samples, LAMP gave positivity of 20% with the concordance of 89% with conventional PCR. CONCLUSION: To conclude, a rapid, efficacious, sensitive LAMP PCR targeting the bcsp 31 gene was developed. The existing LAMP PCR can be used as a point of care screening test in various low resource endemic setting in lieu of conventional PCR for estimation of prevalence data, diagnosis and treatment of brucellosis.

Cell and Tissue Tropism of Brucella spp.

Brucella spp. are facultatively intracellular bacteria that can infect, survive, and multiply in various host cell types in vivo and/or in vitro. The genus Brucella has markedly expanded in recent years with the identification of novel species and hosts, which has revealed additional information about the cell and tissue tropism of these pathogens. Classically, Brucella spp. are considered to have tropism for organs that contain large populations of phagocytes such as lymph nodes, spleen, and liver, as well as for organs of the genital system, including the uterus, epididymis, testis, and placenta. However, experimental infections of several different cultured cell types indicate that Brucella may actually have a broader cell tropism than previously thought. Indeed, recent studies indicate that certain Brucella species in particular hosts may display a pantropic distribution in vivo. This review discusses the available knowledge on cell and tissue tropism of Brucella spp. in natural infections of various host species, as well as in experimental animal models and cultured cells.