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Brucella species are facultative intracellular bacterial pathogens that cause the contagious zoonotic disease, brucellosis. Brucella spp. infect a wide range of animals, including livestock, wild animals, and marine mammals. Compared with other invasive bacterial pathogens, partial information is available on the virulence factors of Brucella that enable them to survive in the host. Here, we performed transposon-based random mutagenesis of B. neotomae and identified the arginine/ornithine binding protein, ArgT, as one of the crucial virulence determinants of Brucella . Deleting ArgT from B. neotomae or B. melitensis resulted in its attenuation in macrophages, which was restored upon complementation with an ArgT expression plasmid. We observed that macrophages infected with Δ ArgT-B. neotomae produced elevated levels of NO due to the inability of these mutants to deplete the host intracellular arginine through their importer. Furthermore, defective survival of Δ ArgT B. neotomae and B. melitensis was observed in the infected mice, which correlated with enhanced NO production in the mice. Our studies revealed that ArgT plays a vital role in preventing intracellular killing and contributes to the chronic persistence of B. neotomae/B. melitensis in the host. This study highlights the essential role of arginine in clearing intracellular infections and the subversion of this host defense mechanism by intracellular pathogens for their chronic persistence.

Brucella relies on the type IV secretion system (T4SS) to establish replication niches within host cells. However, the Brucella T4SS effectors and their functions have not been fully identified. In this study, we investigated the function of Brucella RS15060, a novel T4SS effector discovered in our previous study, on the bacterial biological characteristics and pathogenesis by construction of the gene deletion and complementation strains. We found that deletion of the rs15060 gene weakened abilities of Brucella to replicate within host cells and establish chronic infection in mice but enhanced abilities to adhere/invade HeLa cells and evade lysosomal degradation in the early stage of infection. In addition, the rs15060 deletion Brucella strain showed significant changes in bacterial shape, cell wall thickness, and sensitivity to bactericidal factors. Furthermore, the rs15060 deletion strain showed an increased synthesis of bacterial lipopolysaccharide core and induced a stronger host's inflammatory response. The Brucella rs15060 complementation strain restored the altered biological characteristics. Moreover, BLASTP prediction and 3D structure simulation revealed that the Brucella RS15060 contains NAD(P)-binding and active motifs in structure, which are important for proteins to exert NAD dependent epimerase/dehydratase activity. The complementation strain with mutation on NAD(P)-binding and/or active motifs of RS15060 did not restore the altered characteristics, suggesting that the Brucella RS15060 is a potential NAD dependent epimerase/dehydratase, and the predicted NAD(P)-binding and/or active motifs play an important role on bacterial cell wall and LPS core synthesis, which is crucial for maintaining bacterial morphology and exerting virulence., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Disclosure statement The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Brucella spp. are small aerobes non-motile Gram-negative coccobacilli that act as facultative intracellular pathogens responsible for zoonotic infections. B. melitensis can survive and replicate within host macrophages, the molecular phenomena of this host: pathogen interaction remain totally unknown. The aim of this work was to evaluate the differences in the response between human macrophages infected with different B. melitensis strains. Comparison of transcriptome data was carried out for identifying differentially expressed genes among different strain infection. We evaluated the THP-1 macrophage molecular response at early stages of infection to different strains of Brucella melitensis (B. melitensis wild- type 133 (BM133), B. melitensis ATCC 23456 (BM16M) and a B. melitensis 133 omp31 mutant (LVM31)). Our analysis revealed intriguing differences in the host cell response to two virulent strains (BM16M and BM133), infection with BM16M led to an over-expression of anti-inflammatory pathways, such as cAMP signaling and PI3K-Akt pathway, and down regulation of inflammatory pathways involving IL1A and IL10 compared to BM133. Mutant strain BMLVM31 induced an activation of the apoptotic process and the absence of Omp31, impaired the inhibition of CASP1 and CASP9 expression. Additionally, the mutation of BMLV31 impairs the evasion of cathepsin D in early stages of the infection. These findings shed light on the intricate molecular interactions between B. melitensis strains and human macrophages, providing valuable insights for understanding the pathogenesis of brucellosis.

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barbieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rev1Δwzm is protected under a CSIC-UPNA patent WO/2019/101993 (PCT/EP2018/082539) and both are public institutions without any conflict of interests with this publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Brucellosis is one of the most common global zoonoses and is caused by facultative intracellular bacteria of the genus Brucella. Numerous studies have found that MyD88 signaling contributes to protection against Brucella; however, the underlying mechanism has not been entirely defined. Here, we show that MyD88 signaling in hematopoietic cells contributes both to inflammation and to control of Brucella melitensis infection in vivo . While the protective role of MyD88 in Brucella infection has often been attributed to promotion of gamma interferon (IFN-γ) production, we found that MyD88 signaling restricts host colonization by B. melitensis even in the absence of IFN-γ. In vitro , we show that MyD88 promotes macrophage glycolysis in response to B. melitensis. Interestingly, a B. melitensis mutant lacking the glucose transporter, GluP, was more highly attenuated in MyD88 -/- than in wild-type mice, suggesting MyD88 deficiency results in an increased availability of glucose in vivo , which Brucella can exploit via GluP. Metabolite profiling of macrophages identified several metabolites regulated by MyD88 in response to B. melitensis, including itaconate. Subsequently, we found that itaconate has antibacterial effects against Brucella and also regulates the production of proinflammatory cytokines in B. melitensis-infected macrophages. Mice lacking the ability to produce itaconate were also more susceptible to B. melitensis in vivo . Collectively, our findings indicate that MyD88-dependent changes in host metabolism contribute to control of Brucella infection.

Brucella, a facultative intracellular bacterium, can survive and replicate in various cell types such as epithelial cell, fibroblasts and macrophage. Macrophage is the most important sites for the survival of Brucella in vivo. The mechanisms of pathogenesis are difficult to address, since the unknown virulence genes are still exist. RNA-seq is available to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Here we described and analyzed the transcriptional change of avirulent strain Brucella melitensis M5-90 (B. melitensis M5-90) during macrophage infection using RNA-seq technology. We detected 601 significant changed genes of which 428 were upregulated after infection. The upregulated gene L31 which involved in ribosome KEGG pathway was selected to illustrate its effect on virulence in a vaccine strain B. melitensis M5-90 and a virulent strain B. melitensis M28. Deletion of L31 significant attenuates the spleen colonization in model of M5-90 or M28 infection mouse at 7, 21 and 35 days post-infection (P < 0.05). We further examine the role of L31 in a macrophage cell infection model, and the result showed a significant reduction of intracellular M28ΔL31 cells at 48 h post-infection (P < 0.001). In total, our study provided a view of transcriptional landscape of B. melitensis M5-90 intracellular, and found L31 gene is required for the full virulence of B. melitensis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Brucella species are facultative intracellular bacteria that cause brucellosis, a zoonotic world-wide disease. The live attenuated B. melitensis Rev.1 vaccine strain is widely used for the control of brucellosis in the small ruminant population. However, Rev.1 induces antibodies against the O-polysaccharide (O-PS) of the smooth lipopolysaccharide thus, it is difficult to differentiate between infected and vaccinated animals. Hence, rough Brucella strains lacking the O-PS have been introduced. In the current study, we conducted a comprehensive comparative analysis of the genome sequence of two natural Rev.1 rough strains, isolated from sheep, against that of 24 Rev.1 smooth strains and the virulent reference strain B. melitensis 16M. We identified and characterized eight vital mutations within highly important genes associated with Brucella lipopolysaccharide (LPS) biosynthesis and virulence, which may explain the mechanisms underlying the formation of the Rev.1 rough phenotype and may be used to determine the mechanism underlying virulence attenuation. Further complementation studies aimed to estimate the specific role of these mutations in affecting Brucella morphology and virulence will serve as a basis for the design of new attenuated vaccines for animal immunization against brucellosis.

UDP-glucose pyrophosphorylase (UGPase) is an important pyrophosphatase that reversibly catalyzes the synthesis of UDP-glucose during glucose metabolism. We previously found that the deletion of UGPase may affect structure, growth, the virulence of Brucella, and the activation of cellular NF-κB. However, the exact mechanism of activation of NF-κB regulated by Brucella UGPase is still unclear. Here, we found for the first time that UGPase can regulate the expression of virB proteins (virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11) of type IV secretion system (T4SS) as well as effectors (vceC, btpA, btpB, ricA, bspB, bspC, and bspF) of Brucella by promoting the expression of ribosomal S12 protein (rpsL), BMEI1825, and quinone of 2,4,5-trihydroxyphenylalanine (topA) proteins, and further inhibits the activation of cellular NF-κB and affects the virulence of Brucella. Our findings provide new insights into the mechanism used by Brucella to escape the immune recognition, which is expected to be of great value in the designing of Brucella vaccines and the screening of drug targets., Competing Interests: Declaration of competing interest The authors declare no competing interests. All authors have approved the final version of the manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this paper, we first used recombinant influenza viral vector (rIVV) subtype H5N1 expressing from the open reading frame of NS1 80 and NS1 124 amino acids of Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7/L12, and Cu-Zn superoxide dismutase (SOD) proteins to develop a human brucellosis vaccine. We made 18 combinations of IVVs in mono-, bi-, and tetravalent vaccine formulations and tested them on mice to select the safest and most effective vaccine samples. Then, the most effective vaccine candidates were further tested on guinea pigs. Safety of the rIVV-based vaccine candidate was evaluated by a mouse weight-gain test. Mice and guinea pigs were challenged with the virulent strain B. melitensis 16M. The protective effect of the rIVV-based vaccine candidate was assessed by quantitation of Brucella colonization in tissues and organs of challenged animals. All vaccine formulations were safe in mice. Tested vaccine formulations, as well as the commercial B. melitensis Rev.1 vaccine, have been found to protect mice from B. melitensis 16M infection within the range of 1.6 to 2.97 log 10 units ( P < 0.05). Tetravalent vaccine formulations from the position of NS1 80 amino acids (0.2 ± 0.4), as well as the commercial B. melitensis Rev.1 vaccine (1.2 ± 2.6), have been found to protect guinea pigs from B. melitensis 16M infection at a significant level ( P < 0.05). Thus, tetravalent vaccine formulation Flu-NS1-80-Omp16+Flu-NS1-80-L7/L12+Flu-NS1-80-Omp19+Flu-NS1-80-SOD was chosen as a potential vaccine candidate for further development of an effective human vaccine against brucellosis. These results show a promising future for the development of a safe human vaccine against brucellosis based on rIVVs., Competing Interests: No potential conflict of interest was reported by the authors., (Copyright © 2020 Dina Bugybayeva et al.)

Brucella , the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in Brucella remain unclear. We report here that disruption of Tat increases the sensitivity of Brucella melitensis M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by H 2 O 2 Further, loss of Tat significantly attenuates B. melitensis infection in murine macrophages ex vivo Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of B. melitensis M28. Genome-wide in silico prediction combined with an in vivo amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to Brucella virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in Brucella virulence., (Copyright © 2020 American Society for Microbiology.)

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16 M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors. Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS/MS. A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines. Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Reproductive failure is the hallmark of brucellosis in animals. An uncommon but important complication in pregnant women who become acutely infected with Brucella melitensis is spontaneous pregnancy loss or vertical transmission to the fetus. Unfortunately, the mechanism behind reproductive failure is still obscure, partially due to the lack of a proper study model. Recently, it was demonstrated that intratracheal (IT) inoculation of nonpregnant guinea pigs would replicate features of clinical disease in humans. To determine if IT inoculation would induce reproductive disease, guinea pigs were infected at mid-gestation and monitored daily for fever and abortions. Fever developed between day 14 to 18 postinoculation, and by 3 weeks postinoculation, 75% of pregnant guinea pigs experienced stillbirths or spontaneous abortions mimicking natural disease. Next, to investigate the guinea pig as a model for evaluating vaccine efficacy during pregnancy, nonpregnant guinea pigs were vaccinated with S19, 16MΔ vjbR + Quil-A, or 100 μl PBS + Quil-A (as control). Guinea pigs were bred and vaccinated guinea pigs were challenged at mid-gestation with B. melitensis IT inoculation and monitored for fever and abortions. Vaccination with both vaccines prevented fever and protected against abortion. Together, this study indicates that pregnant guinea pigs are an appropriate animal model to study reproductive disease and offer an improved model to evaluate the ability of vaccine candidates to protect against a serious manifestation of disease., (Copyright © 2020 American Society for Microbiology.)

The intracellular pathogenic bacteria belonging to the genus Brucella must cope with acidic stress as they penetrate the host via the gastrointestinal route, and again during the initial stages of intracellular infection. A transcription-level regulation has been proposed to explain this but the specific molecular mechanisms are yet to be determined. We recently reported a comparative transcriptomic analysis of the attenuated vaccine Brucella melitensis strain Rev.1 against the virulent strain 16M in cultures grown under either neutral or acidic conditions. Here, we re-analyze the RNA-seq data of 16M from our previous study and compare it to published transcriptomic data of this strain from both an in cellulo and an in vivo model. We identify 588 genes that are exclusively differentially expressed in 16M grown under acidic versus neutral pH conditions, including 286 upregulated genes and 302 downregulated genes that are not differentially expressed in either the in cellulo or the in vivo model. Of these, we highlight 13 key genes that are known to be associated with a bacterial response to acidic stress and, in our study, were highly upregulated under acidic conditions. These genes provide new molecular insights into the mechanisms underlying the acid-resistance of Brucella within its host.

Iron is a fundamental element required by most organisms, including Brucella . Several researchers have suggested that the iron response regulator ( irr ) and rhizobial iron regulator ( rirA ) genes regulate iron acquisition by Brucella abortus , influencing heme synthesis by and virulence of this pathogen. However, little is known about another Brucella species, Brucella melitensis . In this research, we successfully constructed two mutants: M5-90Δ irr and M5-90Δ rirA . The adhesion, invasion, and intracellular survivability of these two mutants were evaluated in RAW264.7 cells infected with 1 × 10 6 CFU of M5-90Δ irr , M5-90Δ rirA , or M5-90. We also tested the sensitivity of cells to hydrogen peroxide and their ability to grow. In addition, the virulence of these two mutants was evaluated in BALB/c mice. The results showed that the ability of these two mutants to invade and adhere inside the murine macrophages RAW264.7 was attenuated but their ability to replicate intracellularly was strengthened, enhancing the resistance to hydrogen peroxide. The M5-90Δ irr mutant showed stronger growth ability than the parental strain under iron-limiting conditions. No differences were observed in the number of bacteria in spleen between M5-90 and M5-90Δ irr at 7 or 15 days postinfection. However, the number of M5-90Δ rirA in spleen reduced significantly at 15 days postinfection. The splenic index of the M5-90Δ irr group is evidently lower than that of M5-90. This is the first report that irr and rirA genes of B. melitensis are associated not only with virulence but also with growth ability. Together, our data suggest that M5-90Δ irr is a promising Brucella vaccine candidate.

For control of brucellosis in small ruminants, attenuated B. melitensis Rev1 is used but it can be virulent for animals and human. Based on these aspects, it is essential to identify potential immunogens to avoid these problems in prevention of brucellosis. The majority of OMPs in the Omp25/31 family have been studied because these proteins are relevant in maintaining the integrity of the outer membrane but their implication in the virulence of the different species of this genus is not clearly described. Therefore, in this work we studied the role of Omp31 on virulence by determining the residual virulence and detecting lesions in spleen and testis of mice inoculated with the B. melitensis LVM31 mutant strain. In addition, we evaluated the conferred protection in mice immunized with the mutant strain against the challenge with the B. melitensis Bm133 virulent strain. Our results showed that the mutation of omp 31 caused a decrease in splenic colonization without generating apparent lesions or histopathological changes apparent in both organs in comparison with the control strains and that the mutant strain conferred similar protection as the B. melitensis Rev1 vaccine strain against the challenge with B. melitensis Bm133 virulent strain. These results allow us to conclude that Omp31 plays an important role on the virulence of B. melitensis in the murine model, and due to the attenuation shown by the strain, it could be considered a vaccine candidate for the prevention of goat brucellosis.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Brucella melitensis Rev.1 is the live attenuated Elberg-originated vaccine strain of the facultative intracellular Brucella species, and is widely used to control brucellosis in small ruminants. However, Rev.1 may cause abortions in small ruminants that have been vaccinated during the last trimester of gestation, it is pathogenic to humans, and it induces antibodies directed at the O-polysaccharide (O-PS) of the smooth lipopolysaccharide, thus making it difficult to distinguish between vaccinated and infected animals. Rough Brucella strains, which lack O-PS and are considered less pathogenic, have been introduced to address these drawbacks; however, as Rev.1 confers a much better immunity than the rough mutants, it is still considered the reference vaccine for the prophylaxis of brucellosis in small ruminants. Therefore, developing an improved vaccine strain, which lacks the Rev.1 drawbacks, is a highly evaluated task, which requires a better understanding of the molecular mechanisms underlying the virulence attenuation of Rev.1 smooth strains and of natural Rev.1 rough strains, which are currently only partly understood. As the acidification of the Brucella-containing vacuole during the initial stages of infection is crucial for their survival, identifying the genes that contribute to their survival in an acidic environment versus a normal environment will greatly assist our understanding of the molecular pathogenic mechanisms and the attenuated virulence of the Rev.1 strain. Here, we compared the transcriptomes of the smooth and natural rough Rev.1 strains, each grown under either normal or acidic conditions. We found 12 key genes that are significantly downregulated in the Rev.1 rough strains under normal pH, as compared with Rev.1 smooth strains, and six highly important genes that are significantly upregulated in the smooth strains under acidic conditions, as compared with Rev.1 rough strains. All 18 differentially expressed genes are associated with bacterial virulence and survival and may explain the attenuated virulence of the rough Rev.1 strains versus smooth Rev.1 strains, thus providing new insights into the virulence attenuation mechanisms of Brucella. These highly important candidate genes may facilitate the design of new and improved brucellosis vaccines., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: To investigate the risk factors for brucellosis in suspected cases of the disease., Methods: A self-designed questionnaire was developed to collect data from 3557 people whose initial visit site was the Songyuan Center for Disease Control and Prevention (CDC) from January 1st, 2009 to December 31st, 2012. After collecting blood samples, a plate agglutination test (PAT) and serum agglutination test (SAT) were used to distinguish the patients with brucellosis from the suspected cases., Results: Sex, occupation (farmers and herdsmen), contact with abortion products, and contact with feces were the main risk factors for brucellosis in the suspected cases (all P < 0.05). No difference existed between the confirmed cases and suspected cases in the demographic characteristics, contact with animals (except swine), contact with substances, or clinical symptoms (except fever). However, the confirmed cases showed significant differences from people without brucellosis in demographic characteristics, contact with animals (except cattle and swine), contact with substances, and clinical symptoms. Suspected cases exhibited significant differences from people without brucellosis in the demographic characteristics (except education), contact with animals (except swine), contact with substances (except dust), and clinical symptoms (except chills and acratia). Brucella was cultured from the blood samples of three of 30 suspected cases with fever. Using AMOS-PCR and agarose electrophoresis, the detailed species of Brucella strain was identified as Brucella melitensis., Conclusions: Abortion products and feces are the main risk factors for brucellosis in suspected cases of the disease. Pyrexia in suspected cases with a history of contact with abortion products or feces should raise suspicion for the disease.

Introduction: Brucellosis is a zoonotic disease with variable clinical manifestations and atypical presentation in humans. Human brucellosis cases are not seen often in Malaysia., Case Report: This is a case report of 19 years old gentleman who presented with fever, lower limb redness, pain and swelling. He was initially treated as cellulitis. However, based on the recovery of Brucella melitensis from his blood culture, he was later diagnosed to have brucellosis. He had a history of consumption of fresh goat's milk and uncooked meat which could have been the possible modes of transmission. Brucella serology IgM and IgG were both positive, and anti-Brucella immunocapture agglutination test (BrucellaCapt) was also positive with a titer of 1:2560. He was treated with six weeks of oral doxycycline 100 mg twice daily and oral rifampin 450 mg twice daily., Discussion: This is a case of human brucellosis with atypical cutaneous involvement.

Brucella melitensis is one of the leading zoonotic pathogens with significant economic implications in animal industry worldwide. Lipopolysaccharide, however, remains by far the major virulence with substantial role in diseases pathogenesis. Nonetheless, the effect of B. melitensis and its lipopolysaccharide on immunopathophysiological aspects largely remains an enigma. This study examines the effect of B.melitensis and its lipopolysaccharide on immunopathophysiological parameters following experimental infection using mouse model. Eighty four (n = 84) mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into three groups. Group 1-2 (n = 72) were orally inoculated with 0.4 mL containing 10 9  CFU/mL of B. melitensis and its LPS, respectively. Group 3 (n = 12) was challenged orally with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-infection. We hereby report that B.melitensis infected group demonstrated significant clinical signs and histopathological changes than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1β and IL-6) and antibody levels (IgM and IgG) with varying degrees of predominance in LPS infected group than B. melitensis infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS groups throughout the study period. Moreover, in B. melitensis infected group, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems thereby confirming the infection and transmission dynamics. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in a mouse model after oral inoculation with B. melitensis and its lipopolysaccharide., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Brucella melitensis infection causes acute necrotizing inflammation in pregnant animals; however, the pathophysiological mechanisms leading to placentitis are unknown. Here, we demonstrate that high-mobility group box 1 (HMGB1) acts as a mediator of placenta inflammation in B. melitensis-infected pregnant mice model. HMGB1 levels were increased in trophoblasts or placental explant during B. melitensis infection. Inhibition of HMGB1 activity with neutralising antibody significantly reduced the secretion of inflammatory cytokines in B. melitensis-infected trophoblasts or placenta, whereas administration of recombinant HMGB1 (rHMGB1) increased the inflammatory response. Mechanistically, this decreased inflammatory response results from inhibition of HMGB1 activity, which cause the suppression of both mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Moreover, neutralising antibody to HMGB1 prevented B. melitensis infection-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in trophoblasts. In contrast, in vitro stimulation of trophoblasts with rHMGB1 caused activation of NADPH oxidase and increased the production of ROS, which contributes to high bacterial burden within trophoblasts or placenta. In vivo, treatment with anti-HMGB1 antibody increases the number of Brucella survival within placenta in B. melitensis-infected pregnant mice but successfully reduced the severity of placentitis and abortion., (© 2019 John Wiley & Sons Ltd.)

Brucellar spondylodiscitis, the most prevalent and significant osteoarticular presentation of human Brucellosis, is difficult to diagnose and usually yields irreversible neurologic deficits and spinal deformities. However, no animal models of Brucellar spondylodiscitis exist, allowing for preclinical investigations. The present study investigated whether intraosseous injection of attenuated Brucella melitensis vaccine into rabbits' lumbar vertebrae imitates the radiographic and histopathological characteristics of human Brucellar spondylodiscitis. Radiographic and histopathological analyses at 8 weeks postoperatively revealed radiographic changes within vertebral bodies and intervertebral discs, abscesses formation within the paravertebral soft tissue, and typical prominent inflammation response without caseous necrosis, which were largely comparable to human Brucellar spondylodiscitis. Such a medium-sized, surgically feasible rabbit model provides a promising in vivo setting for further preclinical investigation of Brucellar spondylodiscitis., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Xiaoyu Cai et al.)

Background: Human brucellosis is endemic in China and commonly occurs through contact with infected animals from working with livestock or consumption of unpasteurized dairy products. Although rare, human-to-human, and possible sexual transmission, of Brucella has been reported. In this report, we describe a case of likely mother-to-child transmission of Brucella in Hunan Province, China., Case Presentation: Between June and October 2016, a 28-year old man sought care for testicular swelling and pain at several health facilities. His 26-year old wife developed intermittent fever along with right thigh and hip pain between November 2016 and February 2017 respectively. On April 5, 2017, the female patient delivered a male neonate at 34 weeks of gestation through natural labor. The child's venal blood sample was cultured on April 5, 2017. Brucella was isolated and identified on April 12, 2017. On the same date, serum antibodies of the father and mother were above 1:100 (based on the serum agglutination test [SAT]). The strains isolated from the mother and neonate were identified as Brucella melitensis biotype 1., Conclusions: This report highlights a family cluster of brucellosis. Culture results strongly support mother-to-child transmission, and a high probability of sexual transmission from husband to wife.

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu-like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes., (© 2019 John Wiley & Sons Ltd.)

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens., (Copyright © 2019. Published by Elsevier GmbH.)

Background and Objective: The economic losses due to brucellosis as well as its potential public health in human worldwide encourage more researches to find novel pathways for effective control methods of the disease. The objective of this study was to investigate the most prevalent Brucella strains obtained from cattle and their virulence genes., Materials and Methods: Three hundred small-holders cows in Menoufia governorate, Egypt, were screened for brucellosis using rose bengal test (RBT) and confirmed by complement fixation test (CFT). Milk samples and supra-mammary lymph nodes of serologically positive cows were collected for bacteriological isolation and identification. The obtained isolates were genotyped using PCR and their virulence genes (omp25, omp31, manA, manB, virB and znuA) were screened., Results: The prevalence rate of bovine brucellosis was 15 (5%), 11 (3.6%) and 7 (2.33%) by RBT, CFT and bacteriological examination, respectively. The seven isolates were identified and genotyped as Brucella melitensis biotype3. Furthermore, the molecular detection of substantial virulence genes revealed that manA, manB, omp25 and omp31 genes were detected in all tested B. melitensis strains. Meanwhile, the virB genes were detected in 4 strains and the znuA genes were detected in 3 strains among the isolated B. melitensis strains., Conclusion: It was concluded that B. melitensis biotype3 was the pre-dominant Brucella spp. as well as omp25, omp31, manA and manB were the most common related-virulence genes which assumed to play a worthy function in the pathogenesis of brucellosis.

Objective: This study investigated the association of Brucella seropositivity and spontaneous abortions in human population in the city of Mwanza, Tanzania., Results: A comparative cross sectional study which used 148 sera from women with spontaneous abortion and 250 sera from full-term delivered women was conducted in July 2017. Detection of Brucella abortus and Brucella melitensis antibodies was done using slide agglutination test. Data were analyzed using STATA version 13 software. The median age of the study participants was 25 (interquartile range 21-30) years. The overall seropositivity of Brucella antibodies was significantly higher among sera from women with spontaneous abortion than full term delivered women; (86/148, 58.1%: 95% CI 50-66 vs. 65/250, 26%: 95% CI 18-33, P < 0.001). Seropositivity of B. abortus was significantly higher among sera from women with spontaneous abortion than full-term delivered women (31.8% vs. 10.8%, P < 0.001). Women with abortion had 3.59 odds of being brucella seropositive compared to full term women (OR: 3.59, 95% CI; 2.25-5.74, P < 0.001). Seropositivity of Brucella antibodies is significantly higher among women with spontaneous abortion than full-term delivered women necessitating a need to investigate the relationship between Brucellosis and adverse pregnancy outcomes.

Susceptibilities of 66 Brucella isolates were tested in vitro. All isolates were susceptible to doxycycline, gentamic in and streptomycin. In addition, propyl paraben, cresol and benzalkoniumchloride were found to be the most powerful tested preservative, disinfectant and antiseptic, respectively. All isolates adhered to and invaded into Vero cells by variable degrees. Adherence and invasion of most isolates were significantly reduced by: (1) pretreatment of test isolates with trypsin and sodium metaperiodate; (2) pretreatment of Vero cells with lipase, neuraminidase and sodium metaperiodate; (3) Presence of Ca ++ , Mg ++ and 200mM mannose in the assay medium and (4) growth of test isolates in half MICs of different antimicrobial agents. On the other hand, pretreatment of Vero cells with trypsin increased the adherence and invasion of most test isolates. No significant change in adhesion and invasion by changing the temperature from 27°C to 42°Cor the pH from 6 to 8. Log phase cultures showed higher adherence and invasion than stationary phase cultures.

This study aimed to develop direct PCR methods, which enable the diagnosis of brucellosis agents from ruminant aborted fetus samples at species and genus levels, and determine the applicability of the newly developed methods. For this purpose, 137 lung, 137 liver, and 52 fetal stomach fluid samples belonging to 166 ruminant aborted fetuses (326 samples in total) were examined. Firstly, agent isolation and identification were performed and species-specific multiplex PCR (m-PCR) from the culture was applied to the samples. In addition, the Mayer-Scholl m-PCR method was modified and termed 'modified Mayer-Scholl', and genus specific Bcsp31 PCR was also modified with minor changes. Four different methods were applied to direct examination samples and the obtained results were compared. The conventional culture method was set as the standard method to which sensitivities and specificities of the molecular methods were calculated. According to the assessments on the basis of fetus (n = 166), sensitivity and specificity values for modified Mayer-Scholl m-PCR method were 94.11% and 98.76%, and the same indicators for the modified Bcsp31 PCR were 95.29% and 98.76%, respectively. When all organ samples were taken into account (n = 326), sensitivity and specificity values for the modified Mayer-Scholl m-PCR method were 85.38% and 98.06%, and for the modified Bcsp31 PCR, they were 83.62% and 98.06%, respectively. As a result, it was found that the diagnostic power of the tests were 'high' when results were evaluated at fetus level. On the other hand, it was found to be 'clinically useful' when evaluated at organ level. We concluded that species level identifications can be made through the modified Mayer-Scholl method, which is a direct m-PCR method, with a high diagnostic power by specifying DNAs belonging to Brucella species directly from clinical samples., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brucellosis, caused by Brucella spp., is one of the most serious zoonotic bacterial diseases. Small RNAs (sRNAs) are recognized as a key player in bacterial post-transcription regulation, since they participate in many biological processes with high efficiency and may govern the intracellular biochemistry and virulence of some pathogenic bacteria. Here, a novel small regulatory RNA, Bmsr1 (Brucella melitensis M28 small RNA 1), was identified in a virulent Brucella melitensis M28 strain based on bioinformatic analysis, reverse transcription PCR (RT-PCR), and Northern blot. The Bmsr1 expression level was highly induced after infection of macrophage cells RAW264.7 at 48 h, suggesting a role for Bmsr1 during in vitro infection. Indeed, bmsr1 deletion mutant of M28 attenuated its intracellular survival in RAW264.7 at 24 h and 48 h post-infection. In a mouse model of chronic infection, bmsr1 deletion strain displayed decreased colonization in the spleen while Bmsr1-overexpressed strain showed higher colonization levels than wild type pathogen. Isobaric tags for relative and absolute quantification (iTRAQ) revealed that 314 proteins were differentially expressed in M28Δbmsr1 compared with wild type. Functional annotation analysis demonstrated that most of those proteins are involved in biological processes and those proteins in the ribosome and nitrogen metabolism pathways were enriched. iTRAQ results combined with target prediction identified several potential target genes related to virulence, including virB2, virB9, virB10, virB11, and vjbR and many metabolism genes. Taken together, this study revealed the contribution of a novel sRNA Bmsr1 to virulence of B. melitensis M28, probably by influencing genes involved in T4SS, virulence regulator VjbR and other metabolism genes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR) sensor IRE1α (inositol-requiring enzyme 1) and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1) conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella .

Background: Brucellosis is one of the most widespread zoonoses worldwide. It can affect any organ system, particularly the gastrointestinal system, but there is no report of acute liver failure as a brucellosis complication., Case Presentation: We present a case of acute liver failure secondary to brucellosis infection. A 75-year-old Hispanic man presented to a University Hospital in Chía, Colombia, with a complaint of 15 days of fatigue, weakness, decreased appetite, epigastric abdominal pain, jaundice, and 10 kg weight loss. On examination in an emergency room, abdomen palpation was normal with hepatosplenomegaly and the results of a liver function test were elevated. The diagnosis of brucellosis was confirmed by epidemiological contact and positive Rose Bengal agglutination with negative enzyme-linked immunosorbent assay immunoglobulin M for Brucella. He was then treated with doxycycline plus trimethoprim/sulfamethoxazole, with a favorable clinical outcome., Conclusions: The clinical presentation of brucellosis can be very imprecise because it can affect any organ system; however, there is no report of acute liver failure as a brucellosis complication. This is the first reported case in the Colombian literature of acute liver failure due to brucellosis. We found this case to be of interest because it could be taken into account for diagnosis in future appearances and we described adequate treatment and actions to be taken at presentation.

Brucella melitensis is a major zoonotic pathogen in which lipopolysaccharide (LPS) is believed to play a major role in the diseases pathogenesis. To study the immunopathophysiological aspects, we established a mouse model experimentally infected with whole cell of B. melitensis and its lipopolysaccharide via subcutaneous route of exposure. Eighty four mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into 3 groups. Group 1 (n = 36) were subcutaneoulsy inoculated with 0.4 mL 10 9 of B. melitensis while group 2 (n = 36) were subcutaneously challenged with 0.4 mL 10 9 of LPS. Group 3 (n = 12) was challenged subcuatneously with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-inoculation. Our results revealed that B. melitensis infected group demonstrated significant clinical signs and histopathological evidence than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1β & IL6), antibody levels (IgM & IgG) as early as 3 days post-infection with predominance in LPS infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS challenged groups throughout the study period. Moreover, in B. melitensis infected groups, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems; thereby confirming the possible transmission of the disease dynamics. Moreover, LPS stimulated significantly the innate and acquired immune system without significant systemic dysfunction suggesting the potentiality of the protective properties of this component as an alternative vaccine for brucellosis infection. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in mouse model after subcutaneous inoculation with B. melitensis and its lipopolysaccharide., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Brucellosis remains a disease that is very difficult to control and eradicate in Greece. Information exchange between the responsible authorities is crucial in order to support public health infrastructure in the sense of the 'One-Health' strategy model., Methods: The data for 2007-2012 were retrieved from the notifiable diseases system and analysed statistically for correlations between human brucellosis cases and the disease in small ruminants. Disease-related risk factors were also estimated with parallel exploitation mapping software., Results: In Greece the dominant strain for brucellosis is Brucella melitensis. The average incidence in Greece was estimated to be 1.43/100,000. The majority of human cases were males (67.60%). The age distribution of brucellosis patients differs significantly between men and women. Brucellosis in male patients was related to high risk jobs and animal contact, while brucellosis in females was related to recent consumption of dairy products. Seasonality of the disease was different in relation to the European countries an observation attributed to the traditional customs. There was a statistically significant difference in human brucellosis incidence between the eradication and vaccination zones., Conclusion: The updated information on brucellosis in Greece revealed differences in seasonality and transmission patterns. A more active cooperation between the involved public health-related sectors should be followed in order to effectively fight brucellosis as there are still foci of brucellosis in Greece.

Background: Brucellosis, also known as undulant, Mediterranean or Malta fever, is a systemic infection that causes fever, sweats, arthralgias and myalgias. A globally important disease, brucellosis is re-emerging in Australia in association with feral pig hunting activities., Objective: This article aims to provide clinicians with an overview of brucellosis, covering epidemiology, clinical features, diagnosis, management and prevention., Discussion: Brucellosis should be suspected in all patients with non-specific, flu-like illness who fall into one of the major risk groups (feral pig hunters, overseas travellers and migrants). Depression is common and often severe, relative to other symptoms. Early diagnosis and treatment are important for preventing complications, which include osteoarticular, genitourinary or, more rarely, neurological or cardiovascular diseases. Diagnosing acute infections is&nbsp;based on serology and blood cultures; imaging and biopsy may be required for diagnosis of focal infections. Dual therapy with doxycycline and gentamicin is the recommended treatment. Relapse occurs in up to 10% of patients. Prevention is achieved through the use of protective gear during hunting and avoidance of unpasteurised dairy products in countries where occur in animals.

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella -containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella -containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus -infected HeLa cells and trophoblasts derived from B. melitensis -infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The live attenuated Brucella melitensis Rev.1 Elberg-originated vaccine strain has been widely used to control brucellosis in small ruminants. However, despite extensive research, the molecular mechanisms underlying the attenuation of this strain are still unknown. In the current study, we conducted a comprehensive comparative analysis of the whole-genome sequence of Rev.1 against that of the virulent reference strain, B. melitensis 16M. This analysis revealed five regions of insertion and three regions of deletion within the Rev.1 genome, among which, one large region of insertion, comprising 3,951 bp, was detected in the Rev.1 genome. In addition, we found several missense mutations within important virulence-related genes, which may be used to determine the mechanism underlying virulence attenuation. Collectively, our findings provide new insights into the Brucella virulence mechanisms and, therefore, may serve as a basis for the rational design of new Brucella vaccines.

Brucellosis is a group of closely associated zoonotic bacterial illnesses caused by members of the genus Brucella. B. melitensis Omp31 is a promising candidate for a subunit vaccine against brucellosis. This study surveyed the immunogenicity of Omp31 alone and with incomplete Freund's adjuvant (Omp31-IFA) and N -trimethyl chitosan (TMC/Omp31) nanoparticles (NPs), as well as the effect of Omp31 immunization route on immunological responses and protection. After expression and purification, the recombinant Omp31 (rOmp31) was loaded onto TMC NPs by ionic gelation. The particle size, loading efficiency and in vitro release of the NPs were examined. Omp31-IFA was administered intraperitoneally, while TMC/Omp31 NPs were administered orally and intraperitoneally. According to the antibody subclasses and cytokine profile, intraperitoneal immunization by Omp31-IFA and TMC/Omp31 NPs induced T helper 1 (Th1) and Th1-Th2 immune responses, respectively. On the other hand, oral immunization with TMC/Omp31 NPs elicited a mixed Th1-Th17 immune response. Data obtained from the cell proliferation assay showed that vaccination with Omp31 stimulated a vigorous antigen-specific cell proliferative response, which could be further increased after oral immunization with TMC/Omp31 NPs. Vaccinated groups of mice when challenged with B. melitensis 16M were found to be significantly protected in the orally administered group in comparison with the intraperitoneally immunized mice. Results of this study indicated that the reason for high protection after oral vaccination can be via elicited Th17 response., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

In a context of (re)emerging infectious diseases with wildlife reservoirs, understanding how animal ecology shapes epidemiology is a key issue, particularly in wild ungulates that share pathogens with domestic herbivores and have similar food requirements. For the first time in Europe, brucellosis (Brucella melitensis), a virulent zoonosis, persisted in an Alpine ibex (Capra ibex) population and was transmitted to cattle and humans. To better understand disease dynamics, we investigated the relationships between the spatial ecology of ibex and the epidemiology of brucellosis. Combining home range overlap between 37 GPS-collared individuals and visual observations of 148 visually-marked individuals monitored during the 2013-2016 period, we showed that females were spatially segregated in at least 4 units all year round, whereas males were more prone to move between female units, in particular during the rutting period. In addition to ibex age, the spatial structure in females largely contributed to variation in seroprevalence in the whole population. These results suggest that non-sexual routes are the most likely pathways of intraspecific transmission, crucial information for management. Accounting for wildlife spatial ecology was hence decisive in improving our ability to better understand this health challenge involving a wildlife reservoir.

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209 + MZ macrophages (MZMs) and the CD169 + marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection., (Copyright © 2017 American Society for Microbiology.)

Brucella melitensis is the causative agent of brucellosis in small ruminants and is of considerable economic and public health importance in many countries worldwide. The control of disease in humans depends on the control of disease in livestock; however, few counties with endemic B. melitensis infection have been able to successfully eradicate this pathogen. This underscores the need for further research on the pathogenesis of both virulent and vaccine strains of B. melitensis in the small ruminant host. The aim of the present study was to characterize clinical effects, tissue colonization, shedding, and humoral immune response following B. melitensis infection in goats. Both virulent (16M) and reduced virulence (Rev. 1) strains of B. melitensis were studied. Pregnant goats were infected at 11-14 weeks of gestation with 8 x 106 or 8 x 107 CFU of B. melitensis. Infection of goats with B. melitensis 16M resulted in an 86% abortion rate. This strain disseminated widely in pregnant does post-infection with none of the 15 sampled tissues spared from colonization. Importantly, we report the first isolation of B. melitensis from muscle tissue in ruminants. Pathogenesis of Rev. 1 infection was variable with two does showing minimal colonization and one doe exhibiting disease similar to that of animals infected with fully virulent 16M. Shedding of B. melitensis in milk occurred in all 16M- and Rev. 1- infected goats. In pregnant animals challenged with virulent B. melitensis, median time to seroconversion was 21 days; however, 2 animals did not seroconvert until after abortion.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

Brucellosis is an infectious disease that affects practically all species of mammals, including human, and is a major zoonosis worldwide. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in phagocytic and nonphagocytic cells such as trophoblast and epithelial cells. Among the six recognized species of the genus Brucella, Brucella melitensis is the main etiological agent involved in goat brucellosis and is also the most pathogenic for human. It causes significant losses in livestock production as a result of abortions, metritis, infertility, and birth of weak animals. Outer membrane proteins (OMPs) are exposed on the bacterial surface and are in contact with cells and effectors of the host immune response, whereby they could be important virulence factors of Brucella species. To evaluate this hypothesis, the gene encoding for the major outer membrane protein Omp31 was amplified, cloned into pUC18 plasmid, and inactivated by inserting a kanamycin cassette, rendering pLVM31 plasmid which was transformed into B. melitensis wild-type strain to obtain LVM31 mutant strain. The Outer membrane (OM) properties of the mutant strain were compared with B. melitensis Bm133 wild-type and B. melitensis Rev1 vaccine strains, in assessing its susceptibility to polymyxin B, sodium deoxycholate, and nonimmune serum. The mutant strain was assessed in vitro with survival assays in murine macrophages J774.A1 and HeLa cells. Our results demonstrate that LVM31 mutant is more susceptible to polymyxin B, sodium deoxycholate, and nonimmune serum than control strains; moreover, Omp31 mutation caused a decrease in the internalization and a significant decrease in the intracellular survival compared with the reference strains in both cell lines. These results allow us to conclude that Omp31 is important for maintaining OM integrity, but also it is necessary for bacterial internalization, establishment and development of an optimal replication niche, and essential for survival and intracellular multiplication.

In this study, we developed a mouse model and characterized the effects of intranasal inoculation of virulent Brucella melitensis strain 16M and its lipopolysaccharide (LPS). The effects of the exposure were compared with respective control groups. Both Brucella melitensis-infected and LPS-infected groups showed no significant clinical presentation with minor relevance in the mortality associated with the infection. In Brucella melitensis-infected group, significant histopathological changes in comparison to the LPS infected group with increase bacterial burden in the lungs, reproductive and reticuloendothelial organs were observed. However, both infected groups showed elevated levels of pro-inflammatory cytokine expression (IL-1β and IL6) and antibody production (IgM an IgG) as early as 3 days post-infection with predominance in LPS infected group. In contrast, low levels of sex related hormonal changes was recorded in both infected groups throughout the experimental period. This is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in mouse model after intranasal inoculation with B. melitensis and its lipopolysaccharide. The study revealed a significant difference between infected and control groups with overlap in clinical, pathological, and immunological responses as well as sex related hormonal changes resulting from the infections., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Brucella is Gram-negative intracellular bacterial pathogen that infects humans and animals and contributes to great economic losses in developing countries. Presently, live attenuated Brucella vaccines (Brucella melitensis M5-90) are the most effective means of brucellosis control and prevention in animals. However, these vaccines have several drawbacks, such as an inability to distinguish between a natural infection and immunization and an association with abortions in pregnant animals. Therefore, this study constructed a Brucella M5-90Δbp26 mutant and evaluated its virulence. The survival of the M5-90Δbp26 mutant was attenuated in human placenta trophoblastic 8 cells (HPT-8 cells) and in BALB/c mice, with a high immunoprotectivity noted in mice. Furthermore, safety tests showed that the M5-90Δbp26 mutant was less virulent than the M5-90 vaccine strain. Additionally, an indirect enzyme-linked immunosorbent assay (ELISA) screening was shown to detect the presence of Brucella protein 26 (BP26) with high sensitivity, with M5-90Δbp26 inoculation accompanied with a lack of BP26 expression, and was further confirmed by western blotting. Together, the M5-90Δbp26 mutant and the indirect ELISA can be employed to distinguish vaccinated livestock from infected animals.

Brucellosis is known as one of important zoonosis. Studying the histological and biochemical effects of the disease could help to increase our knowledge about it. The aim of the present study was to evaluate changes of plasma parameters after intraperitoneal injection of two species of Brucella (Brucella melitensis and Brucella abortus) and two vaccines (Rev-1, RB-51) in the rat. Forty male rats were divided into five groups (n = 8 in each group). Two groups received suspensions of Brucella abortus and Brucella melitensis and two other groups were injected intraperitoneally with two mentioned vaccines and the last group received only distilled water. The results showed a significant increase in sphingosine 1-phosphate, Malondialdehyde, hepcidin, homocysteine, cardiac troponin I and copper levels and a considerable decrease in the levels of iron and zinc (P ≤ 0.01) in infected groups compared to the control animals. In vaccinated groups, hepcidin was increased but other parameters were not changed in comparison to the control group. It can be concluded that increase of homocysteine and cardiac troponin I in brucellosis could be a warning for cardiac adverse effects. Besides, increase of sphingosine 1-phosphate probably indicates its stimulant and modulatory effects in anti- Brucellosis biochemical pathways of the host., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Brucella is zoonotic pathogen that induces abortion and sterility in domestic mammals and chronic infections in humans called Malta fever. It is a facultative intracellular potential pathogen with high infectivity. The virulence of Brucella is dependent upon its potential virulence factors such as enzymes and cell envelope associated virulence genes. The aim of this study was to investigate the Brucella virulence factors among strains isolated from humans and animals in different parts of Iran. Seventy eight strains of Brucella species isolated from suspected human and animal cases from several provinces of Iran during 2015-2016 and identified by phenotypic and molecular methods. The multiplex-PCR (M-PCR) assay was performed in order to detect the ure, wbkA, omp19, mviN, manA and perA genes by using gene specific primers. Out of 78 isolates of Brucella spp., 57 (73%) and 21 (27%) isolates were detected as B. melitensis and B. abortus, respectively, by molecular method. The relative frequency of virulence genes ure, wbkA, omp19, mviN, manA and perA were 74.4%, 89.7%, 93.6%, 94.9%, 100% and 92.3%, respectively. Our results indicate that the most of Brucella strains isolated from this region possess high percent of virulence factor genes (ure, wbkA, omp19, mviN, manA and perA) in their genome. So, each step of infection can be mediated by a number of virulence factors and each strain may have a unique combination of these factors that affected the rate of bacterial pathogenesis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)