Type Brucella Research Articles

Minimum Core Genome Sequence Typing of Brucella From China

Abstract The Gram-negative bacterial genus Brucella includes six classic species based on host specificity, pathogenicity and phenotypic differences. Four more Brucella species were identified in 2007. Although many Brucella genomes have been sequenced, genome sequences and analysis of Brucella strains isolated in China are still scarce. An efficient genome-based Brucella typing method is also needed. In this study, we used the minimum core genome (MCG) typing method to identify and type Brucella strains. Twenty Brucella isolates from China were newly sequenced. The genome sequences of 55 representative Brucella strains were downloaded. Among the 75 genomes, 1089 genes and 52,030 single nucleotide polymorphisms (SNPs) shared by all isolates were considered as the MCG genes and MCG SNPs. Using these 52,030 MCG SNPs, Brucella was divided into six MCG groups. In addition, average nucleotide identity (ANI) values and the distributions of 184 virulence genes were all computed. The proportions of virulence genes were 90.96%, 93.56%, 95.89%, 86.04%, 85.78% and 91.87% for MCG groups 1 to 6, respectively. The intragroup ANI values were higher than the intergroup values, further confirming the validity of the MCG taxonomy classification. Brucella melitensis and Brucella abortus, the two main Brucella species pathogenic to humans, were well separated from other species. With the development and cost reduction of next-generation sequencing, the MCG typing method can be used for rapid identification of Brucella, which can contribute to the rapid diagnosis of brucellosis and ensure timely and effective treatment.

Deletion of the LuxR-type regulator VjbR in Brucella canis affects expression of type IV secretion system and bacterial virulence, and the mutant strain confers protection against Brucella canis challenge in mice

Brucella spp. are facultative intracellular pathogens and zoonotic agents which pose a huge threat to human health and animal husbandry. The B. melitensis, B. abortus, and B. suis cause undulant fever and influenza-like symptoms in humans. However, the effects of B. canis have not been extensively studied. The quorum sensing-dependent transcriptional regulator VjbR influences the Brucella virulence in smooth type Brucella strains, such as B. melitensis, B. abortus and rough type Brucella ovis. However, the function of VjbR in the rough-type B. canis is unknown. In the present study, we discovered that deletion of this regulator significantly affected Brucella virulence in macrophage and mice infection models. The expression levels of virB operon and the ftcR gene were significantly altered in the vjbR mutant strain. We further investigated the protective effect of different doses of the vjbR mutant in mice and the results indicated that VjbR conferred protection against the virulent B. canis strain. This study presents the first evidence that the transcriptional regulator VjbR has important function in B. canis. In addition, according to its reduced virulence and the protective immunity it induces in mice, it can be a potential live attenuated vaccine against B. canis.

Partial protection induced by Salmonella based Brucella vaccine candidate in pregnant guinea pigs

Residual virulence is a major drawback in current Brucella vaccines. Live vaccines induce abortions in pregnant animals. Hence, a novel anti-Brucella vaccine was developed utilizing rough Salmonella delivering four Brucella antigens. Safety implications during pregnancy, humoral immune responses, and protective efficacy against wild type Brucella was investigated in guinea pig model. The vaccine did not induce abortions or severe complications in pregnant guinea pigs when administered 4 × 108 CFU via intraperitoneal route. Systemic IgG determination against antigen components reveals induction of immunity via the Salmonella delivery. Protection efficacy against abortions was 33.3% (2/6) when midterm sow challenged with virulent Brucella 544 strain while none was protected in control group. Lower Brucella recovery in spleen and liver and reduced histopathological burden were also noticed. Although abortion induced by Brucella challenge was not completely prevented, the vaccine candidate may perform better with optimization of vaccination such as inoculation dose optimization.

Geographical distribution of Brucella melitensis inferred from rpoB gene variation.

Currently available tests have limitations for the identification of Brucella species and strains, and their genetic lineage. The genome sequence of the rpoB gene encoding the β-subunit of DNA-dependent RNA polymerase was investigated for its use in genotyping Brucella melitensis. Complete rpoB gene sequences of globally distributed Brucella melitensis strains were analyzed. Single nucleotides polymorphisms (SNPs) of the rpoB gene sequences were identified and used to type Brucella melitensis strains. Six DNA polymorphisms were identified, of which two (nucleotides 3201 and 558) were novel. Analysis of the geographical distribution of the strains revealed a spatial clustering pattern with rpoB type 1 representing European and American strains, rpoB type 2 representing European, African, and Asian strains, rpoB type 3 representing Mediterranean strains, and rpoB type 4 representing African (C3201T) and European (C3201T/T558A) strains. We report the discovery of two novel SNPs of rpoB gene that can serve as useful markers for epidemiology and geographical tracking of B. melitensis.

Genetic Diversity of Brucella Reference and Non-reference Phages and Its Impact on Brucella-Typing.

Virulent phages have been used for many years to type Brucella isolates, but until recently knowledge about the genetic makeup of these phages remains limited. In this work the host specificity and genomic sequences of the original set (deposited in 1960) of VLA Brucella reference phages Tb, Fi, Wb, Bk2, R/C, and Iz were analyzed and compared with hitherto described brucellaphages. VLA phages turned out to be different from homonymous phages in other laboratories. The host range of the phages was defined by performing plaque assays with a wide selection of Brucella strains. Propagation of the phages on different strains did not alter host specificity. Sequencing of the phages TbV, FiV, WbV, and R/CV revealed nucleotide variations when compared to same-named phages previously described by other laboratories. The phages Bk2V and IzV were sequenced for the first time. While Bk2V exhibited the same deletions as WbV, IzV possesses the largest genome of all Brucella reference phages. The duplication of a 301 bp sequence in this phage and the large deletion in Bk2V, WbV, and R/CV may be a result of recombination caused by repetitive sequences located in this DNA region. To identify new phages as potential candidates for lysotyping, the host range and Single Nucleotide Polymorphisms (SNPs) of 22 non-reference Brucella phages were determined. The phages showed lysis patterns different from those of the reference phages and thus represent novel valuable candidates in the typing set.

Brucella melitensis Methionyl-tRNA-Synthetase (MetRS), a Potential Drug Target for Brucellosis.

We investigated Brucella melitensis methionyl-tRNA-synthetase (BmMetRS) with molecular, structural and phenotypic methods to learn if BmMetRS is a promising target for brucellosis drug development. Recombinant BmMetRS was expressed, purified from wild type Brucella melitensis biovar Abortus 2308 strain ATCC/CRP #DD-156 and screened by a thermal melt assay against a focused library of one hundred previously classified methionyl-tRNA-synthetase inhibitors of the blood stage form of Trypanosoma brucei. Three compounds showed appreciable shift of denaturation temperature and were selected for further studies on inhibition of the recombinant enzyme activity and cell viability against wild type B. melitensis strain 16M. BmMetRS protein complexed with these three inhibitors resolved into three-dimensional crystal structures and was analyzed. All three selected methionyl-tRNA-synthetase compounds inhibit recombinant BmMetRS enzymatic functions in an aminoacylation assay at varying concentrations. Furthermore, growth inhibition of B. melitensis strain 16M by the compounds was shown. Inhibitor-BmMetRS crystal structure models were used to illustrate the molecular basis of the enzyme inhibition. Our current data suggests that BmMetRS is a promising target for brucellosis drug development. However, further studies are needed to optimize lead compound potency, efficacy and safety as well as determine the pharmacokinetics, optimal dosage, and duration for effective treatment.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

Early transcriptional responses of bovine chorioallantoic membrane explants to wild type, ΔvirB2 or ΔbtpB Brucella abortus infection.

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion.

Clinical Responses and Reproductive Pathological Changes Associated with &lt;em&gt;Brucella melitensis&lt;/em&gt; and it?s Lipopolysaccharides in Female Mice

Brucellosis is a bacterial disease caused by the genus Brucella, in small ruminants it is caused by Brucella melitensis and is a Gram negative, facultative intracellular coccobacillus bacterium. It is characterized by significant reproductive problems and lead to massive economical loses. Hence, this study is designed to observed clinical responses and pathological changes in reproductive organs in female mice via intraperitoneal inoculation. Twenty four healthy female mice were divided into three groups. The treatment groups were inoculated with 1.0 mL of 10 9 wild type Brucella melitensis and 1.0 mL of 10 9 Brucella LPS intraperitoneally while; the control group was inoculated intraperitoneally with 1.0 mL of sterile phosphate buffer. The mice were observed for clinical signs for 10 days of post inoculation. Female reproductive organs were collected after 10 days for histopathological study. Mice in the Brucella group developed severe clinical signs compared to mice infected with LPS. The clinical signs observed were ruffled fur, movement, responsiveness and eye conditions. The pathological changes in the reproductive organs were moderate to severe in the Brucella group in relation to inflammatory cells, mild to moderate necrosis, degeneration; congestion and hemorrhage were also observed. The most affected reproductive organ post inoculation with the wild type Brucella was the ovary especially in relation to the infiltration of inflammatory cells and congestion/hemorrhage. LPS group developed similar lesions with the Brucella group except that the LPS group developed normal to mild necrosis and degeneration. The ovary of the mice infected with LPS developed moderate to severe lesions of inflammation with congestion/hemorrhage but lesser necrosis. In general, the oviducts were moderately inflamed with mild congestion. Some part of the oviduct also showed necrotic and degenerated lesions. Besides that, the vulva of mice in LPS group developed more necrotic lesions compared to the Brucella group. This finding suggests that, Brucella immunogen (LPS) is a good candidate for the development of Brucellosis vaccine.

Transcriptome analysis of HeLa cells response to Brucella melitensis infection: a molecular approach to understand the role of the mucosal epithelium in the onset of the Brucella pathogenesis

Brucella spp. infect hosts primarily by adhering and penetrating mucosal surfaces, however the initial molecular phenomena of this host:pathogen interaction remain poorly understood. We hypothesized that characterizing the epithelial-like human HeLa cell line molecular response to wild type Brucella melitensis infection would help to understand the role of the mucosal epithelium at the onset of the Brucella pathogenesis. RNA samples from B. melitensis-infected HeLa cells were taken at 4 and 12 h of infection and hybridized in a cDNA microarray. The analysis using a dynamic Bayesian network modeling approach (DBN) identified several pathways, biological processes, cellular components and molecular functions altered due to infection at 4 h p.i., but almost none at 12 h p.i. The in silico modeling results were experimentally tested by knocking down the expression of MAPK1 by siRNA technology. MAPK1-siRNA transfected cell cultures decreased the internalization and impaired the intracellular replication of the pathogen in HeLa cells after 4 h p.i. DBN analysis provides important insights into the role of the epithelial cells response to Brucella infection and guide research to novel mechanisms identification.

Analyses of Brucella Pathogenesis, Host Immunity, and Vaccine Targets using Systems Biology and Bioinformatics

Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning.

Use of Amplified Fragment Length Polymorphism To Identify and Type Brucella Isolates of Medical and Veterinary Interest

Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.