Genomic insights into Brucella

Brucellosis is a zoonotic disease caused by certain species of Brucella. Each species has its preferred host animal, though it can infect other animals too. For a longer period, only six classical species were recognized in the genus Brucella. No vaccine is available for human brucellosis. Therefore, human brucellosis can be controlled only by controlling brucellosis in animals. The genus is now expanding with the newly isolated atypical strains from various animals, including marine mammals. Presently, 12 species of Brucella have been recognized. The first genome of Brucella was released in 2002, and today, we have more than 1500 genomes of Brucella spp. isolated worldwide. Multiple genome sequences are available for the major zoonotic species, B. abortus, B. melitensis, and B. suis. The Brucella genome has two chromosomes with the approximate sizes of 2.1 and 1.2 Mbp. The genome of Brucella is highly conserved across all the species at the nucleotide level. One of the unanswered questions is what makes host preference in different species of Brucella. Here, I summarize the recent advancements in the Brucella genomics research.     

Development and evaluation of a core genome multilocus sequence typing (cgMLST) scheme for Brucella spp.

Brucellosis is a zoonotic disease caused by Brucella spp. Brucella spp. can be sub-typed by multilocus sequence typing (MLST) method, which targets a set of housekeeping genes. We have developed a core genome MLST (cgMLST) typing scheme to distinguish and differentiate species of Brucella up to biovar level. A total of 407 whole (complete and draft) genome sequences of different Brucella strains were used in this study. Genome sequences were filtered using the BLAST score ratio (BSR)-based allele calling algorithm, and we found that 164 cgMLST target loci are shared in all the 407 genome sequences. These 164 loci were used to develop the cgMLST scheme and further evaluated to sub-type different species of Brucella. Based on our cgMLST scheme, Brucella spp. were classified into 287 sequence types (STs). A phylogenetic tree was constructed based on the STs derived from the cgMLST analysis. The phylogenetic tree differentiated all the 11 Brucella spp. and five biovars of B. suis. B. vulpis formed the outmost clade followed by B. inopinata and B. microti. Among the four subgroups of B. abortus, group A and B were differentiated based on their geographic origins. Similarly, three subgroups of B. melitensis were separated based on their geographical origins with few exceptions. B. neotomae that infect rodents were distinguished from other Brucella spp. B. canis showed the closest relationship with B. suis bv. 4, followed by B. suis bv. 3 and bv. 1. Brucella spp. associated with the marine mammals, such as B. ceti and B. pinnipedialis were closely related. Of these, B. ceti strains isolated from dolphins and porpoise were differentiated into two groups. We incorporated our cgMLST tool in BrucellaBase (http://www.dbtbrucellosis.in/brucella_cgmlst.html), which will be helpful to predict the cgMLST allelic profile and the ST of a newly sequenced genome.     

Emerging diversity and ongoing expansion of the genus Brucella

Remarkable genetic diversity and breadth of host species has been uncovered in the Brucella genus over the past decade, fundamentally changing our concept of what it means to be a Brucella. From ocean fishes and marine mammals, to pond dwelling amphibians, forest foxes, desert rodents, and cave-dwelling bats, Brucella have revealed a variety of previously unknown niches. Classical microbiological techniques have been able to help us classify many of these new strains but at times have limited our ability to see the true relationships among or within species. The closest relatives of Brucella are soil bacteria and the adaptations of Brucella spp. to live intracellularly suggest that the genus has evolved to live in vertebrate hosts. Several recently discovered species appear to have phenotypes that are intermediate between soil bacteria and core Brucella, suggesting that they may represent ancestral traits that were subsequently lost in the traditional species. Remarkably, the broad relationships among Brucella species using a variety of sequence and fragment-based approaches have been upheld when using comparative genomics with whole genomes. Nonetheless, genomes are required for fine-scale resolution of many of the relationships and for understanding the evolutionary history of the genus. We expect that the coming decades will reveal many more hosts and previously unknown diversity in a wide range of environments.     

Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544

Brucellosis is a disease affecting various domestic and wild life species, and is caused by a bacterium Brucella. Keeping in view the serious economic and medical consequences of brucellosis, efforts have been made to prevent the infection through the use of vaccines. Cell-mediated immune responses [CMI] involving interferon gamma and cytotoxic CD4⁺ and CD8⁺ T cells are required for removal of intracellular Brucella. Omp25 has been reported to be involved in virulence of Brucella melitensis, Brucella abortus and Brucella ovis. In our previous study, we have shown the protective efficacy of recombinant Omp25, when administered intradermally. In this study, the recombinant Omp25 was formulated in PC–PE liposomes and PLGA microparticles, to enhance the protective immunity generated by it. Significant protection was seen with prime and booster liposome immunization in Balb/c mice against virulent B. abortus 544 as it was comparable to B. abortus S-19 vaccine strain. However, microparticle prime and booster immunization failed to give better protection when compared to B. abortus S-19 vaccine strain. This difference can be attributed to the stimulation of cell mediated immune response in PC–PE liposome immunized mice even after challenge which converted to cytotoxicity seen in CD4⁺ and CD8⁺ enriched lymphocytes. However, in PLGA microparticle immunized mice, cell mediated immunity was not generated after challenge as observed by decreased cytotoxicity of CD4⁺ and CD8⁺ enriched lymphocytes. Our study emphasizes on the importance of liposome encapsulating Omp25 immunization in conferring protection against B. abortus 544 challenge in Balb/c mice with a single dose immunization regimen.     

Genetic diversity of Brucella abortus and Brucella melitensis in Kazakhstan using MLVA-16

Brucellosis is an endemic disease in Central Asia characterized by high infection rates in humans and animals. Currently, little is known about the genetic diversity of Brucella spp. circulating in the region, despite the high prevalence of brucellosis. This study aimed to analyze the genetic diversity of Brucella melitensis and Brucella abortus strains circulating in the Republic of Kazakhstan. We genotyped 128 B. melitensis and 124 B. abortus strains collected in regions with the highest prevalence of brucellosis. Genotyping was performed using multi-locus variable-number tandem-repeat analysis (MLVA). Analysis of a subset of 8 loci (MLVA-8) of 128 B. melitensis strains identified genotypes 42 (n = 108), 43 (n = 2), and 63 (n = 19) related to the ‘East Mediterranean’ group. An MLVA-16 assay sorted 128 B. melitensis strains into 25 different genotypes. Excluding one variable locus, MLVA-15 of B. melitensis was distinct from strains originating in the Mediterranean region; however, 77% of them were identical to strains isolated in China. A minimum spanning tree for B. melitensis using MLVA-15 analysis clustered the local strains together with strains previously collected in China. MLVA-8 analysis of 124 B. abortus strains identified them as genotype 36, suggesting Eurasian distribution of this lineage. Complete MLVA-16 assay analysis clustered the strains into five genotypes, revealing little diversity of B. abortus when compared on the global scale. A minimum spanning tree for B. abortus obtained using MLVA-15 analysis clustered the 2 most prevalent genotypes (n = 117) together with strains previously collected in China. Thus, MLVA analysis was used to characterize 252 strains of Brucella collected in Kazakhstan. The analysis revealed genetic homogeneity among the strains. Interestingly, identical MLVA-15 profiles were found in seemingly unrelated outbreaks in China, Turkey, and Kazakhstan. Further analysis is needed for better understanding of the epidemiology of brucellosis in Asia.     

Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice

The immunogenicity of a DNA vaccine containing an open reading frame (ORF) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the open reading frame with homology to an ABC-type transporter (pV278a) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pV278a had a dominant immunoglobulin G2a (IgG2a) response. This DNA vaccine elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 278a protein. Upon stimulation with an appropriate recombinant protein or crude Brucella protein, the vaccine did not induce IL-4, suggesting a typical T-helper (TH1) response. Furthermore, the vaccine induced protection in BALB/c mice when challenged with the virulent strain Brucella abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery of the homologous of an ABC-type transporter antigen, and provides the first evidence of a protective effect of this antigen in the construction of vaccines against B. abortus.     

Efficacy of vaccination strategies against intranasal challenge with Brucella abortus in BALB/c mice

Brucellosis is a zoonotic disease affecting 500,000 people worldwide annually. Inhalation of aerosol containing a pathogen is one of the major routes of disease transmission in humans. Currently there are no licensed human vaccines available. Brucella abortus strain RB51 is a USDA approved live attenuated vaccine against cattle brucellosis. In a mouse model, strain RB51 over-expressing superoxide dismutase (SOD) administered intraperitoneally (IP) has been shown to be more protective than strain RB51 against an IP challenge with B. abortus pathogenic strain 2308. However, there is lack of information on the ability of these vaccine strains to protect against intranasal challenge. With the long-term goal of developing a protective vaccine for animals and people against respiratory challenge of Brucella spp., we tested a number of different vaccination strategies against intranasal infection with strain 2308. We employed strains RB51 and RB51SOD to assess the efficacy of route, dose, and prime-boost strategies against strain 2308 challenge. Despite using multiple protocols to enhance mucosal and systemic protection, neither rough RB51 vaccine strains provided respiratory protection against intranasal pathogenic Brucella infection. However, intranasal (IN) administration of B. abortus vaccine strain 19 induced significant (p ≤ 0.05) pulmonary clearance of strain 2308 upon IN challenge infection compared to saline. Further studies are necessary to address host-pathogen interaction in the lung microenvironment and elucidate immune mechanisms to enhance protection against aerosol infection.     

The use of green fluorescent protein as a marker for Brucella vaccines

Brucellosis is an important malady of productive and wildlife animals and a worldwide zoonosis. The use of effective vaccines and the corresponding diagnostic tests that allow differentiating infected from vaccinated animals are essential tools to control the disease. For this, a prototype of Brucella abortus S19 vaccine expressing green fluorescent protein (S19-GFP) was constructed. The S19-GFP was readily identified under ultraviolet light by macroscopic and microscopic examination and maintained all the biochemical characteristics of the parental S19 vaccine. S19-GFP replicated ex vivo and in vivo, and protected mice against challenge with virulent B. abortus to the same extent as the isogenic S19. An immunoenzymatic assay designed to measure anti-GFP antibodies allowed the discrimination between mice vaccinated with S19-GFP and those immunized with S19. Both vaccines raised antibodies against lipopolysaccharide molecule to similar levels. This experimental model constitutes a “proof of concept” for the use of Brucella-GFP vaccines and associated diagnostic tests to distinguish vaccinated from naturally Brucella infected animals.     

An influenza viral vector Brucella abortus vaccine induces good cross-protection against Brucella melitensis infection in pregnant heifers

Brucella melitensis can be transmitted and cause disease in cattle herds as a result of inadequate management of mixed livestock farms. Ideally, vaccines against Brucella abortus for cattle should also provide cross-protection against B. melitensis. Previously we created a novel influenza viral vector B. abortus (Flu-BA) vaccine expressing the Brucella ribosomal proteins L7/L12 or Omp16. This study demonstrated Flu-BA vaccine with adjuvant Montanide Gel01 provided 100% protection against abortion in vaccinated pregnant heifers and good cross-protection of the heifers and their calves or fetuses (90–100%) after challenge with B. melitensis 16 M; the level of protection provided by Flu-BA was comparable to the commercial vaccine B. abortus S19. In terms of the index of infection and colonization of Brucella in tissues, both vaccines demonstrated significant (P = 0.02 to P < 0.0001) protection against B. melitensis 16 M infection compared to the negative control group (PBS + Montanide Gel01). Thus, we conclude the Flu-BA vaccine provides cross-protection against B. melitensis infection in pregnant heifers.     

Immunization with viable Brucella organisms: Results of a safety test in humans

In many parts of the world contact with infected livestock may involve a serious risk of the spread of human brucellosis. Partial control of bovine brucellosis has been achieved by slaughter of infected herds and immunization of cattle with Brucella abortus strain 19 living vaccine. However, in areas where such measures are unpractical there remains a need for protection of humans. This study compares the safety of two living Brucella vaccine preparations in human volunteers.     

Ability of Brucella abortus rough vaccine strains to elicit DC and innate immunity in lung using a murine respiratory model

Brucella abortus strains RB51 and RB51SOD are live attenuated vaccine strains which protect mice against virulent B. abortus strain 2308 intraperitoneal challenge. By comparison, limited information is available on how Brucella vaccines stimulate pulmonary immunity against respiratory infection, another route of exposure in humans. Therefore, in this study, we assessed the ability of intranasally delivered vaccine strains RB51 and RB51SOD to induce innate immunity. Based on parameters assessed, rough strain RB51 induces a better innate immune response in lung versus strain RB51SOD. Additional studies to further delineate strain RB51's ability to stimulate DC and adaptive immunity are warranted.     

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortusΔwbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-α when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. ΔwbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence.     

Investigating genetic diversity of Brucella abortus and Brucella melitensis in Italy with MLVA-16

Despite the eradication of brucellosis from most of Europe, the disease remains relatively common in a variety of livestock in southern European countries. It is therefore surprising that with such high prevalence rates, there have been few genetic characterizations of brucellosis outbreaks in this region. We conducted a genetic assessment of 206 isolates of Brucella abortus and B. melitensis from Italy using Variable Number Tandem Repeats (VNTRs). We determined genetic diversity and geographic distribution of these Brucella VNTR genotypes from 160 farms in eight regions of Southern Italy in a fine-scale analysis using 16 VNTR loci in a MLVA-16 methodology. In a broad scale analysis, we then used a reduced dataset of 11 VNTR loci (MLVA-11) to compare genotypes from Italy to a global database. In the 84 isolates of B. melitensis, there were 56 genotypes using MLVA-16; 43 of these genotypes were found only once. At a broad scale, 81 of these isolates were part of an Italian sub-group within the West Mediterranean group. One of the two B. melitensis isolates from a human patient shared the same genotype as a livestock isolate, suggesting a possible epidemiological connection. In 122 B. abortus isolates, there were 34 genotypes by MLVA-16; 16 of these genotypes were found only once. At a broad scale with MLVA-11, one genotype was predominant, comprising 77.8% of the isolates and was distributed throughout Southern Italy. These data on the current lineages of Brucella present in Italy should form the basis for epidemiological studies of Brucella throughout the country, while placing these strains in a global context.     

Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice

The immunogenicity of two DNA vaccines encoding open reading frames (ORFs) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the BAB1_0263 and BAB1_0278 genes (pVF263 and pVF278, respectively) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pVF263 or pVF278 had a dominant immunoglobulin G2a (IgG2a) response. In addition, both DNA vaccines elicited a T-cell-proliferative response, but only pVF263 induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 263 protein. Neither DNA vaccine induced interleukin (IL)-10, nor IL-4, upon stimulation with an appropriate recombinant protein or crude Brucella protein, suggesting the induction of a typical T-helper 1 (Th1)-dominated immune response. Furthermore, the pVF278 DNA vaccines induced protection in BALB/c mice against challenge with the virulent strain B. abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery strategy for the BAB1_0278 antigen, and provide the first evidence of a protective effect of this antigen.     

Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2–3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species.     

A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2–30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2–30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2–30.     

Immunotherapeutics to prevent the replication of Brucella in a treatment failure mouse model

Outer membrane vesicles (OMVs) from Brucella melitensis and irradiated Brucella neotomae have been shown to be effective vaccines against a B. melitensis challenge in a mouse model. The present study evaluates the efficacy of these two vaccines as immuno-therapeutics in combination with conventional antibiotics against a B. melitensis infection. BALB/c mice chronically infected with B. melitensis were treated for 4 weeks with doxycycline and gentamicin and vaccinated twice during the course of therapy. Antibiotics in sub-therapeutic concentrations were chosen in such a way that the treatment would result in a therapeutic failure in mice. Although no additive effect of vaccines and antibiotics was seen on the clearance of B. melitensis, mice receiving vaccines along with antibiotics exhibited no Brucella replication post-treatment compared to mice treated only with antibiotics. Administration of irradiated B. neotomae along with antibiotics led to higher production of IFN-γ ex vivo by splenocytes upon stimulation with heat inactivated B. melitensis while no such effect was seen by splenocytes from mice vaccinated with OMVs. OMV vaccinated mice developed significantly higher anti-Brucella IgG antibody titers at the end of the treatment compared to the mice that received only antibiotics. The mice that received only vaccines did not show any significant clearance of Brucella from spleens and livers compared to non-treated control mice. This study suggests that incorporating OMVs or irradiated B. neotomae along with conventional antibiotics might be able to improve therapeutic efficacy and control the progression of disease in treatment failure cases.     

Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: Evaluation of protection in pregnant heifers

The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n = 10) or subcutaneous (n = 10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n = 10) or B. abortus RB51 (n = 10) and a negative (PBS + Montanide Gel01; n = 10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS + Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51.     

Delta-pgm,a new live-attenuated vaccine against Brucella suis

Brucellosis is one of the most widespread zoonosis in the world affecting many domestic and wild animals including bovines, goats, pigs and dogs. Each species of the Brucella genus has a particular tropism toward different mammals being the most relevant for human health Brucella abortus, Brucella melitensis and Brucella suis that infect bovines, goats/camelids and swine respectively. Although for B. abortus and B. melitensis there are vaccines available, there is no efficient vaccine to protect swine from B. suis infection so far. We describe here the construction of a novel vaccine strain that confers excellent protection against B. suis in a mouse model of infection. This strain is a clean deletion of the phosphoglucomutase (pgm) gene that codes for a protein that catalyzes the conversion of glucose-6-P to glucose-1-P, which is used as a precursor for the biosynthesis of many polysaccharides. The Delta-pgm strain lacks a complete lipopolysaccharide, is unable to synthesize cyclic beta glucans and is sensitive to several detergents and Polymyxin B. We show that this strain replicates in cultured cells, is completely avirulent in the mouse model of infection but protects against a challenge of the virulent strain inducing the production of pro-inflammatory cytokines. This novel strain could be an excellent candidate for the control of swine brucellosis, a disease of emerging concern in many parts of the world.