Current understanding of the genetic diversity of Brucella,an expanding genus of zoonotic pathogens

Brucella species are responsible for brucellosis, one of the world's most widespread zoonotic diseases causing abortion in domestic animals and a potentially debilitating infection of man. Despite the identification of a number of distinct species within the genus with differing host preferences and pathogenicity it has been known for many years that members of the genus are genetically homogeneous. However, since the start of the millennium the application of new technologies to the group has resulted in rapid advances in the understanding of Brucella diversity and, after many years of inactivity, a process of expansion of the genus is underway. This review summarises the current state of knowledge in this area and outlines how this informs understanding of the taxonomy and evolution of the group and is enabling the development of increasingly sophisticated molecular typing tools.     

Identification of genetic variants of Brucella spp. through genome-wide association studies

Brucellosis is an important zoonotic disease caused by Brucella spp. We present a phylogeny of 552 strains based on genome-wide single nucleotide polymorphisms (SNPs) determined by an alignment-free k-mer approach. A total of 138,029 SNPs were identified from 552 Brucella genomes. Of these, 31,152 and 106,877 were core and non-core SNPs, respectively. Based on pan-genome analysis 11,937 and 972 genes were identified as pan and core genome, respectively. The pan-genome-wide analysis studies (Pan-GWAS) could not identify the group-specific variants in Brucella spp. Therefore, we focused on SNP based genome-wide association studies (SNP-GWAS) to identify the species-specific genetic determinants in Brucella spp. Phylogenetic tree representing eleven recognized Brucella spp. showed 16 major lineages. We identified 143 species-specific SNPs in Brucella abortus that are conserved in 311 B. abortus genomes. Of these, 141 species-specific SNPs were confined in the positively significant SNPs of B. abortus using SNP-GWAS. Since conserved in all the B. abortus genomes studied, these SNPs might have originated very early during the evolution of B. abortus and might be responsible for the evolution of B. abortus with cattle as the preferred host. Similarly, we identified 383 species-specific SNPs conserved in 132 Brucella melitensis genomes. Of these 379 species-specific SNPs were identified as positively associated using GWAS. Interestingly, > 98% of the SNPs that are significantly, positively associated with the traits showed 100% sensitivity and 100% specificity. These identified species-specific core-SNPs identified in Brucella genomes could be responsible for the speciation and their respective host adaptation.     

BrucellaBase: Genome information resource

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html.     

Identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against Brucella melitensis through reverse vaccinology and immunoinformatics approach

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis.     

Immunization of mice with gamma-irradiated Brucella neotomae and its recombinant strains induces protection against virulent B. abortus, B. melitensis, and B. suis challenge

Human brucellosis, a zoonotic disease of major public health concern in several developing countries, is primarily caused by Brucella abortus, Brucella melitensis, and Brucella suis. No brucellosis vaccine is available for human use. The aim of this study was to determine if Brucella neotomae, a bacterium not known to cause disease in any host, can be used for developing brucellosis vaccines. B. neotomae and its recombinant strains overexpressing superoxide dismutase and a 26 kDa periplasmic protein were rendered non-replicative through exposure to gamma-radiation and used as vaccines in a murine brucellosis model. All three vaccines induced antigen-specific antibody and T cell responses. The vaccinated mice showed significant resistance against challenge with virulent B. abortus 2308, B. melitensis 16 M, and B. suis 1330. These results demonstrate that the avirulent B. neotomae is a promising platform for developing a safe and effective vaccine for human brucellosis.     

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.     

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.     

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.     

Molecular taxonomy,phylogeny and biogeography of nematodes belonging to the Trichinella genus

Studying parasites of the genus Trichinella provides scientists of today many advantages. This is a group of zoonotic nematodes that circulates freely among wildlife hosts with one in particular, Trichinella spiralis that is exceptionally well adapted to domestic swine. Recent reports suggest that human infections from hunted animals are on the rise worldwide and numerous countries still experience problems with T. spiralis in their domestic food supplies. Trichinella is a genus whose members are easily propagated in the laboratories, have been used as models to investigate host–parasite relationships and parasitism among clade I organisms, and represent a poorly investigated link between the phylum Nematoda and other Metazoans. The importance of T. spiralis in better understanding the tree of life was so recognized that in 2004, its genome was carefully selected as one of only nine key non-mammalian organisms to be sequenced to completion. Since it was first discovered in 1835, this genus has expanded from being monospecific to eight species including four other genotypes of undetermined taxonomic rank. Inasmuch as discriminating morphological data have been scant, our understanding of the genus has been relegated to a compilation of molecular, biochemical and biological data. Herein, we provide a collection of information and up-to-date interpretations on the taxonomy, diagnostics, systematics, micro- and macroevolution, and the biogeographical and biohistorical reconstruction of the genus Trichinella.     

Fresh white cheese as a source of Brucella infection.

Human and animal brucellosis is a disease of public health and economic significancein Iran. The consumption of unpasteurized dairy products is one of the important sources of human brucellosis. Fresh white cheese which is produced locally and sometimes privately in country houses from raw goat and sheep milk is probably the most important way of transmission.Out of 1220 fresh white cheese specimens, 86(7%) were found infected with Brucellamelitensis biotype I. These specimens were collected from retail shops in three highly infected villages over a period of 12 months. The average number of organisms per gram of infected cheese was 2120 after collection, which decreased until the ninth week when no Brucella could be isolated. About 40% of infected cheese became free of Brucella one week after collection and in weekly culture 50% of the specimens continued to be infected up to the third week. At the eighth week, only 1·1 % of the 86 infected cheeses showed infection with B. melitensis. The cheese collected from retail shops had a low pH and during weekly culture in the laboratory, B. melitensis was isolated from the specimens within a wide range of pH (5·0–7·6).The seasonal fluctuation of infected cheese corresponds to human brucellosis inwhich most cases are seen from April through August with the maximum rate in May and June.     

The biological stability of the genus Brucella

The constancy of the three species of Brucella (Br. abortus, Br. suis and Br. melitensis) is discussed with regard to both the historical background and current research, and the pertinent literature is reviewed and interpreted. The authors maintain that members of the genus Brucella are not labile nor are they subject to spontaneous species alteration; stability of the recognized species is confirmed when test reactions used in speciation are standardized and controlled. Particular stress is laid on the epidemiological value of retaining the original species designations. The conclusion that Brucella species are biologically stable is substantiated by the authors'' combined experience of more than 30 years in the isolation and typing of Brucella derived from a variety of sources.     

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.     

海南省布鲁氏菌病疫情特征研究

目的 分析海南省布鲁氏菌病（布病）流行病学特征。方法 对2012-2017年海南省收集的16株布鲁氏菌采用Vitek 2 compact进行布鲁氏菌初步鉴定,再用传统生物学分型方法进行确证,结合饲养家畜血清学和病原学检测结果分析患者的流行病学特征。结果 Vitek 2 compact鉴定12株为羊种布鲁氏菌,4株为人苍白杆菌。传统生物学分型方法鉴定11株为羊种布鲁氏菌3型,5株为猪种布鲁氏菌3型。菌株对应的16例病例中2012年1例,2013年2例,2014年4例,2015年1例,2016年2例,2017年6例,分布在东方市、临高县、海口市、万宁市、乐东县、定安县等地。同时对疫区东方市745份羊血清进行布鲁氏菌血清抗体检测,阳性47例（6.3%）,从东方市病羊采集的标本中分离到羊种布鲁氏菌3型。结论 Vitek 2 compact是一种简单、方便的布鲁氏菌鉴定方法,但不能替代传统生物学分型方法;海南地区布病主要流行的菌种为羊种3型及猪种3型,通过东方市2017年布病疫情,说明海南省有布病疫畜传染人的疫情,布病防控形势严峻。     

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.     

Identification of the causative agents and the epidemiology of porcine brucellosis

Brucella organisms that had been isolated from swine or from human beings exposed to infected swine, and that had been previously identified as members of the species Br. melitensis, were examined for their oxidative metabolism, for their growth patterns on media containing basic fuchsin and thionin, and for their hydrogen sulfide production. These organisms displayed the oxidative metabolic pattern that characterizes and identifies the members of the species Br. suis. They also are identical to Br. suis, type 1, in their tolerance to increased concentrations of thionin in the growth media. They are similar to Br. suis, type 2, in their inability to produce hydrogen sulfide, and share with other members of the species Br. suis the characteristic preference for a porcine host. These findings are evidence that these organisms are not Br. melitensis but are a biotype of Br. suis. Their correct identification is important to understanding the epidemiology of brucellosis.     

Clinical Manifestations,Laboratory Findings,and Therapeutic Regimen in Hospitalized Children with Brucellosis in an Iranian Referral Children Medical Centre

Brucellosis is considered a known widespread zoonotic disease and is endemic in Mediterranean region, like Iran. This study reviewed the clinical manifestations, laboratory findings, and therapeutic regimen in childhood brucellosis in Iran. In this retrospective study, we reviewed hospital-records of 34 consecutive children with a confirmed diagnosis of brucellosis among a total number of 10,864 patients admitted to Children''s Medical Center, Tehran, Iran, between 2002 and 2010. Among the patients diagnosed with brucellosis, 22 (65%) were admitted during spring and summer. Clinical findings of these patients at admission were arthritis, splenomegaly, hepatomegaly, lymphadenopathy, maculopapular skin rashes, and fever. Anaemia (53%) and leukopenia (33%) were the most common findings in the children. Only one patient had presented with leukocytosis. Four children (12%) were thrombocytopenic, and none of patients had pancytopenia. Blood cultures were positive in 5 patients (23%). Only one patient underwent bone-marrow aspiration and had positive culture for Brucella spp. Positive titres were found in 33 cases (97%) in Wright test, 23 cases (96%) in Coombs test, and 16 patients (72.7%) in 2ME (2-Mercaptoethanol) test. In one case, Wright and Coombs test titres were below 1:80 while Brucella spp. were isolated from blood at the same time. It is concluded, prolonged fever with joint involvement and organomegaly may increase possibility of infection with Brucella spp. Appropriate treatment regimen by more tolerable oral drugs, with a duration of at least 8 weeks, is recommended.Key words: Brucellosis, Child, Diagnosis, Treatment, Iran     

Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P < 0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.     

A new member of the Pteropine Orthoreovirus species isolated from fruit bats imported to Italy

A novel member of the Pteropine Orthoreovirus species has been isolated and sequenced for the whole genome from flying foxes (Pteropus vampyrus) imported to Italy from Indonesia. The new isolate named Indonesia/2010 is genetically similar to Melaka virus which has been the first virus of this species to be shown to be responsible for human respiratory disease. Our findings highlight the importance of flying foxes as vectors of potentially zoonotic viruses and the biological hazard that lies in the import of animals from geographical areas that are ecologically diverse from Europe.     

Brucella abortus S19 vaccine protects dairy cattle against natural infection with Brucella melitensis

Brucellosis is a zoonotic disease that can cause severe illness in humans and considerable economic loss in the livestock industry. Although small ruminants are the preferential host for Brucella melitensis, this pathogen has emerged as a cause for Brucella outbreaks in cattle. S19 vaccination is implemented in many countries where B. abortus is endemic but its effectiveness against B. melitensis has not been validated. Here we show that vaccine effectiveness in preventing disease transmission between vaccinated and unvaccinated cohorts, as determined by seroconversion, was 87.2% (95% CI 69.5–94.6%). Furthermore, vaccination was associated with a reduced risk for abortion. Together, our data emphasize the role S19 vaccination could play in preventing B. melitensis outbreaks in areas where this pathogen is prevalent in small ruminant populations.     

Increased Host Species Diversity and Decreased Prevalence of Sin Nombre Virus

Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.