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。     

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.     

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.     

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.     

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.     

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.     

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.     

湖南省人间布鲁氏菌病流行特征及布鲁氏菌溯源调查

目的 分析2010－2018年湖南省人间布鲁氏菌病（布病）的流行特征,并追溯该地区布病的感染来源,为该地区人间布病防控提供参考。方法 采用Excel 2016和ArcGIS 10.5等软件对湖南省2010－2018年人间布病监测数据进行流行病学分析,采用例数、构成比和率描述疫情特征。采用常规分型方法对临床分离的2株布鲁氏菌的种型进行鉴定,采用UTS-PCR复核种型鉴定结果,并对菌株的6个毒力基因进行检测。采用多位点可变数目串联重复序列分析（MLVA）方法对临床分离布鲁氏菌进行聚类分析。结果 2010－2018年湖南省共报告人间布病728例,年均发病率为0.12/10万,郴州市和永州市发病率居前,发病率分别为2.50/10万和1.90/10万。发病县（市、区）从2010年的5个增加到2018年的69个。45～54岁年龄组病例最多（279例）,占38.32%（279/728）,农民占59.07%（430/728）,男女性别比为2.75 ∶ 1。5－7月报告病例最多,占45.33%（330/728）,发病呈明显的夏秋季高发特征,发病高峰在5月。常规鉴定表明临床分离的2株布鲁氏菌是羊种1型布鲁氏菌,UTS-PCR鉴定显示菌株为羊种布鲁氏菌。6个毒力基因均被检出,菌株为强毒株。MLVA分析表明2株菌与来自内蒙古自治区从绵羊和骆驼分离的菌株具有完全相同的基因型。结论 湖南省人间布病趋于严峻,疫情向非疫区和普通人群蔓延。湖南省分离的2株羊种布鲁氏菌起源于内蒙古地区。应加强动物转运的检疫检测,防止布病暴发流行。     

Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.     

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.     

B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections

Brucellosis is one of the most serious zoonoses all over the world, with B. melitensis, B. abortus and B. suis being the most pathogenic species for humans. Vaccination of domesticated livestock still represents the most efficient way to prevent human infection. However, the available Brucella vaccines retain an important residual virulence and induce antibodies interfering with surveillance programs. Moreover, each vaccine shows different protective effects versus different Brucella species and different animal hosts.Nowadays, while B. melitensis and B. suis infections in cattle are emerging as a significant problem, there are no available vaccines to overcome such issue.B. melitensis strain B115, a natural, attenuated rough strain in our previous studies proved to be highly protective against B. melitensis and B. ovis infections in mice, without inducing interfering antibodies. In this study, we tested the efficiency of B115 as vaccine against B. abortus and B. suis. Vaccination of mice with 10⁸ CFU/mouse of B. melitensis B115 conferred a satisfactory protection against B. abortus 2308. On the contrary, mice vaccinated once with 10⁸ or 10⁹ CFU/mouse of B115 were weakly protected against B. suis infection. Conversely, when mice were vaccinated twice with 10⁹ CFU B115/mouse, the protective activity significantly increased. Unlike its rough phenotype, B115 showed an adequate persistence in mice accompanied to a solid humoral and cell-mediated immunity. All together, these findings suggest the potential usefulness of B115 to control brucellosis in animal hosts due to heterologous challenges.     

内蒙古自治区通辽市2004-2018年人间布鲁氏菌病流行病学特征分析

目的 分析内蒙古自治区通辽市2004-2018年人间布鲁氏菌病（布病）流行特点和菌株的起源与进化特征,为制定布病防控策略提供依据。方法 对中国疾病预防控制信息系统中的通辽市布病报告数据进行分析,采用构成比和率描述流行病学特征。用常规鉴定方法鉴定菌株的种型,用AMOS-PCR对菌株的种型进行复核,采用多位点可变数目串联重复序列分析（MLVA）对布鲁氏菌进行聚类分析,探讨菌株间的亲缘关系。结果 2004-2018年通辽市共报告布病16 704例,年均发病率为35.41/10万。扎鲁特旗和库伦旗的发病率较高,分别为110.51/10万和67.84/10万。40～54岁年龄组病例最多,占48.75%（8 143/16 704）;农民14 873例,占89.04%。男女性别比为2.40：1。3-5月为发病高峰期,占56.30%（9 405/16 704）,发病高峰在4月。常规鉴定表明临床分离菌株全部为羊种布鲁氏菌,羊1型3株,羊3型3株。AMOS-PCR鉴定显示全部为羊种布鲁氏菌。6株布鲁氏菌分为2个MLVA-11基因型（111和116）,属于东地中海血统。MLVA-16聚类分析表明通辽地区的菌株与吉林省和黑龙江省的菌株具有较近的亲缘关系。结论 2004-2018年通辽市布病疫情极为严重,有向周边地区蔓延的风险。应加强检测防控,防止疫情扩散。     

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.     

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.     

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.     

Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x10⁹ CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.     

Identification of Brucella melitensis Rev.1 vaccine-strain genetic markers: Towards understanding the molecular mechanism behind virulence attenuation

Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers.The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains.We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings.     

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.     

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.     

The degree of protection given by living vaccines against experimental infection with Brucella melitensis in goats

It has been shown by a number of workers that Brucella melitensis Rev. 1 vaccine confers good protection against infection with Br. melitensis in sheep and goats. Although Br. abortus strain 19 vaccine is widely used in some countries to protect sheep, it has not been evaluated for caprine brucellosis. The purpose of the experiment reported in this paper was to determine and compare the degree of protection given by living vaccines prepared from Rev. 1 and strain 19 and by a non-agglutinogenic strain of Br. melitensis against graded, experimental challenge in goats.