Major outer membrane proteins of Brucella spp.: past,present and future

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s and characterised as potential immunogenic and protective antigens. They were classified according to their apparent molecular mass as 36–38 kDa OMPs or group 2 porin proteins and 31–34 and 25–27 kDa OMPs which belong to the group 3 proteins. The genes encoding the group 2 porin proteins were identified in the late 1980s and consist of two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of identity (>85%). In the 1990s, two genes were identified coding for the group 3 proteins and were named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. The recent release of the genome sequence of B. melitensis 16 M has revealed the presence of five additional gene products homologous to Omp25 and Omp31. The use of recombinant protein technology and monoclonal antibodies (MAbs) has shown that the major OMPs appear to be of little relevance as antigens in smooth (S) B. abortus or B. melitensis infections i.e. low or no protective activity in the mouse model of infection and low or no immunogenicity during host infection. However, group 3 proteins, in particular Omp31, appear as immunodominant antigen in the course of rough (R) B. ovis infection in rams and as important protective antigen in the B. ovis mouse model of infection. The major OMP genes display diversity and specific markers have been identified for Brucella species, biovars, and strains, including the recent marine mammal Brucella isolates for which new species names have been proposed. Recently, Omp25 has been shown to be involved in virulence of B. melitensis, B. abortus and B. ovis. Mutants lacking Omp25 are indeed attenuated in animal models of infection, and moreover provide levels of protection similar or better than currently used attenuated vaccine strain B. melitensis Rev.1. Therefore, these mutant strains appear interesting vaccine candidates for the future. The other group 3 proteins identified in the genome merit also further investigation related to the development of new vaccines.     

The Brucella genome at the beginning of the post-genomic era

The year 2002 began with the publication of the first complete genome sequence for a Brucella species, that of the two replicons of B. melitensis 16M. Hopefully in 2002, the complete genome of B. suis 1330, and, perhaps, a B. abortus strain will be published. This is the culmination of over 30 years investigation of the composition, structure, organisation and evolution of the Brucella genome. Brucella research must now adapt to the new challenges of the post-genomic era.     

Incidence and control of brucellosis in the Near East region

In countries of the Near East region, brucellosis was reported in almost all domestic animals, particularly cattle, sheep and goats. Brucellosis in camels has been reported in Saudi Arabia, Kuwait, Oman, Iraq, Iran, Sudan, Egypt, Libya and Somalia. It has been reported even in racing camels in the United Arab Emirates. In Egypt, brucellosis has been reported also in buffaloes, equines and swine. Brucella melitensis biovar 3 is the most commonly isolated species from animals in Egypt, Jordan, Israel, Tunisia and Turkey. B. melitensis biovar 2 was reported in Turkey and Saudi Arabia, and B. melitensis biovar 1 in Libya, Oman and Israel. B. abortus biovar 1 was reported in Egypt, biovar 2 in Iran, biovar 3 in Iran and Turkey, and biovar 6 in Sudan. The countries with the highest incidence of human brucellosis are Saudi Arabia, Iran, Palestinian Authority, Syria, Jordan and Oman. Bahrain is reported to have zero incidence. Most human cases are caused by B. melitensis, particularly biovar 3. However, B. abortus has been responsible for an increasing number of cases in recent years, e.g. in Yemen, where B. abortus was identified in 45 cases and B. melitensis in 7 cases out of 330 cultures performed in 1995. Concerning control of brucellosis in animals, there is a controversy on the choice of policy. In some countries, the test and slaughter policy together with the vaccination of young females is adopted, in others, particularly with regard to sheep and goats; mass vaccination has been recently started. The most commonly used vaccines are B. abortus S19 and B. melitensis Rev.1 vaccines. B. abortus RB51 vaccine is used in some countries on small scale. Vaccination is limited to cattle and small ruminants.     

Brucella proteomes--a review

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.     

Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: laboratory aspects and field observations

Brucellosis vaccines are essential elements in control programs. Since first developed in the mid-1950s, the Brucella melitensis vaccine strain Rev.1 has been used worldwide and its significant value in protecting sheep and goats in endemic areas recognized. This review provides historical background on the development of the vaccine, its use and field complications arising in Israel following changes in the strain’s pathogenicity. The urgent need for resolving cases of vaccine strain excretion in the milk, horizontal transfer and a unique case of human infection has led to identification of an atypical B. melitensis biovar 1 strain that resembles strain Rev.1 in susceptibility to penicillin and dyes. An omp2 based PCR method has been developed that traced the lineage of Israeli B. melitensis biovar 1 strains. This locus serves as an epidemiological tag for the Rev.1 vaccine strain. Despite the rapid development of new approaches in the field of vaccination, it is anticipated that in the near future the Rev.1 vaccine would remain the only accepted vaccine in national control programs.     

Brucellosis vaccines: past,present and future

The first effective Brucella vaccine was based on live Brucella abortus strain 19, a laboratory-derived strain attenuated by an unknown process during subculture. This induces reasonable protection against B. abortus, but at the expense of persistent serological responses. A similar problem occurs with the B. melitensis Rev.1 strain that is still the most effective vaccine against caprine and ovine brucellosis. Vaccines based on killed cells of virulent strains administered with adjuvant induced significant protection but also unacceptable levels of antibodies interfering with diagnostic tests. Attempts were made to circumvent this problem by using a live rough strain B. abortus 45/20, but this reverted to virulence in vivo. Use of killed cells of this strain in adjuvant met with moderate success but batch to batch variation in reactogenicity and agglutinogenicity limited application. This problem has been overcome by the development of the rifampicin-resistant mutant B. abortus RB51 strain. This strain has proved safe and effective in the field against bovine brucellosis and exhibits negligible interference with diagnostic serology. Attempts are being made to develop defined rough mutant vaccine strains that would be more effective against B. melitensis and B. suis. Various studies have examined cell-free native and recombinant proteins as candidate protective antigens, with or without adjuvants. Limited success has been obtained with these or with DNA vaccines encoding known protective antigens in experimental models and further work is indicated.     

An overview of the epidemiology and epizootology of brucellosis in selected countries of Central and Southeast Europe

The objective of this paper is to give an overview of the epidemiologic and epizootic status of brucellosis in selected countries of Central and Southeast Europe (Balkan region). Based on dimension of the disease problem, there is a need to establish collaboration in the eradication and prevention of brucellosis between all countries in the region. Although there were no readily accessible data concerning epidemiology and epizootology of brucellosis in these countries, the limited official and published data were analyzed.

The incidence of brucellosis caused by Brucella melitensis in sheep, goats and humans is a very significant problem in Macedonia and Greece. In Greece, cattle are also affected either by B. melitensis or B. abortus. The disease is an endemic problem in some regions of Yugoslavia and includes B. suis biovar 2 in pigs and in Croatia, B. melitensis in sheep, goats and human is found occasionally. No problem appears to exist with brucellosis in Bulgaria.

Financial well-supported brucellosis control programs of the European Union that will include all countries, regardless of the magnitude of brucellosis incidence, are needed for eradication and control of brucellosis.     

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 μg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to β-lactam antibiotics. PBP2 complements the activity of PBP1. New β-lactam antibiotics and improved vaccines might be developed based on this property.     

Epidemiology and control of brucellosis in China

The paper describes the history and evolvement of brucellosis in China. It presents the variation of epidemic situation, epidemiological characteristics, application of vaccines and control in brief. Before 1980s, human and animal brucellosis was quite severe; during 1980s, the incidence of human and animal brucellosis was relatively low, and seemed to decrease during the decade. During 1990s, there were no obvious changes in the incidence of animal brucellosis, but the incidence of human brucellosis increased, especially from 1995 to 2001. There are not only some common characteristics but also some differences in brucellosis epidemiology relative to that reported in the rest of the world. For the entire country, B. melitensis was the predominant strain associated with outbreaks, and the epidemic peak is from February to June. Several Brucella vaccines have been used in China for prevention and control of brucellosis. such as B. abortus 104 M in humans, B. suis S2 in animals. The introduction of comprehensive measures has allowed great progress in the prevention and control of brucellosis in China. Surveillance points were set-up countrywide to estimate the epidemic situation. In addition, we discussed the new characteristics of brucellosis in China, the influence of the El Nino phenomenon on brucellosis epidemic situation, the phenomenon of antigenic interference between Brucella species and some disadvantages of live Brucella vaccines.     

Brucellosis in the European Union and Norway at the turn of the twenty-first century

Control and eradication programs of brucellosis in cattle, sheep, goats and pigs have been more or less successfully implemented within the Member States (MS) of the European Union (EU) and Norway after Word War II. As a result, the epidemiological situation of animal brucellosis is extremely diverse among different MS or regions within a MS and among the different animal species. Some MS, mainly North European countries, and Norway are declared “officially bovine brucellosis free” and/or “officially ovine and caprine (Brucella melitensis) free”. The situation is less favorable in Southern European countries, particularly as far as sheep and goat brucellosis are concerned. This situation has important zoonotic consequences as reflected in the number of human brucellosis cases due to B. melitensis that are still encountered in those countries. Brucellosis in swine has re-emerged as a result of spillover from the wild boar brucellosis (Brucella suis biovar 2) reservoir, particularly in outdoor reared pigs. Besides the actual challenge to eradicate brucellosis, further issues have to be addressed: (1) the management of false positive serological results that occur in the course of brucellosis testing, particularly in cattle; (2) the impact of wildlife brucellosis, particularly wild boar brucellosis in domestic animals; and (3) the importance of B. melitensis infection in cattle that are in contact with infected sheep.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

A review of Brucella sp. infection of sea mammals with particular emphasis on isolates from Scotland

Brucellae recovered from sea mammals were first reported in 1994. In the years since both culture and serological analysis have demonstrated that the infection occurs in a wide range of species of marine mammals inhabiting a vast amount of the world’s oceans. Molecular studies have demonstrated that the isolates differ from those found amongst terrestrial animals and also distinguish between strains which have seals and cetaceans as their preferred hosts. At the phenotypic level seal and cetacean strains can also be differed with respect to their CO₂ requirement, primary growth on Farrells medium and metabolic activity on galactose. Two new species B. cetaceae and B. pinnipediae have been proposed as a result. This paper provides a review of Brucella in sea mammals and updates findings from the study of sea mammals from around the coast of Scotland.     

Antibody response and antigen-specific gamma-interferon profiles of vaccinated and unvaccinated pregnant sheep experimentally infected with Brucella melitensis

It is well known that the immune response in sheep against Brucella melitensis is subject to individual variation, depending on diverse factors. It bears asking whether these factors (e.g. clinical disease, active infection, state of previous immunity), when affecting a group, can cause variation in the performance of different diagnostic tests. To clarify some of the circumstances in which this immune response can vary, we examine the immune-response profile of sheep protected against the clinical disease by prior vaccination with strain Rev. 1 in comparison with the profile of unprotected females showing the classical brucellosis symptoms. An experimental infection was provoked at midpregnancy under controlled conditions of both non-vaccinated (n=7) and previously Rev.1-vaccinated ewes (n=5). Their immune response was monitored from 7 to 9 weeks before abortion or normal birth to 30 weeks afterwards. Antibody response was assessed by classical tests (Rose Bengal test, complement fixation test (CFT)) in comparison with other diagnostic tests (indirect ELISA (iELISA), competitive ELISA (cELISA), fluorescence polarization assay (FPA), immunocapture test (ICT)). In addition, the cell-mediated immune response was indirectly evaluated by the in vitro antigen-specific release of gamma-interferon. The antibody levels and antigen-specific gamma-IFN profile of the non-vaccinated ewes having the disease and excreting the pathogen was notably high and differed significantly (P<0.05 or P<0.01) from those of vaccinated ewes that neither contracted brucellosis nor excreted the pathogen. In general, all the tests detect the infection in the non-vaccinated ewes with substantial effectiveness. It can be concluded that the high levels of circulating antibodies and of antigen-specific gamma-IFN are related to active Brucella infection. Similarly, the state of protection against the disease, but not necessarily against infection, due to a previous immunization with the Rev. 1 vaccination, appears to be responsible for a low level of detectable immune response. Nevertheless, the design of the study limits conclusions to pregnant ewes and cannot be extrapolated to non-pregnant ewes or rams. Likewise, the study provides no information on animals which are carriers of B. melitensis.     

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10¹⁰ colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.     

Characterization of the caprine model for ruminant brucellosis

The relationship between man, the goat, and brucellosis is historical. Today Brucella melitensis and Brucella abortus pose a serious economic and public health threat in many countries throughout the world. Infection of pregnant goats and sheep with B. melitensis results in abortion during the third trimester of pregnancy. Although nearly eradicated in the US, bovine brucellosis is still a problem in many countries and the potential for re-infection of domestic stock from wildlife reservoirs in this country is a regulatory nightmare. Humans infected with this pathogen develop undulant fever, which is characterized by pyrexia, arthritis, osteomyelitis, and spondylitis. Although available for both organisms, currently available vaccines have problems ranging from false positive serological reactions to limited efficacy in different animal species. With the continued need for new and better vaccines, we have further developed a goat model system to test new genetically derived strains of B. melitensis and B. abortus for virulence as measured by colonization of maternal and fetal tissues, vaccine safety, and vaccine efficacy.     

Regulation of Brucella virulence by the two-component system BvrR/BvrS

The Brucella BvrR/BvrS two-component regulatory system is highly similar to the regulatory and sensory proteins of Sinorhizobium and Agrobacterium necessary for endosymbiosis and pathogenicity in plants, and very similar to a putative system present in the animal pathogen Bartonella. Mutations in the bvrR or bvrS genes hamper the penetration of B. abortus in non-phagocytic cells and impairs intracellular trafficking and virulence. In contrast to virulent Brucella, BvrR/BvrS mutants do not recruit small GTPases of the Rho subfamily required for actin polymerization and penetration to cells. Dysfunction of the BvrR/BvrS system alters the outer membrane permeability, the expression of several group 3 outer membrane proteins and the pattern of lipid A acylation. Constructs of virulent B. abortus chimeras containing heterologous LPS from the bvrS⁻ mutant demonstrated an altered permeability to cationic peptides similar to that of the BvrR/BvrS mutants. We hypothesize that the Brucella BvrR/BvrS is a system devoted to the homeostasis of the outer membrane and, therefore in the interface for cell invasion and mounting the required structures for intracellular parasitism.     

3株非典型羊源布鲁菌的基因分型研究

[目的]探讨采用基因分型方法对非典型布鲁菌鉴定的实用性,为非典型菌株的鉴别提供参考。[方法]采用常规鉴定方法和VITEK 2.0全自动细菌鉴定分析系统对菌株进行初步鉴定,利用AMOS-PCR进行种型鉴定,应用多位点可变数目串联重复序列分析方法(MLVA)确定菌株的基因型。[结果]常规鉴定结果显示试验菌株为疑似布鲁菌;VITEK 2.0全自动细菌鉴定系统显示3株试验菌株均为布鲁菌;AMOS-PCR扩增表明试验菌株均为羊种菌,MLVA聚类分析表明试验菌株与羊种2型布鲁菌紧密地聚为一类,属东地中海基因型,并在Panel 2B中发现了新的基因型(4-4-3-7-5),命名为CN2B-45。[结论]MLVA基因分型方法对非典型布鲁菌具有极高的分辨力,是非典型布鲁菌分型鉴别的最佳策略。     

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals.

Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.