A cellular basis of immunity in experimental Brucella infection

Brucella suis, Brucella abortus, and Brucella melitensis were shown by microscopic and cultural procedures to multiply extensively within normal rat, mouse, and guinea pig monocytes maintained in vitro in cell cultures for 3 days. Intracellular growth of brucellae had no observable toxic effects on most monocytes, although many of the cells became completely engorged with brucellae within 3 days. Non-smooth brucellae and strain 19 multiplied slowly within normal monocytes. In contrast, "immune" monocytes) i.e. those derived from animals previously infected with smooth brucellae, greatly restricted the intracellular growth of smooth and non-smooth brucellae and strain 19. Growth of smooth Brucella, within either normal or "immune" monocytes, was not influenced by addition of Brucella antiserum to the culture medium. Desensitization of immunized guinea pigs did not diminish the refractory state of their monocytes. Cellular resistance did not develop when animals were vaccinated with heat-killed brucellae, though these animals did produce agglutinating antibody. Similarly, vaccination of animals with living, rough B. suis failed to induce a refractory state in their monocytes, even though the vaccinated animals developed delayed hypersensitivity to smooth Brucella antigen. In vivo studies of Brucella survival in the spleens of normal and vaccinated mice (treated with streptomycin to prevent extracellular survival) gave strong support to the in vitro demonstrations of acquired "cellular immunity." Some implications of these results are discussed.     

Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.     

OBSERVATIONS ON THE INFECTION OF CHICK EMBRYOS WITH BACTERIUM TULARENSE,BRUCELLA, AND PASTEURELLA PESTIS

1. Comparison of the infections of chick embryos by the chorio-allantoic route indicates that Bacterium tularense and Brucella suis, abortus, and melitensis exhibit varying degrees of facultative intracellular parasitism. Pasteurella pestis is adapted to rapid proliferation and spread in the intercellular fluids. 2. In the early stages of infection Bacterium tularense has a marked affinity for growth within ectodermal epithelial cells. Brucella suis and Brucella abortus differ in their selectivity for cells of mesodermal derivation and especially in their effect on vascular endothelium. The strain of Brucella melitensis studied is limited in its intracellular growth to ectodermal epithelium. 3. Many of the features characteristic of these infections in the natural hosts are reproduced in the chick embryo and its membranes. 4. The possible implications regarding the differences in behavior of these microorganisms in relation to the problem of infection and pathogenesis of these diseases are discussed.     

Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16 M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors.Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS/MS.A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines.Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays.     

Indirect fluorescent antibody test versus enzyme-linked immunosorbent assay and agglutination tests in the serodiagnosis of patients with brucellosis

Anti-Brucella IgG, IgM and IgA in sera from patients with blood culture positive for B. melitensis and controls were measured by indirect fluorescent antibody (IFA) test and the findings compared with those of enzyme-linked immunosorbent assay (ELISA) and microagglutination test (MAT). Brucella melitensis and B. abortus antigens from three vendors (BioMerieux, Wellcome and Oxoid) and from reference strains (Ames, Iowa) were used in IFA and MAT while a whole cell heat-killed B. melitensis antigen was used in ELISA. Statistical analysis showed comparable results when using B. melitensis or B. abortus antigen, in IFA, from the same manufacturer but there were subtle differences among antigens from different manufacturers. Correlation between IFA and ELISA titers was poor, due to differences in the levels of these titers. However, the percentage of sensitivity, specificity, predictive positive, and predictive negative at different titers indicated the most reliable discriminative titers to be as follows: ELISA IgG 1 : 800 (100% for all), IgM 1 : 400 (100%, 93%, 100%, 100%, respectively) and IgA 1 : 200 (95%, 100%, 100%, 94%, respectively); IFA IgG 1 : 320 (95%, 93%, 95%, 93%, respectively) and IgM 1 : 80 (95%, 100%, 100%, 94%, respectively). IFA IgA showed either poor sensitivity or specificity at all titers. These findings and the subjective reading of IFA limit its value in Brucella diagnosis while the MAT showed high false negatives (5%–40%). Thus, ELISA proves to be the most reliable test for the diagnosis of patients with brucellosis.     

Development of BruAb2_0168 based isothermal polymerase spiral reaction assay for specific detection of Brucella abortus in clinical samples

Bovine brucellosis, predominantly caused by Brucella abortus is one of the most neglected zoonotic diseases causing severe economic losses in the dairy industry. The early and precise diagnosis of the disease is required to reduce the transmission of infection in humans as well as animals. In the current study, a rapid and novel isothermal amplification-based polymerase spiral reaction (PSR) was developed for the specific detection of Brucella abortus by targeting the BruAb2_0168 gene. The assay could be conducted at 65 °C in a water bath and results can be obtained after 60 min. The detection limit of the PSR assay was found to be 1.33 fg. The sensitivity of the assay was found to be 10⁴ fold higher than conventional PCR and equivalent to real-time PCR (RT-PCR). The assay didn't exhibit cross-reaction with selected pathogenic non-Brucella bacteria and Brucella spp. other than B. abortus. Forty clinical samples were also tested using this novel assay and it was able to detect 25 samples as positive, however, conventional PCR could detect the targeted organism in 22 samples only.To the extent of our knowledge, this is the first report towards the development of a PSR assay for specific detection of B. abortus. The assay can be used as a quick, sensitive and accurate test for the diagnosis of bovine brucellosis in the field setting. Relatively one of the paradigm-shifting aspects of this assay would be it does not require any expensive equipment and the results can be easily visualized by the unaided eye, therefore making PSR a valuable diagnostic tool in field conditions.     

脉冲场电泳分析布鲁菌标准菌株

目的探索一种布鲁菌分型的快速、准确、可靠的分子生物学方法。方法建立脉冲场凝胶电泳(PF-GE)的分子生物学分型方法,比较染色体限制性内切酶图谱,确定菌株的亲缘关系。结果在分析的19株布鲁菌标准菌株中,6种布鲁菌的PFGE图谱各不相同,但猪种菌和犬种菌的PFGE图谱只有1条带不同。羊种菌和猪种菌内各生物型均可在各自种内被区别,但牛种菌各生物型被区分为3类。从系统发生的观点来看,羊、牛、猪种菌是从共同祖先进化来的,犬种菌可认为是猪种菌的主要株或是刚从该种布鲁菌进化而来的株。沙林鼠种菌与牛种菌关系密切,而绵羊附睾种菌与猪种5型菌遗传距离也较近。结论PFGE方法可用于布鲁菌的基因分型的研究,是对传统分型方法一种较好的补充。     

Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Real-time PCR-based assays specific for Brucella abortus, Brucella melitensis and Brucella suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS 711, whereas the downstream primers and probes are designed from signature sequences specific to a species or a biovar. The PCR reactions were monitored for fluorescence resonance energy transfer by including two adjacent labeled probes that hybridize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the downstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The assays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis. Therefore, specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies.     

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.     

1950－2018年山西省布鲁氏菌病病原学特征分析

目的系统分析1950 — 2018年山西省布鲁氏菌病病原学特征,为深入分析布鲁氏菌病的流行病学特征提供参考信息。方法对全省69年间检出布鲁氏菌资料按照1950 — 1981、1982 — 1991和1992 — 2018年3个阶段特别对分离到布鲁氏菌按照时间、地区、宿主材料分离情况及优势菌株进行统计分析。结果1950 — 2018年山西省从5种宿主的不同材料中分离到牛、羊、猪种3种共417株布鲁氏菌,其中羊种布鲁氏菌331株、牛种1株、猪种1株、布鲁氏菌非典型菌株1株、未定种83株。结论山西省是羊种为主的羊、牛、猪种布鲁氏菌混合疫区,1950 — 1981、1982 — 1991年以羊1型、羊2型为优势菌种演变为当前以羊种3型为优势菌种,羊种布鲁氏菌可能有宿主转移现象。     

Epidemiological survey of rifampicin resistance in clinic isolates of Brucella melitensis obtained from all regions of Turkey

The aim of the present study was to assess the antimicrobial susceptibility of Brucella melitensis isolates to rifampicin (RIF) depending on time and regional differences. A total of 94 human Brucella isolates collected in an 8-year period from the beginning of 2002 to the end of 2009 throughout Turkey were investigated. The isolates were identified at species and biovar levels by conventional methods, and minimum inhibitory concentrations (MIC) of RIF was determined by using the E test method. All isolates were identified as B. melitensis (93 isolates, biovar 3; 1, biovar 1), and MIC₅₀ and MIC₉₀ values of RIF were 1 and 1.5 μg/ml, respectively (MIC range, 0.25–1.5 μg/ml). All isolates were sensitive to RIF except 2 isolates, which had intermediate susceptibility to RIF. These findings indicated that B. melitensis biovar 3 may be the most frequently agent responsible for human brucellosis in Turkey. None of the isolates in our region was resistant to RIF.     

Bioluminescence Imaging of Colonization and Clearance Dynamics of <Emphasis Type="Italic">Brucella Suis</Emphasis> Vaccine Strain S2 in Mice and Guinea Pigs

Purpose

The goal of this study was to develop a plasmid-based lux bio-reporter for use to obtain in vivo images of Brucella suis vaccine strain 2 (B.suis S2) infection with high resolution and good definition.

Procedures

The pBBR-lux (pBBR1MCS-2-lxCDABE) plasmid that carries the luxCDABE operon was introduced into B. suis S2 by electroporation yielding B. suis S2-lux. The spatial and temporal transit of B. suis S2 in mice and guinea pigs was monitored by bioluminescence imaging.

Results

The plasmid pBBR-lux is stable in vivo and does not appear to impact the virulence or growth of bacteria. This sensitive luciferase reporter could represent B. suis S2 survival in real time. B. suis S2 mainly colonized the lungs, liver, spleen, and uterus in mice and guinea pigs as demonstrated by bioluminescence imaging.

Conclusion

The plasmid-based lux bioreporter strategy can be used to obtain high resolution in vivo images of B. suis S2 infection in mice and guinea pigs.

     

Phospholipase A1 Modulates the Cell Envelope Phospholipid Content of Brucella melitensis,Contributing to Polymyxin Resistance and Pathogenicity

A subset of bacterial pathogens, including the zoonotic Brucella species, are highly resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed primarily to the modification of lipopolysaccharide; however, it is unknown what additional mechanisms mediate high-level resistance against this class of drugs. This work identified a role for the Brucella melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated that it is a phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, lipidomic analysis of B. melitensis revealed an excess of PE lipids in the bacterial membranes isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell envelope, BveA increases the resistance of B. melitensis to polymyxin B. BveA was required for survival and replication of B. melitensis in macrophages and for persistent infection in mice. BveA family esterases are encoded in the genomes of the alphaproteobacterial species that coexist with the polymyxin-producing bacteria in the rhizosphere, suggesting that maintenance of a low PE content in the bacterial cell envelope may be a shared persistence strategy for association with plant and mammalian hosts.     

The use of polyvinylpyrrolidone as a stabilizer in the lyophilization of Brucella]

Stabilizing effects of 5 percent aqueous polyvinylpyrrolidone (PVP) solution and sucrose-agar-gelatin medium (SAG) were examined in lyophilization of 4 Brucella types. The number of viable cells immediately after lyophilization in PVP has made up 48.1 percent on an average; this value was different with different Brucella species: 70.9 percent with B. abortus, 50.4 percent with B. melitensis, 41.2 percent with B. rangiferi, 30.2 percent with B. suis. Only 27.5 percent of cells were viable after Brucella lyophilization in SAG medium. Gradual death of lyophilized cells was observed in the course of strain storage at 5 degrees C. After two-year storage the number of viable cells after lyophilization in PVP was 41.8, after three-year storage 35.1 percent. The number of viable cells after SAG lyophilization was still lower. Biological characteristics of the strains lyophilized in both media corresponded to the initial ones over the three-year follow-up.     

AGGLUTINATION AFFINITIES OF THE ABORTUS-MELITENSIS GROUP OF BACTERIA WITH SPECIAL REFERENCE TO TWO HUMAN STRAINS

The conclusions to be drawn from this study are that while there appears to be evidence of serological distinctions between B. abortus and B. melitensis cultures as studied by earlier workers, the experiments reported in this paper show no serological distinctions among the six strains used in the study. B. melitensis II and B. melitensis III were found to be identical with the bovine and swine strains by means of agglutination and agglutinin absorption reactions in unheated sera, by agglutination in heated sera, and with the use of heated cultures in unheated and heated sera.     

Cross-immunity between Brucella melitensis and Mycobacterium tuberculosis; intracellular behavior of Brucella melitensis in monocytes from vaccinated animals

A non-specific element has been demonstrated in the resistance of monocytes derived from immunized rabbits. Vaccination by BCG or by an effective anti-brucellosis reagent induces protection in either case against both Mycobacterium tuberculosis and Brucella melitensis when studied by the monocyte culture method. The activity of the antiserum required to demonstrate the resistance of the monocyte is not affected when the agglutinating action of the anti-Brucella rabbit serum is removed by absorption. The ability of the monocytes from specifically immunized rabbits to retard the growth of virulent Brucella was demonstrated, not as an all-or-none phenomenon, but in the light of the unrestricted bacterial multiplication which occurs in monocytes from normal animals.     

In vitro susceptibility of clinical isolates of Brucella melitensis to fucidic acid

Brucella species are facultative intracellular bacteria, and therefore a limited number of antibiotics are effective against these organisms. The side effects of drug combination schemes, and the incidences of relapses and therapeutic failures, have led to investigations of new drugs to treat brucellosis. The purpose of this study was to test the in vitro susceptibility of 50 Brucella melitensis isolates to fucidic acid, which has not previously been used for the treatment of brucellosis. The minimum inhibitory concentrations (MICs) of fucidic acid to 50 B. melitensis isolates that were obtained from blood and bone marrow cultures of patients with brucellosis were studied by the broth microdilution method. The MIC₅₀ and MIC₉₀ values for the 50 B. melitensis strains susceptibility to fucidic acid were determined to be 0.5 and 2µg/ml, respectively, and the MIC range was 0.125–2.0µg/ml. Further experiments are needed to reassess the activity of fucidic acid against intracellular Brucella spp.     

Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use.In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1.MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis’ monitoring systems contributing in the effective control of the disease.     

Preliminary studies on the use of carbon substrate utilization patterns for identification of Brucella species.

A commercial system (Biolog, Hayward, CA) that uses reduction of an indicator dye to determine the oxidation of 95 different carbon substrates contained in a defined minimal medium was tested with 35 isolates of Brucella spp. to determine if the system could be used in place of respirometric methods to identify species. Of 95 substrates contained in this system, three were oxidized by all the Brucella strains tested, 48 were oxidized by none of the strains tested, and 44 were oxidized differentially. Brucella melitensis, B. abortus, and B. suis could be distinguished from each other on the basis of their oxidation reactions in seven in these substrates; epidemiologically related strains could not be unambiguously differentiated. This carbon substrate utilization method may prove to be a useful alternative to respirometry as a means to identify strains of Brucella spp. to species level, provided that personnel are protected from exposure to this highly infectious agent.     

Recombinant VirB5 protein as a potential serological marker for the diagnosis of bovine brucellosis

The molecular tag vaccine against Brucella abortus and serological testing are the main methods of prevention of brucellosis used currently. They can discriminate vaccinated animals and humans from those naturally infected. In this study, we constructed a gene deletion mutant strain, B. abortus S19 virB5 with a molecular tag. Recombinant VirB5 was expressed and purified for evaluation as a diagnostic reagent for bovine brucellosis. In total, 400 sera samples were tested using a VirB5 antigen-based enzyme-linked immunosorbent assay (ELISA) and the results were compared with those of the standard tube agglutination test (SAT). This showed that the sensitivity was 88.2%, specificity was 97.8% and accuracy was 94.8%. Recombinant VirB5 could also be used to discriminate B. abortus-infected mice from mice infected with the B. abortus S19 virB5 mutant strain. It was concluded that recombinant VirB5 could be used as a potential antigen and serological marker for the diagnosis of bovine brucellosis.