Immunochemical studies on a Brucella ovis specific protein antigen

A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).     

Biological properties of RB51; a stable rough strain of Brucella abortus

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.     

Monoclonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide complex

Hybridomas producing antibodies to determinants associated with the lipopolysaccharide (LPS) of Brucella abortus and B melitensis were obtained by polyethylene glycol fusion of the SP2/0 myeloma cell line with B lymphocytes harvested from a Sprague-Dawley-derived rat previously immunized with whole B abortus strain 1119 organisms. Two clones, BRU38 and BRU28 , were selected for their ability to react with whole B abortus organisms and purified smooth-LPS ( f5p ). The BRU38 monoclonal antibodies were absorbed with live, rough strain 45/20 and smooth strains of B abortus and B melitensis organisms, whereas only smooth strains absorbed the antibody activity from BRU28 . Complete inhibition of the monoclonal's activity could be achieved with crude smooth-LPS, a purified f5p fraction, and a water-soluble acid degraded polysaccharide. Absorption of BRU38 and BRU28 with rough Brucella LPS, polysaccharide-B antigen, keto- deoxyoctanoic acid, or with several sugars and fatty acids known to be components of the Brucella LPS complex had no effect on the monoclonals. The data indicate that antigenic determinants are associated with the smooth LPS complex, probably with the O-side chain, and are expressed patchwise and in different quantities on several strains of B abortus and B melitensis. The B abortus rough strain 45/20 contains surface determinants which lead to the agglutination of smooth strain 1119 organisms. The potential use of monoclonals in competitive enzyme-linked immunosorbent assays for diagnostic purposes is discussed.     

Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy

Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains.     

Characterization of Brucella ovis surface antigens

A rough antigen (SRA) extracted from Brucella ovis in hot saline by Myers procedure, showed three precipitation lines when tested in immunodiffusion against sera from experimentally infected rams. The components responsible for the lines could be isolated by ultracentrifugation or gel filtration which gave 3 fractions, named PI, PII and PIII. The lipopolysaccharide (LPS) appeared in the pellet (SRA-pp) after ultracentrifugation as judged by the presence of lipids, sugar composition, 2 keto-2deoxyoctulosonic acid (KDO), and its characteristic immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns. SRA-pp contained the antigen responsible for one of the immunoprecipitation lines of SRA and the supernatant (SRA-sn) contained only antigens responsible for the other two. Gel filtration of SRA-pp showed the presence of PI, while SRA-sn gave PII with high protein content and PIII with high carbohydrate content. Immunological activity in gel diffusion (GD) of the Fraction PII and PIII was specific for sera of B. ovis infected rams. Sera from rams experimentally infected with smooth strains (Brucella abortus and melitensis), were not able to react with these antigens.     

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.     

Mechanism of serum resistance among Brucella abortus isolates.

It was shown in this study that complement-resistant Brucella abortus used were unable to activate complement in the absence of specific antibody. Complement-resistant isolates possessed O-antigen, but complement-sensitive organisms used are O-antigen deficient. Since B. abortus LPS does not activate the alternative pathway of complement, we concluded that activation of bovine complement must be due to some other mechanism. In this study, it was shown that bovine C1 binds to the outer membrane proteins of B. abortus. Isolated outer membrane proteins of both smooth (O-antigen positive) and rough (O-antigen negative) B. abortus used bind to C1q. However, only rough isolates were killed by complement. All of the O-antigen positive B. abortus isolates were complement-resistant. We propose that O-antigen shields outer membrane proteins and blocks C1q binding.     

Monoclonal antibodies directed against Brucella abortus cell surface antigens

A panel of monoclonal antibodies (MAb) has been raised against Brucella abortus cell surface antigens from mice immunized with either heat/phenol treated or UV killed bacterial suspensions of B. abortus. The hybridomas were screened by either a microagglutination procedure or by an indirect enzyme immunoassay (EIA) on sonicated bacterial preparations. From a large number of MAb generated by various procedures, two distinct types of MAb emerged. The most numerous type was capable of agglutinating B. abortus and reacting with a soluble preparation of lipopolysaccharide (LPS). A second type was not capable of agglutinating the bacterial suspensions or of binding to the soluble LPS preparation but reacted with an antigen present in bacterial sonicates. Two MAb of this type react differentially with sonicates prepared from virulent and avirulent strains of B. abortus. There appeared to be sufficient evidence from our analysis of the relative degree of cross reaction with antigens present on a range of B. abortus strains and Brucella and xenogenic bacterial species to conclude that each of the seven MAb was recognising a separate antigenic site on the B. abortus cell surface.     

Characterization of an atypical biotype of Brucella abortus.

Brucella abortus strains were isolated from bovine tissue and milk samples from seven Ontario herds. The isolates were characterized by colonial morphology, requirement of CO2 for growth, lysis by Tbilisi phage, biochemical tests and agglutination in monospecific sera. They resembled B. abortus biotype 2 (on the basis of sensitivity to thionin and basic fuchsin) and biotype 4 (on the basis of agglutination with anti-Brucella "M" but not anti-Brucella "A" absorbed sera). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of these isolates and B. abortus biotypes 1, 2 and 4 showed similar profiles. Immunoblots with anti-A and anti-M absorbed sera showed different antigenic regions reacting with the specific sera and also confirmed that the atypical B. abortus isolates were serologically similar to biotype 4.     

Biotypes of Brucella abortus and their value in epidemiologic studies of infected cattle herds.

Four Texas cattle herds containing cows infected with either Brucella abortus biotype 1, 2, or 4 were studied to determine the probability of transmission of Brucella between adjacent cattle herds, the most probable means by which Brucella was introduced into the herds, and the relative frequency of strain 19 isolation from vaccinated cattle. A total of 1,935 cattle in the four herds were tested for brucellosis; 339 reactors were identified, and isolations of B abortus were made from 143. The biotype of B abortus was used to determine that purchased cattle or reentry of bred heifers into the herds was probably responsible for introducing B abortus and that the biotype was not readily transmitted to adjacent herds. Three (9%) of 32 B abortus isolations from adult-vaccinated cattle were strain 19. The data supported the hypothesis that biotypes can be useful in determining the source of B abortus for cattle and in differentiating field and vaccine strain infections in adult-vaccinated cattle.     

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis.     

Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages

Chronic bovine brucellosis is characterized by persistent infection of the mammary gland. The interaction of live Brucella abortus with bovine mammary gland macrophages was studied in vitro. Opsonization of smooth B abortus strain 2308 and rough strain 45/20 was required for phagocytosis by mammary gland macrophages. When opsonized with specific antiserum, strains 2308 and 45/20 stimulated a considerable oxidative burst when phagocytized by mammary gland macrophages. Intracellular survival rates for strain 2308 were significantly higher than those for strain 45/20. After being phagocytized, B abortus localized in phagosomes and phagolysosomes of mammary gland macrophages.     

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy

Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.     

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Characterisation of a new phage lytic for both smooth and non-smooth Brucella species

A brucella phage of the Izatnagar series, designated Iz1, was lytic for all Brucella species that are normally smooth, although the efficiency of plating varied between biovars and species. The phage was also lytic for rough strains of B melitensis and B suis and, to a lesser extent, B ovis. It displayed negligible lytic activity towards B canis and rough B abortus cultures. In its morphological and serological properties and its stability to inactivating agents, the Iz1 phage resembled other brucella phages.     

Bovine monoclonal antibody specific for Brucella abortus lipopolysaccharide

The development of a bovine monoclonal antibody against Brucella abortus smooth lipopolysaccharide (BM-8) by interspecies fusion of bovine peripheral lymphocytes from an immunized cow and a murine plasmacytoma cell line is described. The twice cloned cell line secreted bovine IgG1 subclass antibody. Ascites fluid was prepared in pristane treated nu/nu mice by intraperitoneal injection. The pooled ascites fluid was purified by affinity chromatography and the functions of the antibody assessed in various serological tests. The BM-8 antibody did not agglutinate well at a neutral pH, however, under acid conditions it was efficient at agglutinating B. abortus cells. The antibody did not precipitate B. abortus LPS in double agar gel immunodiffusion but was very active in the direct complement fixation test and the indirect enzyme immunoassay, although it was unable to compete with a murine monoclonal antibody in a competitive enzyme immunoassay.     

中国牛种布鲁菌弱毒疫苗株A19 SNP位点的研究

为鉴别我国牛种布鲁菌弱毒疫苗株A19与野生菌株,运用生物信息学方法结合基因测序,对疫苗株A19基因组SNP位点分析筛选,选取其中部分SNP位点,通过与布鲁菌常见种、生物型标准参考菌株和弱毒疫苗株基因组SNP位置核苷酸测序比较,验证SNP位点的A19特异性。结果表明,共筛选获得A19基因组29个SNP位点,验证ClpX G825-C825、LysR A605-C605、Omp2b G503-A503这3个SNP位点为A19（或S19）特异,揭示了A19基因组SNP位点分布情况,为疫苗株 A19与野生菌株鉴别提供了分子依据。