Use of western immunoblot analysis for testing moose serum for Brucella suis biovar 4 specific antibodies.

To determine if 12 moose (Alces alces) from northern Alaska with agglutinating antibodies specific for Brucella spp. had been exposed to either B. suis biovar 4 or B. abortus biovar 1, western immnnoblot serologic analysis was performed. Differential serologic responses to strain specific A and M antigenic variances of the lipopolysaccharide O-polysaccharide sugar allowed strain identification. Prior to examination, test sera were absorbed with killed whole cells from either B. abortus biovar 1, containing predominately A antigen (A+ M-); B. melitensis biovar 1, containing essentially M antigen (A- M+); or B. suis biovar 4, containing both antigenic tyes (A+ M+). The resulting sera were then examined by western immunoblot for recognition of either B. abortus biovar 1, B. melitensis biovar 1, or B. suis biovar 4 cell lysates. The results of this study indicate that these moose were exposed to B. suis biovar 4, a known pathogen of caribou (Rangifer tarandus) from arctic Alaska.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

Experimental infection of white-tailed deer with rangiferine brucellosis

Experimental infections of white-tailed deer (Odocoileus virginianus) with Brucella suis biovar 4 were evaluated over a period of 6 wk. Five adult male hand-raised white-tailed deer were inoculated with 1 x 10(7) colony forming units of B. suis biovar 4 in the conjunctiva and serologically evaluated over 6 wk by the card test (CARD), rivanol test (RIV), serum agglutination test (SAT), complement fixation test (CFT), particle concentration fluorescence immunoassay (PCFIA), and competitive enzyme linked immunosorbent assay (cELISA), as routinely used for bovine samples. Six weeks postinoculation the animals were euthanized and cultured for B. suis biovar 4. One deer became serologically positive 4 wk postinoculation on CFT, CARD, PCFIA, and cELISA. At 6 wk postinoculation, CFT was positive in four infected deer, CARD was positive in three deer; RIV, SAT, and PCFIA was positive in two deer; and cELISA was positive in one deer. Only the CFT was 100% sensitive. At necropsy B. suis biovar 4 was isolated from four of five deer, and representative colonies were biologically similar to the challenge organism.     

Brucella suis infections in collared peccaries in Venezuela

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.     

上海地区散发布氏杆菌感染的细菌学及分子鉴定

目的 本研究对我院的1例散发布氏杆菌病患者进行细菌学及分子生物学的分析,并在国内首次尝试了用数目可变串联重复单元(VNTR)分子指纹分析法对其进行了基因分型并和国际流行株进行了分子流行病学比较分析。方法 对临床疑似布氏杆菌病病例作血液细菌培养与生化鉴定,进一步作布氏杆菌特异性基因片段的序列分析鉴定以及利用布氏杆菌基因组中的8个位点构建VNTR指纹图谱,参照国际布氏杆菌VNTR数据库,构建布氏杆菌基因系统树。结果 用细菌学方法确定散发疑似布氏杆菌病病例体内分离到的为布氏杆菌,通过基因序列分析进一步得到证实,但不能鉴定到生物种和生物型。对分离株作VNTR指纹分析提示该散发布氏杆菌病为猪2型布氏杆菌感染所致。结论 通过传统细菌培养方法与布氏杆菌VNTR指纹分析可用于我国布氏杆菌病分子流行病学的系统调查。     

Type III secretion homologs are present in Brucella melitensis,B. ovis,and B. suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.     

Identification of species of Brucella using Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. The marked tendency of Brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. We studied the type strains of the different species and biovars of Brucella and 11 isolates of human origin of Brucella melitensis, six corresponding to biovar 1, one to biovar 2 and five to biovar 3. The results of linear discriminant analysis performed using the data provide an above 95% likelihood of correct classification, over half of which are in fact above 99% for the vast majority of Brucella strains. Only one case of B. melitensis biovar 1 has been incorrectly classified. The rest of the microorganisms studied (Staphylococcus aureus, Strteptococcus pyogenes, Enterococcus faecalis, Corynebacterium pseudodiphtheriticum, Clostridium perfringens, Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas aeruginosa) have been classified correctly in all cases to a likelihood of over 80%. In the graphic representation of the analysis, a grouping of these can be seen in clusters, which include the different species. One of these comprises B. melitensis, another Brucella abortus, and another wider one is made up of Brucella suis. The Brucella canis, Brucella ovis and Brucella neotomae strains appear separate from the previously described groups.     

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.     

Complete genome sequence of Brucella suis VBI22, isolated from bovine milk

Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.     

Brucella suis biotype 4: a case of granulomatous nephritis in a barren ground caribou (Rangifer tarandus groenlandicus L.) with a review of the distribution of rangiferine brucellosis in Canada

Severe granulomatous nephritis caused by Brucella suis biotype 4 was found in a barren ground caribou (Rangifer tarandus groenlandicus) from Northwest Territories, Canada. A review of the distribution of human and animal cases of brucellosis in northern Canada indicated that B. suis biotype 4 is distributed widely and is probably enzootic in most Canadian caribou herds.     

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.     

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.     

DNA polymorphism in strains of the genus Brucella

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.     

Complete Genome Sequence of Brucella melitensis 133, an Isolate of Biovar 1 of Sequence Type 32

Brucellosis is highly epidemic in China. Of the six classical species, Brucella melitensis and biovar 1 are the most represented species and biovar that cause human brucellosis in China. Here, we report the genome sequence of Brucella melitensis strain 133, a strain of biovar 1 of sequence type 32.     

Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.     

Taxonomic Position in the Genus Brucella of the Causative Agent of Canine Abortion

The gram-negative organism causing abortion in dogs was examined in parallel with cultures representative of the Brucella species and with Bordetella bronchiseptica. The organism fits into the genus Brucella and most closely resembles B. suis on the basis of its growth characteristics. It is of rough colonial morphology and is agglutinated by antisera prepared against rough Brucella. In mouse toxicity tests, no endotoxic activity could be demonstrated. In contrast to most Brucella cultures, it does not utilize erythritol. Electron microscopy showed a cell wall structure similar to that of other gram-negative organisms. The question of whether the organism should be designated Brucella canis, as proposed by Carmichael and Bruner, or Brucella suis biotype 5 is discussed. The authors favor the designation Brucella canis because the organism lacks the lipopolysaccharide antigen associated with the smooth agglutinogen and endotoxin, and it does not utilize erythritol.     

Revised genome sequence of Brucella suis 1330

Brucella suis is a causative agent of porcine brucellosis. We report the resequencing of the original sample upon which the published sequence of Brucella suis 1330 is based and describe the differences between the published assembly and our assembly at 12 loci.     

Brucellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis) caused by Brucella abortus biovar 4

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep (Ovis canadensis) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk (Cervus elaphus) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella-infected elk or bison (Bison bison) should be cognizant of the possibility of this disease in bighorn sheep.     

BmaC, a novel autotransporter of Brucella suis, is involved in bacterial adhesion to host cells

Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.