DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers

Abstract

The omp-37 gene, encoding a major outer-membrane protein in Bnrcella melitensis, was PCR-amplif ied from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-37 published sequence. Amplification of omp-37 was achieved from DNA of all Bnrcella species with the exception of Brucella abortus, the only Brucella species where expression of omp-37 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (44-1 7 kb) containing B. melitensis 16M omp-37 and adjacent DNA of different sizes, with Hindllldigested total DNA showed that a large fragment, comprising the entire omp-37 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specif ic markers for B. abortus and B. melitensis. A t the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-37 revealed specific markers for Bnrcella ovis, Bnrcella canis and Bnrcella suis bv. 2. Using a combination of omp-37 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1,3,4 and 5. Results presented in this paper demonstrate the potential of omp-37 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-37 and flanking DNA. In such a large deletion, other genes in addition to omp-37 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Bnrcella species.

Minor Nucleotide Substitutions in the omp31 Gene of Brucella ovis Result in Antigenic Differences in the Major Outer Membrane Protein That It Encodes Compared to Those of the Other Brucella Species

“…The omp31 gene was PCR amplified and sequenced from all recognized Brucella species and biovar reference strains. The seven biovars of B. abortus were not included in this study, as they lack omp31 (36). The alignment of the Omp31 amino acid sequences, deduced from the determined nucleotide sequence, is shown in Fig.…”

“…In the present work we have determined the nucleotide sequence of omp31 from all the Brucella species and biovar reference strains, with the exception of Brucella abortus, which lacks omp31 (36). We have shown that the nucleotide substitutions found in the B. ovis omp31 gene result in antigenic differences as measured by reactivity with MAbs specific for Omp31 and sera from B. ovis-infected rams.…”

Comparison of Multiple-Locus Variable-Number Tandem-Repeat Analysis with Other PCR-Based Methods for Typing Brucella suis Isolates

“…With the objective of improving the typing of these biovars, different PCR-based assays have been proposed. The most widely used are the PCR-restriction fragment length polymorphism (RFLP) analysis of genes omp2a and omp2b, which can differentiate between reference biovars 1, 2, and 3 (6), and of gene omp31, which is able to differentiate biovars 1 and 3 from biovar 2 (15). In addition, a modification of the original AMOS-PCR multiplex assay (4), including a new pair of primers derived from the ery locus (AMOS-ery-PCR [14]), can differentiate biovar 1 from others.…”

“…Growth and harvesting of Brucella cells and bacterial DNA were performed as described elsewhere (2,8,12). All isolates were subjected to five different PCR-based typing techniques: PCR-RFLP analysis for omp2a, omp2b, and omp31 genes (6,15), multiplex AMOS-ery-PCR (14), and Brucella MLVA using 15 genetic markers (8,12). Figure 1 summarizes the molecular typing results and represents a dendrogram construct from MLVA genotyping assay data on the B. suis isolates.…”

Using combined recombinant protein in the diagnosis of bovine brucellosis

“…Bioinformatics research and planning genetic engineering studies were carried out using the Vector NTI 11.5 software package (Invitrogen, USA). Based on the studies of Vizcaino N. et al (38)(39)(40), the two most immunodominant regions of Brucella Omps were selected. The first region is in a position from 48 to 83 amino acids of Omp31, and the second one is in the position from 180 to 224 amino acids of Omp25.…”