Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella

Abstract

Seventy-seven Bmcella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an N 50 bp deletion a t the 3' terminal end of the gene in all Brucella owis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Bmcella species and within the Bmcella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. owis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Bmcella species as was previously suggested by Ficht et a/. (1990; Mo/ Microbiol4,1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucella species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.

Molecular Typing of Brucella melitensis and B. abortus from Human Blood Samples Using PCR-RFLP Method

“…Due to differences in the pathogenicity of Brucella species and biovars, with a view to epidemiology of brucellosis, recognition of Brucella biovars is important and hence typing of various strains is the main task of control centers of brucellosis and must be performed continuously (9,13,15). Distinction between the Brucella species and biovars in the past was mainly based on phenotypic characteristics.…”

“…Therefore, in subsequent studies especially in Cloackaert et al study, omp2a and omp2b fragments and several restriction enzymes were used (15). These primers and enzymes were used in different parts of the world by different people, also in central parts of Iran they were used by Salehi et al for molecular typing of common Brucella biovars (15)(16)(17)22).…”

“…After 2 hours, products of RFLP were electrophoresed on 2% gel agarose. Finally the size of the fragments which were resulted from enzymatic digestion of all positive PCR samples compared with each other and according to the results of Cloackaert et al (15), the biovars were identified.…”

“…In this method, by omp25, omp2 and omp31 loci and all Brucella species can be differentiated and their biovars identified (14). Several studies use these genes to differentiate Brucella species and biovars performed around the World (12,(15)(16)(17), but in Iran, no study was done especially on clinical samples by this method, and it is the first time that a study is done on molecular typing of B. melitensis and B. abortus isolated from blood samples.…”

Epidemiologic Survey for Brucella suis Biovar 2 in a Wild Boar (Sus scrofa) Population in Northwest Italy

“…Samples were inoculated onto modified Thayer Martin Medium (Martin et al, 1996), and media plates were enriched with 10% horse serum and selective supplements; cultures were incubated for at least 10 days. If Brucella was suspected during Stamp's staining, colonies were identified to genus by classical techniques (Alton et al, 1988) and typed by means of biochemical (Alton et al, 1988) and molecular tests (Corbel et al, 1983;Bricker and Halling, 1994;Cloeckaert et al, 1995) by the laboratories of the National Reference Centre of Brucellosis (Teramo, Italy). Epi Info TM 6 for DOS Current Version 6.04d (Centers for Disease Control and Prevention, Atlanta, Georgia, USA) was used to test for associations between serologic and culture results with year of sampling, season, gender, and age by chi-square tests.…”