Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3

• Published in: Veterinary microbiology Vol. 110; no. 1; pp. 41 - 51
• Main Authors: Ocampo-Sosa, Alain A., Agüero-Balbín, Jesús, García-Lobo, Juan M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 30.09.2005; Elsevier Science
• Subjects: animal diseases; Animals; Bacteriology; Base Sequence; Biological and medical sciences; Biovar; Blotting, Southern - veterinary; Brucella - classification; Brucella - genetics; Brucella - isolation & purification; Brucella abortus; Brucella abortus - classification; Brucella abortus - genetics; Brucella abortus - isolation & purification; Brucella melitensis biovar Abortus; brucellosis; Brucellosis - diagnosis; Brucellosis - microbiology; Brucellosis - veterinary; disease diagnosis; DNA Fragmentation; DNA, Bacterial - analysis; Fundamental and applied biological sciences. Psychology; gene deletion; Microbiology; Miscellaneous; molecular sequence data; Molecular typing; nucleotide sequences; pathogen identification; PCR; Phylogeny; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - veterinary; strains; Spain; Molecular typing; Brucella abortus; Biovar; PCR; Microbiology; Genotype; Identification; Polymerase chain reaction; Brucellaceae; Molecular epidemiology; Bacteria; Veterinary; Brucella melitensis
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2005.06.007

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS 711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS 711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS 711 copy. Based on these data, we designed a new primer, which together with the IS 711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.