Use of amplified fragment length polymorphism to identify and type Brucella isolates of medical and veterinary interest.
Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.
Whatmore
Adrian M
AM
Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency, Addlestone, Surrey, KT15 3NB UK. a.whatmore@vla.defra.gsi.gov.uk
Murphy
Terry J
TJ
Shankster
Stephen
S
Young
Emma
E
Cutler
Sally J
SJ
Macmillan
Alastair P
AP
D000818:Animals; D015373:Bacterial Typing Techniques; D002002:Brucella / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D002006:Brucellosis / Q000175:diagnosis* / Q000382:microbiology* / Q000662:veterinary; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D011817:Rabbits; D013045:Species Specificity
D004269:DNA, Bacterial; D015246:Deoxyribonuclease EcoRI; C055927:endodeoxyribonuclease MseI; D015252:Deoxyribonucleases, Type II Site-Specific