LAUSR

Abstract Despite the intensive implementation of control programmes goat, sheep and human brucellosis remains endemic in Greece. As the discrimination between field endemic strains and vaccine strain Rev.1 is not feasible, it is essential to develop new diagnostic tools for brucellosis diagnosis. Moreover, effective disease control requires enhanced epidemiological surveillance in both humans and animals including robust laboratory support. Two new multiplex (duplex) polymerase chain reactions (PCRs) were developed and the results were compared with those obtained by real‐time PCR and bacteriological biotyping. A total of 71 Brucella spp. Greek endemic strains were identified at species and biovar level, using both molecular and conventional techniques. Their discrimination from the vaccine strain Rev.1 was achieved, using polymerase chain reaction‐restriction fragment length polymorphism assay (PCR‐RFLP). All 71 strains were identified as Brucella melitensis by multiplex PCR as well as by real‐time PCR and conventional biotyping. Sixty‐two (87.3%) out of 71 strains were identified as B. melitensis biovar 3, eight (11,3%) strains as biovar 1 and only one (1,4%) as biovar 2. Digestion with PstI restriction enzyme revealed that all strains were field endemic strains, as they gave different patterns from the vaccine strain Rev.1. Brucella melitensis biovar 3 appears to be the predominant type in Greece. The novel multiplex PCR produced results concordant to ones obtained by real‐time PCR and conventional biotyping. This technique could support and facilitate the surveillance of Brucellosis in Greece contributing in the control of the disease.