Characterization of Genomic Island 3 (GI3) and Genetic Variability of Chilean field

21 One of the capabilities developed by bacteria is the ability to gain large fragments of DNA 22 from other bacteria or lose portions of their own genome. Among these exchangeable 23 fragments are the genomic islands (GIs). Nine GIs have been identified (GI) in Brucella, 24 being the genomic island 3 (GI-3) shared by two pathogenic species: B. melitensis and B. 25 abortus. GI-3 encodes mostly for unknown proteins. One of the aims of this study was to 26 perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. abortus from Chile, 27 to determine whether these isolates are clonally related. Furthermore, we focused on the 28 GI-3 characterization, studying its organization and the genetic conservation of the GI-3 29 sequence using techniques such as Tiling-path PCR and restriction fragment length 30 polymorphisms-PCR (RFLP-PCR). Our results, after performing PFGE on 69 field isolates 31 of B. abortus from Chile, showed that the strains were genetically homogeneous. To 32 increase the power of genetic discrimination among these strains, we used the Multi Locus 33 VNTR Analysis-16 (MLVA-16). The results obtained by MLVA-16 showed that the strains 34 of B. abortus were genetically heterogeneous, and that most of them clustered according to 35 their geographic origin. Of the genetic loci studied, Panel 2B was the one describing the 36 highest diversity in the analysis, as well as the locus Bruce19 in Panel 2A. In relation to 37 the study of GI-3, our experimental analysis by TP-PCR identified and confirmed that the 38 GI-3 is present in all wild strains of B. abortus, demonstrating the high stability of gene 39 cluster GI-3 in Chilean field strains. 40