Methods Bacterial strains and DNA preparation A total of 104 B. melitensis strains used in the study were isolated from clinical samples (102 from blood, and 2 from bone marrow). The samples were collected as part of standard patient care between 1957 and 2010 and were fully de-identified. So any ethical approval was not required for the use of these samples. B. melitensis

biovar 1 vaccine strain M5 was also included in this study (Table 2). Bacterial isolates were cultured on Trypticase soy agar containing 5% sheep blood (BD Diagnostic Systems, China Ltd., China) at 37°C for 48 h. All isolates were identified as Brucella species (biovar) on the basis of classical identification procedures: CO2 requirement, H2S production, inhibition of growth by basic fuchsin and thionin, agglutination with monospecific antisera and phage typing [17]. Total genomic DNA was extracted with the DNeasy Blood & Tissue check details Kit (Qiagen China Ltd., China) by following the manufacturer’s protocol for extraction of genomic DNA from Gram-negative Casein Kinase inhibitor bacteria. Species-level identification was undertaken by the AMOS-PCR assay [18]. Table 2 The 105 B. melitensis isolates examined in this study Geographical origin Year No. of isolates Panel 1 Genotypes* Inner Mongolia 1955-2006 26 42,63 Qinghai 1965 1 42 Henan 1963,1982

2 42,43 Shanxi 1979-2009 11 42,43,45,63 Shandong 1973,2005 3 42 Shan’xi 1962,2008 5 42 Hebei 2009 1 42 Liaoning 2005 2 42 Guangxi 1961 1 58 Zhejiang 2005,2009 3 42 Fujian 2009 3 42,58 Yunnan 2009 2 58 Beijing 2006 1 42 Guangdong 2006-2010 39 42, 43, 63, CN-1 Hunan 2008 1 42 Jilin 1971 1 42 Tianjin 2010 1 42 Shanghai 2007 1 63 Heilongjiang 1962 1 42 *genotype 42 (1-5-3-13-2-2-3-2), genotype 43 (1-5-3-13-3-2-3-2), genotype 45 (1-5-3-12-2-2-3-2), genotype 58 (1-5-3-13-3-1-3-2) genotype 63 (1-5-3-13-2-3-3-2), genotype CN-1 (1-5-3-13-2-1-3-2)

MLVA-16 genotyping scheme MLVA was performed as previously described [11]. The sixteen primer pairs were divided into three groups as previously described: panel 1 (8 loci including bruce06, bruce08, Selleck HKI 272 bruce11, bruce12, Carteolol HCl bruce42, bruce43, bruce45, and bruce55), panel 2A (3 loci including bruce18, bruce19, and bruce21), and panel 2B (5 loci including bruce04, bruce07, bruce09, bruce16, and bruce30). PCR conditions were as follows: initial denaturation at 94°C for 3 min, and then 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 50 s. Five microliters of the amplification products were loaded in to 2% (panel 1) and 3% (panels 2A and 2B) agarose gels containing ethidium bromide (0.5 μg/ml), visualized under UV light, and photographed. The reference strain B. melitensis 16 M, for which the precise molecular mass is known for each primer pair locus, was used for size comparison.