pseudomallei, especially given the noted

inaccuracies and high background of indirect hemagglutination assays [29]. Little work has examined the seropositive rates in Australia, AZD9291 mouse but two studies in Northern Queensland returned rates of 2.5-5.7% [30, 31]. The high clinical relevance of B. MLN2238 solubility dmso pseudomallei expressing type B or B2 O-antigen, along with the new apparent abundance of these types in Australian near-neighbors, suggest similar exposures may result in false positive diagnoses, as is likely the case in Thailand. These near-neighbor species are avirulent, B. mallei excepted, and as such are not limited to the biosafety regulations that B. pseudomallei is as a biosafety level 3 (BSL-3) organism. Few laboratories worldwide are properly equipped to handle BSL-3 work and so the finding of B. pseudomallei type LPS in these non-pathogenic Burkholderia species will allow many additional laboratories the opportunity to

work towards vaccine development for melioidosis. Conclusions B. thailandensis type A O-antigen has been used with some success to vaccinate mice against B. pseudomallei[7–10]. This O-antigen is indistinguishable between these two species in backbone and side group modifications [12, 16, 22]. Given the high genetic similarity between types B and B2 in near-neighbors and B. pseudomallei, it is likely at least one species will be identical in backbone and side group modifications, GANT61 mouse as well. In such a case, it is possible that particular strain or strains will confer comparable host immunity upon subsequent challenge with type B or B2 B. pseudomallei in much the same way B. thailandensis protects against type A B. pseudomallei challenge. Methods Bacterial strains, DNA, and LPS preparations A total of 113 strains of B. pseudomallei near-neighbors were used in this study. These included 23 B. mallei, 4 B. oklahomensis, 12 B. thailandensis, 5 B. thailandensis-like

species, 44 B. ubonensis, and other 25 Burkholderia strains (Tables 1 and Additional file 1: Table S1). Species identification was made on the basis of recA and 16S rRNA sequences [17, 18]. B. pseudomallei strains K96243, 576, MSHR840, and MSHR1655 were used as references for the O-antigen types A, B, B2, and rough, respectively [11]. All strains were grown on Luria-Bertani P-type ATPase (LB) agar (Difco, USA) for DNA and LPS extractions. DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. LPS was extracted using whole-cell lysis according to a previous method [11, 20] and separated by SDS-PAGE (Invitrogen, USA). PCR analysis Strains were genotyped for B. pseudomallei O-antigen types via multiplex-SYBR-Green real-time PCR in accordance with as previously reported [11]. As the previously published sequences did not detect all near-neighbors expressing type A, this primer pair was redesigned.