Abstract
Melioidosis caused by gram negative bacteria, B. pseudomallei, is a fatal disease in the tropical and sub-tropical regions. However, sporadic cases have been reported in elsewhere. Early detection is imperative to reduce the mortality rate. Serological tests have being substantially developed using recombinant proteins as specific targeted antigens to melioidosis antibodies. In the present study, we focus on a truncated flagellin fragment (FLAG300) and outer membrane protein A (OmpABT) of B. thailandensis E264 as potential antigens for developing indirect ELISA to improve the serodiagnosis of melioidosis. Recombinant proteins were overexpressed and purified by immobilized metal affinity chromatography with denaturing conditions. The sensitivity and specificity of individual test were calculated within culture-confirmed melioidosis sera (n = 42) and non-melioidosis serum samples (n = 241) using the cut-off point at average of absorbance plus 2 standard deviations. The results demonstrated that a FLAG 300 based indirect ELISA showed 90.48 % sensitivity and 87.14 % specificity and an OmpABT based this assay revealed sensitivity of 80.95 % and specificity of 89.21 %. Their use in a double-antigen ELISA resulted in improve specificity (92.95 %) and still high degree of sensitivity (85.71 %). These data suggest a facile method for serodiagnosis of melioidosis by the use of antigens from a non-pathogenic strain.
Highlights
Burkholderia pseudomallei infection, melioidosis, is an infectious disease that is mainly described in the major endemic areas of SE Asia and Northern Australia
We focused on outer membrane proteins (Omps), especially outer membrane protein A (OmpA) and truncated flagellin fragment (FLAG300) of B. thailandensis E264
OmpA gene cloning and DNA sequencing The amplified product, 680 bps, was isolated from genomic DNA of B. thailandensis E264 by specific primers designed based on the ompA sequence of B. pseudomallei K96243
Summary
Burkholderia pseudomallei infection, melioidosis, is an infectious disease that is mainly described in the major endemic areas of SE Asia and Northern Australia. There have been reported cases outside these endemic regions such as in Brasil, India and United States of America (Corral et al 2008; Deshmukh and Mundhada 2013; Kunnathuparambil et al 2013; Miralles et al 2004) It is a major public health problem in Southeast Asia and Northern Australia (Dance 1991; Leelarasamee and Bovornkitti 1989), because the mortality rate is still high even with treatment, and no Wajanarogana and Kritsiriwuthinan SpringerPlus (2016)5:1814 areas (Ashdown and Guard 1984; Kanaphun et al 1993). Some biological properties of B. pseudomallei have been explained by using B. thailandensis as a non-pathogenic model (Haraga et al 2008)
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