Recent advances in biomedical sciences of Burkholderia pseudomallei (basonym: Pseudomonas pseudomallei).

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

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Bacterial Expression of the scFv Fragment of a Recombinant Antibody Specific for Burkholderia pseudomallei Exotoxin

Publisher Summary The aetiological agents of glanders and melioidosis are closely related because they cause similar diseases and have antigenic affinity. Genetic techniques, however, have resulted in rationalization and greater precision in the taxonomy of the pseudomonads. Seven organisms in homology group II are sufficiently different from the others to assign them to a new genus, Burkholderia. DNA hybridization studies of Burkholderia have shown that B. mallei and B. pseudomallei are the most closely related species, with a DNA homology of about 90%. Serological differentiation of the two species is difficult, because of marked antigenic cross-reaction. Fermentation results are slow to determine, hence the interest in molecular methods to differentiate the two species. This has already had important implications for bacterial evolution, taxonomy and patterns of regulatory behavior. Genetic techniques are currently being applied to B. mallei and B. pseudomallei to elucidate their taxonomy, to construct molecular maps, to determine virulence factors, to improve diagnostic tests, and to assist in epidemiological studies of the diseases they cause.

Burkholderia mallei and Burkholderia pseudomallei

Sera from 420 military personnel serving in Sabah and Sarawk, Malaysia, were tested for antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens by enzyme-linked immunosorbent assay procedure (ELISA). Data showed that 54.4% of serum samples were positive for antibodies to P. pseudomallei exotoxin and 65.7% were positive for antibodies to the whole cell antigens. Samples gave much lower titers for anti-exotoxin antibodies compared to titers against crude whole cell antigens. The incidence of antibody to exotoxin was highest in the age groups ranging from 26 to 32 years, where the positive rates were higher than 40% and 30% for military personnel serving in Sarawak and Sabah, respectively.

Prevalence of antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens in military personnel in Sabah and Sarawak, Malaysia.

A nation-wide survey was conducted to see the prevalence of serosensitivity to Pseudomonas pseudomallei antigens by indirect hemagglutination (IHA) and indirect immunofluorescent assay (IFA) for IgG and IgM. Serum samples were collected from blood donors in eight selected areas and bacteriologically confirmed melioidosis patients in Ubon Ratchathani province. The distribution patterns of antibody titers were compared among the survey areas with cut-off points set at 1:160 for IHA, 1:4 for IFA-IgM and 1:32 for IFA-IgG. These cut-off points were decided by ROC (Receiver Operating Characteristics) analysis. The specificity (% true negative reactions) of each serological test in the general population differed significantly among survey areas, possibly reflecting the extent of inapparent infection in each community. IFA was more successful than IHA in differentiating between negative from positive reactions. The survey classified the areas into endemic (Khon Kaen, Ubon Ratchathani), transported (Bangkok), and non-endemic (other provinces) types.

H.S.M. Sharifah

Papers

Antibody to Pseudomonas pseudomallei exotoxin in sheep exposed to natural infection