Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the wholecell proteomebased paradigm

Background and Objectives: Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIEnotifiable disease of equidae. Despite the importance of B. mallei, little is known about serodiagnosis of glanders. The present study aimed to develop an immunoblotting assay based on wholecell proteome of B. mallei to enable accurate serodiagnosis of glanders. #xD; Materials and Methods: Three farm horses were subcutaneously immunized with a crude suspension (106 cfu/ml) of heatinactivated B. mallei formulated with incomplete Freund s adjuvant (IFA) to achieve a hyperimmune sera panel. The immunization was done for 1, 14 and 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The hyperimmunity of sera was confirmed by CFT. B. mallei wholecell proteome was prepared through sonication and the protein content was visualized by SDSPAGE and quantified by Western blot using HRPconjugated rabbit antihorse IgG. A comprehensive set of positive and negative horse sera validated the test. #xD; Results: A ladder pattern of the B. mallei immunoreactive antigens was seen within the region of 2090 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders. #xD; Conclusion: The wholecell proteomebased immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in lessdeveloped countries.