Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm

Background and Objectives: Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-no- tifiable 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 whole-cell proteome of B. mallei to enable accurate serodiagnosis of glanders. Materials and Methods: Three farm horses were subcutaneously immunized with a crude suspension (106   cfu/ml) of heat-inactivated 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 whole-cell proteome was prepared through sonication and the protein content was visu- alized by SDS-PAGE and quantified by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative horse sera validated the test. Results: A ladder pattern of the B. mallei immunoreactive antigens was seen within the region of 20-90 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. Conclusion: The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared an- tigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appro- priate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries.