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.