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

Abstract

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.

Validation of a Commercial Glanders ELISA as an Alternative to the CFT in International Trade of Equidae

“…However, the lack of specificity and the resulting obstacles to the trade of equids on the one hand, and the difficulties in standardization of the CFT on the other hand, have stimulated research for alternative serological methods ( 15 – 17 ). In addition to Western blot methods ( 13 , 21 , 24 ), several ELISA based on crude B. mallei antigen preparations and various recombinant proteins have already been developed and evaluated ( 14 , 19 , 20 , 25 , 26 ).…”

Applied Proteomics in ‘One Health’

“…Whole-cell proteome of the organism was prepared through sonication and the protein content was visualised by SDS-PAGE. A ladder pattern of the B. mallei immunoreactive antigens was clearly evident within the region of 20 to 90 kDa [ 122 ].…”

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing

“…Iran is on the unintended path of regional glanders due to the existence of geographical and economical commonalities with the neighboring countries possessing endemic diseases and consequently, they have been strengthening the source tracking on B. mallei Iranian strains. Furthermore, the weakness of neighboring countries in controlling and preventing the diseases and the passage of the pack animals, beast of burden, at the country's borders have made Iran the major center of glanders in the world (6).…”