Use of a Western blot technique for the serodiagnosis of glanders

Abstract

Background: The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT). Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS) containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas.

Results: The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories.

Conclusions: The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

Figure 1

Silver stained SDS-PAGE gel. Silver stain of SDS-PAGE of mixtures of purified LPS of B. mallei strains protein standard (lane 1), Mukteswar (lane 2); Bogor (lane 3); Zagreb (lane 4), mixture of all (lane 5).

Figure 2

Score of immunoblot. Score of immunoblot: lane 1: negative, lane 2: suspicious, lane 3: positive.

Figure 3

Western blot analysis of sera from Pakistan. Lane 1: negative control; lane 2: positive control; lane 3-12 sera of acutely infected horses from Pakistan.

Figure 4

Western blot analysis of rabbit hyperimmunesera (HIS) against B. mallei LPS. Lane 1: negative control serum, lane 2: anti B. mallei HIS, lane 3: anti B. cepacia HIS, lane 4: anti P. aeruginosa HIS, lane 5: anti B. pseudomallei HIS.