DEVELOPMENT OF A NEW IMMUNOBLOTTING TECHNIQUE FOR SERODIAGNOSIS OF BURKHOLDERIA MALLEI INFECTION

Background and Aim:Burkholderia mallei is a causative agent of glanders, a highly contagious disease in equines which is notifiable to the World Organisation of Animal Health (OIE). The disease is still endemic in our country, Iran. The present study was conducted to develop a new immunoblotting method based on lipopolysaccharide (LPS) antigen of B. mallei for glanders serodiagnosis.Methods:One horse was immunized subcutaneously using a mixture of crude suspensions of heat inactivated B. mallei adjuvanted with aluminium hydroxide gel to prepare a panel of positive sera. Immunizations were performed weekly for seven weeks with 7 doses of 1.5 ml antigen containing increasing cell concentrations. The sera was confirmed by ELISA. Antigen from B. mallei was prepared by formaldehyde 12% and a typical LPS ladder was proofed by SDS-PAGE. A partly purified LPS of B. mallei was assayed by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative sera obtained from horses validated the test.Results:A ladder pattern of the B. mallei LPS was seen within the region of 20 to 60 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders.Conclusion:The developed immunoblot technique based on LPS-preparation testified is highly practicable in our study. The prepared antigen demonstrated to be suitable for its use in immunoblotting. The developed method is a sensitive and specific for glanders serodiagnosis in endemic areas in less developed countries.

Keywords:

Burkholderia mallei , immunoblotting , glanders , LPS