Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm.
Abstract: Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-notifiable 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. 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 visualized 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. Conclusions: The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries.
Publication Date: 2019-09-17 PubMed ID: 31523407PubMed Central: PMC6711873
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article is about the development of an immunoblotting assay for the serodiagnosis of Burkholderia mallei, the cause of glanders in horses. The study presents the whole-cell proteome-based immunoblotting process as an effective diagnostic tool for glanders.
Research Methods
- The study involved three farm horses that were subcutaneously immunized with a crude suspension (10 cfu/ml) of heat-inactivated Burkholderia mallei formulated with incomplete Freund’s adjuvant (IFA) with the aim of generating a hyperimmune sera panel.
- The immunization was done for 1, 14, and 28 days with 1 dose of 1 ml antigen containing 10 cfu/ml.
- The hyperimmunity of the sera was then confirmed by Complement Fixation Test (CFT).
- Burkholderia mallei whole-cell proteome was prepared using sonication and the protein content was visualized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and quantified by Western blot using Horseradish Peroxidase (HRP)-conjugated rabbit anti-horse IgG.
- Positive and negative horse sera were used to validate the test.
Results
- The results showed a ladder pattern of the Burkholderia mallei immunoreactive antigens within the region of 20-90 kDa.
- The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders.
Conclusions
- The whole-cell proteome-based immunoblot proved reliable and straightforward in the study.
- The prepared antigen was found suitable for application in immunoblotting.
- The researchers concluded that this improved immunoblotting system provides appropriate sensitivity and specificity expected in the serodiagnosis of glanders especially in less-developed countries and endemic areas.
Cite This Article
APA
Yazdansetad S, Mosavari N, Tadayon K, Mehregan I.
(2019).
Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm.
Iran J Microbiol, 11(3), 232-238.
Publication
Researcher Affiliations
- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
- Department of Tuberculin and Mallein, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
- Department of Veterinary Aerobic Bacterial Vaccines, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
References
This article includes 24 references
- Smith ME, Gossman WG. Glanders And Melioidosis. StatPearls 2018.
- Cárdenas NC, Galvis JOA, Farinati AA, Grisi-Filho JHH, Diehl GN, Machado G. Burkholderia mallei: The dynamics of networks and disease transmission. Transbound Emerg Dis 2019; 66:715–728.
- Khan I, Wieler LH, Melzer F, Elschner MC, Muhammad G, Ali S. Glanders in animals: a review on epidemiology, clinical presentation, diagnosis and countermeasures. Transbound Emerg Dis 2013; 60: 204–221.
- Van Zandt KE, Greer MT, Gelhaus HC. Glanders: an overview of infection in humans. Orphanet J Rare Dis 2013; 8: 131.
- Dohre SK, Kamthan A, Singh S, Alam SI, Kumar S. Identification of a new diagnostic antigen for glanders using immunoproteome analysis. Comp Immunol Microbiol Infect Dis 2017; 53: 26–32.
- Hornstra H, Pearson T, Georgia S, Liguori A, Dale J, Price E. Molecular epidemiology of glanders, Pakistan. Emerg Infect Dis 2009; 15: 2036–2039.
- Khaki P, Mosavari N, Khajeh NS, Emam M, Ahouran M, Hashemi S. Glanders outbreak at Tehran Zoo, Iran. Iran J Microbiol 2012; 4: 3–7.
- Thibault FM, Hernandez E, Vidal DR, Girardet M, Cavallo JD. Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents. J Antimicrob Chemother 2004; 54: 1134–1138.
- Saqib M, Muhammad G, Naureen A, Hussain MH, Asi MN, Mansoor MK. Effectiveness of an antimicrobial treatment scheme in a confined glanders outbreak. BMC Vet Res 2012; 8: 214.
- Khan AS, Sage MJ. Biological and chemical terrorism: strategic plan for preparedness and response. Morb Mortal Wkly Rep 2000; 49: 1–26.
- Inglis TJJ, Merritt AJ. Burkholderia pseudomallei and Burkholderia mallei. Molecular Medical Microbiology 2015; pp. 769–791.
- . Manual of diagnostic tests and vaccines for terrestrial animals. OIE World Organization for Animal Health 2018.
- Naureen A, Saqib M, Muhammad G, Hussain MH, Asi MN. Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders. J Vet Diagn Invest 2007; 19: 362–367.
- Casey WT, McClean S. Exploiting molecular virulence determinants in Burkholderia to develop vaccine antigens. Curr Med Chem 2015; 22: 1719–1733.
- Elschner MC, Scholz HC, Melzer F, Saqib M, Marten P, Rassbach A. Use of a Western blot technique for the serodiagnosis of glanders. BMC Vet Res 2011; 7: 4.
- Neubauer H, Sprague LD, Zacharia R, Tomaso H, Al Dahouk S, Wernery R. Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives. J Vet Med B Infect Dis Vet Public Health 2005; 52: 201–205.
- Sprague LD, Zachariah R, Neubauer H, Wernery R, Joseph M, Scholz HC. Prevalence-dependent use of serological tests for diagnosing glanders in horses. BMC Vet Res 2009; 5: 32.
- Pal V, Kumar S, Malik P, Rai GP. Evaluation of recombinant proteins of Burkholderia mallei for serodiagnosis of glanders. Clin Vaccine Immunol 2012; 19: 1193–1198.
- Malik P, Singha H, Goyal SK, Khurana SK, Tripathi BN, Dutt A. Incidence of Burkholderia mallei infection among indigenous equines in India. Vet Rec Open 2015; 2(2): e000129.
- Khan I, Wieler LH, Melzer F, Gwida M, Santana VL, de Souza MMA. Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders. Vet Rec 2011; 169: 495.
- Khan I, Elschner MC, Melzer F, Gwida M, Wieler LH, Ali R. Performance of complement fixation test and confirmatory immunoblot as two-cascade testing approach for serodiagnosis of glanders in an endemic region of South East Asia. Berl Munch Tierarztl Wochenschr 2012; 125: 117–121.
- Tamigney Kenfack M, Mazur M, Nualnoi T, Shaffer TL, Ngassimou A, Blériot Y. Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens. Nat Commun 2017; 8: 115.
- Hatcher CL, Muruato LA, Torres AG. Recent advances in Burkholderia mallei and B. pseudomallei research. Curr Trop Med Rep 2015; 2: 62–69.
- Dominguez M, Münstermann S, de Guindos I, Timoney P. Equine disease events resulting from international horse movements: Systematic review and lessons learned. Equine Vet J 2016; 48: 641–653.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
