TheScientificWorldJournal2014; 2014; 469407; doi: 10.1155/2014/469407

Optimization and validation of indirect ELISA using truncated TssB protein for the serodiagnosis of glanders amongst equines.

Abstract: To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. Methods: In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein-a type 6 secretory effector protein--were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity of the assay was assessed with equine disease control serum and human melioidosis positive serum. Results: In comparison to CFT, diagnostic sensitivity and specificity of ELISA were 99.7% and 100%, respectively. Conclusions: The indirect ELISA method using the truncated TssB offered safer and more rapid and efficient means of serodiagnosis of glanders in equines. These data highlight the use of TssB as potential diagnostic antigen for serological diagnosis of glanders.
Publication Date: 2014-02-03 PubMed ID: 24672321PubMed Central: PMC3932216DOI: 10.1155/2014/469407Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Validation Study

Summary

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This study successfully developed an optimized and validated indirect ELISA method that uses a truncated TssB protein for glanders diagnosis amongst equines, exhibiting an impressive sensitivity and specificity.

Introduction and Objective

  • The motivation of this research is to diagnose glanders, a disease that affects equines, using serological detection.
  • Specifically, the researchers aimed to express the truncated TssB protein of Burkholderia mallei and evaluate its usability for diagnostic purposes.

Methods

  • In their attempt to develop a recombinant protein-based enzyme-linked immunosorbent assay (ELISA), the scientists expressed the N-terminal 200 amino acid sequences of B. mallei’s TssB protein. This protein is known as a type 6 secretory effector protein.
  • Next, they evaluated the diagnostic potential of the recombinant TssB protein via indirect ELISA. This was performed using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811).
  • The researchers also checked the cross-reactivity of the assay with equine disease control serum and human melioidosis positive serum to ensure that the ELISA does not mistake other diseases for glanders.

Results

  • When compared to the complement fixation test (CFT), which is another method of diagnosing glanders, the diagnostic sensitivity and specificity of the ELISA were 99.7% and 100%, respectively.
  • Demonstrating such high sensitivity and specificity means that the new ELISA method can very accurately detect true positive cases and true negative cases of glanders.

Conclusions

  • Using the truncated TssB protein, the indirect ELISA method offers a safer and more rapid and efficient means of diagnosing glanders amongst equines compared to other current methods.
  • These data highlight that the TssB could serve as a powerful diagnostic antigen for the serological diagnosis of glanders.

Cite This Article

APA
Singha H, Malik P, Goyal SK, Khurana SK, Mukhopadhyay C, Eshwara VK, Singh RK. (2014). Optimization and validation of indirect ELISA using truncated TssB protein for the serodiagnosis of glanders amongst equines. ScientificWorldJournal, 2014, 469407. https://doi.org/10.1155/2014/469407

Publication

ISSN: 1537-744X
NlmUniqueID: 101131163
Country: United States
Language: English
Volume: 2014
Pages: 469407

Researcher Affiliations

Singha, Harisankar
  • National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
Malik, Praveen
  • National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India ; Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
Goyal, Sachin K
  • National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
Khurana, Sandip K
  • National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.
Mukhopadhyay, Chiranjay
  • Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka 576104, India.
Eshwara, Vandana K
  • Department of Microbiology, Kasturba Medical College, Manipal University, Manipal, Karnataka 576104, India.
Singh, Raj K
  • National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India.

MeSH Terms

  • Animals
  • Base Sequence
  • Blotting, Western
  • Burkholderia mallei / genetics
  • Enzyme-Linked Immunosorbent Assay / methods
  • Glanders / diagnosis
  • Horse Diseases / diagnosis
  • Horses
  • Molecular Sequence Data
  • Sequence Homology, Nucleic Acid
  • Serologic Tests

References

This article includes 23 references
  1. DeShazer D, Waag D. Glanders: new insights into an old disease. In: Lindler L, Lebeda F, Korch GW, editors. Biological Weapons Defense: Infectious Diseases and Counterbioterrorism. Totowa, NJ, USA: The Humana Press; 2004. pp. 209u2013237.
  2. Galyov EE, Brett PJ, DeShazer D. Molecular insights into Burkholderia pseudomallei and Burkholderia mallei pathogenesis.. Annu Rev Microbiol 2010;64:495-517.
  3. Office International des Epizooties (OIE) Glanders , Chapter 2. 5. 11. OIE Terrestrial Manual. Paris, France: OIE; 2008.
  4. Verma RD, Sharma JK, Venkateswaran KS, Batra HV. Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines.. Vet Microbiol 1990 Oct;25(1):77-85.
    pubmed: 2247938doi: 10.1016/0378-1135(90)90095-dgoogle scholar: lookup
  5. Sprague LD, Zachariah R, Neubauer H, Wernery R, Joseph M, Scholz HC, Wernery U. Prevalence-dependent use of serological tests for diagnosing glanders in horses.. BMC Vet Res 2009 Sep 1;5:32.
    pmc: PMC2745380pubmed: 19723336doi: 10.1186/1746-6148-5-32google scholar: lookup
  6. Teles JAA, Campos AC, Silva KPC, et al. Standardization and evaluation of an indirect ELISA for the serological diagnosis of glanders in horses. Pesquisa Veterinu00e1ria Brasileira. 2012;32(9):838u2013842.
  7. Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, Chen D, Lipscomb L, Kim HS, Mru00e1zek J, Nierman WC, Deshazer D. Type VI secretion is a major virulence determinant in Burkholderia mallei.. Mol Microbiol 2007 Jun;64(6):1466-85.
  8. Schwarz S, West TE, Boyer F, Chiang WC, Carl MA, Hood RD, Rohmer L, Tolker-Nielsen T, Skerrett SJ, Mougous JD. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.. PLoS Pathog 2010 Aug 26;6(8):e1001068.
  9. Chen Y, Wong J, Sun GW, Liu Y, Tan GY, Gan YH. Regulation of type VI secretion system during Burkholderia pseudomallei infection.. Infect Immun 2011 Aug;79(8):3064-73.
    pmc: PMC3147588pubmed: 21670170doi: 10.1128/IAI.05148-11google scholar: lookup
  10. Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens.. FEBS Lett 1990 Dec 10;276(1-2):172-4.
    pubmed: 1702393doi: 10.1016/0014-5793(90)80535-qgoogle scholar: lookup
  11. Malik P, Singha H, Khurana SK, Kumar R, Kumar S, Raut AA, Riyesh T, Vaid RK, Virmani N, Singh BK, Pathak SV, Parkale DD, Singh B, Pandey SB, Sharma TR, Chauhan BC, Awasthi V, Jain S, Singh RK. Emergence and re-emergence of glanders in India: a description of outbreaks from 2006 to 2011.. Vet Ital 2012 Apr-Jun;48(2):167-78.
    pubmed: 22718333
  12. Siegel S, Castellan NJ. Nonparametric Statistics for the Behavioral Sciences. 2nd edition. New York, NY, USA: McGraw-Hill; 1998.
  13. Gardner IA, Greiner M. Advanced Method for Test Validation and Interpretation in Veterinary Medicine. Berlin, Germany: Freie Universitat; 2000.
  14. Neubauer H, Sprague LD, Zacharia R, Tomaso H, Al Dahouk S, Wernery R, Wernery U, Scholz HC. Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives.. J Vet Med B Infect Dis Vet Public Health 2005 Jun;52(5):201-5.
  15. 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 Jul;19(4):362-7.
    pubmed: 17609344doi: 10.1177/104063870701900404google scholar: lookup
  16. Hagebock JM, Schlater LK, Frerichs WM, Olson DP. Serologic responses to the mallein test for glanders in solipeds.. J Vet Diagn Invest 1993 Jan;5(1):97-9.
    pubmed: 8466990doi: 10.1177/104063879300500121google scholar: lookup
  17. Elschner MC, Scholz HC, Melzer F, Saqib M, Marten P, Rassbach A, Dietzsch M, Schmoock G, de Assis Santana VL, de Souza MM, Wernery R, Wernery U, Neubauer H. Use of a Western blot technique for the serodiagnosis of glanders.. BMC Vet Res 2011 Jan 19;7:4.
    pmc: PMC3034690pubmed: 21247488doi: 10.1186/1746-6148-7-4google scholar: lookup
  18. Bowers JR, Engelthaler DM, Ginther JL, Pearson T, Peacock SJ, Tuanyok A, Wagner DM, Currie BJ, Keim PS. BurkDiff: a real-time PCR allelic discrimination assay for Burkholderia pseudomallei and B. mallei.. PLoS One 2010 Nov 12;5(11):e15413.
  19. Koh SF, Tay ST, Sermswan R, Wongratanacheewin S, Chua KH, Puthucheary SD. Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex.. J Microbiol Methods 2012 Sep;90(3):305-8.
    pubmed: 22705921doi: 10.1016/j.mimet.2012.06.002google scholar: lookup
  20. Kumar S, Malik P, Verma SK, Pal V, Gautam V, Mukhopadhyay C, Rai GP. Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.. Clin Vaccine Immunol 2011 Sep;18(9):1456-61.
    pmc: PMC3165212pubmed: 21752949doi: 10.1128/CVI.05185-11google scholar: lookup
  21. Pal V, Kumar S, Malik P, Rai GP. Evaluation of recombinant proteins of Burkholderia mallei for serodiagnosis of glanders.. Clin Vaccine Immunol 2012 Aug;19(8):1193-8.
    pmc: PMC3416092pubmed: 22695165doi: 10.1128/CVI.00137-12google scholar: lookup
  22. Mukhopadhyay C, Kaestli M, Vandana KE, Sushma K, Mayo M, Richardson L, Tuanyok A, Keim P, Godoy D, Spratt BG, Currie BJ. Molecular characterization of clinical Burkholderia pseudomallei isolates from India.. Am J Trop Med Hyg 2011 Jul;85(1):121-3.
    pmc: PMC3122355pubmed: 21734136doi: 10.4269/ajtmh.2011.11-0166google scholar: lookup
  23. Shalom G, Shaw JG, Thomas MS. In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages.. Microbiology (Reading) 2007 Aug;153(Pt 8):2689-2699.
    pubmed: 17660433doi: 10.1099/mic.0.2007/006585-0google scholar: lookup

Citations

This article has been cited 6 times.
  1. Elschner MC, Melzer F, Singha H, Muhammad S, Gardner I, Neubauer H. Validation of a Commercial Glanders ELISA as an Alternative to the CFT in International Trade of Equidae.. Front Vet Sci 2021;8:628389.
    doi: 10.3389/fvets.2021.628389pubmed: 33665218google scholar: lookup
  2. Wang G, Glaser L, Scott NE, Fathy Mohamed Y, Ingram R, Laroucau K, Valvano MA. A glycoengineered antigen exploiting a conserved protein O-glycosylation pathway in the Burkholderia genus for detection of glanders infections.. Virulence 2021 Dec;12(1):493-506.
    doi: 10.1080/21505594.2021.1876440pubmed: 33509023google scholar: lookup
  3. Erdemsurakh O, Ochirbat K, Gombosuren U, Tserendorj B, Purevdorj B, Vanaabaatar B, Aoshima K, Kobayashi A, Kimura T. Seroprevalence of equine glanders in horses in the central and eastern parts of Mongolia.. J Vet Med Sci 2020 Sep 24;82(9):1247-1252.
    doi: 10.1292/jvms.20-0219pubmed: 32641602google scholar: lookup
  4. Elschner MC, Laroucau K, Singha H, Tripathi BN, Saqib M, Gardner I, Saini S, Kumar S, El-Adawy H, Melzer F, Khan I, Malik P, Sauter-Louis C, Neubauer H. Evaluation of the comparative accuracy of the complement fixation test, Western blot and five enzyme-linked immunosorbent assays for serodiagnosis of glanders.. PLoS One 2019;14(4):e0214963.
    doi: 10.1371/journal.pone.0214963pubmed: 30951554google scholar: lookup
  5. Malik P, Singha H, Goyal SK, Khurana SK, Tripathi BN, Dutt A, Singh D, Sharma N, Jain S. Incidence of Burkholderia mallei infection among indigenous equines in India.. Vet Rec Open 2015;2(2):e000129.
    doi: 10.1136/vetreco-2015-000129pubmed: 26457190google scholar: lookup
  6. Hatcher CL, Muruato LA, Torres AG. Recent Advances in Burkholderia mallei and B. pseudomallei Research.. Curr Trop Med Rep 2015 Jun;2(2):62-69.
    doi: 10.1007/s40475-015-0042-2pubmed: 25932379google scholar: lookup