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PLoS neglected tropical diseases2021; 15(12); e0010007; doi: 10.1371/journal.pntd.0010007

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Abstract: Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.
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Publication Date: 2021-12-21 PubMed ID: 34932554PubMed Central: PMC8691619DOI: 10.1371/journal.pntd.0010007Google Scholar: Lookup
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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.

This research outlines the development of a highly sensitive competitive enzyme-linked immunosorbent assay (cELISA) for detecting Burkholderia mallei, the bacteria behind a potentially serious contagious disease known as Glanders. The method is based on identifying the presence of a specific monoclonal antibody against lipopolysaccharide (LPS) of B. mallei, which was found to have a high sensitivity and specificity in different animals.

Creation of the cELISA

  • The researchers created a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei, a bacteria that causes the highly contagious disease known as Glanders.
  • The optimal conditions for the cELISA were determined and involved B. mallei-LPS (2 ng) for microtiter plate coating and sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5.
  • The cELISA’s cutoff value was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. The calculated percentage inhibition (PI) values from these samples served to establish the threshold of the cELISA, below which a negative result is presumed.

Diagnostic Applications and Evaluations

  • Initially, the researchers checked the sensitivity of the LPS-based cELISA using sera from donkeys and mice that had been inoculated with B. mallei.
  • These results showed an increasing trend of the PI values, suggesting that the anti-LPS antibodies had been detected. This indicated that the cELISA was effective in identifying the presence of B. mallei.

Further Testing on Real-Life Samples

  • Upon initial success, the team went on to evaluate the sensitivity and specificity of the cELISA by testing 31 positive horse sera from glanders outbreaks in Bahrain and Kuwait. Of these, 30 tested positive by the cELISA.
  • Moreover, 21 seronegative horse sera and 20 seronegative donkey sera from Dubai were all also tested, and each was shown to be negative by the cELISA.
  • The sensitivity (97.2%) and specificity (100%) of the developed cELISA was thus established, meaning that the assay is highly efficient in distinguishing true positives and true negatives in regards to the presence of B. mallei antibodies in different animals.

Cite This Article

APA
Wernery U, Chan E, Raghavan R, Teng JLL, Syriac G, Siu SY, Joseph M, Yeung ML, Jia L, Cai JP, Chiu TH, Lau SKP, Woo PCY. (2021). Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent. PLoS Negl Trop Dis, 15(12), e0010007. https://doi.org/10.1371/journal.pntd.0010007

Publication

PLoS neglected tropical diseases
ISSN: 1935-2735
NlmUniqueID: 101291488
Country: United States
Language: English
Volume: 15
Issue: 12
Pages: e0010007

Researcher Affiliations

Wernery, Ulrich
  • Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Chan, Elaine
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Raghavan, Rekha
  • Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Teng, Jade L L
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
  • State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.
  • Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong.
Syriac, Ginu
  • Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Siu, Sing-Yung
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Joseph, Marina
  • Central Veterinary Research Laboratory, Dubai, United Arab Emirates.
Yeung, Man-Lung
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
  • State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.
  • Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong.
Jia, Lilong
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Cai, Jian-Piao
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Chiu, Tsz-Ho
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
Lau, Susanna K P
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
  • State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.
  • Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong.
Woo, Patrick C Y
  • Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong.
  • State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong.
  • Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong.

MeSH Terms

  • Animals
  • Antibodies, Bacterial / blood
  • Burkholderia mallei / immunology
  • Burkholderia mallei / isolation & purification
  • Enzyme-Linked Immunosorbent Assay / methods
  • Equidae
  • Glanders / blood
  • Glanders / diagnosis
  • Glanders / microbiology
  • Horse Diseases / blood
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology
  • Horses
  • Mice
  • Sensitivity and Specificity
  • Serologic Tests / methods

Conflict of Interest Statement

The authors have declared that no competing interests exist.

References

This article includes 34 references
  1. 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(3):204–21.
    doi: 10.1111/j.1865-1682.2012.01342.xpubmed: 22630609google scholar: lookup
  2. World Organization for Animal Health (OIE). Manual of diagnostic tests and vaccines for terrestrial animals, 8th Ed.. OIE, Paris, France pp. 1350–1362; 2019.
  3. Wernery U, Wernery R, Joseph M, Al-Salloom F, Johnson B, Kinne J. Natural Burkholderia mallei infection in Dromedary, Bahrain.. Emerg Infect Dis 2011;17(7):1277–9.
    doi: 10.3201/eid1707.110222pmc: PMC3381376pubmed: 21762586google scholar: lookup
  4. Wilkinson L. Glanders: medicine and veterinary medicine in common pursuit of a contagious disease.. Med Hist 1981;25(4):363–84.
    doi: 10.1017/s0025727300034876pmc: PMC1139069pubmed: 7038356google scholar: lookup
  5. Jelesijevic T, Zimmerman SM, Harvey SB, Mead DG, Shaffer TL, Estes DM. Use of the common marmoset to study Burkholderia mallei infection.. PLoS One 2015;10(4):e0124181.
    doi: 10.1371/journal.pone.0124181pmc: PMC4393281pubmed: 25860021google scholar: lookup
  6. Srinivasan A, Kraus CN, DeShazer D, Becker PM, Dick JD, Spacek L. Glanders in a military research microbiologist.. N Engl J Med 2001;345(4):256–258.
    doi: 10.1056/NEJM200107263450404pubmed: 11474663google scholar: lookup
  7. Centers for Disease Control and Prevention. Security Plan Guidance: Section 11(f)–Tier 1 Security. 2020 August 26. [Cited 2021 Nov 5] Available from: https://www.selectagents.gov/compliance/guidance/security-plan/section11f.htm
  8. Kinoshita Y, Cloutier AK, Rozak DA, Khan MSR, Niwa H, Uchida-Fujii E. A novel selective medium for the isolation of Burkholderia mallei from equine specimens.. BMC Vet Res 2019;15(1):133.
    doi: 10.1186/s12917-019-1874-0pmc: PMC6505306pubmed: 31064357google scholar: lookup
  9. Scholz HC, Pearson T, Hornstra H, Projahn M, Terzioglu R, Wernery R. Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.. PLoS Negl Trop Dis 2014;8(9):e3195.
    doi: 10.1371/journal.pntd.0003195pmc: PMC4177748pubmed: 25255232google scholar: lookup
  10. Raghavan R, Syriac G, Wernery R, Elschner M, Neubauer H, Mawhinney I. Comparative test performance of different serological tests for glanders.. In: 10th International Equine Infectious Disease Conference (IEIDC X); 2016 Apr 4–8; Buenos Aires, Argentina.
  11. Laroucau K, Aaziz R, Vorimore F, Varghese K, Deshayes T, Bertin C. A genetic variant of Burkholderia mallei detected in Kuwait: Consequences for the PCR diagnosis of glanders.. Transbound Emerg Dis 2020;00:1–4.
    doi: 10.1111/tbed.13777pubmed: 33245616google scholar: lookup
  12. 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.
    doi: 10.1186/1746-6148-5-32pmc: PMC2745380pubmed: 19723336google scholar: lookup
  13. Scholz HC, Joseph M, Tomaso H, Al Dahouk S, Witte A, Kinne J. Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay.. Diagn Microbiol Infect Dis 2006;54(4):241–247.
    doi: 10.1016/j.diagmicrobio.2005.09.018pubmed: 16466896google scholar: lookup
  14. Altemann D, Bauerfeind R, Wernery U. Pathogenesis of glanders in experimentally infected feral donkeys using different infection routes and doses.. J Equine Vet Sci 2012;32:S82.
  15. Yokoyama WM. Production of monoclonal antibody supernatant and ascites fluid.. Curr Protoc Mol Biol Chapter 11:Unit 11.10;2008.
    doi: 10.1002/0471142727.mb1110s83pubmed: 18633992google scholar: lookup
  16. Amemiya K, Bush GV, DeShazer D, Waag DM. Nonviable Burkholderia mallei induces a mixed Th1- and Th2-like cytokine response in BALB/c mice.. Infect Immun 2002;70(5):2319–2325.
    doi: 10.1128/IAI.70.5.2319-2325.2002pmc: PMC127890pubmed: 11953366google scholar: lookup
  17. Martelli P, Teng JLL, Lee FK, Yeong KY, Fong JYH, Hui SW. Influenza A(H1N1)pdm09 Virus Infection in a Captive Giant Panda, Hong Kong.. Emerg Infect Dis 2019;25(12):2303–2306.
    doi: 10.3201/eid2512.191143pmc: PMC6874238pubmed: 31742520google scholar: lookup
  18. Wernery U, Tsang CC, Hebel C, Damerau A, Kinne J, Cai JP. Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p-based enzyme-linked immunosorbent assay.. Mycoses 2018;61(8):600–609.
    doi: 10.1111/myc.12776pubmed: 29611232google scholar: lookup
  19. Khaki P, Mosavari N, Khajeh NS, Emam M, Ahouran M, Hashemi S. Glanders outbreak at Tehran Zoo, Iran.. Iran J Microbiol 2012;4(1):3–7.
    pmc: PMC3391557pubmed: 22783454
  20. Alibasoglu M, Yesildere T, Calislar T, Inal T, Calsikan U. Malleus-Ausbruch bei Löwen im Zoologischen Garten Istanbul.. Berl Münch Tierärztl Wochenschr 1986;99(2):57–63.
    pubmed: 3964210
  21. Bertani B, Ruiz N. Function and Biogenesis of Lipopolysaccharides.. EcoSal Plus 2018;8(1).
    doi: 10.1128/ecosalplus.ESP-0001-2018pmc: PMC6091223pubmed: 30066669google scholar: lookup
  22. Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity.. J Endotoxin Res 2001;7(3):167–202.
    pubmed: 11581570
  23. Brett PJ, Burtnick MN, Snyder DS, Shannon JG, Azadi P, Gherardini FC. Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes.. Mol Microbiol 2007;63(2):379–90.
    doi: 10.1111/j.1365-2958.2006.05519.xpmc: PMC1974782pubmed: 17163980google scholar: lookup
  24. West TE, Ernst RK, Jansson-Hutson MJ, Skerrett SJ. Activation of Toll-like receptors by Burkholderia pseudomallei.. BMC Immunol 2008;9:46.
    doi: 10.1186/1471-2172-9-46pmc: PMC2527550pubmed: 18691413google scholar: lookup
  25. Charuchaimontri C, Suputtamongkol Y, Nilakul C, Chaowagul W, Chetchotisakd P, Lertpatanasuwun N. Antilipopolysaccharide II: an antibody protective against fatal melioidosis.. Clin Infect Dis 1999;29(4):813–8.
    doi: 10.1086/520441pubmed: 10589895google scholar: lookup
  26. Thibault FM, Valade E, Vidal DR. Identification and discrimination of Burkholderia pseudomallei, B. mallei, and B. thailandensis by real-time PCR targeting type III secretion system genes.. J Clin Microbiol 2004;42(12):5871–4.
    doi: 10.1128/JCM.42.12.5871-5874.2004pmc: PMC535269pubmed: 15583328google scholar: lookup
  27. Brett PJ, DeShazer D, Woods DE. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei-like species.. Int J Syst Bacteriol 1998;48 Pt 1:317–20.
    doi: 10.1099/00207713-48-1-317pubmed: 9542103google scholar: lookup
  28. 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(1):115.
    doi: 10.1038/s41467-017-00173-8pmc: PMC5524647pubmed: 28740137google scholar: lookup
  29. Anuntagool N, Sirisinha S. Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei.. Microbiol Immunol 2002;46(3):143–50.
    doi: 10.1111/j.1348-0421.2002.tb02679.xpubmed: 12008922google scholar: lookup
  30. Sengyee S, Yoon SH, West TE, Ernst RK, Chantratita N. Lipopolysaccharides from Different Burkholderia Species with Different Lipid A Structures Induce Toll-Like Receptor 4 Activation and React with Melioidosis Patient Sera.. Infect Immun 2019;87(12):e00692–19.
    doi: 10.1128/IAI.00692-19pmc: PMC6867861pubmed: 31548320google scholar: lookup
  31. Wittig MB, Wohlsein P, Hagen RM, Al Dahouk S, Tomaso H, Scholz HC. Ein Ubersichtsreferat zur Rotzerkrankung [Glanders—a comprehensive review].. Dtsch Tierarztl Wochenschr 2006;113(9):323–330.
    pubmed: 17009807
  32. Lertpatanasuwan N, Sermsri K, Petkaseam A, Trakulsomboon S, Thamlikitkul V, Suputtamongkol Y. Arabinose-positive Burkholderia pseudomallei infection in humans: case report.. Clin Infect Dis 1999;28(4):927–8.
    doi: 10.1086/517253pubmed: 10825075google scholar: lookup
  33. Glass MB, Gee JE, Steigerwalt AG, Cavuoti D, Barton T. Pneumonia and septicemia caused by Burkholderia thailandensis in the United States.. J Clin Microbiol 2006;44(12):4601–4.
    doi: 10.1128/JCM.01585-06pmc: PMC1698378pubmed: 17050819google scholar: lookup
  34. Gee JE, Elrod MG, Gulvik CA, Haselow DT, Waters C, Liu L. Burkholderia thailandensis Isolated from Infected Wound, Arkansas, USA.. Emerg Infect Dis 2018;24(11):2091–2094.
    doi: 10.3201/eid2411.180821pmc: PMC6199988pubmed: 30334705google scholar: lookup

Citations

This article has been cited 2 times.
  1. He G, Zeng Y, He Q, Liu T, Li N, Lin H, Zeng M, Li Y, Peng M, Cheng J, Liu W, Yao W. A Case Report of Burkholderia mallei Infection Leading to Pneumonia. Comb Chem High Throughput Screen 2023;26(1):241-245.
    doi: 10.2174/1386207325666220509152221pubmed: 35579163google scholar: lookup
  2. Lau SKP, He Z, Tsang CC, Chan TTY, Luk HKH, Chan E, Li KSM, Fung J, Chow FWN, Tam AR, Chung TWH, Wong SCY, Que TL, Fung KSC, Lung DC, Wu AKL, Hung IFN, Teng JLL, Wernery U, Hui SW, Martelli P, Woo PCY. A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals. Microorganisms 2021 May 10;9(5).
    doi: 10.3390/microorganisms9051019pubmed: 34068581google scholar: lookup
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