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The Journal of veterinary medical science2025; 87(4); 394-401; doi: 10.1292/jvms.25-0039

Comparison of immunogenicity of 17 Burkholderia mallei antigens and whole cell lysate using indirect ELISA.

Abstract: Glanders is a World Organization for Animal Health (WOAH)-notifiable equine disease caused by the infection of Burkholderia mallei, and is endemic in Mongolia, South Asia, Africa, and South America. While the complement fixation test (CFT) has been widely used for serodiagnosis of glanders and is considered a standard serological test, it has several limitations. These limitations include poor specificity, labor intensive techniques, variability in antigen and protocol. Consequently, indirect enzyme-linked immunosorbent assays (iELISAs) based on recombinant proteins have been developed as alternative serodiagnostic assays to address some of the challenges associated with the CFT. The accuracy of iELISA relies on the B. mallei proteins used as an antigen. Hence, to determine the best diagnostic candidate in iELISA, in terms of sensitivity and specificity, a comparison of 17 immunogenic B. mallei proteins and detergent-based whole cell lysate (WCL) was performed. According to the sensitivity and specificity on the sera from glanderous and non-glanderous Mongolian native horses, iELISA using Hcp1, GroEL, and detergent-based WCL represented the highest diagnostic accuracy. These three candidates did not have cross-reactivity to horse sera with several other equine diseases. WCL, Hcp1, and GroEL showed considerable potential as antigens for iELISA in the serodiagnosis of glanders in Mongolia. Detergent-based WCL extraction offers a consistent approach for the preparation of reliable B. mallei antigen. WCL-iELISA should be further validated in a large-scale study to meet WOAH demands.
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Publication Date: 2025-03-04 PubMed ID: 40044168PubMed Central: PMC11964862DOI: 10.1292/jvms.25-0039Google Scholar: Lookup
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  • Journal Article
  • Comparative Study

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.

Overview

  • This study compares the immune response triggered by 17 different Burkholderia mallei proteins and a whole cell lysate in order to identify the most accurate antigen for diagnosing glanders in horses using indirect ELISA tests.
  • The goal is to find better diagnostic tools than the traditionally used complement fixation test (CFT), which has several drawbacks.

Background

  • Glanders is a contagious and serious disease in horses caused by the bacterium Burkholderia mallei.
  • It is notifiable to the World Organization for Animal Health (WOAH), meaning it has significant animal health and trade implications.
  • The disease is endemic in specific regions such as Mongolia, South Asia, Africa, and South America.
  • Current standard testing uses the complement fixation test (CFT), which faces challenges such as:
    • Poor specificity — leading to false positives or negatives.
    • Labor-intensive procedures.
    • Variability due to differences in antigen preparations and testing protocols.
  • Indirect ELISA (iELISA) methods have been developed to overcome limitations of the CFT by using specific antigens from B. mallei proteins.

Research Objective

  • To evaluate and compare the immunogenicity (ability to provoke an immune response) of 17 recombinant B. mallei proteins and a detergent-based whole cell lysate (WCL) as antigens.
  • To identify which antigen(s) provide the best balance of sensitivity and specificity in diagnosing glanders via iELISA.
  • To assess potential cross-reactivity of these antigens with sera from horses suffering from other equine diseases, which would affect test accuracy.

Methodology

  • Seventeen different immunogenic B. mallei proteins were selected based on previous data and recombinant protein production.
  • A detergent-based WCL was prepared, which extracts a broad spectrum of bacterial proteins but in a consistent manner.
  • Indirect ELISA tests were performed using sera from:
    • Horses clinically diagnosed with glanders.
    • Non-glanderous Mongolian native horses serving as controls.
    • Horses with other equine diseases to test cross-reactivity.
  • Sensitivity (ability to correctly identify infected horses) and specificity (ability to correctly identify non-infected horses) were calculated for each antigen.

Key Findings

  • Among the 17 proteins tested, the Hcp1 and GroEL proteins showed the highest sensitivity and specificity when used in iELISA.
  • The detergent-based whole cell lysate (WCL) also demonstrated high diagnostic accuracy comparable to the best recombinant proteins.
  • None of these top candidates (Hcp1, GroEL, WCL) cross-reacted with sera from horses infected with other common equine diseases, ensuring good test specificity.
  • The detergent-based WCL provides a reproducible method of extracting a comprehensive antigen profile from B. mallei, which is advantageous for assay consistency.

Implications

  • The WCL-iELISA, along with Hcp1 and GroEL based iELISAs, have strong potential to replace or supplement CFT for serodiagnosis of glanders, particularly in Mongolia where the disease is endemic.
  • Use of recombinant proteins allows for more standardized and less labor-intensive testing compared to traditional methods.
  • The consistent preparation of WCL antigen means that it could be scaled up for routine testing.
  • Further large-scale validation studies are needed to meet WOAH standards before these tests can be adopted widely for international health reporting and control measures.

Conclusions

  • This study identifies Hcp1, GroEL, and detergent-based WCL as highly immunogenic and specific antigens suitable for iELISA detection of glanders antibodies in horse sera.
  • The findings support continuing development and validation of these antigens, potentially improving glanders diagnosis worldwide.

Cite This Article

APA
Ichikawa Y, Iinuma Y, Okagawa T, Shimbo R, Enkhtuul B, Khurtsbaatar O, Kinoshita Y, Niwa H, Aoshima K, Kobayashi A, Batbaatar V, Ohashi K, Kimura T. (2025). Comparison of immunogenicity of 17 Burkholderia mallei antigens and whole cell lysate using indirect ELISA. J Vet Med Sci, 87(4), 394-401. https://doi.org/10.1292/jvms.25-0039

Publication

The Journal of veterinary medical science
ISSN: 1347-7439
NlmUniqueID: 9105360
Country: Japan
Language: English
Volume: 87
Issue: 4
Pages: 394-401

Researcher Affiliations

Ichikawa, Yoshiki
  • Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Iinuma, Yukiko
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Okagawa, Tomohiro
  • Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Shimbo, Ryo
  • Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Enkhtuul, Batchuluun
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Sciences, Ulaanbaatar, Mongolia.
Khurtsbaatar, Ochirbat
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Sciences, Ulaanbaatar, Mongolia.
Kinoshita, Yuta
  • Equine Research Institute, Japan Racing Association, Tochigi, Japan.
Niwa, Hidekazu
  • Equine Research Institute, Japan Racing Association, Tochigi, Japan.
Aoshima, Keisuke
  • Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Kobayashi, Atsushi
  • Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Batbaatar, Vanaabaatar
  • Laboratory of Infectious Disease and Immunology, Institute of Veterinary Medicine, Mongolian University of Life Sciences, Ulaanbaatar, Mongolia.
Ohashi, Kazuhiko
  • Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
  • International Affairs Office, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
Kimura, Takashi
  • Laboratory of Comparative Pathology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.

MeSH Terms

  • Burkholderia mallei / immunology
  • Animals
  • Enzyme-Linked Immunosorbent Assay / veterinary
  • Enzyme-Linked Immunosorbent Assay / methods
  • Glanders / diagnosis
  • Glanders / immunology
  • Glanders / microbiology
  • Horses
  • Antigens, Bacterial / immunology
  • Sensitivity and Specificity
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology
  • Horse Diseases / immunology
  • Serologic Tests / veterinary
  • Antibodies, Bacterial / blood

Conflict of Interest Statement

The authors declare that there is no conflict of interest.

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Citations

This article has been cited 1 times.
  1. Badten AJ, Oaxaca-Torres S, Torres AG. Antibodies to Burkholderia pseudomallei Outer Membrane Proteins Coupled to Nanovaccines Exhibit Cross-Reactivity to B. cepacia Complex and Pseudomonas aeruginosa Homologues. Microorganisms 2026 Jan 17;14(1).
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