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Development of a microsphere-based immunoassay for the serological detection of glanders in equids
K Laroucau1, M Saqib2, B Martin1
1Paris-Est University, ANSES, Animal Health Laboratory, Bacterial Zoonosis Unit, EURL for equine diseases/Glanders, Maisons-Alfort, France.
Abstract:
Burkholderia mallei is the etiologic agent of glanders, an infectious disease of solipeds, with renewed scientific interest due to its increasing incidence in different parts of the world. More rapid, sensitive and specific assays are required by laboratories for confirmatory testing of this disease. A microsphere-based immunoassay consisting of beads coated with B. mallei recombinant proteins (BimA, GroEL, Hcp1, and TssB) has been developed for the serological diagnosis of glanders. The proteins' performance was compared with the OIE reference complement fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (iELISA) on a large panel of sera comprised of uninfected horses (n=198) and clinically confirmed cases of glanders from India and Pakistan (n=99). Using Receiver Operating Characteristics (ROC) analysis and adjusting the cutoff levels, Hcp1 (Se=100%, Sp=99.5%) and GroEL (Se= 97%, Sp=99.5%) antigens exhibited the best specificity and sensitivity. Neither Hcp1 and GroEL proteins, nor iELISA reacted with doubtful and positive CFT samples from glanders free countries which further confirmed the false positive reactions seen in CFT.
Insights
A new microsphere immunoassay using Burkholderia mallei recombinant proteins Hcp1 and GroEL offers highly sensitive and specific detection for glanders, a significant advancement for equine disease diagnosis.
Area of Science:
- Veterinary Microbiology
- Immunodiagnostics
- Equine Infectious Diseases
Background:
- Glanders, caused by Burkholderia mallei, is an infectious disease affecting solipeds with increasing global incidence.
- Accurate and rapid diagnostic assays are crucial for effective glanders control and surveillance.
- Current diagnostic methods like CFT and iELISA have limitations requiring improved alternatives.
Purpose of the Study:
- To develop and evaluate a novel microsphere-based immunoassay for the serological diagnosis of glanders.
- To compare the performance of recombinant Burkholderia mallei proteins (BimA, GroEL, Hcp1, TssB) in a diagnostic assay.
- To assess the sensitivity and specificity of the developed assay against established methods.
Main Methods:
- Development of a microsphere-based immunoassay using beads coated with B. mallei recombinant proteins (BimA, GroEL, Hcp1, TssB).
- Testing a large panel of sera from uninfected horses (n=198) and confirmed glanders cases (n=99) from India and Pakistan.
- Performance comparison with the OIE reference complement fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (iELISA) using ROC analysis.
Main Results:
- Recombinant proteins Hcp1 and GroEL demonstrated superior performance, with Hcp1 showing 100% sensitivity and 99.5% specificity, and GroEL showing 97% sensitivity and 99.5% specificity.
- The microsphere immunoassay showed high specificity, with no cross-reactivity with sera from glanders-free countries, unlike potential false positives in CFT.
- The developed assay effectively distinguished between infected and uninfected animals, confirming its diagnostic potential.
Conclusions:
- The microsphere-based immunoassay utilizing Hcp1 and GroEL antigens is a highly sensitive and specific tool for diagnosing glanders.
- This novel assay offers a reliable alternative for laboratory confirmation of glanders, addressing the need for improved diagnostic methods.
- The findings support the use of this assay in global surveillance and control programs for glanders in equids.
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