Development of lateral flow assays for the rapid diagnosis of glanders in equids.

Overview

• This study developed and evaluated rapid lateral flow assays (LFAs) to detect glanders, a contagious disease in horses, donkeys, and mules, providing a practical diagnostic tool for use in the field, especially in regions with limited laboratory infrastructure.

Background

• Glanders is a zoonotic disease caused by the bacterium Burkholderia mallei that primarily affects equids such as horses, donkeys, and mules.
• The disease remains endemic in regions of Asia, Africa, the Middle East, and South America.
• It poses major economic and health risks due to its contagious nature and impact on animal health.
• Traditional diagnostic methods for glanders are expensive, time-consuming, and require well-equipped laboratories and trained personnel, limiting their applicability in resource-poor settings.
• There is a pressing need for accessible, rapid, and user-friendly diagnostic tools suitable for field use.

Objective

• To develop and validate lateral flow assays (LFAs) that can rapidly detect antibodies against Burkholderia mallei antigens, facilitating quick diagnosis of glanders in equids without the need for complex equipment.

Methods

• Three lateral flow assays were developed, targeting antibodies against three different antigens: Hcp1, GroEL, and whole-cell lysate (WCL) of B. mallei.
• These antigen targets were previously identified as promising markers in indirect enzyme-linked immunosorbent assays (ELISAs).
• Recombinant streptococcal protein G was used as the colloidal gold conjugate in the LFAs to capture equid antibodies.
• The diagnostic performance of the LFAs was tested using serum samples collected in Mongolia, comprising:
  • 31 glanders-positive equids
  • 51 glanders-negative equids
  • 60 horses with other infections from Japan to assess cross-reactivity
• Stability testing of the LFAs was conducted by assessing their functionality after storage at 37°C for 16 weeks.

Results

• The Hcp1-based LFA demonstrated:
  • 100% sensitivity (correctly identifying all infected animals)
  • 100% specificity (no false positives among uninfected animals)
• The WCL-based LFA showed:
  • 100% sensitivity
  • 98.0% specificity (very few false positives)
• The GroEL-based LFA exhibited:
  • 100% sensitivity
  • Lower specificity at 90.2%, indicating more false positives.
• Both Hcp1- and WCL-LFAs were effective at detecting antibodies even in asymptomatic horses, which is crucial for early diagnosis and disease control.
• No cross-reactivity was observed with sera from horses infected with other diseases, indicating good assay specificity.
• All LFAs were stable and functional after prolonged storage at an elevated temperature (37°C) for 16 weeks, demonstrating suitability for field conditions in warm climates without cold chain requirements.

Significance

• The developed LFAs provide a rapid, easy-to-use, and cost-effective alternative to conventional glanders diagnostic methods.
• The assays require minimal training and no specialized equipment, making them ideal for use in low-resource and endemic settings.
• Rapid field diagnosis facilitates early detection and timely intervention, which is critical to preventing the spread of glanders among equine populations.
• The stability of the LFAs at high temperatures enhances their practicality in tropical and subtropical regions.

Limitations and Future Work

• The study sample size, while adequate for initial validation, remains relatively limited; further validation with larger and more diverse sample sets across different geographic regions is necessary.
• Continued evaluation under actual field conditions will help confirm operational effectiveness and guide potential improvements.
• Exploring integration with other disease surveillance systems could extend their utility.

Conclusion

• Lateral flow assays based on the Hcp1 and whole-cell lysate antigens represent promising tools for the rapid and reliable diagnosis of glanders in equids.
• Their simplicity, stability, and affordability make them well suited for deployment in endemic regions with constrained resources.
• These assays have the potential to significantly improve disease control efforts and reduce the adverse economic and health impacts of glanders worldwide.