Real-time fluorometric isothermal assays for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus in horses: Validation, comparison and evaluation of their clinical application.
Abstract: Infectious diseases significantly impact equine health and welfare, causing illness and death, and loss of productivity globally. One such disease is 'strangles', a highly contagious upper respiratory condition in horses caused by Streptococcus equi subspecies equi (SEE). Diagnostic methods for this pathogen include sensitive molecular assays and less reliable bacterial isolation and biochemical testing. However, the presence of closely related streptococci, such as Streptococcus equi subspecies zooepidemicus (SZOO), may confound diagnosis. Rapid assays for SEE are crucial for outbreak control. This study aimed to validate species-specific loop-mediated isothermal amplification (LAMP) assays for SEE and SZOO using a portable real-time fluorometer performed in a clinical setting and to compare their performance with laboratory-based conventional API20 Strep strip and qPCR assays. Rapid sample processing methods were also evaluated with a range of clinical samples, including nasopharyngeal and guttural pouch lavage, purulent exudate, mucosal swabs and bacterial isolates. The LAMP assays demonstrated an analytical sensitivity of 50 genome copies per reaction and showed high congruence with external diagnostic methods. For SEE, the agreement was 96.55% (Kappa 0.88), while for SZOO, it was 94.87% (Kappa 0.87). When applied to rapidly processed clinical samples, SEE and SZOO LAMPs achieved agreements of 97.01% (Kappa 0.70) and 94.94% (Kappa 0.87), respectively, compared with qPCR assays. Testing in an equine clinical setting revealed a 93.84% agreement between LAMP and qPCR results, and 86.96% between LAMP and API20 Strep assays. This study highlights LAMP assays as effective, rapid diagnostic tools, offering reliable options for clinical settings and enabling appropriate and more timely biosecurity interventions.
© 2025 The Author(s). Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association.
Publication Date: 2025-09-29 PubMed ID: 41024428PubMed Central: PMC12961256DOI: 10.1111/avj.70022Google Scholar: Lookup
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- Journal Article
- Comparative Study
- Validation Study
- Animal Health
- Animal Species
- Diagnostic Technique
- Disease
- Disease Diagnosis
- Disease Management
- Disease Prevention
- Disease Surveillance
- Disease Transmission
- Equine Diseases
- Equine Health
- Equine Medicine
- Infection
- Infectious Disease
- Laboratory Methods
- Microbiology
- Molecular biology
- Polymerase Chain Reaction
- Veterinary Medicine
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.
Objective Overview
- This study validates and evaluates rapid, species-specific LAMP assays for detecting Streptococcus equi subspecies equi (SEE) and subspecies zooepidemicus (SZOO) in horses, comparing them to conventional laboratory tests for clinical diagnostic use.
Background and Importance
- Infectious diseases like strangles—caused by SEE—pose significant health risks to horses worldwide by causing illness, death, and loss of productivity.
- Strangles is highly contagious and affects the upper respiratory tract of horses, necessitating rapid and accurate diagnosis to control outbreaks effectively.
- Existing diagnostic methods include:
- Sensitive molecular assays such as qPCR (quantitative Polymerase Chain Reaction).
- Traditional bacterial culture and biochemical testing using API20 Strep strips, which are less reliable and slower.
- The presence of closely related bacteria like SZOO can complicate diagnosis due to similarities.
- Rapid, field-deployable diagnostic tools are therefore needed to enable timely biosecurity measures and treatment decisions.
Aims and Objectives
- To validate LAMP (loop-mediated isothermal amplification) assays that target SEE and SZOO DNA, enabling real-time detection using a portable fluorometer device.
- To compare the clinical performance of LAMP against established diagnostic methods: qPCR (laboratory-based molecular assay) and API20 Strep (biochemical test).
- To assess various rapid sample preparation methods suitable for clinical settings using different clinical specimens (nasopharyngeal swabs, guttural pouch lavage, purulent exudate, mucosal swabs, and bacterial culture isolates).
- To test the feasibility and accuracy of using these LAMP assays directly in equine clinical environments.
Methodology
- Design of species-specific LAMP assays targeting genetic sequences unique to SEE and SZOO.
- Use of a portable real-time fluorometer to conduct LAMP at the point-of-care or clinical settings, bypassing extensive lab infrastructure.
- Collection of a broad range of clinical samples, including:
- Nasopharyngeal swabs
- Guttural pouch lavage fluids
- Purulent exudates
- Mucosal swabs
- Bacterial isolates cultured from clinical samples
- Rapid sample processing protocols were applied to prepare these samples for LAMP testing without elaborate purification.
- Comparison of LAMP assay results with those from qPCR and API20 Strep biochemical tests.
- Statistical analysis of diagnostic agreement was performed using percent agreement and Cohen’s Kappa coefficient to measure concordance beyond chance:
- Kappa values closer to 1 indicate excellent agreement.
Key Results
- LAMP assay sensitivity reached as low as 50 genome copies per reaction, reflecting high analytical sensitivity.
- Agreement rates between LAMP and external diagnostic methods were very high:
- SEE detection: 96.55% agreement with Kappa 0.88 (almost perfect agreement).
- SZOO detection: 94.87% agreement with Kappa 0.87 (almost perfect agreement).
- When testing rapidly processed clinical samples, LAMP vs. qPCR agreement remained high:
- SEE: 97.01% agreement, Kappa 0.70 (substantial agreement).
- SZOO: 94.94% agreement, Kappa 0.87 (almost perfect agreement).
- Testing performed in an equine clinical setting showed:
- 93.84% agreement between LAMP and qPCR results.
- 86.96% agreement between LAMP and API20 Strep biochemical results.
Conclusions and Implications
- LAMP assays provide a rapid, reliable alternative for detecting both SEE and SZOO in various clinical samples related to equine respiratory disease.
- The assays are highly sensitive and show excellent agreement with established molecular tests, validating their accuracy for clinical use.
- Use of a portable fluorometer and simple sample preparation makes the method well suited for field or clinic settings where quick results are critical.
- Faster diagnosis using LAMP could enable quicker biosecurity and treatment responses during outbreaks, improving equine health outcomes and controlling disease spread.
- This study supports implementing LAMP assays as practical diagnostic tools to complement or replace slower, less reliable biochemical methods in equine veterinary medicine.
Cite This Article
APA
Jelocnik M, Hall C, Dennis S, Mitchell K, Blishen A, Mashkour N, Anstey SI, Jenkins C, Jeffers K, El-Hage C, McMillan D, Gilkerson J.
(2025).
Real-time fluorometric isothermal assays for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus in horses: Validation, comparison and evaluation of their clinical application.
Aust Vet J, 104(3), 158-166.
https://doi.org/10.1111/avj.70022 Publication
Researcher Affiliations
- School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Centre for Equine Infectious Disease, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Asia Pacific Centre for Animal Health, The Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Scone Equine Group, Scone, New South Wales, Australia.
- Scone Equine Group, Scone, New South Wales, Australia.
- Australia & New Zealand IDEXX Laboratories Pty Ltd, Eight Mile Plains, Queensland, Australia.
- School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Elizabeth Macarthur Agricultural Institute, NSW Department of Primary Industries, Menangle, New South Wales, Australia.
- Centre for Equine Infectious Disease, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Asia Pacific Centre for Animal Health, The Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Centre for Equine Infectious Disease, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Asia Pacific Centre for Animal Health, The Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
- Centre for Equine Infectious Disease, Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
- Asia Pacific Centre for Animal Health, The Melbourne Veterinary School, The University of Melbourne, Parkville, Victoria, Australia.
MeSH Terms
- Animals
- Horses
- Horse Diseases / diagnosis
- Horse Diseases / microbiology
- Streptococcal Infections / veterinary
- Streptococcal Infections / diagnosis
- Streptococcal Infections / microbiology
- Nucleic Acid Amplification Techniques / veterinary
- Nucleic Acid Amplification Techniques / methods
- Sensitivity and Specificity
- Molecular Diagnostic Techniques / veterinary
- Molecular Diagnostic Techniques / methods
- Streptococcus equi / isolation & purification
- Fluorometry / veterinary
- Fluorometry / methods
- Streptococcus / isolation & purification
- Real-Time Polymerase Chain Reaction / veterinary
Grant Funding
- PRO-015486 / Agrifutures Australia
Conflict of Interest Statement
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
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