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

M Jelocnik; C Hall; S Dennis; K Mitchell; A Blishen; N Mashkour; S I Anstey; C Jenkins; K Jeffers; C El-Hage; D McMillan; J Gilkerson

doi:10.1111/avj.70022

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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

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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

M Jelocnik, C Hall, S Dennis, K Mitchell, A Blishen, N Mashkour, S I Anstey, C Jenkins, K Jeffers, C El-Hage, …

Australian Veterinary Journal, Vol.104(3), pp.158-166

2026

DOI: https://doi.org/10.1111/avj.70022

PMID: 41024428

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Abstract

Strangles

Equine

Streptococci

Isothermal amplification

Point‐of‐care application

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.

Details

Title: 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
Authors: M Jelocnik (Corresponding Author) - University of the Sunshine Coast, Queensland, School of Science, Technology and Engineering
C Hall - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
S Dennis - The University of Melbourne
K Mitchell - Scone Equine Hospital (Australia)
A Blishen - Scone Equine Hospital (Australia)
N Mashkour - IDEXX-AU
S I Anstey - University of the Sunshine Coast, Queensland, School of Science, Technology and Engineering
C Jenkins - New South Wales Department of Primary Industries
K Jeffers - The University of Melbourne
C El-Hage - The University of Melbourne
D McMillan - University of the Sunshine Coast, Queensland, School of Science, Technology and Engineering
J Gilkerson - The University of Melbourne
Publication details: Australian Veterinary Journal, Vol.104(3), pp.158-166
Publisher: Wiley-Blackwell Publishing Ltd.
Date published: 2026
DOI: 10.1111/avj.70022
ISSN: 1751-0813
PMID: 41024428
Copyright note: © 2025 The Author(s). Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Data Availability: The data that supports the findings of this study are available in the supplementary material of this article.
Grant note: PRO-015486 / Agrifutures Australia
Organisation Unit: Academic Support Unit; School of Science, Technology and Engineering; Centre for Bioinnovation
Language: English
Record Identifier: 991166461502621
Output Type: Journal article

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