A new single-antigen serological test accurately detects horses infected with Streptococcus equi subsp equi.

Overview

• This study developed and validated a new, highly accurate blood test to detect horses infected with Streptococcus equi subsp equi (SEE), the causative agent of strangles.
• The new test uses a single protein, called P92, identified through analysis of many SEE proteins to improve detection accuracy over existing methods.

Background and Objectives

• Strangles is a contagious infection in horses caused by Streptococcus equi subsp equi (SEE).
• Accurate, early detection of infected horses is essential for controlling outbreaks and preventing transmission.
• Existing serological tests, such as those targeting the M protein of SEE (SeM), have limitations in sensitivity and specificity.
• The objective was to develop a more accurate serological test by identifying novel antigenic proteins of SEE that elicit detectable antibody responses in infected horses.

Methodology

• Protein Selection and Microarray Construction:
  • 89 proteins from SEE, either surface-expressed or secreted, were cloned and expressed using a cell-free system.
  • These proteins were printed onto a microarray slide to allow simultaneous testing of immune responses against all candidates.
• Sample Collection:
  • Serum samples were collected from 78 Texas horses between 2020 and 2021:
    • 30 horses with confirmed strangles (SEE infection)
    • 30 control horses with no infection or immunization history
    • 18 horses infected with Streptococcus equi subsp zooepidemicus (SEZ), which is a related but distinct bacterium
• Microarray Screening:
  • The serum samples were tested against the microarray proteins to identify which proteins triggered antibody binding specific to SEE infection.
  • The statistical analysis highlighted one novel protein, named protein 92 (P92), as a promising antigen for serological testing.
• Validation by ELISA:
  • Enzyme-linked immunosorbent assays (ELISAs) were developed using purified P92 protein and separately, the known SeM protein.
  • Serum from a larger, diverse population of horses collected in 2022-2023 across multiple US states was tested:
    • 102 horses with strangles
    • 104 control horses
    • 24 horses infected with SEZ
• Data Analysis:
  • Diagnostic accuracy was evaluated by calculating the area under the receiver operating characteristic curve (AUC-ROC) for each ELISA.
  • Generalized linear modeling and pairwise comparisons (Tukey method) assessed differences in antibody responses between groups.

Key Findings

• The microarray effectively identified P92 as a novel antigen specific to SEE infection.
• The P92 ELISA showed significantly improved diagnostic accuracy compared to the SeM ELISA:
  • P92 ELISA AUC-ROC was 95% (Confidence Interval: 92%-98%)
  • SeM ELISA AUC-ROC was 88% (Confidence Interval: 83%-92%)
• This indicates better sensitivity and specificity of the P92 test in distinguishing strangles-infected horses from uninfected controls and those infected with related bacteria (SEZ).

Implications and Conclusions

• The study identified a new, single protein antigen (P92) that substantially improves serological detection of strangles in horses.
• The P92-based ELISA test provides veterinarians and researchers with a more accurate diagnostic tool for identifying infected horses.
• Early and precise detection of SEE infection helps in timely treatment and controlling outbreaks.
• The approach demonstrates the utility of high-throughput protein microarrays to discover novel diagnostic targets for infectious diseases.
• The improved test can potentially replace or supplement assays based on existing antigens such as SeM.

Summary

• This research successfully developed a novel serological test based on the newly identified P92 protein that outperforms traditional diagnostics in detecting strangles infection in horses.
• The method combines modern molecular techniques, comprehensive protein screening, rigorous validation, and robust statistical analysis to enhance veterinary diagnostic capabilities.