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Equine veterinary journal2026; doi: 10.1002/evj.70222

Diagnosis of bacteraemia in neonatal foals using 16S rRNA high-throughput sequencing.

Abstract: Sepsis is an important cause of morbidity and mortality in foals. Early diagnosis can improve outcome but is complicated by non-specific clinical signs and delayed confirmation of infection via blood culture. Molecular assays represent a rapid and more sensitive alternative. Objective: To evaluate and compare bacterial load and composition in blood and blood culture media (BCM) of sick and healthy foals using molecular assays and to compare results with traditional bacterial culture. Methods: Cross-sectional observational clinical study. Methods: Thirteen septic foals, 10 sick non-septic and 8 healthy foals were included. Bacterial load and composition from whole blood (WB), pre-enriched BCM and contamination controls were analysed by quantitative PCR and sequencing of the universal bacterial 16S rRNA marker gene. Results: WB sequencing yielded more positive samples (25/31) than BCM (6/62; p < 0.01). Positive blood culture and WB sequencing were comparable in only 3/12 foals. Sequencing samples were at high potential for contamination, with most samples having high relative abundances (RAs) of Paucibacter and Ralstonia. High RAs of Actinobacillus and Staphylococcus in septic foals may, however, suggest true pathogen detection. No significant differences in bacterial RAs, 16S rRNA gene load (qPCR), or other sequencing-based metrics were found between foal groups and between WB and contamination controls. Conclusions: Inherent inaccuracies of classification schemes for septic and sick non-septic foals and the impact of contamination when sequencing low biomass samples. Conclusions: High-throughput sequencing represents a possible avenue for the diagnosis of bacteraemia in foals but has high potential for environmental and extraction-related contamination and cannot replace blood culture at this time. Further research to optimise detection yield and sensitivity on WB samples is warranted.
© 2026 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.
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Publication Date: 2026-07-01 PubMed ID: 42381558DOI: 10.1002/evj.70222Google Scholar: Lookup
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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.

Overview

  • This study investigates the use of 16S rRNA high-throughput sequencing to diagnose bacteraemia (bacterial infection in the bloodstream) in neonatal foals, comparing its effectiveness and bacterial detection with traditional blood culture methods and molecular assays.

Background and Objective

  • Sepsis is a leading cause of illness and death in newborn foals.
  • Early and accurate detection of sepsis is challenging due to:
    • Non-specific symptoms
    • Delayed results from conventional blood cultures
  • Molecular assays, such as sequencing of the universal bacterial 16S rRNA gene, are promising for faster and potentially more sensitive diagnosis.
  • The primary objective was to:
    • Evaluate bacterial load and composition in whole blood (WB) and blood culture media (BCM) of sick and healthy foals using molecular methods
    • Compare results with traditional bacterial culture outcomes

Methods

  • Study design: Cross-sectional observational clinical study.
  • Participants:
    • 13 septic foals (with suspected or confirmed sepsis)
    • 10 sick but non-septic foals
    • 8 healthy foals (controls)
  • Sample types analyzed:
    • Whole blood (WB)
    • Pre-enriched blood culture media (BCM)
    • Contamination controls (to assess background noise and contaminants)
  • Analytical methods:
    • Quantitative PCR (qPCR) for measuring bacterial 16S rRNA gene load
    • High-throughput sequencing of the universal bacterial 16S rRNA gene to identify bacterial species present
    • Traditional bacterial culture to compare pathogen detection

Key Findings

  • Whole blood sequencing detected bacterial DNA in a significantly higher number of samples (25 out of 31) compared to blood culture media (6 out of 62), suggesting higher sensitivity of sequencing in direct blood samples.
  • There was limited concordance between positive blood cultures and sequencing results; only 3 out of 12 foals tested positive by both methods, indicating potential discrepancies.
  • High relative abundance (RA) of bacteria commonly associated with contamination, such as Paucibacter and Ralstonia species, was found in many sequencing samples.
  • Some foals, particularly septic ones, showed higher RAs of likely pathogenic bacteria such as Actinobacillus and Staphylococcus, suggesting true infection detection in those cases.
  • No significant differences were found between foal groups (septic, non-septic sick, healthy) or between whole blood samples and contamination controls based on:
    • Bacterial relative abundances
    • Quantitative 16S rRNA gene loads
    • Other sequencing-based diversity or abundance metrics

Interpretation and Limitations

  • Challenges in precisely classifying foals as septic vs. non-septic complicate the interpretation of sequencing results.
  • Sequencing low-biomass samples like blood is prone to contamination from:
    • Environmental sources
    • DNA extraction procedures
  • This contamination can obscure true infection signals and generate false-positive results.
  • Conventional blood culture, although slower and possibly less sensitive, remains the gold standard for diagnosing bacteraemia in foals for now.

Conclusions and Future Directions

  • High-throughput 16S rRNA gene sequencing shows promise as a diagnostic tool for detecting bacteraemia in neonatal foals due to its rapidity and sensitivity.
  • However, current methodologies are limited by contamination risks and lack of perfect correlation with blood cultures.
  • Further method development is needed to:
    • Reduce contamination effectively
    • Improve specificity and sensitivity for detecting true pathogens directly from whole blood
    • Enhance clinical classification criteria to better distinguish septic from non-septic foals

Cite This Article

APA
Payette F, Long AE, Hu W, Bittinger K, Moustafa AM, Stefanovski D, Abraham M, Aitken MR. (2026). Diagnosis of bacteraemia in neonatal foals using 16S rRNA high-throughput sequencing. Equine Vet J. https://doi.org/10.1002/evj.70222

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Payette, Flavie
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.
Long, Alicia E
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.
Hu, Weiming
  • Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Bittinger, Kyle
  • Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Moustafa, Ahmed M
  • Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Stefanovski, Darko
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.
Abraham, Michelle
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.
Aitken, Maia R
  • Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania, USA.

Grant Funding

  • 580-5805-1-400666-XXXX-2000-5351 / Firestone/Tamworth/Raker-Tulleners Grant from the Department of Clinical Studies, New Bolton Center, University of Pennsylvania

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