Exhaled breath condensate in horses: a first microbiological insight.

Overview

• This study explores the types of bacteria found in the exhaled breath condensate (EBC) of healthy horses kept in different environments—either in stalls or on pasture—to better understand the microbial profiles present in the airway fluids using a non-invasive sampling method.

Background

• Exhaled breath condensate (EBC) is a non-invasive technique that collects airway lining fluid by condensing moisture from exhaled air.
• While EBC has been used in research to monitor respiratory conditions, its microbiological composition, especially in horses, remains largely unknown.
• This study aims to fill that gap by characterizing the culturable bacteria present in EBC from clinically healthy horses.

Study Design and Methods

• Subjects: 30 clinically healthy mares divided into two equal groups based on their housing conditions:
  • Racetrack group: Horses housed in stalls.
  • Farm group: Horses maintained on pasture.
• Sampling procedure:
  • Used a validated device designed by the research group to collect EBC.
  • Nasal cleaning with dry gauze was performed prior to sampling to minimize contamination.
  • Condensed droplets of exhaled breath were collected using sterile swabs and stored in Stuart transport medium for microbiological analysis.
• Microbiological analysis:
  • Samples were cultured on blood agar (supports growth of many bacteria including Gram-positive) and MacConkey agar (selective for Gram-negative bacteria).
  • Colony counting was performed to quantify bacterial growth.
  • Gram staining distinguished Gram-positive from Gram-negative bacteria.
  • Biochemical tests were used to identify bacterial species or groups.
  • Control samples of collection surfaces were tested to rule out contamination.

Key Findings

• No bacterial growth was found on the control samples, confirming the sampling process was not contaminated.
• Both groups of horses showed significantly higher numbers of Gram-positive bacteria compared to Gram-negative bacteria in EBC samples:
  • Farm group: Approximately 4,013 Gram-positive colonies vs. 1,713 Gram-negative colonies.
  • Racetrack group: Approximately 2,967 Gram-positive colonies vs. 213 Gram-negative colonies.
• Environmental samples from both locations showed a similar pattern, with more Gram-positive colonies:
  • Farm environment: 16 Gram-positive colonies vs. 0 Gram-negative colonies.
  • Racetrack environment: 1,220 Gram-positive colonies vs. 38 Gram-negative colonies.
• The racetrack environment had fewer Gram-negative bacteria both in environmental samples and EBC compared to the farm environment.

Interpretation and Conclusions

• This research gives the first insights into the types and quantities of culturable bacteria present in the breath condensate of healthy horses.
• The predominance of Gram-positive bacteria suggests these microorganisms might be typical constituents of the equine airway microbiome or may reflect environmental exposure.
• Differences between horses kept in stalls (racetrack) and those on pasture (farm) imply that management practices and environmental factors influence the microbial profiles detected in exhaled breath.
• The study validates EBC collection as a feasible, non-invasive method to sample the airway lining and study respiratory microbiology in horses.
• Future research may explore the clinical significance of these bacterial profiles, their role in respiratory health or disease, and how environmental factors shape the airway microbiome.