Species diversity and within-host tropism for mixed equine strongyle infections using a cytochrome c oxidase subunit I metabarcoding approach.

Overview

• This study investigated the species diversity and distribution of strongyle parasites in horses using a DNA-based technique (COI metabarcoding) to accurately identify different species within mixed infections.
• The research compared parasite communities across different sample types and intestinal locations, revealing insights into species prevalence and habitat preference within the horse gut.

Background

• Strongyles, particularly non-migratory Strongylidae, are the most common parasites inhabiting the equine gut.
• Individual horses often carry mixed infections containing multiple strongyle species, typically between 10 and 20 species per host.
• Knowledge on the prevalence of specific strongyle species and their preferred locations (within-host tropism) in the intestinal tract remains limited.
• Traditional methods have difficulty distinguishing closely related species, especially cryptic species complexes.

Research Objectives

• Determine the species composition of mixed strongyle infections in untreated young horses by sampling different stages and gut locations.
• Assess the agreement in species diversity data derived from different sample types: faecal eggs, cultured larvae, and adult worms from distinct gut regions (ventral vs dorsal colon).
• Employ a cytochrome c oxidase subunit I (COI) metabarcoding approach to differentiate closely related species, including cryptic taxa, within strongyle complexes.
• Explore within-host tropism by comparing species prevalence between ventral and dorsal colon.

Methodology

• Samples were collected from 12 naturally infected, untreated young horses, including:
• Strongyle eggs isolated from faeces.
• Third stage larvae (L3) cultured from faecal samples.
• Adult worms collected post-mortem from the ventral and dorsal colon.
• DNA was extracted from these samples and subjected to COI metabarcoding, a genetic barcoding technique targeting the cytochrome c oxidase I gene sequence.
• Operational taxonomic units (OTUs) were defined to classify and distinguish closely related species, including cryptic species difficult to separate morphologically.
• Data analysis included estimation of alpha diversity (within-sample species diversity) using diversity indexes such as Inverse Simpson and Shannon, and beta diversity (between-sample community similarity/difference) using Bray-Curtis and Jaccard dissimilarities.
• Statistical tests including PERMANOVA were applied to assess differences in parasite communities between sample types and gut locations.

Key Findings

• A total of 17 strongyle species were identified: 16 small strongyle species and one large strongyle species.
• Eleven small strongyle species occurred in all horses, including three cryptic species designated as Cylicostephanus calicatus OTU II, Cylicostephanus minutus OTU II and III.
• Species detection rate and diversity measures showed strong agreement between faecal egg DNA and cultured larvae DNA, confirming the reliability of non-invasive sampling methods.
• Alpha diversity was significantly greater in parasite eggs and larvae compared to adult worms recovered from the lumen, indicating possible differences in community composition at life stages or sampling biases.
• Beta diversity analysis revealed:
• Egg and larvae samples clustered closely, reflecting similar species composition.
• Samples from ventral and dorsal colon formed distinct clusters, indicating different parasite communities in these gut regions.
• The difference between ventral and dorsal colon communities was statistically significant (PERMANOVA P < 0.001).
• Five small strongyle species, including the cryptic Cylicostephanus calicatus OTU II, were significantly more prevalent in the ventral colon than the dorsal colon, demonstrating within-host spatial tropism.

Conclusions and Significance

• The study successfully applied a COI metabarcoding approach to resolve species diversity and cryptic species in strongyle infections, overcoming limitations of traditional methods.
• It provides novel prevalence data especially for cryptic species that are difficult to detect morphologically.
• Non-invasive sampling techniques (faecal eggs and cultured larvae) provide species composition data closely matching that from adult worms, supporting their use for epidemiological monitoring.
• Clear evidence of within-host niche preferences (tropism) for different parasite species within the intestinal tract was documented, with certain species favoring the ventral colon.
• Understanding strongyle species diversity and tropism is vital for managing parasite infections, improving diagnosis, and tailoring targeted treatments in horses.