Cytochrome c oxidase I deep amplicon sequencing for metabarcoding of equine strongyle communities: unexpectedly high Strongylus spp. prevalence in treated horses.

Overview

• This study developed and applied a new DNA sequencing method targeting the cytochrome c oxidase subunit I (COI) gene to identify and analyze the parasitic nematode communities (Strongylidae) in horses and related equines.
• The research revealed unexpectedly high prevalence of Strongylus species even in horses that had been regularly treated with anthelmintic drugs, challenging previous assumptions about Strongylus rarity in treated equines.

Background and Rationale

• Equines are commonly infected by complex communities of strongyle nematodes, specifically the family Strongylidae.
• These infections typically involve multiple species simultaneously, complicating diagnosis and treatment.
• Among these nematodes, cyathostomins (small strongyles) are generally the most prevalent, while Strongylus species (large strongyles) are believed to be rare today, especially in horses that receive regular anthelmintic treatment.
• Traditional microscopic methods cannot reliably differentiate nematode eggs and larvae at the species level, except for Strongylus spp., limiting ecological and pathological understanding.
• Previous molecular studies focused on ITS-2 gene regions for species identification, but recent evidence suggests that the COI gene provides better resolution for differentiating strongyle species.

Methodology

• The study developed a metabarcoding approach utilizing deep amplicon sequencing of nematode COI gene fragments, termed a “nemabiome” method.
• Third stage larvae (infective stage) were collected from equines across multiple countries including Brazil, France, Germany, Ukraine, the UK, and the USA.
• Equine hosts included mostly domestic horses, but also Przewalski’s horses, donkeys, and kulans (a type of wild ass).
• The study compared nematode communities from two groups:
  • Regularly treated (RT) equines, which received routine anthelmintic treatments
  • Never treated (NT) equines, which had no history of anthelmintic treatment
• Sequencing data were analyzed to classify reads to species level and assess species diversity, community composition, and prevalence.

Key Findings

• Approximately 87.7% of sequencing reads were identified confidently to species level, indicating the method’s effectiveness.
• Reads from donkeys and kulans had a higher proportion of unclassified sequences, suggesting the presence of undescribed or poorly characterized nematode species in these less-studied hosts.
• No significant differences were found in overall species diversity and richness between RT and NT equine groups, meaning treatment did not reduce the variety of species present.
• However, there were significant differences in species composition between the groups:
  • Strongylus spp., traditionally considered rare in treated horses, were detected at unexpectedly high abundance even in RT horses.
  • Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cyathostomum catinatum were comparatively more abundant in the RT group, implying these species may be more resilient or favored by treatment regimes.
• These findings suggest that regular anthelmintic treatment over decades may have shaped the strongyle community composition, potentially through selective pressures.
• The limited classification success in non-caballine equines highlights the need for expanded genetic reference databases to include more strongyle species from such hosts.

Implications and Conclusions

• The study challenges the assumption that Strongylus species have become rare due to anthelmintic treatments, indicating these parasites remain prevalent even in treated horses.
• This has important implications for equine health management, as Strongylus species can cause severe pathology.
• Using the COI gene for metabarcoding provides improved species-level resolution, allowing better ecological, epidemiological, and pathological insights into equine strongyle communities.
• The presence of potentially novel or uncharacterized strongyle species in non-horse equids emphasizes the need for broader research into parasite biodiversity across all equines.
• Overall, nemabiome sequencing can be a powerful tool to monitor parasite communities and guide more effective parasite control strategies in equine populations worldwide.