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

Equines are parasitized by complex communities of Strongylidae (Nematoda) comprising multi-species infections. Currently, Cyathostominae are most prevalent, while Strongylus species are only rarely detected. Since eggs and, in most cases, infective larvae cannot be differentiated to species level, except for Strongylus spp., species-specific knowledge of the pathology, epidemiology and ecology of these parasitic nematodes is limited. Reference sequence data for several cyathostomin species are limited or missing. Deep amplicon sequencing of internal transcribed spacer 2 (ITS-2) regions of nematodes has been used in equines previously, although barcoding studies demonstrated a better species resolution for the cytochrome c oxidase subunit I (COI) region. The present study introduces a nemabiome method based on sequencing of COI fragments. This method was applied to compare third stage larvae, representing strongyle communities, derived from regularly treated (RT) and never treated (NT) equine populations from Brazil, France (only RT), Germany, Ukraine, the UK, and the USA. Samples were predominantly from horses, but some were obtained from Przewalski's horses (Ukraine), donkeys (Germany, Ukraine) and kulans (Ukraine). Most sequence reads (87.7%) were identified to species level, but unclassified reads occurred more frequently in donkeys and kulans than horses. No obvious difference in species diversity and richness was observed between RT and NT equines. However, there were significant differences in species composition between the RT and NT groups. Strongylus spp. were more common in NT groups but also showed unexpectedly high abundances in RT horses. Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cyathostomum catinatum were more abundant in RT groups, suggesting that strongyle communities in domestic equines may have been shaped by anthelmintic treatments during last decades. The decreased classification success for reads from non-caballine equines suggests that there are more strongyle species specific for this rarely-investigated group which requires additional efforts to improve the sequence database, particularly for these hosts.