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

Equines are parasitized by complex communities of Strongylidae (Nematoda) comprising multi-species infections. Currently, the Cyathostominae are most prevalent, while the Strongylus species are only rarely detected. Since eggs and, in most cases, infective larvae cannot be differentiated to species level, with the exception of 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 demonstrate a better species resolution for the cytochrome c oxidase subunit I (COI) region. The present study introduces a nemabiome method based on the 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 Przewalskis horses (Ukraine), donkeys (Germany, Ukraine) and kulans (Ukraine). Most sequence reads (87.7%) were identified to the 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. While Strongylus spp. were significantly more abundant in the NT groups, Cylicocyclus nassatus, Cylicostephanus longibursatus, and Cyathostomum catinatum were more abundant in the RT group, suggesting that strongyle communities in domestic equines may have been shaped by anthelmintic treatments in the 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 and that additional efforts are needed to improve the sequence database, particularly for these hosts. Author summaryThis study shows that long-term deworming treatments have influenced strongyle nematode communities in equines. Our findings showed that regular deworming does not always reduce species richness and diversity. Noteworthy, we observed that the more pathogenic species such as Strongylus vulgaris and Strongylus edentatus were still present but in low abundance in equines. Due to their low abundance, less sensitive diagnostic methods, such as morphological examination of larval cultures might not detect these species, which would lead to an underestimated threat in equine herds. We applied an effective metabarcoding approach based on a reliable gene marker region to accurately detect these and other strongyle species in equines. The detection of various species was more effective for horse samples than for those from donkeys and kulans. An expansion of the current database that includes more specimens from more rare species obtained from different equine species can improve identification and understanding of these complex multi-species communities in the future. In summary, the study underscores the importance of continuing monitoring equine herds based on sensitive methods such as metabarcoding to evaluate the current nematode communities and to adapt and develop treatment strategies for managing strongyle infections in equines.