Today, the most prevalent gut parasites in equines are non-migratory species from the family Strongylidae, commonly referred to as small strongyles (Bellaw and Nielsen, 2020). They cause multi-species infections, typically with 10 – 20 species detected in a single host (Bull et al., 2025, Halvarsson et al., 2024, Lyons et al., 2011, Sallé et al., 2018, Sargison et al., 2022). While most small strongyle infections remain subclinical, they have been linked to diarrhoea, colic, weight loss and poor growth (Love et al., 1999, Mair et al., 2000). In rare cases, they can cause life-threatening disease known as larval cyathostominosis, which usually affects young horses (Lawson et al., 2023, Love et al., 1999, Mair et al., 2000, Peregrine et al., 2006). In addition to small strongyles, three migratory Strongylidae species (large strongyles), Strongylus (Str.) vulgaris, Str. edentatus and Str. equinus remain relevant to equine management (Nielsen et al., 2022b, Nielsen, 2022, Sallé et al., 2020), of which only Str. vulgaris is considered highly pathogenic (Pihl et al., 2018). Because both small and large strongyles share egg morphology, individual species cannot be detected using faecal egg tests. Broad-spectrum diagnostic tools are therefore required for species-level monitoring.

Based on morphological identification, 47 small strongyle species (morphospecies) are recognized and known to infect equines (Equus ferus caballus) (Lichtenfels et al., 2008). Of those, 35 species have been identified globally in domestic equines since 1975, with some regional differences in species composition (Bellaw and Nielsen, 2020). For North America, Cyathostomum (Cya.) catinatum, Cylicocyclus (Cyc.) nassatus, Cylicostephanus (Cys.) longibursatus, Coronocyclus (Cor.) coronatus and Cylicostephanus (Cys.) goldi, followed by Cylicostephanus (Cys.) calicatus, Cylicostephanus (Cys.) minutus, Cylicocyclus (Cyc.) leptostomus, Cylicocyclus (Cyc.) insigne and Cyathostomum (Cya.) pateratum have been reported as ‘core’ small strongyle species, accounting for up to 98% of all identified specimens (Bellaw and Nielsen, 2020, Reinemeyer et al., 1984). In addition to morphospecies, phylogenetic studies have identified three lineages each within Cys. minutus (Bredtmann et al., 2019b, Gao et al., 2020, Hung et al., 1999) and Cys. calicatus (Bredtmann et al., 2019a, Louro et al., 2021), shown to represent six separate cryptic species named Cys. minutus OTU I – III and Cys. calicatus OTU I – III (Bredtmann et al., 2019b, Diekmann et al., 2025, Louro et al., 2021). Distinguishing these cryptic species is important, as collapsing them under two single morphospecies obscures true species composition and could confound ecological and epidemiological analyses.

Species-specific investigations of equine small strongyles have been hampered by the non-availability of feasible cost-effective sensitive and specific methods in the past, which has resulted in limited availability of species-specific prevalence reports (Bredtmann et al., 2017, Gasser et al., 2004). Prior to the rise of high-throughput sequencing (HTS) technologies, small strongyle species were assessed through laborious and highly specialised morphological identification of adult worms, invasively collected during necropsy or expelled after anthelmintic treatment (Bellaw and Nielsen, 2020, Kuzmina et al., 2005). Currently, HTS metabarcoding approaches present the most feasible solution for species differentiation and for investigating species-specific characteristics (Abbas et al., 2023, Bull et al., 2025, Byrne et al., 2024, Courtot et al., 2023, Diekmann et al., 2025, Ghafar et al., 2023, Hedberg Alm et al., 2023, Mitchell et al., 2019; Nielsen et al., 2022a; Poissant et al., 2021, Rinaldi et al., 2022, Sargison et al., 2022). Moreover, these approaches continue to refine our taxonomic understanding of this diverse nematode subfamily (Bredtmann et al., 2019a, Diekmann et al., 2025, Louro et al., 2021). Metabarcoding targets for mixed strongyle infections currently include the internal transcribed spacer 2 (ITS-2) sequence (Gasser et al., 2004), a short 450 bp cytochrome c oxidase I (COI) sequence (Courtot et al., 2023) and a long 650 bp COI sequence (long COI) (Diekmann et al., 2025). The ITS-2 and long COI methods have been used to identify large strongyles as well (Diekmann et al., 2025, Gasser et al., 2004). The ITS-2 approach, while widely applied (Bull et al., 2025, Byrne et al., 2024, Courtot et al., 2023, Ghafar et al., 2023, Halvarsson et al., 2024, Poissant et al., 2021), cannot discriminate between all small strongyle species. Most notably, two ‘core species’ Cor. coronatus and Cys. calicatus cannot be reliably differentiated using ITS-2 (Bredtmann et al., 2019a, Byrne et al., 2024, Diekmann et al., 2025, Louro et al., 2021). In contrast, the 650 bp COI method is able to discriminate these species (Diekmann et al., 2025) as well as the three cryptic species Cys. calicatus OTU I – III (Bredtmann et al., 2019a, Louro et al., 2021). In a recent methods comparison, the long COI approach showed higher power for nemabiome analysis compared to ITS-2 (Diekmann et al., 2025) and demonstrated its suitability for differentiating recognized morphospecies as well as cryptic species.

A further consideration in equine nemabiome studies is the choice of biological sample type. Metabarcoding approaches enable species differentiation regardless of developmental stage (Avramenko et al., 2015, Courtot et al., 2023, Gasser et al., 2004, Miller et al., 2024), which offers more flexibility in study design and reduces the need for invasive approaches used to collect luminal worms (Bucknell et al., 1995, Collobert-Laugier et al., 2002). Faecal eggs are the most accessible sample type and can be directly isolated from faeces, but they contain the smallest amount of DNA per specimen. Depending on egg-shedding levels and retention during egg isolation, samples may fall short of necessary thresholds for HTS library preparation. Moreover, because egg recovery depends on gravid females, it is possible that some species may go undetected due to differences in infection timing, prepatent period, and egg production numbers (Kuzmina et al., 2012). Larval culture, during which faecal eggs are developed to the L3 stage, may offer a suitable alternative with increased DNA content per specimen. However, development rates may differ between equine strongyle species. In ruminants, for instance, substantial efforts were made to account for uneven development of mixed gastrointestinal nematodes (Avramenko et al., 2015, Borkowski et al., 2020, Redman et al., 2019, Rinaldi et al., 2022). While the suitability of larval culture and faecal eggs has been investigated in many host species (Pafčo et al., 2018), only one other metabarcoding study compared larval cultures to other biological sample types for equines strongyles (Courtot et al., 2023). That study, however, did not include cryptic species and data for many species are still missing. Further studies are needed to validate this approach, particularly with respect to cryptic species.

All small strongyle species are considered to share similar characteristics, including a direct non-migratory life cycle, during which third-stage larvae mature to adults within the host’s hindgut. However, several morphological studies have described differences in species abundance and tropism between different sections of the hindgut (Bellaw et al., 2018a; Gasser et al., 2004; Nielsen et al., 2022a; Ogbourne, 1976). Necropsy studies report three main parasitism sites of small strongyles within the host: caecum, ventral ascending colon (ventral colon) and dorsal ascending colon (dorsal colon) (Love and Duncan, 1992, Ogbourne, 1978). Moreover, while large strongyle larval stages undertake a migration through several organs within the host, they are found as preadult and adult stages in the caecum, ventral and dorsal colon, making these three sections optimal sampling targets for luminal worm stages of mixed strongyle infections. Worm burdens are lowest in the caecum, estimated at only 10% of the total worm burden, while ventral and dorsal colon harbour similar amounts (Collobert-Laugier et al., 2002, Ogbourne, 1976). To date, it remains unclear why certain small strongyle species prefer one location over another and implications for the host are unknown. Species’ tropism within the host could affect their pathogenicity, which may have implications for the clinical relevance of strongyle infections, if characterized to species-level. For the ventral colon, Cys. minutus, Cya. catinatum and Cyc. nassatus have been described as most abundant (Collobert-Laugier et al., 2002, Love and Duncan, 1992, Mfitilodze and Hutchinson, 1985, Ogbourne, 1976), while two publications also reported Cyc. labiatus (Collobert-Laugier et al., 2002, Mfitilodze and Hutchinson, 1985). In the dorsal colon, Cys. longibursatus has been unanimously described as most abundant (Collobert-Laugier et al., 2002, Love and Duncan, 1992, Mfitilodze and Hutchinson, 1985, Ogbourne, 1976). Furthermore, multiple studies reported high abundances of Cys. goldi and Cyc. insigne in the dorsal colon as well (Collobert-Laugier et al., 2002, Mfitilodze and Hutchinson, 1985, Ogbourne, 1976). Studies based on genetic species differentiation have also reported differences in anthelmintic response (Bull et al., 2025, Hedberg Alm et al., 2023), including shortened egg-reappearance periods following macrocyclic lactone treatment (Bellaw et al., 2018a; Kooyman et al., 2016; Nielsen et al., 2022a; Van Doorn et al., 2014) and evidence of pyrantel- and macrocyclic lactone-resistant populations (Bull et al., 2025, Hedberg Alm et al., 2023). Resistance to benzimidazoles is considered widespread in small strongyles (Bellaw et al., 2018b; Lester et al., 2013, Lyons, 2003). However, detailed data for many species are lacking and our understanding of their biology is still incomplete. Furthermore, there is a lack of metabarcoding data for species composition in different hindgut sections.

Considering that geographic differences, treatment history and husbandry conditions shape strongyle species composition (Bellaw and Nielsen, 2020), the present study population deserves special consideration, as several aspects are unique. ‘Population S’ is a long-term research herd at the Gluck Equine Research Center of the University of Kentucky and was established in 1974 (Lyons, 2003). It consists of a herd of Shetland-type ponies and has remained closed, except for the occasional replacement stallion. The history in terms of gastrointestinal parasite infections, anthelmintic treatments, treatment efficacy and husbandry data since its establishment is well documented, as the population has been investigated in several studies over the years (Drudge et al., 1985, Drudge et al., 1983, Drudge et al., 1974, Lyons, 2003, Lyons et al., 2001, Lyons et al., 1996, Lyons et al., 1994, Scare et al., 2020, 2018). Over the course of various anthelmintic treatment regimens, the herd has become subject to small strongyles that are now resistant to both the benzimidazole and tetrahydropyrimidine drug classes, demonstrated in consecutive critical tests (Lyons et al., 2001, Lyons et al., 1996, Scare et al., 2018). While the only remaining available drug class, macrocyclic lactones, has been widely used for decades in horses worldwide and increasing reports of resistance in small strongyles have emerged (Nielsen, 2022), it was first used on this herd in 2018 (Scare et al., 2020). This offers a unique opportunity for strongyle community investigations.

Strongyle communities in this herd have been well characterized in morphological studies, which found 14 to 28 small strongyle species (Lyons, 2003, Lyons et al., 2001, 1996). Four species dominated between 1977 and 1999: Cya. catinatum, Cyc. nassatus, Cys. goldi and Cys. longibursatus. More recently, Cor. coronatus and Cys. calicatus have also shown consistently high prevalence and abundance (Lyons, 2003). In addition, Cys. minutus was among the proven benzimidazole and tetrahydropyrimidine double-resistant species and showed increased occurrence since the introduction of tetrahydropyrimidine drug classes (Lyons et al., 2001). In contrast to the consensus ‘core species’ described for North America, prevalence for Cys. leptostomus and Cyc. insigne were reported low in this population (Lyons et al., 2001). Both Str. vulgaris and Str. edentatus were evident in earlier studies (Drudge et al., 1983) but were subsequently not identified for several decades (Lyons, 2003, Lyons et al., 1996). A recent study, however, detected Str. edentatus through morphological identification of cultured larvae (Scare et al., 2018). The species composition of the strongyle community infecting ‘Population S’ has not been investigated since 2003 (Lyons, 2003) and no metabarcoding approach has been used on this population to date.

The objectives of this study were to differentiate mixed strongyle infections in equines using a novel COI metabarcoding approach, to (1) obtain prevalence data for individual strongyle species, (2) characterize species composition in eggs, cultured larvae and luminal worms, (3) compare detection rates between different sample types, and (4) investigate species within-host tropism across caecum, ventral colon, and dorsal colon.