‘First record of Hsiungia pekingensis (Nematoda: Strongylidae) in North America: Morphological and molecular identification of a rare equine strongyle

Abstract

Equids are infected by a diversity of gastrointestinal nematode parasites, including 64 species of equine strongyle nematodes from19 genera. Despite numerous surveys of horse strongyles worldwide, certain geographic regions and rare species remain understudied. In 1964, a new species of equine strongyle, Cylicocyclus pekingensis, was described from a donkey in China. Subsequently, this species was recorded in horses from Kazakhstan and reclassified as Hsiungia pekingensis (K’ung and Yang, 1964), the only species in this genus. Since then, H. pekingensis has not been reported elsewhere, with limited knowledge on its distribution and phylogeny.

This study documents the first record of H. pekingensis in North America. Adult specimens were recovered from fecal samples of a domestic horse in Alberta, Canada, following treatment with ivermectin. Species identification involved detailed morphological examination, complemented with sequencing of the internal transcribed spacer 1 (ITS1), 5.8S rRNA gene, and the internal transcribed spacer 2 (ITS2) regions of the nuclear genome. Phylogenetic analysis indicated a close evolutionary relationship with species from Poteriostomum and Parapoteriostomum genera. Nemabiome ITS2 sequencing of a paired pre-treatment sample also detected the presence of H. pekingensis in the studied horse. Re-analysis of public equine nemabiome datasets further detected H. pekingensis in feral horses in Alberta, but not in other regions considered. This study expands the known distribution of this rare species and enhances our knowledge of its placement in the phylogeny of equine strongyles. Furthermore, our re-analysis of public nemabiome datasets highlights the value of this approach for studying the global distribution of parasite species.

Introduction

Equids host numerous parasite species, many of which can impact their health and performance (Lichtenfels 1975; Nielsen and Reinemeyer 2018). Most of these parasites belong to a single family of nematodes known as Strongylidae Baird, 1853 (Lichtenfels et al. 2008). Up to now, 64 species in 19 genera have been recognized to infect wild and domestic equids worldwide (Lichtenfels et al. 2008). These parasites are classified into two subfamilies, 14 species belonging to the subfamily Strongylinae Railliet, 1893 (commonly known as large strongyles) and 50 species belonging to the subfamily Cyathostominae Nicoll, 1927 (small strongyles or cyathostomins) (Lichtenfels et al. 1998). Although individual horses can be parasitized by more than 20 strongyle species, the strongyle community in a horse is typically dominated by a few species (Bellaw and Nielsen 2020; Kuzmina et al. 2016; Mfitilodze and Hutchinson 1990; Tolliver 2000). According to prevalence and abundance values, approximately a third of the strongyle species that parasitize an equid host can be considered rare species – as they usually are found in single specimens, and together compose less than 0.05–0.1% of the strongyle community (Kuzmina et al. 2007; Kuzmina et al. 2009; Kuzmina et al. 2016).

In 1964, a new strongyle species was described from a donkey in Beijing, China, and named Cylicocyclus pekingensis (K’ung and Yang 1964). In their study, the authors proposed a new subgenus Cylicocyclus (Hsiungia) K’ung and Yang 1964. Later, this parasite was recorded in horses from the Ural region of Kazakhstan and reclassified as Hsiungia pekingensis, a single representative of a separate genus (Dvojnos and Kharchenko 1988). Since it has not been reported elsewhere, this species is considered extremely rare, and very little is known about its prevalence, biology, and geographical distribution.

As evidenced by its reclassification based on morphological characters, this species has a contentious taxonomy. While H. pekingensis broadly appears to be morphologically similar to species belonging to the genus Poteriostomum, the male bursa and shape of the buccal capsule are more similar to Cylicocyclus (Dvojnos and Kharchenko 1988; Lichtenfels et al. 2008). Since morphology alone may be insufficient for an accurate taxonomic assessment, molecular information is essential for providing a more precise classification (Van Den Ende et al. 2023). However, the rarity of H. pekingensis has prevented its inclusion in public molecular databases, hindering the possibility of studying its diversity and phylogenetic position within the subfamily Cyathostominae.

In recent years, nemabiome DNA metabarcoding (Avramenko et al. 2015) has emerged as a powerful tool for characterizing complex communities of equine strongyles (Poissant et al. 2021). By leveraging high-throughput sequencing methods, nemabiome analysis allows for the simultaneous identification of multiple species from non-invasive samples, making it particularly useful for assessing biodiversity and tracking less common species within host populations. While this technique is gaining popularity in horse parasitology (e.g., Abbas et al. 2024; Forman et al. 2024; Hamad et al. 2024; Mitchell et al. 2019; Nielsen et al. 2022; Sargison et al. 2022), its accuracy relies heavily on the completeness and accuracy of molecular databases. However, many rare strongylid species remain unstudied by molecular methods, and without comprehensive reference data, the full species diversity present in equine strongyle communities, including rare species like H. pekingensis, may go undetected.

In this study, we report the first detection of H. pekingensis in North America confirmed by its morphological identification in a population of adult worms obtained from a domestic horse in Alberta, Canada. We sequenced the region of the nuclear genome encompassing the first internal transcribed spacer (ITS1), the 5.8S ribosomal RNA (rRNA) gene, and the second internal transcribed spacer (ITS2) of a specimen to investigate the species molecular diversity and phylogenetic position and confirmed the ability of the nemabiome technique to detect the species in mixed infections. Finally, we re-analyzed publicly available nemabiome datasets using the newly obtained sequence to determine if H. pekingensis infections have gone undetected in other parts of the world.

Materials and methods

Sample collection and parasitological methods

In spring 2023, fecal samples were obtained opportunistically prior and following owner-administered ivermectin treatment of a 12-year-old mare in Water Valley, Alberta, Canada with an unknown deworming treatment history. Approximately 30 g of feces was incubated at room temperature (~21°C) for 14 days to culture infective larvae (L3) for subsequent nemabiome analysis. Cultured L3 were harvested using a modified Baermann technique, then fixed in 70% ethanol and stored at -20°C until further analysis (Poissant et al. 2021). Approximately 500 grams of fecal sample collected 24 hours after treatment was also carefully examined visually using forceps to collect adult worms. A total of 802 nematodes were retrieved from the sample, rinsed in saline buffer, and fixed in 70% ethanol. For morphological identification, the heads and the tails of every worm were cut and placed on a microscopic slide, then cleared using approximately 2 ml of lactophenol (Sigma-Aldrich, USA). Identification was performed using a light microscope (Olympus, Japan). The middle section of each worm was fixed in 70% ethanol and was stored at -20°C for subsequent molecular analyses.

Morphological identification and description

Morphological identification of nematodes was done using published identification keys and descriptions (Dvojnos and Kharchenko 1988; Lichtenfels et al. 2008). Seven specimens were identified as adult H. pekingensis (four females and three males); two males and two females were used for the morphological examination and redescription. The redescription was performed based on the morphology of the buccal capsule, including the shape and dimensions of the buccal capsule walls, the cephalic papillae, internal leaf-crown (ILC), external leaf-crown (ELC), and esophageal funnel. The posterior ends of males and females were measured, and their morphological characteristics were also considered.

DNA extraction and sequencing of adult worm

DNA was extracted from the middle part of the one H. pekingensis female utilizing the DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer protocols and eluted in 180 μl of molecular grade water. The concentration of extracted DNA was quantified using a Qubit® 1X dsDNA BR Assay Kits on a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA). PCR targeting the ITS1-5.8S-ITS2 genomic region was performed using the NC5 (5´-GTAGGTGAACCTGCGGAAGGATCATT-3´) and NC2 (5´-TTAGTTTCTTTTCCTCCGCT-3´) primers (Gasser and Hoste 1995; Newton et al. 1998) and the Kapa HiFi Hotstart PCR kit (Roche, Switzerland). The PCR reaction included a total volume of 25 μl: 5 μl of KAPA HiFi Buffer, 0.75 μl of each primer (10 μM), 0.75 μl dNTPs (10mM), 0.5 μl of KAPA HiFi Polymerase (0.5U), 15 μl of the template DNA (~10 ng), and 2.25 μl of molecular grade water. Thermocycler conditions consisted of an initial denaturation at 95°C for 2 min followed by 35 cycles of 98°C for 20 s, 62°C for 15 s, and 72°C for 15 s, concluding with a final extension at 72°C for 2 min. The PCR products were then subjected to electrophoresis on a 2 % agarose gel, visualized with a UV transilluminator, and purified using the QIAquick Gel Extraction Kit following manufacturer protocols (Qiagen, Germany). Sanger sequencing of the purified PCR products was conducted by the University of Calgary Centre for Health Genomics and Informatics (CHGI) using both forward and reverse primers on a 3730xl DNA Analyzer (Applied Biosystems, USA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA). Sequences obtained from CHGI were inspected and manually edited, and forward and reverse reads were merged using the UniPro UGENE software (Okonechnikov et al. 2012).

Phylogenetic analysis

The ITS1-5.8S-ITS2 region of 31 species of the family Strongylidae and Trichostrongylus axei (to serve as an outgroup) were obtained from the Nucleotide database of the National Center for Biotechnology Information (Supplementary file 1). T. axei, a member of the same order (Rhabditida) but from a different family (Trichostrongylidae), was selected as the outgroup to provide a shared evolutionary history while maintaining sufficient divergence for accurate tree rooting and determination of relationships within the family Strongylidae. For species with multiple available sequences, a representative sequence was selected based on sequence quality (long and with few ambiguous bases). Sequences were aligned using MAFFT using default parameters (Li et al. 2015), and phylogenetic analysis performed with IQ-TREE (Nguyen et al. 2015) using the Maximum Likelihood (ML) method with the Transition-Intermediary Model 2 with empirical base frequencies and a gamma distribution with four rate categories (TIM2+F+G4). The robustness of the tree topology was assessed through 1,000 bootstrap replicates. Visualization and annotation of the phylogenetic tree was carried out using the Interactive Tree of Life (iTOL) online tool (https://itol.embl.de/) (Letunic and Bork 2021).

DNA extraction and ITS2 nemabiome sequencing of pre-treatment sample

DNA was extracted from an aliquot of ~ 2500 L3s. Briefly, as much ethanol as possible was first drawn out from the sample following centrifugation at 13,000 g for 4 minutes and the remainder removed by vacuum evaporation. Larvae were then re-suspended in 150 μl of lysis buffer and 6 μl of Proteinase K (20 mg/ml), and bead beat for 10 minutes using two 5 mm stainless steel beads (Qiagen, Germany) and a Vortex-Genie 2 fitted with an adapter (Qiagen Vortex Adapter, Germany). Immediately following bead beating, the sample was placed on a plate shaker (300 rpm) in an incubator set to 55°C for 2 hours. Following lysis, Proteinase K was inactivated by incubation at 95°C for 20 min. Finally, a 1:10 dilution of the DNA lysate was prepared with molecular-grade water and stored at -80°C until further processing.

PCR amplification and sequencing of the ITS2 region followed published protocols (Poissant et al. 2021). Briefly, the ITS2 region was first PCR-amplified using the NC1 (5´-ACGTCTGGTTCAGGGTTGTT-3´) and NC2 (5´-TTAGTTTCTTTTCCTCCGCT-3´) primers (Gasser et al. 1993) with three random bases and an adapter for barcode addition. This PCR involved denaturation at 95°C for 3 min, 25 cycles of 98°C for 20 s, 62°C for 15 s, 72°C for 15 s, and a final extension at 72°C for 2 min. Products were purified with AMPure XP beads, followed by a second PCR to add unique dual indices and Illumina adaptors (IDT for Illumina Nextera UD Indexes). The DNA concentration of the final PCR product was quantified using a BioTek Take3 Plate Reader, in duplicate. The sample in this study was pooled in a library including 375 additional equine nemabiome samples from different geographic areas (not presented in this study). The pooled library was quantified using a Qubit® 1X dsDNA BR Assay Kits on a Qubit 4 Fluorometer (Thermo Fisher Scientific, USA), then diluted to 12 pM. Sequencing of the pooled library was conducted on an Illumina MiSeq platform with a MiSeq Reagent Kit v2(500-cycle), using a 20% PhiX spike-in.

Bioinformatic analysis

The raw output from the sequencing run was processed following an updated version (v2) of the bioinformatics pipeline published in (Poissant et al. 2021) available at https://data.mendeley.com/datasets/vhyysw8xt2/2. As in Nielsen et al. 2022, up to 2.5% base mismatches were allowed during the merging of forward and reverse reads. For taxonomic assignments, the ITS2 reference database v1.6.0 (Workentine et al. 2020) accessed at https://www.nemabiome.ca was curated following the steps in Poissant et al. 2021, and the sequence of H. pekingensis was manually added. Taxonomic classification was performed using the DADA2 assignTaxonomy function, with an assignment confidence threshold of 80% or higher. We also repeated this analysis while excluding H. pekingensis from the ITS2 reference database to assess how (if any) H. pekingensis Amplicon Sequence Variants (ASVs) would get classified in that case. In the final step, the processed ASVs were consolidated at the species level.

Re-analysis of published databases

To investigate the global occurrence of H. pekingensis, publicly available equine nemabiome datasets were compiled and re-analyzed. The datasets were sourced from studies of domestic horses in Thailand (Hamad et al. 2024), France (Boisseau et al. 2023), Scotland (Sargison et al. 2022), England (Bull et al. 2025), and the United States (Poissant et al. 2021); feral horses from Sable Island and Alberta in Canada (Ahn et al. 2024; Poissant et al. 2021); and wild asses from the Negev desert in Israel (Forman et al. 2024) (Supplementary file 1). The raw outputs of the sequencing runs were downloaded and processed using the bioinformatics pipeline described above.

Results

Morphological description

Hsiungia pekingensis (K’ung and Yang, 1964)

Morphological descriptions reported herein are based on two males and two females, and measurements are given in micrometers unless otherwise noted (Figures 1 and 2, Table 1).

Figure 1. Hsiungia pekingensis, A) Esophageal region, bar: 400 μm. (B) Female tail, bar: 400 μm. (C) Male tail, bar: 400 μm. (D) Spicule tips bar: 100 μm. (E) Genital cone, ventral view, bar: 200 μm.

Table 1. Morphological characteristics of H. pekingensis. Measurements are given in micrometers unless otherwise noted

* Measured from anterior end.

Figure 2. Hsiungia pekingensis, A) Buccal capsule, dorsoventral view, male specimen. (B) Submedian papilla. (C) Wall of buccal capsule. (D) Esophageal region, ventral view, female sample. (E) Female (top) and Male (bottom), bar: 10 mm.

General. The nematode belongs to the Cyathostominea group, medium to large in size. The mouth collar (MC) is prominently raised, featuring inner and outer rings. The amphids do not significantly protrude through the MC, and the stalk of the submedian papillae extends beyond the surface of the MC. The submedian papillae are bullet-shaped, with a slightly flattened tip, and are approximately twice as long as they are broad (Figure 2B). The lateral cephalic papillae do not project above the surface of the MC. The elements of the external leaf crown (ELC) are inserted at the tips of the internal leaf crown (ILC) (Figure 2A). The number of elements in the ELC ranges from 80 to 86, approximately equal to or slightly fewer than those in the ILC. Both the ELC and ILC exhibit the same length, and their elements are longer than broad, with pointed tips. The insertion of the ILC occurs on the anterior edge of the buccal capsule. The buccal capsule is short and cylindrical and generally wider than deep, and the walls are thinner anteriorly but thicken slightly at the base (Figure 2C). The dorsal gutter is nipple-shaped, and esophageal teeth are noticeable. The esophageal funnel is well-developed. The esophagus is thick and moderately short (Figures 1A, 2D). The excretory pore is posterior to the nerve ring. Deirids are near the middle of glandular esophagus.

Male. Body length 12.5–13.5 mm. Esophagus length 740–882. Buccal capsule (BC) width 122–150, depth 35–44. The distance from the anterior end to the deirids 522–620; to excretory pores 455–486; to nerve ring (NR) 412–433. Spicule length 1.10–1.25 mm. Gubernaculum length 230–259. Dorsal ray length (to the base of externodorsal ray) 321–358. Characteristics of males include a dorsal ray with six branches, with ventral rays longer than the laterals. The lobes, both dorsal and lateral, are of equal length. Externodorsal rays originate at the junction of the dorsal and lateral lobes. Additionally, the gubernaculum displays a prominent size, featuring a dorsal handle and a ventral notch. The genital cone is uniform, short, and conical in shape with a developed dermal collar and does not project outside the bursa. Spicules are blade-shaped with alae and tips that are slightly curved.

Female. Body length 20–24 mm. Esophagus length 863–897. BC width 196–210, depth 49–52. The distance from the anterior end to deirids 686–740; to excretory pores 641–700; to NR 472–523. The distance from vulva to tail tip 1.3–1.8 mm; from anus to tail tip 420–533. Eggs are 120–150 × 55–70. Characteristics of female include a noticeably short vagina and a Y-shaped ovejector. The tail is straight, conical, and elongated, with noticeable separation between the vulva and anus.

Sequence and phylogenetic analysis

An 837-bp segment of the ITS1-5.8S-ITS2 genomic region was successfully amplified from one female H. pekingensis specimen and added to GenBank (accession number PV074874). The ITS1 and ITS2 regions of the amplified fragment were 330 bp and 354 bp in length, respectively. The phylogenetic analysis, employing the ITS1-5.8S-ITS2 region, revealed distinct clades corresponding to the subfamilies Strongylinae and Cyathostominae. H. pekingensis formed a cluster with species of two genera, Poteriostomum and Parapoteriostomum, with strong statistical support (82% bootstrap value, Figure 3). Sequence similarities for the ITS1 region between H. pekingensis and Poteriostomum ratzii (KP693434), Poterioustomum imparidentatum (KY495604), Parapoteriostomum euproctus (KP693692), and Parapoteriostomum mettami (KP693435) were 89.43%, 89.55%, 89.25%, and 91.02%. Sequence similarities for the ITS2 region between H. pekingensis and the same species were 78.40%, 81.93%, 80.42%, and 79.88%, respectively.

Figure 3. Phylogenetic analyses of ITS1-5.8S-ITS2 constructed using nucleotide sequences of 31 species of the family Strongylidae. The tree was developed using the ML method (1,000 bootstrapped alignments) in agreement with the TIM2+F+G4 substitution model. H. pekingensis is in red and T. axei is an outgroup. Bootstrap values (1,000 replicates) of >50% are represented at internodes.

ITS2 nemabiome metabarcoding analysis

A total of 22,081 reads were obtained from the L3 cultured from the pre-treatment sample. After applying quality filtering, a total of 19,516 reads were retained, and 131 ASVs were assigned to species with at least 80% confidence. Overall, 11 strongyle species from 7 different genera were detected in the sample (Figure 4). Notably, 337 reads (1.7%) were classified as H. pekingensis, represented by two ASVs with 100% bootstrap support. When analyzing the data while excluding the H. pekingensis sequence from the ITS2 database, these ASVs were matched to Tridentoinfundibulum gobi with 40% and 26% bootstrap support, respectively.

Figure 4. Relative abundances of ITS2 amplicon reads assigned to different equine parasitic strongyles for a L3 culture from a domestic horse near Water Valley, Alberta, Canada.

Re-analysis of publicly available nemabiome datasets

The re-analysis of publicly available nemabiome datasets revealed the presence of H. pekingensis sequences in two additional samples obtained from feral horses in Alberta, Canada, by Poissant et al. (2021). These samples were from two different horses (out of five) sampled in May 2015, and 0.1–0.2% of the reads were assigned to H. pekingensis. No sequences corresponding to H. pekingensis were detected in nemabiome datasets of horses from Thailand, France, Scotland, England, Kentucky, Sable Island, or wild asses in Israel.

Discussion

In the current study, we report the first detection of H. pekingensis in North America, provide a morphological redescription of the species, and analyze its taxonomic position based on the ITS1-5.8S-ITS2 ribosomal DNA gene region. We also confirmed that the species can be confidently detected using ITS2 nemabiome analyses and re-analyzed publicly available datasets to expand our knowledge of its global distribution.

The species was initially described by K’ung and Yang (1964) as a new species in the genus Cylicocyclus. In their description, they mentioned that the species was one of the larger cyathostomins. Additionally, they offered to divide the Cylicocyclus genus into three subgenera, with one of them, Cylicocyclus (Hsiungia), containing C. pekingensis and C. ultrajectinum (syn. C. ultrajectinus). In contrast, Lichtenfels (1975) mentioned that differences between C. pekingensis and C. ultrajectinus were substantial and argued for a reclassification of the subgenera. Specifically, Lichtenfels (1975) recognized the species to be more similar to species of Poteriostomum and Cylicocyclus in morphology except for the genital bursa of the males and considered this a species inquirenda. In a later redescription of specimens from Kazakhstan by Dvojnos and Kharchenko (1988), the species was reclassified to the separate genus Hsiungia, with H. pekingensis being its only known species. In the current study, we redescribed H. pekingensis from a horse in Canada based on morphological characteristics of four specimens. Although the differences between our findings and previous descriptions are minor, they are nonetheless important for accurate species identification and taxonomic clarity. In particular, the original description of K’ung and Yang (1964) left room for refinement regarding the morphological details. The size of the female specimens in our study aligns more closely with K’ung and Yang’s (1964) description, while the male specimens more closely resembled those described by Dvojnos and Kharchenko (1988).

Our phylogenetic analysis using the ITS1-5.8S-ITS2 region suggests that H. pekingensis is most closely related to species of the Poteriostomum and Parapoteriostomum genera, supporting Lichtenfels’ (1975) suggestion of a closer affinity of this taxon with the genus Poteriostomum than the genus Cylicocyclus. These results highlight the utility of molecular techniques to inform the taxonomic classification of nematode species where morphological differentiation is challenging. Moreover, the distinct clades observed for the subfamilies Strongylinae and Cyathostominae validate the usefulness of ribosomal DNA regions in resolving higher-level taxonomic distinctions (Hung et al. 2000).

The detection of H. pekingensis in Alberta significantly expands our knowledge of the geographical distribution of this species outside of Asia, indicating a wider distribution than previously thought. The fact that this species has been found outside its originally documented range suggests the possibility of either natural dispersal mechanisms or anthropogenic factors contributing to its spread, such as the global movement of horses (Vasileiou et al. 2015). Notably, H. pekingensis had up to now very rarely and possibly never been detected in morphology-based surveys of equine strongyles following the original descriptions from K’ung and Yang (1964) and Dvojnos and Kharchenko (1988) despite considerable global effort (Bellaw and Nielsen 2020). However, while morphology-based surveys have been conducted in many parts of the world (Bellaw and Nielsen 2020), they are usually limited in scope due to the time and expertise required. For example, published morphology-based surveys of equine strongyles in North America have so far been limited to a handful of regions of the United States (Kentucky, Ohio, and Louisiana; reviewed by Bellaw and Nielsen 2020). Given the efficiency of molecular methods, such as nemabiome DNA metabarcoding, in identifying species using non-invasive samples (Avramenko et al. 2015), it is likely that rare species will get detected more often moving forward. This, in turn, should improve our understanding of the ecological and evolutionary history of equine strongyles.

Our re-analysis of publicly available equine nemabiome datasets revealed that H. pekingensis is present in both domestic and feral horse populations in Alberta. In the previous nemabiome analysis of five feral horses from Alberta, Poissant et al. (2021) had detected 31 species of strongyles but failed to detect H. pekingensis due to its absence from ITS2 databases. This finding illustrates the value of sharing and re-analyzing metabarcoding datasets when reference databases get updated. Presence of H. pekingensis in Alberta may be explained by the presence of a large unmanaged feral horse population in the region, which is known to harbor a great diversity of strongyle species (Poissant et al. 2021). The unmanaged nature of these feral horses and their relatively frequent adoption (Zomorodi and Walker 2019) may facilitate both the maintenance of rare strongyles and their transmission to the domestic horse population. While H. pekingensis was not detected in other parts of the world, we note that the scope of currently available equine nemabiome datasets is fairly modest. Additional research and datasets will therefore be needed to assess the exact geographical distribution of H. pekingensis.

In conclusion, the discovery of H. pekingensis in North America expands its known distribution and prompts a reevaluation of its geographical range. The newly generated molecular data helps clarify its taxonomic position and will aid in identifying the species in new geographical regions and hosts globally. Additionally, the detailed redescription and microscopic images provided here are invaluable for future identification efforts and species classification. We believe that this work encourages further studies on the identification, distribution, genetic diversity, and taxonomic positions of rare equine strongyles worldwide.