Introduction
The badger hookworm was first observed and described by the German zoologist Johann August Ephraim Goeze (1731–1793), who mentioned it in his 1782 monograph ‘Versuch einer Naturgeschichte der Eingeweidewürmer thierischer Körper.’ Goeze (Reference Goeze1782) described worms found at the terminal end of the intestine of the Eurasian badger, originally referred to as Ursus meles and now classified as Meles meles (Linnaeus, 1758). He named the worms Ascaris criniformis and used the term ‘uncinate’ to describe their shape. The Latin word uncinatus means ‘hooked’ or ‘having a hooked shape’, and the accompanying illustrations of both male and female worms reflect this morphology. Notably, the female worm appears to possess buccal capsule with a characteristic form that would today be recognized as typical of hookworms. In 1798, Josef Aloys Frölich (1766–1841) reviewed intestinal worms and introduced the genus Uncinaria for hookworms. The first species Frölich (Reference Frölich1789) assigned to this genus was Uncinaria melis, from the Eurasian badger, which he considered synonymous with Ascaris criniformis (Goeze, Reference Goeze1782), and Strongylus melis Müller, 1787. According to the principle of priority in zoological nomenclature, the earliest valid name must be used; therefore, Uncinaria criniformis (Goeze, Reference Goeze1782) is the valid name. In 1809, Karl Asmund Rudolphi (1771–1832), a Swedish-born German naturalist often regarded as the father of helminthology, referenced the badger hookworm in Volume II of his Entozoorum, sive vermium intestinalium: historia naturalis, under the name Strongylus criniformis. Rudolphi (Reference Rudolphi1809) included a comprehensive list of synonyms, such as U. melis (Frölich, Reference Frölich1789), and Ascaris criniformis (Goeze, Reference Goeze1782). However, Goeze’s original description was considered sparse, and later authors found it too ambiguous to definitively identify the species. Looss (Reference Looss1905) criticized Frölich’s description of Uncinaria as inadequate for recognizing the type species of the genus. Similarly, Railliet (Reference Railliet1900) referred to Frölich’s Uncinaria species as ‘formes spécifiquement indeterminables’ (species of indeterminable specificity).
Morphological comparisons of hookworms from Eurasian badgers with those from domestic dogs and foxes have generated ongoing controversy (Cameron, Reference Cameron1924; Fülleborn, Reference Fülleborn1924; Ransom, Reference Ransom1924). Over time, the debate has narrowed to focus on whether 2 species are distinct orconspecific: (1) U. criniformis (Goeze, Reference Goeze1782) from the Eurasian badger, and (2) U. stenocephala (Railliet, 1884) from dogs and foxes (Baylis, Reference Baylis1933; Wolfgang, Reference Wolfgang1956). In addition to questions of species identity, the genus name has also been a source of debate. Cameron (Reference Cameron1924) proposed abandoning the name Uncinaria in favour of a new genus, Dochmoides, for these hookworms. However, the use of Dochmoides has largely fallen out of favour in more recent literature. The name and species U. criniformis continue to be used in contemporary studies of Eurasian badger parasites (Seguel and Gottdenker, Reference Seguel and Gottdenker2017; Byrne et al., Reference Byrne, Fogarty, Mooney, Harris, Good, Marples and Holland2020). To date, no molecular markers have been generated for U. criniformis, and thus no molecular comparisons with other hookworm species have been possible.
Molecular diagnostics for wildlife parasite surveillance, particularly through non-invasive methods such as metabarcoding, are only effective when reference databases are complete (Redman et al., Reference Redman, Queiroz, Bartley, Levy, Avramenko and Gilleard2019; Ilik et al., Reference Ilik, Schwarz, Noskova and Pafco2024). While barcode libraries are now well-developed for veterinary-relevant hosts like ruminants, horses and dogs, they remain incomplete for many wildlife species, despite the availability of morphological records (Cháves-González et al., Reference Cháves-González, Morales-Calvo, Mora, Solano-Barquero, Verocai and Rojas2022; Antonopoulos et al., Reference Antonopoulos, Gilleard and Charlier2024; Mejías-Alpízar et al., Reference Mejías-Alpízar, Porras-Silesky, Rodríguez, Quesada, Alfaro-Segura, Robleto-Quesada, Gutiérrez and Rojas2024; Šlapeta et al., Reference Šlapeta, Vande Velde, Martinez-Valladares, Canton, Claerebout and Gilleard2024). To enable reliable studies beyond mainstream hosts, it is critical to update and generate molecular barcodes, especially ITS rDNA and cox1, from morphologically verified specimens.
The aim of this study was to molecularly characterize U. criniformis from the Eurasian badger in order to resolve the current taxonomic ambiguity of this species. This gap was addressed by sequencing and assembling a reference mitochondrial genome and ITS rDNA from hookworms morphologically consistent with the description of U. criniformis as provided by Ransom (Reference Ransom1924). The availability of these molecular markers enabled us to compare and distinguish U. criniformis from other hookworm species.
Materials and methods
An adult female Eurasian badger (Meles meles) was found dead on the roadside in September 2024 in Bihor County, Romania (47°06′37.9″N, 21°50′35.4″E). Collecting road-kills was approved by Ethical Committee Decision no. 232/23.11.2020 (University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Cluj-Napoca, Romania). The intestinal contents were examined for the presence of hookworms. Recovered hookworms were preserved in 70% ethanol (v/v) and transported to the University of Sydney.
Genomic DNA (gDNA) was extracted from individual hookworms after allowing the ethanol to evaporate, using the Monarch Genomic DNA Purification Kit (New England Biolabs, Melbourne, Australia). The extracted gDNA was stored at −20°C.
The gDNA from 2 adult hookworms was used for genome skimming via next-generation sequencing (NGS) on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads with approximately 3 Gb of raw sequence data (Novogene, Singapore). The resulting FastQ files were analysed using the Artemis High Performance Computing (HPC) system at the Sydney Informatics Hub, University of Sydney.
The internal transcribed spacer (ITS) rDNA region – comprising the 5′ end of the small subunit ribosomal RNA gene, ITS1, 5.8S rRNA gene, ITS2, and the 3′ end of the large subunit rRNA gene – was assembled from the FastQ data using the MITObim v1.9.1 pipeline (Hahn et al., Reference Hahn, Bachmann and Chevreux2013); https://github.com/chrishah/MITObim, with the U. stenocephala sequence (AF194145) as a reference. The assembled ITS rDNA sequences were aligned with available ITS rDNA sequences from other hookworm species using CLC Main Workbench 25.0.1 (CLC bio, Qiagen, Clayton, Australia). ITS2 is a commonly used barcode for nematodes that is used in ‘nemabiome’ studies (Avramenko et al., Reference Avramenko, Redman, Lewis, Yazwinski, Wasmuth and Gilleard2015).
The complete mitochondrial genome (mitogenome) was assembled from the FastQ data using the GetOrganelle v1.7.5.3 pipeline (Jin et al., Reference Jin, Yu, Yang, Song, Depamphilis, Yi and Li2020); https://github.com/Kinggerm/GetOrganelle. The circularized mitogenome was aligned with available complete mitogenomes of other hookworm species using CLC Main Workbench. The mitogenome of Strongylus vulgaris was used as the outgroup.
Phylogenetic analysis was based on 12 mitochondrial protein-coding genes, comprising 3,394 aligned amino acid positions. Analyses were conducted in MEGA11 (Tamura et al., Reference Tamura, Stecher and Kumar2021). The phylogenetic tree was inferred using the Maximum Likelihood (ML) method with the JTT matrix-based model, incorporating a discrete Gamma distribution to model rate variation among sites (5 categories, + G, parameter = 0.2729), and allowing for a proportion of invariable sites (+I, 35.96%). A second tree was inferred using the Minimum Evolution (ME) method, with distances computed using the JTT model and rate variation modelled with a Gamma distribution (shape parameter = 0.7). Bootstrap analysis was performed to assess node support (1,000 replicates for ME; 100 replicates for ML).
Results
The recovered hookworms measured 6–8 mm in length and closely resembled U. criniformis as described by Ransom (Reference Ransom1924). In lateral view, the buccal capsule appeared only slightly curved (Figure 1A). The buccal capsule formed an elongate cone with an anterior opening bearing 2 cutting plates and no visible teeth. Despite examining the buccal capsule at multiple focal planes, we were unable to identify distinct articulated plates. Due to ethanol preservation, the relaxation of the specimens was suboptimal, particularly affecting the observation of the male bursa. As a result, the digitation of the dorsal rays could not be determined. However, the lateral rays showed a distinctly narrower externo-lateral ray compared to the medio-lateral and postero-lateral rays, with the medio-lateral ray being slightly thicker than the postero-lateral ray (Figure 1B). Male spicules measured 590–600 µm, and female tails were 130–150 µm long, each ending in a terminal bristle (Figure 1C).
Figure 1. Uncinaria criniformis Goetze, 1872 from Eurasian badger (Meles meles). (A) buccal capsule in 6 different consecutive lateral focal planes. (B) Male bursa from the lateral view showing the lateral rays (externo-lateral, e.L.; medio-lateral, m.L.; postero-lateral, p.L.). (C) Female caudal end showing a tail with terminal bristle. All images at the same scale, scale bar = 100 µm.
Genomic DNA was extracted from 2 U. criniformis specimens (one male and one female), yielding 0.74 ng/μl (JS6867) and 0.49 ng/μl (JS6869). Sequencing of gDNA from voucher JS6867 (GD01-1) produced 27,638,320 raw 150 bp reads, totaling 4.1 Gb of data (Q30 = 91.04%; GC = 42.66%). Voucher JS6869 (GD01-3) yielded 22,037,344 reads, totaling 3.3 Gb (Q30 = 91.01%; GC = 43.43%).
The ITS rDNA region (5′ end of SSU rRNA – ITS1 – 5.8S rRNA – ITS2 – 3′ end of LSU rRNA) was assembled from both FastQ datasets. Each sequence was 757-nt long, differing by 2 mismatches in the ITS1 region. Sequence comparison revealed a close match to 2 ITS rDNA sequences (MN078169 and MK348041), annotated as Arthrostoma sp. ex Meles leucurus (isolate LK-01-3, China), with 99.7% identity. These sequences differed from our samples by a single indel in ITS2 and one mismatch in ITS1 per sample. In contrast, ITS rDNA sequences from U. stenocephala (AF194145, PQ316553, MT345056) showed only 92.4–92.8% identity.
Complete circular mitochondrial genomes (mitogenomes) were assembled for both specimens: 13,749 nt (JS6867) and 13,748 nt (JS6869). Each mitogenome encoded 12 protein-coding genes (COX1–3, NAD1–6, NAD4L, ATP6, CYTB), 2 rRNA genes, and 22 tRNA genes (Figure 2A). All genes were transcribed in the same direction (5′→3′), with gene boundaries inferred by comparison to related hookworm mitogenomes. The 2 mitogenomes were 99.7% identical, differing by 46 positions, including 3 gaps.
Figure 2. Circular mitogenomes (mtDNA) of two hookworms (Uncinaria criniformis) from Eurasian badger (Meles meles). (A) all amino acid coding genes are coded by the same DNA strand, transcribed clockwise and labelled by their protein name (yellow). Transfer RNA genes (red) are identified by a 3-letter amino acid code. Two ribosomal RNA genes (rRNA) are indicated in blue. The inner circle consisting of individual radiating lines represents polymorphism between the two genomes (each line represents a single-nucleotide polymorphism). the start of mtDNA is arbitrarily set at the start (1) of the cox1 coding for COX1, in addition the positions of 2,000 to 12,000 nucleotides are indicated. The red inner most box within cox1 gene region represents hookworm sequence (MW517832) from the Asian badger (Meles leucurus) with each bolded line represents polymorphism against U. criniformis cox1 sequence. (B) Phylogenetic tree reconstructed using maximum likelihood (ML) method and JTT matrix-based model (log likelihood = −20813.94). the model included gamma distribution (+G, parameter = 0.2729) and allowed for some sites to be evolutionarily invariable ([+I], 35.96% sites). the tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test, 100 replicates for ML and 1,000 for minimum evolution [ME], are shown next to the branches. ME analysis included evolutionary distances computed using the JTT matrix-based method a gamma distribution (shape parameter = 0.7). this analysis involved 12 amino acid sequences and a total of 3,394 positions in the final dataset. Branches with uncinaria spp. are drawn in red. Strongylus vulgaris mitogenome served as an outgroup.
Partial cox1 sequences (1,209 nt) showed the closest match to Arthrostoma sp. ex Meles leucurus (MW517831 and MW517832, isolates M1F and M2M, China), with 99.5–99.8% identity and 3–6 synonymous substitutions. The 2 U. criniformis sequences (JS6867 and JS6869) differed by 2 synonymous substitutions (99.8% identity).
Comparison with U. stenocephala cox1 sequences (PP916662, MW682884, PQ555179) over a 333 nt region revealed only 88.0–88.5% identity, with 38–40 mismatches. In contrast, U. stenocephala sequences were 99.1–99.4% identical to each other over the same region.
The newly assembled U. criniformis mitogenomes were aligned with available hookworm mitogenomes (Ancylostoma spp., Uncinaria sanguinis, Bunostomum phlebotomum, Necator americanus). Phylogenetic analysis was based on amino acid sequences of 12 protein-coding genes (3,394 aligned positions). Both Maximum Likelihood (ML) and Minimum Evolution (ME) methods produced identical tree topologies (Figure 2B). Uncinaria criniformis formed a strongly supported (100%) monophyletic group with Ancylostoma spp. In contrast, U. sanguinis formed a polyphyletic group relative to U. criniformis. The branching support between U. criniformis, U. sanguinis, and the Necator–Bunostomum clade was weak in both ML (48%) and ME (51%) analyses (Figure 2B).
Discussion
Hookworm infections in Eurasian badgers (M. meles) are commonly reported, with the species typically identified as U. criniformis (Magi et al., Reference Magi, Banchi, Barchetti and Guberti1999; Torres et al., Reference Torres, Miquel and Motje2001; Seguel and Gottdenker, Reference Seguel and Gottdenker2017; Byrne et al., Reference Byrne, Fogarty, Mooney, Harris, Good, Marples and Holland2020). During the early to mid-20th century, taxonomists debated the morphological distinctiveness of U. stenocephala, a species mainly found in canids such as dogs and foxes, compared to hookworms found in badgers, which are mustelids (Looss, Reference Looss1905; Cameron, Reference Cameron1924; Ransom, Reference Ransom1924; Baylis, Reference Baylis1933; Wolfgang, Reference Wolfgang1956). Although consensus has not been universally reached, U. criniformis is now widely used in studies of European wildlife diseases and epidemiology (Seguel and Gottdenker, Reference Seguel and Gottdenker2017; Kelly et al., Reference Kelly, Marples, Byrne, Fogarty, Kenny, Cameron, Griffin and Holland2022).
To address this taxonomic uncertainty using molecular tools, Górski et al (Reference Górski, Długosz, Bartosik, Bąska, Łojek, Zygner, Karabowicz and Wiśniewski2023) recently examined hookworms from Eurasian badgers in Poland and concluded that their specimens belonged to U. stenocephala, showing molecular identity with U. stenocephala from dogs, red foxes, and raccoon dogs. However, this study demonstrates that the ITS rDNA sequence from U. stenocephala (OP811914) reported by Górski et al (Reference Górski, Długosz, Bartosik, Bąska, Łojek, Zygner, Karabowicz and Wiśniewski2023) is clearly distinct from the ITS rDNA sequences obtained here from hookworms identified as U. criniformis. This provides strong evidence that Eurasian badgers are parasitized by a hookworm species, U. criniformis, that is molecularly distinct from U. stenocephala.
Numerous authors have attempted to identify consistent morphological differences between these U. stenocephala and U. criniformis, but results have often been inconclusive, especially when host species was used as a key differentiating factor (Cameron, Reference Cameron1924; Fülleborn, Reference Fülleborn1924; Ransom, Reference Ransom1924). If Eurasian badgers can host both species as supported by Górski et al (Reference Górski, Długosz, Bartosik, Bąska, Łojek, Zygner, Karabowicz and Wiśniewski2023), then host-based assumptions may have confounded earlier analyses. It would be prudent to assess the prevalence of U. stenocephala in badgers using molecular tools such as ITS rDNA or cox1 markers. Host species should no longer be used to determine hookworm identity, as hookworms can act as generalists, infecting and completing their life cycles in a wide range of hosts. Therefore, host association is not a reliable indicator of species identity.Our DNA sequence comparisons revealed a high degree of similarity (>99% identity) between U. criniformis and sequences annotated as Arthrostoma sp. from the Asian badger (Meles leucurus) in China. Specifically, the cox1 sequences (MW517831 and MW517832) are reference sequences for Arthrostoma leucurus, recently described by Liu et al (Reference Liu, Wang, Liang, Hornok, Zhao, Tan, Liu, Gu and Wang2022). This high-sequence identity strongly suggests that these hookworms are conspecific. This is not surprising, given the close evolutionary relationship between M. meles and M. leucurus (Kinoshita et al., Reference Kinoshita, Kosintsev, Raichev, Haukisalmi, Kryukov, Wiig, Abramov, Kaneko and Masuda2017). The distribution boundary between these 2 badger species lies near the Volga River in Russia, with M. meles to the west and M. leucurus to the east. It is therefore plausible that the hookworm species parasitized a common ancestral badger species prior to their divergence.
The morphology of the buccal capsule in A. leucurus led Liu et al (Reference Liu, Wang, Liang, Hornok, Zhao, Tan, Liu, Gu and Wang2022) to classify it within the genus Arthrostoma, which is characterized by the presence of articulated buccal plates (Cameron, Reference Cameron1927; Liu et al., Reference Liu, Wang, Liang, Hornok, Zhao, Tan, Liu, Gu and Wang2022). These plates, typically 8 to 10 in number, may not always be fully articulated (Cameron, Reference Cameron1927; Liu et al., Reference Liu, Wang, Liang, Hornok, Zhao, Tan, Liu, Gu and Wang2022). Comparing the published morphology of A. leucurus with U. criniformis as described by Ransom (Reference Ransom1924) reveals similarities, particularly in the male bursa. Liu et al (Reference Liu, Wang, Liang, Hornok, Zhao, Tan, Liu, Gu and Wang2022) described the medio-lateral ray as slightly thicker than the postero-lateral ray and much thicker than the externo-lateral ray, so consistent with Ransom’s (Reference Ransom1924) description of U. criniformis. While our specimens matched Ransom’s description and showed similar buccal capsule morphology, we could not confirm the presence of articulated plates without direct examination of A. leucurus material. Cameron (Reference Cameron1924) provided a detailed revision of U. criniformis and U. stenocephala, concluding they were the same species and proposing the new genus Dochmoides. Cameron (Reference Cameron1927) introduced the genus Arthrostoma without linking it to Dochmoides (=Uncinaria). Based on our findings, we interpret that hookworms in Meles species are conspecific. Therefore, A. leucurus should be considered a junior synonym of U. criniformis unless future evidence demonstrates distinct species in Eurasian and Asian badgers.
Our mitogenome-based phylogenetic analysis revealed that the genus Uncinaria is polyphyletic. Currently, the only other Uncinaria species with a sequenced mitogenome is U. sanguinis, a hookworm of sea lions (Haynes et al., Reference Haynes, Marcus, Higgins, Gongora, Gray and Šlapeta2014; Marcus et al., Reference Marcus, Higgins, Šlapeta and Gray2014). To better resolve hookworm evolutionary relationships, further mitogenomic characterization is needed for additional Uncinaria species, particularly U. stenocephala, as well as for related genera such as Globocephalus, Hypodontus, Agriostomum, Galoncus, Arthrocephalus, Placoconus, Tetragomphius and Arthrosoma (Ilik et al., Reference Ilik, Schwarz, Noskova and Pafco2024). Ideally, the type species of each of these genera should be reviewed and their mitogenomes sequenced, including that of the canine hookworm U. stenocephala, to clarify their phylogenetic relationships.
In this study, we characterized U. criniformis from the Eurasian badger and generated reference sequence data for 2 key molecular markers: the complete ITS rDNA region and the mitochondrial genome. We employed genome skimming via NGS as a cost-effective method to recover these markers, enabling robust species identification. The ITS rDNA region is widely used in molecular surveys, such as nemabiome or amplicon-based metabarcoding, to profile nematode communities in samples like faeces (Stocker et al., (Reference Stocker, Scott and Šlapeta2023); Abdullah et al., (Reference Abdullah, Stocker, Kang, Scott, Hayward, Jaensch, Ward, Jones, Kotze and Šlapeta2025) (Avramenko et al., Reference Avramenko, Redman, Lewis, Yazwinski, Wasmuth and Gilleard2015; Redman et al., Reference Redman, Queiroz, Bartley, Levy, Avramenko and Gilleard2019). Our demonstration that U. criniformis is molecularly distinct from U. stenocephala provides a reliable basis for distinguishing these species in future parasitological surveys.
Data availability
The vouchers of U. criniformis have been deposited at the IPCAS Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budějovice, Czech Republic under IPCAS N-1292. Sequence data were deposited in GenBank under the following accession numbers: PV763276-PV763277 and PV766792- PV766793. Raw FastQ sequence data were deposited at SRA NCBI BioProject: PRJNA1273505. Intermediate data for the samples and analyses are available at LabArchives: https://dx.doi.org/10.25833/yap8-7191.
Author contributions
G.D. and J.Š. designed the study. G.D. collected the material. J.Š. performed the analyses. G.D. and J.Š. wrote the article.
Financial support
Funding, in part, was provided by the Margo Roslyn Flood Bequest (Sydney School of Veterinary Science, The University of Sydney, Australia).
Competing interests
The authors declare there are no conflicts of interest.
Ethical standards
Ethical Committee Decision no. 232/23.11.2020 (University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Cluj-Napoca, Romania).
Open access
