Opening a can of lungworms: Molecular characterization of Dictyocaulus (Nematoda: Dictyocaulidae) infecting North American bison (Bison bison).

Dictyocaulus is a globally distributed genus of lungworms of domestic and wild ungulates. Dictyocaulus adults inhabit the bronchi, frequently causing subclinical and clinical disease, and that impacts animal health and production. North American bison (Bison bison) and cattle (Bos taurus) share various parasitic nematode species, particularly in areas where co-grazing occurs. The current assumption is that North American bison share the lungworm D. viviparus with cattle, but this has not been confirmed on a molecular basis. The aim of this study was to molecularly characterize Dictyocaulus lungworm isolates from North American plains bison (Bison bison bison). Fecal samples were collected from 5 wild conservation bison herds located in Iowa, North Dakota, Oklahoma, Colorado, and Montana in 2019 and 2020, and from ranched and feedlot bison from 2 herds in Oklahoma and Texas. First-stage lungworm larvae (L1) were isolated via Baermann technique. Genomic DNA was extracted from L1s of up to 3 samples per herd and followed by PCR and sequencing targeting the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA and the partial cytochrome oxidase c subunit 1 (cox1) of mitochondrial DNA. Phylogenetic analyses were performed in MEGA X 10.1. Sequences of North American plains bison Dictyocaulus belong to a single, uncharacterized species, clustering in well-supported clades (100% and 100% bootstrap support for ITS2 and cox1, respectively), differing from D. viviparus of cattle in North America and Europe, and European bison (Bison bonasus). Our results contradict previous assumptions regarding parasite identity, highlighting the need for characterization of this species through morphological and molecular methods, elucidating its biology and host range, and potential impact on host health. Further investigation into the biodiversity of Dictyocaulus species infecting bovids and cervids in North America is warranted.

Introduction

DictyocaulusRailliet and Henry, 1907 (Nematoda: Dictyocaulidae) is a globally distributed genus of lungworm parasites of domestic and wild ungulates, including an array of bovid and cervid hosts (Gibbons and Khalil, 1988; Anderson, 2000). Dictyocaulus adults inhabit the bronchi and have a direct life cycle. Briefly, the first-stage larvae (L1) are passed in feces and will develop to third-stage larvae (L3) in 1–4 weeks in the environment. Susceptible ungulate species are infected by ingestion of L3s on pasture, after which larvae migrate to the lungs. In the lungs, larvae develop into adult males and females, which produce eggs after sexual reproduction. In many Dictyocaulus species associated with ruminant hosts, the eggs hatch and the resulting L1 larvae are coughed up, swallowed, and deposited in the feces, completing the transmission to the environment (Anderson, 2000; Panuska, 2006). The pre-patent period is 3–4 weeks, however, some Dictyocaulus species, specifically Dictyocaulus viviparus (Bloch, 1782) of cattle, may undergo hypobiosis in the case of overwintering which can increase the pre-patent period to 150 days.

Dictyocaulus infection has been shown to cause subclinical and clinical disease in domestic and wild ungulate hosts (Panuska, 2006; Kutz et al., 2012). For example, previous studies with D. viviparus in cattle have revealed a negative impact on animal health and decreased milk production (Panuska, 2006; Dank et al., 2015; May et al., 2018). In high intensity infections, Dictyocaulus leads to verminous pneumonia, associated with inflammation, obstruction of bronchioles, emphysema, and pneumonia resulting in clinical signs including coughing, dyspnea, nasal discharge, or occasionally mortality (Panuska, 2006). Information on the impact of Dictyocaulus infection in wild ungulates remains scarce.

The North American bison (Bison bison (Linnaeus, 1758)) is the largest terrestrial mammal of North America, and is listed as “near threatened” in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species (Aune et al., 2017). Bison were driven to near extinction in the late 1800s, resulting in a genetic bottleneck (Hedrick, 2009), and possibly including the parasites they carry. Today, an estimated number of 11,000 to 13,000 wild individuals remain in severely fragmented populations (Aune et al., 2017). In addition, the North American bison has been kept and bred as livestock, with an estimated slaughter of 66,000 bison in the US in 2020, with an increasing trend in recent years (United States Department of Agriculture, 2020). Previous studies have suggested that bison grazed on range more likely to have been previously occupied by cattle have different parasite communities than bison grazing ranges without shared cattle history (Avramenko et al., 2018). The current paradigm is that North American bison share the lungworm D. viviparus with cattle, likely due to range overlap, but this has not been confirmed using molecular methods (Dikmans, 1936; Frick 1951; Locker, 1953; Boddicker and Hugghins, 1969; Wade et al., 1979). However, Dictyocaulus hadweni Chapin (1925) was historically described as a species in North American bison from western Canada (Chapin, 1925; Roudabush 1936). The validity of this species was later questioned by Dikmans (1936), who considered it a junior synonym of D. viviparus, influencing subsequent reports.

The aim of this study was to molecularly characterize Dictyocaulus lungworm isolates from plains bison across the United States and understand its phylogenetic relationships with other species within the genus. This study highlights the need to elaborate on the uncharacterized species of Dictyocaulus present in North American bison and investigate other lungworm infections in North American ungulates.

Materials and methods

Collection

Bison fecal samples were collected from five US Fish and Wildlife Service National Wildlife Refuge herds and 2 ranched herds across the US in 2019–2020: Wichita Mountains Wildlife Refuge (Oklahoma), National Bison Range (Montana), White Horse Hill National Game Preserve (North Dakota), Neal Smith National Wildlife Refuge (Iowa), Rocky Mountain Arsenal National Wildlife Refuge (Colorado), Cheyenne and Arapaho tribes (Oklahoma), and a privately-owned Texas feedlot (containing a herd originally from Oklahoma) (Table 1). Wild bison samples were collected from each refuge herd by US Fish and Wildlife Service personnel using techniques to approach and collect samples from the herd in a short period of time to reduce the potential for duplicate sample collection from any individual animal. Ranched and feedlot bison samples were collected either during routine handling and vaccination operations of from the field. In addition, a bovine calf (Bos taurus Linnaeus, 1758) fecal sample originating from Marion County, northeastern Texas, was included for comparison. The samples were refrigerated in either 50 mL plastic tubes or plastic bags for no more than 12 days prior to shipment in insulated containers with coolant packs to the Texas A&M University Parasitology Diagnostic Laboratory via overnight courier service (Fig. 1).

Table 1

Description of bovid herds samples, and larvae included in PCR, sequencing, and alignment.

Herd	State	Animals Sampled	Sequenced L1 (ITS2)	Sequenced L1 (cox1)
Bovine	Texas	1	2/3	1/3
Cheyenne and Arapaho	Oklahoma	4	10/10	8/9
National Bison Range	Montana	3	8/8	8/8
Neal Smith National Wildlife Refuge	Iowa	3	9/9	9/9
Privately owned feedlot	Texas	3	8/8	8/8
Rocky Mountain Arsenal National Wildlife Refuge	Colorado	3	9/9	9/9
White Horse Hill National Game Preserve	North Dakota	6	17/18	16/17
Wichita Mountains Wildlife Refuge	Oklahoma	3	9/9	9/9
Total		25 bison, 1 cattle	70/71 bison, 2/3 cattle	67/69 bison, 1/3 cattle

Fig. 1

Sites of bison fecal collections; WHH: White Horse Hill National Game Preserve; RMA: Rocky Mountain Arsenal National Wildlife Refuge; NBR: National Bison Range; NSM: Neal Smith National Wildlife Refuge; WMW: Wichita Mountains Wildlife Refuge.

Baermann technique

The Baermann technique was used to isolate Dictyocaulus lungworm larvae from bison and cattle fecal samples of bison or cattle using a modification of a previously protocols (Zajac and Conboy, 2012; Verocai et al., 2020). Briefly, we utilized a plastic 50 mL conical tube and 5 g of feces wrapped in cheesecloth and a Kimwipe submerged in water for 24 h. The following day, after removal of the wrapped feces, the bottom 1 mL was collected and microscopically examined at 4× magnification on a glass slide. Any Baermann filtrate containing Dictyocaulus L1 was collected and stored in cryogenic vials at −80 °C for subsequent processing. Archived samples were thawed to room temperature and Dictyocaulus larvae were isolated with the aid of micropipettes under a compound microscope at 4× magnification.

DNA extraction and PCR

We have extracted DNA of Dictyocaulus L1 recovered from a total of 25 bison fecal samples were analyzed from 7 herds, including up to 4 larvae from each sample per year (Table 1). DNA extraction of single larva was performed using DirectPCR® lysis reagent (Cell) (Viagen Biotech, Inc., Los Angeles, CA, USA) with Proteinase K (QIAGEN®, Hilden, Germany) (0.5 mg/mL) as per manufacturer's instructions. The extracted DNA was kept frozen at −20 °C. To amplify the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA, NC1 and BD3R primers were utilized as stated in Höglund et al. (1999) and Pyziel et al. (2017), respectively (Table 2). Additionally, to amplify the partial cytochrome oxidase c subunit 1 (cox1) region of mitochondrial DNA, LCO1490 and HCO2198 were utilized according to Folmer et al. (1994). Reactions were carried out in a volume of 25 μL containing 1x GoTaq® Green Master Mix (Promega Corporation, Madison, WI, USA), 0.625 μL of each primer at 0.25 μM concentration and 1 μL of DNA template. Extracted DNA of Pseudostertagia bullosa (Ransom and Hall, 1912) was included as a positive control and water as the negative control. Cycling conditions for ITS2 amplification were an initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 51 °C for 45 s, and 72 °C for 1 min, and a final extension step at by 72 °C for 5 min. Cycling conditions for cox1 were identical except for the annealing temperature, which was lowered to 50 °C. PCR products were visualized utilizing gel electrophoresis and purified using E.Z.N.A. Cycle Pure Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's protocol. The purified DNA products were sequenced in both directions using the original PCR primers in 3730xl DNA Analyzer at Eurofins Genomics (Louisville, KY, USA).

Table 2

Primers used for polymerase chain reaction amplification and sequencing of the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA and cytochrome c oxidase subunit 1 (cox1) of mitochondrial DNA in Dictyocaulus isolates.

Gene	Primer	Forward/Reverse	Sequence	Reference
ITS2	NC1	Forward	5′-ACGTCTGGTTCAGGGTTGTT-3′	Höglund et al. (1999)
	BD3R	Reverse	5′-TATGCTTAAGTTCAGCGGGT-3′	Pyziel et al. (2017)
cox1	LCO1490	Forward	5′-GGTCAACAAATCATAAAGATATTGG-3′	Folmer et al. (1994)
	HCO2198	Reverse	5′-TAAACTTCAGGGTGACCAAAAAATCA-3′	Folmer et al. (1994)

Phylogenetic analyses

Sequencing data were analyzed using SnapGene software (GSL Biotech LLC, San Diego, CA, USA) as chromatograms to verify sequence quality. Initially, Dictyocaulus isolate sequences were compared with other Dictyocaulus sequences available in the GenBank database through BLAST searches. Sequences were aligned and analyzed using MEGA X 10.1 (Kumar et al., 2018). Gaps and point mutations were verified referring to the chromatograms. Forward and reverse sequences were analyzed to create a composite sequence. A maximum composite likelihood model was utilized to generate pairwise distances. ITS2 was run with partial deletion penalties while cox1 was run with complete deletion penalties based on the degree to which gaps persisted in the alignment. Maximum likelihood phylogenetic trees were generated in MEGA X 10.1 (Kumar et al., 2018) using 1,000 bootstrap replicates. The best fit DNA/protein models for ITS2 and cox1 analyses were Tamura (1992) with invariant sites, and Tamura-Nei (Tamura and Nei, 1993) with gamma distribution, respectively. Angiostrongylus vasorum (Baillet, 1866) was included as an outgroup for both analyses.

Results

Samples analyzed

Representative pooled L1 from each wild bison herd were deposited at the Division of Parasites of the Museum of Southwestern Biology, University of New Mexico, under the accession numbers: MSB:Para:32452–7 (arctos.database.museum).

For the ITS2 region of the nuclear ribosomal DNA, 86.6% (71/82) of larvae were successfully amplified by PCR, with 98.6% (70/71) of those successfully sequenced and included in the phylogenetic analysis. A total of 70 Dictyocaulus ITS-2 sequences (229–235 base pairs) of individual L1 from North American plains bison (Accession Numbers: OK575977-OK576046) and 2 from cattle (OM417137-OM417138) were produced and accessioned in GenBank.

For cox1 of mitochondrial DNA, 84.1% (69/82) of the larvae obtained were successfully amplified by PCR, with 97% (67/69) of those successfully sequenced for subsequent phylogenetic analysis. A total of 67 Dictyocaulus partial cox1 sequences (621 base pairs) from North American plains bison (Accession Numbers: OK562219-OK562285) and 1 from cattle (OM368333) were generated and accessioned in GenBank.

Pairwise distance analysis

Maximum identity within and between species for the ITS2 region can be found in Table 3. The Dictyocaulus from North American plains bison shared an average of 99.9% identity among each other. Dictyocaulus viviparus from our North American cattle isolate, European cattle, and European bison averaged 88.9%, 90.6%, and 90.7% shared identity with Dictyocaulus of North American plains bison, respectively. The lowest shared identity with North American plains bison Dictyocaulus was 62.9% with Dictyocaulus capreolus Gibbons and Höglund, 2002.

Table 3

Pairwise identity among Dictyocaulus species based on sequences of the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA.

Dictyocaulus species	Dictyocaulus sp., US bison	D. viviparus, US cattle	D. viviparus, European cattle	D. viviparus, European bison	D. cervi	D. capreolus	D. eckerti
Dictyocaulus sp.,	99.0–100	–	–	–	–	–	–
US bison	(99.9 ± 0.20)
D. viviparus,	88.3–89.0	100	–	–	–	–	–
US cattle	(88.87 ± 0.21)
D. viviparus,	90.0–90.7	98.9–99.0	100a	–	–	–	–
European cattle	(90.6 ± 0.21)	(99.0 ± 0)
D. viviparus,	90.1–90.8	99.0	100	100	–	–	–
European bison	(90.7 ± 0.2)
D. cervi	74.5–75.2	74.6	73.8	74	100a	–	–
European red deer	(74.5 ± 17)
D. capreolus	62.9–62.9	56.4	53.5	53.9	59.6	100	–
European red deer	(62.9 ± 0)
European roe deer
D. eckerti	81.7–82.4	81.2	79.0	79.1	91.5	69.5	100a
European fallow deer	(81.7 ± 0.16)

Single sequence.

Calculated maximum identities were obtained from cox1 sequence data between species of Dictyocaulus (Table 4). The species of Dictyocaulus found in the present study from North American plains bison had an average of 99.9% shared identity. When compared to D. viviparus of our North American cattle isolate, the Dictyocaulus samples of our plains bison had an average of 88.8% shared identity. The other Dictyocaulus species assessed averaged a shared identity with Dictyocaulus of our plains bison ranging between 85.5% (D. capreolus) and 88.9% (D. viviparus).

Table 4

Pairwise identity among Dictyocaulus species based on sequences of the partial cytochrome oxidase c subunit 1 (cox1) of the mitochondrial DNA.

Dictyocaulus species	Dictyocaulus sp., US bison	D. viviparus, US cattle	D. viviparus, European cattle	D. viviparus, European bison	D. cervi	D. capreolus	D. eckerti
Dictyocaulus sp.,	99.2–100	–	–	–	–	–	–
US bison	(99.9 ± 0.12)
D. viviparus,	87.9–88.9	100a	–	–	–	–	–
US cattle	(88.8 ± 0.12)
D. viviparus,	87.7–89.1	99.83	99.7–100	–	–	–	–
European cattle	(88.9 ± 0.23)		(99.8 ± 0.20)
D. viviparus,	87.9–88.8	96.3	96.1–96.5	100	–	–	–
European bison	(88.8 ± 0.12)		(96.4 ± 0.15)
D. cervi	85.8–88.0	83.5–85.1	83.2–85.3	85.5–87.3	96.5–99.8	–	–
European red deer, European fallow deer, European roe deer	(87.2 ± 0.38)	(84.4 ± 0.35)	(84.4 ± 0.41)	(86.28 ± 0.45)	(98.3 ± 0.70)
D. capreolus	84.0–85.9	86.8–87.5	86.6–87.7	87.0–87.7	82.4–85.9	97.9–100	–
European roe deer	(85.5 ± 0.30)	(87.2 ± 0.27)	(87.3 ± 0.34)	(87.3 ± 0.25)	(84.1 ± 0.62)	(99.1 ± 0.50)
D. eckerti	86.9–87.6	85.3	85.1–85.5	85.4	89.4–90.7	83.4–84.8	100a
European red deer	(87.6 ± 0.10)		(85.4 ± 0.26)		(90.0 ± 0.34)	(84.3 ± 0.40)

Single sequence.

Phylogenetic analysis

Maximum likelihood analysis of ITS2 sequence data demonstrated that Dictyocaulus of North American plains bison demonstrated distinction from other Dictyocaulus species. A well-supported clade (83% bootstrap support) containing North American Dictyocaulus isolates and European D. viviparus was further divided into two equally well-supported subclades (100% and 99% bootstrap support respectively), one containing only North American plains bison Dictyocaulus isolates, and one including D. viviparus of cattle and European bison (Fig. 2). Within D. viviparus, there were two well-supported subclades, one containing North American cattle isolates and another of European bison (D. viviparus bisontis) and cattle. Basal to this bovine Dictyocaulus clade, was a clade containing D. eckerti Skrjabin, 1931 of moose in Sweden. Basal was a clade containing Dictyocaulus cervi Pyziel, Laskowski, Demiaszkiewicz and Höglund, 2017 and D. eckerti of fallow deer and red deer of European origin. Basal to the above clades were D. capreolus of roe deer and red deer also with a European origin.

Fig. 2

Maximum likelihood analysis of internal transcribed spacer 2 (ITS2) sequence data of Dictyocaulus spp. Analysis was run with T92 as best nucleotide substitution model and 1,000 bootstraps. Angiostrongylus vasorum = outgroup.

Maximum likelihood analysis of cox1 sequence data further demonstrated the reciprocal monophyly of isolates of Dictyocaulus of North American plains bison with 100% bootstrap support (Fig. 3). The North American plains bison isolates clustered with D. viviparus and D. capreolus, but with moderate (72%) bootstrap support. However, the subclade containing D. viviparus from North American cattle, European cattle, European bison, and D. capreolus from European roe deer had 91% bootstrap support. Within D. viviparus, cattle and European bison isolates also formed well-supported clusters. A separate clade contained Dictyocaulus spp. of red deer from New Zealand and Europe, roe deer from Europe, and fallow deer from Europe.

Fig. 3

Maximum likelihood analysis of cytochrome oxidase c subunit 1 (cox1) sequence data of Dictyocaulus spp. Analysis was run with TN93 + G as best nucleotide substitution model and 1,000 bootstraps. Angiostrongylus vasorum = outgroup.

Discussion

Since 1936, the species of Dictyocaulus in North American bison has been presumed to be D. viviparus (Dikmans, 1936). The results of our study contradict this long-held assertion with strong support from pairwise distance and phylogenetic analyses of both a nuclear and mitochondrial gene. Instead, both wild and ranched North American plains bison across the United States are parasitized by a genetically distinct Dictyocaulus species. Notably, we found this same uncharacterized species across diverse geographies with minimal variation between genetic sequences. This finding may be explained by the historic genetic bottleneck experienced by wild bison in the last century, with modern wild and ranched herds established from small remnant herds gathered in the late 1800s (Hartway et al., 2020). Furthermore, the broad geographic distribution of those small remnant herds across the United States suggests that this genetically distinct Dictyocaulus species may have been historically widespread prior to the introduction of European cattle, occurring commonly enough to survive the bottleneck with their bison host. Although our study revealed surprising homology across bison herds under different management models across different geographic areas, the history of bison translocations among wild herds in North America is well documented and can be utilized for further study of genetic structure within this uncharacterized Dictyocaulus species. Bison interactions with other North American ungulates, including a larger sampling of domestic cattle, as well as cervids such as white-tailed deer, elk, and moose, should be further investigated to assess if this newly identified Dictyocaulus species infects other sympatric hosts. While these isolates are distinct from other Dictyocaulus species and it is the only species found in all of the North American plains bison herds sampled in our study, its phylogenetic relationships among other Dictyocaulus species warrants further investigation.

Phylogenetic relationships

While variation in clades identified in our analysis are minimal, most notably, cox1 of mitochondrial DNA clusters D. capreolus with D. viviparus, while it is represented by a separate clade under ITS2 of the nuclear ribosomal DNA. Additional sequencing of other genetic markers is required to further elucidate phylogenetic relationships among the North American plains bison Dictyocaulus isolate and other species within the genus.

Novel species or Dictyocaulus hadweni?

The lack of adult Dictyocaulus specimens isolated from North American plains bison for detailed morphologic assessment hampers direct comparison with the description of D. hadweni, a species that has been considered invalid by Dikmans (1936), following reassessment of its type-specimens. The original description of D. hadweni is relatively poor, contains confusing if not erroneous morphological comparisons with other Dictyocaulus species, and male specimens were considered virtually indistinguishable from those of D. viviparus from cattle. The most recent revision of the genus Dictyocaulus also failed to recognize D. hadweni as a valid species (Gibbons and Khalil, 1988). Nevertheless, the morphological differentiation among adult male and female can be rather challenging, and recent studies integrating classical and molecular approaches have recognized two new species in Eurasian cervids, namely D. capreolus and D. cervi, and a proposed susbspecies infecting the European bison, D. v. viviparus (Gibbons and Khalil, 1988; Gibbons and Höglund, 2002; Pyziel et al., 2017, 2020). Overall, these recent advances around the biodiversity of the genus Dictyocaulus suggest that cryptic species may yet to be recognized, especially since our analysis identified a previously uncharacterized species genetically distinct from both D. viviparus and the proposed subspecies D. v. bisontis described in European bison (Pyziel et al., 2020).

Our laboratory has begun acquiring adult nematode specimens from North American plains bison for a detailed morphological characterization in tandem with the confirmation of the species identity based on our ITS2 and cox1 data. Future studies should include experimental infections with Dictyocaulus of cattle to elucidate transmissibility as well as additional fecal samples from cattle which co graze with bison.

Host-specificity of Dictyocaulus

Recent studies have shown that some cervid and bovid-associated Dictyocaulus may not be host-specific, especially in areas of sympatry of susceptible hosts (Pyziel et al., 2017). For instance, through molecular characterization, D. eckerti has been reported from various cervids including red deer (Cervus elaphus Linnaeus, 1758), Eurasian moose (Alces alces (Linnaeus, 1758)), and roe deer (Dama dama (Linnaeus, 1758)); D. capreolus in roe deer and moose (Gibbons and Khalil, 1988; Gibbons and Höglund, 2002; Pyziel et al., 2017), and D. viviparus in cattle and European bison (Höglund et al., 2003; Pyziel, 2014). In North America, reports of Dictyocaulus in various wild ungulates were identified or assumed to be associated with D. eckerti (revised in Kutz et al., 2012). Currently, it is unknown if the North American plains bison may be infected by D. viviparus, and if the bison Dictyocaulus isolate we have characterized can infect cattle or other ungulate hosts. Further molecular screening of Dictyocaulus in different hosts, along with cross transmission studies should be conducted to further elucidate host-specificity. While it is unknown if the bison Dictyocaulus identified in our study may infect cervids, Chapin (1925) reported that D. hadweni of bison was morphologically indistinguishable from isolates from moose (Alces americanus (Linnaeus, 1758)) and elk or wapiti (Cervus canadensis (Erxleben, 1777)) from North America. Elucidating the host range of Dictyocaulus associated with the North American plains bison will have implications for future management practices, specifically where bison co-graze with other wild ungulates and domestic cattle.

Potential impacts of Dictyocaulus infection in bison

Studies in cattle show that subclinical D. viviparus infections can cause significant losses in milk production, with a reduction ranging from 1.01 to 1.68 kg/cow/day of milk (Dank et al., 2015). Dank et al. (2015) also demonstrated that cows with subclinical D. viviparus infections produced milk with 0.14% less milk fat, potentially reducing growth in calves. Dictyocaulosis has been also associated with reduction of yearly calf survival and weakening of individuals in cattle (Panuska, 2006; David, 1999). Currently, the impact of Dictyocaulus infection in North American plains bison is unknown, although previous reviews suggest that subclinical or clinical disease may develop in infected individuals managed under high density production models (Haigh et al., 2002; Berezowski et al., 2018). While dictyocaulosis could cause direct economic impact to the bison industry, the effects on wild herds managed at low densities on natural range are likely minimal, and especially if our newly identified species of Dictyocaulus evolved in North American plains bison. In contrast with many production management models, wild bison health is determined by the resilience and sustainability of this species to native pathogens and parasites (Stephen, 2014; Jones et al., 2020).

Future explorations of the historical biogeography of Dictyocaulus in North American bison

The substantial genetic divergence of North American plains bison Dictyocaulus isolates in relation to those found in cattle and European bison allows us to infer on its historical biogeography. Assuming B. bison bison is the primary host for this species, one would expect a deeper association with this wild bovine species – the larger remnant bovine in the Nearctic fauna. The genus Bison arrived in North America through the Bering Land Bridge in two waves: one 195-135kybp (thousand years before present) and another 45-21kybp in the Pleistocene Epoch, likely as Bison priscus. This species later evolved into Bison latifrons, and later as Bison antiquus and Bison occidentalis with the latter existing until 1730ybp (Froese et al., 2017; Zver et al., 2021). According to fossil records, B. bison only appeared around 10kybp within the Holocene, and is currently subdivided into two subspecies, the plains bison, B. b. bison, and the wood bison, B. b. athabascae (Heintzman et al., 2016; Geist, 1991). To explore beyond our identification of this distinct Dictyocaulus species in North American plains bison, sampling of wood bison and additional plains bison herds may further elucidate the geographic distribution of this lungworm. Moreover, a deeper understanding of the phylogeography of this newly identified Dictyocaulus may be attained by means of comparative mitogenome and whole genome sequencing and molecular clock analysis (Gasser et al., 2012; McNulty et al., 2016).

Conclusions

Our recent discovery of a previously uncharacterized Dictyocaulus isolate contradicts previous assumptions regarding parasite identity, necessitating further research of Dictyocaulus infecting wild bovids and cervids in North America. Investigations should include studies into host health impact and how to preserve this unique host-parasite assemblage, especially in systems likely to have co-evolved over long periods of time. Thought-provoking studies have stated the importance of parasite conservation and how vital it is to keeping ecosystems in balance. In addition, taxonomic description of parasites is crucial to enhance monitoring and quantifying of named species, especially in the face of climate change and other anthropogenic processes (Carlson et al., 2020). This study adds to the knowledge of Dictyocaulus and thereby contributes to overall parasite conservation, including in the fragile ecosystems in which wild North American plains bison exist.

Declaration of competing interest

The authors declare no conflict of interest.