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International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
Available online 30 April 2022
2213-2244/© 2022 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND
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Opening a can of lungworms: Molecular characterization of Dictyocaulus
(Nematoda: Dictyocaulidae) infecting North American bison (Bison bison)
Hannah A.Danks
a
, Caroline Sobotyk
a
, Meriam N.Saleh
a
, Matthew Kulpa
a
, Joe L.Luksovsky
a
,
Lee C Jones
b
, Guilherme G. Verocai
a
,
*
a
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA
b
Wildlife Health Ofce, Natural Resource Program Center, United States Fish and Wildlife Service, Bozeman, MT, USA
ARTICLE INFO
Keywords:
Bison bison
dictyocaulosis
Nearctic fauna
Phylogenetic relationships
Verminous pneumonia
Lungworm
ABSTRACT
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 conrmed 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 cat-
tle in North America and Europe, and European bison (Bison bonasus). Our results contradict previous as-
sumptions 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.
1. Introduction
Dictyocaulus Railliet 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. Briey, the rst-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, specically
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
* Corresponding author. Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station,
TX, 77843, USA.
E-mail address: gverocai@cvm.tamu.edu (G. G. Verocai).
Contents lists available at ScienceDirect
International Journal for Parasitology: Parasites and Wildlife
journal homepage: www.elsevier.com/locate/ijppaw
https://doi.org/10.1016/j.ijppaw.2022.04.011
Received 7 April 2022; Received in revised form 26 April 2022; Accepted 26 April 2022
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
129
high intensity infections, Dictyocaulus leads to verminous pneumonia,
associated with inammation, 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 Agricul-
ture, 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 conrmed 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, inuencing subsequent reports.
The aim of this study was to molecularly characterize Dictyocaulus
lungworm isolates from plains bison across the United States and un-
derstand its phylogenetic relationships with other species within the
genus. This study highlights the need to elaborate on the uncharac-
terized species of Dictyocaulus present in North American bison and
investigate other lungworm infections in North American ungulates.
2. Materials and methods
2.1. Collection
Bison fecal samples were collected from ve 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 eld. 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).
2.2. Baermann technique
The Baermann technique was used to isolate Dictyocaulus lungworm
larvae from bison and cattle fecal samples of bison or cattle using a
modication of a previously protocols (Zajac and Conboy, 2012; Verocai
et al., 2020). Briey, 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×magnication on
a glass slide. Any Baermann ltrate containing Dictyocaulus L1 was
collected and stored in cryogenic vials at −80 ◦C for subsequent pro-
cessing. Archived samples were thawed to room temperature and Dic-
tyocaulus larvae were isolated with the aid of micropipettes under a
compound microscope at 4×magnication.
2.3. 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 H¨
oglund 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
μ
L containing 1x
GoTaq® Green Master Mix (Promega Corporation, Madison, WI, USA),
0.625
μ
L of each primer at 0.25
μ
M concentration and 1
μ
L 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 amplication were an initial dena-
turation 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 nal 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 puried using E.Z.N.A. Cycle Pure Kit
(Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s
protocol. The puried DNA products were sequenced in both directions
using the original PCR primers in 3730xl DNA Analyzer at Eurons
Genomics (Louisville, KY, USA).
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
H. A.Danks et al.
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
130
2.4. 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 veried
referring to the chromatograms. Forward and reverse sequences were
analyzed to create a composite sequence. A maximum composite like-
lihood model was utilized to generate pairwise distances. ITS2 was run
with partial deletion penalties while cox1 was run with complete dele-
tion 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 t 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.
3. Results
3.1. 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 amplied 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
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.
Table 2
Primers used for polymerase chain reaction amplication and sequencing of the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA and cy-
tochrome c oxidase subunit 1 (cox1) of mitochondrial DNA in Dictyocaulus isolates.
Gene Primer Forward/Reverse Sequence Reference
ITS2 NC1 Forward 5′-ACGTCTGGTTCAGGGTTGTT-3′H¨
oglund 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)
H. A.Danks et al.
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
131
and accessioned in GenBank.
For cox1 of mitochondrial DNA, 84.1% (69/82) of the larvae ob-
tained were successfully amplied 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.
3.2. 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. Dictyo-
caulus 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 H¨
oglund, 2002.
Calculated maximum identities were obtained from cox1 sequence
data between species of Dictyocaulus (Table 4). The species of Dictyo-
caulus 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).
3.3. Phylogenetic analysis
Maximum likelihood analysis of ITS2 sequence data demonstrated
that Dictyocaulus of North American plains bison demonstrated distinc-
tion 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 con-
taining 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 H¨
oglund, 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.
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.
4. 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 uncharac-
terized species across diverse geographies with minimal variation be-
tween genetic sequences. This nding 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 Eu-
ropean 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 spe-
cies. 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 identied Dictyocaulus species infects other sym-
patric 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.
4.1. Phylogenetic relationships
While variation in clades identied in our analysis are minimal, most
notably, cox1 of mitochondrial DNA clusters D. capreolus with
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 98.9–99.0 100
a
– – – –
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 74 100
a
– –
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 69.5 100
a
European fallow deer (81.7 ±0.16)
a
Single sequence.
H. A.Danks et al.
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
132
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.
4.2. 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 molec-
ular 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 H¨
oglund, 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
identied 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 character-
ization in tandem with the conrmation 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.
4.3. Host-specicity of Dictyocaulus
Recent studies have shown that some cervid and bovid-associated
Dictyocaulus may not be host-specic, 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
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, 87.9–88.9 100
a
– – – – –
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 83.4–84.8 100
a
European red deer (87.6 ±0.10) (85.4 ±0.26) (90.0 ±
0.34)
(84.3 ±
0.40)
a
Single sequence.
Fig. 2. Maximum likelihood analysis of internal transcribed spacer 2 (ITS2) sequence data of Dictyocaulus spp. Analysis was run with T92 as best nucleotide sub-
stitution model and 1,000 bootstraps. Angiostrongylus vasorum =outgroup.
H. A.Danks et al.
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
133
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 H¨
oglund, 2002; Pyziel et al., 2017), and D. viviparus in
cattle and European bison (H¨
oglund et al., 2003; Pyziel, 2014). In North
America, reports of Dictyocaulus in various wild ungulates were identi-
ed 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 molec-
ular screening of Dictyocaulus in different hosts, along with cross
transmission studies should be conducted to further elucidate
host-specicity. While it is unknown if the bison Dictyocaulus identied
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, specically
where bison co-graze with other wild ungulates and domestic cattle.
4.4. Potential impacts of Dictyocaulus infection in bison
Studies in cattle show that subclinical D. viviparus infections can
cause signicant 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 in-
fections 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 identied species of Dictyocaulus evolved in
North American plains bison. In contrast with many production man-
agement 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).
4.5. 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 re-
cords, 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 identication of this distinct Dictyocaulus
species in North American plains bison, sampling of wood bison and
additional plains bison herds may further elucidate the geographic dis-
tribution of this lungworm. Moreover, a deeper understanding of the
phylogeography of this newly identied Dictyocaulus may be attained by
means of comparative mitogenome and whole genome sequencing and
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.
H. A.Danks et al.
International Journal for Parasitology: Parasites and Wildlife 18 (2022) 128–134
134
molecular clock analysis (Gasser et al., 2012; McNulty et al., 2016).
5. 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 con-
servation and how vital it is to keeping ecosystems in balance. In addi-
tion, 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 conict of interest.
Acknowledgments
This research did not receive any specic grant from funding
agencies in the public, commercial, or not-for-prot sectors. HAD was
supported by Boehringer Ingelheim Veterinary Scholars Program
through the Veterinary Medical Scientist Research Training Program at
the Texas A&M University, College of Veterinary Medicine and
Biomedical Sciences.
The authors would like to thank Dr. Tracy Vemulapalli for her
valuable comments on the manuscript.
This work would not have been possible without the dedicated ef-
forts of US Fish and Wildlife Service personnel who collect biological
samples to evaluate and monitor wild bison herd health.
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