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High frequency of benzimidazole resistance polymorphisms and age-class
differences in trichostrongyle nematodes of ranched bison from the
south-central United States
Kaylee R. Kipp
a
, Elizabeth M. Redman
b
, Joe L. Luksovsky
a
, Dani Claussen
a
,
John S. Gilleard
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
Department of Comparative Biology and Experimental Medicine, Host-Parasite Interactions Program, Faculty of Veterinary Medicine, Hospital Drive, 3330, University
of Calgary, Calgary, Alberta, Canada
ARTICLE INFO
Keywords:
Anthelmintic drug resistance
Haemonchus contortus
Metabarcoding
North American bison
Ostertagia ostertagi
Trichostrongyloidea
ABSTRACT
Bison production is a growing sector of the United States agriculture, with more consumers choosing bison
products. Commercial bison are kept on smaller pastures and treated with anthelmintics for gastrointestinal
nematodes (GIN) to maintain production. However, there is a lack of information regarding the GIN parasite
communities in ranched bison or the extent of their resistance to anthelmintics. Our objectives were: i) to
determine the GIN species present and the extent of resistance to the benzimidazole drug class in commercial
bison herds in the southern US and ii) to assess age class differences in GIN species composition and BZ resis-
tance. Composite coprocultures from bison in Texas (n =14) and Oklahoma (n =2), and individual bison of
different age classes from a single ranch in central Texas (n =43) were analyzed using ITS2 rDNA nemabiome
metabarcoding to determine the trichostrongylid species composition. For both the composite and individual
samples, the most common trichostrongylid species found were Haemonchus contortus, Haemonchus placei, and
Ostertagia ostertagi. Among the known canonical isotype-1 β-tubulin BZ resistance polymorphisms (at codons 200,
198, 167), the 200Y (TTC >TAC) substitution was the most widespread across the two southern states, with a
prevalence of 81.3 %. Other polymorphisms, such as 167Y (TTC >TAC) and 198L (GAA >TTA), were also
detected, and both had prevalences of 62.5 %. Ostertagia ostertagi was found to have very high frequencies
(overall mean frequency =62.6 %; range =28.3–100 %) of the 200Y (TTC >TAC) polymorphism in all age
classes sampled. Overall, benzimidazole resistance polymorphisms were found at moderate to high frequency in
the three major economically important GIN species in ranched bison in Texas and Oklahoma, suggesting a
potential widespread distribution of benzimidazole resistance polymorphisms in the southern United States. This
work has important implications for all other grazing livestock and illustrates the importance of early detection
of anthelmintic resistance and the need for mitigation strategies.
1. Introduction
The North American bison (Bison bison) is a keystone species across
grassland ecosystems of North America (Knapp et al., 1999) and has
become a unique and high-valued livestock species in the United States
and Canada. The popularity of bison in both husbandry and consump-
tion has led to more ranchers owning and raising bison for meat pro-
duction to meet consumer demand (Bison Producers’ Handbook, 2015;
National Bison Association, 2019). It is estimated that over 500,000
bison are farmed in North America, with a large majority owned and
managed on private ranches (Bison Producers’ Handbook, 2015; Na-
tional Bison Association, 2021). Though often thought of as being
mainly found across the northern plains, many bison ranches are found
in the southern United States, including Texas and Oklahoma. In fact,
the 2022 Agriculture Census estimated a total of 8187 bison across 298
ranches in Texas and 7137 bison across 77 ranches in Oklahoma (USDA:
Animal and Plant Health Inspection Service, 2019). Although many of
these ranched bison are on smaller hobby farms, there are many
* Corresponding author. Department of Veterinary Pathobiology, School 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:
Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
https://doi.org/10.1016/j.ijpddr.2025.100594
Received 9 January 2025; Received in revised form 11 April 2025; Accepted 14 April 2025
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
Available online 14 April 2025
2211-3207/© 2025 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY license
( http://creativecommons.org/licenses/by/4.0/ ).
large-scale production ranches whose goal is to produce marketable
animals for consumer consumption. Despite not being domesticated,
bison ranching has followed many of the same management strategies
used in cattle production (Bison Producers’ Handbook, 2015; USDA:
Animal and Plant Health Inspection Service, 2019). These bison can be
raised in smaller, over-grazed, and over-stocked pastures leading to
higher parasitic burdens (USDA: Animal and Plant Health Inspection
Service, 2019). With this increase in parasitism throughout herds, pro-
ducers have become more reliant on anthelmintics to control these
production-limiting parasitic nematodes.
Gastrointestinal nematodes (GIN) within the order Strongylida often
exist in complex communities within a host and can vary in their level of
pathogenicity, drug sensitivity, and production effect (Avramenko et al.,
2017; Charlier et al., 2022). These GIN, specically those of the tri-
chostrongylid group, are a major cause of disease and production loss in
livestock species (Corwin, 1997; Fleming et al., 2006; Sutherland and
Leathwick, 2011; Kaplan, 2020), including bison (Frick, 1951; Wade
et al., 1979; Woodbury et al., 2014; Avramenko et al., 2018; Chelladurai
et al., 2024). Anecdotal evidence and clinical cases suggest that bison
herds often report GIN parasite infection intensities which can lead to
more signicant clinical signs and production loss compared to cattle
(Frick, 1951; Wade et al., 1979; Woodbury et al., 2014; Avramenko
et al., 2018). Though parasitism of GIN often causes subclinical disease,
clinical disease, and mortality has been reported in ranched bison,
causing greater economic losses within the industry (Frick, 1951; Wade
et al., 1979; Tessaro, 1989; Eljaki et al., 2016; Avramenko et al., 2018).
Unfortunately, the control of GIN in grazing livestock can be chal-
lenging as regions of the country experience different obstacles based on
climate, geographical distributions, and management strategies.
Regional parasite diversity differences are evident in cattle throughout
North America and are associated with climatic conditions (Stromberg
et al., 2015; Hildreth and McKenzie, 2020; Navarre, 2020; De Seram
et al., 2022). While information is available on parasites of bison in
central and northern US (Frick, 1951; Locker, 1953; Boddicker and
Hugghins, 1969; Wade et al., 1979; Eljaki et al., 2016; Chelladurai et al.,
2024) as well as Canada (Tessaro, 1989; Dies and Coupland, 2001;
Woodbury et al., 2014; Avramenko et al., 2018), knowledge on bison
parasites across the southern United States is largely unstudied. How-
ever, in the southern states, warmer climates create favorable conditions
for GIN transmission, thus, parasite transmission seasons are often
longer in the southern states compared to northern states and Canada
(Williams and Loyacano, 2001; Hildreth and McKenzie, 2020; Navarre,
2020). These climatic differences necessitate different parasite man-
agement strategies, including anthelmintic administration timing and
different refugia management approaches (Morgan and Van Dijk, 2012;
Hildreth and McKenzie, 2020; Navarre, 2020).
Studies from Canada and the northern United States show that bison
share most of the common trichostrongylid GIN with cattle and small
ruminants including species within the Haemonchus, Ostertagia, Coop-
eria, and Trichostrongylus genera (Tessaro, 1989; Van Vureni and Scott,
1995; Marley et al., 1995; Dies and Coupland, 2001; Woodbury et al.,
2014; Eljaki et al., 2016; Avramenko et al., 2017). These trichostrongyle
nematodes produce eggs that are morphologically indistinguishable
making their identication virtually impossible at this stage. Feces can
be cultured to allow trichostrongyle eggs to hatch and develop into their
third-stage larvae (L3) allowing morphological identication to the
genus level (Verocai et al., 2020; Van Wyk and Mayhew, 2013). How-
ever, this technique is very specialized, time-consuming, requires
intensive training, and can often lead to false identication. These
challenges have led to the development of a powerful molecular
approach, referred to as “nemabiome” metabarcoding, which allows for
species-level determination of GIN in individual samples through
deep-amplicon sequencing of the ITS-2 nuclear ribosomal DNA locus
(Avramenko et al., 2015).
Another largely understudied aspect is the level of anthelmintic
resistance in the GIN in grazing ruminants, especially bison, in the
southern United States. With all grazing ruminants harboring some level
of parasitism, many livestock producers heavily rely on anthelmintics to
control GIN burdens and maximize production. Overreliance and misuse
of these drugs apply selective pressure, which is a potential risk factor
for the emergence of anthelmintic resistance. While these impacts are
relatively well-studied in cattle and small ruminants (Fleming et al.,
2006; Sutherland and Leathwick, 2011; Gasbarre, 2014; Avramenko
et al., 2019, 2020; Kaplan, 2020), information regarding the anthel-
mintic resistance in internal parasites in ranched bison is relatively
scarce (Woodbury and Lewis, 2011; Avramenko et al., 2020). The
emergence of anthelmintic resistance further impacts livestock pro-
duction, causing economic losses to individual producers and the overall
industry (Eljaki et al., 2016; Kaplan, 2020). Determining resistance is
classically based on the fecal egg count reduction test, which can be
difcult to perform when working with undomesticated animals. It is
also an insensitive technique. Consequently, by the time resistance has
been identied within a herd, it could be too late for mitigation strate-
gies. Molecular testing has the ability to identify anthelmintic resistance
early and with only one sampling time. Currently, the most
well-characterized anthelmintic resistant mechanism is for the benz-
imidazole drug class (Gilleard, 2006; Avramenko et al., 2019; Kaplan,
2020). Avramenko et al. (2019) developed a high-throughput molecular
assay to screen for polymorphisms (200Y [TTC >TAC], 167Y [TTC >
TAC], 198L [GAA >TTA], and 198A [GAA >GCA]) in the isotype-1
β-tubulin gene that is associated with resistance to benzimidazoles in
various GIN of sheep and later, cattle and bison (Avramenko et al., 2019,
2020). While resistance is relatively common and widespread in
abomasal nematodes of cattle from the US (Gasbarre et al., 2009;
Edmonds et al., 2010; Sutherland and Leathwick, 2011; Gasbarre, 2014;
Avramenko et al., 2020), there are very few reports that specically look
at bison. When examining Canadian bison herds Ostertagia ostertagi
benzimidazole resistance alleles were found at low frequency in a few
herds (7/51) (Avramenko et al., 2020).
As well as anthelmintic resistance status, the overall parasite di-
versity remains to be explored in bison in the southern United States,
where management practices, climate, and parasite species composition
differ from the northern United States and Canada. In this paper, we
apply nemabiome ITS2 metabarcoding and screen for benzimidazole
resistance polymorphisms in the isotype-1 β-tubulin gene to investigate
the species composition of parasite communities and the frequency level
of benzimidazole resistance in composite bison herds in Texas and
Oklahoma as well as individual bison of three different age classes
(calves, yearlings, and mature) from a single ranch in east-central Texas.
This is one of the rst in-depth papers assessing the species composition
and level of resistance in GIN of bison from the southern United States,
and the rst to assess bison parasites in Texas, using next-generation
sequencing techniques.
2. Materials and methods
2.1. Collection of fecal samples
2.1.1. Archival composite samples
Samples were collected from bison herds in Texas (n =14) and
Oklahoma (n =2) from 2018 to 2022 (Fig. 1). Many of the sampled
herds encompass north and central Texas, characterized by a humid
subtropical climate with mild winters and hot summers. Other areas of
sample collection in Texas include the Panhandle, which is a semi-arid
climate with hot summers and cold winters (ClimateDataTexas.org).
The two samples from Oklahoma were from central and northeastern
regions and are characterized as having a subtropical climate with mild
winters with the northern regions having a colder winter compared to
the south (ClimateDataOklahoma.org). The two Oklahoma herds and
four of the Texas herds were sampled at multiple time points giving the
total number of samples selected to be 11 and 22, respectively. Herd
composites comprised an average of 10 individual bison in Texas (range
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
2
=5–21) and 16 in Oklahoma (range =7–20). Samples were sent either
in the Spring (March–May; 15.6 %), Summer (June–August; 28.1 %),
Fall (September–November; 43.8 %), or Winter (December–February;
12.5 %). Individual fecal samples were collected either per rectum by
producers in a chute or from the ground after visual conrmation and
animal identication, placed in plastic bags devoid of air, and shipped
on ice to the Texas A&M University Parasitology Diagnostic Laboratory
for general routine diagnostic analysis. Because of the highly variable
management practices among herds, it is not possible to give a full
description of each. However, each herd was given a routine anthel-
mintic to maintain production. Fecal egg counts (FEC) were determined
using a modied Wisconsin technique with Sheather’s sucrose oatation
solution (specic gravity: 1.26). For each FEC 5 g of feces were used to
give a theoretical detection threshold of 0.2 eggs per gram (EPG) of
feces. The remaining fecal samples for each herd were then pooled
together by age class (e.g., young [<3 years], mature [>3 years]) and
cultured for 14 days at room temperature (~20 ◦C) to hatch and develop
the trichostongyle nematodes. Larvae were then estimated and ali-
quoted into 1000 larvae per herd if possible, however in some cases
fewer than 1000 larvae were collected (range =200–1000). Larvae were
xed in 70 % ethanol and stored at −80 ◦C until further analysis.
2.1.2. Samples to assess GIN species composition among different age
classes
To assess GIN species composition differences associated with age
class, individual fecal samples were rectally collected from bison (n =
43) from a ranch located in the Brazos Country, central Texas (30◦38
′
5
″
N, 96◦20
′
31
″
W) in April of 2022. Bison samples were classied ac-
cording to their reported age classes: calves (n =14; <1 year), yearlings
(n =3; 1-2 year-old), and mature (n =26; >3 years). This herd was
selected due to the known over reliance of albendazole which has been
administered twice each year for the last +5 years, in combination with
an injectable macrocyclic lactone. It should be noted that this herd ro-
tates pastures but does not co-graze with cattle. The bison rotationally
graze on four pastures that average 40 acres and maintain a relatively
low stocking rate. The continued use of albendazole within the herd is
largely due to the known presence of infection by liver uke, Fasciola
hepatica. Fecal egg counts were determined using the Mini-FLOTAC
technique with sodium nitrate solution (FECA-MED, VDCO; specic
gravity =1.25–1.30), and a detection level of 5 EPG (Cringoli et al.,
2017; Paras et al., 2018). The remaining fecal samples were individually
cultured for 14 days for the collection of trichostrongylid L3 using the
same protocol described above. The number of L3 obtained from each
individual coproculture was estimated and aliquoted into 1000 L3 per
herd, if possible (range =100–1000), xed in 70 % ethanol, and stored
at −80 ◦C until further analysis.
2.2. Molecular analysis
2.2.1. First round PCR for ITS-2 rDNA nemabiome metabarcoding
The species composition of GIN in each herd from both Texas and
Oklahoma were determined using ITS-2 nemabiome metabarcoding of
L3 larval populations using the Illumina Miseq platform (Avramenko
et al., 2015). For the preparation of genomic DNA, ethanol-xed larvae
were washed in lysis buffer (50 mM KCl, 10 mM Tris (pH 8.3), 2.5 mM
MgCl
2
, 0.45 % Nonidet P-40, 0.45 % Tween 20, 0.01 % (w/v) gelatin)
three times by centrifugation before using the QIAamp PowerFecal Pro
DNA kit for DNA extraction. After DNA extraction, the ITS-2 rDNA
amplicon was PCR-amplied using previously published primers that
consisted of four forward (NC1Adp, NC1Adp1N, NC1adp2N,
NC1Adp3N) and four reverse (NC2Adp, NC2Adp1N, NC2Adp2N,
NC2Adp3N) primers in equal proportions (Avramenko et al., 2015). The
high-delity polymerase KAPA HiFi HotStart (KAPA Biosystems, USA)
was used to minimize PCR error rates. The PCR consisted of 5
μ
L x5
Buffer, 15.15
μ
L of ddH
2
O, 0.75
μ
L dNTPs (10 mM), 0.75
μ
L
NC1+Adapter Primer (10 mM), 0.75
μ
L NC2+Adapter Primer (10 mM),
0.1
μ
L BSA, and 2
μ
L of the DNA template. The thermocycling param-
eters for the PCR were 95 ◦C for 2 min, followed by 35 cycles of 98 ◦C for
Fig. 1. (A) Geographical distribution of commercial bison ranches in various counties, highlighted in red, in Texas and Oklahoma that sent in fecal samples. (B)
Geographical location of Texas and Oklahoma in reference to the United States.
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
3
20 s, 62 ◦C for 15 s, 72 ◦C for 15 s, followed by the nal extension of
72 ◦C for 2 min. Following amplication, PCR products were puried
using AMPure XP magnetic beads (ratio 1:1; Beckman Coulter, Inc.),
following the manufacturer’s recommended protocol.
2.2.2. First round PCR for Isotype-1 β-tubulin deep-amplicon sequencing
The same samples were also screened for the presence and frequency
of three independent SNP in the isotype-1 β-tubulin gene that are
associated with benzimidazole resistance, namely 200Y (TTC >TAC),
167Y (TTC >TAC), 198L (GAA >TTA), and 198A (GAA >GCA)
(Avramenko et al., 2020). The isotype-1 β-tubulin gene was
PCR-amplied using previously reported primer sets which included a
mix of 7 forward primers and 11 reverse primers (Avramenko et al.,
2019; Supplemental Table 1) and the Q5 HotStart polymerase (New
England Biolabs). The PCR consisted of 5
μ
L x5 Buffer, 11.65
μ
L of
ddH
2
O, 0.5
μ
L dNTPs (10 mM), 1.25
μ
L Forward Primers (10
μ
M), 0.75
μ
L Reverse Primers (10
μ
M), 0.25
μ
L Q5 HotStart Polymerase (0.5 U),
0.1
μ
L BSA, and 5
μ
L of the DNA template. The thermocycling conditions
for the PCR were 98 ◦C for 2 min, followed by 40 cycles of 98 ◦C for 10 s,
60 ◦C for 15 s, 72 ◦C for 25 s followed by the nal extension of 72 ◦C for
5 min. The PCR product was then puried with AMPure XP Magnetic
Beads (ratio 1:1).
2.2.3. Illumina library preparation
For both ITS2 rDNA and isotype-1 β-tubulin bead-puried ampli-
cons, a second limited cycle PCR amplication was completed for the
addition of Illumina indices and P5/P7 sequencing tags. All samples
were barcoded with Illuminas’ Unique Dual Indexes (UDI). This consists
of the addition of 384 unique 12bp indexes to both anks of the
amplicon. The second PCR conditions were as follows: 5
μ
L KAPA HiFi
HotStart Fidelity Buffer (5X), 1.25
μ
L Forward Primers (10
μ
M), 1.25
μ
L
Reverse Primers (10
μ
M), 0.75
μ
L dNTPs (10 mM), 0.5
μ
L KAPA HiFi
Polymerase (0.5 U), 8.75
μ
L H
2
O, and 5
μ
L of the rst round PCR product
as the template. The thermocycling conditions for the second PCR were
95 ◦C for 45 s followed by 11 cycles of 98 ◦C for 20 s, 63 ◦C for 20 s, and
72 ◦C for 2 min. Products were puried using AMPure XP Magnetic
Beads (1X). Puried products were then pooled (~50 ng) to create a
normalized sequencing library and quantied with the KAPA qPCR Li-
brary Quantication Kit (KAPA Biosytems, USA) following the manu-
facturer’s recommended protocol. An Illumina MiSeq Desktop
Sequencer (Illumina Inc., San Diego, CA, USA) using a 500-cycle pair-
end reagent kit (MiSeq Reagent Kits v2, MS-103-2003) with the addi-
tion of a 25 % PhiX Control v3 (Illumina, FC-110-3001) was used to
sequence the prepared pooled library at a concentration of 12.5 nM. An
average read depth of 11,144 ±4107 (range =4230–21,878) was ob-
tained for each sample.
2.3. Bioinformatic and statistical analysis
2.3.1. Nemabiome ITS-2 bioinformatics
A bioinformatics pipeline based on the analysis package DADA2 R
version 1.14.0 (Callahan et al., 2016) was used to process the raw data
(described in detail, https://www.nemabiome.ca). In brief, raw data is
demultiplexed, barcode indices are removed and fastq les for each
sample are generated. The pipeline uses the program Cutadapt version
2.8 (Marcel, 2011) to remove primers and lter the reads based on size
(>200bp) and quality (utilizing the lterAndTrim function), discarding
reads with a maximum of two expected errors in the forward read or ve
in the reverse read. Sequencing errors are removed using a dynamic
error learning approach called “denoising” and the forward and reverse
reads are then merged. Possible chimeric sequences are identied and
removed from the dataset. The resulting Amplied Sequence Variants
(ASV) are classied using IDTaxa (Murali et al., 2018) at a condence
threshold of 60 % against the nematode ITS2 rDNA database 1.1.0
(Workentine et al., 2020). The ASVs were processed further to remove
singletons, potential contaminants, and samples with less than 1000
reads and then converted into species proportions based on the total
number of reads per sample.
2.3.2. Isotype-1 beta tubulin bioinformatics
A bioinformatics pipeline based on the analysis package DADA2 R
version 1.14.0 (Callahan et al., 2016) was used to process the raw data in
a similar approach as the ITS-2 pipeline; the reads were ltered, trim-
med, denoised, and merged using default settings. The resulting ASVs
were aligned against reference sequences (Avramenko et al., 2019)
using a global (Needleman-Wunsch) pairwise alignment algorithm
without end gap penalties. Following alignment, the ASVs were dis-
carded if they were <180 bp or >350 bp long, or if they had a percentage
identity <70 % to any of the reference sequences in the database. The
ASV was also removed if it had fewer than 200 reads in a sample and if
the ASV was only present in a single sample. Additionally, samples with
<1000 reads per sample were excluded from further analysis. The co-
dons 167, 198, and 200 were then analyzed for the presence of any
variants resulting in non-synonymous changes. The frequency of each of
the resistance polymorphisms for each species was estimated as a pro-
portion of reads with the resistance polymorphisms out of the total
number of mapped reads for a given species.
2.3.3. Statistical analysis of variables
The resulting data from the nemabiome was statistically analyzed
using the Statistical Analysis System (SAS; Version 9.4, SAS Institute,
Cary, North Carolina, USA) to determine the prevalence for each GIN
species and the three known benzimidazole resistance markers across
herds. To assess the effect of age differences on FEC and sequence
polymorphism frequencies a generalized linear mixed model (GLMM)
was determined using the PROC GLIMMIX procedure in SAS. In this
model age group, sex, year, and state were xed effects, and individuals
or herds were the random effect to account for repeated measures at
multiple time points. Because EPG data is skewed, a gamma distribution
with a log link function was specied within the model. Other descrip-
tive statistics were also determined including mean, standard deviation,
and standard error. Alpha diversity was calculated for each age class in
the central Texas herd using the inverse Simpson index.
3. Results
3.1. Fecal egg counts of GIN in archival composite samples
Fecal egg counts were performed on individual animals submitted
from each herd for diagnostic purposes. The overall EPG mean for tri-
chostrongyles in Texas (n =14) and Oklahoma (n =2) herds was 179.1
±59.7 EPG (range =17.1–997.7) and 137.4 ±32.2 EPG (range =
54.4–350.5), respectively (Fig. 2). There were three herds analyzed
(Texas =2; Oklahoma =1) that had submitted samples representing
different age groups, including Herd A which had different age groups
over multiple time points (Fig. 2). Considering these three herds overall
we can observe the differences in EPGs between mature (>3 years; 93.1
±39.2 EPG) and younger bison (<3 years; 419.6 ±125.6 EPG). Other
helminths and protozoa were also recorded and summarized in Sup-
plementary Table 2 by herd and state.
3.2. Species composition of GIN in archival composite samples
We used ITS-2 nemabiome metabarcoding, to determine the species
composition of GIN for each herd composite in Texas and Oklahoma.
The most predominant GIN species found in Texas overall were Hae-
monchus contortus (11/14 herds; mean (±SE) relative abundance =42.2
±7.7 %), Ostertagia ostertagi (13/14 herds, mean relative abundance =
28.4 ±6.4 %), and Haemonchus placei (11/14 herds, mean relative
abundance =25.6 ±7.8 %; Table 1, Fig. 2). Other GIN were also pre-
sent, though in lower relative abundances, throughout Texas herds
including four Cooperia species, Trichostrongylus axei, and
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
4
Oesophagostomum radiatum (Table 1, Fig. 2). However, the distribution
of these GIN species was variable between ranches, with some species
detected only in one (Cooperia spatulata and T. axei) or two (Cooperia
pectinata and O. radiatum) herds (Fig. 2). Despite H. placei being one of
the most predominant GIN species found overall in Texas, higher levels
(>60 %) were only found in 5 of the 16 herds, with most of the herds in
Texas having lower proportions of H. placei, compared to H. contortus
(Fig. 2).
In Oklahoma, H. placei (2/2 herds; mean (±SE) relative abundance =
82 ±3.7 %) was the most abundant in both herds (A =83.9 ±4.2 %; B
=73.2 ±4.0 %), time points, and age groups (herd A: yearling =74.3 ±
4.4 %; mature =91.7 ±4.3 %) (Fig. 2). Cooperia punctata (2/2 herds;
mean relative abundance =10.5 ±3.2 %) and O. ostertagi (2/2 herds;
mean relative abundance =7.1 ±3.1 %) were also present, yet in much
smaller proportions (Table 1). Haemonchus contortus, C. spatulata,
Cooperia oncophora, and O. radiatum comprised an average of <1 % of
the parasite community in Oklahoma herds (Table 1, Fig. 2). In fact,
H. contortus, C. spatulata, and C. oncophora were only present in a single
herd (i.e., herd A) (Fig. 2).
3.3. Benzimidazole resistance polymorphisms in GIN in archival
composite samples
Benzimidazole resistance was determined by screening the trichos-
trongylid L3 for isotype-1 β-tubulin single nucleotide polymorphisms
(SNPs) known to be associated with benzimidazole resistance at codons
200, 198, and 167 using deep amplicon sequencing. There was an
overall high mean (±SE) frequency of the 200Y (TTC >TAC) poly-
morphism in O. ostertagi (mean frequency =71.7 ±3.1 %) and
H. contortus (mean frequency =70.6 ±5.5 %) in almost every Texas
bison herd, and low to moderate levels in H. placei (mean frequency =
20.6 ±6.8 %) (Fig. 3; Table 2). The 167Y (TTC >TAC) polymorphism
was also detected in H. contortus and O. ostertagi, with an overall mean
frequency of 12.2 ±2.0 % and 3.2 ±0.8 %, respectively. Lastly, the
198L (GAA >TTA) polymorphism was only detected in O. ostertagi and
had a mean frequency of 23.0 ±3.9 % (Fig. 3; Table 2). Using the data
available in the Texas herds, there was a signicant difference (P =
0.007) in the overall 200Y (TTC >TAC) polymorphism frequency in
O. ostertagi over the years (2019–2022) with a gradual increase (2019 =
36 %; 2020 =70 %; 2021 =60 %; 2022 =83 %).
The 200Y (TTC >TAC) resistance polymorphism was also the most
predominant resistant polymorphism found in the Oklahoma samples,
Fig. 2. Infection intensities and species composition of parasitic gastrointestinal nematodes from commercial bison herds in Texas (n =14) and Oklahoma (n =2) are
shown. Texas herds are shown as herds 1–14 and Oklahoma herds are depicted as A and B. Herds that repeat in number or letter are across multiple time points. The
date of each time point is represented at the top of the gure. The upper portion of the chart shows the mean fecal egg counts (FEC) of trichostrongyles in each bison
herd across Texas and Oklahoma. The lower portion of the chart shows the relative proportion of each parasite species present as determined by the nemabiome
sequencing. Each color represents a different trichostrongyle as indicated by the legend. Age classes (Mature =>3 years; Young =<3 years) within herds are
characterized by their afliated shape at the bottom of the gure and are indicated in the legend.
Table 1
Average relative abundance of individual parasite species detected from ITS-2
rDNA nemabiome metabarcoding from Texas (14 herds) and Oklahoma (2
herds).
Species Texas Oklahoma
Mean (%) ±SE Mean (%) ±SE
Haemonchus placei 25.6 ±7.8 82.0 ±3.7
Haemonchus contortus 42.2 ±7.7 0.1 ±0.1
Cooperia punctata 3.1 ±1.4 10.5 ±3.2
Cooperia spatulata 0.1 ±0.1 0.3 ±0.2
Cooperia pectinata 0.2 ±0.2 0.0 ±0.0
Cooperia oncophora 0.2 ±0.1 0.002 ±0.002
Ostertagia ostertagi 28.4 ±6.4 7.1 ±3.1
Trichostrongylus axei 0.1 ±0.1 0.0 ±0.00
Oesophagostomum radiatum 0.1 ±0.1 0.1 ±0.1
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
5
though at a low frequency. The 200Y (TTC >TAC) polymorphisms in
H. placei was detected in both herds (A =6.03 %; B =77.9 %), across all
time points (Fig. 3.), and in both age classes (herd A: yearlings [ylg] =
5.4 %; mature =6.5 %). Though only found in low frequencies the
frequency of the 200Y polymorphism, in both age classes in herd A, were
observed to slowly increase over the months and years as follows: May
2019 (ylg =2.9 %; mature =4.2 %), July 2019 (ylg =5.4 %; mature =
6.7 %), October 2019 (ylg =6.2 %; mature =6.8 %), and June 2020
(ylg =7.3 %; mature =8.2 %). Ostertagia ostertagi in herd B were found
to have both the 200Y (TTC >TAC) (34.6 %) and 198L (GAA >TTA)
Fig. 3. Benzimidazole resistance mutations in Texas and Oklahoma commercial bison herds. The allele frequency at codons 200, 198, and 167 of the β-tubulin
isotype-1 gene is shown for Haemonchus contortus (A), Haemonchus placei (B), and Ostertagia ostertagi (C) derived from 16 composite herds samples from Texas (14)
and Oklahoma (2) across multiple time points, as determined by deep-amplicon sequencing. Susceptible alleles are displayed in blue, while documented resistance
alleles 200Y (TTC >TAC), 167Y (TTC >TAC), and 198L (GAA >TTA) are represented in the legend as red, blue, and green, respectively. Blank bars indicate that the
species was either not present in the sample, or there were too few sequences (<200) assigned to the species to assess the allele frequency.
Table 2
200Y (TTC >TAC), 167Y (TTC >TAC), and 198L (GAA >TTA) resistant polymorphism frequency in Texas and Oklahoma commercial bison herds.
Polymorphism Species # Herds # Herds SNP detected Range (%)
Species detected
Texas (14 herds) 200Y Haemonchus contortus 11 11 20.8–95.2
Haemonchus placei 11 9 3.5–67.9
Ostertagia ostertagi 13 9 46.8–87.3
167Y Haemonchus contortus 11 10 4.1–37.9
Haemonchus placei 11 0 N/A
Ostertagia ostertagi 13 4 1.4–5.6
198L Haemonchus contortus 11 0 N/A
Haemonchus placei 11 0 N/A
Ostertagia ostertagi 13 9 3.5–51.0
Oklahoma (2 herds) 200Y Haemonchus contortus 1 1 52.0
Haemonchus placei 2 2 2.85–78.2
Ostertagia ostertagi 2 1 20.0–49.3
167Y Haemonchus contortus 1 0 N/A
Haemonchus placei 2 0 N/A
Ostertagia ostertagi 2 0 N/A
198L Haemonchus contortus 1 0 N/A
Haemonchus placei 2 0 N/A
Ostertagia ostertagi 2 1 2.1
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
6
(2.1 %) polymorphisms n, though the latter at a much lower frequency.
Herd A was the only herd in Oklahoma that detected the 200Y (TTC >
TAC) codon in H. contortus (52 %), which was found in high frequency
only in yearlings from the last time point analyzed (June 2020) (Fig. 3).
3.4. Fecal egg counts of GIN in individual bison of different age classes
The average strongyle FEC in each age class was as follows; calves =
922.1 ±162.5 EPG (14/14; range =5–3385 EPG), yearlings =340.0 ±
325.0 EPG (3/3; range =20–975 EPG), and mature =70.8 ±110.4 EPG
(16/26; range =0–1020) (Fig. 4; S2. 3). There was an overall signicant
difference between the age groups with calves reporting higher EPG
compared to mature bison (P =0.0001). Other helminths and protozoa
were also recorded in the Supplementary Table 3 by age class.
3.5. Assessing age class species composition of GIN in the single east-
central Texas ranch
ITS-2 nemabiome metabarcoding was used to determine the species
composition of GIN for individual animals in each age class (Fig. 4).
There was a stark difference between the calves and mature bison (p <
.0001) with H. contortus being more predominant in calves (mean fre-
quency [±SE] =90.4 ±1.8 %), compared to yearlings (43.9 ±25.8 %)
and mature (18.6 ±5.3 %) (Table 3). However, O. ostertagi was the
predominant species in the mature bison (mean frequency =65.7 ±6.4
%) followed by yearlings (52.9 ±28.0 %; calves =4.5 ±0.95 %), with a
signicant difference (p <.0001) between mature and calves (Fig. 4;
Table 3). Other GIN species present within the herd were as follows, in
order of overall prevalence: H. placei (6.7 %), O. radiatum (0.29 %),
C. punctata (2.5 %), C. spatulata (1.7 %), C. oncophora (0.19 %), and
C. pectinata (0.04 %) (Table 3). The alpha diversities for each age class
were calculated using the inverse-Simpson index and were as follows:
calves (0.18), yearling (0.53), mature (0.52).
3.6. Benzimidazole resistance polymorphisms in GIN in the single east-
central Texas ranch
Benzimidazole resistance polymorphisms in the isotype-1 β-tubulin
gene at codons 200, 167, and 198 were screened using deep amplicon
sequencing. There was a very high mean frequency of the 200Y (TTC >
TAC) resistance polymorphism in H. contortus (59.3 %) in all age groups
(calves =83 %; yearling =59.8 %, mature =40.4 %) and O. ostertagi
(44.4 %; calves =61.9 %; yearlings =53.8 %; mature =64.1 %) (Fig. 5;
Table 4). The 200Y (TTC >TAC) resistance polymorphism was also
detected in low frequency in H. placei but was only detected in mature
bison (3.01 %) (Fig. 5; Table 4). The second most predominant allele was
the 198L (GAA >TTA) resistance polymorphism which, though was
Fig. 4. Infection intensities and species composition of parasitic gastrointestinal nematodes rectally collected from a single commercial bison herd in east-central
Texas, along with three different age classes (calves, yearlings, and mature), are shown. Age classes within herds are characterized by their afliated shape at
the bottom of the gure and are indicated in the legend. The upper portion of the chart shows the mean fecal egg counts (FEC) of trichostrongyles in each individual
bison within the central Texas ranch. The lower portion of the chart shows the relative proportion of each parasite species present as determined by the nemabiome
sequencing. Each color represents a different trichostrongyle as indicated by the legend.
Table 3
Average relative abundance of individual parasite species detected from ITS-2
rDNA nemabiome metabarcoding across different age classes from a single
commercial bison herd from east-central Texas.
Species Calves Yearlings Mature
Mean (%) ±SE Mean (%) ±SE Mean (%) ±SE
Haemonchus placei 1.70 ±0.35
a
2.0 ±2.0
a
9.87 ±2.59
a
Haemonchus contortus 90.36 ±1.83
a
43.88 ±25.75
ab
18.61 ±5.34
b
Cooperia punctata 2.89 ±0.99
a
1.22 ±0.54
a
2.52 ±1.03
a
Cooperia spatulata 0.07 ±0.07
a
0.00 ±0.00
a
2.73 ±1.75
a
Cooperia pectinata 0.00 ±0.00
a
0.00 ±0.00
a
0.06 ±0.05
a
Cooperia oncophora 0.39 ±0.25
a
0.00 ±0.00
a
0.11 ±0.07
a
Ostertagia ostertagi 4.46 ±0.95
a
52.90 ±28.03
ab
65.69 ±6.35
b
Oesophagostomum
radiatum
0.13 ±0.06
a
0.00 ±0.00
a
0.41 ±0.32
a
a,b
within a row, means without a common superscript differ (P <.0001).
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
7
only detected in O. ostertagi, had a moderately high mean frequency of
32.7 % (calves =38.15 %; yearlings =42.3 %; mature =28.8 %),
compared to the 198A allele (1 %), which was also found in both 1-2
year-olds (0.3 %) and mature (1.1 %) but in low frequencies (Fig. 5;
Table 4). Lastly, the 167Y (TTC >TAC) resistance polymorphism was
detected in H. contortus (5.1 %) in all three age classes (calves =6.8 %;
yearlings =2 %; mature =4.6 %), while 167Y (TTC >TAC) in
O. ostertagi was only detected mature bison (1.2 %) (Fig. 5; Table 4).
4. Discussion
Using metabarcoding and deep amplicon sequencing approaches, we
were able to look at “snapshots” of 16 different ranched bison herds from
two southern states in the United States, Texas and Oklahoma, from
2018 to 2021 to determine the species composition and screen for
benzimidazole resistance polymorphisms. We investigated the species
diversity and benzimidazole resistance in cohorts of individuals over
different time points and, in some cases, between age classes for a subset
of herds. Management strategies between the different ranches vary
greatly with little to no prior knowledge of anthelmintic history. How-
ever, the common use of albendazole as an oral drench as well as the
ease of giving fenbendazole range cubes, suggested there would be
potentially a high selection pressure for benzimidazole resistance in
many of the herds.
All nine GIN species found in the sampled bison are primarily those
commonly associated with cattle and/or small ruminants. Our ndings
are similar to previous studies in ranched bison from Canada, and the
northern United States (Avramenko et al., 2018; Eljaki et al., 2016;
Avramenko et al., 2020). Nemabiome metabarcoding found the pre-
dominant GIN parasite species in Texas herds to be H. contortus, H.
placei, and O. ostertagi. The higher prevalence and relative abundance of
H. contortus, a parasite most often associated with small ruminants, in
Texas bison contrast with what has been previously reported in other
bison herds where H. placei, associated with cattle, was predominant
(Avramenko et al., 2018, 2020). However, Texas is number one in the
United States for the total number of sheep and goats produced (Sheep
Production-2024.pdf; Cooperative Extension, 2019). Though pro-
ducers of sheep and goats are often localized in the Edwards Plateau
region of west-central Texas, there are many small ruminant herds
throughout Texas. The two Oklahoma samples, including every time
point in herd A, were H. placei with few Cooperia species. and O. ostertagi.
A much lower number of herds were sampled in Oklahoma (n =2) than
in Texas (n =14) and should be acknowledged that associations be-
tween states were not the goal of this current study, rather we aimed to
report descriptive ndings of herds that have never been reported pre-
viously. Although it was not unexpected to nd Haemonchus spp. and
O. ostertagi in southern regions of the United States, like Texas and
Oklahoma, many herds sampled had high percentages of these
production-limiting parasites. Previous studies conducted in the north-
ern United States and Canada found Cooperia spp. to be the most
abundant trichostrongylid nematode overall in bison herds, followed by
either Trichostrongylus (Dies and Coupland, 2001) or O. ostertagi
(Avramenko et al., 2018). However, Haemonchus spp. is known to be
more adaptable to warmer weather and is often found in higher preva-
lence in subtropical and temperate climates, like found in the southern
United States, compared to the northern United States and Canada
(Terrill et al., 2012; Gasser and von Samson-Himmelstjerna, 2016;
Charlier et al., 2022). These ndings are of importance due to the
pathogenicity of both Haemonchus spp. and O. ostertagi in all grazing
ruminants (Terrill et al., 2012), including bison (Cameron, 1923; Frick,
Fig. 5. Benzimidazole resistance mutations in individual bison separated by age class (Calves, Yearlings, and Mature) from a single east-central Texas commercial
bison ranch. The allele frequency at codons 200, 198, and 167 of the β-tubulin isotype-1 gene is shown for Haemonchus contortus (A), Haemonchus placei (B), and
Ostertagia ostertagi (C) as determined by deep-amplicon sequencing. Susceptible alleles are displayed in blue, while documented resistance alleles 200Y (TTC >TAC),
167Y (TTC >TAC), 198L (GAA >TTA), and 198A (GAA >GCA) are represented in the legend as red, blue, green, and orange respectively. Blank bars indicate that
the species was either not present in the sample, or there were too few sequences (<200) assigned to the species to assess the allele frequency.
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
8
1951; Wade et al., 1979; Tessaro, 1989).
Selection pressure on GIN from anthelmintic drug use will be a risk
factor for the emergence of anthelmintic resistance in any livestock
species, including bison. We screened each compositely sampled bison
herd for the presence of different benzimidazole drug resistance poly-
morphisms in the isotype-1 β-tubulin gene in 200Y (TTC >TAC), 167Y
(TTC >TAC), 198A (GAA >GCA), and 198L (GAA >TTA). Each of these
polymorphisms has been previously identied in one or more trichos-
trongylid nematodes in small ruminants (Redman et al., 2015; Chagas
et al., 2016; Avramenko et al., 2019), cattle (Chaudhry et al., 2014;
Avramenko et al., 2020), and bison (Avramenko et al., 2020). However,
resistance levels in ranched bison in the southern United States have
never been investigated. Avramenko et al. (2020) did look at cattle herds
in the central and southern United States, which included cattle from
Oklahoma. The 200Y (TTC >TAC) polymorphism was detected in
H. placei (frequency range =0.57–27.45 %) and O. ostertagi (frequency
range =0.29–12.03 %), which is similar to what was found in the
present study, where the 200Y (TTC >TAC) substitution was the most
prevalent in H. contortus (frequency range =5.78–95.24 %), H. placei
(frequency range =2.85–78.18 %), and O. ostertagi (frequency range =
20.02–87.33 %), both in Texas and Oklahoma. Although this is consis-
tent with previous studies, resistance levels were much lower in fre-
quency overall (15.2 %) between herds (Avramenko et al., 2020).
However, for the herds in Texas, there were high frequencies of the 200Y
(TTC >TAC) polymorphism in H. contortus (mean frequency [±SE] 57.7
±7.5 %) and O. ostertagi (mean frequency 52.1 ±7.3 %). Detecting the
200Y (TTC >TAC) polymorphism in such high levels in highly patho-
genic and economically important parasites should be of great concern
to not just the bison industry, but all grazing production animals. The
frequency levels in the polymorphisms 198L (GAA >TTA) and 167Y
(TTC >TAC) were considerably lower than the 200Y (TTC >TAC)
polymorphism in herds from Oklahoma, as seen in Fig. 3. The 167Y
(TTC >TAC) codon in H. contortus was the only other polymorphism
detected. However, in O. ostertagi, codons 198L (GAA >TTA) and 167Y
(TTC >TAC) were also found, but at a lower frequency. Despite their
relatively lower frequency levels, 198L (GAA >TTA) was found in more
than half of the bison herds in Texas. This is similar to what has previ-
ously been reported where the 200Y (TTC >TAC) polymorphism is more
predominant compared to 198L (GAA >TTA), 198A (GAA >GCA), and
167 (TTC >TAC) in various livestock parasite species (Garg and Yadav,
2009; Gilleard, 2006, Avramenko et al., 2019; Avramenko et al., 2020).
Using the same molecular methods described previously for the
composite archival samples from Texas and Oklahoma, we also inves-
tigated species composition and the presence of BZ resistance poly-
morphisms in GIN of individual bison within a single herd in east-central
Texas. This provided the ability to look at individuals in more detail
within three different age classes co-grazing in the same pastures.
Though there was a lack of yearling data due to the limited sampling,
there was a striking difference between the calves and the older bison
with H. contortus being signicantly higher in the younger animals (p <
.0001) but O. ostertagi predominating in the older animals (p <.0001)
(Fig. 2; Table 3). Though this is the rst paper to look at the GIN species
composition in different age classes in bison herds, others have observed
age-related differences in cattle. It has been reported that calves often
will have a higher abundance of Haemonchus spp., and as they mature
O. ostertagi will become more prominent. This is thought to be due to
age-related acquired immunity and species competition (Charlier et al.,
2022). This age-related species difference is of great importance due to
Table 4
200Y (TTC >TAC), 167Y (TTC >TAC), 198L (GAA >TTA), 198A (GAA >GCA)
resistant mutation frequency in three age classes from a single commercial bion
herd from east-central Texas.
Polymorphism Species # Herds # Herds
SNP
detected
Range
(%)
Species
detected
Calves (n
=14)
200Y Haemonchus
contortus
14 13 76.9–88.0
Haemonchus
placei
12 0 N/A
Ostertagia
ostertagi
13 13 28.3–100
167Y Haemonchus
contortus
14 13 5.3–7.9
Haemonchus
placei
12 0 N/A
Ostertagia
ostertagi
13 0 N/A
198L Haemonchus
contortus
14 0 N/A
Haemonchus
placei
12 0 N/A
Ostertagia
ostertagi
13 11 14.6–71.7
198A Haemonchus
contortus
14 0 N/A
Haemonchus
placei
12 0 N/A
Ostertagia
ostertagi
13 0 N/A
Yearlings
(n =3)
200Y Haemonchus
contortus
2 2 82.7–96.8
Haemonchus
placei
1 0 N/A
Ostertagia
ostertagi
3 3 28.9–64.9
167Y Haemonchus
contortus
2 1 6.0
Haemonchus
placei
1 0 N/A
Ostertagia
ostertagi
3 0 N/A
198L Haemonchus
contortus
2 0 N/A
Haemonchus
placei
1 0 N/A
Ostertagia
ostertagi
3 3 27.0–71.1
198A Haemonchus
contortus
2 0 N/A
Haemonchus
placei
1 0 N/A
Ostertagia
ostertagi
3 1 0.8
Mature
(n =
26)
200Y Haemonchus
contortus
14 13 61.5–100
Haemonchus
placei
13 7 3.3–19.5
Ostertagia
ostertagi
26 26 48.1–88.4
167Y Haemonchus
contortus
14 11 3.3–37.1
Haemonchus
placei
13 0 N/A
Ostertagia
ostertagi
26 9 0.9–9.3
198L Haemonchus
contortus
14 0 N/A
Haemonchus
placei
13 0 N/A
Ostertagia
ostertagi
26 26 3.6–48.2
198A Haemonchus
contortus
14 0 N/A
Haemonchus
placei
13 0 N/A
Table 4 (continued )
Polymorphism Species # Herds # Herds
SNP
detected
Range
(%)
Species
detected
Ostertagia
ostertagi
26 10 0.6–6.3
K.R. Kipp et al.
International Journal for Parasitology: Drugs and Drug Resistance 28 (2025) 100594
9
the high pathogenesis of H. contortus in young animals. Bison calves are
not often separated at weaning; rather, they remain with the herd their
whole life. However, calves that are continually grazing in pastures with
mature bison increase their exposure to pathogenic GIN species within
the herd. Coupled with higher resistance in these parasites, this can
prove to be problematic for calves battling high parasitic burdens with
little reduction after treatment, compared to mature bison that often
experience lower parasitic burdens compared to calves. The east-central
Texas ranch sampled for assessing age-class differences was of particular
interest due to some management practices implemented. Specically,
the usage of albendazole which has been administered twice each year
for the last +5 years, in combination with an injectable macrocyclic
lactone. The continued use of albendazole within the herd is largely due
to the known presence of infection by liver uke, Fasciola hepatica.
Consequently, we hypothesized that higher frequencies of benzimid-
azole resistance polymorphisms would be found, and this was supported
by the detection of very high frequencies of benzimidazole resistance
polymorphisms in both H. contortus and O. ostertagi across all age classes
in this herd. Indeed, when considering the polymorphisms at codons
167, 198, and 200 combined in this population, the overall frequency of
benzimidazole resistance polymorphisms for O. ostertagi was 94.48 %
(frequency range =48.2–100 %) in this herd. The nding of four
different resistance polymorphisms 200Y (TTC >TAC), 167Y (TTC >
TAC), 198A (GAA >GCA), and 198L (GAA >TTA), at such a high
combined frequency is striking and suggests a high level of drug selec-
tion has occurred in this herd resulting in effectively every O. ostertagi
worm in the population having a benzimidazole resistant genotype, This
is the rst paper to report such high levels of benzimidazole resistance
polymorphisms in O. ostertagi. Polymorphisms associated with benz-
imidazole resistance have been reported in O. ostertagi before but
generally at a much lower frequency (Avramenko et al., 2020). Knap-
p-Lawitzke et al. (2015) were able to create high frequencies of the
β-tubulin codon 200 using experimental infections of calves. There have
been previous reports of benzimidazole resistance in H. contortus and
H. placei. Similar to what was found in the present study, Chaudhry et al.
(2014), reported a low frequency of the β-tubulin codon 200 in H. placei
in cattle in the southern United States. The high-frequency levels could
be due to the over and misuse of anthelmintics in this bison herd. In
addition, the bison screened rotate pastures with cattle which are also
given anthelmintic treatment twice a year. Though it can be speculated,
it is impossible to know who the selection was through originally, the
overuse of anthelmintics could have started in the cattle herd and
resistant GIN were subsequently picked up by the bison herds and is
further fueled by their own continual dosage of anthelmintics over the
years, leading to increased levels of resistance.
Further investigation should be made to determine how widespread
benzimidazole resistance is in the central and southern United States,
where favorable conditions for GIN occur most of the year. These nd-
ings have major implications for not just the bison industry but all
susceptible grazing livestock. With increased animal movement, pro-
ducers could unknowingly be purchasing bison harboring resistant GIN
onto their property, potentially infecting other grazing animals that
rotate the pasture. This could have a major economic impact on live-
stock industries such as cattle, sheep, and goats, which rely on anthel-
mintics to maintain production.
5. Conclusion
The misuse and overuse of anthelmintics in grazing livestock have
contributed to increased levels of anthelmintic resistance in GIN pop-
ulations which are known to transmit cross-species. While the bison
industry is relatively new, benzimidazole resistance polymorphisms
have already been reported at low frequency, in several GIN species in
commercial bison in Canada and different parts of the United States.
This is the rst paper to examine the species composition and benz-
imidazole resistance in bison herds in the southern United States as well
as to investigate age-related differences in individual bison using next-
generation deep-amplicon sequencing. Our ndings show that not
only are SNPs at codons 167, 198, and 200 present in bison herds, but
most appear to be widespread and present in high frequencies in
economically important trichostrongyle nematodes, in special
O. ostertagi and H. contortus. More specically, our nding of such a high
frequency of benzimidazole resistance in O. ostertagi is of great concern
due to its effects on adult bison and minimal previous reports. This
highlights the importance of early detection of resistance, proper stew-
ardship of anthelmintics, and the implementation of mitigation
strategies.
CRediT authorship contribution statement
Kaylee R. Kipp: Writing – review & editing, Writing – original draft,
Methodology, Investigation, Formal analysis, Data curation. Elizabeth
M. Redman: Writing – review & editing, Methodology, Formal analysis,
Data curation. Joe L. Luksovsky: Writing – review & editing, Meth-
odology, Formal analysis, Data curation. Dani Claussen: Writing – re-
view & editing, Methodology, Data curation. John S. Gilleard: Writing
– review & editing, Supervision, Resources, Methodology, Investigation,
Formal analysis. Guilherme G. Verocai: Writing – review & editing,
Supervision, Resources, Project administration, Methodology, Funding
acquisition, Conceptualization.
Conict of interest
The authors declare no conict of interest.
Acknowledgement
We would like to thank all bison producers who contributed through
collection and submission of samples. We would also like to thank all
personnel from Texas A&M University Parasitology Diagnostic Labora-
tory who helped with sample processing. Additionally, we would like to
thank those at the University of Calgary for their assistance in molecular
diagnostics.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpddr.2025.100594.
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