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International Journal for Parasitology: Drugs and Drug Resistance logoLink to International Journal for Parasitology: Drugs and Drug Resistance
. 2023 Apr 22;22:27–35. doi: 10.1016/j.ijpddr.2023.04.002

Integration of ITS-2 rDNA nemabiome metabarcoding with Fecal Egg Count Reduction Testing (FECRT) reveals ivermectin resistance in multiple gastrointestinal nematode species, including hypobiotic Ostertagia ostertagi, in western Canadian beef cattle

Eranga L De Seram a,e, Fabienne D Uehlinger a,∗∗, Camila de Queiroz b,f, Elizabeth M Redman b, John R Campbell a, Drue Nooyen b, Arianna Morisetti b, Colleen M Pollock c, Samantha Ekanayake a, Gregory B Penner d, John S Gilleard b,∗
PMCID: PMC10165142  PMID: 37119733

Abstract

A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8–90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information.

Keywords: Cooperia, ivermectin resistance, Haemonchus, Hypobiotic larvae, Ostertagia, nemabiome metabarcoding

Graphical abstract

Image 1

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Highlights

  • •

    Integration of nemabiome metabarcoding to enhance fecal egg count reduction tests in cattle.

  • •

    Multiple ivermectin-resistant cattle gastrointestinal nematode species in western Canadian beef cattle.

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    Ivermectin-resistant hypobiotic O. ostertagi larvae in western Canadian beef cattle.

1. Introduction

Anthelmintic resistance is a global issue and a serious threat to sustainable control of gastrointestinal nematode (GIN) infections in livestock operations (Gasbarre, 2014; Rose Vineer et al., 2020; Gilleard et al., 2021). Although the problem was identified much earlier in the sheep industry, anthelmintic resistance is now also an escalating problem in the cattle industry. For instance, a beef study conducted in the USA between 2007 and 2008 identified suboptimal anthelmintic efficacy in one-third of the participating cow-calf operations (Gasbarre et al., 2015). Compromised production performance associated with anthelmintic-resistant GIN has been reported in young beef cattle, demonstrating the practical significance of the problem (Borges et al., 2013; Candy et al., 2018).

Macrocyclic lactones (ML) are the most common anthelmintic drug class used in beef operations worldwide, with numerous reports of individual and multi-drug resistant GIN (Mejía et al., 2003; Gasbarre et al., 2009; Ramos et al., 2016). Macrocyclic lactone resistance in Cooperia spp. has been commonly reported in beef cattle from multiple countries, including the USA (Mejía et al., 2003; Waghorn et al., 2006; Gasbarre et al., 2015; Ramos et al., 2016). Although there are increasing reports of ivermectin resistance in Ostertagia ostertagi in several countries, only one peer-reviewed report from North America is currently available (Edmonds et al., 2010; Geurden et al., 2015; Waghorn et al., 2016). The risk of ivermectin resistance in O. ostertagi is of particular concern as it is the most pathogenic GIN species in North American beef cattle (Hildreth and McKenzie, 2020).

The use of ML to control GIN infections is also predominant in western Canadian cow-calf operations (Wills et al., 2020a). In a recent study, suboptimal efficacy of pour-on ML (at 95% threshold for the herd-level fecal egg count (FEC) reduction) against GIN was detected in 20/31 (64.5%) western Canadian cow-calf operations (Avramenko et al., 2017). That study's results were based on the efficacy data of producer-applied pour-on ML; therefore, the reduced anthelmintic efficacies could be associated with actual anthelmintic resistance or other factors such as inappropriate dosing and product administration or storage (Avramenko et al., 2017). Cooperia oncophora was the main GIN species present following the pour-on ivermectin treatment in that study (Avramenko et al., 2017). Those findings suggested further studies to confirm whether poor treatment efficacy was due to anthelmintic resistance and how widespread this might be.

Demonstration of anthelmintic resistance in beef cattle, particularly in the northern USA and Canada, is a practical challenge due to the difficulty of designing a study with sufficient statistical power due to low pre-treatment FEC (Stromberg et al., 2015; Avramenko et al., 2017; Wills et al., 2020b). Also, current guidelines for cattle fecal egg count reduction testing (FECRT) are not standardized, and many limitations have been identified (Kaplan and Vidyashankar, 2012; Levecke et al., 2012a). However, one approach to overcome the issue of low FEC is to undertake a large-scale FECRT to fulfill the statistical power requirements (Levecke et al., 2012a, 2012b; Torgerson et al., 2014). In addition, integrating nemabiome metabarcoding to provide species-specific information potentially adds further evidence to support resistance detection and provide GIN species-specific information (Queiroz et al., 2020).

The primary objective of the work presented here was to determine if anthelmintic-resistant GIN are present in beef cattle in western Canada and which GIN species are involved. Spring-born calves in cow-calf operations acquire GIN parasite populations while grazing on pastures over the summer before entering feedlots for finishing. Consequently, to investigate the presence of anthelmintic-resistant GIN on western Canadian pastures, a large-scale FECRT was conducted in feedlot pens on first-season calves purchased from an auction market in Saskatchewan. ITS-2 rDNA nemabiome metabarcoding was integrated into the FECRT to identify the GIN species resistant to anthelmintics. Further, to detect the presence of ML-resistant hypobiotic larvae without resorting to necropsy procedures, monthly fecal sampling and nemabiome metabarcoding over six months were undertaken to identify any eggs that reappeared in fecal samples from adults developed from reactivated hypobiotic larvae.

2. Material and methods

2.1. Experimental animal management

The University of Saskatchewan's Animal Care Committee approval was obtained to use steers in this study (Protocol number: AUP, 20170028). The guidelines of the Canadian Council on Animal Care 2009 (Ottawa, ON, Canada) were followed in caring for and handling steers.

Two hundred and thirty-four fall-weaned steer calves bought at an auction market in Saskatchewan (Saskatoon Livestock Sales Ltd, Saskatoon, SK, Canada) were placed in a research feedlot facility (University of Saskatchewan, Saskatoon, SK, Canada) in October 2017. The farms of origin of these steers were in western Canada, but their exact locations were unknown. There was a three to five-week acclimatization period for steers based on their arrival date at the feedlot before the trial started. Upon feedlot arrival, steers were weighed, ear-tagged, vaccinated with clostridial and Histophilus somni bacterin-toxoid (Vision 8 somnus, Merck Animal Health, Kirkland, QC, Canada) and bovine respiratory disease complex (Vista once SQ, Merck Animal Health, Kirkland, QC, Canada) vaccines, implanted with growth stimulating anabolic agent (Ralgro, Merck Animal Health, Kirkland, QC, Canada), and given metaphylactic antibiotics (Oxyvet 200 LA, Vetoquinol, QC, Canada) as per routine feedlot practice. During the acclimatization period, steers were housed in feedlot pens in groups of 13 according to their arrival sequence. The acclimatization period ended two days before (day −2) the commencement of the trial. Steers were fed a standard barley-based feedlot ration, and feedlot staff regularly monitored their health.

2.2. Experimental design

After the acclimatization period, steers were strategically randomized into three treatment groups of 78 steers. Each treatment group consisted of six replicated pens with 13 steers per pen. Treatment groups consisted of an untreated control (CTRL) group, an injectable ivermectin-only (IVM) treatment group, and a combination of injectable ivermectin and oral fenbendazole (IVM + FZ) treatment group.

Steer allocation to pens was based on strategic randomization based on individual FEC obtained at feedlot arrival and BW measured on day −1 to ensure a similar median FEC and mean BW across each pen. A detailed depiction of the randomization process of steer and treatment allocations to pens is shown in Supplementary Figs. S1 and S2.

The following randomization procedure was used to assign treatments to pens. Pen numbers were arranged in ascending sequential order and categorized into six groups with three pens each. Each treatment was assigned a number from 1 to 3 (e.g., no treatment = 1, ivermectin = 2, combination treatment of ivermectin and fenbendazole = 3). Then, a treatment number was randomly pulled ‘out of the hat,’ and that number was assigned to the first pen of the first group of pens (e.g., if number 1 were pulled out of the hat, then pen number 1 would receive no treatment, pen 2 would be treated with ivermectin, and pen 3 would be treated with the treatment combination). Randomization was repeated for each group of pens.

Anthelmintics were administered on day 0 of the trial, with the dose calculated from individual body weights measured on day −1 and according to the manufacturer's recommendations. Injectable ivermectin (Ivomec injection 1%, Merial Canada inc., QC, Canada) was administered subcutaneously at 0.2 mg/kg of body weight. The oral drench of fenbendazole (Safe-guard suspension 10%, Merck Animal Health, Kirkland, QC, Canada) was administered at 5 mg/kg of body weight. The control group received a pour-on pyrethroid ectoparasiticide (lambda-cyhalothrin) at 10 mL (if < 275 kg) or 15 mL (if > 275 kg) per steer (Saber, Merck Animal Health, Kirkland, QC, Canada).

2.3. Sampling timeline and animal availability during the sampling period

Fecal samples were collected from the rectum of each steer on arrival (to obtain a FEC for strategic randomization as previously mentioned), on days 0 (immediately before anthelmintic administration), 14, 28, and then monthly until steers were sent to slaughter (on day 181). Days 28, 61, 90, 120, 149, and 177 samplings were considered months 1–6.

All steers were clinically healthy and available from arrival to month 1 sampling. One steer was removed from the CTRL group due to lameness before the month 2 sampling. Consequently, 77, 78, and 78 steers were sampled in months 2 and 3, in the CTRL, IVM, and IVM + FZ groups respectively. Four steers that died due to non-infectious diseases were unavailable from month 4 sampling onwards meaning that 76, 76, and 77 steers were sampled in months 4, 5, and 6 CTRL, IVM, and IVM + FZ groups respectively.

2.4. Fecal sample collection, fecal egg counting, and coproculturing

A modified Wisconsin sugar floatation method was used to obtain GIN FEC from 5 g of feces from individual samples at a theoretical minimum detection limit of 0.2 eggs per gram of feces (EPG) (Ito, 1980). Based on the morphology, GIN eggs were identified and counted as strongyle-type, Nematodirus spp., and Trichuris spp. Throughout the trial, fecal egg counting was performed by a single person blinded to the anthelmintic treatment to prevent between-person variability of egg counts and minimize potential observer bias.

Individual coprocultures from each sampling (except at feedlot arrival) were prepared to isolate third stage GIN larvae (L3) for species identification, according to a modified method of Roberts and O’Sullivan (1950). Fifty grams of feces were mixed with vermiculite and tap water in a 250 mL glass. Coprocultures were incubated at 27 °C for 14 days, and larvae were harvested as described by Roberts and O’Sullivan (1950). Subsequently, L3 harvested from each sample were counted under the dissecting microscope, pooled by pen, and the sample collection date. Larvae in pooled samples were concentrated by centrifugation (3725×g, 4 °C, 10 min), fixed by adding 0.7 mL of 95% ethanol to a total volume of 1 mL, and stored at −80 °C for genomic DNA preparation and archiving.

2.5. Determination of gastrointestinal nematode species identity and relative abundance

The relative abundance of GIN species in each sample pool was determined using ITS-2 rDNA nemabiome metabarcoding (Avramenko et al., 2015). The genomic DNA lysates were prepared from three independent aliquots of approximately 150 L3 from each pen-level pool. Amplification of the ITS-2 marker using NC1 and NC2 primers and metabarcoding library preparation was conducted as previously described, with details available at https://www.nemabiome.ca/sequencing.html (Avramenko et al., 2015).

2.6. Data analysis

2.6.1. Fecal Egg Count Reduction Test (FECRT)

The mean (arithmetic) percentage reduction in strongyle-type FEC (95% confidence interval (CI)) of each anthelmintic treatment group was calculated. For these calculations, the eggCounts add-on package to the R statistical software was used (Torgerson et al., 2014). Data with zero egg counts were omitted from the calculation. The pre-treatment (day 0) and post-treatment (day 14) FEC of all steers were considered in calculating the percentage reduction of FEC as per the following formula (Geurden et al., 2015):

Reduction in FEC (%) = [(pre-treatment FEC – day 14 post-treatment FEC) / (pre-treatment FEC)] × 100

Anthelmintic resistance was confirmed when all of the following criteria were met: mean reduction in FEC <95%, upper 95% confidence limit <95% FEC reduction, and lower 95% confidence limit <90% FEC reduction. If one or two of the three criteria were not met, the efficacy status was considered inconclusive (Geurden et al., 2015). Fecal egg count reduction tests were not conducted for Nematodirus and Trichuris spp. due to very low pre-treatment FEC.

2.6.2. Bioinformatic analysis

Samples were analyzed using the Mothur bioinformatic tool version 1.36.1 (Schloss et al., 2009) as previously described (Avramenko et al., 2015). After removing reads <200 bp or >450 bp, paired-end reads were assembled into single contigs aligned to a bespoke ITS-2 database (Nematode ITS2 database version 1.3, https://www.nemabiome.ca/mothur_workflow/) and assigned to reference sequences using the k-nearest-neighbor method with k = 3. The percentage species composition of samples was calculated by dividing the total reads assigned to each species by the total number of reads per sample to obtain the relative percentage of each species. The number of species-specific sequence reads was divided by the total number of reads in the sample and then multiplied by a species-specific correction factor (to account for species-specific biases in the assay) to determine the relative species proportions (Avramenko et al., 2015). Detailed ITS-2 rDNA nemabiome sequencing and bioinformatic analysis information are available at https://www.nemabiome.ca/analysis.html.

Mean inverse Simpson indexes (± standard error of the mean) were calculated to determine alpha diversity, the overall mean species diversity of GIN populations, in pre- and day 14 post-IVM and CTRL groups. Alpha diversity was not determined in the IVM + FZ group because L3 were not recovered from the day 14 post-treatment coprocultures. The analysis was conducted in Mothur v.1.36.1 using the built-in inverse Simpson calculation (Avramenko et al., 2015). The statistical difference between mean inverse Simpson indexes of pre- and day 14 post-treatment populations was determined by a t-test assuming non-equal variances using the SPSS software (IBM Corp. Released 2012. IBM SPSS Statistics for Macintosh, Version 21.0. Armonk, NY, USA).

The beta diversity (± standard error of the mean) analysis was conducted to determine the difference between mean relative abundances of a particular GIN species in the treatment groups (including CTRL) at different sampling occasions (White et al., 2009). The MetaStats plugin in 275 Mothur v. 1.36.1, which used 1000 permutations and default parameters, was used for beta diversity calculations. A modified non-parametric t-test was used for the pairwise comparisons of the beta diversity estimations assuming data were not normally distributed. If the relative species percentages were present at <2% in both comparison groups, statistical significance was not claimed to avoid potential overestimation of GIN species proportions. Significance were declared if P < 0.05.

A particular GIN species was considered resistant to the anthelmintic drug or drug combination if its mean relative abundance at day 14 post-treatment was significantly higher compared to its relative abundance at pre-treatment and if the mean FEC reduction for that anthelmintic drug met resistance criteria.

3. Results

3.1. A large-scale fecal egg count reduction test reveals ivermectin resistance in gastrointestinal nematode parasites in western Canadian beef calves

The individual FEC at days 0 (pre-treatment) and day 14 (post-treatment) sampling are shown in Fig. 1.

Fig. 1.

Fig. 1

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Fecal egg count data from the auction market-derived, fall-weaned feedlot steers in the untreated control (CTRL) group, injectable ivermectin (IVM) group, and the combination of injectable ivermectin and oral fenbendazole (IVM + FZ) group pre-treatment (day 0) and day 14 post-treatment. Each dot in scatter-violin plots represents an individual steer's fecal egg count and the solid line represents the median. Each treatment group consisted of 78 steers allocated to 6 pens, with 13 steers per pen. “*" in the scatter-violin plot related to Pen 9 of the Day 0 IVM group represents an outlier fecal egg count that was removed to allow clear data plotting.

The percentage reduction of the paired FEC at day 0 pre-treatment and day 14 post-treatment FEC for each steer in each pen for IVM and IVM + FZ groups are shown in Fig. 2. The mean percentage reduction in FEC and 95% confidence interval (CI) was calculated for each pen and the IVM and IVM + FZ treatment groups overall, using the eggCounts package (Torgerson et al., 2014) (Fig. 2). For the IVM treatment group, the overall percentage mean FEC reduction was 82.4% (95% CI 67.8–90.4), with mean FEC reductions of the individual pens of 89.1% (95% CI 63.8–96.7), 58.2% (95% CI 5.3–81.5), 80.0% (95% CI 15.6–95.2), 82.9% (95% CI 42.8–94.9), 85.0% (95% CI 52.5–95.3), and 93.4% (95% CI 82.6–97.5), respectively (Fig. 2). For the IVM + FZ combination treatment group, the overall mean FEC reduction was 100%, with a 100% reduction in FEC in each of the six individual pens.

Fig. 2.

Fig. 2

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Fecal Egg Count Reduction Test Data for the auction market-derived, fall-weaned feedlot steers in the injectable ivermectin-treated (IVM) pens and the injectable ivermectin and oral fenbendazole combination (IVM + FZ) pens 14 days after treatment. The horizontal dotted line in red indicates the 95% threshold fecal egg count reduction for the ivermectin treatment. Each dot represents the fecal egg count reduction for an individual steer. For four individuals, the number of eggs after treatment was higher than before treatment (one individual in Pen 7 and three individuals in Pen 8), and for those steers, the fecal egg count reduction is shown as zero. The grey bars indicate mean percentage reduction and 95% confidence intervals in strongyle-type fecal egg counts as calculated by the eggCounts package (Torgerson et al., 2014). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

As a separate analytical approach, the overall mean FEC reduction of the day 14 post-IVM and the IVM + FZ groups relative to the day 14 mean FEC of the CTRL group was also calculated using the method described by Coles et al. (1992). According to this calculation, the overall percentage mean FEC reduction was 78.1% (95% CI 61.687.5) and 100% for the IVM and IVM + FZ treatment groups, respectively.

3.2. Integration of ITS-2 nemabiome metabarcoding with the FECRT indicates ivermectin resistance in C. oncophora, C. punctata, and H. placei adult worms

Ostertagia ostertagi and C. oncophora were the most abundant species in all pre-treatment L3 pools, with C. punctata, H. placei, and N. helvetianus being less abundant (Fig. 3). For the CTRL group, there was no significant difference (P = 0.40) in the alpha diversity of the GIN populations between day 0 (2.08 ± 0.11) and day 14 (1.96 ± 0.09) as measured by the Simpson index. However, the mean Simpson indices of the GIN populations in the pre- and day 14 post-IVM group were significantly different (2.31 ± 0.12 and 1.61 ± 0.19, respectively; P = 0.02), demonstrating a change in species composition following the ivermectin treatment. These analyses could not be performed for the IVM + FZ treatment group as no larvae were recovered from day 14 post-treatment coprocultures.

Fig. 3.

Fig. 3

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Relative species abundance of gastrointestinal nematode communities, determined by ITS-2 rDNA nemabiome metabarcoding, in pen-level pools of third stage larvae harvested from individual coprocultures of auction market-derived, fall-weaned feedlot steers in the untreated control (CTRL) group, the injectable ivermectin only treatment (IVM) group, and the injectable ivermectin and oral fenbendazole combination treatment (IVM + FZ) group at days 0 (pre-treatment) and 14 (post-treatment). Each panel's narrow upper stacked bar chart shows the arithmetic mean fecal egg counts of strongyle-type, Nematodirus spp., and Trichuris spp. for each pen in each treatment group (colour code above figure). EPG = eggs per gram of feces. The main bar chart in each panel shows the species composition of larval cultures determined by ITS-2 rDNA metabarcoding (colour key below figure). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

For the CTRL group, there was no significant difference in relative abundance between day 0 and day 14 samples for O. ostertagi (P = 0.25), C. oncophora (P = 0.25), or H. placei (P = 0.91) as determined by beta diversity analysis (Fig. 3 and Table 1). The mean relative abundances of C. punctata, N. helvetianus, O. radiatum, and Oesophagostomum venulosum were <2% in the CTRL group on days 0 and 14; therefore, statistical significance was not claimed. In contrast, for the IVM treatment group, there was a significant decrease between day 0 and day 14 post-treatment samples in the relative abundance of O. ostertagi (P = 0.001) and a significant increase for C. oncophora (P = 0.002), demonstrating ivermectin resistance for the latter species. Although the change did not reach statistical significance, there was also an increase in the relative abundance of both H. placei and C. punctata in day 14 post-IVM treatment group (Table 1). However, there was a significant difference in the relative abundance of H. placei (P = 0.04) and C. punctata (P = 0.03) between the day 14 post-IVM and day 14 CTRL group suggesting positive selection indicative of resistance for these species. Such changes were not evident in the relative abundance of H. placei (P = 0.54) and C. punctata (P = 0.14) in the day 0 CTRL group compared to the day 0 IVM group.

Table 1.

Beta diversity comparisons of mean gastrointestinal nematode relative species compositions of day 0 (pre-treatment), day 14 (post-treatment), and Months 1, 5, and 6 pen-level third stage larvae pools obtained from auction market-derived, fall-weaned feedlot steers from Saskatchewan in the untreated control (CTRL) group and the injectable ivermectin group (IVM).

Relative species composition (%) (±SEM)a
O. ostertagi C. oncophora H. placei C. punctata N. helvetianus
CTRL
Day 0 60.1 (1.95) 34.4 (1.71) 3.1 (1.94) 0.35 (0.19) 1.2 (0.09)
Day 14 64.0 (2.45) 31.8 (1.46) 3.4 (1.81) 0.08 (0.04) 0 (0)
Month 1 93.6 (1.78) 2.4 (0.92) 2.7 (1.47) 0 (0) 0 (0)
Month 5 67.2 (6.48) 13.1 (4.09) 19.5 (6.28) 0.03 (0) 0 (0)
Month 6 68.2 (5.56) 5.1 (1.68) 26.5 (5.78) 0 (0) 0 (0)
IVM
Day 0 55.2 (2.67) 32.9 (4.49) 7.4 (2.92) 1.3 (0.61) 3.1 (1.75)
Day 14 0.88 (0.32) 75.5 (7.65) 20.6 (7.72) 2.4 (0.09) 0.52 (0.52)
Month 1 7.0 (6.13) 74.0 (8.63) 15.8 (6.11) 2.3 (1.01) 0 (0)
Month 5 51.9 (0.11) 11.1 (3.34) 36.5 (12.95) 0.5 (0.03) 0 (0)
Month 6 52.8 (3.60) 3.1 (1.03) 43.8 (4.04) 0 (0) 0 (0)
P-value
Day 0 vs day 14 CTRL 0.25 0.25 0.91 0.18 0.18
Day 0 vs day 14 IVM 0.001 0.002 0.09 0.48 0.12
Day 0 CTRL vs day 0 IVM 0.02 0.95 0.54 0.14 0.54
Day 14 CTRL vs day 14 IVM < 0.001 0.001 0.04 0.03 0.30
Month 1 vs. month 5 CTRL 0.051 0.06 0.06 0.25 NEb
Month 1 vs. month 6 CTRL 0.02 0.14 0.02 NE NE
Month 1 vs. month 5 IVM 0.01 0.01 0.13 0.01 NE
Month 1 vs. month 6 IVM 0.001 0.001 0.001 0.01 NE
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The number of steers available for fecal sampling in the CTRL and IVM was as follows per each group: days 0 and 14 and month 1: n = 78 each; months 5 and 6: n = 76 each.

a

Standard error of the mean.

b

Not estimated as relative species compositions were zero in both groups in respective comparisons.

3.3. ITS-2 nemabiome metabarcoding of monthly samples for six months post-treatment indicates the ivermectin resistance in O. ostertagi hypobiotic fourth-stage larvae

For the CTRL group, O. ostertagi was the most abundant GIN species found throughout the trial, but there was a significant increase in the relative abundance of H. placei by month 6 relative to month 1 (P = 0.02) after the commencement of the trial (Fig. 4 and Table 1). For the IVM group, C. oncophora remained the predominant GIN until four months post-treatment, after which time there was an increase in both the O. ostertagi and H. placei species abundance. This increase in the relative abundance of both O. ostertagi (P < 0.001) and H. placei (P = 0.001) was significant in month 6 compared to month 1. For the IVM + FZ group, the FEC remained extremely low following treatment such that insufficient L3 were recovered from the coprocultures until five months post-treatment; therefore, nemabiome sequencing data was unavailable for months 1–4. However, in the IVM + FZ group, FEC started to rise at months 5 and 6, and nemabiome metabarcoding revealed the GIN population to be almost exclusively O. ostertagi.

Fig. 4.

Fig. 4

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Relative species abundance of gastrointestinal nematode communities, determined by ITS-2 rDNA deep amplicon nemabiome sequencing, in pen-level pools of third stage larvae (L3) harvested from individual coprocultures of auction market-derived, fall-weaned feedlot steers in the untreated control (CTRL) group, injectable ivermectin only treatment (IVM) group, and the combination of injectable ivermectin and oral fenbendazole treatment (IVM + FZ) group at Months 1–6 sampling across the feedlot period. Each panel's narrow upper stacked bar chart shows the arithmetic mean fecal egg counts of strongyle-type, Nematodirus spp., and Trichuris spp. for each pen in each treatment group (colour code above figure). EPG = eggs per gram of feces. The main bar chart in each panel shows the species composition of larval cultures determined by ITS-2 rDNA metabarcoding (colour key below figure). All pools contained at least 150 L3 larvae for the CTRL and IVM groups. However, the high efficacy of the IVM + FZ combination treatment meant insufficient L3 were recovered for nemabiome metabarcoding at sampling months 1–4. However, at sampling months 5 and 6, L3 were recovered from coprocultures for each IVM + FZ-treated pen (Month 5: 110, 169, 213, 33, 25, and 24 L3; Month 6: 363, 522, 607, 91, 37, and 73 L3 for Pens 13–18, respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

Although the suboptimal efficacy of producer-applied pour-on ivermectin is widespread in Canadian beef cattle, it does not necessarily indicate the presence of anthelmintic-resistant parasites (Avramenko et al., 2017). Poor efficacy can occur for various reasons, including inappropriate product administration, storage or incorrect dosage calculation, grooming behavior, or product washing off due to rainfall following application (Bousquet-Mélou et al., 2011; Leathwick and Miller, 2013). Consequently, we performed a large-scale, well-controlled FECRT to investigate if ivermectin-resistant cattle GIN are present in western Canada. The study used an injectable ivermectin product with dosage calculated by individual cattle weights and also had the novel approach of integrating ITS-2rDNA nemabiome metabarcoding to identify the ivermectin-resistant GIN species.

One of the major challenges of undertaking FECRTs in cattle in northern climatic zones is very low egg counts of individual cattle. To address this challenge, we used a study design with a large number of calves (78 per treatment group) and internal replication within each treatment group of 6 pens of 13 calves each. The FECRT data on total strongyle egg counts definitively confirmed the presence of ivermectin-resistant GIN in western Canadian beef cattle. The overall mean FEC reduction for the IVM group was 82.4% (95% CI 67.8–90.4) when comparing pre-treatment and day 14 post-treatment samples and 78.1% (95% CI 61.6–87.5) when comparing day 14 post-IVM and untreated control groups. Consequently, the FEC reductions and the lower confidence intervals were below 90% for pre-IVM treatment vs. day 14 post-IVM treatment and day 14 post-IVM treatment vs. control. In contrast, the overall FEC reduction for the IVM + FZ combination treatment was 100% for both comparisons. Further, 5 out of 6 separate pens in the IVM group (13 calves in each pen) exceeded this resistance threshold. Consequently, resistance is diagnosed based on the criteria of the original guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (Coles et al., 1992), as well as Geurden et al. (2015).

ITS-2 rDNA metabarcoding was used to determine which GIN species were present in L3 populations harvested from coprocultures at day 0 (pre-treatment) and day 14 post-treatment. This comparison determined the species identity of the adult GIN surviving ivermectin treatment in the calves. There was a significant decrease in the relative abundance of O. ostertagi between day 0 and day 14 post-IVM treatment (P = 0.001), with very few larvae present in coprocultures post-treatment. This finding indicates that the adult O. ostertagi were susceptible to the drug without evidence of ivermectin resistance (Fig. 3 and Table 1). In contrast, there was a large and significant increase for C. oncophora on day 14 post-IVM treatment compared to the pre-IVM treatment (P = 0.002) and the day 14 untreated control (P = 0.001), confirming its resistance to ivermectin. There was also an increase in the relative abundance of H. placei and C. punctata on day 14 post-IVM treatment compared to pre-IVM treatment and untreated control. Although these increases were not statistically significant when comparing pre- and post-treatment samples, they were statistically significant when comparing day 14 post-IVM treatment and untreated control groups (P = 0.04 and P = 0.03 for H. placei and C. punctata, respectively), suggesting the likely presence of ivermectin-resistant adult worms for these two species.

Statistical modeling suggests that the minimum number of eggs that are physically counted in the pre-treatment group is an important criterion when determining the statistical robustness of an anthelmintic resistance diagnosis when using the FECRT in ruminants (Dobson et al., 2012). Bayesian modeling has been used to calculate the minimum number of eggs that should be counted pre-treatment for reliable detection of reduced drug efficacy for different group sizes and expected drug efficacies, and this approach is currently being incorporated into the newly updated WAAVP guidelines on the diagnosis of anthelmintic resistance (Levecke et al., 2018; Kaplan et al., in preparation). It has been suggested that the minimum number of eggs that need to be counted to determine a true reduction in efficacy (<95%), when the expected efficacy was 99%, was 113 or 97 for group sizes of 10 or 15 animals, respectively (Levecke et al., 2018). In this study, the total number of eggs counted on slides for the pre-treatment samples of the IVM treatment group (comprising 78 calves) was 1812 and so easily exceeds these minimum values making the diagnosis of ivermectin-resistant strongyle GIN statistically robust. Indeed, the suggested minimum number of eggs counted pre-treatment was exceeded for each IVM-treated pen of 13 calves (Supplementary Table S1).

Statistical robustness of the resistance diagnosis for the individual GIN species based on combining the FECRT and ITS-2 rDNA metabarcoding data can also be considered. In the case of C. oncophora, its relative abundance in the pre-IVM treatment group was 32.9%, so approximately 596 of the 1812 stronglye eggs counted on slides for the pre-IVM treatment group are predicted to have been C. oncophora which easily fulfills the minimum number of eggs counted criteria. In the case of H. placei, its relative abundance in the pre-IVM treatment group was 7.4%; therefore, approximately 134 of the 1812 stronglye eggs counted on slides for the pre-IVM treatment group are predicted to have been H. placei, again fulfilling the criteria. The relative abundance of C. punctata was only 1.3%, meaning that only 24 of the 1812 strongyle eggs counted on slides for the pre-IVM treatment group are expected to be this species, making the diagnosis of resistance less robust.

Cooperia oncophora is the dose-limiting GIN species for macrocyclic lactone products and was the first cattle GIN parasite to be reported as developing resistance to ivermectin (Coles et al., 1998; Vercruysse and Rew, 2002). In our study, we found C. oncophora as the predominant ivermectin-resistant GIN species, consistent with previous reports from other Northern temperate climatic regions where ivermectin resistance in this GIN species is increasingly common (Coles et al., 1998; Edmonds et al., 2010). Although this is a milder pathogen than O. ostertagi, it can still cause production loss, so the emergence of ivermectin resistance in this species is of concern (Candy et al., 2018). Identifying ivermectin resistance in H. placei and C. punctata in the western Canadian beef calves is perhaps even a greater concern given their higher level of pathogenicity (Stromberg et al., 2012). Ivermectin resistance has been previously reported in both H. placei and C. punctata in warmer southern regions of the USA and Brazil, where these GIN species predominate, but this is the first report in a more northerly temperate region (Gasbarre et al., 2009; Felippelli et al., 2014). Although these GIN species were a relatively minor component of the parasite populations in this study pre-treatment, recent work suggests that their geographical range may be expanding, and they are becoming more abundant in Canada (Avramenko et al., 2017; De Seram et al., 2022). Indeed, C. punctata was recently found to be the predominant GIN species in several beef herds in Manitoba (De Seram et al., 2022). The establishment of ivermectin-resistant H. placei and C. punctata is potentially a major threat to cattle health and production in western Canada over the coming years.

The almost complete absence of O. ostertagi L3 in coprocultures from calves at 14 post-IVM treatment indicates that O. ostertagi adult worms in these calves were not ivermectin resistant (Fig. 3). However, ITS-2 rDNA nemabiome metabarcoding revealed a return of O. ostertagia L3 to coprocultures taken from 4 months of feedlot residence onwards in both the IVM treatment and IVM + FZ combination treatment groups (Fig. 4). This result strongly indicates the presence of ivermectin-resistant O. ostertagi hypobiotic fourth stage larvae (L4) for a number of reasons. Firstly, O. ostertagi L3 do not survive in the feedlot environment and so the return of O. ostertagia eggs in fecal samples 4–5 months after ivermectin or IVM + FZ treatment is highly unlikely to be due to reinfection by ingested L3. Further, the timing of this experiment was favorable for winter hypobiosis of O. ostertagi L4 in the abomasal mucosa, and their typical recrudescence and subsequent development into adults in the spring in northern temperate regions coincides with their return at 4–5 months (Armour and Duncan, 1987; Ranjan et al., 1992; Almería et al., 1996). Hypobiosis is much more a feature of O. ostertagi than the other cattle GIN species, so the recrudescence of O. ostertagi, and not the other species, is further consistent with remerging hypobiotic larvae rather than oral transmission of L3 in the feedlot. As per the product label, a subcutaneous dose of 0.2 mg/kg of body weight injectable ivermectin should be highly effective against O. ostertagi hypobiotic L4 and so their survival and recrudescence strongly indicates ivermectin resistance in this parasite stage. The failure of the IVM + FZ combination to kill ivermectin resistant hypobiotic L4 is expected, as fenbendazole has variable efficacy against O. ostertagi inhibited L4 (Williams et al., 1995, 1997).

Although it appears to have been slower to develop than for Cooperia and Haemonchus species, ivermectin resistance in O. ostertagi is now increasingly reported in different parts of the world (Waghorn et al., 2016; Rose Vineer et al., 2020). Ivermectin-resistant O. ostertagi has only been previously reported in North America once before in the peer-reviewed literature (Edmonds et al., 2010). Interestingly, as in our study, resistance was only detected in hypobiotic L4 and not in adult worms, as revealed by necropsies in that large-scale controlled efficacy study in California (Edmonds et al., 2010). Ivermectin resistance in O. ostertagia hypobiotic L4 may already be widespread in North America since it would escape detection using standard fecal egg count reduction testing, where sampling occurs 14 days post-treatment. However, ivermectin-resistant O. ostertagia hypobiotic L4 has now been found in California and western Canada in study designs capable of detecting it. Unsurprisingly, resistance may occur in hypobiotic L4 before it occurs in adult worms as the former is a dose-limiting stage. It seems likely that the emergence of resistance in hypobiotic larvae may be a precursor to resistance in adult worms, although that has yet to be experimentally determined. The emergence of ivermectin resistance in O. ostertagi is of particular concern since it is the predominant GIN in western Canada and the most pathogenic cattle GIN species (Gasbarre, 1997; Avramenko et al., 2017).

The results of this study suggest ivermectin resistance to multiple cattle GIN species is likely to be widespread in western Canada. The reasoning for this is that the calves in this FECRT study were not derived from a single grazing location but instead purchased over ten days from a Saskatchewan auction market sourcing cattle from multiple farms within the province. It is common for Saskatchewan producers to graze cattle at multiple locations before gathering them for weaning and sale. Given that the calves were randomly allocated to treatment groups regardless of their origin, the parasite populations we tested are likely admixtures derived from multiple Saskatchewan beef herds and pastures. Consequently, our results suggest that ivermectin resistance in the multiple GIN species is likely widespread across the province. Still, more individual FECRT studies from different locations in the region will be needed to define this more precisely and assess any geographical variation within the region.

This study illustrates the value of integrating ITS-2 rDNA nemabiome metabarcoding into the FECRT in cattle, as previously described for sheep (Queiroz et al., 2020). In this case, it has not only allowed determining different cattle GIN species resistant to ivermectin but has also enabled the detection of resistance in O. ostertagi hypobiotic larvae without the need for necropsies and laborious adult worm and mucosal larval recovery and counting.

Data submission

The raw Fastq sequencing files generated during the current study are available in the SRA database under the BioProject accession reference number PRJNA950954 (https://www.ncbi.nlm.nih.gov/sra/PRJNA950954).

Declaration of the interest

None.

Declaration of competing interest

The authors have no conflicts of interest to disclose.

Acknowledgments

This research was funded by the Government of Saskatchewan's Agriculture Development Fund (ADF, grant number 20150268), Merck Animal Health, Kirkland, QC, Canada, the University of Saskatchewan, the Beef Cattle Research Council (project ANH.04.17), and the Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC grant number 2021-02489).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpddr.2023.04.002.

Contributor Information

Eranga L. De Seram, Email: eranga.seram@usask.ca, eranga.seram@vet.pdn.ac.lk.

Fabienne D. Uehlinger, Email: f.uehlinger@usask.ca.

Camila de Queiroz, Email: camila.dequeiroz@ucalgary.ca.

Elizabeth M. Redman, Email: libbyredman@hotmail.co.uk.

John R. Campbell, Email: john.campbell@usask.ca.

Drue Nooyen, Email: drue.nooyen1@ucalgary.ca.

Arianna Morisetti, Email: ariannamorosetti@gmail.com.

Colleen M. Pollock, Email: canada.AH@merck.com.

Samantha Ekanayake, Email: samantha.ekanayake@usask.ca.

Gregory B. Penner, Email: greg.penner@usask.ca.

John S. Gilleard, Email: jsgillea@ucalgary.ca.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (214.2KB, docx)

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