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International Journal for Parasitology: Drugs and Drug Resistance logoLink to International Journal for Parasitology: Drugs and Drug Resistance
. 2021 Mar 22;15:162–167. doi: 10.1016/j.ijpddr.2021.03.003

Monepantel pharmaco-therapeutic evaluation in cattle: Pattern of efficacy against multidrug resistant nematodes

Candela Canton 1,∗, Lucila Canton 1, Adrian Lifschitz 1, María Paula Domínguez 1, Juan Torres 1, Carlos Lanusse 1, Luis Alvarez 1, Laura Ceballos 1, Mariana Ballent 1
PMCID: PMC8044591  PMID: 33799058

Abstract

The goal of the current work was to perform an integrated evaluation of monepantel (MNP) pharmacokinetics (PK) and pharmacodynamics, measured as anthelmintic efficacy, after its oral administration to calves naturally infected with GI nematodes resistant to ivermectin (IVM) and ricobendazole (RBZ) on three commercial farms. On each farm, forty-five calves were randomly allocated into three groups (n = 15): MNP oral administration (2.5 mg/kg); IVM subcutaneous (SC) administration (0.2 mg/kg); and RBZ SC administration (3.75 mg/kg). Eight animals from the MNP treated group (Farm 1) were selected to perform the PK study. Drug concentrations were measured by HPLC. The efficacy was determined by the faecal egg count reduction test (FECRT). MNP and MNP-sulphone (MNPSO2) were the main analytes recovered in plasma. MNPSO2 systemic exposure was markedly higher compared to that obtained for MNP. Higher Cmax and AUC values were obtained for the active MNPSO2 metabolite (96.8 ± 29.7 ng/mL and 9220 ± 1720 ng h/mL) compared to MNP (21.5 ± 4.62 ng/mL and 1709 ± 651 ng h/mL). The MNPSO2 AUC value was 6-fold higher compared to the parent drug. Efficacies of 99% (Farm 1), 96% (Farm 2) and 98% (Farm 3) demonstrated the high activity of MNP (P < 0.05) against GI nematodes resistant to IVM (reductions between 27 and 68%) and RBZ (overall efficacy of 75% on Farm 3). While IVM failed to control Haemonchus spp. and Cooperia spp., and RBZ failed to control Coooperia spp. and Ostertagia spp., MNP achieved 100% efficacy against Haemonchus spp., Cooperia spp. and Ostertagia spp. However, a low efficacy of MNP against Oesophagostomum spp. (efficacies ranging from 22 to 74%) was observed. In conclusion, oral treatment with MNP should be considered for dealing with IVM and benzimidazole resistant nematode parasites in cattle. The work described here reports for the first time an integrated assessment of MNP pharmaco-therapeutic features and highlights the need to be considered as a highly valuable tool to manage nematode resistant to other chemical families.

Keywords: Monepantel, Cattle, Resistant nematodes, Pharmaco-parasitological assessment

Graphical abstract

Image 1

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Highlights

  • •

    MNP and its anthelmintically active metabolite MNPSO2 were the main analytes recovered in plasma.

  • •

    The MNPSO2 AUC value was 6-fold higher compared to the parent drug.

  • •

    MNP obtained overall efficacies of 96–99% against IVM and BZD resistant nematode parasites in cattle.

  • •

    MNP failed to control Oesophagostomum spp.

  • •

    The work described here reports for the first time an integrated assessment of MNP pharmaco-therapy features.

1. Introduction

Considering the increasing prevalence and worldwide dissemination of gastrointestinal (GI) nematodes resistant to most of the available anthelmintic families, drug resistance is considered one of the main sanitary problems in extensive cattle production systems today (Kaplan, 2020). During the last decades, chemical control has been mainly based on the use of only three anthelmintic chemical families: macrocyclic lactones (ML), benzimidazoles (BZD) and imidazothiazoles. Furthermore, since GI parasitism has a high impact on animal production, these anthelmintic drugs have been intensively used at short intervals in different cattle production grazing systems worldwide. This heavy reliance on anthelmintics to control parasitism and the limited implementation of refugia-based sustainable control programmes have led to the development of resistance to all the available chemical groups. Unfortunately, resistance is becoming a worldwide serious problem, particularly in countries such as New Zealand (Waghorn et al., 2006), Brazil (Ramos et al., 2016), Australia (Rendell, 2010), Uruguay (Mederos et al., 2019), United States (Kaplan, 2020) and Argentina (Cristel et al., 2017) among many others. Despite the complex current situation regarding the widespread development of anthelmintic resistance, dependence on chemically-based control continues to be high since it is still the most practical option for parasite control on commercial beef cattle farms.

The increasing levels of resistance to all traditional drug classes and the still high dependence on anthelmintics for controlling parasitic nematodes, have encouraged the introduction of new molecules with different modes of action into the veterinary pharmaceutical market. The compound monepantel (MNP) is a compound of a new family of anthelmintics, the amino-acetonitrile derivatives, developed to treat ruminants infected with GI nematodes (Kaminsky et al., 2008). Its mode of action is different from the other available anthelmintic families since it acts as a positive allosteric modulator of the nematode specific acetylcholine receptor MPTL-1 (Rufener et al., 2009, 2010). MNP binding to this receptor results in a constant uncontrolled flux of ions and finally in a depolarization of muscle cells leading to nematode paralysis (Epe and Kaminsky, 2013). The cellular target of MNP, the MPTL-1 receptor, is so far only present in nematodes, which might explain the excellent tolerability of MNP in mammals and its high efficacy against multidrug-resistant parasites to other anthelmintic classes in sheep and cattle (Baker et al., 2012; King et al., 2015). The first formulation of MNP, launched in 2009, was licensed for exclusive use in sheep, and some years later was also introduced in a limited number of countries as an oral formulation for use in cattle (King et al., 2015). The disposition kinetics and distribution to target tissues of MNP have been previously described in sheep (Lifschitz et al., 2014), and some data on plasma profiles in dairy cows have been also reported (Ballent et al., 2017). However, until now there have been no published reports regarding the relationship between MNP pharmacokinetics and its efficacy against resistant GI nematodes in beef cattle.

The goal of the work described here was to perform an integrated evaluation of MNP pharmacokinetics (PK) and pharmacodynamics (PD), assessed as anthelmintic efficacy, after its oral administration to calves naturally infected with GI nematodes resistant to ivermectin (IVM) and ricobendazole (RBZ) on three commercial farms.

2. Material and methods

2.1. Field trial

This study was conducted on three cattle commercial farms located in the Humid Pampean Region, Argentina. All farms (Farms 1, 2 and 3) had a grazing system of meat production representative of Argentina bovine production. The resistance status of the nematode population characteristic of each farm was previously determined by the faecal egg count reduction test (FECRT) (Canton et al., 2019). In this way, the study included two farms with a predominance of IVM and RBZ-resistant nematode population (Farms 1 and 3) and one farm with only an IVM-resistant nematode population (Farm 2).

2.2. Animals

All the farms involved in the trial raise calves acquired from other producers. The herd on each farm from which the animals were selected were treated with levamisole prior to the study to remove their worm infections. It is important to point out that resistance to levamisole has not been reported in this region of Argentina (Cristel et al., 2017). They had then grazed on the study farms for at least two months prior to the study, which ensured that their parasite burden was native from each Farm. All the animals had free access to water.

On day −1, 60 (Farms 1 and 3) or 80 (Farm 2) male Aberdeen Angus calves, aged 9–11 months old, naturally infected with GI nematodes resistant to IVM and RBZ (Farms 1 and 3) or resistant to IVM (Farm 2), were checked for worm egg per gram (EPG) counts, ear-tagged, and the individual body weights were recorded. The animals for inclusion in the trial were then selected based on the EPG counts. Forty-five (45) animals on each farm, with at least 100 EPG on day −1, were selected for inclusion in the study. Experimental animals had an average of 508 EPG counts ranging from 100 to 2440 on Farm 1, 274 EPG counts ranging from 100 to 660 on Farm 2, and 450 EPG counts ranging from 140 to 1440 on Farm 3.

Animal procedures and management protocols were approved by the Ethics Committee (act 11/2020) of the Facultad de Cs. Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Argentina.

2.3. Treatments

On each farm (1, 2 and 3), all parasitized animals (n = 45) were ranked according to EPG counts, and then randomly assigned into three groups of 15 animals each: MNP: animals were treated with MNP (Zolvix®, 2.5% solution, Elanco, Argentina) by the oral route at a dose of 2.5 mg/kg; IVM: animals were treated with IVM (Ivomec®, 1% solution, Boehringer Ingelheim, Argentina) by the subcutaneous (SC) route at 0.2 mg/kg and RBZ: animals were treated with RBZ (Bayverm PI®, 15% solution, Bayer, Argentina) by the SC route at 3.75 mg/kg. The mean EPG were similar (P > 0.05) across all groups on each farm at the beginning of the trial.

2.4. Monepantel PK trial

The PK trial was carried out on Farm 1. Eight randomly selected animals from the MNP treated group were used in the PK trial. Blood samples (10 mL) were taken from the jugular vein in heparinised Vacutainer® tubes (Becton Dickinson, NJ, USA) before treatment and at 2,4, 6, 8 and 10 h and 1, 2, 3, 5, 7 and 9 days post-treatment. Plasma was separated by centrifugation at 3000g for 15 min, placed into plastic tubes and frozen at −20 °C until analysis by High Performance Liquid Chromatography (HPLC).

2.5. Anthelmintic efficacy trial: faecal egg count reduction test and coprocultures

Faecal samples were individually collected directly from the rectum of each calf during pre-treatment (day −1) and again on day 15 post-treatment. A modified McMaster technique with a sensitivity of 10 EPG (Roberts and O'sullivan, 1950) was used to analyse the faecal samples and estimate EPG counts. Additionally, 10 g of faeces (obtained from an individual animal and/or from a pool of each experimental group) was used to prepare coprocultures on each sampling day. The nematode genera and species were identified through the third-stage larvae recovered from these coprocultures (MAFF, 1986). Third stage larvae (L3) were collected by the Baermann technique and approximately 100 L3 were differentiated from each sample. Thus, the relative participation of each genus per experimental group was determined.

The anthelmintic efficacy of the different treatments was assessed by the faecal egg count reduction test (FECRT), calculated according to the following formula (McKenna, 1990):

FECRT (%) = 100 (1 - [T2/T1])

where T2 is the arithmetic mean EPG count in each treated group at 15 days post-treatment, and T1 is the arithmetic mean EPG count in each treated group on day −1. The 95% confidence intervals were calculated as reported by Coles et al. (1992). Besides, efficacy against different genera was calculated by dividing the mean faecal egg count of each treatment group at day −1 and 15 post-treatment, by the proportion of L3 of each genus in the associated coproculture (McKenna, 1990).

2.6. Analytical procedures

MNP and its metabolite, MNP-sulphone (MNPSO2), concentrations were determined in plasma by HPLC with UV detection. Briefly, MNP/MNPSO2 were extracted from plasma (0.5 mL) by the addition of 1 mL of acetonitrile. The preparation was mixed with a high-speed shaker (Multi Tube Vortexer, VWR Scientific Products, West Chester, PA) for 15 min at room temperature to allow phase separation. The solvent-sample mixture was centrifuged at 2000 g for 10 min at 4 °C and the supernatant was manually transferred into a clean tube. This volume was evaporated to dryness under a gentle stream of dry nitrogen at 56 °C in a water bath. Finally, the dried residue was reconstituted with 250 μL of mobile phase (acetonitrile:methanol:water 60:8:32, v/v/v) and 200 μL of this solution was injected directly into the chromatography system.

MNP plasma concentration was determined by HPLC (Shimadzu 10 A-HPLC System, Kyoto, Japan) with a UV detector set at 230 nm following a method previously developed (Ballent et al., 2017; Lifschitz et al., 2014). A C18 reversed-phase column (Kromasil, Eka Chemicals, Bohus, Sweden, 5 μm, 4.6 × 250 mm) was used for separation. Elution of MNP and MNPSO2 from the stationary phase was carried out at a flow rate of 0.8 mL/min (MNP) using acetonitrile/methanol/water (60:8:32, v/v/v). Under the described chromatographic conditions, the retention times (min) were established at 9.3 (MNPSO2) and 12.5 (MNP). There was no interference of endogenous compounds in any of the chromatographic determinations. A calibration curve in the range between 4 and 400 ng⁄mL was prepared for both molecules. The plasma calibration curve had a correlation coefficient ≥0.998. Mean absolute recovery percentages for concentrations ranging between 4 and 400 ng⁄mL (n = 6) were 74.9% (MNP) and 74.1% (MNPSO2) with coefficients of variation (CV) of 14.1% and 15.7, respectively. Accuracy (expressed as the relative error) and precision (expressed as the coefficient of variation) were 10% and 5.2%, respectively. The limit of quantification (LOQ) was established at 4 ng⁄mL for MNP and MNPSO2, which is the lowest concentration measured with a recovery higher than 70% and a CV < 20%. In all cases, concentration values below the LOQ were not considered for the kinetic analysis of experimental data.

2.7. Pharmacokinetic analysis of the data

The concentration vs. time curves for MNP and MNPSO2 in plasma for each animal after the different treatments was fitted with the PK Solution 2.0 software (Summit Research Service, CO, USA). The peak concentration (Cmax) and time to peak concentration (Tmax) were recorded directly from the measured concentration data. The elimination half-life (T½el) and absorption half-life (T½abs) were calculated as ln2/λel and ln 2/kabs, respectively, where λel is the elimination rate constant and kabs represents the first-order absorption rate constant. The rates were calculated by performing regression analysis using data points belonging to the terminal or absorption phase concentration-time plot. The area under the plasma concentration-time curve from zero up to the quantificationlimit (AUC0-LOQ) was calculated using the trapezoidal rule (Gibaldi and Perrier, 1982) and further extrapolated to infinity (AUC0-∞) by dividing the last experimental concentration by the terminal elimination rate constant (λel). Statistical moment theory was applied to calculate the mean residence time (MRT) according to Perrier and Mayersohn (1982). PK analysis of the experimental data was performed using a non-compartmental model method.

2.8. Statistical analysis of the data

The PK parameters and concentration data are reported as arithmetic mean ± Standard Deviation (SD). PK parameters for MNP and MNPSO2 were statistically compared using Student t-test. Faecal egg counts (reported as arithmetic mean ± SD) were compared by non-parametric Kruskal–Wallis test. A value of P < 0.05 was considered statistically significant. The statistical analysis was performed using the Instat 3.0 software (Graph Pad Software, CA, USA).

3. Results

MNP and MNPSO2 were the main analytes recovered in plasma after oral administration of MNP to cattle. The mean (±SD) plasma concentrations profiles of MNP and its MNPSO2 metabolite are shown in Fig. 1. MNPSO2 systemic exposure was markedly higher compared to that obtained for MNP. It accounted for >80% of the total amount of the analytes recovered in plasma. While low concentrations of MNP were measured in plasma only up to 120 h (5 days) post-administration, the persistence of the sulphone metabolite was longer in the bloodstream, being recovered up to 216 h (9 days). These differences were reflected in the values estimated for the main PK parameters. Table 1 summarizes the plasma PK parameters for MNP and MNPSO2 obtained after the oral administration of MNP to cattle. Higher Cmax and greater AUC values were obtained in plasma for MNPSO2 compared to MNP. In fact, the AUC value for MNPSO2 were 6-fold higher compared to those reported for the parent drug (MNPSO2/MNP AUC ratio = 5.99 ± 2.08).

Fig. 1.

Fig. 1

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Plasma concentration profiles of monepantel (MNP) and monepantel sulphone (MNPSO2) obtained after the oral administration of monepantel (2.5 mg/kg) to parasitized calves (n = 8). The insert shows the chemical structures of MNP and an its anthelmintically active metabolite MNPSO2.

Table 1.

Plasma pharmacokinetic parameters (mean ± SD) for monepantel (MNP) and monepantel sulphone (MNPSO2) obtained after the oral administration of MNP (2.5 mg/kg) to naturally parasitized calves.

MONEPANTEL
Pharmacokinetic parameters MNP MNPSO2
Tmax (h) 8.00 ± 1.51a 41.3 ± 17.9b
Cmax (ng/mL) 21.5 ± 4.62a 96.8 ± 29.7b
AUC0-LOQ (ng.h/mL) 1709 ± 651a 9220 ± 1720b
AUC0-∞ (ng.h/mL) 2174 ± 783a 10242 ± 1405b
MRT (h) 112 ± 40.8a 99.3 ± 21.0a
T½el (h) 81.0 ± 31.0a 57.6 ± 13.9a
T½abs (h) 1.74 ± 0.66a 9.79 ± 4.06b
Ratio of the AUC
MNPSO2/MNP 		– 5.99 ± 2.08
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Tmax: time to peak plasma concentration; Cmax: peak plasma concentration; AUC0-LOQ: area under the plasma concentration vs. time curve from 0 to the quantification limit; AUC0-∞: area under the concentration-time curve extrapolated to infinity; MRT: mean residence time; T½el: elimination half-life; T½abs: absorption half-life (the value express the metabolite formation half-life for MNPSO2).

Pharmacokinetic parameters with different superscript letters are statistically different (P < 0.05).

Table 2 shows the overall faecal egg counts (arithmetic mean) and reduction percentages of faecal egg counts (FECR) (undifferentiated) with its 95% lower and upper confidence intervals obtained for all experimental groups on Farms 1, 2 and 3. The results of the FECRT with 99%, 96% and 98% of reduction for MNP on Farms 1, 2, and 3, respectively, demonstrated the high efficacy of this amino-acetonitrile derivative against GI nematodes resistant to IVM and RBZ in cattle. In fact, the low efficacies obtained for IVM (43%, 68% and 27% of reduction) confirm the presence of resistant parasites to this anthelmintic. On the other hand, the overall efficacy for RBZ on Farm 2 was 98%, demonstrating that this farm was the only one included in the study with a predominance of a RBZ-susceptible nematode population. Although the total efficacy for RBZ on Farm 1 was 94%, the 95% lower confidence interval for this anthelmintic was less than 90%, indicating an initial level of resistance. Finally, a higher level of resistance for RBZ was reported on Farm 3, where an overall reduction of 75% confirms the presence of resistant GI nematodes. In this context, whilst on Farms 1 and 2 significant (P < 0.05) differences were only observed between EPG counts post-IVMand MNP treatments, on Farm 3, the EPG counts after MNP were significantly (P < 0.05) lower than the egg counts after both IVM and RBZ.

Table 2.

Nematode egg per gram counts (EPG, arithmetic mean, range) and reduction percentages of faecal egg counts (FECR) (undifferentiated) with its 95% lower and upper confidence intervals, after the oral administration of monepantel (MNP, 2.5 mg/kg), and the subcutaneous administration of ivermectin (IVM, 0.2 mg/kg) and ricobendazole (RBZ, 3.75 mg/kg) to naturally parasitized calves.

Experimental Group FARM 1
 FARM 2
 FARM 3
EPG Counts (range)
 FECRa (CI) EPG Counts (range)
 FECRa (CI)
 EPG Counts (range)
 FECRa (CI)
Day −1 Day 15 Day −1 Day 15 Day −1 Day 15
MNP (oral) 547a (100–2440) 5.6a (0–20) 99% (97–99) 188a (100–400) 8a (0–20) 96% (90–98) 374a (140–740) 7a (0–20) 98% (95–99)
IVM (sc) 469a (100–1460) 269b (0–1060) 43% (0–73) 351a (100–660) 111b (0–320) 68% (42–83) 498a (140–1360) 362b (20–1520) 27% (0–69)
RBZ (sc) 508a (140–1380) 31a (0–120) 94% (85–97) 283a (120–580) 3a (0–20) 98% (94–99) 480a (140–1140) 115b (0–320) 75% (45–89)
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EPG counts on each column with different superscript letters are statistically different (P < 0.05).

a

FECR estimated according to McKenna (1990). CI: lower and upper confidence intervals.

The anthelmintic efficacies against Cooperia spp., Haemonchus spp., Ostertagia spp. and Oesophagostomum spp. for the different treatments on Farms 1, 2 and 3, are shown in Table 3. On Farms 1 and 3 IVM failed to control Haemonchus spp. and Cooperia spp., showing efficacies ranging from 0% to 80%. In the case of Farm 2, only IVM-resistant Cooperia spp. was present, being the others GI nematode genera susceptible to RBZ. The BZD treatment failed to control Cooperia spp. and Ostertagia spp. on Farms 1 and 3 (FECR below 90% for both nematode genera). In contrast, MNP was the only treatment that achieved 100% efficacy against Cooperia spp., Haemonchus spp. and Ostertagia spp., including against resistant parasites (99% against Ostertagia spp. on Farm 3). However, MNP failed to control Oesophagostomum spp., showing low efficacies of 74%, 22% and 64% against this genus on Farms 1, 2, and 3, respectively.

Table 3.

Reduction percentages of faecal egg counts (FECR) for Cooperia, Haemonchus, Ostertagia and Oesophagostomum spp. after the oral administration of monepantel (MNP, 2.5 mg/kg), and the subcutaneous administration of ivermectin (IVM, 0.2 mg/kg) and ricobendazole (RBZ, 3.75 mg/kg) to naturally parasitized calves.

Genus -Treatment FECRa Day 15
FARM 1 FARM 2 FARM 3
Cooperia spp.
MNPoral 100% 100% 100%
IVMsc 80% 56% 43%
RBZsc 86% 99% 54%
Haemonchus spp.
MNPoral 100% 100% 100%
IVMsc 19% 100% 0%
RBZsc 99% 95% 98%
Ostertagia spp.
MNPoral 100% 100% 99%
IVMsc 100% 100% 100%
RBZsc 89% 100% 0%
Oesophagostomum spp.
MNPoral 74% 22% 64%
IVMsc 100% 100% 100%
RBZsc 100% 100% 100%
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a

FECR estimated according to McKenna (1990).

Finally, no adverse events were observed in any of the cattle treated with MNP.

4. Discussion

Since GI parasitism negatively affects weight gain in grazing animals (Charlier et al., 2014a), parasite control is necessary to ensure adequate production levels on beef cattle farms. Alternative nematode control strategies, such as grazing management, host genetic resistance and helminth vaccines, are now being developed for further reduce reliance on chemically-based parasite control (Charlier et al., 2014b). However, dependence on anthelmintics continues to be high, since it is still being the most practical tool for parasite control on large scale commercial beef cattle farms. Due to the enormous difficulties involved in the development of novel anthelmintic molecules, such as the lastly introduced amino-acetonitrile derivative MNP, it is essential to understand its pharmacological behaviour to optimize its use in cattle under natural field conditions. The work described here reports for the first time an integrated assessment of MNP pharmacokinetics and pharmacodynamics (measured as anthelmintic efficacy), in cattle naturally infected with GI nematodes resistant to IVM and RBZ on a field trial performed on three different commercial farms.

The MNP plasma disposition kinetics has not been described in beef cattle. However, in line with previous PK studies in sheep (Karadzovska et al., 2009; Lifschitz et al., 2014) and dairy cows (Ballent et al., 2017), a rapid decline in the plasma profiles of the parent drug and the recovery of the MNPSO2 metabolite as the main analyte detected in the bloodstream, were observed in beef calves in the current trial. The metabolic conversion of MNP into MNPSO2 also involves the production of an intermediate sulphoxide derivative (Karadzovska et al., 2009), which is rapidly and almost completely converted into MNPSO2, being undetectable in plasma of MNP treated animals. In fact, the Cmax of the sulphone metabolite was four times higher than the corresponding parent concentration (21.5 vs 96.8 ng/mL for MNP and MNPSO2, respectively). Moreover, when MNP reached the Cmax (at 8 h post-oral treatment), the MNPSO2 metabolite was already about twice as high. Since MNPSO2 is an active metabolite against nematodes (Karadzovska et al., 2009), its high plasma and GI exposure greatly contribute to the overall MNP nematodicidal efficacy. In fact, the ratio of the total plasma AUC of MNPSO2 over the total AUC of MNP in both species, exhibited higher systemic exposure for MNPSO2 compared to the parent drug after the oral administration of MNP. However, interspecies differences in MNPSO2 systemic availability were observed between cattle and sheep. While Lifschitz et al. (2014) reported a MNPSO2/MNP AUC ratio of about 12 in sheep, a 50% lower value is described for that ratio after oral administration of MNP in cattle (Table 1). This finding may be explained by the different patterns of MNP liver metabolism (S-oxidation) between sheep and cattle. The rate of MNP conversion into MNPSO2 was five-fold higher in sheep compared to cattle (Ballent et al., 2016). While in sheep, the formation of the sulphone metabolite is based on the enzymatic activity of both flavin-monooxygenase (FMO) and cytochrome P- 450 (CYP), in cattle MNP is converted into MNPSO2 only in a CYP- mediated metabolic reaction (Ballent et al., 2016). These interspecies differences do not necessarily imply lower exposure of worms to the active drug. Moreover, considering MNP anthelmintic activity may be mainly based on a considerable drug/metabolite accumulation in the GI tissues and fluid contents during the first 2–3 days post-treatment, the different patterns of MNP liver metabolism between sheep and cattle should not affect its efficacy against GI nematodes (Lifschitz et al., 2014).

The results of the current PK assessment in cattle and those reported in sheep by Lifschitz et al. (2014) on the characterization of MNP accumulation in target tissues, give strong pharmacological support to the anthelmintic efficacy findings. The increasing worldwide prevalence of GI nematodes resistant to most of the traditional anthelmintic groups such as ML and BZD, therapeutic failures associated with anthelmintic resistance has enormous economic importance of global significance, particularly in countries where weather and production conditions contribute to a high incidence of parasitism. For instance, resistance to IVM was diagnosed in 93% of the farms tested in Argentina, while resistance to RBZ was diagnosed in 28% of the farms included in a nation-wide survey (Cristel et al., 2017). The main resistant genera were Cooperia spp. and Haemonchus spp. to IVM, and Ostertagia spp. and Cooperia spp. to RBZ (Cristel et al., 2017). Therefore, the efficacy of MNP was evaluated in scenarios where the nematode population was representative of the real situation on most commercial cattle farms. In this context, the efficacy results showed 99%, 96% and 98% of reduction for MNP on Farms 1, 2 and 3, respectively. These results demonstrated the high efficacy of MNP against resistant GI nematodes in cattle. Only limited information is available on MNP efficacy against GI nematodes in cattle (King et al., 2015). In that particular trial, MNP was administered in a combined formulation with abamectin. However, the reported efficacy results are consistent with those observed in our current trial with efficacies measured by FECR ranging from 98.3 to 99.9%. Similarly, the efficacy results observed in the present work are consistent with several studies in sheep (Bustamante et al., 2009; Hosking et al., 2009; Kaminsky et al., 2009; Sager et al., 2009). Bustamante et al. (2009) also evaluated MNP efficacy against IVM resistant nematode parasites. The low IVM efficacies obtained in the current work (43%, 68% and 27% of reduction on Farms 1, 2 and 3), confirm the presence of resistant nematode populations to this ML anthelmintic. Additionally, MNP was the only treatment that achieved >95% both in the overall efficacy and in the 95% lower confidence interval.

It should be considered that GI parasitism in cattle always involves different parasite genera.

In this sense, while on Farms 1 and 3 IVM failed to control Cooperia spp. and Haemonchus spp., on Farm 2 Cooperia spp. was the only genus resistant to IVM. Cooperia spp. is commonly present in the cases of IVM resistance in cattle. In fact, resistant Cooperia spp. was recovered in 100% of the farms where resistance to IVM were present in a survey carried out in Argentina in 2017 (Cristel et al., 2017). Cooperia spp. is one of the genera in which resistance to IVM is more frequent not only because it is a “dose-limiting” parasite for IVM (Benz et al., 1989), but also because routine IVM treatments are administered in the absence of any significant larval population in refugia (Sauermann and Leathwick 2018). However, similarly to our findings, some studies have also reported both Cooperia spp. and Haemonchus spp. resistant to IVM (Anziani et al., 2004; Ramos et al., 2016; Canton et al., 2018). Although RBZ achieved higher overall efficacies than IVM, the BZD treatment did not show effective control against all the GI nematodes present on Farms 1 and 3. Indeed, on these farms, RBZ failed to control Cooperia spp. and Ostertagia spp. (FECR below 90% for both nematode genera). In contrast, MNP was the only treatment that achieved 100% efficacy against Cooperia spp., Haemonchus spp. and Ostertagia spp. Similar results were found in different studies in sheep against resistant GI nematodes. Hosking et al. (2008) and Sager et al. (2009) demonstrated high (>95%) efficacy of MNP administered orally to sheep against GI nematodes resistant to either BZ or levamisole. Furthermore, Steffan et al. (2011) and Baker et al. (2012) showed almost 100% efficacy of MNP against GI nematodes multiple resistant to BZ, levamisole and ML. Although those studies were performed in sheep, their results and resistance scenarios were comparable with the current trial of MNP in cattle.

Efficacy of MNP against Oesophagostomum spp. is a particularly relevant issue due to efficacy results failed to meet an adequate reduction. The findings of the present study in cattle demonstrated that MNP failed only to control Oesophagostomum spp., with efficacies ranging from 22% to 74%. Similarly, it has been reported in sheep that Oesophagostomum was only reduced by 88% (Sager et al., 2009) and 61.9% (Bustamante et al., 2009). Furthermore, Hosking et al. (2009) also found efficacies below 90% against this nematode in sheep. In fact, the dose of 2.5 mg/kg was established as a suitable minimum dose rate (Kaminsky et al., 2009), because lower doses failed to control Oesophagostomum spp., which was established as the dose-limiting nematode for MNP (Hosking et al., 2010). Although a reduced sensitivity of this genus to MNP may explain its low efficacy, Lifschitz et al. (2014) suggested that a PK-related issue should contribute to this limited therapeutic response in sheep. The lower concentration of MNP achieved in the large intestine mucosa (225 ng/g) compared to that measured in the small intestine mucosa (562 ng/g in the ileum and 762 ng/g in the duodenum) may explain the efficacy levels obtained against Oesophagostomum spp. (Lifschitz et al., 2014), situation that could also occur in cattle. The PK/PD of MNP against GI nematodes may suggest that the high concentrations of MNP parental drug achieved in the GI contents and mucosa during 48–72 h after its oral administration are relevant to the effectiveness of this compound (Lifschitz et al., 2014).

The activity of MNP against multidrug-resistant parasites, which is based on its novel mode of action, is a highly favorable element. However, resistance to MNP has occurred on the field within less than 2 years of the product first being used in sheep and goats in New Zealand. In this first report of resistance in goats excessively treated with the amino-acetonitrile derivative, MNP was ineffective against at least two GI nematode species, Teladorsagia circumcincta and Trichostrongylus colubriformis (Scott et al., 2013). Moreover, Mederos et al. (2014) found Haemonchus contortus resistant to MNP on sheep farms in Uruguay. Lack of efficacy of MNP was also reported on sheep farms in the Netherlands (van den Brom et al., 2015), Brazil (Cintra et al., 2016), Australia (Sales and Love, 2016), Argentina (Illanes et al., 2018) and the United Kingdom (Hamer et al., 2018; Bartley et al., 2019). Considering that resistance to MNP has already been reported in sheep in different countries, it is essential to understand the mechanisms of resistance to this compound. In this way, the presence of multiple separate mutations in theMPTL-1 gene in field-derived H. contortus and T. circumcincta isolates may at least partly explain MNP resistance (Bagnall et al., 2017; Turnbull et al., 2019). The reports of resistance highlight the need to learn from the use of this anthelmintic on sheep farms. It is essential to maintain the awareness on the possibility of development of resistance to MNP in cattle nematode parasites, which includes the need to follow appropriate guidelines of parasite control (Bartley et al., 2019).

Overall, there is no published reports on the simultaneous assessment of the relationship between the PK performance and the anthelmintic therapeutic response to MNP in cattle. The results of the current work determined that the oral route is a very efficient administration route for MNP in beef cattle. This is particularly relevant when the described high systemic exposure of the anthelmintically active MNP and MNPSO2 exposure is considered. MNP achieved effective control of GI nematodes with multiple anthelmintic resistance to ML and BZD. The widespread appearance of resistant parasites highlights the need for novel anthelmintics acting at novel target sites to be used in cattle, such as MNP. However, it is now crucial to accomplish adequate management of this novel compound to prolong its lifespan and optimize parasite control based on diagnosis and treatment strategies implemented on an individual cattle farm basis. The findings described here contribute to the knowledge on MNP pharmacology and efficacy against resistant GI nematodes in beef cattle.

Declaration of competing interest

There are no potential conflicts of interest associated with this study.

Acknowledgements

This study was funded by Agencia Nacional de Promoción Científica y Técnica (ANPCyT) (PICT 2014–0683) from Argentina. The authors would like to thank the farmers for collaborating with this study.

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