Cyathostomin resistance to moxidectin and combinations of anthelmintics in Australian horses.

BACKGROUND: Cyathostomins are the most important and common parasitic nematodes of horses, with > 50 species known to occur worldwide. The frequent and indiscriminate use of anthelmintics has resulted in the development of anthelmintic resistance (AR) in horse nematodes. In this study we assessed the efficacy of commonly used anthelmintics against cyathostomins in Australian thoroughbred horses. METHODS: Two drug efficacy trials per farm were conducted on two thoroughbred horse farms in the state of Victoria, Australia. In the first trial, the horses on Farm A were treated with single and combinations of anthelmintics, including oxfendazole (OFZ), abamectin (ABM), abamectin and morantel (ABM + MOR), moxidectin (MOX) and oxfendazole and pyrantel (OFZ + PYR), at the recommended doses, whereas the horses on Farm B only received MOX, at the recommended dose. The faecal egg count reduction test (FECRT) was used to determine the efficacy and egg reappearance period (ERP) of anthelmintics. Based on the results of the first trial, the efficacies of MOX and a combination of ABM + MOR were reassessed to confirm their activities against cyathostomins. RESULTS: Of the five anthelmintic products tested on Farm A, resistance against OFZ, ABM and OFZ + PYR was found, with efficacies of - 41% (- 195% lower confidence limit [LCL]), 73% (60% LCL) and 82% (66% LCL) at 2 weeks post-treatment, respectively. The FECRT showed high efficacies of MOX and ABM + MOR (100%) at 2 week post-treatment and shortened ERPs for these anthelmintics (ABM + MOR: 4 weeks; MOX: 5 weeks). Resistance to MOX was found on Farm B, with a reduced efficacy of 90% (70% LCL) and 89% (82% LCL) at 2 weeks post-treatment in trials one and two, respectively. CONCLUSIONS: This study provides the first evidence of MOX- and multidrug-resistant (ABM and combinations of anthelmintics) cyathostomins in Australia and indicates the need for continuous surveillance of the efficacy of currently effective anthelmintics and large-scale investigations to assess the ERP for various anthelmintics.

Anthelmintic resistance and homeostatic plasticity (Brugia malayi).

Homeostatic plasticity refers to the capacity of excitable cells to regulate their activity to make compensatory adjustments to long-lasting stimulation. It is found across the spectrum of vertebrate and invertebrate species and is driven by changes in cytosolic calcium; it has not been explored in parasitic nematodes when treated with therapeutic drugs. Here we have studied the adaptation of Brugia malayi to exposure to the anthelmintic, levamisole that activates muscle AChR ion-channels. We found three phases of the Brugia malayi motility responses as they adapted to levamisole: an initial spastic paralysis; a flaccid paralysis that follows; and finally, a recovery of motility with loss of sensitivity to levamisole at 4 h. Motility, calcium-imaging, patch-clamp and molecular experiments showed the muscle AChRs are dynamic with mechanisms that adjust their subtype composition and sensitivity to levamisole. This homeostatic plasticity allows the parasite to adapt resisting the anthelmintic.

Competitive inhibition of the high-affinity choline transporter by tetrahydropyrimidine anthelmintics.

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 µM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.

The cholinomimetic morantel as an open channel blocker of the Ascaris suum ACR-16 nAChR.

Nematode parasite infections pose a significant threat in human and veterinary medicine. At least a third of the world's population is at risk from nematode parasite infections. These infections not only cause health problems, but also cause loss of livestock production and hence, economic losses. Anthelmintic drugs are the mainstay by which control of nematode parasite infections is achieved. Many of the currently available anthelmintics act on nicotinic acetylcholine receptors (nAChRs). However, the detailed mode of action (MOA) of these anthelmintics is not clearly understood. Elucidation of the MOA of anthelmintics is highly desirable; an in-depth knowledge of the MOA will better inform on mechanisms of resistance development and on ways to slow down or overcome resistance. The cholinomimetic anthelmintic, morantel, has a complex MOA involving the activation and block of levamisole-sensitive single nAChR channels (L-type nAChR or L-nAChR). More recently, morantel has been demonstrated to activate Haemonchus contortus and Parascaris equorum ACR-26/ACR-27 nAChRs expressed in Xenopus laevis oocytes. Previous studies in our laboratory, however, have shown morantel does not activate the nicotine-sensitive nAChR (N-type nAChR or N-nAChR), Ascaris suum ACR-16 (Asu-ACR-16). In this study, we used two-electrode voltage-clamp (TEVC) electrophysiology to investigate the inhibitory effects of morantel, on expressed Asu-ACR-16 nAChRs in X. laevis oocytes. Our results show that morantel acts as a non-competitive antagonist on Asu-ACR-16. This non-competitive antagonism by morantel was further demonstrated to be voltage-sensitive. We conclude based on our findings that morantel is a non-competitive voltage-sensitive open channel blocker of Asu-ACR-16.

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

Intra-subunit flexibility underlies activation and allosteric modulation of neuronal nicotinic acetylcholine receptors.

Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3ß2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of ß sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer ß sheet meets the ß sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these ß sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function.

The positive allosteric modulator morantel binds at noncanonical subunit interfaces of neuronal nicotinic acetylcholine receptors.

We are interested in the positive allosteric modulation of neuronal nicotinic acetylcholine (ACh) receptors and have recently shown that the anthelmintic compound morantel potentiates by enhancing channel gating of the alpha3beta2 subtype. Based on the demonstration that morantel-elicited currents were inhibited by the classic ACh competitor dihydro-beta-erythroidine in a noncompetitive manner and that morantel still potentiates at saturating concentrations of agonist (Wu et al., 2008), we hypothesized that morantel binds at the noncanonical beta2(+)/alpha3(-) subunit interface. In the present study, we created seven cysteine-substituted subunits by site-directed mutagenesis, choosing residues in the putative morantel binding site with the aid of structural homology models. We coexpressed the mutant subunits and their respective wild-type partners in Xenopus oocytes and characterized the morantel potentiation of ACh-evoked currents, as well as morantel-evoked currents, before and after treatment with a variety of methanethiosulfonate (MTS)-based compounds, using voltage-clamp recordings. The properties of four of the seven mutants, two residues on each side of the interface, were changed by MTS treatments. Coapplication with ACh enhanced the extent of MTS modification for alpha3A106Cbeta2 and alpha3beta2S192C receptors. The activities of two mutants, alpha3T115Cbeta2 and alpha3beta2T150C, were dramatically altered by MTS modification. For alpha3beta2T150C, while peak current amplitudes were reduced, potentiation was enhanced. For alpha3T115Cbeta2, both current amplitudes and potentiation were reduced. MTS modification and morantel were mutually inhibitory: MTS treatment decreased morantel-evoked currents and morantel decreased the rate of MTS modification. We conclude that the four residues showing MTS effects contribute to the morantel binding site.

Glutamine 57 at the complementary binding site face is a key determinant of morantel selectivity for {alpha}7 nicotinic receptors.

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal alpha7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high efficacy and more potent agonist than ACh of alpha7 receptors. The EC(50) for activation by morantel of both alpha7 and alpha7-5HT(3A) receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate alpha7-5HT(3A) channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of alpha7 and alpha7-5HT(3A), and both drugs are very low efficacy agonists of muscle AChRs. The replacement of Gln(57) in alpha7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (epsilonG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to Trp(149) of the principal face of the binding site, whereas the other cyclic group is proximal to Gln(57) of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for alpha7. These results provide new information for further progress in drug design.

Morantel allosterically enhances channel gating of neuronal nicotinic acetylcholine alpha 3 beta 2 receptors.

We studied allosteric potentiation of rat alpha3beta2 neuronal nicotinic acetylcholine receptors (nAChRs) by the anthelmintic compound morantel. Macroscopic currents evoked by acetylcholine (ACh) from nAChRs expressed in Xenopus laevis oocytes increase up to 8-fold in the presence of low concentrations of morantel (< or =10 microM); the magnitude of the potentiation depends on both agonist and modulator concentrations. It is noteworthy that the potentiated currents exceed the maximum currents achieved by saturating (millimolar) concentrations of agonist. Studies of macroscopic currents elicited by prolonged drug applications (100-300 s) indicate that morantel does not increase alpha3beta2 receptor activity by reducing slow (> or =1 s) desensitization. Instead, using outside-out patch-clamp recordings, we demonstrate that morantel increases the frequency of single-channel openings and alters the bursting characteristics of the openings in a manner consistent with enhanced channel gating; these results quantitatively explain the macroscopic current potentiation. Morantel is a very weak agonist alone, but we show that the classic competitive antagonist dihydro-beta-erythroidine inhibits morantel-evoked currents noncompetitively, indicating that morantel does not bind to the canonical ACh binding sites.

Activation of single nicotinic receptor channels from Caenorhabditis elegans muscle.

Nicotinic acetylcholine receptors (nAChRs) are pentameric neurotransmitter-gated ion channels that mediate synaptic transmission throughout the nervous system in vertebrates and invertebrates. Caenorhabditis elegans is a nonmammalian model for the study of the nervous system and a model of parasitic nematodes. Nematode muscle nAChRs are of considerable interest because they are targets for anthelmintic drugs. We show single-channel activity of C. elegans muscle nAChRs for the first time. Our results reveal that in the L1 larval stage acetylcholine (ACh) activates mainly a levamisole-sensitive nAChR (L-AChR). A single population of 39 pS channels, which are 5-fold more sensitive to levamisole than ACh, is detected. In contrast to mammalian nAChRs, open durations are longer for levamisole than for ACh. Studies in mutant strains reveal that UNC-38, UNC-63, and UNC-29 subunits are assembled into a single L-AChR in the L1 stage and that these subunits are irreplaceable, suggesting that they are vital for receptor function throughout development. Recordings from a strain mutated in the LEV-1 subunit show a main population of channels with lower conductance (26 pS), prolonged open durations, and reduced sensitivity to levamisole. Thus, although LEV-1 is preferentially incorporated into native L-AChRs, receptors lacking this subunit can still function. No single-channel activity from levamisole-insensitive nAChRs is detected. Thus, during neuromuscular transmission in C. elegans, the majority of ACh-activated current flows through L-AChRs. This study contributes to the understanding of the molecular mechanisms underlying functional diversity of the nAChR family and offers an excellent strategy to test novel antiparasitic drugs.

Acid phosphatase activity in excretion/secretion products from Heligmosomoides polygyrus adults: an indicator of the physiological status of the worms.

Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus. Female and male excretion/secretion products showed similar specific activity. For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate. The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin). These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms.

Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken alpha7 and ACR-16 from the nematode Caenorhabditis elegans.

Two homomer-forming nicotinic acetylcholine receptor subunits with 47% identity in their amino acid sequences were employed to compare the actions of cholinergic anthelmintics and ivermectin on expressed vertebrate and nematode nicotinic receptors of known molecular composition. Voltage-clamp electrophysiology was used to study recombinant nicotinic receptors expressed in Xenopus laevis oocytes following nuclear injection of cDNA encoding either chicken alpha7 or Caenorhabditis elegans ACR-16 (Ce21) subunits. Butamisole, morantel and metyridine were without agonist actions on either alpha7 or ACR-16 nicotinic receptors in the range 10nM-1mM. However, butamisole (pIC(50)=4.9 for both alpha7 and ACR-16) and morantel (pIC(50)=5.6 for alpha7 and 5.7 for ACR-16) antagonized responses of both alpha7 and ACR-16 receptors to acetylcholine. Metyridine (1mM) did not affect responses to acetylcholine of either receptor. Oxantel was without agonist actions on ACR-16, but was an acetylcholine antagonist (pIC(50)=5.4). In contrast, it was found to have low efficacy agonist action (pEC(50)=4.4) on alpha7 at concentrations in the range 10-300microM. In agreement with a previous study, ivermectin (30microM), an agonist of L-glutamate-gated chloride channels, enhanced the amplitude of responses to acetylcholine of alpha7 nicotinic receptors. However, this same concentration of ivermectin (30microM) did not potentiate the acetylcholine-induced responses of ACR-16, but rather resulted in a slight attenuation. We conclude that oxantel and ivermectin have identified new pharmacological differences between the chicken alpha7 nicotinic receptor and its C. elegans homologue ACR-16.

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics.

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Fumarate metabolism and the microaerophily of Campylobacter species.

(1) The role of fumarate metabolism in the microaerophily of the Campylobacter genus and the effects of therapeutic agents against it were investigated. (2) NMR spectroscopy was employed to determine the properties of Campylobacter fumarase (Fum) and fumarate reductase (Frd). Radiotracer analysis was used to determine the production of carbon dioxide by Campylobacter cells. Standard microbiological techniques were used to measure the effects of environmental conditions and inhibitors on bacterial growth. (3) All Campylobacter species tested showed both Fum and Frd activities. Frd activity was observed with or without the addition of an exogenous electron donor in the particulate fractions obtained from lysates. Fumarate was oxidized to carbon dioxide via the acetyl-CoA cleavage pathway. The genes encoding proteins involved in fumarate metabolism were identified in the Campylobacter jejuni genome. Cells grew better in atmospheres with 5 and 10% oxygen levels. Fum activity was the same in cultures grown under different oxygen tensions and did not vary with the age of cultures. Frd activity was higher in cultures which grew at faster rates and decreased with the age of cultures. Four Frd inhibitors showed bactericidal effects against Campylobacter spp. with different potencies. The relative strengths of inhibition of the compounds followed the same order as the bactericidal effects. (4) The results suggested that Frd and Fum are constitutive and play a fundamental role in these microaerophiles which show characteristics of anaerobic metabolism, and that the Frd inhibitors tested would not be of therapeutic use.

The in vitro motility response to various anthelmintics of third-stage larvae of Oesophagostomum spp. from pigs.

The in vitro activities of thiabendazole, levamisole, pyrantel, morantel and ivermectin against Oesophagostomum spp., the nodular worm of pigs, were determined and compared. The study was carried out using isolates of O. dentatum and O. quadrispinulatum, which had been defined in vivo. Infective larvae were exposed to the anthelmintics for 24 h and then placed in a micromotility meter. All the treatments significantly reduced the motility of the ensheathed L3 larvae, but the micromotility meter was not able to differentiate between anthelmintic resistant and anthelmintic susceptible isolates.

Efficacy of moxidectin and other anthelmintics against small strongyles in horses.

OBJECTIVE: To compare the efficacy of moxidectin to ivermectin, oxibendazole and morantel against some gastrointestinal nematodes in horses. DESIGN: Faecal egg count reduction after treatment. PROCEDURE: A farm was selected where the population of small strongyles in horses was known to be resistant to oxibendazole. Horses were allocated to treatment groups based on faecal egg counts. After treatment, faecal samples were taken up to 109 days after treatment and faecal egg counts estimated. Faecal cultures were used to estimate the contribution of small and large strongyles to the faecal egg counts at each sampling. RESULTS: Moxidectin (0.4 mg/kg) suppressed faecal egg counts for 109 days after treatment in most horses compared to 40 days with ivermectin (0.2 mg/kg), 13 days with morantel (9.4 mg/kg) and less than 13 days with oxibendazole (10 mg/kg). Most of the faecal egg count was attributable to small strongyles based on faecal culture, although Strongylus vulgaris was present in some samples in low numbers. Oxibendazole resistance in small strongyles was confirmed and a less than expected efficacy of morantel was also seen. CONCLUSION: Moxidectin was highly effective in reducing faecal egg counts after treatment for at least 12 weeks and up to 16 weeks in most horses. These horses were infected with a population of small strongyles known to be resistant to oxibendazole and possibly morantel. The duration of the reduction in faecal egg counts after treatment with moxidectin (0.4 mg/kg) was at least twice that of ivermectin (0.2 mg/kg) and greater than four times that for morantel and oxibendazole.

Activation and cooperative multi-ion block of single nicotinic-acetylcholine channel currents of Ascaris muscle by the tetrahydropyrimidine anthelmintic, morantel.

1. We have investigated activation and block, by the tetrahydropyrimidine anthelmintic, morantel, of nicotinic-acetylcholine receptor (AChR) currents in membrane vesicles isolated from somatic muscle cells of the nematode parasite Ascaris suum. Standard single-channel recording techniques were employed. Morantel in the pipette (6 nM to 600 microM), activated single nicotinic AChR currents. 2. Kinetic properties of the main-conductance state of morantel-activated currents were investigated in detail throughout the concentration range, 0.6 microM to 600 microM. Open-time distributions were best fitted by a single exponential. Mean open-times were slightly voltage-dependent, increasing from 0.9 ms at +75 mV to 1.74 ms at -75 mV in the presence of 0.6 microM morantel. At low concentrations, closed-time distributions were best fitted by the sum of two or three exponential components. 3. As the concentration of morantel was increased (100-600 microM), fast-flickering open channel-block was observed at positive potentials, even though morantel, a cation, was only present at the extracellular surface of the membrane. The block rate was dependent on morantel concentration and both block rate and duration of block increased as the potential became less positive. A simple channel-block mechanism did not explain properties of this block. 4. At negative potentials, as the morantel concentration increased, a complex block was observed. With increases in morantel concentration two additional gap components appeared in closed-time distributions: one was short with a duration (approximately 13 ms) independent of morantel concentration; the other was long with a duration that increased with morantel concentration (up to many minutes). In combination, these two components produced a marked reduction in probability of channel opening (Po) with increasing morantel concentration. The relationship between the degrees of block and morantel concentration had a Hill coefficient of 1.6, suggesting the involvement of at least two blocking molecules. The data were analysed by use of a simple sequential double block kinetic model.

In vitro characterization of anthelmintic susceptibility of field isolates of the pig nodular worm Oesophagostomum spp., susceptible or resistant to various anthelmintics.

A larval development assay (LDA) and an egg hatch paralysis assay (EHPA) were used to measure the sensitivity to anthelmintics of eggs and larvae of nodular worms (Oesophagostomum spp.) in pigs. The tests were carried out using in vivo defined resistant and susceptible isolates of Oesophagostomum dentatum, O. quadrispinulatum and Oesophagostomum spp. For measurement of pyrantel/morantel and levamisole sensitivity the LDA was found able to distinguish between susceptible or resistant isolates of Oesophagostomum. The EHPA was able to detect levamisole resistance, but the test failed to show differences in response to pyrantel between pyrantel susceptible and resistant lines. The possible routine application of LDA and EHPA in the diagnosis of anthelmintic resistance in Oesophagostomum spp. is discussed.