Further investigation of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [³H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.     

Temperature dependent binding of mebendazole to tubulin in benzimidazole-susceptible and -resistant strains of Trichostrongylus colubriformis and Caenorhabditis elegans

The binding of [³H]mebendazole ([³H]MBZ) to tubulin in benzimidazole-susceptible (BZ-S) and benzimidazole-resistant (BZ-R) strains of Trichostrongylus colubriformis and Caenorhabditis elegans was examined in order to investigate the biochemical changes to tubulin that result in BZ resistance in parasitic and free-living nematodes. In both species the extent of [³H]MBZ binding to tubulin was significantly reduced in the BZ-R strain compared with the BZ-S strain. The decrease in [³H]MBZ binding in the BZ-R strain of each species was the result of a significant reduction in the amount of charcoal stable [³H]MBZ-tubulin complexes and was not related to a change in the association constant of the [³H]MBZ-tubulin interaction. [³H]MBZ binding to tubulin was temperature dependent, reaching maximum levels at 37°C in BZ-S T. colubriformis and 10°C in BZ-R T. colubriformis. Both the BZ-S and BZ-R strains of C. elegans displayed maximum [³H]MBZ binding at 4°C. Resistance ratios derived from the amount of [³H]MBZ binding in the BZ-S and BZ-R strains and in vitro development assays demonstrated that the temperature dependence and extent of drug binding was indicative of BZ resistance status and was species specific in the BZ-S isolates. These results indicate that biochemical differences exist in the binding of benzimidazole carbamates to tubulin in nematode species, and suggest that the susceptibility of the parasitic nematodes to the benzimidazole anthelmintics is the result of a unique high affinity and/or high capacity interaction ofbenzimidazole carbamates with tubulin.     

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.     

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 β-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 β-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 β-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.     

Structural characterization of the papaya cysteine proteinases at low pH

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.     

Nippostrongylus brasiliensis: effect of dexamethasone upon anthelmintic efficacy

A study has been made to determine the chemotherapeutic susceptibility of natural and “steroid sustained” Nippostrongylus brasiliensis infections in rats. Dexamethasone in the drinking water (2.5 × 10^?4 mg/ml) aided establishment of greater parasite populations but restricted growth of young rats. Comparison trials with thiabendazole, cambendazole, and parbendazole, as well as 10 candidate drugs, showed that the parasiticidal activity of these materials was not significantly altered between the 10-day-old naturally maintained and the 17-day-old steroid-treated parasitic infections. As expected, 17-day-old naturally maintained parasite populations were highly susceptible to the anthelmintic action of these materials. In one instance, the toxic response elicited by a candidate drug was apparently potentiated by dexamethasone.     

SLO,SLO, quick,quick, slow: calcium-activated potassium channels as regulators of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> behaviour and targets for anthelmintics

Large-conductance calcium and voltage-activated potassium channels, termed SLO-1 (or BK), are pivotal players in the regulation of cell excitability across the animal phyla. Furthermore, emerging evidence indicates that these channels are key mediators of a number of neuroactive drugs, including the most recent new anthelmintic, the cyclo-octadepsipeptide emodepside. Detailed reviews of the structure, function and pharmacology of BK channels have recently been provided (Salkoff et al. in Nat Rev Neurosci 7:921–931, 2006; Ghatta et al. in Pharmacol Ther 110:103–116, 2006) and therefore these aspects will only briefly be covered here. The purpose of this review is to discuss how SLO-1 channels might function as regulators of neural transmission and network activity. In particular, we focus on the role of SLO-1 in the regulation of Caenorhabditis elegans behaviour and highlight the role of this channel as an effector for pleiotropic actions of neuroactive drugs, including emodepside. On the premise that C. elegans is a ‘model nematode’ with respect to many aspects of neural function, the intention is that this might inform a broader understanding of the role of these channels in the nematodes and their potential as novel anthelmintic targets.     

Unresolved issues in anthelmintic pharmacology for helminthiases of humans

Helminth infections are an important constraint on the health and development of poor children and adults. Anthelmintic treatment programmes provide a safe and effective response, and increasing numbers of people are benefitting from these public health initiatives. Despite decades of clinical experience with anthelmintics for the treatment of human infections, relatively little is known about their clinical pharmacology. All of the drugs were developed initially in response to the considerable market for veterinary anthelmintics in high- and middle-income countries. In contrast, the greatest burden caused by these infections in humans is in resource-poor settings and as a result there has been insufficient commercial incentive to support studies on how these drugs work in humans, and how they should best be used in control programmes. The advent of mass drug administration programmes for the control of schistosomiasis, lymphatic filariasis, onchocerciasis and soil-transmitted helminthiases in humans increases the urgency to better understand and better monitor drug resistance, and to broaden the currently very narrow range of available anthelmintics. This provides fresh impetus for developing a comprehensive research platform designed to improve our understanding of these important drugs, in order to bring the scientific knowledge base supporting their use to a standard equivalent to that of drugs commonly used in developed countries. Furthermore, a better understanding of their clinical pharmacology will enable improved therapy and could contribute to the discovery of new products.     

Anthelmintic efficacy on UK Thoroughbred stud farms

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14–17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n = 429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8–100%; 95% lower confidence intervals range: 96.8–100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7–100%; 95% lower confidence intervals range: 65–100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0–73%; 95% lower confidence intervals range: 0–59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98–99.4%; 95% lower confidence intervals range: 91.8–99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4–41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5–99.9%; 95% lower confidence intervals range: 96.3–99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5–91.2%; 95% lower confidence intervals range: 6.7–82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.     

Flavones from Struthiola argentea with anthelmintic activity in vitro

Parasitic diseases caused by helminthes lead to significant health hazards to animals resulting in enormous economic impact. While a number of anthelmintics are currently available, all are encountering resistance and ones with a mode of action are needed. We report herein bioassay-guided isolation of three anthelmintic flavones 1-3, including the flavone, 5,6,2',5',6'-pentamethoxy-3',4'-methylenedioxyflavone (3) from the methanol extract of Struthiola argentea (Thymelaeaceae). The structure of 3 was elucidated by analysis of its 1D and 2D NMR and MS data. The two major flavones produced by this plant were also isolated and identified as yuankanin (4) and amentoflavone (5). A number of flavones related to the compounds isolated from S. argentea were acquired and tested to ascertain structure activity relationships. The isolation, structure, anthelmintic activity and structure activity relationships of the flavones are described. Compound 3 exhibited the most potent in vitro activity with 90% inhibition of larval motility at 3.1 microg/mL and compound 15 showed modest in vivo activity.