Interaction of benzimidazole anthelmintics with Haemonchus contortus tubulin: binding affinity and anthelmintic efficacy

The ability of various benzimidazoles (BZs) to bind tubulin under different conditions was assessed by determining their IC50 values (the concentration of unlabeled drug required to inhibit 50% of the labeled drug binding), Ka (the apparent equilibrium association constant) and Bmax (the maximum binding at infinite [BZ] = [drug-receptor]). The ability of unlabeled benzimidazoles--fenbendazole, mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ), rycobendazole (albendazole sulfoxide, ABZSO), albendazole sulfone, oxfendazole (OFZ), and thiabendazole--to bind tubulin was determined from their ability to inhibit the binding of [3H]MBZ or [3H]OBZ to tubulin in supernatants derived from unembryonated eggs or adult worms of Haemonchus contortus. The binding constants (IC50, Ka, and Bmax) correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZ compounds except for OFZ and ABZSO whose Ka values were lower than could be expected from anthelmintic potency. The binding of [3H]ABZ or [3H]OFZ to tubulin in supernatants derived from BZ-susceptible and BZ-resistant H. contortus was compared. [3H]ABZ demonstrated saturable high-affinity binding but [3H]OFZ bound with low affinity. The high-affinity binding of [3H]ABZ was reduced for the R strain. Tubulin bound BZ drugs at 4 degrees C with lower apparent Ka than at 37 degrees C.     

Inhibited development in Haemonchus contortus.

Inhibited development of Haemonchus contortus was studied in single experimental infections of worm-free lambs. Chilling of the infective larvae at +4 degrees C was without effect on the percentage of larvae subsequently becoming inhibited and a period of exposure to autumnal conditions was unnecessary to induce a high rate of inhibition. It was concluded that seasonal inhibition of H. contortus in East Anglia is brought about primarily by an environmental stimulus acting upon the preparasitic stages but that, unlike Obeliscoides cuniculi and Ostertagia ostertagi, this was not cold. It could be provided in a culture kept in the dark at 25 degrees C for 12 days. While the age of the host did influence the phenomenon, in that larvae were less inclined to inhibition in very young animals, it was concluded that this was not a primary factor in the aetiology.     

Detection of resistance to ivermectin in Haemonchus contortus.

Infective, third-stage (L3) larvae of Haemonchus contortus isolates resistant to ivermectin (IVM) show a decreased sensitivity to IVM-induced paralysis in vitro. The inhibition of larval motility by IVM can be detected in L3 larvae incubated in the dark on an agar matrix containing IVM, by the failure of affected larvae to move when stimulated by exposure to light. Optimally, avermectin (AVM) potency is quantified after three cycles, each involving storage in the dark for 24 h followed by a brief exposure to light. For IVM-susceptible isolates, a 50% inhibition of motility (LP50) was achieved with IVM concentrations between 0.30 and 0.49 microM, while LP50 values in IVM-resistant isolates ranged from 0.8 to 2.6 microM depending on the in vivo resistance status of the isolate. A limited study of structure-activity relationships within the AVM class indicated that in vitro inhibition of L3 motility was consistent with the known in vivo efficacy of each analogue. Resistance factors for IVM-resistant isolates were dependent on AVM structure with the more polar AVM B2 analogue being a particularly sensitive probe of IVM-resistance status.     

The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the blood of treated hosts. Closantel binds specifically to ovine serum albumin (K(a) of 9. 3x10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier.     

The inheritance of thiabendazole resistance in Haemonchus contortus.

Haemonchus contortus worm populations isolated from naturally infected sheep at the Pastoral Research Laboratory, Armidale, N.S.W., were found to contain approximately 20% of worms resistant to a 50 mg/kg dose of thiabendazole. Following 3 generations of selection with 50 mg/kg thiabendazole the number of worms removed by the anthelmintic was too small to detect differences between treated and control groups. After more than 15 generations of selection, matings between males from the selected strain and non-resistant females produced resistant males and females in equal numbers. Thus, thiabendazole resistance does not appear to be sex-linked. A dose--response assay on the F2 adults indicated that worms from female resistant x male non-resistant crosses were more resistant than F2 adults of the reciprocal cross. An in vitro technique that identified thiabendazole-resistant eggs by their ability to hatch in a solution containing thiabendazole and 0.1% NaCl solution was also used to study the inheritance of resistance. F1 eggs had similar LC50's to the resistant parents. F2 and back-cross eggs from an original mating of thiabendazole-resistant females x non-resistant males had a higher LC50 than F2 and back-cross eggs from the reciprocal mating, indicating a degree of matroclinous inheritance of resistance. However, the resistant parents had tolerances to thiabendazole exceeding those of F2. F3 eggs had a resistance distribution that ranged from that of the resistant to the non-resistant parent. No significant deviation from linearity was observed in any of the dose--response lines. These results indicate that thiabendazole resistance in H. contortus worms is inherited as an autosomal and semi-dominant trait.     

No adaptation of Haemonchus contortus to genetically resistant sheep.

A composite population of Haemonchus contortus was established with larvae from seven diverse sources, then maintained in Merino sheep bred to have either increased or decreased resistance to Haemonchus. After five, seven and 14 parasite generations, the two resulting lines of parasites were used to infect sheep from the increased resistance line, an unselected control line and the decreased resistance line. Line of sheep had a highly significant effect on average faecal egg counts 4 and 5 weeks after infection (geometric means 329, 735 and 1490 epg, respectively after the 14th generation), but the two lines of parasites yielded similar egg counts. There was no significant interaction between line of sheep and line of parasite, indicating that the parasite populations had not diverged significantly in their reproductive fitness, as measured by faecal egg count.     

Haemonchus contortus: sequence heterogeneity of internucleotide binding domains from P-glycoproteins

P-Glycoproteins are transmembrane proteins associated with acquired multidrug resistance in mammalian cells and some protozoan parasites by a process of active drug export. P-glycoproteins contain two nucleotide binding domains which couple ATP to the drug transport process. The region between the nucleotide binding domains of P-glycoproteins, termed the internucleotide binding domain (IBD), was PCR-amplified from adult and larval cDNA libraries using degenerate primers. The 11 clones isolated by this method fall into several distinct groups, with one group of alleles displaying between 82 and 99% identity at the nucleotide level. This sets a baseline for sequence variation of transcribed alleles from a parasitic nematode. Northern blotting showed that P-glycoprotein genes are transcribed in a developmentally regulated fashion in Haemonchus contortus. Southern blots of H. contortus drug-resistant isolates with an IBD probe revealed a pattern consistent with the involvement of P-glycoprotein in resistance to avermectin/milbemycin anthelmintics.     

Somatic proteome of Haemonchus contortus

Currently, there is a dearth of proteomic data to underpin fundamental investigations of parasites and parasitism at the molecular level. Here, using a high throughput LC–MS/MS-based approach, we undertook the first reported comprehensive, large-scale proteomic investigation of the barber's pole worm (Haemonchus contortus) – one of the most important parasitic nematodes of livestock animals worldwide. In total, 2487 unique H. contortus proteins representing different developmental stages/sexes (i.e. eggs, L3s and L4s, female (Af) and male (Am) adults) were identified and quantified with high confidence. Bioinformatic analyses of this proteome revealed substantial alterations in protein profiles during the life cycle, particularly in the transition from the free-living to the parasitic phase, and key groups of proteins involved specifically in feeding, digestion, metabolism, development, parasite-host interactions (including immunomodulation), structural remodelling of the body wall and adaptive processes during parasitism. This proteomic data set will facilitate future molecular, biochemical and physiological investigations of H. contortus and related nematodes, and the discovery of novel intervention targets against haemonchosis.     

Field studies on inhibition of Haemonchus contortus.

The epidemiology of Haemonchus contortus was studied in north east England in lambs reared under field conditions with infected ewes. The parasite population was monitored by pasture larval counts, faecal egg counts and lamb postmortems. From 25 June to 11 November, eight lambs were slaughtered each month, together with eight worm-free tracer lambs. Postmortem worm counts showed that on 25 June, small numbers of adult H. contortus were present but no inhibited larvae. However, subsequently the number of worms recovered rose rapidly to 1000-5000 and the percentage inhibition increased to 57% in July, 75% in August and virtually 100% in September, October and November. On each sampling occasion no significant differences in worm count or percentage inhibition were recorded between experimental and tracer lambs. These results suggest that neither autumn climatic effects nor host immunity were responsible for inhibition in this strain of H. contortus.     

Resistance to ivermectin by Haemonchus contortus in goats and calves.

Efficacy of ivermectin on susceptible or resistant populations of the parasitic nematode Haemonchus contortus was determined in cattle and goats held in a barn. Goats were each infected with 3000 infective, ivermectin-susceptible or -resistant H. contortus larvae on day 0 and reinfected with 2000 infective larvae on day 24. Goats were treated orally with 600 micrograms kg-1 ivermectin on day 31. No significant differences were detected in blood packed cell volume (PCV) or total protein (TP), prepatent period, or epg among the four groups of goats that were each infected with one of four parasite strains (one susceptible, three resistant). There were no differences among the four parasite strains in the numbers of infective larvae that developed to the third larval stage from fecal cultures or in the viability of cultured infective larvae when held in the laboratory at 27 +/- 1 degrees C for 14 weeks. After treatment with ivermectin, there were significant differences among the parasite strains in PCV, TP, and epg. Total worm counts were reduced by 94 to 97% with three times the recommended dose. Immature and adult Skrjabinema ovis were also present in two treated goats. In a second test, one goat infected once with 10,000 infective larvae of a resistant strain of H. contortus and then treated with nine doses of ivermectin, increasing from 500 to 2000 micrograms kg-1 over a period of 133 days, had 35 adult worms at necropsy. In a third test, three calves were readily infected with an ivermectin-resistant strain of H. contortus from goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Haemonchus contortus: enzymes. 3. Glutamate dehydrogenase

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.     

Laboratory selection of Haemonchus contortus for resistance to ivermectin

The eighth generation of adult Haemonchus contortus, selected by subjecting infected pairs of sheep to suboptimal ivermectin treatment once per generation from parent (P; BBH isolate) through F7 (IV-A; selected isolate), required an approximate 4-fold increase in the ivermectin dose to produce 95% efficacy compared with its contemporary parent isolate. In a dose titration experiment the dose-response curve of the drug pressure-derived isolate, IV-A, was significantly (0.02 less than P less than 0.05) less steep than was the response curve of the parent, BBH, isolate. Potency estimates based upon these nonparallel dose-response curves would not remain constant over a range of efficacy levels but would decrease rapidly at efficacies greater than 95%. Passage of a closed population of the F8 generation of IV-A sequentially through pairs of sheep for an additional 11 generations (F8A-F8K) without additional drug pressure being applied produced no reversion to sensitivity to ivermectin relative to the F7 generation, thus suggesting that the selected "resistance" was stable.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

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.     

The avermectin receptors of Haemonchus contortus and Caenorhabditis elegans

Most of the recent evidence suggests that the avermectin/milbemycin family of anthelmintics act via specific interactions with glutamate-gated chloride channels. These channels are encoded by a small family of genes in nematodes, though the composition of the gene family and the function of the individual members of the family may vary between species. We review our current knowledge concerning the properties of the glutamate-gated chloride channels from Caenorhabditis elegans and the related parasite, Haemonchus contortus. We conclude that the biological effects of the avermectins/milbemycins can be largely explained by the known pharmacology and distribution of the glutamate-gated chloride channels and that differences between the glutamate-gated chloride channels from different nematodes may underlie species-specific variations in anthelmintic action.