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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.

Animals↗

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)

Animals↗

Differential stability of the benzimidazole (BZ)-tubulin complex in BZ-resistant and BZ-susceptible isolates of Haemonchus contortus and Trichostrongylus colubriformis.

The binding of [3H]mebendazole ([3H]MBZ) to tubulin from BZ-susceptible (BZ-S) and BZ-resistant (BZ-R) isolates of Haemonchus contortus and Trichostrongylus colubriformis was investigated using charcoal extraction and gel filtration techniques. The amount of [3H]MBZ bound at infinite free ligand concentration (Bmax) was significantly reduced for the BZ-R isolate compared with the BZ-S isolate in both species when assayed by charcoal extraction. However, Bmax was increased to comparable levels for both BZ-S and BZ-R isolates of each species when assayed by the less stringent gel filtration technique. These results indicate that the BZ-tubulin interaction in trichostrongylid nematodes is comprised of a minimum of two components. As similar levels of total [3H]MBZ binding were observed for both BZ-S and BZ-R isolates of each species when assayed by gel filtration, it is suggested that the reduction in the pseudo-irreversible BZ binding component in BZ-R isolates results in an increase in the level of reversible BZ binding and therefore provides a survival advantage to BZ-R nematodes.

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Cytochrome P450 monooxygenase activity in Haemonchus contortus (Nematoda).

Cytochrome P450 monooxygenase activities towards aldrin and 7-ethoxycoumarin were detected in microsomes prepared from L1 and L3 larvae of Haemonchus contortus, and very low levels of activity towards aldrin were detected in adults. Larval activities were NADPH-dependent, suppressed by carbon monoxide (CO) and piperonyl butoxide, and induced (up to 60-fold) by exposure to phenobarbital. Different patterns of expression of activities towards the 2 substrates in various life-stages, as well as different sensitivities to piperonyl butoxide, suggested the presence of more than 1 cytochrome P450 enzyme. Cytochrome P450 itself could only be detected by spectral assay in phenobarbital-treated L3 larvae. It is most likely that the observed shift of several nanometres in the position of the spectral peak of cytochrome P450 was due to the presence of other CO-reactive haemoproteins (probably cytochrome oxidases). It is apparent that H. contortus possesses cytochrome P450 monooxygenase activities; however, they may be important only in the free-living stages, and be of little significance in parasitic stages existing in oxygen-poor environments.

7-Alkoxycoumarin O-Dealkylase↗

Haemonchus contortus: the uptake and metabolism of closantel.

Closantel is an anthelmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms.

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Characterization of haemolytic activity from adult Haemonchus contortus.

Adult Haemonchus contortus contain a detergent-soluble factor that haemolyses sheep red blood cells in a time- and concentration-dependent manner. This factor had comparable haemolytic activity at pH 5.0 and 8.0; activity was lower at pH 6.0 and 7.0. The activity was heat-stable, unaffected by proteolytic inhibitors, and inhibited by 20 mM polyethyleneglycol. Haemolytic activity was associated with the particulate fraction of the isolated intestine, suggesting an essential role for this activity in the acquisition of nutrients by disrupting host red blood cells. The data are consistent with the hypothesis that the mechanism of action of the haemolytic factor is as a pore-forming agent.

Abomasum↗

The interaction of Trypanosoma congolense and Haemonchus contortus in Djallonké sheep.

The interaction between Trypanosoma congolense and Haemonchus contortus was studied in 5 groups of 8 Djallonké sheep. Two groups received a single infection with either H. contortus or T. congolense, and 2 groups were infected with T. congolense followed by H. contortus (TH) or vice versa (HT). One group was kept as uninfected controls. Mortality due to infection was observed only in the dual infection groups. In the TH group, the effects were more acute whereas in the HT group they were more chronic. No significant differences in weight gain could be demonstrated between infected and control groups. Djallonké sheep are able to withstand a single infection with either T. congolense or H. contortus, which confirms their trypanotolerant nature and provides preliminary indication of resistance against helminth infections. However, when exposed to successive infections with both parasites, some of the animals lose this tolerance.

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Selection for high levamisole resistance in Haemonchus contortus monitored with an egg-hatch assay.

To investigate the characteristics of selection for levamisole resistance in Haemonchus contortus, the consecutive nematode generations of an in vivo selection were monitored with a newly developed egg-hatch assay. The in vivo selection was started with a population not previously exposed to any anthelmintics (SHS). At first, the levamisole resistance progressed gradually in successive nematode generations by treating sheep with increasing doses of levamisole, the initial dose being 1 mg kg-1. Treatment with 5 mg kg-1 levamisole resulted, however, in a steep increase of resistance. The selection was ended after six generations, since a level of 30 mg kg-1 levamisole, which is not far from the toxic level for sheep, was reached. The final population, RHS6, was studied in a controlled test. Treatment of RHS6-infected sheep with 30 mg kg-1 levamisole caused an 80% decrease of faecal egg output, and a reduction of 34% in worm numbers. It was remarkable that only the number of female adults was reduced. RHS6 showed a reduced viability, but a fertility similar to the starting population SHS.

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Inheritance of levamisole and benzimidazole resistance in an isolate of Haemonchus contortus.

Reciprocal crosses between an isolate of Haemonchus contortus resistant to both benzimidazole and levamisole anthelmintics and a susceptible isolate were performed in order to determine the mode of inheritance of these resistances. F1 and F2 generations and parent isolates were assayed for susceptibility to thiabendazole and levamisole in vitro. For each drug all of the filial generations were intermediate in susceptibility between the parent isolates, and analysis indicated that resistance was inherited as an incompletely recessive character determined by more than one gene in each case. There was no evidence of maternal inheritance. Results of both the in vitro assays and in vitro selection, followed by determination of sex ratio in the survivors, as well as studies on adult worms, provided no evidence for sex-linkage. This work illustrates that in vitro assays coupled with minimal studies in sheep are useful for determining inheritance of resistance, yet use fewer experimental animals than traditional studies.

Animals↗

Levamisole binding sites in Haemonchus contortus.

Larval and adult extracts from isolates of Haemonchus contortus were assayed for specific [3H]levamisole binding activity. All of the tissue preparations displayed [3H]levamisole binding sites. The sensitive isolate SE and resistant isolate RJ showed no differences in larval and adult binding data. Larval SE extracts had higher receptor density (Bmax = 648 fmol mg-1) and dissociation constant (Kd = 1.28 microM) for [3H]levamisole than larval extracts of the American isolate RUSA (Bmax = 87 fmol mg-1 and Kd = 0.15 microM). Extracts of adult SE and RUSA isolates contain as much as 327 fmol mg-1 of protein and 205 fmol mg-1 of protein, respectively, and similar dissociation constants (Kd = 0.77 microM and Kd = 0.81 microM, respectively). There was a good correlation between specific binding activity of larval and adult extracts in both SE and RUSA isolates. The nicotinic cholinergic antagonist alpha-bungarotoxin had no effects in either isolate on [3H]levamisole binding activity. The results confirm that levamisole acts at a cholinergic receptor in H. contortus, and suggest that target site modification could be involved in the development of levamisole resistance.

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Evidence of multiple mechanisms of avermectin resistance in haemonchus contortus--comparison of selection protocols.

Three isolates of Haemonchus contortus selected for avermectin resistance in sheep were compared in three in vitro pharmacological tests previously shown to discriminate between field isolates of H. contortus resistant and susceptible to the avermectins. Two isolates, F7-A and IVC, were selected for avermectin resistance in the laboratory from a reference susceptible isolate using suboptimal doses of ivermectin (LD95) for 7 and 16 generations, respectively. In these isolates avermectin resistance was not associated with a decreased sensitivity to avermectin inhibition of larval development or L3 motility but was associated with an increased sensitivity to paraherquamide. The third isolate, Warren, was derived from an overwhelmingly avermectin-susceptible, mixed species field isolate in a single generation by propagating the small number of survivors of a 0.2 mg/kg ivermectin treatment (i.e. 10 x LD95). This isolate, like previously characterised avermectin-resistant H. contortus isolates derived from the field in South Africa and Australia, showed a markedly reduced sensitivity to avermectin inhibition of larval development and L3 motility, as well as an increased sensitivity to paraherquamide. These results suggest that avermectin resistance can manifest itself in different ways and that the two selection protocols used to generate the F7-A, IVC and Warren isolates have resulted in the selection of different resistance phenotypes.

Animals↗

Binding of [3H]m-aminolevamisole to receptors in levamisole-susceptible and -resistant Haemonchus contortus.

M-aminolevamisole, a potent analogue of the commercial anthelmintic levamisole, was used to investigate ligand-binding properties of homogenates of larval and parasitic stages of the nematode parasite of sheep, Haemonchus contortus. Kinetics of the binding of [3H]m-aminolevamisole to homogenates was measured in a drug-susceptible isolate and compared with a levamisole-resistant isolate. Equilibrium binding studies and kinetic studies revealed a high affinity binding component with a KD of 3 nM. A low affinity component (KD = 2.4 microM) was also apparent in equilibrium studies. High affinity [3H]m-aminolevamisole binding was displaced in a concentration-dependent manner by levamisole analogues and cholinergic agonists. Compared with the susceptible isolate, binding in a levamisole-resistant isolate of the parasite, was quantitatively similar over a range of developmental stages and binding conditions. However, under the conditions of binding there was a reduced affinity (larger KD) and more binding sites (larger Bmax) at the low affinity site in the resistant compared with the susceptible isolate. It was concluded that the ligand was binding to acetylcholine receptor populations of the nematode and that resistance may be associated with alterations in the low affinity site of this receptor.

Animals↗

Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus.

A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and closantel. No polymorphism could be correlated with any of the different resistances. A P-glycoprotein gene probe was also isolated from the human nematode parasite Onchocerca volvulus and an Onchocerca-specific PCR was developed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

The intramammary inflammatory response of genetically resistant Merino ewes infected with Haemonchus contortus.

The mammary glands of 103 pasture-reared non-lactating, non-pregnant Merino ewes were infused via the teat canal with antigens prepared from the nematode Haemonchus contortus, and the inflammatory response to infusion assessed by washing the gland of its contents after 24 h and 14 days. The ewes were of two genotypes: one with proven high levels of resistance to infection with the nematode H. contortus, the other random-bred animals with relative susceptibility to infection. On day 0 of a H. contortus infection, one gland of the subgroups of both genotypes was infused with the antigen preparation. At the same time, the other gland of the random-bred ewes was infused with sterile physiological saline. A third group of infected random-bred ewes was infused with only sterile physiological saline. Similar infusions were performed on other subgroups on days 12, 21 and 35 of infection, which was then terminated with anthelmintic. A fourth group of uninfected random-bred control ewes was given both infusions 35 days after the other groups were infected. Sheep of the resistant genotype had lower worm egg counts and smaller reductions in blood packed cell volumes from day 21 of infection. Infusion of antigen had no effect on the course of infection and no effect on the response of the other gland, which had been infused with saline alone. The dominant leukocyte response from the antigen-infused gland was eosinophilia. On all days of infusion, and after both 24 h and 14 days, eosinophil counts from the resistant genotype were higher than those from their random-bred counterparts. The sheep mammary gland provides a source of eosinophils whose number is related to host genotype and stage of infection and may provide a model for the investigation of cellular responses in mucosal immunity to nematode infections.

Animals↗

Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus.

The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.

7-Alkoxycoumarin O-Dealkylase↗

Selection for anthelmintic resistance by macrocyclic lactones in Haemonchus contortus.

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.

Animals↗

Inheritance of avermectin resistance in Haemonchus contortus.

A larval development assay was used to compare the responses of the Chiswick Avermectin Resistant (CAVRS) isolate of Haemonchus contortus, an avermectin-susceptible isolate (VRSG) and their crosses to avermectins. The F(1) and F(2) generations of reciprocal crosses between CAVRS and VRSG were denoted as CAVRS malesxVRSG females=CXV, and VRSG malesxCAVRS females=VXC. The levels of avermectin resistance in the developing larvae of the F(1) of both CXV and VXC were indistinguishable from that in the avermectin-resistant parent, indicating that the resistance trait is completely dominant. Avermectin dose-response curves for the CXV F(1) did not show a 50% mortality rate at low concentrations, indicating that avermectin resistance is not sex-linked. This conclusion was confirmed when adult male worms of the F(1) of the CXV mating were found to have survived treatment of the host with 200microgkg(-1) ivermectin. This dose rate (200microgkg(-1) ivermectin) caused a 50% reduction in the number of adult males in the F(1) from both CXV and VXC crosses, but only a non-significant reduction in the number of adult females in the F(1). Dose-response curves obtained for the F(2) generations in the larval development assay indicated the presence of 25% of avermectin-susceptible individuals, suggesting that a single major gene largely controls the avermectin-resistance trait. This genetic analysis of avermectin resistance in an Australian H. contortus isolate indicates that the expression of the gene for avermectin resistance is an autosomal, complete dominant in the larvae; however, in adults its expression is sex-influenced, with males having a lower resistance to avermectin than females.

Animals↗

Immune exclusion of Haemonchus contortus larvae in the sheep: effects on gastric mucin of immunization, larval challenge and treatment with dexamethasone.

The rapid expulsion of the gastric nematode Haemonchus contortus from primed sheep is a complex mechanism which probably involves immediate hypersensitivity reactions and mucus. Quantitative techniques were employed to determine the content and distribution of mucin in the gastric mucosae of sheep of varying immune status, following a single large challenge with H. contortus. Substantial depletion of both neutral and acidic mucin, following challenge, was noted in the gastric mucosae of naive sheep. When compared with normal controls, animals rendered immune by daily oral challenge had significant reductions in neutral mucin at the mucosal surface and increased quantities of acidic mucin deeper in the mucosa. Mucin distribution in immune sheep was not significantly altered 48 h after challenge. Treatment of immune animals with the corticosteroid dexamethasone, abrogated the protective response with gross depletion of mucus in challenged sheep, but had little measurable effect on the mucin profile of unchallenged animals, except for a slight increase in acidic mucin at the mucosal surface and the absence of any detectable mucin in the zone adjacent to the sub-mucosa. Immunity to H. contortus diminishes with time after immunization. Animals still immune 6 weeks after immunization were found to have mucin profiles which did not differ significantly from those of freshly immunized animals, whereas animals susceptible to re-infection 12 weeks after immunization had mucin profiles more closely resembling those seen in naive controls. Immunization by daily oral challenge was also accompanied by hyperplasia of the gastric mucosa which persisted for at least 12 weeks after immunization had ceased.

Animals↗