Jennifer H. Gill

Avermectin inhibition of larval development in Haemonchus contortus ― effects of ivermectin resistance

Avermectin (AVM) inhibition of the development of the free-living stages of Haemonchus contortus has been quantified in an assay in which nematode eggs are placed on an agar matrix containing serial dilutions of a drug in the wells of a microtitre plate. Development is allowed to proceed for 6 days by which time larvae in control wells (no drug) have reached the infective, third (L3) stage. At high concentrations (> 30 nM) ivermectin (IVM) paralyses L1 larvae soon after hatching, however, much lower concentrations (approximately 1 nM) are sufficient to inhibit development to the L3 stage which suggests that effects of the drug other than those relating to gross motor activity are responsible for the latter effect. The larval stages of IVM-susceptible H. contortus isolates from both Australia and South Africa, including isolates known to be resistant to levamisole or rafoxanide and/or the benzimidazoles, were equally sensitive to inhibition by AVMs. In contrast, 6 isolates of H. contortus resistant to IVM in vivo showed a reduced sensitivity to AVM inhibition of development. The order of potency of a limited range of AVMs as inhibitors of larval development was consistent with in vivo efficacy. Resistance ratios for IVM-resistant isolates were dependent on AVM structure, with AVM B2 the most sensitive probe for IVM resistance in the isolates tested.

Management of anthelmintic resistance: Inheritance of resistance and selection with persistent drugs

Resistance to the benzimidazole (BZ) anthelmintics is inherited as an incomplete dominant/ incomplete recessive trait and is now widespread in populations of gastrointestinal nematode parasites of sheep. Unlike benzimidazole resistance, which is common in Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta, resistance to levamisole is relatively rare in H. contortus, although common in the other 2 species. One explanation for the slow spread of resistance to levamisole in H. contortus is that it is inherited as an autosomal recessive trait, while in T. colubriformis levamisole resistance is inherited as a recessive sex-linked trait. With the introduction of the avermectin/milbemycin class resistance has developed to the relatively short-acting ivermectin, but this time it is inherited as a completely dominant trait. The potentially more serious situation of a persistent anthelmintic selecting a dominant resistance gene was investigated using a simulation model. Efficacy against incoming infective larvae (L3) was assumed to decline or remain high over the period of drug persistence (3 days to 4 weeks), thus allowing the estimation of the relative importance of selecting resistant L3s on the development of resistance in the worm population. These factors were also examined against a background of initial efficacy levels, against adults, and mode of inheritance. Persistence and initial efficacy were found to be far more important in determining the rate of selection for resistance than was selection of resistant L3 as drug efficacy declined.

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.

Characterisation of an avermectin resistant strain of Australian Haemonchus contortus

A strain of Haemonchus contortus (CAVR) isolated in Australia was found to be resistant to ivermectin (IVM) with 0.4 mg kg-1 of the anthelmintic failing to significantly reduce worm burdens. Resistance to IVM was sex-influenced in the CAVR strain with adult males showing a greater sensitivity to IVM. Cross resistance to moxidectin was evident with approximately 15% of the population surviving a dose of 0.1 mg kg-1. The free-living stages of the CAVR isolate had a reduced sensitivity to avermectin (AVM) inhibition of development and motility. Similar structure-activity patterns and resistance factors were obtained for a series of related AVMs as inhibitors of larval development and L3 motility in CAVR and White River II, an IVM-resistant H. contortus isolate from South Africa. Further, both isolates were found to be 3 times more sensitive to paraherquamide than a susceptible H. contortus isolate. This suggest that the same resistance mechanism is operating in both isolates. The CAVR strain is susceptible to the benzimidazoles, levamisole and closantel.

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.

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.

Marine nematocides: Tetrahydrofurans from a southern Australian brown alga, Notheia anomala

Chemical analysis of N. anomala collected off rock platforms along the southern coast of Australia yielded a cis-dihydroxytetrahydrofuran (2), the structure for which was assigned by spectroscopic analysis, chemical derivatization and biomimetic synthesis. Tetrahydrofurans from Notheia anomola are reported for the first time as potent and selective inhibitors of the larval development of parasitic nematodes. SAR observations are made on a selection of natural, semi-synthetic and synthetic tetrahydrofurans

Binding of [3H]benzimidazole carbamates to mammalian brain tubulin and the mechanism of selective toxicity of the benzimidazole anthelmintics

The binding of tritiated benzimidazole carbamates ([3H]BZCs) to mammalian brain tubulin was examined to investigate the kinetics of the BZC-tubulin interaction and to establish the mechanism of the selective toxicity of the BZC based anthelmintics. [3H]BZC binding to tubulin was markedly greater at 4 degrees than at 37 degrees for all ligands. The association constant (Ka) and maximum amount of [3H]BZC bound (Bmax) were temperature dependent for [3H]mebendazole ([3H]MBZ), [3H]oxibendazole ([3H]-OBZ) and [3H]oxfendazole ([3H]OFZ). The Ka and Bmax values obtained for [3H]MBZ, [3H]OBZ and [3H]OFZ, and the comparatively weak binding of [3H]carbendazim, reflected the known in vitro potency of these compounds as microtubule inhibitors. Dissociation of the [3H]MBZ-tubulin complex was also temperature dependent, the first order dissociation rate constant being reduced by two orders of magnitude at 4 degrees compared with that observed for 37 degrees. These results indicate that the binding of BZCs to mammalian brain tubulin is temperature dependent and suggest that temperature induced conformational changes in the tubulin dimer influence the ability of the BZCs to form a stable BZC-tubulin complex. The temperature dependence of BZC binding and the affinity of the BZCs for mammalian tubulin are therefore unlike the BZC-tubulin interaction observed for parasitic nematodes, where optimum BZC binding occurs at 37 degrees and results in the formation of a pseudo-irreversible complex.

Aspergillicins A–E: five novel depsipeptides from the marine-derived fungus Aspergillus carneus

A search for new antiparasitic agents from a strain of the fungus Aspergillus carneus isolated from an estuarine sediment collected in Tasmania, Australia, yielded the known terrestrial fungal metabolite marcfortine A (1) as an exceptionally potent antiparasitic agent. This study also yielded a series of new depsipeptides, aspergillicins A-E (2-6) and the known terrestrial fungal metabolite acyl aszonalenin (7). Marcfortine A (1) and acyl aszonalenin (7) were identified by spectroscopic analysis, with comparison to literature data. Complete stereostructures were assigned to aspergillicins A-E (2-6) on the basis of detailed spectroscopic analysis, together with ESIMS analysis of the free amino acids generated by acid hydrolysis, and HPLC analysis of Marfey derivatives prepared from the acid hydrolysate. The peptide amino acid sequence for all aspergillicins was unambiguously assigned by MS(n) ion-trap ESI mass spectrometry.

Acremolides A-D, Lipodepsipeptides from an Australian Marine-Derived Fungus, Acremonium sp

An Australian estuarine isolate of an Acremonium sp. (MST-MF588a) yielded the two known compounds 19- O-acetylchaetoglobosin D ( 1) and 19- O-acetylchaetoglobosin B ( 2), as the sole cytotoxic principles, along with the known aromatic metabolite RKB 3564S ( 3), and a novel family of lipodepsipeptides, acremolides A-D ( 4- 7). Structures were assigned to 4- 7 on the basis of detailed spectroscopic analysis and chemical derivatization and by application of a new C 3 Marfeys method for amino acid analysis.