Ernest Lacey

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.

Interactions of benzimidazoles (BZ) with tubulin from BZ-sensitive and BZ-resistant isolates of Haemonchus contortus

The binding of tritiated benzimidazoles (BZs)-albendazole, parbendazole, oxibendazole, mebendazole, oxfendazole and fenbendazole-to crude tubulin extracts from BZ-susceptible and -resistant Haemonchus contortus has been examined. For all BZs, the binding was substantially lower in the resistant isolate. The extent of this reduction was dependent on the structure of the BZ, with mebendazole demonstrating superior binding to the resistant isolate than the other BZs. Enrichment of the crude tubulin extract using polylysine-linked agarose demonstrated that for both isolates more than 85% of the observed binding was to protein eluted in the tubulin-containing fraction. Based on biochemical kinetics, the change in tubulin associated with resistance is reduced capacity in resistant tubulin to bind BZ with little or no reduction in the association constant of the BZ-tubulin complex. Comparative egg hatch assay demonstrated a similar structural specificity with the resistance factor of mebendazole observed to be lower than that of albendazole, parbendazole, oxibendazole and thiabendazole. The results of both studies support the hypothesis that BZ resistance is due to a change in tubulin and that this change is dependent on the structure of the BZ.

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.

Comparison of inhibition of polymerisation of mammalian tubulin and helminth ovicidal activity by benzimidazole carbamates.

The correlation between the inhibition of hatching of Haemonchus contortus eggs and inhibition of mammalian tubulin polymerisation by benzimidazole carbamates has been investigated. The hatching process was observed to be independent of the biomass (eggs plus debris) over a 6-fold range and the early (E1-E3) stages of egg development, but was dependent on the concentration of co-solvent (DMSO) and time of incubation. Benzimidazole carbamates with strong inhibitory activity against mammalian tubulin were potent inhibitors of egg hatch, while non-inhibitors failed to prevent hatching. It is postulated that the primary mode of action of these drugs on nematode eggs is the inhibition of microtubule-dependent processes within the developing egg. The implications and limitations of this correlation are discussed.

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters, Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m3) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (Fv/Fm) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.

Tubulin and benzimidazole-resistance in Trichostrongylus colubriformis (Nematoda).

Benzimidazole treatment produced greater effects on microtubule-dependent acetylcholinesterase secretion, the presence of microtubules in intestinal cells, and colchicine binding in susceptible compared with benzimidazole-resistant Trichostrongylus colubriformis. In addition, the binding of benzimidazoles was markedly reduced in preparations from the latter strain, indicating that the mechanism of resistance to benzimidazoles in this nematode involves a reduced affinity of tubulin for benzimidazoles.

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Background: Ras proteins must be plasma membrane-localized for biological activity. Results: A high content screen identified staurosporines as inhibitors of Ras plasma membrane localization and K-Ras signal transmission by disrupting endosomal recycling of phosphatidylserine. Conclusion: Staurosporines are novel inhibitors of phosphatidylserine trafficking. Significance: Ras trafficking pathways and Ras spatiotemporal organization on the plasma membrane are valid targets for anti-Ras drug development. Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.

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.

A routine diagnostic assay for the detection of benzimidazole resistance in parasitic nematodes using tritiated benzimidazole carbamates

Resistance to benzimidazoles (BZs) in parasitic nematodes has recently been shown to be due to a reduction in the ability of BZs to bind the structural protein, tubulin, in resistant isolates. Based on these observations the development and standardisation of a routine diagnostic assay has been undertaken by measuring the binding of tritiated mebendazole to crude supernatants of L3 larvae. The assay is rapid, requiring less than 2 h, and is robust, highly reproducible and sensitive to minor changes in the resistance status of parasite populations. Investigation of the routine application and validity of this assay has been documented using 24 isolates of known resistance status from the species Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta: In all cases the observed binding and calculated susceptibility factors were in accordance with their respective resistance status.