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Heartworm disease, caused by Dirofilaria immitis, can be lethal in dogs and cats. It is transmitted by mosquitoes, and occurs in many parts of the world. Prevention relies on macrocyclic lactones. Macrocyclic lactones used are ivermectin, selamectin, abamectin, eprinomectin, milbemycin oxime and moxidectin, administered at 30-day intervals during the transmission season. Some moxidectin formulations are long-acting injectables. In the USA, preventives are recommended throughout the year. Loss of efficacy of macrocyclic lactone preventives was reported in 2005 and proof of resistance in the USA was published a decade later. Understanding factors which promote resistance is important to maintain control. Factors important for resistance development are discussed. Better, inexpensive tests to confirm resistance are needed. Infection in animals under chemoprophylaxis per se does not imply resistance because lack of compliance in preventive use could be the reason. In vivo confirmation of resistance is expensive, slow and ethically questionable. A microfilariae suppression test can be a surrogate test, but requires a high dose of a macrocyclic lactone and repeated blood microfilaria counts 2-4 weeks later. DNA single nucleotide polymorphism markers have been successfully used. However, the specific genetic changes which cause resistance are unknown. Surveys to map and follow the extent of resistance are needed. Long acting mosquito repellants and insecticides can play a useful role. High dose rate formulations of moxidectin, coupled with mosquito biting mitigation may reduce transmission of resistant genotypes. Doxycycline, daily for 28 days, as anti-Wolbachia treatment, can reduce transmission and remove adult parasites. However, new classes of heartworm preventives are needed. While any preventive strategy must be highly effective, registration requirements for 100% efficacy may hinder development of useful new classes of preventives. Continued reliance on macrocyclic lactone preventives, when they do not work against resistant genotypes, will spread resistance, and allow for more disease., (Copyright © 2021. Published by Elsevier Ltd.)

Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the 'R' site for Rhodamine 123 and the 'H' site for Hoechst 33342. The avermectins appear to bind the 'R' binding site unlike the milbemycins, whereas both sub-classes of macrocyclic lactones might interact with the 'H' site of D. immitis PGP-11., (Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Haemonchus contortus is a nematode of livestock that can cause severe disease and mortality. Ivermectin, an anti-parasitic drug that targets glutamate-gated chloride channels, is widely used in humans, livestock, companion animals and agriculture. Although an association between genetic changes to β-tubulin and exposure to ivermectin has been previously reported, direct binding between ivermectin and tubulin has not been demonstrated to date. Tubulin/microtubules are key targets for many anti-mitotic drugs used in anti-parasite and cancer therapies. We now report that ivermectin exposure increased the rate and extent of polymerisation of H. contortus recombinant α- and β-tubulin, and protected the parasitic α- and β-tubulins from limited trypsin proteolysis. Direct binding between ivermectin and the tubulin monomers exhibited low micromolar affinities, as determined using surface plasmon resonance. Subsequent equilibrium dialysis indicated that ivermectin and Taxol compete for binding to tubulin, supporting our molecular modelling that predicts ivermectin interacts with the Taxol binding pocket of both parasitic and mammalian tubulins. Collectively, our data indicate that ivermectin can bind to and stabilise microtubules (i.e., alter the tubulin polymerisation equilibrium) and this can then lead to mitotic arrest. This work extends the range of known pharmacological effects of ivermectin, and reveals its potential as an anti-mitotic agent., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm's intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

A levamisole-sensitive acetylcholine receptor has recently been described in the parasitic nematode Haemonchus contortus. The pentameric receptor is produced from different subunit proteins, one of which is Hco-ACR-8. A truncated transcript, Hco-acr-8b, has been identified in six levamisole-resistant H. contortus isolates and was found to be absent in four levamisole-susceptible isolates, indicating Hco-acr-8b could be a potential marker for levamisole resistance. The Hco-acr-8b transcript contains exons 1 and 2 and terminates with 347bp from within the intron 2. In this work, we investigated genomic DNA sequences of the Hco-acr-8 gene, in a region including exon 2 and exon 3, from a wide range of levamisole-susceptible and resistant H. contortus isolates. Sequences potentially involved in generating the truncated splice variant within the second intron were analysed from individuals and pools of parasites. We found an insertion/deletion (indel) of 63bp located just downstream from the splice acceptor site for the alternative third exon. The sequence of the indel, when present, was similar in the 12 isolates examined. The presence or absence of this indel was statistically (Chi(2) test) correlated with levamisole resistance status. A correlation was also demonstrated between the absence of the indel and the expression of the Hco-acr-8b transcript. We believe this is the first report of a putative DNA marker for levamisole resistance detection. Using this new knowledge, we have developed a novel DNA-based assay for the detection and monitoring of levamisole resistance in parasitic nematodes of animals., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Serotonin and dopamine play important roles in the biology of nematodes where they exert their effect on feeding, locomotion and reproductive behavior. Haemonchus contortus, a parasitic nematode which infects small ruminants, is responsible for considerable economic losses in agriculture. In the current study we have mapped the localisation of these two neurotransmitters in this parasite using immuno-staining. Serotonin localised in amphidial and pharyngeal neurons in both adult female and male worms. Serotonin was also found in ray sensory neurons as well as in a few ventral cord motor neurons exclusively in adult males. Surprisingly, dopamine was only detected in the neuronal commissures linking the lateral and sub-lateral nerve cords in both sexes. We also studied the effect of these two molecules on female adult worms in vitro. Serotonin mainly inhibited movement whereas dopamine had a profound paralytic effect on the mid-body of the worms., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

The activity of glutathione S-transferase (GST) decreased progressively in Schistosoma mansoni from mice treated with oltipraz (OPZ). However, the peroxidase activity of GST (selenium-independent) and selenium-dependent glutathione peroxidase was not affected by OPZ treatment. Purification and quantification of GST from worms after OPZ treatment indicated that the decrease in enzyme activity was greater than could be accounted for by the decrease in GST protein content. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with GST isoenzyme specific antisera revealed a slight decrease in the quantity of both 26 and 28 kDa GSTs. Fractionation of cytosolic GSTs from male S. mansoni by chromatofocusing resolved three major isoenzymes (SmI, II and III) and a minor form which eluted first from the column. SmI, II and III all had a molecular weight of about 28 kDa on SDS-polyacrylamide gel electrophoresis. However, on electrophoresis in the absence of SDS, the three GST forms exhibited different mobilities. The pattern of SmI, II and III was similar in untreated and OPZ-treated worms, but the activities of the isoenzymes from treated worms were lower. The results suggest that OPZ interacts with the GST isoenzymes SmI, II and III in a similar manner; thus, the effects are not isoenzyme specific. Taken together, these results suggest that OPZ and/or its metabolites interact directly with GST resulting in inhibition of activity and reduction in total enzyme protein. This mechanism may be important in the antischistosomal action of OPZ.

Three monoclonal antibodies specific to alpha- and beta-tubulin were used to examine the expression of tubulin isoforms in the intestine, reproductive tract and body wall muscle of A. suum. The tubulins were found to be different in their isoelectric points, number of isoforms and peptide maps with Western blot analysis of one-dimensional polyacrylamide gel confirming the presence of alpha-, beta 1- and beta 2-tubulin. Commercial cross-reactive anti-alpha and anti-beta MAbs 356 and 357 recognized tubulin from A. suum tissues as well as from pig brain, whereas anti-A. suum beta-tubulin specific MAb P3D6 recognized tubulin from the A. suum tissues only. Two-dimensional gel analysis showed different isoform patterns in different A. suum tissues with anti-A. suum beta-tubulin MAb P3D6 and cross-reactive beta-tubulin MAb 357 recognizing 2-4 beta-tubulin isoforms and anti-alpha-tubulin MAb 356 recognizing 1-6 alpha-tubulin isoforms. Different peptide maps of tubulin were observed in the three tissues, when subjected to limited proteolysis followed by SDS-PAGE. The data indicate that different tubulins are found in different tissues of adult A. suum.

Resistance has now been reported to all of the broad spectrum anthelmintic types currently available, namely to the benzimidazoles, levamisole/morantel and to ivermectin. The problem causes most concern for parasite control in sheep, but anthelmintic resistance has also been reported in nematodes of horses, goats, pigs and more recently cattle. Our understanding of the factors which select rapidly for resistance has increased and programmes of worm control which minimize selection for anthelmintic resistance are being developed and tested. One of the greatest problems encountered in attempting to reduce the selection for overt drug resistance is the need for more sensitive tests for developing resistance. In the long term, new approaches to chemotherapy and to overcoming anthelmintic resistance problems will arise from improving our understanding of the modes of action of, and mechanisms of resistance to, anthelmintics at the level of the receptor proteins and their genes.

This experiment was designed to study, over a 5-year-period, the effect of different frequencies of treatment with three different anthelmintic groups, namely, benzimidazoles, levamisole and ivermectin, and different frequencies of alternation between them, on existing levels of anthelmintic resistance in the nematode parasites Haemonchus contortus and Trichostrongylus colubriformis of grazing sheep. No evidence of ivermectin resistance emerged, even in suppressively treated groups. Likewise, H. contortus failed to develop resistance to levamisole under a similar selection regimen. Thiabendazole was shown to select positively against levamisole resistance in T. colubriformis resulting in significantly greater susceptibility to this drug than for the natural reversion which occurred in the untreated control. There was no evidence that an anthelmintic treatment combined with a movement of sheep to pastures of low infectivity selected more rapidly for resistance than where the same number of treatments were given to set-stocked sheep. Rotation between anthelmintic groups at yearly intervals appeared to be more beneficial in delaying resistance than rotation of drugs with each treatment.