Macrocyclic Lactone Anthelmintics Research Articles

Delayed canine heartworm (Dirofilaria immitis) microfilarial reduction following Advocate™ for dogs (imidacloprid, moxidectin) treatment

Macrocyclic lactone (ML) anthelmintics are currently the only class of drugs available for canine heartworm prevention. Recent reports of Dirofilaria immitis infection occurring in dogs reportedly receiving ‘rigorous’ prevention in Queensland, Australia, coupled with the confirmation of ML-resistant isolates in the USA, has led to speculation about the potential emergence of ML-resistance in Australia. In this study, we describe two cases (Dog 1 and 2) of asymptomatic canine heartworm disease in Townsville, Australia, that were reportedly receiving ‘rigorous’ heartworm prevention according to the owners’ claims. We aimed to deploy currently available tools to assess the phenotypic and genotypic ML-resistance status of these two dogs. For phenotypic testing, we performed an in-vivo 7-day microfilariae suppression test using a dose of spot-on moxidectin (Advocate™ for Dogs, 100 g/L imidacloprid + 25 g/L moxidectin). This formulation is marketed as Advantage Multi® for Dogs in the USA, which claims a D. immitis microfilaricidal effect. For genetic testing, an Illumina amplicon metabarcoding approach was used to target single nucleotide polymorphisms (SNPs) previously associated with ML-resistance in D. immitis from the USA. Dog 1 and Dog 2 demonstrated <10 % and <40 % reductions in circulating microfilariae seven days after moxidectin treatment, respectively. These phenotypes were not corroborated by genetic SNP testing, as both dogs were classified as susceptible across all examined markers. To streamline testing of D. immitis SNPs, we developed a rhAmp™ SNP qPCR approach for rapidly genotyping suspect cases of ML-resistant infections at the two major loci (L15709_A and L30575). These findings illustrate a phenomenon shown in some heartworm cases outside the USA, whereby infected dogs are failing to see marked reductions in microfilaraemia after ML treatment but possess an ML-susceptible genotype.

Simultaneous resistance to multiple anthelmintic classes in nematode parasites of cattle in New Zealand

Resistance to the benzimidazole and macrocyclic lactone anthelmintics is widespread in Cooperia spp. on cattle farms in New Zealand. Since this was first documented in 2006 little has changed in cattle farming systems except for the widespread use of levamisole to control Cooperia spp. in young cattle (i.e., parasite control has maintained an almost total reliance on use of anthelmintics). Here we report the emergence of simultaneous resistance to the benzimidazole, macrocyclic lactone and levamisole anthelmintics in Cooperia spp. and in Ostertagia spp. Anthelmintic efficacy against nematode parasites of cattle was investigated on four commercial farms following reports of poor animal growth rates and welfare, and positive faecal egg counts, despite routine treatment with combination anthelmintics, which included levamisole. Faecal egg count reduction tests involved 15 animals per treatment group, individual egg counts (paired samples) conducted pre- and post-treatment, with eggs counted to ≤ 15 eggs per g faeces and larval cultures for morphological identification. Actives tested varied between farms but always included levamisole alone and several combination products containing levamisole. Of the 20 tests conducted (i.e., 5 products on each of 4 farms) only 3 exceeded 90% efficacy against Cooperia spp. even though 8 of the products tested were combinations containing levamisole and at least one other broad-spectrum anthelmintic. Levamisole used alone achieved efficacies between 44% and 71% against Cooperia spp. across the four trials. The only product to exceed 95% efficacy against Cooperia spp. was a combination of monepantel + abamectin which was 100% effective against all parasites. Resistance to oxfendazole in Ostertagia spp. was indicated on 3 farms, while on one farm efficacy of all the tested products was ≤75% against this parasite. All the farms involved in this study were farming intensive cattle operations with an almost total reliance on anthelmintics to control parasitism. The results clearly demonstrate the emergence of simultaneous resistance to oxfendazole, levamisole and the macrocyclic lactone anthelmintics. Despite years of advice and recommendations to change farming practices away from intensive monocultures, many farmers have continued with the practice, and some are now faced with the very real possibility of being unable to control cattle parasites on their farms.

Dose-dependent in vivo effects of formulated moxidectin on seedling emergence of temperate grassland species

Sheep function as effective endozoochorous seed vectors in grasslands. Recent laboratory-based studies showed that this important function can be impaired by macrocyclic lactone anthelmintics, which are used to control parasites and enter into the environment mainly via faeces; however, there is a lack of in vivo studies. We conducted a seed-feeding experiment with sheep that included four temperate grassland species from four different families (Achillea ptarmica, Asteraceae; Agrostis capillaris, Poaceae; Dianthus deltoides, Caryophyllaceae; Plantago lanceolata, Plantaginaceae). A series of three feeding trials was carried out after one of two groups of sheep received a single administration of a common oral formulation of the macrocyclic lactone moxidectin. Faeces were collected to determine seedling emergence rate and emergence timing as well as moxidectin concentration via HPLC. Seedling emergence differed significantly between the anthelmintic-treated sheep and the control group. This impact depended on time of seed uptake after anthelmintic administration. Number of emerging seedlings was significantly reduced (27.1 %) when faeces moxidectin concentrations were high (on average 3153 ng g−1; 1 d post treatment) and significantly increased (up to 68.8 %) when moxidectin concentrations were low (≤86 ng g−1; 7, 14 d pt). Mean emergence time was significantly lowered at low moxidectin concentrations. These results demonstrate dose-related effects of deworming on seedling emergence which might affect endozoochory and eventually plant population dynamics in grasslands.

Effects of macrocyclic lactone anthelmintics on seed germination of temperate grassland species.

Macrocyclic lactone anthelmintics are widely used to control invertebrate pests in livestock, such as sheep. While anthelmintic effects on non-target animals, such as dung-dwelling insects, are well studied, effects on seed germination are largely unknown. Seeds can come into contact with anthelmintics either during passage through the gastro-intestinal tract of grazing animals or when anthelmintics are excreted with their dung into the environment, which may result in changed germination patterns. We used four commonly applied macrocyclic lactones to assess their effects on germination: moxidectin, ivermectin, abamectin and doramectin as pure substances; moxidectin and ivermectin also in formulated form. We tested these pharmaceuticals on 17 different temperate grassland species from five plant families. Seeds were exposed to three concentrations of macrocyclic lactones (0.1, 1.0 and 10.0 mg·l-1 ) under controlled conditions, and germination was assessed over a 6-week period. From these data, we calculated germination percentage, mean germination time and germination synchrony. Most of the tested species were significantly affected in germination percentage and/or mean germination time by at least one of the tested pharmaceuticals, with formulated moxidectin having the largest impact. In general, the effects found were species- and pharmaceutical-specific. While formulated substances generally reduced germination percentage and increased mean germination time, pure substances increased germination percentage. Synchrony showed less clear patterns in all pharmaceuticals. Although effect size and sign varied between species, our study shows that non-target effects of macrocyclic lactones commonly occur in terrestrial plants. This may impede successful seed exchange between habitats via sheep, and even translate into profound changes to grazed ecosystems.

Shortened strongylid egg reappearance periods in horses following macrocyclic lactone administration – The impact on parasite dynamics

Over the past three decades, equine strongylid egg reappearance periods (ERPs) have shortened substantially for macrocyclic lactone anthelmintics. The ERPs of ivermectin and moxidectin were originally reported in the 8–10 and 12–16 week ranges, respectively, but several recent studies have found them to be around 4–5 weeks for both actives. This loss of several weeks of suppressed strongylid egg output could have substantial implications for parasite control. This study made use of a computer simulation model to evaluate the impact of shortened ERPs on the anthelmintic performance of ivermectin and moxidectin against equine cyathostomins. The original ERPs were set to 7.1 and 15.4 weeks for ivermectin and moxidectin, respectively, while the reduced ERP was set to 4.6 weeks for both actives. Simulations were set to compare model outputs between original and reduced ERP scenarios and results expressed as percent increase in strongylid egg output, infective third stage larvae on herbage (L3h), and encysted early third stage larvae (EL3). For each drug, simulations were evaluated for two different treatment scenarios (2 and 4 treatments annually), two different age groups (yearlings and adults), and for four different climates (cold humid continental, temperate oceanic, humid subtropical, and hot/cold semi-arid). Across all simulations, there was a substantial increase of the three evaluated parameters. With the ivermectin simulations, all three parameters increased in the 100–300% range across climates, age groups and treatment intensities. The moxidectin simulations displayed a wider range of results with parameters increasing from a few hundred to several thousand percent. The increases were most pronounced for L3h in the two cooler climates, reaching as high as 6727%. Overall, the loss of anthelmintic performance was at a magnitude of 10 times larger for moxidectin compared to ivermectin. This performance loss was climate dependent, and was also affected by treatment intensity, but not by horse age. This is the first study to evaluate consequences of shortened ERPs in horses and demonstrated a substantial loss in anthelmintic performance resulting from this development. The results illustrate that anthelmintic efficacy is more than the percent reduction of fecal egg counts at 14 days post treatment, and that substantial anthelmintic performance can be lost despite FECRTs remaining at 100%.

Comparison of the efficacy of macrocyclic lactone anthelmintics, either singly or in combination with other anthelmintic(s), in nine beef herds in southern NSW.

Anthelmintic resistance (AR) is a well-recognized challenge in farmed ruminants. The use of anthelmintics in combination is one of the strategies recommended to slow the rate of AR development. Two studies were undertaken in 2017 and 2019 to assess the efficacy of single-dose macrocyclic lactone (ML) anthelmintic and ML combination drenches. In total, 11 Faecal Egg Count Reduction Trials (FECRTs) were set up in 10 different beef herds, with results available from 10 of those FECRTs (9 herds). AR to a single ML anthelmintic was detected in all 9 herds, with resistance to Cooperia and Haemonchus spp on 9 farms, and resistance to Ostertagia and Trichostrongylus spp on 2 farms. In contrast, for the ML combination anthelmintics, all FECRTs resulted in efficacies of 99%-100%. The results suggest that cattle producers should strongly consider using combination drenches in their herds in preference to single actives.

Temperature moderates impact of formulated moxidectin on seed germination of three temperate grassland species.

Formulations of macrocyclic lactone anthelmintics such as moxidectin are regularly administered to sheep to combat parasites. A disadvantage of these pharmaceuticals are their side effects on non-target organisms when entering the environment. Little is known about anthelmintic effects on plant reproduction and whether the effects depend on environmental factors. For ecological and methodological reasons, we aimed at testing whether temperature affects the efficacy of a common moxidectin-based formulation on seed germination. We carried out a germination experiment including three typical species of temperate European grasslands (Centaurea jacea, Galium mollugo, Plantago lanceolata). We applied three temperature regimes (15/5, 20/10, 30/20°C), and a four-level dilution series (1:100-1:800) of formulated moxidectin (i.e., Cydectin oral drench). These solutions represent seed-anthelmintic contacts in the digestive tract of sheep shortly after deworming. In addition, a control was carried out with purified water only. We regularly counted emerging seedlings and calculated final germination percentage, mean germination time and synchrony of germination. Formulated moxidectin significantly reduced percentage, speed and synchrony of germination. A 1:100 dilution of the formulation reduced germination percentage by a quarter and increased mean germination time by six days compared to the control. Temperature moderated effects of the anthelmintic drug on germination in all response variables and all species, but in different patterns and magnitudes (significant anthelmintic x temperature x species interactions). In all response variables, the two more extreme temperature regimes (15/5, 30/20°C) led to the strongest effects of formulated moxidectin. With respect to germination percentage, G. mollugo was more sensitive to formulated moxidectin at the warmest temperature regime, whereas P. lanceolata showed the highest sensitivity at the coldest regime. This study shows that it is important to consider temperature dependencies of the effects of pharmaceuticals on seed germination when conducting standardised germination experiments.

Route of administration affects the efficacy of moxidectin against Ostertagiinae nematodes in farmed red deer (Cervus elaphus)

The influence of route of administration on the pharmacokinetics and efficacy of macrocyclic lactone anthelmintics has been a subject of interest due to its potential to influence the development of anthelmintic resistance. For most parasite species studied so far, oral administration results in the highest concentrations of drug in the parasites and the highest efficacy against resistant genotypes. However, a recent study in cattle measured the highest levels of ivermectin in the abomasal Ostertagia ostertagi following subcutaneous injection, but it was not possible to correlate these elevated levels with efficacy. Therefore, the current study was initiated to determine whether injectable delivery might be optimal for attaining high efficacy against this important group of parasites.Three on-farm trials were conducted to measure the efficacy of moxidectin administered by the oral, injectable, and pour-on routes against Ostertagiinae parasites in farmed red deer. Groups of rising 1-year old stags (red or red-wapiti crossbreds) in the 84−104 kg weight range were randomised on liveweight into treatment groups of 6 (1 farm) or 8 (2 farms). Animals were treated to individual liveweight with moxidectin oral (0.2 mg/kg), injectable (0.2 mg/kg), pour-on (0.5 mg/kg) or remained untreated. Twelve days later all animals were euthanised and abomasa recovered for worm count. Adult worms were counted in a 2% aliquot of abomasal washings, and adult and fourth stage larvae in a 10 % aliquot following mucosal incubation in physiological saline. In addition, blood was collected from the same 5 animals in each of the treatment groups on days 0, 1, 2, 3, 5, 7 and 12 after treatment and moxidectin levels in plasma were determined using a mass spectrometer.The number of Ostertagiinae surviving treatment was significantly different for each of the treatment groups with injectable administration being most effective, oral administration being the next most effective and pour-on administration the least effective. This applied to both adult worms and fourth stage larvae. A similar pattern was seen in the levels of moxidectin in plasma with both the peak value and area under the concentration curve being highest following injectable administration and lowest following pour-on treatment.Although undertaken in a different host species, the results support the proposition that injectable administration of macrocyclic lactone anthelmintics is likely to be optimal for efficacy against Ostertagiinae parasites and potentially useful in slowing the emergence of resistance in these parasites.

Whole-genome reference of Dirofilaria immitis from Australia to determine single nucleotide polymorphisms associated with macrocyclic lactone resistance in the USA

For the past 30 years, chemoprophylaxis with macrocyclic lactone (ML) anthelmintics has been the primary strategy for canine heartworm (Dirofilaria immitis) control in both the USA and Australia. ML-resistant D. immitis isolates have been confirmed to exist in the USA and studies have shown that 42 single nucleotide polymorphisms (SNPs) are associated with phenotypic ML-resistance. Currently, ML-resistance has not been reported in any Australian clinical cases of canine heartworm. The aim of the study is to determine whether the 42 SNPs associated with resistance to MLs in the isolates from the USA are present in adult heartworms from a clinical case in Australia. Five adult D. immitis obtained from a dog at post-mortem (Sydney, Australia) were sequenced using the Illumina sequencing technology. The genomic analyses revealed 6 out of the 42 SNPs associated with ML-resistance to be present in our samples, 3 out of the 6 SNPs identified were nonsynonymous SNPs but not in candidate genes for ML-resistance. ML-susceptibility profile was mixed using the 42-SNP and 10-SNP models, but the 5-SNP, 3-SNP and 2-SNP models demonstrated ML susceptibility for all five individuals. In this study, the first whole-genome reference of D. immitis from Australia establishes a new baseline for comparative studies and will be valuable for tracking ML-resistance emergence.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Equine helminth prevalence and management practices on Australian properties as shown by coprological survey and written questionnaire

Context Parasite control is an essential part of a broader equine health-management strategy and is often completely administered by the horse owner, with little or no supporting evidence on which to base decisions. Practical and sound advice relies on knowledge of the resident parasite species, the anthelmintic resistance status of important species, and the strategies currently being utilised by owners and managers of horses. Much of this farm-level information is lacking in the Australian literature. Aims The present study aimed to gather both farm- and horse-level prevalence data for four important equine helminth species and to gather information on the current worm-management practices conducted on Australian horse farms. Methods We conducted a coprological survey of cyathostomins, Strongylus vulgaris, Parascaris spp. and equine tapeworm on 102 horse properties, using a combination of classical and molecular parasitological methods, including a tapeworm polymerase chain reaction developed as part of the study. A questionnaire canvasing horse owners on internal parasite-control practices was also distributed. Key results Cyathostomin were present on all farms surveyed and S. vulgaris, despite being considered rare, was present on 7.8% (95% CI: 3.9–15.0) of farms. The prevalence of Parascaris spp. and equine tapeworm was 33.3% (95% CI: 19.6–50.6) and 3.9% (95% CI: 1.5–10.1) respectively. Questionnaire responses showed that the majority (85.0%) of horse owners administer anthelmintics at regular intervals of 12 weeks or less, and only 2.6% utilise faecal egg counts to inform treatment decisions. Conclusions Prevalence data confirmed the endemic nature of cyathostomin and P. equorum infections, as well as low levels of tapeworm and S. vulgaris infections on Australian horse farms. Worm-management practices were reminiscent of traditional interval-style treatment regimens that rely heavily on macrocyclic lactone anthelmintics. Implications These results suggest a need for more strategic approaches to internal-parasite control in horses to slow the development of anthelmintic resistance. Such programs need to consider the risk of re-establishment of the pathogenic S. vulgaris in significant numbers.

IMPROVING SOME PARAMETERS OF ANTIBODY RESPONSE IN HORSES BY PROBIOTIC AGENTS AGAINST BACKGROUND USE OF MACROCYCLIC LACTONE ANTHELMINTIC

Multiple researches demonstrate that both invasive diseases and use of anthelmintcs adversely affect horse immunity harming the gut flora. Macrocyclic lactone anthelmintics are proved to be highly effective agents but there is insufficient knowledge of their effect on the horse antibody immunity. The issue of immune system correction by the probiotic agents is also underreported. The paper demonstrates the results of the effect made by parascaridosis and strongylidosis invasion and deworming using the macrocyclic lactone drug on some parameters of antibody response in horses. Data on effect of probiotic Bioxymin horse on blood serum bacteriocidal and lysozyme activity were reported for the first time. The research was performed from December 2018 to January 2019. The results demonstrated that helminthic invasion and anthelmintic drug Univerm make depressive effect on some parameters of antibody response in horses. It also has an impact on the extensive efficacy of the anthelmintic agent. Probiotic Bioxymin horse makes a stimulating effect on the antibody response in horses.

Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Adaptation of a 96-well plate larval migration inhibition test for measuring the sensitivity of cyathostomins to macrocyclic lactone anthelmintics

The use of macrocyclic lactone drugs for control of equine cyathostomins is threatened by increasing levels of resistance. Detection of changes in drug sensitivity is important for effective and sustainable management of cyathostomins, however, at present such detection relies on the use of the faecal egg count reduction test, which is known to be an insensitive method. The present study therefore aimed to examine the use of a 96-well plate larval migration inhibition test for detection of resistance to macrocyclic lactone drugs in cyathostomins. We optimised conditions for migration of larvae, and examined the effects of larval storage time on drug dose responses. The modified test was able to define the sensitivity of cyathostomin isolates to ivermectin and eprinomectin in terms of dose response curves, and IC50 and IC95 values. The IC95 showed much greater consistency than the IC50 with larvae that had been stored for different periods prior to the test. Comparisons between two isolates, which had both been defined previously as susceptible using faecal egg count reduction tests, showed more variation at the IC50 compared to the IC95. Limitations of the test included the degree of variation in control-well migration despite optimisation of migration incubation conditions, and the need to incorporate a method to determine the species composition of the larval populations to account for possible species differences in drug sensitivity among cyathostomins. Validation of the technique on reference susceptible and resistant isolates of known species composition is still required.

Strongyloides spp. infections of veterinary importance.

This paper reviews the occurrence and impact of threadworms, Strongyloides spp., in companion animals and large livestock, the potential zoonotic implications and future research. Strongyloides spp. infect a range of domestic animal species worldwide and clinical disease is most often encountered in young animals. Dogs are infected with Strongyloides stercoralis while cats are infected with different species according to geographical location (Strongyloides felis, Strongyloides tumefaciens, Strongyloides planiceps and perhaps S. stercoralis). In contrast to the other species, lactogenic transmission is not a primary means of infection in dogs, and S. stercoralis is the only species considered zoonotic. Strongyloides papillosus in calves has been linked to heavy fatalities under conditions of high stocking density. Strongyloides westeri and Strongyloides ransomi of horses and pigs, respectively, cause only sporadic clinical disease. In conclusion, these infections are generally of low relative importance in livestock and equines, most likely due to extensive use of macrocyclic lactone anthelmintics and/or improved hygiene. Future prevalence studies need to include molecular typing of Strongyloides species in relation to different hosts. More research is urgently needed on the potential zoonotic capacity of Strongyloides from dogs and cats based on molecular typing, information on risk factors and mapping of transmission routes.

Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein

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.

Characterization of Haemonchus contortus P-glycoprotein-16 and its interaction with the macrocyclic lactone anthelmintics

Anthelmintic resistance in veterinary nematodes, including Haemonchus contortus, has become a limitation to maintaining high standards of animal health. Resistance in this parasite, to all drug families including the macrocyclic lactones (MLs) is a serious issue worldwide. Mechanisms of resistance to the MLs appear to be complex and to include the elimination of these compounds by ABC transporter-like proteins present in nematodes. In order to investigate the potential involvement of ABC transporters in ML resistance in H. contortus, we have characterized the functionality of the ABC transporter H. contortus P-glycoprotein-16 (Hco-PGP-16) expressed in mammalian cells. This has included a study of its interaction with different MLs, including the avermectins, abamectin (ABA) and ivermectin (IVM), and the milbemycin, moxidectin (MOX). Hco-PGP-16 transport activity was studied using the fluorophore Rhodamine 123 (Rho 123). Transfected cells expressing Hco-PGP-16 accumulated less than 50% of Rho 123 than control cells, suggesting an active transport of this tracer dye by Hco-PGP-16. The influence of the MLs on the Rho123 transport by Hco-PGP-16 was then investigated. A marked inhibition of Rho123 transport by ABA and IVM was observed. In contrast, MOX showed less effect on inhibition of Rho123 transport by Hco-PGP-16, and the inhibition was not saturable. The difference in the interaction of the avermectins and MOX with Hco-PGP-16 may help explain the slower rate of development of resistance to MOX compared with the avermectins in H. contortus.

Relative potency of macrocyclic lactones in in vitro assays with larvae of susceptible and drug-resistant Australian isolates of Haemonchus contortus and H. placei

The present study used in vitro assays to determine the relative potency of commercial macrocyclic lactone (ML) anthelmintics against larvae of drug-susceptible and drug-resistant Australian isolates of important parasites of sheep and cattle, Haemonchus contortus and Haemonchus placei, respectively. Cattle pour-on products containing abamectin, ivermectin, eprinomectin, doramectin or moxidectin were diluted in DMSO and used in larval development assays. Abamectin was the most potent chemical (lowest IC50 value) towards the drug-susceptible H. contortus Kirby isolate. The abamectin IC50 was approximately 2-fold lower than those for ivermectin, moxidectin, eprinomectin and doramectin. Moxidectin and abamectin were the most potent chemicals towards the resistant H. contortus Wallangra isolate. This isolate showed resistance ratios up to 70-fold towards eprinomectin. The resistance ratio for this species was lowest with moxidectin (ratio of 4.0-fold). Abamectin was also the most potent chemical towards both drug-susceptible (Bremner) and drug-resistant (Dayboro) H. placei isolates. The larval development assay only showed low levels of resistance for the drug-resistant H. placei, with resistance ratios ranging from 1.7 to 2.0 fold for moxidectin and abamectin, up to 3.3-fold for eprinomectin. This study examined the readily-accessible larval life stages of these parasites in in vitro assays, and, hence, the relationship between our findings and relative drug efficacies in vivo remains to be determined. Despite this, the study accords with some evidence from the use of these anthelmintics in the field in demonstrating the potency of moxidectin and abamectin against ML-resistant H. contortus. The study also highlights the usefulness of eprinomectin as a readily-available compound which is a more sensitive marker for ML resistance in in vitro larval development assays than the other commercial ML compounds examined.

Faecal worm egg count analysis for targeting anthelmintic treatment in horses: Points to consider

Equine gastrointestinal nematodes are ubiquitous; in horses that graze contaminated pasture and that are not treated appropriately, large numbers of worms can accumulate, which can lead to serious clinical disease. Nematode control has traditionally followed interval treatment regimens, which involve regular anthelmintic administration to all horses based on the strongyle egg reappearance periods of each drug, usually defined around the time of licensing. Interval treatment programmes have resulted in substantial reductions in large strongyle disease, but have made major contributions to the development of anthelmintic resistance, particularly in cyathostomins. Cyathostomin resistance to 2 of the 3 available anthelmintic classes is widespread, and resistance to both classes in single populations is not uncommon. Reduced efficacy of the most commonly used macrocyclic lactone anthelmintics, as measured by shortened egg reappearance periods after treatment, is emerging in cyathostomins. Macrocyclic lactone resistance is also now commonly reported in Parascaris equorum on stud farms. Faecal worm egg counts (FWEC) are increasingly being used as part of targeted approaches to parasite control, whereby only those horses with moderate to high FWEC within a group are treated with an anthelmintic. The objective of this approach is to reduce environmental contamination, while leaving a proportion of the worm population in some horses unexposed to selection pressure for anthelmintic resistance. This article reviews recent findings in equine parasitology research that will underpin guidelines for control, with a particular focus on how to optimise the value of FWEC methodologies and anthelmintic efficacy analyses.