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The sheep blowfly, Lucila cuprina, is a myiasis-causing parasite responsible for significant production losses and welfare issues for the Australian sheep industry. Control relies largely on the use of insecticides. The pyrimidine compound, dicyclanil, is the predominant control chemical, although other insecticides also are used, including imidacloprid, ivermectin, cyromazine and spinosad. We investigated in vitro resistance patterns and mechanisms in field-collected blowfly strains.The Walgett 2019 strain showed significant levels of resistance to both dicyclanil and imidacloprid, with resistance factors at the ICThis study indicates that in vitro resistance to both dicyclanil and imidacloprid in this field-collected blowfly strain is likely mediated by cytochrome P450, with Cyp12d1 implicated as the enzyme responsible; however, it remains possible that another P450 also may be involved. A common resistance mechanism for the two drugs has important implications for drug rotation strategies designed to prolong the useful life of flystrike control chemicals. © 2022 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

Anthelmintic resistance is a significant threat to livestock production systems worldwide and is emerging as an important issue in companion animal parasite management. It is also an emerging concern for the control of human soil-transmitted helminths and filaria. An important aspect of managing anthelmintic resistance is the ability to utilise diagnostic tests to detect its emergence at an early stage. In host-parasite systems where resistance is already widespread, diagnostics have a potentially important role in determining those drugs that remain the most effective. The development of molecular diagnostics for anthelmintic resistance is one focus of the Consortium for Anthelmintic Resistance and Susceptibility (CARS) group. The present paper reflects discussions of this issue that occurred at the most recent meeting of the group in Wisconsin, USA, in July 2019. We compare molecular resistance diagnostics with in vivo and in vitro phenotypic methods, and highlight the advantages and disadvantages of each. We assess whether our knowledge on the identity of molecular markers for resistance towards the different drug classes is sufficient to provide some expectation that molecular tests for field use may be available in the short-to-medium term. We describe some practical aspects of such tests and how our current capabilities compare to the requirements of an ‘ideal’ test. Finally, we describe examples of drug class/parasite species interactions that provide the best opportunity for commercial use of molecular tests in the near future. We argue that while such prototype tests may not satisfy the requirements of an ‘ideal’ test, their potential to provide significant advances over currently-used phenotypic methods warrants their development as field diagnostics., Graphical abstract Image 1, Highlights • Management of anthelmintic resistance requires reliable, sensitive and cost-effective diagnostic methods. • Molecular anthelmintic resistance tests have a number of advantages over currently-used phenotypic tests. • However, there are a number of challenges impacting on the use of molecular tests in the field. • There are also clear opportunities for proof-of-concept use of molecular tests in the short term.

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species.

This paper reports the use of near infrared and mid-infrared spectroscopy to detect the presence and quantity of eggs of the gastrointestinal nematode Haemonchus contortus in sheep faeces. Haemonchus contortus eggs were quantified in dried, finely ground sheep faeces and in moist, coarsely ground faeces using near infrared and mid-infrared bench top spectrometers and a portable near infrared spectrometer. When Haemonchus contortus eggs were presented without faecal medium, it was found that the wavelength region of 1880–2100 nm was most important for detection. Broad classes of chemical properties found in the near infrared region were identified for dried Haemonchus contortus eggs using a mid-infrared spectrometer. However, when Haemonchus contortus eggs were mixed into the complex matrix of sheep faeces, the development of a robust calibration model for egg detection proved to be challenging (R2

Flystrike remains a serious financial and animal welfare issue for the sheep industry in Australia despite many years of research into control methods. The present paper provides an extensive review of past research on flystrike, and highlights areas that hold promise for providing long-term control options. We describe areas where the application of modern scientific advances may provide increased impetus to some novel, as well as some previously explored, control methods. We provide recommendations for research activities: insecticide resistance management, novel delivery methods for therapeutics, improved breeding indices for flystrike-related traits, mechanism of nematode-induced scouring in mature animals. We also identify areas where advances can be made in flystrike control through the greater adoption of well-recognised existing management approaches: optimal insecticide-use patterns, increased use of flystrike-related Australian Sheep Breeding Values, and management practices to prevent scouring in young sheep. We indicate that breeding efforts should be primarily focussed on the adoption and improvement of currently available breeding tools and towards the future integration of genomic selection methods. We describe factors that will impact on the ongoing availability of insecticides for flystrike control and on the feasibility of vaccination. We also describe areas where the blowfly genome may be useful in providing impetus to some flystrike control strategies, such as area-wide approaches that seek to directly suppress or eradicate sheep blowfly populations. However, we also highlight the fact that commercial and feasibility considerations will act to temper the potential for the genome to act as the basis for providing some control options.

This review article provides an historical perspective on some of the major research advances of relevance to ruminant livestock gastrointestinal nematode control over the last 50 years. Over this period, gastrointestinal nematode control has been dominated by the use of broad-spectrum anthelmintic drugs. Whilst this has provided unprecedented levels of successful control for many years, this approach has been gradually breaking down for more than two decades and is increasingly unsustainable which is due, at least in part, to the emergence of anthelmintic drug resistance and a number of other factors discussed in this article. We first cover the remarkable success story of the discovery and development of broad-spectrum anthelmintic drugs, the changing face of anthelmintic drug discovery research and the emergence of anthelmintic resistance. This is followed by a review of some of the major advances in the increasingly important area of non-pharmaceutical gastrointestinal nematode control including immunology and vaccine development, epidemiological modelling and some of the alternative control strategies such as breeding for host resistance, refugia-based methods and biological control. The last 50 years have witnessed remarkable innovation and success in research aiming to improve ruminant livestock gastrointestinal nematode control, particularly given the relatively small size of the research community and limited funding. In spite of this, the growing global demand for livestock products, together with the need to maximise production efficiencies, reduce environmental impacts and safeguard animal welfare – as well as specific challenges such as anthelmintic drug resistance and climate change– mean that gastrointestinal nematode researchers will need to be as innovative in the next 50 years as in the last.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans. Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M−PONTX−Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8–5.6 μM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

BackgroundExisting diagnostic methods for the parasitic gastrointestinal nematode,Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, thereby preventing losses in production and flock welfare. We previously demonstrated the ability of visible–near-infrared (Vis–NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we report our investigation of whether variation in sheep type and environment affect the prediction accuracy of Vis–NIR spectroscopy in quantifying blood in faeces.MethodsVisible–NIR spectra were obtained from worm-free sheep faeces collected from different environments and sheep types in South Australia (SA) and New South Wales, Australia and spiked with various sheep blood concentrations. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387–609 nm) using partial least squares regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected sheep faeces from Queensland (QLD). Samples from QLD were quantified using Hemastix® test strip and FAMACHA© diagnostic test scores.ResultsPrincipal component analysis showed that location, class of sheep and pooled versus individual samples were factors affecting the Hb predictions. The models successfully differentiated ‘healthy’ SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity 57–94%, specificity 44–79%). The models were not predictive for blood in the naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of the QLD samples, however, identified a difference between samples containing high and low quantities of blood.ConclusionThis study demonstrates the potential of Vis–NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture sufficient environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas whereH. contortusis endemic.Graphical Abstract

Background: Existing diagnostic methods for the parasitic gastrointestinal nematode, Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, preventing production and welfare loss in the flock. We previously demonstrated the ability of visible-near infrared (vis-NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we investigate whether variation in sheep type and environment affect the prediction accuracy of vis-NIR spectroscopy in quantifying blood in faeces.Methods: Vis-NIR spectra were obtained from worm-free sheep faeces from different environments in South Australia (SA) and New South Wales (NSW), Australia and spiked with various sheep blood concentrations collected. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387 – 609 nm) using partial least squares (PLS) regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected Queensland (QLD) faeces. Naturally occurring blood in QLD samples was quantified using Hemastix® and FAMACHA© scores.Results: PCA showed that location, class of sheep and pooled/individual samples were factors affecting the Hb predictions in sheep faeces. The calibration models successfully differentiated ‘healthy’ SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity: 57 – 94%, specificity: 44 – 79%). The models were not predictive for naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of QLD samples, however, identified a difference between samples containing high and low quantities of blood.Conclusion: This study demonstrates the potential of vis-NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture enough environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas where H. contortus is endemic for the accurate prediction of H. contortus infections in these regions.

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula, exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus, the fruit fly Drosophila melanogaster, and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure&ndash, activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types.

Heavy infestations of the Barber’s pole worm, Haemonchus contortus, can cause severe wasting, morbidity and mortality in animals if not promptly treated. The current detection methods for this blood-sucking parasite involve faecal worm egg counts and diagnosis of anaemia, both of which are time consuming and require expertise. As blood is detected in sheep faeces sooner during infection than worm eggs, quantitative evaluation of blood may serve as a sensitive indicator of H. contortus infection. Here we investigated the feasibility of rapid estimation of haemoglobin (Hb) in sheep faeces using visible near-infrared spectroscopy. Haemoglobin (Hb) was assessed at various concentrations in moist sheep faeces using portable visible near-infrared (vis-NIR) spectrometers. Calibration models were developed for the region of 400–600 nm, where Hb absorption bands can be found. Within this wavelength region, Hb in sheep faeces can be estimated with minimal interference from background moisture (970 nm) or chlorophyll (670 nm), suggesting that difference in diets in sheep will have minimal effect on prediction accuracy. Predictions for sheep faeces based on a drenching threshold of 3 µg Hb/mg faeces showed high levels of accuracy with minimal sample preparation (Sensitivity = 89%, specificity = 80%). The success in detecting Hb in sheep faeces indicates the potential of vis-NIR spectroscopy as a rapid, on-farm diagnostic method for predicting blood in sheep faeces, and timing treatment of H. contortus infections.

The larval development assay has been used for many years to measure the sensitivity of the free-living life stages of trichostrongylid nematodes to anthelmintics. The assay has applications in both drug discovery and the diagnosis of drug resistance. We revisited the usefulness of the larval development assay for diagnosis of resistance to levamisole using field-derived isolates of Haemonchus contortus and Trichostrongylus colubriformis showing varying levels of resistance to this drug in vivo. Each of the resistant isolates showed a plateau in their larval development assay dose-response at the highest drug concentrations tested, representing a highly-resistant fraction, amounting to between 6.9 and 55.1% of the populations. The remaining population fractions for the resistant isolates showed IC50 values from 1.4- to 17.8-fold higher than their corresponding susceptible isolate of the same species. We used a data set from the DrenchRite® test user manual to derive equations describing the relationship between the IC50 values for these low / moderate resistance components of each population and in vivo drug efficacy, and then used these equations to estimate the expected in vivo efficacy of levamisole against this population component of each isolate. A combination of this expected efficacy, with the known zero efficacy of the drug in vivo against the highly-resistant population fractions in each isolate, allowed us to calculate a predicted drug efficacy for the whole population for each isolate. The predicted levamisole efficacies for the three resistant H. contortus isolates were 88.8, 84.1 and 43.7%. These compared favourably with the actual efficacy of the drug against these isolates as determined in faecal egg count reduction tests or total worm count studies: 79, 66.3 and 40.6%, respectively. Similarly, for T. colubriformis, predicted efficacies of 82.0 and 1.8% compared favourably with the actual efficacies of 65-92 % and 0%, respectively. This study illustrates the usefulness of the larval development assay as a diagnostic tool for predicting in vivo efficacy of levamisole against H. contortus and T. colubriformis.

There is a need to investigate new methods of controlling cyathostomins in horses due to increasing anthelmintic resistance amongst these parasites. In a previous study we identified the Australian plant Alectryon oleifolius as having anthelmintic activity towards cyathostomins. This study aimed to isolate and identify the bioactive compound(s) responsible for all or part of this anthelmintic activity and quantify its activity in vitro. The condensed tannin procyanidin A2 was isolated from the plant through a process of bioassay guided fractionation and identified using 1D and 2D nuclear magnetic resonance spectroscopy and high performance liquid chromatography with mass spectrometry. Procyanidin A2 demonstrated significant anthelmintic activity in larval development assays, completely inhibiting development from egg to third larval stage at concentrations as low as 50μg/mL and having an IC50 value of 12.6μg/mL. Procyanidin A2 also significantly inhibited larval migration at concentrations of 25μg/mL. This study indicates that procyanidin A2 is the principal anthelmintic compound in extracts from A. oleifolius, and further highlights the potential for the use of this plant as a component of cyathostomin control programs in the future.

Resistance to the anthelmintic drug monepantel (Zolvix®) has emerged in parasitic worms infecting sheep and goats. The mechanism of resistance in these cases is unknown. The drug targets nicotinic acetylcholine receptors belonging to the nematode-specific DEG-3 subfamily. We examined the receptor gene, Hco-mptl-1, in a highly Zolvix®-resistant and a -susceptible isolate of the parasitic nematode Haemonchus contortus. cDNA coding for the full length receptor protein (Hco-MPTL-1) was present in all clones prepared from a pool of susceptible larvae (21/21 clones) and approximately 50% of those from the resistant isolate (17/33). On the other hand, the remaining clones from the resistant isolate showed various mutations that resulted in truncated predicted proteins, missing at least one transmembrane domain. The most common mutation (11/33 clones) resulted in the retention of intron 15, a premature stop codon, and a truncated protein. Sequencing of intron 15 genomic DNA showed very few SNPs in susceptible larvae and in 12/18 clones from resistant larvae, alongside the presence of at least 17 SNPs in the remaining resistant clones. The present study shows that the highly resistant isolate has a number of mutations in the drug target gene that would most-likely result in a non-functional receptor, thus rendering the larvae insensitive to the drug. The presence of many wild-type sequences in this highly-resistant population suggests that there was a significant presence of heterozygotes in the survivors of the field drench treatment from which the isolate was derived, and hence that at least some of the mutations may be dominant. Alternatively, their presence may be due to the additional influence of mutations at another locus contributing to the resistance phenotype. The presence of multiple separate mutations in the Hco-mptl-1 gene in this viable field-derived worm isolate may at least partly explain why resistance to Zolvix® has arisen rapidly in the field., Graphical abstract Image 1, Highlights • Hco-mptl-1 gene compared in susceptible and monepantel-resistant Haemonchus contortus. • Six separate mutations detected in the resistant isolate. • Most common mutation resulted in retention of intron and truncated predicted protein. • Many separate mutations in the drug target site in the field-derived resistant isolate.

The macrocyclic lactone (ML) drugs are central to the control of equine strongyles but recent international reports raise concerns about reduced efficacy of these drugs against cyathostomins. The objectives of the present study were firstly, to evaluate the efficacy of ML drugs against cyathostomins on a cross-section of Australian horse farms, and secondly, to determine the egg reappearance period (ERP) following treatment of horses with MLs. A total of 419 horses on 43 properties were treated orally with ivermectin, abamectin or moxidectin, at recommended dose rates and drug efficacy was determined using the faecal egg count reduction test. Efficacy of 100% at 14 days post-treatment was reported on all of the 43 farms. ERP following ivermectin treatment was 6 weeks on two properties and ERP following moxidectin treatment was 12 weeks on a third property. These ERPs are shorter than those reported at the time of commercial release of these drugs which likely reflects changing drug susceptibility of the cyathostomin populations tested. Ongoing surveillance of drug efficacy and ERPs should be part of an integrated management approach to equine worm control that prioritises the preservation of anthelmintic efficacy.

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the classification of M&HI infections. Validation studies are required before they can be recommended for general use in assessing M&HI infections in programmatic settings., Author summary The prevalence of moderate-to-heavy intensity (M&HI) infections is a key indicator for measuring the success of large-scale deworming programs for intestinal worms because they account for the majority of the worm-attributable morbidity. Currently, intestinal worm infections are classified as M&HI when the number of worm eggs that are microscopically detected in stool using a standard diagnostic method exceeds a threshold set by the World Health Organization. Over the years, a variety of new promising diagnostic methods have been introduced for the diagnosis of intestinal worms. Although they have some important advantages over the current standard method, it is not clear whether they can reliably classify M&HI infections. This is because their test results either systematically indicate lower egg counts or are expressed in a unit other than eggs per gram of stool (e.g, concentration of worm DNA), warranting the need for method-specific thresholds. We defined method-specific thresholds and verified whether they increased the correct classification of M&HI infections. Overall, our results indicate that method-specific thresholds improved the classification of M&HI infections, but that further validation is required before they can be recommended for evaluating the occurrence M&HI infections in large-scale deworming programs.

Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC50 4.9 μM and 16.1 μM, respectively, in a drug-susceptible isolate) with no existing cross-resistance in two multidrug resistant isolates and a monepantel-resistant isolate. Combinations of pentamidine with commercial anthelmintics showed additive activity, with no significant synergism detected. Pentamidine and diminazene showed different life-stage patterns of activity; both were active against early stage larvae in development assays, but only diminazene was active against the infective L3 stage in migration assays. This suggests some differences in uptake of the two drugs across the nematode cuticle, or differences in the nature and expression patterns of their molecular targets. As pentamidine and diminazene have been reported to be potent inhibitors of mammalian acid-sensing ion channels (ASIC), we tested the activity of known ASIC inhibitors against H. contortus to probe whether these channels may represent potential anthelmintic targets in nematodes. Remarkably, the spider-venom peptide Hi1a, a potent inhibitor of ASIC1a, inhibited H. contortus larval development with an IC50 of 22.9 ± 1.9 μM. This study highlights the potential use of diarylamidines as anthelmintics, although their activity needs to be confirmed in vivo. In addition, our demonstration that ASIC inhibitors have anthelmintic activity raises the possibility that this family of ion channels may represent a novel anthelmintic target.

Breeding for resistance to gastrointestinal nematodes (GIN) in sheep relies largely on the use of worm egg counts (WEC) to identify animals that are able to resist infection. As an alternative to such measures of parasite load we aimed to develop a method to identify animals showing resistance to GIN infection based on the impact of the infection on blood parameters. We hypothesized that blood parameters may provide a measure of infection level with a blood-feeding parasite through perturbation of red blood cell parameters due to feeding behaviour of the parasite, and white blood cell parameters through the mounting of an immune response in the host animal. We measured a set of blood parameters in 390 sheep that had been exposed to an artificial regime of repeated challenges with Trichostrongylus colubriformis followed by Haemonchus contortus. A simple analysis revealed strong relationships between single blood parameters and WECs with correlation coefficients −0.54 to −0.60. We then used more complex multi-variate methods based on supervised classifier models (including Bayesian Network) as well as regression models (Lasso and Elastic Net) to study the relationships between WECs and blood parameters, and derived algorithms describing the relationships. The ability of these algorithms to classify sheep GIN resistance status was tested using the WEC and blood parameters collected from a different group of 418 sheep that had acquired natural infections of H. contortus from pasture. We identified the most resistant and most susceptible animals (10% percentiles) of this group based on WECs, and then compared the identities of these animals to the identities of animals that were predicted to be most resistant and most susceptible by our algorithms. The models showed varying abilities to predict susceptible and resistant sheep, with up to 65% of the most susceptible animals and 30% of the most resistant animals identified by the Elastic Net model algorithms. The prediction algorithms derived from female sheep data performed better than those for male sheep in some cases, with the predicted animals accounting for up to 50–60% of the actual resistant and susceptible female animals. Heritability values were calculated for blood parameters and the aggregate trait descriptions defined by the novel prediction algorithms. The aggregate trait descriptions were moderately heritable and may therefore be suitable for use in genetic selection strategies. The present study indicates that multivariate models based on blood parameter data showed some ability to predict the resistance status of sheep to infection with H. contortus.

About 80% of the world’s cattle are affected by ticks and tick-borne diseases, both of which cause significant production losses. Cattle host resistance to ticks is the most important factor affecting the economics of tick control, but it is largely neglected in tick-control programs due to technical difficulties and costs associated with identifying individual-animal variation in resistance. The present paper reviews the scientific literature to identify factors affecting resistance of cattle to ticks and the biological mechanisms of host tick resistance, to develop alternative phenotype(s) for tick resistance. If new cost-effective phenotype(s) can be developed and validated, then tick resistance of cattle could be genetically improved using genomic selection, and incorporated into breeding objectives to simultaneously improve cattle productive attributes and tick resistance. The phenotype(s) could also be used to improve tick control by using cattle management. On the basis of the present review, it is recommended that three possible phenotypes (haemolytic analysis; measures of skin hypersensitivity reactions; simplified artificial tick infestations) be further developed to determine their practical feasibility for consistently, cost-effectively and reliably measuring cattle tick resistance in thousands of individual animals in commercial and smallholder farmer herds in tropical and subtropical areas globally. During evaluation of these potential new phenotypes, additional measurements should be included to determine the possibility of developing a volatile-based resistance phenotype, to simultaneously improve cattle resistance to both ticks and biting flies. Because the current measurements of volatile chemistry do not satisfy the requirements of a simple, cost-effective phenotype for use in commercial cattle herds, consideration should also be given to inclusion of potentially simpler measures to enable indirect genetic selection for volatile-based resistance to ticks.

The natural extract pyrethrum is an insecticidal oil derived from Tanacetum cinerariifolium that is commonly used in domestic and agricultural pesticides. The major constituents of the extract are the Pyrethrins, six esters that provide pyrethrum with its insecticidal properties. These Pyrethrins readily degrade through several environmental means and as such, there can be significant Pyrethrin losses during processing and long-term storage of pyrethrum-based insecticides. This work attempts to alleviate the effect of these degradative processes through the pursuit of stabilised Pyrethrins by chemically removing oxidatively sensitive functionality. Several reduced Pyrethrin analogues were produced and a method to convert the more sensitive Pyrethrins present in the pyrethrum concentrate into their respective more stable jasmolin counterparts, as a mixture with the over-reduced tetrahydropyrethrins, was developed. All other reduction processes abolished insecticidal activity against Lucilia cuprina larvae, whereas some isomerised analogues showed comparable potency with the individual natural pyrethrin esters. This work has revealed new insights into the structure–activity relationships in this unique class of insecticide.

Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a -susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC50), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with 'resistance-breaking' effects being much more marked in migration assays, suggest that some life-stage-specific aspects may exist in the interaction of ivermectin with P-gps in the two worm isolates.

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate., Graphical abstract, Highlights • We studied interaction of anthelmintics with ABC transporters in Haemonchus contortus. • pgp-1, 2, -9.1, -10, and -11 and haf-6 up-regulated after 3 h exposure to ivermectin. • Up-regulation occurred in a drug-resistant isolate but not in a -susceptible isolate. • A proportion of the drug exposed larvae showed tolerance to subsequent drug treatment. • Rapid up-regulation of transporters may be component of resistance in parasitic nematodes.

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.

P-glycoproteins (P-gps) play an important role in the sensitivity of nematodes to anthelmintic drugs. They have been implicated in a number of anthelmintic resistances, particularly for macrocyclic lactone drugs. Hence, inhibition of nematode P-gps has been suggested as a means of reversing some types of anthelmintic resistance. The present study aimed to investigate the ability of the most-recently developed group of P-gp inhibitors (the so-called 'third generation' of inhibitors) including tariquidar, zosuquidar and elacridar, to increase the sensitivity of Haemonchus contortus larvae to various anthelmintics (ivermectin, levamisole and thiabendazole) in vitro. We compared these compounds to some older P-gp inhibitors (e.g. verapamil and valspodar). Larval migration and development assays were used to measure the sensitivity of larvae to anthelmintics alone, or in combination with P-gp inhibitors. Significant increases in sensitivity to ivermectin were observed with zosuquidar and tariquidar in larval migration assays (synergism ratios up to 6-fold). Several of the inhibitors increased the sensitivity of both the drug-resistant and -susceptible isolates (e.g. tariquidar with ivermectin in migration assays, zosuquidar with ivermectin in larval development assays), while others had significant effects on the resistant isolate only (e.g. zosuquidar with ivermectin in migration assays, verapamil with ivermectin in development assays). This suggests that some of the inhibitors interact with P-gps representing intrinsic pathways present across nematode populations with quite different drug sensitivities, while other inhibitors interact with P-gps of significance only to resistant nematodes, and hence most likely representing an acquired resistance mechanism. The study highlights the potential of the third generation of P-gp inhibitors for increasing the sensitivity of nematodes to anthelmintics.

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S -transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC 50 ) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field.

Graphical abstract, Highlights • We report on the Consortium for Anthelmintic Resistance and Susceptibility 2013 meeting. • Recent advances in the identification of markers for anthelmintic resistance are described. • The use of markers for benzimidazole resistance in field studies with veterinary and human nematodes. • The application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance., Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

There is an increasing interest in the use of combination anthelmintic products for the control of intestinal nematode parasites of livestock. These products are seen as attractive options for parasite control in the face of increasing levels of resistance to the different anthelmintic drug classes, as well as a means to slow the rate at which resistance develops to the individual components of the combination. With the recent introduction of an anthelmintic combination product containing abamectin and monepantel (at 1:12.5), we were interested in measuring the response of drug-susceptible and drug-resistant isolates of Haemonchus contortus to these two drugs alone and in combination, using larval development assays. The GWBII isolate showed resistance to abamectin (12-fold) alongside susceptibility to monepantel. The resistance ratio was reduced from 12- to 3.2-fold when the two drugs were combined. The MPL-R isolate was resistant to both drugs, with resistance factors of 6-fold towards abamectin, and 10.6- and 1008-fold towards monepantel in two sub-populations present in the isolate. This isolate showed 6.4-fold resistance to the drug combination. Hence, for both GWBII and MPL-R, the level of resistance towards the combination was reduced compared to the resistance towards abamectin or monepantel alone, respectively, but was not abolished. However, for GWBII, this in vitro resistance to the drug combination would be expected to have no impact on the in vivo efficacy of the combination drench product as the isolate is resistant to only the abamectin component of the drench, with monepantel remaining effective. On the other hand, the observed in vitro resistance to the combination shown by the MPL-R isolate is derived from significant levels of resistance towards both components separately, and hence may impact on in vivo efficacy of the combination. Isobologram analysis did not find any evidence for a synergistic interaction between the two drugs in larval development assays. We examined the predicted effects of varying the abamectin:monepantel ratio in drug combinations, assuming that the two drugs acted in an additive fashion. For GWBII, resistance to the drug combination was reduced to almost zero as the abamectin:monepantel ratio increased from 1:12.5 to 1:100, reflecting its resistance to only the abamectin component of the combination. For MPL-R, on the other hand, the resistance increased as the relative proportion of monepantel in the combination was increased, reflecting the extreme level of in vitro resistance shown by this isolate to monepantel.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach–potato aphid ( Myzus persicae ). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

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.

We examined faecal egg count reduction tests (FECRTs) conducted with hookworm-infected humans in Ghana in 2007 (study 1) and 2010 (study 2) in order to explore aspects of the test analysis. Some subjects showed increased FEC following drug treatment. This occurred mostly in

Graphical abstract, Highlights • A Haemonchus contortus isolate subdivided according to level of resistance to levamisole in vitro. • Increased P-glycoprotein gene expression in larvae showing low level resistance. • Decreased expression of nAChR subunit and receptor assembly genes in larvae showing higher levels of resistance. • Results suggest drug efflux mediated low level resistance, with target site changes conferring higher level resistance., While there is some evidence that changes in nicotinic acetylcholine receptor (nAChR) subunits confer resistance to levamisole in gastrointestinal helminth parasites, the exact nature of the resistance mechanism(s) is unclear. We utilised the presence of a resistant fraction within the Wallangra 2003 isolate of Haemonchus contortus larvae in order to subdivide the population into three subpopulations of larvae able to survive increasing concentrations of the drug. We then measured gene expression levels in the subpopulations and the larval population as a whole, focusing on genes encoding the subunit components of levamisole-sensitive receptors, genes encoding ancillary proteins involved in receptor assembly, and P-glycoprotein (P-gp) genes. The subpopulation surviving the lowest levamisole concentration showed increases of 1.5- to 3-fold in a number of P-gp genes (Hco-pgp-3, -4, -10, and -14) alongside unchanged receptor genes, compared to the whole Wallangra larval population. On the other hand, the subpopulation surviving the intermediate levamisole concentration showed an increase in only a single P-gp (Hco-pgp-14), alongside decreases in some receptor subunit (Hco-unc-63a) and ancillary protein genes (Hco-unc-50, Hco-ric-3.1 and 3.1). The subpopulation surviving the highest levamisole concentration showed further decreases in receptor subunit genes (Hco-unc-63a and Hco-unc-29 paralogs) as well as genes involved in receptor assembly (Hco-unc-74, Hco-unc-50, Hco-ric-3.1 and 3.1), alongside no increased P-gp gene levels. This suggests a biphasic pattern of drug resistance in the larvae of this worm isolate, in which a non-specific P-gp-mediated mechanism confers low levels of resistance, while higher level resistance is due to altered receptor subunit composition as a result of changes in both subunit composition and in the levels of proteins involved in receptor assembly.

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non-specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5-30-fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active-site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.

Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ecto-parasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC-907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc-binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides., Graphical abstract Image 1, Highlights • We measured the insecticidal effects of histone deacetylase inhibitors against the sheep blowfly. • Insecticidal activity correlated with inhibition of HDAC enzyme activity in nuclear extracts. • Romidepsin showed equivalent or greater potency than commercial blowfly insecticides. • Some insights gained into structural requirements for insecticidal HDAC inhibitors. • Potential for HDAC inhibitors as insecticides.

Macrocyclic lactone (ML) drugs inhibit pharyngeal pumping, motility and egg laying in parasitic nematodes. Previous work has indicated that in vitro effects on worm feeding occurred at lower ivermectin concentrations than effects on worm motility, suggesting that the pharynx musculature was a more important target site for the ML drugs than somatic musculature. We have reassessed this issue of relative sensitivity by examining the response of drug-susceptible and -resistant adult Haemonchus contortus worms to abamectin in vitro using both feeding and motility assays. The motility assay involved observation of changes in the form and degree of movement of individual worms in response to the drug. A comparison of the data from the two assays indicated that worm motility was affected at drug concentrations below those required to inhibit feeding. Analysis of the motility data using different levels of sensitivity (varying in the degree to which they accounted for subtle vs. more profound changes in worm motility) provided an explanation as to why earlier reports had observed feeding to be the more sensitive target. Motility IC50 values shifted from being less than feeding IC50s to being greater than the feeding IC50s as the motility assay analysis method became less sensitive. The present study indicates that when sensitive worm motility assessment methods are utilised, worm motility is affected at lower abamectin concentrations than worm feeding, and hence highlights somatic musculature as a more important target site for this ML drug, and most likely for ML drugs in general.

Background There is some evidence that ATP binding cassette (ABC) transporters play a role in resistance to anthelmintics, particularly against macrocyclic lactones. Some anthelmintics, including ivermectin (IVM), have been shown to induce transcription of multiple ABC transporters in nematodes; however, the effects of monepantel (MPL) on transcription of these transporter genes has not been studied. Methods Larvae of two MPL-susceptible isolates of Haemonchus contortus were exposed to MPL at two concentrations (2.5 and 250 μg/ml) for periods of 3, 6 and 24 h. Transcription levels of sixteen ABC transporter genes were measured at the end of the incubation periods. The consequences of MPL exposure were examined by measuring rhodamine-123 efflux from the larvae, and their sensitivity to subsequent treatment with IVM or levamisole. Results Multiple ABC transporter genes showed significantly higher transcription in both worm isolates following exposure to MPL at 250 μg/ml for 3, 6 or 24 h, particularly the P-glycoprotein (P-gp) genes pgp-11, pgp-12 and pgp-14. Of these, only pgp-11 maintained the elevated levels 24 h after the end of the drug exposure period. In contrast, there was only a single instance of low-level upregulation as a result of exposure to MPL at 2.5 μg/ml. Larvae exposed to MPL at 250 μg/ml showed an increased efflux of rhodamine-123 and a proportion of the larval population showed an ability to subsequently tolerate higher concentrations of IVM in migration assays. There was no increased tolerance to IVM following pre-exposure to MPL at 2.5 μg/ml. Conclusions Exposure of H. contortus larvae to 250 μg/ml MPL results in increased transcription of multiple transporter genes and increased R-123 efflux. The subsequent ability of a proportion of the larvae to tolerate IVM suggests a protective role of ABC transporters across different chemical entities. However, these observations were only made at a concentration of MPL well above that experienced by parasitic life stages in vivo, and hence their significance remains unclear. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1806-9) contains supplementary material, which is available to authorized users.

The dual oxidases (DUOX1 and DUOX2) are NADPH-dependent hydrogen peroxide-producing enzymes that are reported to function in a physiological capacity and as a component of the mucosal immune response. We have previously reported increased expression of the DUOX2 gene in the gut mucosa of sheep in response to gastrointestinal nematode (GIN) challenge. In this paper, we report the cloning of the full-length ovine DUOX2 transcript, using a PCR based strategy. The ovine DUOX2 transcript includes an ORF of 4644 bases, and encodes a protein with 97% identity to the bovine sequence. We also cloned a fragment of DUOX1 (encompassing nucleotides 2692-2829), and the proximal promoter sequence of DUOX2. Through analysis of sequence data we have confirmed that DUOX1 and DUOX2 are co-located in a head to tail arrangement conserved across many species. Alignment of the sequences to the ovine genome predicts a location of this gene cluster on ovine chromosome 7. We quantified the expression of ovine DUOX1 and DUOX2 transcripts in 24 different sheep tissues, and discovered tissue specific expression signatures. DUOX2 was found to be most highly expressed in tissues of the gastrointestinal tract, while expression of DUOX1 predominated in the bladder. Rapid amplification of cDNA ends (RACE) analysis identified the existence of multiple 5' UTR variants in DUOX2, ranging in size from 32 to 242 nucleotides, with 3 distinct transcribed regions. Real time PCR quantification of the DUOX2 UTR variants revealed that these were differentially expressed between tissues, and at various stages of the response to GIN parasite infection. The collective evidence suggested a complex regulation of DUOX2, prompting a bioinformatic analysis of the proximal promoter regions of ovine DUOX2 to identify potential transcription factor binding sites (TFBS) that may explain the differences in the observed expression of the transcript variants of DUOX2. Possible transcription factor families that may regulate this process were identified as Kruppel-like factors (KLF), ETS-factors, erythroid growth receptor factors (EGRF) and myogenic differentiation factors (MYOD).