The current status and outlook for insecticide, acaricide and anthelmintic resistances across the Australian ruminant livestock industries: assessing the threat these resistances pose to the livestock sector.

The Australian ruminant livestock industries are faced with the need to control parasitic infectious diseases that can seriously impact the health of animals. However, increasing levels of resistance to insecticides, anthelmintics and acaricides are substantially reducing the ability to control some of these parasites. Here we review the current situation with regard to chemical resistances in parasites across the various sectors of the Australian ruminant livestock industries and assess the level of threat that these resistances pose to the sustainability of these sectors in the short to long terms. We also look at the extent to which testing for resistance occurs across the various industry sectors, and hence how well-informed these sectors are of the extent of chemical resistance. We examine on-farm management practices, breeding of parasite-resistant animals, and non-chemical therapeutics that may act as short to long term means to reduce the current reliance on chemicals for parasite control. Finally, we look at the balance between the prevalence and magnitude of current resistances and the availability and adoption rates of management, breeding and therapeutic alternatives in order to assess the parasite control outlook for the various industry sectors.

Control of sheep flystrike: what's been tried in the past and where to from here.

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.

Procyanidin A2 in the Australian plant Alectryon oleifolius has anthelmintic activity against equine cyathostomins in vitro.

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.

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.

A survey of macrocyclic lactone efficacy in Australian cyathostomin populations.

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 14days post-treatment was reported on all of the 43 farms. ERP following ivermectin treatment was 6weeks on two properties and ERP following moxidectin treatment was 12weeks 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.

Anthelmintic Resistance in Haemonchus contortus: History, Mechanisms and Diagnosis.

Haemonchus contortus has shown a great ability to develop resistance to anthelmintic drugs. In many instances, resistance has appeared less than 10years after the introduction of a new drug class. Field populations of this species now show resistance to all major anthelmintic drug classes, including benzimidazoles (BZs), imidazothiazoles and macrocyclic lactones. In addition, resistance to the recently introduced amino-acetonitrile derivative class (monepantel) has already been reported. The existence of field populations showing resistance to all three major drug classes, and the early appearance of resistance to monepantel, threatens the sustainability of sheep and goat production systems worldwide. This chapter reviews the history of the development of resistance to the various anthelmintics in H. contortus and examines the mechanisms utilized by this species to resist the effects of these drugs. Some of these mechanisms are well understood, particularly for BZ drugs, while our knowledge and understanding of others are increasing. Finally, we summarize methods available for the diagnosis of resistance. While such diagnosis currently relies largely on the faecal egg count reduction test, which suffers from issues of expense and sensitivity, we describe past and current efforts to utilize cheaper and less laborious phenotypic assays with free-living life stages, and then describe progress on the development of molecular assays to provide sensitive resistance-detection tests.

Suspected ivermectin resistance in a south-east Queensland Parascaris equorum population.

BACKGROUND: There have been several international reports of macrocyclic lactone (ML)-resistant Parascaris equorum over the past decade, but the resistance status of Australian P. equorum populations is largely unknown. A case of apparent reduced efficacy of ivermectin against P. equorum in Australia was investigated. METHODS AND RESULTS: A faecal egg count reduction test carried out on a group of weanling foals in south-east Queensland showed the efficacy of ivermectin to be 65%. CONCLUSION: The case highlights the need to review current worm control strategies, especially for young horses.

Cloning, recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly, Lucilia cuprina.

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.

Australian plants show anthelmintic activity toward equine cyathostomins in vitro.

Anthelmintic resistance in gastrointestinal parasites of horses is an increasing problem, particularly in cyathostomins, and there is a need to find alternative means for the control of these parasites. We screened crude extracts from 37 species of Australian native plants for their anthelmintic activity in vitro against cyathostomin larvae (development from egg to third larval stage), with the aim of identifying those species that may be suitable for incorporation into sustainable parasite management programs. Water extracts from seven species, namely Acacia baileyana, Acacia melanoxylon, Acacia podalyriifolia, Alectryon oleifolius, Duboisia hopwoodii, Eucalyptus gomphocephala and Santalum spicatum completely inhibited larval development (100% inhibition compared to the control), while another 10 species caused 90% inhibition at the initial screening concentration of 1400 µg of extractable solids/mL. The seven most potent extracts produced IC50 values (concentration of extract which resulted in a 50% inhibition of development) in the range 30.9-196 µg/mL. Fourteen extracts were incubated with polyvinylpolypyrrolidone (PVPP) before the assays, which removed the anthelmintic activity from 12 of these extracts, indicating that tannins were likely to be the bioactive compound responsible for the effect, while in two species, i.e. A. melanoxylon and D. hopwoodii, compounds other than tannins were likely to be responsible for their anthelmintic action. Our results suggest that a number of Australian native plants have significant anthelmintic activity against cyathostomin larval development in vitro. There is potential for these plants to be used as part of sustainable parasite control programs in horses, although more research is needed to identify the compounds responsible for the anthelmintic effects and confirm their activity in vivo.

Molecular cloning and characterisation of ovine dual oxidase 2.

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).

Silencing of essential genes by RNA interference in Haemonchus contortus.

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

Target-based and whole-worm screening approaches to anthelmintic discovery.

Experimental approaches for identifying new anthelmintics include target-based and whole-worm screening methods. The former involves basic research into characterising and validating new targets, mostly proteins, followed by identification of inhibitors or agonists through the use of target-based screening assays and/or in silico drug design. The latter experimental approach uses whole-worm assays to identify anthelmintic agents with unknown modes of action, or where the primary interest lies in whether analogues are able to kill (or disable) worms rather than in measuring their direct impact on their likely target. This paper focuses initially on the intestine and external layers of nematodes as potential drug targets. Specific anthelmintic agents targeting either tissue are discussed to illustrate the impact of disruption to these structures. In both cases, the activity of these agents against insects was known, and activity against nematodes was identified using whole worm screening assays. Recent literature identifying ecdysone signalling pathway receptors in nematodes is then used to provide an example of basic research into a specific target that may lead to the development of high-throughput target-based drug screening assays. Finally, the role of whole-worm screening approaches versus target-based screening is discussed briefly.

Dual oxidase 2 and glutathione peroxidase gene expression are elevated in hyperimmunized sheep challenged with Haemonchus contortus.

A sequential biopsy sampling method was used to investigate oxidant and antioxidant gene responses in resistant sheep challenged with Haemonchus contortus larvae or a sham saline challenge. The expression of key sheep oxidant and antioxidant producing genes were measured in sequential samples removed from the abomasums at days 0, 1, 2, 3, 5, 7 and 28 post challenge. Gene expression levels at each time point were compared to expression at day 0, and levels of the various genes were also correlated to other markers of infection including immune cell counts and cytokine gene expression. The early response to larval challenge infection in resistant animals was marked by a divergence of two groups of host oxidant producing genes: the dual oxidase group (DUOX2/DUOXA2) showing increases in expression to day 7, while members of the phagocytic NADPH oxidase (PHOX) group showed significant decreases in expression. The change in DUOX2 expression between days zero and seven, when host resistance to infection is mediated, was negatively correlated to final worm burden suggesting NADPH oxidase expression may play a role in parasite expulsion. Expression of the DUOX group oxidants was positively correlated to expression of the Th2 cytokine IL4. Changes in host antioxidant pathways between different members of the glutathione peroxidase family (intestinal and plasma GPX) and genes involved in glutathione metabolism were also observed. This first study of the putative roles of oxidant production by the dual oxidase group, antioxidant glutathione pathways, immune cell populations, and cytokine profiles, in the development of resistance to infection by hyperimmune sheep are discussed.

Animal grazing selectivity and plant chemistry issues impact on the potential of Rhagodia preissii as an anthelmintic shrub.

Rhagodia preissii had shown significant in vitro anthelmintic activity in a previous study, we examined the effect of including this shrub in the diet of sheep infected with Trichostrongylus colubriformis. Worm-infected merino wethers were grazed for 7 weeks on either R. preissii or annual pasture, and faecal egg counts (FECs) were conducted weekly. Plant material was collected weekly from eaten and uneaten plants, and analysed for levels of plant secondary metabolites (tannins, oxalates, saponins) and in vitro anthelmintic activity. While mean FECs were consistently lower in sheep grazing R. preissii compared to pasture (reductions of 20-74%), the differences were not significant. There was no relationship between grazing preference (eaten or uneaten) and in vitro anthelmintic activity of plant extracts. The levels of saponins and oxalates did not correlate with grazing preference or in vitro anthelmintic activity, while tannins were not responsible for the anthelmintic activity. While the identity of the grazing deterrent and in vitro anthelmintic compounds remain unknown, the presence of plants which were both highly preferred by the sheep and showed in vitro anthelmintic activity indicates a potential to develop the species as an anthelmintic shrub through selection of shrub populations dominated by such plants.

Evaluation of reference genes for real-time PCR quantification of gene expression in the Australian sheep blowfly, Lucilia cuprina.

The Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), is an important pest of sheep in Australia and other parts of the world. However, the paucity of information on many fundamental molecular aspects of this species limits the ability to exploit functional genomics techniques for the discovery of new drug targets for its control. The present study aimed to facilitate gene expression studies in this species by identifying the most suitable reference genes for normalization of mRNA expression data. Quantitative real-time polymerase chain reaction (PCR) was performed with 11 genes across RNA samples from eggs, L1, L3, pupae and adult life stages, and two normalization programs, Normfinder and geNorm, were then applied to the data. The results showed an ideal set of genes (18S rRNA, 28S rRNA, GST1, beta-tubulin and RPLPO) for data normalization across all life stages. The most suitable reference genes for studies within specific life stages were also identified. GAPDH was shown to be a poor reference gene. The reference gene recommendations in this study will be of use to laboratories investigating gene expression in L. cuprina and related blowfly species.

The use of DNA markers to map anthelmintic resistance loci in an intraspecific cross of Haemonchus contortus.

The use of DNA markers to track the development of anthelmintic resistance in parasites of livestock would allow informed choices for the management of this important problem. We describe a genetic mapping approach for the discovery of DNA markers for anthelmintic resistance in Haemonchus contortus. We crossed a multi-drug resistant field isolate of H. contortus with a well-characterized laboratory strain susceptible to 4 drug classes. The F2 were separately selected with 5 anthelmintics from 4 drug classes, producing drug-resistant populations carrying gene variants derived from both the field isolate and the laboratory strain. Individual F2 worms were analysed using amplicon length polymorphisms (ALPs). We looked for field isolate alleles over- or under-represented in F2 populations compared to the unselected F2 and/or the laboratory strain. The data we obtained suggest that marker association can be used to link neutral markers with resistance, but also that more markers and perhaps more inbred laboratory strains would make the procedure more likely to succeed.

Exploring the anthelmintic properties of Australian native shrubs with respect to their potential role in livestock grazing systems.

We measured in vitro anthelmintic activity in extracts from 85 species of Australian native shrub, with a view to identifying species able to provide a degree of worm control in grazing systems. Approximately 40% of the species showed significant activity in inhibiting development of Haemonchus contortus larvae. The most active extracts showed IC50 values of 60-300 microg/ml. Pre-incubation with polyvinylpolypyrrolidine removed the activity from some extracts, implicating tannins as the bioactive agent, while in other cases the pre-incubation had no effect, indicating the presence of other anthelmintic compounds. Plant reproductive maturity (onset of flowering or fruiting) was associated with increasing anthelmintic activity in some species. Variability was observed between plants of the same species growing in different environments, while variation between individual plants of the same species within a single field suggests the existence of distinct chemotypes. Significant activity against adult H. contortus worms in vitro was also demonstrated in a limited number of extracts tested against this life stage. Our study indicates that there is potential for Australian native shrubs to play an anthelmintic role in grazing systems, and highlights some plant biology factors which will need to be considered in order to maximize any anthelmintic effects.

Strategies for the storage of Ancylostoma caninum third-stage larvae.

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.

The requirement for early exposure of Haemonchus contortus larvae to Bacillus thuringiensis for effective inhibition of larval development.

The potential for a nematocidal Bacillus thuringiensis (Bt) to target the free-living larval stages of Haemonchus contortus was examined using in vitro larval development and migration assays. Bt toxicity in larval development assays decreased as the time period between egg hatch and initial exposure to the Bt was increased; a time lag of 48 h resulted in a 350-fold increase in the IC(50) (from 2.6 ng/ml to 910 ng/ml). The effects on larval migration largely paralleled the effects on larval development, indicating that the larvae which reached the infective stage after exposure to Bt were generally as 'fit' as control worms in terms of migration ability. However, a comparison of the two assays also showed the presence of a level of Bt exposure which showed significantly more toxicity in migration assays than development assays, indicating that, in some cases, fully developed Bt-exposed larvae were less able to migrate than controls, and hence may be compromised in their ability to infect sheep. The rapid decrease in toxicity when exposure to the Bt is delayed highlights a significant issue concerning the use of Bt for control of the free-living larval stages of animal-parasitic nematodes. Targeting the larvae by delivering bacterial spores to the faeces through the host animal's digestive tract would require the spores to germinate upon defecation, grow through a vegetative phase, to then produce crystal toxin protein upon subsequent sporulation. This period of bacterial development will introduce a time lag between worm egg hatching and initial exposure of the larvae to the Bt, which, as demonstrated in the present study, may allow the worm larvae to develop to late larval stages which are relatively insensitive to the toxin.

A modified larval migration assay for detection of resistance to macrocyclic lactones in Haemonchus contortus, and drug screening with Trichostrongylidae parasites.

We have developed a modified migration assay system in 96-well plate format which is able to detect resistance to the macrocyclic lactone group of drugs in Haemonchus contortus. The assay involves exposure of infective stage larvae to drug for a 24 h period, then counting the numbers of larvae that are able to migrate through an agar and filter mesh system over a further 48 h. The agar barrier greatly increased the sensitivity of the assay for resistance detection compared to use of filter mesh alone. The assay was able to detect the presence of 10% resistant worms in an otherwise susceptible background. However, the assay was ineffective with Trichostrongylus colubriformis and Ostertagia circumcincta indicating that its usefulness for field monitoring will be restricted to situations where H. contortus is of most significance. A small-scale drug screening exercise showed that the assay identifies some anthelmintic activities distinct from those identified by larval development assays. The assay therefore also has a potential role in drug discovery programmes in screening for new anthelmintics.