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Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae from fecal samples from an experimental herd in Kentucky, USA and two feral horse populations from Sable Island and Alberta, Canada. In addition to reporting the detection of 33 different species with high confidence, we illustrate the assay's repeatability by comparing results generated from aliquots from the same fecal samples and from individual horses sampled repeatedly over multiple days or months. We also validate the quantitative potential of the assay by demonstrating that the proportion of amplicon reads assigned to different species scales linearly with the number of larvae present. This new tool significantly improves equine strongyle diagnostics, presenting opportunities for research on species-specific anthelmintic resistance and the causes and consequences of variation in mixed infections.

Graphical abstract, Highlights • Exlana breed ewes were monitored for gastrointestinal nematodes during the peri-parturient period. • Ewes selected for resistance when lambs produced fewer eggs as adults. • There was no observed reproductive cost to resistance. • Simulations predict that lambs of resistant ewes are exposed to reduced infection pressure. • Nematode resistance in the female line could help mitigate the impact of climate change on infection pressure., Global livestock production is facing serious new challenges, including climate-driven changes in parasite epidemiology, and anthelmintic resistance, driving a need for non-chemotherapeutic methods of parasite control. Selecting for genetic resistance to gastrointestinal nematode infection could reduce reliance on chemical intervention and mitigate increases in parasite challenge due to climate change. Ewes of the composite Exlana breed with a range of estimated breeding values (EBVs) based on nematode faecal egg counts (FECs) were monitored during the peri-parturient period on two farms in southwestern England. Ewes with low EBVs (“resistant”) had lower FECs during the peri-parturient period than those with high EBVs (“susceptible”): the mean FEC was reduced by 23% and 34% on Farms 1 and 2, respectively, while the peak FEC was reduced by 30% and 37%, respectively. Neither EBV nor FEC were correlated with key performance indicators (estimated milk yield, measured indirectly using 8 week lamb weight, and ewe weight loss during lactation). Simulations predict that the reduced FECs of resistant ewes would result in a comparable reduction in infection pressure (arising from eggs shed by ewes) for their lambs. Furthermore, although the reduced FECs observed were modest, simulations predicted that selecting for nematode resistance in ewes could largely offset predicted future climate-driven increases in pasture infectivity arising from eggs contributed by these ewes. Selective breeding of the maternal line for nematode resistance therefore has potential epidemiological benefits by reducing pasture infectivity early in the grazing season and alleviating the need for anthelmintic treatment of ewes during the peri-parturient period, thus reducing selection pressure for anthelmintic resistance. These benefits are magnified under predicted future climate change. The maternal line warrants more attention in selective breeding programmes for nematode resistance.

Aelurostrongylus abstrusus and Troglostrongylus brevior are snail-transmitted helminths causing respiratory diseases in infected cats. The shedding of feline lungworm L1s and their infectivity to the snail intermediate host, after administration of anthelminthic products to cats, are poorly documented. To assess the efficacy of 8.3% fipronil, 10% (S)-methoprene, 0.4% eprinomectin and 8.3% praziquantel (i.e. eprinomectin formulation) and 10% imidacloprid/1% moxidectin (i.e. moxidectin formulation) against these nematodes and to determine the number of days post-treatment until viable L1s are released in the faeces, 384 animals were screened by faecal examination. Of the 54 positive animals (i.e., 14.1%; 7.3% A. abstrusus, 6.2% T. brevior and 0.5% coinfected), 36 were randomly allocated to four groups. Groups A and B were composed of cats positive for T. brevior and treated with the eprinomectin and with the moxidectin formulations, respectively, whereas cats in groups C and D were positive to A. abstrusus and treated with the eprinomectin and the moxidectin formulations, respectively. Prior to and every day after treatment, faecal samples were analysed by the Baermann technique and the number of larvae per gram of faeces determined, and again four weeks after treatment, to assess the efficacy of a single administration of the products. In addition, to evaluate the pre- and post-treatment infectivity of L1s to snail intermediate hosts, one/two snails per cat were infected with 100 L1s collected from the faeces of enrolled animals and then digested 28 days p.i. Based on L1s faecal counts, the efficacy of the eprinomectin and the moxidectin formulations at 28 days was 100% for both A. abstrusus and T. brevior, with a mean number of days of 7.9 ± 1.2 in group A, 7.8 ± 1.9 in B, 6.9 ± 1.6 in C and 8.9 ± 2.0 in D to become negative. Following the artificial digestion, active L3s of T. brevior and A. abstrusus were found in 160 (87.4%) experimentally infected snails. The results of this study demonstrate that a single administration of the two formulations is effective in the treatment of A. abstrusus and T. brevior infections and that during the post-treatment period live L1s are shed for up to 8.9 ± 2.0 days. L1s of both lungworm species released in the faeces after drug administration are still able to reach the infective larval stage in the infected snails. Hence, preventative measures after the treatment of infected animals should include keeping cats indoors and disposal of their faeces for approximately 10 days to avoid environmental contamination and infection of gastropod intermediate hosts.

Cyathostomins are ubiquitous in grazing horses across the world, and anthelmintic resistance has been reported with increasing levels over past decades. The aims of the present study were (i) to investigate the efficacy against encysted larval stages of moxidectin (0.4 mg/kg) and fenbendazole (10 mg/kg daily for five consecutive days) and compare these regimens at 2 and 5 weeks post-treatment, (ii) to investigate individual cyathostomin species associated with shortened egg reappearance periods, and (iii) to document species exhibiting decreased susceptibility to the evaluated compounds. Thirty-six ponies were allocated to treatment groups with half euthanatized 2 weeks post-treatment, and the remainder necropsied after 5 weeks. Luminal and mucosal worm counts were conducted and strongyle egg counts were determined at weekly intervals. At 2 weeks, mean reductions of early L3s were 50.4% and 73.8% for fenbendazole and moxidectin, respectively. At 5 weeks, the respective efficacies were 51.3% and 71.8%. Two week efficacies against late L3s and L4s (LL3s/L4s) were 70.8% and 74.6% for fenbendazole and moxidectin, respectively, whereas very low numbers were found in all three groups at 5 weeks. None of the mucosal counts were significantly different between treatment groups. Fenbendazole and moxidectin reduced luminal worm counts by 93.2% and 98.3% at 2 weeks following administration, with moxidectin group adult counts being significantly lower than the other two groups (P 0.0001). Both treatment groups had increased counts 3 weeks later (P = 0.0415). A moxidectin ERP of 4 weeks was associated with surviving luminal L4s, and adult species contributing to this were Cyathostomum catinatum, Cylicostephanus longibursatus, Cylicocyclus ashworthi and Cylicocyclus nassatus. This study documented (i) larvicidal efficacy of fenbendazole much lower than historical standards, (ii) survival of luminal immatures (L4) following moxidectin administration, and (iii) new information about cyathostomin species associated with these phenomena.

The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotation protocols exist, little is known about the characteristics of these eggs with respect to their movement through a flotation solution. In this study, we have demonstrated a novel method for the observation and quantification of microscopic (65–100 µm) objects as they experience unassisted flotation. This also represents, to our knowledge for the first time, that the flotation of parasite eggs has been observed and their movement characteristics quantified as they float through solution. Particle tracking and video analysis software were utilised to automatically detect and track the movement of individual eggs as they floated. Three 30 s videos and one 2 min video of each egg type were analysed. If the first 30 s of video were discounted, the differences in mean flotation speed among all videos was statistically significant between egg types (P = 0.0004). Strongyle type eggs (n = 201) moved the fastest with a mean 51.08 µm/s (95% confidence interval: 47.54–54.62). This was followed by Parascaris spp. (n = 131) and Anoplocephala perfoliata eggs (n = 322), with mean speeds of 44.43 µm/s (95% confidence interval: 39.47–49.4) and 31.11 µm/s (95% confidence interval: 29.6–32.61), respectively. This method for evaluating the mean speed of passive flotation may represent a first step towards further optimizing fecal egg flotation and be of interest to parasitologists and veterinary practitioners.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the β-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous β-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.

The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small ruminants.

Due to major problems with drug resistance in parasitic nematodes of animals, there is a substantial need and excellent opportunities to develop new anthelmintics via genomic-guided and/or repurposing approaches. In the present study, we established a practical and cost-effective whole-organism assay for the in vitro-screening of compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). The assay is based on the use of exsheathed L3 (xL3) and L4 stages of H. contortus of small ruminants (sheep and goats). Using this assay, we screened a panel of 522 well-curated kinase inhibitors (GlaxoSmithKline, USA; code: PKIS2) for activity against H. contortus by measuring the inhibition of larval motility using an automated image analysis system. We identified two chemicals within the compound classes biphenyl amides and pyrazolo[1,5-α]pyridines, which reproducibly inhibit both xL3 and L4 motility and development, with IC50s of 14-47 μM. Given that these inhibitors were designed as anti-inflammatory drugs for use in humans and fit the Lipinski rule-of-five (including bioavailability), they show promise for hit-to-lead optimisation and repurposing for use against parasitic nematodes. The screening assay established here has significant advantages over conventional methods, particularly in terms of ease of use, throughput, time and cost. Although not yet fully automated, the current assay is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation. The current assay is highly adaptable to many parasites of socioeconomic importance, including those causing neglected tropical diseases. This aspect is of major relevance, given the urgent need to deliver the goals of the London Declaration (http://unitingtocombatntds.org/resource/london-declaration) through the rapid and efficient repurposing of compounds in public-private partnerships.

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

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50μg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50μg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14-17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n=429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8-100%; 95% lower confidence intervals range: 96.8-100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7-100%; 95% lower confidence intervals range: 65-100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0-73%; 95% lower confidence intervals range: 0-59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98-99.4%; 95% lower confidence intervals range: 91.8-99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4-41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5-99.9%; 95% lower confidence intervals range: 96.3-99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5-91.2%; 95% lower confidence intervals range: 6.7-82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.

Parasitic nematodes are found in almost all wild vertebrate populations but few studies have investigated these host-parasite relationships in the wild. For parasites with free-living stages, the external environment has a major influence on life-history traits, and development and survival is generally low at sub-zero temperatures. For reindeer that inhabit the high Arctic archipelago of Svalbard, parasite transmission is expected to occur in the summer, due to the extreme environmental conditions and the reduced food intake by the host in winter. Here we show experimentally that, contrary to most parasitic nematodes, Marshallagia marshalli of Svalbard reindeer is transmitted during the Arctic winter. Winter transmission was demonstrated by removing parasites in the autumn, using a novel delayed-release anthelmintic bolus, and estimating re-infection rates in reindeer sampled in October, February and April. Larval stages of nematodes were identified using molecular tools, whereas adult stages were identified using microscopy. The abundance of M. marshalli adult worms and L4s increased significantly from October to April, indicating that reindeer were being infected with L3s from the pasture throughout the winter. To our knowledge, this study is the first to experimentally demonstrate over-winter transmission of a gastro-intestinal nematode parasite in a wild animal. Potential mechanisms associated with this unusual transmission strategy are discussed in light of our knowledge of the life-history traits of this parasite.

Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay to promote good clinical practice. Using a fluorescent chitin-binding protein, we show that this structural carbohydrate is present and accessible in shells of ova of strongyle, ascarid, trichurid and coccidian parasites. Furthermore, we show that a cellular smartphone can be used as an inexpensive device to image fluorescent eggs and, by harnessing the computational power of the phone, to perform image analysis to count the eggs. Strongyle egg counts generated by the smartphone system had a significant linear correlation with manual McMaster counts (R(2)=0.98), but with a significantly lower coefficient of variation (P=0.0177). Furthermore, the system was capable of differentiating equine strongyle and ascarid eggs similar to the McMaster method, but with significantly lower coefficients of variation (P

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The genetic changes underlying resistance to anthelmintics, particularly ivermectin (IVM), remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of changes in gene expression. In this study, we investigated the expression of putative drug transporter molecules, P-glycoproteins (Pgps), in Teladorsagia circumcincta, the predominant parasitic nematode species of sheep in the UK and the major anthelmintic resistant species. Utilising a degenerate PCR approach, 11 partial Pgp sequences were identified. Constitutive differences in gene expression between an IVM-susceptible (MTci2) and a multidrug-resistant (MTci5) isolate were determined for 10 of the Pgps using the ΔΔCt TaqMan® real-time PCR method. Gene expression differences were particularly marked in one of these genes, namely Tci-pgp-9. In the MTci5 isolate, statistically significant increases in Tci-pgp-9 expression, at the mRNA level, were observed across all life-cycle stages and most notably in eggs (55-fold increase). Comparison of the partial Tci-pgp-9 nucleotide sequences from MTci2 and MTci5 also identified high levels of polymorphism. This work has shown that constitutively increased expression in Tci-pgp-9, coupled with increased sequence polymorphism, could play a role in allowing multidrug-resistant T. circumcincta to survive IVM exposure. The genetic changes underpinning these gene expression changes remain to be elucidated and need to be investigated in other isolates. These changes could form the basis of an IVM resistance marker to monitor the spread of resistance and to evaluate management practices aimed at delaying its spread.

The free-living nematode Caenorhabditis elegans is a useful model for studying the pharmacology of anthelmintics. Currently approved anthelmintics have various mechanisms of action, including activity at nematode nicotinic acetylcholine receptors (nAChRs). Classical anthelmintic agonists of these receptors (nicotine, levamisole, pyrantel and bephenium) caused intact specimens of C. elegans to undergo contracted paralysis. The nAChR antagonist mecamylamine paralysed intact worms and blocked the actions of the agonists. The time to onset of effects of these drugs was enhanced when worms bisected between the mid- and anterior-portions were tested. The novel anthelmintic nAChR antagonist derquantel (2-desoxoparaherquamide, 2-DOPH) was weakly active in intact specimens of C. elegans at concentrations of 50 μM over several days. No antagonism of the nAChR agonists was observed with this drug in intact worms. However, derquantel had direct and marked effects on motility in cut worms and blocked the effects of nAChR agonists in this preparation. A representative of the new amino-acetonitrile derivative (AAD) class of nAChR agonists was not antagonised by derquantel in cut C. elegans, suggesting that these two anthelmintics may not demonstrate unfavourable drug–drug interactions at the receptor level if used to treat livestock infected with parasitic nematodes. The permeability properties of the C. elegans cuticle may be more restrictive than those of adult parasites, calling into question primary anthelmintic screening strategies that rely on this model organism.

Monepantel (AAD 1566) is the first compound from the recently discovered amino-acetonitrile derivative (AAD) class of anthelmintics to be developed for use in sheep. Three dose determination studies were conducted in Australia and Europe to identify the therapeutic dose of monepantel, when formulated for the oral treatment of sheep, to control adult gastro-intestinal nematodes. In each study, sheep infected with various nematode species were treated with either 1.25, 2.5 or 5.0 mg monepantel/kg bodyweight. Following euthanasia and worm counting, their worm burdens were compared with those from untreated control groups. At a dose rate of 1.25 mg/kg, monepantel showed efficacy above 91.9% against all major nematode species, with the exception of Chabertia ovina and Oesophagostomum venulosum. Efficacy against these two species was 93.6% and 94.0%, respectively, at a dose of 2.5 mg/kg. At this dose, efficacy was above 99.2% against nine other nematode species including Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp. and Nematodirus spp. It was concluded that 2.5 mg/kg would be a suitable dose rate for a commercial product.

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.

Ivermectin and moxidectin are closely related avermectin/milbemycin anthelmintics and available data suggest that side resistance occurs with these two drugs. However, moxidectin remains effective against many species of ivermectin-resistant worms due to its higher potency. The larval development assay (LDA) is routinely used to diagnose ivermectin resistance in Haemonchus contortus but laboratory diagnosis of moxidectin resistance is hampered by the lack of any validated in vitro tests. The objective of this study was to measure the relative susceptibility/resistance of H. contortus to moxidectin on goat farms in Georgia, and to validate the DrenchRite LDA for detecting resistance to moxidectin. Fecal egg count reduction tests (FECRT) were performed at five different moxidectin dose levels and DrenchRite LDAs were performed in duplicate on nine meat goat farms in Georgia, USA. To improve our ability to make inferences on the relative levels of resistance between farms, FECRT data were first analysed using a linear mixed model, and then Tukey's sequential trend test was used to evaluate the trend in response across dose levels. LDA data were analysed using log-dose logit-response and probit models. Using these statistical results, we were able to rank the nine farms from the least to the most resistant, and to develop a set of criteria for interpreting DrenchRite LDA results so that this assay can be used to diagnose both clinically apparent moxidectin resistance, as well as sub-clinical emerging resistance. These results suggest that our novel approach for examining these types of data provides a method for obtaining an increased amount of information, thus permitting a more sensitive detection of resistance. Based on results of the LDA, moxidectin-resistant farms had resistance ratios, compared with an ivermectin-sensitive farm, ranging from 32 to 128, and had resistance ratios of 6-24 compared with an ivermectin-resistant/moxidectin naive farm. Moxidectin resistance was diagnosed both in Haemonchus and Trichostrongylus on almost half of the farms tested, despite this drug only being used on these farms for 2-3 years.

Emodepside, a cyclooctadepsipeptide, is a broad-spectrum anthelmintic previously shown to paralyse body wall muscle and pharyngeal muscle in the model nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans L4 are less sensitive than adults to emodepside in two independent assays of locomotor behaviour: body bend generation on agar (adult IC(50) 3.7 nM, L4 IC(50) 13.4 nM) and thrashing behaviour in liquid (thrashing behaviour as a % of controls after 1h in 10 microM emodepside: adults 16%, L4 worms 48%). We also show that continuous exposure of wild-type C. elegans to emodepside throughout the life-cycle from egg onwards, slows worm development, an effect that is emodepside concentration-dependent. The rate of worm-hatching from eggs on agar plates containing emodepside was not significantly different from controls, suggesting that it is development post-hatching rather than hatching itself that is affected by the drug. Emodepside also inhibits wild-type C. elegans egg-laying, with acute exposure to the drug at 500 nM resulting in an almost total inhibition within the first hour. However, the rate of egg production was not inhibited and therefore emodepside-treated worms became bloated with eggs, eventually rupturing. This suggests that the effect of emodepside on reproduction is not due to an inhibition of egg production but rather a paralytic effect on the egg-laying muscles. These results, when coupled with previous research, suggest that emodepside interferes with signalling at the neuromuscular junction on the body-wall muscles (Willson et al., 2003), pharynx (Willson et al., 2004) and egg-laying muscles and thus inhibits three important physiological functions: locomotion, feeding and reproduction.

A collection of Bacillus thuringiensis (Bt) strains (Bts) were screened for activity against the free-living larval stages of nematode parasites of livestock. Two strains were identified with significant activity in inhibiting larval development of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta. These strains were also toxic to the adult parasitic stages of these nematode species in vitro. Adult H. contortus and O. circumcincta showed complete cessation of movement within 2 and 4 days, respectively. Trichostrongylus colubriformis adults were less affected, however, movement was still significantly reduced compared with controls. The in vitro activity against the larval stages was of a magnitude similar to or greater than that seen with the anthelmintic drugs thiabendazole and levamisole. N-terminal amino acid sequencing indicated that the two Bts contained either Cry5A and Cry5B proteins, or a Cry13 protein, and the presence of the corresponding cry5A, cry5B and cry13 genes was confirmed by PCR and sequencing. Bacillus thuringiensis spore-crystal suspensions exposed to acidic pH conditions (pHor=3) showed greatly reduced toxicity in subsequent bioassays with nematode larvae, highlighting the need to protect the toxin from the acidic conditions of the sheep abomasum if it were to be administered per os as an anthelmintic. This study indicates that both the parasitic adult stages and the free-living larval stages of economically significant nematode parasites are susceptible to the effects of Bt, thus identifying this group of toxins as potential biocontrol agents.

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined.

Praziquantel exhibits activity against all major human schistosome parasites and has become the cornerstone for treatment and morbidity control of schistosomiasis. Praziquantel is also active against a wide range of trematodes, human and veterinary cestodes and displays cysticidal effects. To the best of our knowledge anthelminthic properties have never been documented. Here, we report a study among 96 schoolchildren from an area highly endemic for Schistosoma mansoni and hookworm infection, and place particular emphasis on the effect of praziquantel on the prevalence and intensity of hookworm infections. Stool specimens were screened over several consecutive days prior and 4 weeks after systematic administration of praziquantel. We found a significant reduction in the prevalence of hookworm infection from 75.0 to 40.6% (odds ratio (OR)=0.21; 95% confidence interval (CI): 0.11-0.40). Infection intensities, expressed by geometric mean egg counts of all children, were also reduced significantly from 10.7 to 2.0 eggs per gram stool (paired t-test=7.78, P

With the increasing incidence of anthelmintic resistance worldwide, immunological control of worm infections through vaccination is often put forward as a rational and cost-effective alternative for anthelmintic drugs. In this study we report on the evaluation of a double-domain activation-associated secreted protein purified from the excretory-secretory material of the adult stage of the small intestinal parasite Cooperia oncophora as a vaccine antigen against this parasite. In a first experiment, calves were vaccinated three times i.m. with activation-associated secreted protein and Quil A adjuvant or with adjuvant alone, and subsequently challenged with a trickle infection of 25,000 infective larvae in total over 25 days. Vaccinated calves showed a significant reduction of 91% in their cumulative faecal egg counts and a significantly higher number of inhibited L4s present in their intestine compared with control animals. Furthermore, both female and male adult worms were significantly smaller in the vaccinated group than in the control group. In a second experiment, the vaccine antigen was further evaluated under field conditions. Calves were immunised as described above, followed by a natural challenge infection on pasture. Cooperia oncophora faecal egg counts in the vaccinated animals were reduced during the entire grazing period, resulting in a significant reduction in the cumulative faecal egg counts of 58.5%. Numbers of infective C. oncophora larvae were lower on plots grazed by vaccinated calves, with a reduction in mean pasture larval counts of 65% at housing. A significant reduction of 81.6% in total numbers of C. oncophora worms was shown in the vaccinated group compared with the control group. Taken together, the data highlight the protective capacity of the double-domain activation-associated secreted protein and the possibility of controlling C. oncophora infections through vaccination.

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration-response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration-response curves for all isolates to the left, again eliminating differences in EC50 values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis , Acanthocheilonema viteae and Brugia malayi . The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5×100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by ≥95%) determined 3 and 7 days after treatment were 3.125–6.25 and 6.25–12.5 mg/kg, respectively. Using the spot on formulation, doses of 6.25 mg/kg ( L. sigmodontis ), 12.5 mg/kg ( A. viteae ) and 25 mg/kg ( B. malayi ) were required to cause reductions of microfilaraemia levels by ≥95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100 mg/kg independent of the route of administration). Repeated oral treatment (100 mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi . However, single doses of 6.25 and 25 mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi , respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.

The most commonly used modern anthelmintics include the benzimidazoles, the nicotinic agonists. praziquantel, triclabendazole and the macrocyclic lactones. These drugs interfere with target sites that are either unique to the parasite or differ in their structural features from those of the homologous counterpart present in the vertebrate host. The benzimidazoles exert their effect by binding selectively and with high affinity to the beta-subunit of helminth microtubule protein. The target site of the nicotinic agonists (e.g. levamisole, tetrahydropyrimidines) is a pharmacologically distinct nicotinic acetylcholine receptor channel in nematodes. The macrocyclic lactones (e.g. ivermectin, moxidectin) act as agonists of a family of invertebrate-specific inhibitory chloride channels that are activated by glutamic acid. The primary mode of action of other important anthelmintics (e.g. praziquantel, triclabendazole) is unknown. Anthelmintic resistance is wide-spread and a serious threat to effective control of helminth infections, especially in the veterinary area. The biochemical and genetic mechanisms underlying anthelmintic resistance are not well understood, but appear to be complex and vary among different helminth species and even isolates. The major mechanisms helminths use to acquire drug resistance appear to be through receptor loss or decrease of the target site affinity for the drug. Knowledge on the mechanisms of drug action and resistance may be exploitable for the development of new drugs and may provide information on ways to overcome parasite resistance, respectively.

The drug, artemether, has been shown to be active against the juvenile stages of Schistosoma japonicum and Schistosoma mansoni in experimentally infected animals, while it is less effective on adult worms. These findings have been confirmed in randomised controlled trials in humans. Consequently, it could be expected that artemether is also active against Schistosoma haematobium. We present here the first results from experiments assessing the effect of artemether on S. haematobium. Hamsters with a single infection received intra-gastrically an initial dose of 300 mg/kg artemether on day 14, 21 or 28, followed by further doses at varying treatment regimens. In all the treatment groups, the total and female worm reduction rates were highly significant, and ranged from 78 to 100% in hamsters harbouring juvenile schistosomes. Hamsters infected three times with S. haematobium, on days 0, 4 and 9, and repeatedly treated with artemether at the same dose as above, showed highly significant total and female worm reduction rates of between 94 and 99%. Artemether was also active against 77-day-old adult S. haematobium, since its administration on two consecutive days resulted in highly significant total and female worm reduction rates of 76-89%. Our findings confirm that artemether is also active against S. haematobium, especially the schistosomules. These results provide a basis for clinical trials in humans, for further assessment of the potential of artemether for schistosomiasis control.

Naive wethers were used to investigate the long-term effects of dietary condensed tannins from Quebracho extract, during an intestinal parasitic infection in sheep. Sheep were allocated to eight groups; seven groups were daily infected with 3000 L(3) Trichostrongylus colubriformis for 10 weeks and the eighth group was the uninfected control. The 10-week experiment was divided into two periods; Period 1 (P(1), week 1-5) corresponded to high worm establishment and acquisition of immunity, whereas Period 2 (P(2), week 6-10) to the established worm population and expression of host immunity. Three experimental foods with similar composition were formulated: Q0, Q3 and Q6. Their difference was in the content of Quebracho extract which was 0, 30 and 60 g per kg fresh matter, respectively. All foods were offered at an allowance of 3.5% of sheep liveweight. During P(1), parasitised sheep were offered one of the three experimental foods and during P(2) they either remained on the same food or changed food according to the design (P(1)-P(2)): Q0-Q0, Q0-Q3, Q0-Q6, Q3-Q0, Q3-Q3, Q6-Q0, Q6-Q6. Control sheep were offered the allowance of Q0 throughout. Sheep that consumed Q3 and Q6 reduced their faecal egg counts (FEC) compared to sheep offered Q0, during both periods (P0.05). No differences were observed in the FEC between sheep offered Q3 and Q6. The changeover from Q0 in P(1) to either Q3 or Q6 during P(2), was accompanied by a reduction in FEC (P0.05), whereas an increase in FEC was observed when food changed from Q3 or Q6 to Q0 (P0.05). Worm burdens and fecundity at the end of the experiment were reduced in sheep offered foods Q3 and Q6 compared to sheep offered Q0. A significant decrease in liveweight gain and in food conversion efficiency of parasitised sheep offered Q3 and Q6 compared to sheep offered Q0, was observed in P(1) (P0.05) but not in P(2). By the end of the experiment control sheep had achieved higher liveweight and converted food more efficiently than parasitised sheep (P0.05). In conclusion, evidence for a long-term effect of Quebracho extract, during both the initial establishment and on the established T. colubriformis population in sheep, was provided by the present study. It is suggested that the effect observed was a direct anthelmintic effect of the condensed tannins included in sheep diets.

The causative agent of alveolar hydatidosis in humans, the fox tapeworm Echinococcus multilocularis, is extending its geographical range in Europe and has been found in domestic cats in some areas. A dermally applied cestocidal treatment for domestic cats has been developed and the efficacy of this treatment is reported. Thirty purpose-bred cats were experimentally infected each with 10000 protoscoleces of Echinococcus multilocularis. Ten days later one group of ten cats was treated with Droncit(R) Spot-on (Praziquantel) 4% w/v dermally in one place on the dorsal aspect of the neck at a dose of 8 mg/kg. Eleven days later (21 days p.i.) a second group of ten cats was also treated with Droncit(R) Spot-on the same way. One group of ten cats was left untreated as controls. Twenty three days after infection the cats were examined for the presence of E. multilocularis tapeworms. No E. multilocularis were recovered from any of the cats in either of the treated groups. Echinococcus multilocularis were recovered from eight of the ten cats left untreated as controls. The worm burdens in the untreated cats were 0, 0, 5, 15, 75, 110, 220, 815, 2635, and 3045 worms per cat. The worms ranged in development from the three to four segment stage. Many of the E. multilocularis with four segments contained unshelled eggs in the terminal segment. This study indicates that Droncit(R) Spot-on (Praziquantel) 4% w/v applied dermally at 8 mg/kg is highly effective in removing E. multilocularis from the small intestine of cats infected with immature and mature (prepatent) infections of E. multilocularis. In the cats with the mature infections all tapeworms were absent from the small intestine within 2 days of treatment.

The identification of genes associated with anthelmintic resistance can be facilitated in Haemonchus contortus by the ability of this species to hybridise with Haemonchus placei . Although the hybrid males are sterile, the lines can be rescued by backcrossing the females to either parental species. Resistance genes can be retained in Haemonchus hybrids, while the unwanted contortus background is removed through backcrossing to H . placei and anthelmintic selection of the progeny. Under this selection, genes involved in resistance would retain the H . contortus nucleotide sequence, while those that are not would either be H . placei or a random mixture of both, depending on the amount of backcrossing that had occurred. The first candidate gene to be tested in this system was a Haemonchus P-glycoprotein, hcpgp-1. hcpgp-1 was amplified, cloned and sequenced from H. contortus and H. placei . Two restriction sites were then identified in the sequenced product; one specific to H. contortus hcpgp-1 and the other found only in the H. placei gene. These genes were identified from macrocyclic lactone selected and non-selected worms by restricting PCR products from individual worms. Fitted occurrence of the H. contortus allele was 49% of unselected worms and 69% of macrocyclic lactone selected worms. The probability of this percentage occurring by chance was P =0.006. Thus macrocyclic lactone selection was acting to increase the percentage of hcpgp-1 from macrocyclic-lactone-resistant CAVRS.

Two morphologically marked strains of Haemonchus contortus, CAVRS (smooth-macrocyclic lactone resistant) and McMaster (linguiform-macrocyclic lactone susceptible), were used to investigate the selection for anthelmintic resistance following exposure to ivermectin (IVM), a non-persistent anthelmintic. and a more persistent anthelmintic, oral moxidectin (MOX). Three types of selection were investigated: (1) selection of resident worms at the time of treatment (Head selection); (2) selection of incoming-larvae post-treatment (Tail selection); and (3) selection of both resident population and incoming larvae (Head + Tail selection). The experimental animals were adult sheep and lambs. In the controls where there was no anthelmintic selection, the proportion of CAVRS in the adult worm population was the same as the proportion in larvae given to both adults and lambs indicating that CAVRS and McMaster H. contortus were equally infective. There was a significant effect of anthelmintic on total worm numbers in adult sheep with MOX treated adults having less worms, but selection type was non-significant. Anthelmintic type had a significant effect on numbers of resistant worms in adult sheep with less resistant worms in the MOX treated groups, but selection type had no effect. Analysis of variance of arcsine-transformed proportions of resistant worms found that the type of anthelmintic had a highly significant effect, with MOX treated adults having a higher proportion of resistant worms, while type of selection was not significant. In the lambs, nil treated controls and IVM Head + Tail and Tail selected groups had similar geometric mean total worm burdens while Head selected had less total worms. In the MOX treated lamb groups the worm burdens were similar within selection type but less than the IVM treated groups. In the lambs, the types of selection that resulted in more resistant worms were IVM Tail, MOX Head + Tail and MOX Tail. Resistant worm numbers were similar in both adult and lamb groups with Head selection by either MOX or IVM. Moxidectin selected out higher proportions of resistant worms than did IVM in the lambs, with Tail and Head + Tail being stronger selectors than Head. Computer simulations were used to estimate the rate at which resistance developed in the field using the information generated in the present study. The anthelmintic treatments used in the simulation followed a strategic parasite control program for H. contortus in which all sheep receive three Closantel (CLS) treatments in summer. all sheep receive a broad-spectrum (BS) drench or capsule at weaning and lambs receive an additional two BS drenches insummer or no further treatment in the case of the capsule. Moxidectin, IVM-capsule and IVM were the broad spectrum anthelmintics simulated. All simulations were run four times assuming high or low efficacy against resident resistant worms and in the presence or absence of CLS resistance. The simulations indicated that the presence of CLS resistance hastened selection for macrocyclic lactone (ML) resistance. While the IVM-capsule will select most rapidly for ML resistance, IVM oral is expected to be least selective. Moxidectin treatment is intermediate, except in simulations with no CLS resistance and when MOX is assumed to be highly effective against resident ML-resistant worms, in which case MOX can be expected to select more slowly than IVM oral treatments.

Weaner sheep that had been hand-fed on diets containing increasing concentrations of protein for a 9-week period (when uninfected, or infected with Haemonchus contortus) were studied during the next 69 weeks when put on to pasture as a single, unsupplemented flock. During the 9-week period, groups of 12 sheep (six infected, six uninfected) were offered one of five iso-energetic (9.0 MJ kg−1) diets containing 10, 13, 16, 19 or 22% crude protein. All sheep were treated with anthelmintic at the end of the 9 weeks and then put out to pasture for 69 weeks, where they were all subject to the same environmental variables including nematode larval challenge. During the grazing period, animals that had previously received the higher protein diets consistently had higher live-weight gain and wool production, higher antibody responses to both H. contortus and Trichostrongylus colubriformis antigenic challenge in vitro, and lower faecal nematode egg counts than did the lambs previously offered the lower protein diets. Faecal egg counts of the grazing sheep that had been artificially infected with H. contortus while being hand-fed were similar to those of the uninfected sheep and there was no interaction between previous infection and dietary protein concentration. We conclude that short periods of enhanced post-weaning nutrition can have long-term and perhaps life-long effects on production.

The ability of lactating Romney ewes to resist establishment of ingested infective-stage larvae (L3) of Ostertagia circumcincta and Trichostrongylus colubriformis was measured in the field. Three groups of seven single-lamb-bearing ewes were selected on the basis of uniformity of lambing date from a large flock held on pasture. Either 2, 4 or 6 weeks after parturition, groups of ewes were dosed with 24000 L3 of known oxfendazole-resistant parasite strains; 12000 of each species. Ten to 14 days later the ewes, along with their lambs, were transferred from the field to indoor pens. Twenty-five days after the challenge dose the ewes were drenched with oxfendazole to remove any field-derived infection and 3 days later slaughtered for worm counts. Mean establishment of the resistant parasites was low at all times, with the highest rate recorded being 6.1% for O. circumcincta 2 weeks after parturition. Establishment of O. circumcincta 4 and 6 weeks after parturition, and of T. colubriformis at all times, never exceeded 2%. By comparison, mean establishment in lambs held indoors and parasite free for 13 weeks prior to infection, was 24.9% and 47.1% for O. circumcincta and T. colubriformis, respectively. These results indicate that the lactating ewes were exhibiting a substantial ability to prevent establishment of ingested larvae. The results of this and other similar studies suggest that the dynamics of parasitism in lactating Coopworth and Romney ewes in New Zealand is substantially different to that in Merino ewes in Australia, and that these differences influence optimal strategies for the management of anthelmintic resistance in the two countries.

New organometallic complexes having protozoocidal properties were evaluated for their in vitro antifilarial activity using two models: infective larvae of Molinema dessetae and adult females of Brugia pahangi. The compound most active on the M. dessetae model was Ir(I)-COD-pentamidine tetraphenylborate with an EC50 = 6 +/- 1 microM after 7-day-incubation. In the 2-aminobenzothiazole series, Ruthenium was more potent than Iridium for antifilarial activity. A dithiocarbamate function significantly enhanced the antifilarial activity. The compounds derived from benzimidazole were inactive whatever the metal (Iridium or Rhodium). The other compounds exhibited EC50 ranging from 10 to 31 microM. On adult female Brugia pahangi in vitro, Pt-DDH-N-acetylleucine, Pt-diminazene and Pd-Cl4-piperazine at 20 microM began to kill both microfilariae and the developing embryos within the mothers on day 2. The compounds, except for Pd-Cl4-piperazine, killed the adults after 5 days. Rh-Cl-2-chloropyridine caused obvious slowing of the adults from day 3 onward but did not affect the viability of adults, microfilariae or developing embryos. In vivo antifilarial investigations are necessary to appreciate the real advantage of heavy metal complexes in the experimental treatment of filariasis.

The ability of young Merino lambs to achieve protective immunity following vaccination via viable nematode infections was assessed. Lambs were infected from 1 month of age by repeated continuous low dose (trickle) administration of Trichostrongylus colubriformis or Haemonchus contortus infective larvae (L3), or by truncated infections with high doses of viable T. colubriformis L3. After 7 weeks all groups were drenched with anthelmintic and at 3 months of age they were re-infected with the homologous species. Protection was assessed by faecal egg counts at 3, 4, 5, 6 and 7 weeks after challenge, and worm count at 7 weeks after challenge. Young lambs were partially protected by 3 months of age against Trichostrongylus by trickle infection. This protection correlated with local mast cell and T-cell priming, increased numbers of local antigen-presenting cells and T-cells and increased worm-specific antibody titres in the intestine. However, there was no evidence that young lambs were capable of immunologically recognising H. contortus antigens following trickle infection, nor did trickle infection significantly protect young lambs against Haemonchus challenge.

The effects of a reduction of parasite burdens were determined in adult female Mountain Brushtail Possums, Trichosurus caninus, on the birth, mortality and growth rates of pouch-dependent young, as well as the haematological and serum biochemical values of the mothers. The efficacy of the anthelmintic drug ivermectin for reducing parasite burdens in this host was assessed using faecal and necropsy examinations of a small number of animals. Ivermectin began to reduce parasite burdens by 48 h after treatment. In the second stage of the experiment, animals were treated or sham injected (control individuals) with ivermectin and praziquantel at 8–10-week intervals throughout the breeding season to the time of emergence of young from the pouch. Treatment with ivermectin and praziquantel had no significant effect on the proportion of females giving birth, or on the survival of young to emergence. An effect of treatment was recorded for absolute eosinophil counts in adult females, which, in spring, were higher among control animals than those that were treated.