[show abstract][hide abstract] ABSTRACT: The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.
Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.
The H. contortus genome and transcriptome provides an essential platform for postgenomic research in this and other important strongylid parasites.
[show abstract][hide abstract] ABSTRACT: Cyathostomins are considered to be the most important group of helminths to affect equids due to their high prevalence, potential pathogenicity and ability to develop anthelmintic resistance. Their control relies almost exclusively on frequent anthelmintic use. Currently, fenbendazole (FBZ), pyrantel embonate (PYR), ivermectin (IVM) and moxidectin (MOX) are licensed for use in horses in the UK. With no new anthelmintics likely to be licensed in the near future, it is essential that investigations into the efficacy of current anthelmintics in different locations are performed to help inform control programmes. Here, efficacy of FBZ, PYR, IVM and MOX in horse populations in the South of England was investigated. Horses with a strongyle faecal egg count (FEC) of ≥50eggs per gram (EPG) were enrolled onto a faecal egg count reduction test (FECRT) study. Efficacy was determined by calculating the percentage reduction in FEC between the group mean at Day 0 and 14 days post-treatment. Efficacy was indicated when a group arithmetic faecal egg count reduction (FECR) of ≥90% was recorded for FBZ and PYR, and ≥95% for IVM and MOX. Between March and December 2012, 404 FECRT were performed on 12 yards examining 101, 110, 93 and 100 equids for FBZ, PYR, IVM, and MOX, respectively. FBZ resistance was identified on all yards (mean FECR range 0-65.8%). On 10 of 12 yards, PYR efficacy was >90% (91.0-99.4%) and on two yards, PYR resistance was suspected (86.8-87.2%). IVM (96.4-100%) and MOX (99.9-100%) were >95% efficacious on all yards. As the prevalence of FBZ resistance was 100%, the future use of this anthelmintic for the control of strongyles should be questioned. PYR should be used strategically to reduce reliance on the macrocyclic lactone class products. Over-dispersion of FEC between horses was observed (average k=0.21) with 80% of the strongyle eggs counted measured in 15% of horses tested, strongly supporting the application of targeted helminth control programmes in this host species.
[show abstract][hide abstract] ABSTRACT: Infection of humans and livestock with parasitic nematodes can have devastating effects on health and production, affecting food security in both developed and developing regions. Despite decades of research, the development of recombinant sub-unit vaccines against these pathogens has been largely unsuccessful. We have developed a strategy to identify protective antigens from Teladorsagia circumcincta, the major pathogen causing parasitic gastroenteritis in small ruminants in temperate regions, by studying IgA responses directed at proteins specific to post-infective larvae. Antigens were also selected on the basis of their potential immunomodulatory role at the host/parasite interface. Recombinant versions of eight molecules identified by immunoproteomics, homology with vaccine candidates in other nematodes and/or with potential immunoregulatory activities, were therefore administered to sheep in a single vaccine formulation. The vaccine was administered three times with Quil A adjuvant and the animals subsequently subjected to a repeated challenge infection designed to mimic field conditions. Levels of protection in the vaccinates were compared to those obtained in sheep administered with Quil A alone. The trial was performed on two occasions. In both trials, vaccinates had significantly lower mean fecal worm egg counts (FWECs) over the sampling period, with a mean reduction in egg output of 70% (Trial 1) and 58% (Trial 2). During the period of peak worm egg shedding, vaccinates shed 92% and 73% fewer eggs than did controls in Trials 1 and 2, respectively. At post mortem, vaccinates had 75% (Trial 1) and 56% (Trial 2) lower adult nematode burdens than the controls. These levels of protection are the highest observed in any system using a nematode recombinant sub-unit vaccine in the definitive ruminant host and indicate that control of parasitic helminths via vaccination with recombinant subunit vaccine cocktails is indeed an alternative option in the face of multi-drug resistance.
[show abstract][hide abstract] ABSTRACT: Refugia-based drenching regimes have been widely recommended to slow development of anthelmintic resistance but there are few comparisons between different treatment approaches in the UK. The impact of four ivermectin treatment regimes on drug efficacy, lamb body weight and nematode contamination during a 154 day grazing season were evaluated in a consecutive five year field study. Regimes were whole-flock treatment every 4 weeks (NST), targeted selective treatment (TST) based on individual per- formance, strategic whole-flock treatments at pre-determined times (SPT) or whole-flock treatment when clinical signs were apparent (MT). Mean numbers of ivermectin drenches administered per season were 4.0, 1.8, 2.0 and 1.4 for NST, TST, SPT and MT groups, respectively. The mean anthelmintic efficacy (AE) for each treatment group was based on faecal egg count reduction post-treatment employing a boot- strap sampling based algorithm. Mean AE was 95–98% for all groups in 2006 and mean AE (95% confi- dence limits) for NST declined to 62% (55%, 68%) in 2010. In comparison, AE for TST, SPT and MT in 2010 were 86% (81%, 92%), 86% (83%, 90%) and 83% (78%, 88%), respectively. Body weight in TST and SPT was similar to NST in all years (p > 0.05), however MT lambs were lighter than NST in 2006–2008 (p 6 0.04). Tracer lamb worm burdens was lowest in NST but was not significantly different between other groups. Overall, both the TST and SPT regimes appeared to maintain animal performance and conserve anthelmintic efficacy compared with a neo-suppressive anthelmintic treatment regime.
International Journal for Parasitology: Drugs and Drug Resistance. 01/2013; 3:77-84.
[show abstract][hide abstract] ABSTRACT: The anthelmintic sensitivity of two field-derived isolates (designated FI001 and FI004) of cattle nematodes from beef farms in Scotland were investigated in a controlled efficacy test (CET). Efficacies of ivermectin pour-on (IVM-PO), IVM injectable (IVM-INJ) and moxidectin pour-on (MOX-PO) formulations were assessed. In each group, five helminth-naïve calves were infected experimentally with 50,000 third stage larvae from either isolate and administered with anthelmintic at the manufacturers' recommended dose rate 28 days later. For each isolate, nematode burdens were compared between treatment and control groups to determine efficacy. Nematode species composition, based on data derived from the untreated control groups' burden estimations, were 39 and 14% Cooperia oncophora and 61 and 86% Ostertagia ostertagi for isolates FI001 and FI004, respectively. Macrocyclic lactone resistance in C. oncophora was confirmed for both FI001 and FI004 isolates. Efficacies (as determined by nematode burden analysis) of 4, 21 and 31% for FI001, and 10, 1 and 74% for FI004, were obtained for IVM-INJ, IVM-PO and MOX-PO, respectively. Efficacy based on faecal egg count reduction at seven days post anthelmintic administration were 8, 99 and 100% for FI001, and 37, 20 and 100% for FI004 for IVM-INJ, IVM-PO and MOX-PO, respectively. In summary, this study details two macrocyclic lactone resistant isolates of C. oncophora obtained from cattle from two distinct geographical locales in the UK.
[show abstract][hide abstract] ABSTRACT: Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for over 50 years. However, the continued development of anthelmintic resistance (AR) threatens this control. Measuring early AR is difficult as there many routes that resistance can arise from within multi-nematode populations operating complex metabolism capabilities coupled to different drug management pressures. There is an urgent need to identify and measure early resistance in the field situation. Proteomic profiling of expressed soluble proteins offers a new approach to reveal a drug resistant phenotype within a complex protein pattern. The hypothesis under test was that established differences in drug response phenotypes between nematode isolates can also be measured in their comparative proteomes. As a case study, proteomic differences were measured between an ivermectin resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females were extracted from the abomasa of six lambs. The nematodes had been maintained in the lambs as monospecific isolates of either ivermectin susceptible or ivermectin resistant worms. Comparative analysis of the soluble proteome was completed along with immuno-proteomic analysis using pooled infection sera from the lambs. Following image analysis, spots of interest were excised and analysed by peptide mass fingerprinting and the proteins putatively identified using BLAST. Overall, a relative increase in the expression of proteins involved in the detoxification metabolic area was observed in the resistant isolate. In addition, Western blotting analysis also revealed differences in immuno-reactivity profiles between resistant and susceptible isolates. It can be concluded from this study that proteomic differences can be detectable between ivermectin susceptible and a resistant isolates of H. contortus, which could be further explored using other isolates to confirm if proteomic based fingerprinting offers molecular phenotyping or a new panel of resistance biomarkers.
[show abstract][hide abstract] ABSTRACT: The objective of the present studies was to evaluate the efficacy of a combined formulation (Startect(®) Dual Active Oral Solution for Sheep, Pfizer Animal Health) of derquantel (DQL) and abamectin (ABA) for the treatment of: (1) sheep experimentally infected with a moxidectin (MOX)-resistant isolate of Teladorsagia circumcincta, and (2) multi-drug resistant gastrointestinal nematode parasites under UK field conditions. In the first study, a total of 40 animals were allocated into 4 treatment groups, and were either left untreated or treated with DQL+ABA, MOX or ABA. Faecal samples were collected on days 1-5 and on day 7 after treatment to examine the reduction in faecal egg excretion and to evaluate the egg viability. On day 14 post treatment all animals were euthanised for abomasal worm counts. There was a 100% reduction in geometric mean worm counts for the DQL+ABA treated animals compared to the untreated control animals (P<0.0001), whereas the percentage reduction in worm counts for the MOX- (P>0.05) and ABA-treated (P=0.0004) animals was 12.4% and 71.8%, respectively. The data from the egg hatch assay (EHA) indicated that in the MOX-treated and the ABA-treated animals, the majority of the eggs hatched after treatment. In the field study, performed on four farms, animals were allocated into 6 groups of 11-15 animals each in order to conduct a faecal egg count reduction test (FECRT), based on arithmetic mean egg counts. One group of animals remained untreated, whereas the other animals were treated with DQL+ABA, MOX, fenbendazole (FBZ), levamisole (LV) or ivermectin (IVM). On each of the farms the reduction in egg excretion after treatment with FBZ, LV or IVM was below 95.0%, indicating anthelmintic resistance. The efficacy of DQL+ABA ranged from 99.1 to 100%, yielding significantly lower egg counts compared to the untreated control group (P≤0.003). For MOX the egg counts were significantly (P≤0.003) lower compared to the untreated group at each farm, with reductions varying from 98.2 to 100%. The post-treatment copro-cultures for larva identification indicated that T. circumcincta was the most abundant worm species after treatment (52-99% of the larvae). The results of these studies confirm the high efficacy of the DQL+ABA combination formulation against anthelmintic resistant nematodes in the UK.
[show abstract][hide abstract] ABSTRACT: Disease caused by gastrointestinal (GI) nematodes is arguably one of the most important health constraints affecting productivity in small ruminants. This is of particular importance for many tropical and subtropical countries where goats play a vital role in the agricultural economies. Anthelmintic resistance is an important component of the losses attributable to parasitoses, unfortunately there is ample evidence that it is more common in goats than in other farmed ruminants. The increased prevalence of anthelmintic resistance in goats can largely be explained by unique aspects of their pharmacology, immunology and behaviour, all of which we need to understand and make allowance for if we are to effectively and sustainably manage anthelmintic resistance in goats.
Small Ruminant Research - SMALL RUMINANT RES. 03/2012;
[show abstract][hide abstract] ABSTRACT: Non-specific mechanisms involving ATP-binding cassette drug efflux transporters may play an important role in xenobiotic clearance in ovine gastro-intestinal nematodes. By using transporter inhibitors, the aim of this trial was to assess the possibility of increasing drug bioavailability in the host in an attempt to improve treatment efficacy. Thirty-six lambs were infected with 5000 multiple-drug resistant Haemonchus contortus third stage larvae and separated into six groups (n=6): ivermectin alone (IVM; 0.2 mg/kg body-weight, BW), ketoconazole alone (KET; 10 mg/kg BW), Pluronic 85 alone (P85; 4 mg/kg BW), IVM+KET, IVM+P85 or untreated control. Ivermectin was administered once on day 28 post-infection for all appropriate groups, whereas KET and P85 were administered as five separate doses on day 26-30 post-infection inclusive. The resultant data showed that concomitant administration of KET or P85 with IVM induced increases in plasma and tissue concentrations of IVM in treated animals, resulting in a two-fold increase in the area under the time-concentration curve (p<0.05). Faecal egg counts and worm burdens of the IVM+KET and IVM+P85 groups were lower than in the untreated, KET and P85 alone control animals. Worm burdens were reduced by between 16% and 51% with IVM+KET and IVM+P85 respectively compared to untreated control animals. The co-administration of P85 with IVM increased the efficacy by 34%, compared with IVM alone, in terms of worm count reduction of the multi-resistant isolate of H. contortus.
[show abstract][hide abstract] ABSTRACT: Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a "proof-of-concept" of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci.
[show abstract][hide abstract] ABSTRACT: Anthelmintics in the absence of vaccines have underpinned a parasite control strategy for
over 50 years. However, the continued development of anthelmintic resistance (AR) threatens
this control. Measuring early AR is difﬁcult as there many routes that resistance can
arise from within multi-nematode populations operating complex metabolism capabilities
coupled to different drug management pressures. There is an urgent need to identify and
measure early resistance in the ﬁeld situation. Proteomic proﬁling of expressed soluble
proteins offers a new approach to reveal a drug resistant phenotype within a complex protein
pattern. The hypothesis under test was that established differences in drug response
phenotypes between nematode isolates can also be measured in their comparative proteomes.
As a case study, proteomic differences were measured between an ivermectin
resistant and susceptible adult female Haemonchus contortus. Adult H. contortus females
were extracted from the abomasa of six lambs. The nematodes had been maintained in
the lambs as monospeciﬁc isolates of either ivermectin susceptible or ivermectin resistant
worms. Comparative analysis of the soluble proteome was completed along with immunoproteomic
analysis using pooled infection sera from the lambs. Following image analysis,
spots of interest were excised and analysed by peptide mass ﬁngerprinting and the proteins
putatively identiﬁed using BLAST. Overall, a relative increase in the expression of
proteins involved in the detoxiﬁcation metabolic area was observed in the resistant isolate.
In addition, Western blotting analysis also revealed differences in immuno-reactivity proﬁles
between resistant and susceptible isolates. It can be concluded from this study that
proteomic differences can be detectable between ivermectin susceptible and a resistant
isolates of H. contortus, which could be further explored using other isolates to conﬁrm if
proteomic based ﬁngerprinting offers molecular phenotyping or a new panel of resistance
[show abstract][hide abstract] ABSTRACT: The inhibition of Teladorsagia and other nematode genera at the early fourth-stage is a biological process that allows the parasites to survive in their host in a dormant state when prevailing conditions may otherwise kill them or prevent their progeny from surviving in the external environment. A study was conducted in Scotland to evaluate the efficacy of monepantel, an amino-acetonitrile derivative, against natural infections of inhibited fourth-stage Teladorsagia spp. larvae. At necropsy it was determined that the untreated control sheep were additionally infected with developing fourth-stage Teladorsagia spp. larvae and this is the first published evidence on the efficacy of monepantel against natural infections of this parasite and stage. The study sheep, which had grazed on naturally contaminated pastures since birth, were transferred to indoor housing after a subset of animals was examined to confirm the presence of inhibited larvae within the study population prior to the experiment. After 14 days of housing, monepantel was orally administered at 2.5 mg/kg to half of the animals. The sheep were necropsied seven days later and their parasite burdens recovered for the determination of efficacy, which was 99.7% for the inhibited stages and 99.3% for the developing fourth-stages. In conclusion, monepantel dosed orally at 2.5 mg/kg is a highly effective treatment against naturally acquired infections of inhibited and developing fourth-stage larvae of Teladorsagia spp.
[show abstract][hide abstract] ABSTRACT: Lectins are plant secondary metabolites (PSM) found in many forages and which may confer anthelmintic properties to gastrointestinal parasites through disrupting the development of parasitic larvae throughout its life cycle. In experiment 1, the ability of the plant lectins jacalin (JAC), concanavalin A (Con A), phytohemagglutinin E2L2 (PHA-E2L2), phytohemagglutinin L4 (PHA-L4), phytohemagglutinin E3L (PHA-E3L), kidney bean albumin (KBA), Robinia pseudoacacia agglutinin (RPA), Maackia amurensis lectin (MAA), Maclura pomifera agglutinin (MAA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) to disrupt the feeding of the first stage larvae (L(1)) of the sheep gastro-intestinal nematodes (GIN) Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis was investigated using a larval feeding inhibition test (LFIT). Only PHA-E3L, WGA and Con A had a potent effect on disrupting larval feeding of all of the three species of GIN investigated. The lectin concentration required to inhibit feeding in 50% of L(1) (IC50) was 7.3±1.2, 8.3±1.4 and 4.3±1.7 μg/ml for PHA-E3L; 59.1±32.4, 58.7±11.9 and 8.1±7.0 μg/ml for Con A and 78.9±11.2, 69.4±8.1 and 28.0±14.1 μg/ml for WGA for T. circumcincta, H. contortus and T. colubriformis larvae, respectively (P=0.006). The addition of the lectin inhibitors fetuin, glucose/mannose or N-acetylglucosamine for PHA-E3L, Con A and WGA, respectively, caused an increase in the proportion of larvae that had fed at all concentrations for PHA-E3L only. In experiment 2, the effect of extracts from the tropical plants Azadiractha indica, Trichanthera gigantea, Morus alba, Gliricidia sepium and Leucaena leucocephala on the feeding behaviour of H. contortus L(1,) was examined. A. indica, T. gigantea and M. alba failed to inhibit 50% of larvae from feeding at concentrations up to 10mg plant extract per ml. In contrast, both G. sepium and L. leucocephala demonstrated a dose-dependent effect on larval feeding with respective IC50 estimates (mean±s.e.) of 0.015 mg/ml ±0.001 and 3.465 mg/ml ±0.144, effects which were partly reversed by the inclusion of either the tannin inhibitor polyethylene glycol or the lectin inhibitor Fetuin. These studies demonstrate that plant lectins can have an inhibitory effect on the feeding behaviour of first stage larvae of ovine GIN in vitro. Moreover they also provide novel evidence that lectins may contribute to the anthelmintic properties of some tropical forage plant extracts, such as G. sepium and L. leucocephala.