Parasitic Nematode Haemonchus Contortus Research Articles

Characterization of a secreted cystatin of the parasitic nematode Haemonchus contortus and its immune-modulatory effect on goat monocytes

BackgroundHaemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant’s production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators.MethodsThe recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.ResultsWe cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm’s gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.ConclusionsOur results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.

Screening of a small, well-curated natural product-based library identifies two rotenoids with potent nematocidal activity against Haemonchus contortus

The control of parasitic roundworms (nematodes) is heavily reliant on the use of a limited number of anthelmintic drugs. However, drug resistance is now very widespread and no vaccines are available, such that the discovery of new chemical entities is crucial. Within this context, we screened a library of pure natural products (n=400) against exsheathed third-stage (xL3) larvae of the parasitic nematode Haemonchus contortus using a whole-organism screening method. We identified two plant-derived rotenoids, deguelin and rotenone, with inhibitory activity on xL3 motility. Rotenone was not investigated further, because of its toxicity to some vertebrates. The dose response and cytotoxicity studies showed potent and selective inhibitory activity of deguelin on motility of xL3 larvae of H. contortus. Detailed future work needs to be conducted to explore the mode of action of this compound on H. contortus and related nematodes, and to assess its potential as an anthelmintic candidate.

Mutations in the Hco-mptl-1 gene in a field-derived monepantel-resistant isolate of Haemonchus contortus

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

The impact of a mixture of medicinal herbs on ruminal fermentation, parasitological status and hematological parameters of the lambs experimentally infected with Haemonchus contortus

The aim of this study was to determine the effect of dietary supplementation with a mixture of selected medicinal herbs (Herbmix) on hematological and parasitological parameters and on the inflammatory response of lambs infected experimentally with the gastrointestinal parasitic nematode Haemonchus contortus. Herbmix contained selected medicinal herbs common in central Europe (8.55% each of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis and Melissa officinalis, 5% Foeniculum vulgare and 1% Artemisia absinthium). Herbmix (100g dry matter (DM)/d) was added to the basal diets of meadow hay (600g DM/d) and a commercial concentrate (350g DM/d) per animal. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with Herbmix (Inf+Herb), uninfected control animals (C) and uninfected animals supplemented with Herbmix (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herbmix supplementation for Inf+Herb and C+Herb began on day 0 (D0). Feces were collected and eggs per gram (EPG) of feces were quantified on D0, D20, D32, D50 and D60. Variables differed after 24h of ruminal incubation of the individual medicinal herbs (P<0.001), but the herbs had no negative effect on the fermentation patterns. The mean EPG counts were significantly lower by 45% between D32 and D50 (P<0.05) and by 63% between D32 and D60 (P<0.01) for the infected lambs receiving Herbmix (Inf+Herb). These EPG reductions were only 10 and 30% in infected group (Inf, P>0.05). Mean cumulative gain in live weight was higher in Inf+Herb as compared to Inf by D32 (P<0.01) and D50 (P<0.001). The evaluation of red blood cells identified clinical signs of haemonchosis, such as anemia, in Inf and Inf+Herb. Total antioxidant capacity (P<0.008) and activity of glutathione peroxidase (P<0.005) in serum at D50 were lower in Inf and Inf+Herb than C and C+Herb. The concentration of serum calprotectin differed significantly (P<0.001) in Inf vs. Inf+Herb. The Zn and Fe serum concentrations differed significantly (P<0.05 and P<0.001, respectively) in Inf vs. Inf+Herb, Inf vs. C and Inf vs. C+Herb by D32. The mixture of the medicinal herbs thus contributed to slowing the dynamics of infection and improving the production indicators of the animals.

Meta-analysis of the parasitic phase traits of Haemonchus contortus infection in sheep

BackgroundThe parasitic nematode Haemonchus contortus shows highly variable life history traits. This highlights the need to have an average estimate and a quantification of the variation around it to calibrate epidemiological models.MethodsThis paper aimed to quantify the main life history traits of H. contortus and to identify explanatory factors affecting these traits using a powerful method based on a systematic review and meta-analysis of current literature. The life history traits considered are: (i) the establishment rate of ingested larvae; (ii) the adult mortality rate; (iii) the fertility (i.e. the number of eggs laid/female/day); and (iv) fecundity of female worms (i.e. the number of eggs per gram of faeces).ResultsA total of 37 papers that report single experimental infection with H. contortus in sheep and published from 1960 to 2015, were reviewed and collated in this meta-analysis. This encompassed 115 experiments on 982 animals. Each trait was analysed using a linear model weighted by its inverse variance. The average (± SE) larval establishment rate was 0.24 ± 0.02, which decreased as a function of the infection dose and host age. An average adult mortality rate of 0.021 ± 0.002) was estimated from the literature. This trait varied as a function of animal age, breed and protective response due to prior exposure to the parasite. Average female fertility was 1295.9 ± 280.4 eggs/female/day and decreased in resistant breeds and previously infected hosts. Average faecal egg count at necropsy was 908.5 ± 487.1 eggs per gram of faeces and varied as a function of infection duration and host resistance. The average sex ratio of H. contortus was 0.51 ± 0.006.ConclusionThis work is the first systematic review to summarise the available information on the parasitic phase of H. contortus in sheep. The results of the meta-analysis provide robust estimates of life history traits for parametrization of epidemiological models, their expected variation according to experimental factors, and provides correlations between these.

Modulation of goat monocyte function by HCcyst-2, a secreted cystatin from Haemonchus contortus.

Modulation and suppression of the host immune response by nematode parasites have been reported extensively and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulator. In the present study, we cloned and produced recombinant cystatin protein from nematode parasite Haemonchus contortus (rHCcyst-2) and investigated its immunomodulatory effects on goat monocyte. rHCcyst-2 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B and papain. Immunohistochemical test demonstrated that the native HCcyst-2 protein was predominantly localized at the body surface and internal surface of the worm's gut. We demonstrated that rHCcyst-2 could be distinguished by antisera from goats experimentally infected with H. contortus and could uptake by goat monocytes. The immunomodulatory effects of HCcyst-2 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-2 with goat monocytes. The results showed that the interaction of rHCcyst-2 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 in goat monocytes. After rHCcyst-2 exposure, the expression of MHC-II on goat monocytes was inhibited. Moreover, rHCcyst-2 could up-regulate the LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-2 inhibited the phagocytosis of goat monocytes. Our findings provided potential target as immunoregulator, and will be helpful to illustrate the molecular basis of host–parasite interactions and search for new potential molecule as vaccine and drug target candidate.

Characterization of a secreted macrophage migration inhibitory factor homologue of the parasitic nematode Haemonchus Contortus acting at the parasite-host cell interface.

Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively and the migration inhibitory factor (MIF) is identified as one of the major immunomodulator. In the present study, we cloned and produced recombinant MIF protein from the small ruminant’s nematode parasite Haemonchus contortus (rHCMIF-1), and investigated its immunomodulatory effects on goat monocyte. Enzymatic assays indicated that rHCMIF-1 possessed tautomerase activity. Immunohistochemical test demonstrated that the native HCMIF-1 protein was predominantly localized at the body surface and internal surface of the worm’s gut. We demonstrated that rHCMIF-1 could be distinguished by antisera from goats experimentally infected with H. contortus and could bind by goat monocytes. The immunomodulatory effects of HCMIF-1 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCMIF-1 with goat monocytes. The results showed that the interaction of rHCMIF-1 decreased the production of TNF-α, IL-1β and IL-12p40, where as, it significantly increased the secretion of IL-10 and TGF β in goat monocytes. After rHCMIF-1 exposure, the expression of MHC-II on goat monocytes was inhibited. Moreover, rHCMIF-1 could down-regulate the LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCMIF-1 could inhibit the phagocytosis of goat monocytes. Our findings provided potential targetas immunoregulator, and will be helpful to elucidate the molecular basis of host–parasite interactions and search for new potential protein as vaccine and drug target candidate.

Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour.

Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.

The Pathophysiology, Ecology and Epidemiology of Haemonchus contortus Infection in Small Ruminants.

The parasitic nematode Haemonchus contortus occurs commonly in small ruminants, and it is an especially significant threat to the health and production of sheep and goats in tropical and warm temperate zones. The main signs of disease (haemonchosis) relate to its blood-feeding activity, leading to anaemia, weakness and frequently to deaths, unless treatment is provided. Due to the high biotic potential, large burdens of H.contortus may develop rapidly when environmental conditions favour the free-living stages, and deaths may occur with little prior warning. More chronic forms of haemonchosis, resulting in reduced animal production and eventually deaths, occur with smaller persistent infections, especially in situations of prolonged, poor nutrition. The global distribution of the main haemonchosis-endemic zones is consistent with the critical requirements of the egg and larval stages of H. contortus for moisture and moderate to relatively warm temperatures, but the seasonal propensity for hypobiosis (inhibition of the fourth-stage larvae within the host) largely explains the common, though sporadic, outbreaks of haemonchosis in arid and colder environments. The wide climatic distribution may also reflect the adaptation of local isolates to less favourable ecological conditions, while an apparent increase in the prevalence of outbreaks in environments not previously considered endemic for haemonchosis - especially cold, temperate zones - may be attributable to climatic changes. Although the risk of haemonchosis varies considerably on a local level, even where H. contortus is endemic, the extensive range of ecological investigations provides a sound basis for predictions of the relative geographical and seasonal risk in relation to climatic conditions.

Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus.

The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL-1, and egg hatch assays were conducted at protein concentrations of 0.8, 0.4, 0.2, 0.1 and 0.05 mg mL-1. The effective concentration for 50% hatching inhibition (EC50) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC50 of the CE and TE extracts were 0.48 and 0.33 mg mL-1, respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.

Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode Haemonchus contortus

It is important to understand how anthelmintic drug resistance mutations arise and spread in order to determine appropriate mitigation strategies. We hypothesised that a molecular genetic study of Haemonchus contortus in southern India, a region where resistance may be less advanced than in western Europe and North America, might provide some important insights into the origin and spread of anthelmintic resistance. The F200Y (TAC) isotype-1 β-tubulin benzimidazole resistance mutation is common in H. contortus throughout the world and the F167Y (TAC) and E198A (GCA) mutations, although less common, have been reported in a number of different countries. We have investigated the haplotypic diversity and phylogenetic relationship of isotype-1 β-tubulin benzimidazole resistance alleles for 23 H. contortus populations from small ruminants across southern India. The F200Y (TAC) mutation was most common, being detected in 18/23 populations at frequencies between 9% and 84% and the E198A (GCA) mutation was also detected in 8/23 populations at frequencies between 8% and 18%. The F167Y (TAC) mutation was not detected in any of the 23 populations. Phylogenetic haplotype network analysis suggested that the F200Y (TAC) mutation has arisen multiple independent times in the region with at least three independent origins of resistance alleles across the populations surveyed. In contrast, the E198A (GCA) mutation was present on a single haplotype which, given the high level of haplotypic diversity of the susceptible alleles in the region, suggests this particular mutation has spread from a single origin, likely by anthropogenic animal movement. Population genetic analysis of 12 of the H. contortus populations, using a panel of eight microsatellite markers, revealed extremely low genetic differentiation between populations, consistent with the hypothesis of high gene flow among sites. Additionally, there was no significant genetic differentiation between H. contortus taken from sheep and goats which is consistent with H. contortus populations being freely shared between these two different hosts. Overall, we believe these results provide the first clear genetic evidence for the spread of an anthelmintic resistance mutation to multiple different locations from a single origin.

A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor.

Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(-)-4-amino-3-hydroxybutyric acid [R(-)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(-)- and S(+)-GABOB. The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.

Synthesis of photoreactive ivermectin B1a derivatives and their actions on Haemonchus and Bombyx glutamate-gated chloride channels

Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls.

Monepantel irreversibly binds to and opens Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 receptors.

Monepantel is a recently developed anthelmintic with a novel mode of action. Parasitic nematodes with reduced sensitivity to monepantel have led to the identification of MPTL-1, a ligand-gated ion-channel subunit of the parasitic nematode Haemonchus contortus, as a potential drug target. Homomeric MPTL-1 channels reconstituted in Xenopus oocytes are gated by µM concentrations of betaine and mM concentrations of choline. Measurement of reversal potentials indicated that the channel has a similar conductance for Na(+) and K(+) ions and does not permeate Ca(2+). Concentrations of monepantel (amino-acetonitrile derivative [AAD]-2225) >0.1 μM, but not its inactive enantiomer AAD-2224, induced channel opening in an irreversible manner. Currents elicited by monepantel alone were larger than the maximal current amplitudes achieved with betaine or choline, making monepantel a superagonist. Currents elicited by betaine or choline were allosterically potentiated by nM concentrations of monepantel and to a much smaller degree by AAD-2224. We have also reconstituted the Caenorhabditis elegans homomeric ACR-20 receptor in Xenopus oocytes. The acr-20 sequence has higher similarity to mptl-1 than acr-23, the primary target for monepantel mode of action in C. elegans. The ACR-20 channel is gated similarly as MPTL-1. Monepantel, but not AAD-2224, was able to induce channel opening in an irreversible manner at similar concentrations as for MPTL-1. Interestingly, the allosteric potentiation measured in the presence of betaine was much smaller than in MPTL-1 receptors. Together, these results establish the mode of action of monepantel in H. contortus and contribute to our understanding of the mode of action of this anthelmintic.

Acquired resistance to monepantel in C. elegans: What about parasitic nematodes?

In 2008, Novartis Animal Health developed a new class of anthelmintics, the amino-acetonitrile derivatives (AAD) of which monepantel is the most prominent compound. Monepantel was designed for the treatment of sheep against the parasitic nematode Haemonchus contortus. Because monepantel acts through a different mechanism, it is effective against nematodes that have acquired resistance to long-standing anthelmintics. In order to benefit from a maximum lifespan and efficacy of this new compound, the mode of action of monepantel needs to be understood. Studies on the model nematode Caenorhabditis elegans led to the identification of at least one target of monepantel: the monovalent cation channel ACR-23. Here we comment on the effects of monepantel on C. elegans and on the development of resistant parasitic nematode strains.

Novel assay for the detection and monitoring of levamisole resistance in Haemonchus contortus

A levamisole-sensitive acetylcholine receptor has recently been described in the parasitic nematode Haemonchus contortus. The pentameric receptor is produced from different subunit proteins, one of which is Hco-ACR-8. A truncated transcript, Hco-acr-8b, has been identified in six levamisole-resistant H. contortus isolates and was found to be absent in four levamisole-susceptible isolates, indicating Hco-acr-8b could be a potential marker for levamisole resistance. The Hco-acr-8b transcript contains exons 1 and 2 and terminates with 347bp from within the intron 2. In this work, we investigated genomic DNA sequences of the Hco-acr-8 gene, in a region including exon 2 and exon 3, from a wide range of levamisole-susceptible and resistant H. contortus isolates. Sequences potentially involved in generating the truncated splice variant within the second intron were analysed from individuals and pools of parasites. We found an insertion/deletion (indel) of 63bp located just downstream from the splice acceptor site for the alternative third exon. The sequence of the indel, when present, was similar in the 12 isolates examined. The presence or absence of this indel was statistically (Chi2 test) correlated with levamisole resistance status. A correlation was also demonstrated between the absence of the indel and the expression of the Hco-acr-8b transcript. We believe this is the first report of a putative DNA marker for levamisole resistance detection. Using this new knowledge, we have developed a novel DNA-based assay for the detection and monitoring of levamisole resistance in parasitic nematodes of animals.

Haemonchus contortus P-Glycoproteins Interact with Host Eosinophil Granules: A Novel Insight into the Role of ABC Transporters in Host-Parasite Interaction

Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response.

Evolution of Structure and Mechanistic Divergence in Di-Domain Methyltransferases from Nematode Phosphocholine Biosynthesis

The phosphobase methylation pathway is the major route for supplying phosphocholine to phospholipid biosynthesis in plants, nematodes, and Plasmodium. In this pathway, phosphoethanolamine N-methyltransferase (PMT) catalyzes the sequential methylation of phosphoethanolamine to phosphocholine. In the PMT, one domain (MT1) catalyzes methylation of phosphoethanolamine to phosphomonomethylethanolamine and a second domain (MT2) completes the synthesis of phosphocholine. The X-ray crystal structures of the di-domain PMT from the parasitic nematode Haemonchus contortus (HcPMT1 and HcPMT2) reveal that the catalytic domains of these proteins are structurally distinct and allow for selective methylation of phosphobase substrates using different active site architectures. These structures also reveal changes leading to loss of function in the vestigial domains of the nematode PMT. Divergence of function in the two nematode PMTs provides two distinct antiparasitic inhibitor targets within the same essential metabolic pathway. The PMTs from nematodes, plants, and Plasmodium also highlight adaptable metabolic modularity in evolutionarily diverse organisms.

Thymus capitatus from Tunisian arid zone: Chemical composition and in vitro anthelmintic effects on Haemonchus contortus

The increasing prevalence of anthelmintic resistant strains of helminths, the drug residues in animal products and the high cost of conventional anthelmintics has created an interest in studying medicinal plants as an alternative source of anthelmintics. Thymus capitatus (Lamiales: Lamiaceae) is used traditionally by people as spices and reported to possess some biological effects. The objective of this study is to evaluate the anthelmintic efficacy of T. capitatus in comparison to albendazole against the gastrointestinal nematodes of sheep. To fulfil the objectives, in vitro anthelmintic activities of crude aqueous and crude ethanolic extracts of aerial parts of T. capitatus were investigated on the eggs and adults of the nematode parasite Haemonchus contortus. Both extract types of T. capitatus completely inhibited egg hatching at a concentration close to 2mg/ml. LC50 of ethanolic extract of T. capitatus was 0.368mg/ml while that of aqueous extract was 6.344mg/ml (p<0.05). The ethanolic extract showed higher in vitro activity against adult parasites than the aqueous one in terms of the paralysis and/or death of the worms at different hours post-treatment. Dose dependent effect was observed for both extracts. Chemical analyses revealed that the overall profile of both extracts was dominated by oxygenated constituents. In addition, ethanolic extract is mainly composed of phenols among which thymol (71.22%) and camphor (17.18%).As far as the literature could be ascertained, this is the first publication on anthelmintic activity of T. capitatus. The results of the present study suggest that T. capitatus extracts are a promising alternative to the commercially available anthelmintics like albendazole for the treatment of small ruminants’ gastrointestinal nematodes.

Characterization of cys-loop receptor genes involved in inhibitory amine neurotransmission in parasitic and free living nematodes

We have isolated two genes, Hco-lgc-53 and Hco-mod-1, from the parasitic nematode Haemonchus contortus, which are orthologs of previously characterized genes that encode dopamine and serotonin-gated chloride channels, respectively, in Caenorhabditis elegans. A search of transcriptome data for the filarial nematode parasites Loa loa, Brugia malayi, and Wucheria bancrofti revealed predicted coding sequences for orthologs of acetylcholine, serotonin and dopamine-gated chloride channels, which correspond to the C. elegans clades acc-1, mod-1 and ggr-3, respectively. Genome data for the more distantly related nematode parasite, Trichinella spiralis, contain genes predicted to encode members of the acc-1 clade only, but all three clades were absent from the trematode Schistosoma mansoni. Analysis of the ratio of non-synonymous to synonymous substitutions (ω) for receptor subunit sequences revealed strong selective constraint over the entire protein, consistent with the known highly conserved 3D structure of cys-loop receptors. This constraint was significantly greater for binding loop residues that are predicted to contact bound ligand and residues of the transmembrane domains. The substitution rate for ligand binding residues was significantly higher for branches leading to the acc-1 and mod-1 clades, where the convergent evolution for binding acetylcholine and serotonin, respectively, is thought to have occurred. Homology models of both Hco-MOD-1 and Hco-LGC-53 channels revealed the presence of binding structures typical of the cys-loop receptor family, including the presence of an aromatic box that is important for the formation of the binding pocket. Both receptors contain a tryptophan in loop C that appears to be a key residue important for the binding of amines to ligand-gated chloride channels. As additional ligand-gated chloride-channel sequences become available for a wider range of species the combination of molecular modeling and analysis of sequence evolution should provide an effective tool to understand the wide diversity of neurotransmitters that bind to this unique group of receptors.