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PubMed2001

A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus.

Sheep immunoglobulin (Ig) heavy-chain (V(H)DJ(H)) and lambda light-chain variable region (V(lambda)J(lambda)) nucleotide coding sequence was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from abomasal lymph node (ALN) B cells of immune sheep challenged with the gastrointestinal nematode parasite Haemonchus contortus. Single-chain antibodies (scFv) were then constructed with the purified V(H)DJ(H) and V(lambda)J(lambda) Ig gene region DNA using oligonucleotides to PCR and join the variable regions to a central [Gly(4)Ser](3)-linker. In a similar fashion 5'-SfiI and 3'-NotI restriction endonuclease sites were added for cloning into a phagemid expression vector. Expression of sheep scFv from pHFA phagemid in an amber-suppresser strain of Escherichia coli, after infection with filamentous phage, resulted in 10(9) sheep scFv antibodies displayed as a library on phagemid particles. Western blot analysis demonstrated sheep scFv gene expression in E. coli cell lysate and on purified library phage. In addition, four rounds of scFv-library selection against H. contortus surface antigen resulted in a 300-fold increase in the elution titre of phage recovered from parasite surface antigen. Nearly 1000 of the selected and eluted scFvs were expressed in an attempt to identify monoclonal sheep scFv against parasite antigen. Only low affinity clones were isolated during screening of this sheep scFv-library, suggesting different strategies will be needed for isolation of specific high affinity recombinant antibody in future studies.

Amino Acid Sequence↗
PubMed2000

EST sequencing of the parasitic nematode Haemonchus contortus suggests a shift in gene expression during transition to the parasitic stages.

Expressed sequence tags from the parasitic nematode Haemonchus contortus were generated in order to identify anchor loci for comparative mapping between nematode genomes and candidate targets for future control measures. In total, 370 SL1 trans-spliced cDNAs from different developmental stages representing 195 different genes were partially sequenced. From these expressed sequence tags 50% were similar to genes with a known or predicted function and 19% were similar to nematode sequences with no ascribed function. From the first, free-living L1 and L3 stages relatively many cDNAs matched to housekeeping genes, and 11% (L1) or 23% (L3) of the encoded proteins were predicted to contain signal peptides. In contrast, no function could be ascribed to most of the cDNAs from the early L5 and adult parasitic stages, but for 30% (L5) or 55% (adult) of the encoded proteins a signal sequence was predicted. This limited analysis suggests that during the transition from the free-living to parasitic stages gene expression shifts towards the synthesis of less conserved extracellular proteins. These proteins offer the best perspectives for vaccine development and the development of anthelmintic drugs. In contrast, cDNAs from the first larval stages may be most suitable for comparative mapping with the free-living nematode Caenorhabditis elegans.

Animals↗
PubMed2000

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus.

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.

Animals↗
PubMed2001

High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel.

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluClbeta, Hcgbr-2A and Hcgbr-2B) with high amino-acid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluClalpha, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluClalpha1, alpha2 or alpha3/GBR-2 subunits ( approximately 55% amino-acid identity). The HcGluClalpha cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluClbeta subunits failed to bind [3H]ivermectin. The Hcgbr-2B and HcGluClalpha subunits bound [3H]ivermectin with high affinity; the K(d) values were 70+/-16 and 26+/-12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [3H] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [3H]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70+/-7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary.

Animals↗
PubMed2001

Cathepsin B-like cysteine proteases and Caenorhabditis elegans homologues dominate gene products expressed in adult Haemonchus contortus intestine.

Proteins expressed by nematode intestinal cells are potential targets for parasite control by immune or chemical based strategies. To expand our knowledge on nematode intestinal proteins, expressed sequence tags were generated for 131 cDNA clones from the intestine of adult female Haemonchus contortus. An estimated 55 distinct protein genes or gene families were identified. Predicted proteins represented diverse functions. Several predicted polypeptides were related to H. contortus proteins implicated in inducing protective immunity against challenge infections of this parasite. The dominant intestinal transcripts were represented by cathepsin B-like cysteine protease genes (cbl) (17% of protein coding expressed sequence tags (ESTs) analyzed). An estimated 11 previously undescribed cbl genes were identified, doubling the recognized members of this gene family. Multiple C-type lectin sequences were identified. Other notable sequences included a predicted Y-box binding protein, serine/threonine kinases and a cyclin E-like sequence. Predicted protein homologues were found in Caenorhabditis elegans for all but one H. contortus sequence (99%), while fewer homologues from other parasitic nematodes were found. Many of the proteases, lipase and C-type lectin homologues in C. elegans had apparent signal peptides, suggesting that they are secreted. Several gene products had no obvious similarity outside the phylum Nematoda. The ESTs identified intestinal genes with potential application to immune control, understanding of basic intestinal regulatory processes and refinement of nematode genomic resources.

Animals↗
PubMed2003

Haemonchus contortus: HcGluCla expressed in Xenopus oocytes forms a glutamate-gated ion channel that is activated by ibotenate and the antiparasitic drug ivermectin.

Ion channels are targets for many drugs including insecticides and anthelminthic agents such as ivermectin (IVM) and moxidectin (MOX). IVM has been shown to activate glutamate-gated chloride channels (GluCls) from the free-living nematode, Caenorhabditis elegans. Haemonchus contortus is a parasitic nematode that is also extremely sensitive to IVM. The high sensitivity of H. contortus to IVM is probably the result of the fact that, like C. elegans, H. contortus also express GluCls. To investigate the potential physiological response to IVM in H. contortus we have expressed a GluCl from this parasite (H. contortus glutamate-gated chloride channel, HcGluCla) in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to glutamate and ibotenate as well as the anthelmintics IVM and MOX. The response to glutamate and ibotenate was fast acting and reversible whereas the response to IVM and MOX was a slower activating channel that was essentially irreversible. These results suggest that IVM toxicity in H. contortus is the result of its irreversible activation of GluCls.

Animals↗
PubMed2003

Selection at a gamma-aminobutyric acid receptor gene in Haemonchus contortus resistant to avermectins/milbemycins.

Gamma-aminobutyric acid (GABA) Type A receptors are inhibitory chloride channels in membranes of vertebrate and invertebrate neuromuscular cells. Gating of the channels by GABA leads to an influx of chloride ions into, and hyperpolarisation of, the cell. GABA receptors are believed to form channels by the association of five protein molecules of varying subunit types, with the second transmembrane (M2) domain of each protein molecule forming a central pore through which chloride ions can pass. We have analysed by single-strand conformation polymorphism the genetic variation of a GABA-receptor gene, HG1, from two sets of unselected and anthelmintic-selected strains of the parasitic nematode Haemonchus contortus. Significant differences in allele frequencies were detected between one unselected strain and its derived ivermectin-selected strain and between the other unselected strain and its derived ivermectin- and moxidectin-selected strains. In each set of strains, one allele increased substantially in frequency in the drug-selected strains relative to their respective unselected strains. The selected allele, however, differed between the two sets of strains. Similar analyses were performed on a phosphoenolpyruvate carboxykinase gene and a nicotinic acetylcholine receptor subunit gene. No significant differences were found in allele frequencies between the unselected and their derived anthelmintic-selected strains. These results indicate the GABA receptor as a possible site of action for avermectins and milbemycins, and suggest its involvement in resistance to these anthelmintics.

Animals↗
PubMed1997

Characterization of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance.

The anthelminitic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the molecular mechanism of levamisole resistance in the parasitic nematode Haemonchus contortus was studied by isolating and characterising cDNA clones encoding a putative ligand binding nicotinic acetylcholine receptor subunit, HCAl, of two susceptible and one levamisole resistant population. Hcal is related to unc-38, a nicotinic acetylcholine receptor subunit gene associated with levamisole resistance in Caenorhabditis elegans. Although extensive sequence analyses of hcal sequences revealed polymorphism at amino acid level, no association with levamisole resistance could be detected. Restriction fragment length polymorphism analyses confirmed that, although polymorphism was detected, no selection of a specific allele of hcal has taken place during selection for levamisole resistance in various levamisole resistant populations.

Amino Acid Sequence↗
PubMed1997

Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus.

It has been shown that vaccination with two low molecular mass excretory secretory (ES) antigens of 15 and 24 kDa, respectively, afforded a substantial degree of protection against Haemonchus contortus to sheep. In vitro cultivation of the parasite usually yields a limited amount of these proteins and therefore, recombinant DNA technology was employed to clone the cDNAs encoding the ES proteins of interest and to express them in a convenient vector system. The N-terminal amino acid sequences of the two ES products were determined. Specific 5' primers were used in combination with an oligo (dT) 3' primer to amplify the appropriate cDNAs by polymerase chain reaction (PCR). A lambda ZAPII cDNA library was constructed from mRNA of L5 larvae and subsequently screened with the PCR products. The full length clone of the 15 kDa ES protein coded for a 17.2 kDa precursor molecule of 148 amino acids with a signal peptide of 30 amino acids. The full length clone of the 24 kDa ES protein coded for a 24.6 kDa precursor protein of 222 amino acids with a leader sequence of 19 residues. The expression of both ES products appeared to be developmentally regulated; mRNA encoding occurs only in the parasitic life stages. A cDNA of each ES protein was sub-cloned, without the leader sequence, into a pQE9 expression vector. Both purified recombinant proteins were recognized by sera from H. contortus hyperimmunised sheep as judged by immunoblot analysis, suggesting that antigenic determinants were also present on the recombinant proteins.

Amino Acid Sequence↗
PubMed1997

Microsatellites of the parasitic nematode Haemonchus contortus: polymorphism and linkage with a direct repeat.

To develop tools to analyse parasitic nematode population structures and the effects of selection pressure on the nematode population, we isolated and characterised 13 microsatellite markers of the nematode Haemonchus contortus. The density of CA/GT microsatellites, once in 575 kb, is lower than in mammals. Although the isolated CA/GT repeats were imperfect, the majority of the microsatellites were polymorphic in one or more populations. An extensive genetic diversity both within and between populations could be established. Two-thirds of the CA/GT microsatellites were followed by a variable number of 128 bp direct repeat elements, HcREP1. HcREP1 is a repetitive element in the H. contortus genome, which is homologous to the repetitive TcREP element in the nematode Trichostrongylus colubriformis.

Animals↗
PubMed1998

Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus.

Resistance to anthelmintics that are used to control parasite populations in domestic animals has become a serious problem worldwide. The development of resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. The anthelmintic ivermectin is known to bind to the human membrane transport protein, P-glycoprotein, and P-glycoprotein-deficient mice treated with ivermectin have shown signs of neurotoxicity. P-glycoprotein is believed to be involved in the multidrug resistance phenotype seen in some human cancers and for drug resistance in some protists. We have examined the genetic variation of a P-glycoprotein homologue from the nematode Haemonchus contortus to see if an association exists between specific alleles of this gene and survival to exposure to ivermectin or moxidectin. Two parasite strains passaged without drug treatment and three strains, subjected to anthelmintic selection and derived from the unselected strains, were examined. Allelic variation in the unselected strains showed this locus to be highly polymorphic. chi 2 analyses of allele frequencies showed significant differences between the unselected and the drug-selected derived strains. In all three drug-selected strains, an apparent selection for the same allele was observed. These findings suggest that P-glycoprotein may be involved in resistance to both ivermectin and moxidectin in H. contortus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species.

General methods to conduct tissue specific analysis are largely lacking for nematodes. An approach is described that focused on isolation of membrane and secreted protein genes from the gut of the parasitic nematode Haemonchus contortus. The approach capitalized on a monoclonal antibody that recognizes multiple membrane and secreted worm proteins. Polyclonal antisera made against these proteins were used to screen expression cDNA libraries made either from adult worm gut or whole worm. The genes identified encode predicted or known membrane and secreted proteins from gut, including a cysteine protease, a zinc metallopeptidase and a previously described GA1 protein. Another gene, Hc40, was isolated from the whole worm cDNA library and is nearly identical to a vaccine patent sequence pBTA879. Tissue analysis demonstrated the intended focus on membrane and secreted proteins from parasite gut was achieved. Proteins related to each of those described were identified from other nematode species through data base analysis. Additionally, this analysis led to (1) identification of homologues of each gene in C. elegans; (2) deduction of a dimorphic structure in the Hc40 protein; (3) recognition of both monomorphic and dimorphic families of Hc40-related proteins; and (4) detection of two apparent classes of transcripts (mep1a and mep1b) that would each encode a divergent version of the putative zinc metallopeptidase MEP1. The tissue specific approach and information base described should generally contribute to investigations on nutrient digestion and related secretory processes in nematode gut.

Amino Acid Sequence↗
PubMed1998

Cloning and localisation of an avermectin receptor-related subunit from Haemonchus contortus.

Ivermectin is believed to exert its anthelminthic effects by binding to glutamate-gated chloride channels (Glu-Cl) and several cDNAs encoding subunits of Glu-Cl have been cloned from Caenorhabditis elegans. We report the cloning of cDNAs encoding a putative Glu-Cl subunit (HG4) from the parasite Haemonchus contortus. The HG4 cDNAs were isolated using RT-PCR and the sequence of the predicted polypeptide has 82% amino-acid identity with the C. elegans Glu-Cl beta subunit. Individual HG4 cDNAs showed up to 4% sequence variation at the nucleotide level, but the vast majority of these polymorphisms were translationally silent. A synthetic peptide corresponding to sequence near the N-terminus of the mature polypeptide was used to raise an antiserum that recognised the N-terminal domain of HG4 expressed in E. coli. Affinity purified antibodies reacted with motor neuron commissures in immuno-localisation studies: these commissures were limited to the anterior portion of the worms, from a region level with the nerve ring to just anterior of the vulva. Some possible nerve cord staining was also observed, but no expression of HG4 on pharyngeal muscle could be detected.

Amino Acid Sequence↗
PubMed1999

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus.

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 microM)-induced contractions, with a threshold for activity of 1 microM. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus.

Amino Acid Sequence↗
PubMed1999

Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus.

Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus.

Amino Acid Sequence↗
PubMed1999

Ligand-gated chloride channel subunits encoded by the Haemonchus contortus and Ascaris suum orthologues of the Caenorhabditis elegans gbr-2 (avr-14) gene.

The alternatively-spliced Caenorhabditis elegans gbr-2/avr-14 gene encodes two subunits of the nematode ligand-gated chloride channel family which forms an important molecular target for the avermectin and related anthelminthics. We used reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to isolate cDNAs encoding the products of the gbr-2/avr-14 orthologues from the parasitic nematodes Haemonchus contortus and Ascaris suum. The predicted polypeptides possess all the characteristics of subunits of the ligand-gated chloride channels, sharing greater than 80% amino-acid identity with their counterparts in C. elegans and with partial sequences from the filarial species Onchocerca volvulus and Dirofilaria immitis. The pattern of alternative splicing of the gbr-2/avr-14 gene observed in C. elegans is conserved in H. contortus but may not be in A. suum. Affinity-purified anti-GBR-2 antibodies were used to study the expression of these subunits in adult worms and they reacted specifically with the nerve ring, the ventral and dorsal nerve cords, the anterior portion of the dorsal sub-lateral cord and motor-neuron commissures in H. contortus. Specific immunofluorescence of the nerve cords was confirmed in A. suum; isolated muscle cells did not react with the antibody.

Alternative Splicing↗
PubMed2000

Multiple lethal effects induced by a benzimidazole anthelmintic in the anterior intestine of the nematode Haemonchus contortus.

A mechanism of benzimidazole efficacy against parasitic nematodes is postulated to involve inhibition of intestinal secretory vesicle transport via depolymerization of microtubules. We show that fenbendazole (FBZ) treatment of lambs causes pathology localized to the anterior intestine in the parasitic nematode Haemonchus contortus. The pathology included gross disintegration of the anterior intestine, DNA fragmentation in anterior intestinal nuclei with characteristics of an apoptosis-like process, and inhibition of host erythrocyte digestion. These lethal effects were associated with inhibited transport of apical secretory vesicles in the anterior intestine, and then generalized dispersal of these vesicles contents throughout the intestinal cytoplasm and worm body. Cytoplasmic accumulation of secretory vesicles and undigested erythrocytes preceded DNA fragmentation and vesicle content dispersal. Both DNA fragmentation and vesicle content dispersal were detected in disintegrated intestine and intestine that had not yet undergone disintegration. These pathologic effects in the anterior intestine appear sufficient to explain the efficacy of FBZ against adult H. contortus. The results implicate mechanisms in the anterior intestine that govern dispersal of apical secretory vesicle contents, DNA fragmentation and tissue disintegration as effectors of this pathology.

Animals↗
PubMed2000

Transposon associated markers for the parasitic nematode Haemonchus contortus.

We have previously characterized a Tcl-like transposable element Hctcl, from the parasitic nematode Haemonchus contortus. Here we describe the genetic variation of Hctcl insertion sites in H. contortus populations differing in geographical origin, resistance to chemotherapeutics and level of inbreeding. Clear differences between populations were observed on Southern blots with a Hctcl-specific probe. Sequencing the 5'- or 3'-flanks of individual Hctcl integration sites allowed the design of PCR reactions between a Hctcl-specific primer and the flanking regions. This revealed a considerable variation of integration sites of Hctcl both within and between populations, although several integrations were shared by populations of different geographical origin. For four of the eight markers allele frequencies were shifted during selection for resistance to chemotherapeutics and/or inbreeding. For two positions both the 5' and 3' regions flanking Hctcl were isolated and PCR showed that for these two positions the variation of transposon associated markers between populations were indeed caused by variation in integration of Hctcl. For these two positions co-dominant markers were developed. These results indicate that Hctcl insertions may serve as genetic markers for H. contortus.

Animals↗