Ruurdtje Hoekstra

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.

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.

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.

Amplified fragment length polymorphism analysis of genetic diversity of Haemonchus contortus during selection for drug resistance

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.

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.

Selection for high levamisole resistance in Haemonchus contortus monitored with an egg-hatch assay

To investigate the characteristics of selection for levamisole resistance in Haemonchus contortus, the consecutive nematode generations of an in vivo selection were monitored with a newly developed egg-hatch assay. The in vivo selection was started with a population not previously exposed to any anthelmintics (SHS). At first, the levamisole resistance progressed gradually in successive nematode generations by treating sheep with increasing doses of levamisole, the initial dose being 1 mg kg-1. Treatment with 5 mg kg-1 levamisole resulted, however, in a steep increase of resistance. The selection was ended after six generations, since a level of 30 mg kg-1 levamisole, which is not far from the toxic level for sheep, was reached. The final population, RHS6, was studied in a controlled test. Treatment of RHS6-infected sheep with 30 mg kg-1 levamisole caused an 80% decrease of faecal egg output, and a reduction of 34% in worm numbers. It was remarkable that only the number of female adults was reduced. RHS6 showed a reduced viability, but a fertility similar to the starting population SHS.

Polymorphic DNA markers in the genome of parasitic nematodes.

Polymorphic molecular markers are being identified to characterize the genomes of parasitic nematodes. The aim is to construct a map with markers evenly spread over the six chromosomes. With such a map, regions can be identified that are under selection pressure when attempts are being made to eradicate worms, be it by drugs, vaccines or genetic resistance in the sheep. Several types of markers have been developed, microsatellites, transposon-associated markers, amplified fragment length polymorphism (AFLP) and expressed sequence tag (EST) markers. Linkage groups can be constructed using several genetic crosses between inbred and drug resistant strains. EST markers will be especially important for comparative mapping with the genome of Caenorhabditis elegans, and therefore localization of the linkage group on a chromosome. It will then be possible to identify functional genes close to markers that have changed allele frequencies under selection pressure and identify the mechanisms of resistance to parasite control.

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.

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.

Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.