Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) – stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

With increasing resistance to anti-parasitic drugs, it has become more important to detect and recognize phenotypes of resistant isolates. Molecular methods of detecting resistant isolates are limited at present. Here, we introduce a microfluidic bioassay to measure phenotype using parameters of nematode locomotion. We illustrate the technique on larvae of an animal parasite Oesophagostomum dentatum. Parameters of sinusoidal motion such as propagation velocity, wavelength, wave amplitude, and oscillation frequency depended on the levamisole-sensitivity of the isolate of parasitic nematode. The levamisole-sensitive isolate (SENS) had a mean wave amplitude of 135 μm, which was larger than 123 μm of the levamisole-resistant isolate (LEVR). SENS had a mean wavelength of 373 μm, which was less than 393 μm of LEVR. The mean propagation velocity of SENS, 149 μm s−1, was similar to LEVR, 143 μm s−1. The propagation velocity of the isolates was inhibited by levamisole in a concentration-dependent manner above 0·5 μm. The EC50 for SENS was 3 μm and the EC50 for LEVR was 10 μm. This microfluidic technology advances present-day nematode migration assays and provides a better quantification and increased drug sensitivity. It is anticipated that the bioassay will facilitate study of resistance to other anthelmintic drugs that affect locomotion.

This investigation compared the effect of two common types of diet on worm burdens and faecal egg counts of pigs inoculated with Ascaris suum and Oesophagostomum dentatum. The diets were both considered nutritionally sufficient for pigs of this age, and gave high and comparable growth rates. Pigs were inoculated with both worm species at 14 weeks of age and slaughtered 3 or 12 weeks later. At 3 weeks, A. suum larvae were numerous in the small intestine of pigs fed ground barley plus protein supplement, but were not detected from pigs fed a commercial full-constituent pelleted feed. At 12 weeks, however, average adult worm burdens were low and similar in both groups. At both slaughter times, O. dentatum worm burdens were significantly higher in pigs fed barley plus protein than in pigs given commercial feed, particularly for female worms, and the commercial diet led to worms being located further along the large intestine, and to reduced worm fecundity. The chemical composition of large intestinal contents differed significantly between pigs fed the two diets.

Oesophagostomum dentatum stages were investigated for glutathione S-transferase (GST) expression at the protein and mRNA levels. GST activity was detected in all stages (infectious and parasitic stages including third- and fourth-stage larvae of different ages as well as males and females) and could be dose-dependently inhibited with sulfobromophthalein (SBP). Addition of SBP to in vitro larval cultures reversibly inhibited development from third- to fourth-stage larvae. Two glutathione-affinity purified proteins (23 and 25 kDa) were detected in lysates of exsheathed third-stage larvae by SDS-PAGE. PCR-primers were designed based on peptide sequences and conserved GST sequences of other nematodes for complete cDNA sequences (621 and 624 nt) of 2 isoforms, Od-GST1 and Od-GST2, with 72% nucleotide similarity and 75% for the deduced proteins. Genomic sequences consisted of 7 exons and 6 introns spanning 1296 bp for Od-GST1 and 1579 and 1606 bp for Od-GST2. Quantitative real-time-PCR revealed considerably elevated levels of Od-GST1 in the early parasitic stages and slightly reduced levels of Od-GST2 in male worms. Both Od-GSTs were most similar to GST of Ancylostoma caninum (nucleotides: 73 and 70%; amino acids: 80 and 73%). The first three exons (75 amino acids) corresponded to a synthetic prostaglandin D2 synthase (53% similarity). O. dentatum GSTs might be involved in intrinsic metabolic pathways which could play a role both in nematode physiology and in host-parasite interactions.

Transmission of Oesophagostomum dentatum and Trichuris suis was studied in outdoor reared pigs. Six farrowing paddocks were naturally contaminated in May to mid-June 2001 by experimentally infected seeder pigs. In early July 1 sow farrowed on each paddock and starting at week 3 post-partum (p.p.) the offspring was slaughtered serially every 2 weeks for parasite recovery. Faeces were collected regularly for parasite egg counts and acid-insoluble ash (AIA) content as an indicator of geophagy. Weaning took place at week 7 p.p. by removing the sow. Paddock infection levels were estimated in mid-June (O. dentatum) and late November (O. dentatum and T. suis) using helminth-naïve tracer pigs. Soil and vegetation samples were collected regularly. Despite a high initial contamination by the seeder pigs, O. dentatum paddock infectivity was negligible to low throughout the raising of the experimental piglets, which had a slow accumulation of nodular worms ending with a mean of 422 worms/pig at week 19 p.p. As only few eggs developed to infectivity overall T. suis transmission was minimal. The first T. suis were recovered at week 11 p.p. and the highest mean burden of 21 worms/pig was recorded at week 19 p.p. The experimental pigs initially had a high faecal level of AIA although it decreased over time. The results are discussed in relation to the biological characteristics of the 2 parasites and their occurrence in organic pig production.

In the present study, we constructed gender-enriched cDNA libraries for the adult stage of the parasitic nematode Oesophagostomum dentatum (order Strongylida) using suppressive-subtractive hybridization (SSH), sequenced clones from the female-library and male-library (480 from each) and conducted bioinformatic and microarray analyses of the expressed sequence tags (ESTs). In total, 873 ESTs (440 male and 433 female) were obtained, achieving a sequencing success of 91%The nucleotide sequences reported in this article (Tables 1–5) have been deposited in the EMBL, GenBank and DDJB databases under the Accession nos. AM157797–AM158083. Microarray analyses of 516 unique ESTs representing both gender-enriched libraries revealed differential hybridization for 391 of them (75·8%). Of these, 220 (56·3%) had significantly greater signal intensities in the female than in the male, and 154 (70%) of these were predicted to have homologues in C. elegans. These homologues were predicted to be involved in key biological processes, including embryonic nutrition, gametogenesis, molecular binding/transport or metabolism, nucleic acid synthesis and function, and signal transduction. Of the 171 ESTs with statistically higher signal intensities in male O. dentatum, 43·8% had homologues in C. elegans. These homologues included major sperm proteins (MSPs) or MSP-like molecules, keratin-like molecules, molecules involved in metabolism, PDZ domain-containing proteins, sugar binding proteins, protein kinases, serine proteases or protease inhibitors, molecules involved in proteolysis and other proteins, such as enzymes and various putative proteins. Of the 287 ESTs (from both gender-enriched cDNA libraries) with no known homologues in C. elegans, 50 (17·4%) had homologues in other nematodes, 8 had homologues in various other organisms and 104 (36·2%) had no homology to any sequence in current gene databases. The present study lays a foundation for the isolation and molecular, biochemical and functional characterization of selected genes from the gender-enriched cDNA archives established for O. dentatum.

Major sperm protein (msp) genes were isolated from complementary (cDNA) and genomic DNA libraries prepared from the parasitic nematode, Oesophagostomum dentatum, characterized at the nucleotide and amino acid (aa) levels, and their expression was investigated. Three different msp cDNA and 2 genomic sequences were determinedThe nucleotide sequences reported in this article have been deposited in the EMBL, GenBank and DDJB databases under the Accession numbers AJ627869–AJ627873., each with an open reading frame (ORF) of 381 nucleotides. Nucleotide variation was detected at 30 positions in the ORF among all 5 sequences. Conceptual translation of the full-length msp sequences inferred 4 different MSPs each of 126 aa. These predicted MSPs differed at aa positions 15 (serine<->threonine), 101 (alanine<->glycine), 103 (glutamine<->leucine) and 126 (proline<->leucine). Southern blot analysis of O. dentatum genomic DNA, digested separately with various restriction endonucleases, displayed multiple (n=7–13) bands for each enzyme, providing support for a multigene family. Also, at the genomic level, sequence tracts consistent with a ‘substitute’ TATA box sequence motif were identified within a region (−1 to −123 nt) preceding the 2 msp genes. In contrast to other species of nematode investigated to date, no GATA transcription factor binding motif was detected immediately upstream of the msp coding region. Real-time PCR analysis demonstrated that msp mRNA was expressed exclusively in the males of both fourth-stage larvae (L4s) and adults of O. dentatum (raised in pigs after intragastric inoculation). The magnitude of expression in male O. dentatum raised in pigs in the presence of female worms was the same as in males in the absence of females. Comparative analyses showed aa sequence conservation among MSPs from various nematodes, suggesting similar functional roles for these proteins.

The present study was designed to assess the role of inulin and sugar beet fibres (SBF) on adult O. dentatum in growing pigs. Four experimental diets were formulated based on barley flour with added insoluble fibre from oat husk (Diet 1), a pure carbohydrate source inulin (Diet 2), soluble fibre from sugar beet fibre (SBF) with a high proportion of soluble fibre components (Diet 3) or inulin plus SBF (Diet 4). Thirty-two 10-week-old pigs were divided randomly into 4 groups each of 8 pigs. After 3 weeks adaptation on Diet 1 all pigs were infected with a single dose of 6000 L3O. dentatum. At week 7 p.i. one group was switched to Diet 2, another group to Diet 3 and another group to Diet 4. The remaining 8 pigs continued on Diet 1 until the end of the experiment and served as controls. At week 13, all pigs were necropsied and their worm burdens determined. The worm recoveries from the pigs on the inulin supplemented diet (Diet 2) were reduced by 97% compared to the controls (Diet 1). Further, the inulin-fed pigs exhibited markedly reduced faecal egg counts. The pigs on inulin plus SBF diet (Diet 4) and on SBF diet (Diet 3) had 86% and 70% adult worm reductions compared with the controls, respectively. The results from this study indicate that highly degradable and rapidly fermentable carbohydrates such as dietary inulin have a profound deworming effect on O. dentatum infection.

A cDNA was isolated from an adult male Oesophagostomum dentatum gene library by screening with a male-specific, partial expressed sequence tag (EST) probe identified previously using a differential display technique. The full-length cDNA of 642 bp included 5′ and 3′ untranslated regions of 44 and 121 nucleotides, respectively, and encoded a predicted protein with a putative 18 amino acid signal sequence and a mature polypeptide of 14.7 kDa comprising ∼15% cysteine residues. The amino acid sequence showed similarity with a number of proteins from Caenorhabditis elegans, parasitic nematodes, insects and amphibia, all of which contain a trypsin inhibitor-like cysteine-rich domain. A 3-dimensional structure model constructed for the O. dentatum protein (designated OdmCRP) inferred that it is composed of 2 domains, each with 5 disulfide bonds, which are indicative of the Ascaris family of serine protease inhibitors. These findings indicate that OdmCRP, with 2 structural domains relating to functionally active sites, is a new member of this inhibitor family.

An experiment was undertaken to study the effect of dietary carbohydrates with different digestibility on the populations of Oesophagostomum dentatum in the intestinal tract of pigs. Sixty-four worm-free pigs from a specific pathogen-free farm were randomly divided into 8 equal groups. The animals in 4 groups were assigned to a diet with partially undegradable carbohydrates (diet 1), while the pigs in the 4 remaining groups were given a diet with fermentable carbohydrates (diet 2). Diet 1 was comprised of barley flour, oat husk meal, soybean meal, vitamins and minerals and diet 2 of barley flour, inulin and sugar beet fibre, soybean meal, vitamins and minerals. The pigs in 6 of the groups (n = 48) were inoculated with 6000 infective larvae of O. dentatum. To determine O. dentatum populations at the early stage of infection, 16 pigs were slaughtered 3 weeks p.i., while the remaining 4 groups continued on the diets for a further 9 weeks after which they were slaughtered. In a diet cross-over experiment 6 weeks after inoculation, 8 pigs changed from diet 1 to diet 2 (diet 1>diet 2), and 8 pigs from diet 2 to diet 1 (diet 2>diet 1). The results showed that partially undegradable carbohydrates provided favourable conditions not only for parasite establishment and sustainability, but also for already established O. dentatum infection while, in contrast, the diet composed of highly degradable carbohydrates decreased worm establishment, size and female fecundity. The implications for pastured pigs or pigs fed different complex carbohydrate diets is discussed.

Ten chickens and 2 pigs were fed non-embryonated eggs of Ascaris suum, Trichuris suis and Oesophagostomum dentatum. Each chicken was fed approximately 15000 eggs of each parasite species while approximately 300000 eggs were given to each of the pigs. After passage in chickens 8.3% of O. dentatum eggs were recovered in faeces compared to 61.1% and 41.6% of A. suum and T. suis eggs, respectively. After passage in pigs the percentages were 38.4%, 49.1% and 30.3%, respectively. After embryonation in the laboratory, 1000 eggs of each parasite species having passed through chickens or pigs or having been kept in the laboratory as controls were fed to groups of 6 pigs to check the infectivity. The number of A. suum recovered from pigs was similar in the 3 groups with 34.0, 52.8 and 41.8%, respectively. The recovery of T. suis in the pig passage group was 54.0% which was significantly lower than the recovery in the chicken passage group (81.8%) and the laboratory group (88.0%). The number of O. dentatum recovered was not significantly different among the 3 experimental groups, the percentage recovery being 30.5, 9.2 and 28.5%, respectively. One explanation for the lower infectivity of T. suis in the pig passage group may be that the eggs have been sublethally damaged through their passage. The results demonstrate that chickens and pigs can act as transport hosts for A. suum, T. suis and O. dentatum, and it is highly probable that these domestic animals are able to act also as transport hosts for the human parasite equivalents. This will have important consequences for the environmental and behavioural strategies in human helminth control programmes.

Pats of pig faeces containing known numbers of Oesophagostomum dentatum eggs were placed on plots with bare soil, short or tall herbage on 8 occasions during 1 year. The number of eggs and larvae and the relative distribution of larvae in faeces, soil and herbage was monitored for 1 year after deposition. On 2 occasions soil from 8 selected plots was given to pigs, which were later slaughtered and examined for the presence of adult O. dentatum. Less than 1% of the deposited eggs could be recovered as infective larvae. The highest recoveries were generally made on tall herbage plots. The majority of infective larvae was found within the faecal pats, which indicates that infective O. dentatum larvae, to a large extent, do not disperse onto the herbage or into soil. The infective larval stage was reached only when the mean temperature in the weeks post-deposition was above 10 °C. This stage was reached within 1 week when the mean weekly temperature was above 13 °C. After the winter period no infective larvae could be recovered from any plots and no parasitic worms could be isolated from pigs fed soil from 8 selected plots.

It is pointed out that two of the three major groups of anthelmintic act by opening membrane ion-channels. It is appropriate, therefore, to use electrophysiological methods to study the properties of the sites of action of these drugs and the changes in the properties of these receptor sites associated with resistance. This paper describes the use of the patch- clamp technique to observe the currents that flow through the levamisole-activated channels as they open and close in levamisole-sensitive and levamisole-resistant isolates. It was found that, on average, the proportion of time the channels are open, is less in the resistant isolate. The patch-clamp technique also showed that the ion-channels are heterogeneous and that one of the subtypes is lost with the appearance of resistance. The use of the current clamp technique is illustrated to record a site of action of ivermectin in the pharyngeal muscle of Ascaris.

Living and fixed specimen of Oesophagostomum dentatum were labelled in situ with serum antibodies or a panel of biotin- labelled lectins. Specific binding of antibodies was observed in all parasitic stages – freshly exsheathed 3rd-stage larvae (L3), 3rd- and 4th-stage (L4) larvae cultured in vitro and L3 and L4 and adults isolated from pig intestines. The shedding of the stained layer by motile larvae was inhibited by levamisole-induced paralysis. Larvae cultured in vitro exposed serum-derived proteins on their surface which could be labelled with secondary antibody directed against the respective serum donor species. While freshly exsheathed larvae were recognized by O. dentatum-positive serum only, older larvae and adults cross-reacted with serum from pigs infected with O. quadrispinulatum, a closely related species. Lectin binding varied considerably between stages. While binding was not observed in pre-parasitic stages, Concanavalin A, Soybean Agglutinin, Wheat Germ Agglutinin, Ricinus communis Agglutinin and Peanut Agglutinin bound to developing larvae in varying degrees. Dolichos biflorus Agglutinin only bound to advanced (luminal) larval stages, while adults generally displayed only weak or partial lectin binding (except with Concanavalin A and Wheat Germ Agglutinin). Ulex europaeus Agglutinin only labelled larvae derived from cultures containing 10% pig serum. Cleavage of the carbohydrate residues by sodium periodate treatment resulted in reduction of antibody binding to cultured larvae, but not to freshly exsheathed L3. Concanavalin A, Soybean Agglutinin, and Peanut Agglutinin binding was also reduced by periodate treatment, while binding of Wheat Germ Agglutinin and Ricinus communis Agglutinin was inhibited only in early L3, but not in older stages. The different lectin labelling patterns are related to the different stages of the nematode – infective, invasive, histotropic, and luminal – and may serve as a mode of adaptation for the parasite against the host's immune attack by surface glycoprotein variation, together with antigen shedding (as demonstrated by labelling of motile larvae) and a possible acquisition of host molecules at the parasite's surface. Furthermore, a possible role of this developmental variation in surface carbohydrates in parasite–parasite interactions is discussed.

A trickle infection experiment was undertaken to study in detail the population dynamics of Oesophagostomum dentatum in pigs. Three groups of 32 pigs were inoculated via the feed twice weekly with 100 (Group A), 1000 (Group B) or 10000 (Group C) O. dentatum infective larvae (L3). Five pigs from each group were killed 2, 4, 8, 12, 16, and 20 weeks after the first inoculation (p.i.) to determine their worm burdens. Weekly faecal egg counts were determined. At slaughter, worms were counted, differentiated according to sex and developmental stage, and their length measured. Faecal egg counts ranked with dose rate until week 15, but later were more variable. The proportion of the total number of L3 administered which were recovered at slaughter inversely ranked with dose rate. In group C it decreased over time, whereas in groups A and B there was no consistent pattern. Worm fecundities (epg/female) in groups A and B were higher than in group C. The lengths of the female worms increased over time, whereas the lengths of the male worms remained approximately constant from week 8. The study suggests reduced establishment of incoming larvae and lower fecundity of the female worms at high dose levels.

The impact of diets varying in type and level of carbohydrates resistant to endogenous enzymes and lignin on the establishment and location of Ascaris suum and Oesophagostomum dentatum was investigated experimentally. Fifty worm-free pigs, from a specific pathogen-free farm were used. The animals were assigned randomly to 5 diets and infected with 600 infective A. suum eggs and 6000 infective larvae of O. dentatum per pig. The diets consisted of a traditional ground barley plus protein feed (diet A), commercial full-constituent pelleted feed (diet B), barley flour plus protein (diet C), barley flour, inulin (Raftiline® ST, ORAFTI, Tienen, Belgium), sugar beet fibre plus protein (diet D), and barley flour, wheat bran, and protein (diet E). The faecal egg excretion was followed and the pigs were slaughtered at 8 weeks p.i. and samples taken from the small and large intestine. Intestinal contents were analysed for worm burdens, worm location and female worm fecundity along with the concentration of insoluble (chromic oxide) and soluble (polyethylene glycol-4000) markers, lignin, non-starch polysaccharides (NSP) and organic acids. In all diet groups A. suum worm burdens were low and comparable, whereas the O. dentatum worm burdens were significantly higher in pigs fed the diets with high levels of NSP and lignin (diets A and E) than in pigs fed diets B, C, and D. The present study suggests that a diet rich in lignin and insoluble NSP's provides favourable conditions for the establishment of O. dentatum in the large intestine of pigs while it is unlikely that the concentration of short-chain fatty acids and pH plays any major role.

The interaction between the 2 nodular worm species in the pig, Oesophagostomum dentatum (O.d.) and Oesophagostomum quadrispinulatum (O.q.), was studied by comparing the development and distribution of the species following single or mixed infections. The faecal egg excretion levels were assessed at regular intervals from week 3 post-inoculation, and indicated a strong negative impact of the introduction of O.q. on the continued egg excretion of O.d. All pigs were killed 9 weeks after the first inoculations to determine the composition and location of the worm burdens in the large intestine. O.q. was found more anteriorly located in the intestine than O.d., thus confirming previous descriptions. When both species were present, the distribution of O.d. was moved further posteriorly and was more spread out than in single-species infections. There appeared to be no adverse effect of O.d. on the establishment and fecundity of O.q. However, the worm recoveries corroborated the egg excretion observations, namely reduced worm burdens of O.d. if O.q. was introduced, or if O.q. was already present. It is uncertain whether this effect is caused by differences in host reaction against the two species, or whether a more specific competition occurs between the two nodular worm species in pigs.