Natural nodular worm infection in goats induces eosinophil extracellular DNA trap (EET) formation.

Extracellular DNA Trap (ET) formation by granulocyte is a strong innate immune machinery that plays crucial roles in trapping and killing of pathogens. Here, we show Eosinophil Extracellular DNA Trap (EET) formation in goats naturally infected with nodular worms (Oesophagostomum columbianum, Strongyloidae: Nematoda). By a slaughterhouse based survey, we found that 60% goats were infected with nodular worms. We detected numerous, hard and pale yellow to dark black nodules of variable sizes (0.25-2 cm) in the large intestine and the number of nodules were significantly (p < .05) higher in the cecum (21.7 ± 17.9) than in the colon (10.1 ± 9.9). Histologically, pink colored circumscribed caseous mass was surrounded by a dense zone of infiltration and fibrous proliferation along with massive infiltration of eosinophils in and around the necrotic mass. DAPI staining revealed huge accumulation of extracellular DNA, which formed wide ridge like structure surrounding the necrotic zone. Massive release of eosinophils cationic proteins (ECP), a helmintho-toxic substance, was found into the lesions. Collectively, our results suggest that nodular worm infection induces EETosis and ECP release, and is one of the major parasitic problem affecting Black Bengal goats that causes distortion of normal architecture of the gut wall.

Recent Advances in Elucidating Nematode Moulting - Prospects of Using Oesophagostomum dentatum as a Model.

There are major gaps in our knowledge of many molecular biological processes that take place during the development of parasitic nematodes, in spite of the fact that understanding such processes could lead to new ways of treating and controlling parasitic diseases via the disruption of one or more biological pathways in the parasites. Progress in genomics, transcriptomics, proteomics and bioinformatics now provides unique opportunities to investigate the molecular basis of key developmental processes in parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum, represents a large order (Strongylida) of socioeconomically important nematodes, and provides a useful platform for exploring molecular developmental processes, particularly given that this nematode can be grown and maintained in culture in vitro for periods longer than most other nematodes of this order. In this article, we focus on the moulting process (ecdysis) in nematodes; review recent advances in our understanding of molecular aspects of moulting in O. dentatum achieved by using integrated proteomic-bioinformatic tools and discuss key implications and future prospects for research in this area, also with respect to developing new anti-nematode interventions and biotechnological outcomes.

The influence of endoparasites on selected production parameters in pigs in various housing systems.

The aim of the study was to determine the level of lean meat content and daily gains of 400 fatteners infected by endoparasites and kept in two systems (shallow and deep litter). Slaughter evaluation of the pigs was conducted according to the EUROP carcass classification. In order to evaluate the average daily gains (g) during finishing period, body weights were investigated twice: at the beginning and at the end of the finishing period. The housing system affected the presence of Ascaris suum and Oesophagostomum spp. Infestation was found to be higher on shallow than on deep litter, and it significantly affected selected fattening and slaughter parameters of the fatteners. Infected animals were characterized by gains approximately 60 g lower than those of uninfected ones, while meatiness was higher in fatteners which were not infected at the end of the fattening period compared to animals with parasites (55.2% vs. 52.0%).

Proteomics elucidates key molecules involved in exsheathment in vitro in Oesophagostomum dentatum.

We explored molecules involved in in vitro exsheathment of Oesophagostomum dentatum L3s using a proteomic-transcriptomic-bioinformatic approach. Analysis of L3s before, during and after exsheathment identified 11 proteins that were over-expressed exclusively during exsheathment. These proteins (including key enzymes, heat shock, structural and nematode-specific proteins) were inferred to be involved in development, metabolism, structure, motility and/or host-parasite interactions. Some of these molecules represented homologues linked to entry into and exit from the dauer stage in the free-living nematode Caenorhabditis elegans. The approach established here provides a basis for investigations of ecdysis in other strongylid nematodes.

Trichuris suis and Oesophagostomum dentatum show different sensitivity and accumulation of fenbendazole, albendazole and levamisole in vitro.

BACKGROUND: The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known. METHODOLOGY: We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species. PRINCIPAL FINDINGS: The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6-17.2%) as compared to O. dentatum (0.8-0.9%). CONCLUSION/SIGNIFICANCE: The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.

Parasite population dynamics in pigs infected with Trichuris suis and Oesophagostomum dentatum.

The aim of the present study was to investigate the population dynamics and potential interactions between Trichuris suis and Oesophagostomum dentatum in experimentally co-infected pigs, by quantification of parasite parameters such as egg excretion, worm recovery and worm location. Forty-eight helminth naïve pigs were allocated into four groups. Group O was inoculated with 20 O. dentatum L3/kg/day and Group T with 10 T. suis eggs/kg/day. Group OT was inoculated with both 20 O. dentatum L3/kg/day and 10 T. suis eggs/kg/day, while Group C was kept as an uninfected control group. All inoculations were trickle infections administered twice weekly and were continued until slaughter. Faecal samples were collected from the rectum of all pigs at day 0, and twice weekly from 2 to 9 weeks post first infection (wpi). Six pigs from each group were necropsied 5 wpi and the remaining 6 pigs from each group were necropsied 10 wpi. The faecal egg counts (FEC) and total worm burdens of O. dentatum were dramatically influenced by the presence of T. suis, with significantly lower mean FECs and worm burdens at 5 and 10 wpi compared to single infected pigs. Furthermore, in the presence of T. suis we found that O. dentatum was located more posteriorly in the gut. The changes in the Trichuris population were less prominent, but faecal egg counts, worm counts 5 wpi (57% recovered vs. 39%) and the proportion of infected animals at 10 wpi were higher in Group OT compared to Group T. The location of T. suis was unaffected by the presence of O. dentatum. These results indicate an antagonistic interaction between T. suis and O. dentatum which is dominated by T. suis.

Helminth parasites in pigs: new challenges in pig production and current research highlights.

Helminths in pigs have generally received little attention from veterinary parasitologists, despite Ascaris suum, Trichuris suis, and Oesophagostomum sp. being common worldwide. The present paper presents challenges and current research highlights connected with these parasites. In Danish swine herds, new indoor production systems may favour helminth transmission and growing knowledge on pasture survival and infectivity of A. suum and T. suis eggs indicates that they may constitute a serious threat to outdoor pig production. Furthermore, it is now evident that A. suum is zoonotic and the same may be true for T. suis. With these 'new' challenges and the economic impact of the infections, further research is warranted. Better understanding of host-parasite relationships and A. suum and T. suis egg ecology may also improve the understanding and control of human A. lumbricoides and T. trichiura infections. The population dynamics of the three parasites are well documented and may be used to study phenomena, such as predisposition and worm aggregation. Furthermore, better methods to recover larvae have provided tools for quantifying parasite transmission. Thus, an on-going study using helminth naïve tracer pigs has surprisingly demonstrated that soil infectivity with A. suum and T. suis increases during the first 2-3 years after pasture contamination. Though all three helminth species stimulate the Th2 arm of the immune system, Oesophagostomum seems weakly immunogenic, perhaps via specific modulation of the host immune system. A. suum and T. suis potently modulate the host immune response, up-regulating Th2 and down-regulating Th1. As a consequence, A. suum may compromise the efficacy of certain bacterial vaccines, whereas T. suis, which establish only short-term in humans, is a favourite candidate for down-regulating autoimmune Th1-related diseases in man. Some basic research findings have offered new possibilities for future sustainable control measures. For example, the heredity of host resistance to A. suum and T. suis is so high that breeding for resistant pigs may be a possibility. Experimental studies have demonstrated that fermentable dietary carbohydrates have an antagonistic effect on Oesophagostomum and to a lesser extent on T. suis and A. suum, whereas egg-destroying microfungi may be used to inactivate the hard-shelled A. suum and T. suis eggs in the environment. Helminth control in Denmark has previously relied solely on anthelmintic treatment in herds with low helminth transmission. When indoor transmission rates increase, or in outdoor herds with high pasture contamination levels, medication may advantageously be combined with sustainable control measures, such as selected pig genomes, bioactive forages, and egg-destroying microfungi.

Microfluidic bioassay to characterize parasitic nematode phenotype and anthelmintic resistance.

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.

Construction of gender-enriched cDNA archives for adult Oesophagostomum dentatum by suppressive-subtractive hybridization and a microarray analysis of expressed sequence tags.

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.

Vertical migratory behavior of the infective third-stage larvae of Oesophagostomum dentatum.

The vertical migratory behavior of third-stage infective larvae (L3i) of Oesophagostomum dentatum was investigated using upright truncated agarose cones and equivalent conical depressions in agarose. Geotactic response varied with the age of the infective larvae. Four-day-old L3i showed no preference for the sloping surfaces of either indented or upright cones, while the 8-day-old L3i showed a positive geotactic reaction, migrating down the sloping surface of the depressions.

Survival and viability of Ascaris suum and Oesophagostomum dentatum in ensiled swine faeces.

The survival and viability of eggs from Ascaris suum and Oesophagostomum dentatum and of infective larvae (L3) from O. dentatum were determined in the ensiled solid fraction of swine faeces after 0, 7, 14, 28 and 56 days of ensiling. The experiment had two treatments, un-ensiled and ensiled manure, in a split-plot design. Each of 50 containers was inoculated with 40,000 eggs of both A. suum and O. dentatum, and another 50 containers were inoculated with 32,747 L3 of O. dentatum each. A. suum eggs were not destroyed by the ensiling process, although their viability was diminished. O. dentatum eggs and larvae were destroyed during the first 7-14 days of the ensiling process.

Human oesophagostomiasis: a histomorphometric study of 13 new cases in northern Ghana.

Oesophagostomiasis is an infrequently described and recognised parasitic infection in humans, caused by Oesophagostomum bifurcum. Although the disease is most often found in the northern part of Togo and the neighbouring part of Ghana, sporadic cases have been described in other parts of Africa and in Asia and South America: Uganda, Ivory Coast, Sudan, Kenya, Ethiopia, Indonesia, Malaysia and Brazil. Infection probably occurs by way of the ingestion of L3 larvae. These larvae penetrate the intestinal wall, especially that of the colon. Some of these larvae develop into young adult worms and return to the bowel lumen. Other larvae, however, develop into immature worms, which fail to settle in the lumen, forming abscesses in the bowel wall and causing pathology. In the literature 105 human cases have been described, many originating in the northern regions of Ghana and Togo. The present study was performed to evaluate 13 new cases originating in the northern part of Ghana (7 female and 6 male patients, aged between 2 and 60 years). Histopathologically, the patients could be divided into two groups: the first group showed multinodular disease, while patients in the second group presented with a single, nodular mass. In the first group, abscesses were seen throughout the colonic wall. The mean size of the cavities was 4.3+/-0.7 mm. There was no relation between the size and the localisation in the colonic wall. Abscesses were significantly larger in male patients than in female patients. There was no correlation with age. In the second group, histopathological examination showed a cyst of variable wall thickness with very limited inflammation. These cysts represented older lesions, often encapsulated in the mesentery. In conclusion, in this study we present 13 new cases of human oesophagostomiasis. The abscess formation was found to be organ specific, independent of age, and gender-related, producing a more intense tissue reaction in male patients.

Concurrent Ascaris suum and Oesophagostomum dentatum infections in pigs.

The aim of this study was to examine interactions between Ascaris suum and Oesophagostomum dentatum infections in pigs with regard to population dynamics of the worms such as recovery, location and length; and host reactions such as weight gain, pathological changes in the liver and immune response. Seventy-two helminth-naïve pigs were allocated into four groups. Group A was inoculated twice weekly with 10000 O. dentatum larvae for 8 weeks and subsequently challenge-infected with 1000 A. suum eggs, while Group B was infected with only 1000 A. suum eggs; Group C was inoculated twice weekly with 500 A. suum eggs for 8 weeks and subsequently challenge-infected with 5000 O. dentatum larvae, whereas Group D was given only 5000 O. dentatum larvae. All trickle infections continued until slaughter. Twelve pigs from Group A and B were slaughtered 10 days post challenge infection (p.c.i.) and the remaining 12 pigs from the each of the four groups were slaughtered 28 days p.c.i.. No clinical signs of parasitism were observed. The total worm burdens and the distributions of the challenge infection species were not influenced by previous primary trickle-infections with the heterologous species. Until day 10 p.c.i. the ELISA response between A. suum antigen and sera from the O. dentatum trickle infected pigs (Group A) pigs were significantly higher compared to the uninfected Group B. This was correlated with a significantly higher number of white spots on the liver surface both on Day 10 and 28 p.c.i. in Group A compared to Group B. The mean length of the adult O. dentatum worms was significantly reduced in the A. suum trickle infected group compared to the control group. These results indicate low level of interaction between the two parasite species investigated.

Development and survival of infective larvae of gastrointestinal nematodes of cattle on pasture in central Kenya.

On a series of pasture plots, 2 kg pats of bovine faeces containing known numbers of strongylid (Haemonchus, Cooperia, Oesophagostomum and Trichostrongylus) eggs were deposited at intervals of 4 weeks from July 1995 to June 1996. The plots were sampled every 2 weeks after contamination and infective larvae were identified and counted. Larvae of all the genera developed throughout the year, but the pats exposed during the rainy season yielded more abundant larvae on the herbage. Irrespective of the season of deposition of the pats, larvae were found in larger numbers from 2 to 6 weeks after deposition and generally declined to below detectable levels within 12 to 16 weeks of contamination. The comparatively short survival times noted in this experiment may present opportunities for manipulation of the population dynamics of the gastrointestinal nematodes in the tropical environment of Kenya.

Oesophagostomum spp. in pigs: resistance to reinfection.

For a study on the occurrence of resistance to reinfection with porcine nodular worm species, pigs were infected twice weekly with 1,000 infective larvae (L3) of Oesophagostomum quadrispinulatum for 8 weeks. All pigs, including noninfected controls, were then treated with fenbendazole. At 10 days after treatment, all pigs received a single challenge inoculation of 5,000 L3 of either O. dentatum or O. quadrispinulatum, respectively. Pigs were slaughtered at 6 weeks after the challenge infection for determination of their worm burdens. The pigs trickle- and challenge-infected with O. quadrispinulatum had significantly lower egg excretion levels (P < 0.01) and worm burdens (P < 0.05) than challenge control pigs, thus indicating some degree of host immunity against the homologous challenge infection. No resistance to reinfection was evident for the heterologous challenge infection. This study elucidates further aspects of the interaction between nodular worm species in the pig.

Anthelmintics and ion-channels: after a puncture, use a patch.

Two of three major types of anthelminitic, the avermectins and the nicotinic agonists, exert their therapeutic effect by an action on ligand-gated membrane ion-channels of nematodes. The avermectins, such as ivermectin, open glutamategated chloride channels which have so far been found only in invertebrate preparations; nicotinic anthelmintics, like levamisole, selectively gate nematode nicotinic acetylcholine receptors. We describe recent advances in the knowledge of the molecular structure of these ion-channel receptors in nematodes. Because opening of the ion-channels by these two groups of anthelmintic generates currents across cell membranes of nematodes, we can use electrophysiological methods to examine properties of the channels, the mode of action of the anthelmintics, and changes in the receptors associated with anthelmintic resistance. We illustrate some of our observations on these receptors using a two micro-electrode current-clamp technique to monitor membrane resistance (the puncture); and then some observations using The patch-clamp technique to monitor currents through individual ion-channels (the patch). The receptors for the two major groups of anthelmintics may not be homogeneous. Even in a single membrane patch from one muscle cell, nematode nicotinic acetylcholine receptors show evidence of heterogeneity and the avermectins may have multiple sites-of-action. If separate independent recessive genes are involved in production of different receptor subtypes, and if each subtype has to change to allow the development of resistance by the whole nematode, then the probability of resistance developing would be smaller than for anthelminitics with a single site-of-action. The MISER (multiple independent sites-of-action evading resistance) concept favours the development and use of anthelminitics with more than one site-of-action.

[Migration and transposition of Oesophagostomum quadrispinulatum larvae from feces]. / Untersuchungen zur Migration und Translation von Oesophagostomum-quadrispinulatum-Larven aus dem Kot.

Investigations on the migration and translation of free-living stages of Oesophagostomum quadrispinulatum, using faeces containing eggs as starting material, revealed that mostly third stage larvae and very few second stage larvae migrated out of faeces, whereas first stage larvae remained in the faeces. The emigration rates depended on ambient relative humidity. Compared with the control, third stage larvae emigrated out of faeces at rates of 0.3%, 1.6% and 12.2% at 50%, 75% and 100% relative humidity, respectively. Offering helminth-free faeces, emigrated third stage larvae returned into faces at rates of 0.4-1.2%, 5.8-17.7%, 39.0-52.7%, and 45.2-60.7% after 1 h, 24 h, 5 days and 14 days, respectively. After a period of 1, 2, 3 or 4 weeks staying out of faeces, emigrated third stage larvae returned into faeces at rates of 23.3%, 8.8%, 22.1% and 6.0%, respectively. An examination of the horizontal translation revealed that most of the third stage larvae migrated distances up to 80 cm and a few even up to 150 cm returning into helminth-free faeces.

Effect of time on migration of Oesophagostomum spp. and Hyostrongylus rubidus out of agar-gel.

The agar-gel migration technique has previously been described, however, aspects regarding the effect of timing on worm migration needed further scrutiny. In the first experiment, pigs inoculated with Oesophagostomum dentatum were slaughtered simultaneously and their intestines stored at 21-23 degrees C until processed pairwise 2, 4, 6, 8, 12 and 18 h after slaughter. More than 95% of the worms migrated out of the agar if processed within 6 h. In the second experiment, intestines were treated immediately after slaughter and the migratory speed of adult worms or 4th-stage larvae of O. dentatum or O. quadrispinulatum, or adult Hyostrongylus rubidus were studied. For both Oesophagostomum species, more than 90% of the worms were recovered within 1 h. H. rubidus was significantly slower; however, approximately 98% of the worms had migrated out of the agar-gel by 20 h. This information is essential in planning experiments where recovery of live worms is of value.

Seasonal infection pattern of gastrointestinal nematodes of beef cattle in Minas Gerais State--Brazil.

Tracer calves were used to assess the seasonality of infections of gastrointestinal parasites in beef cattle extensively raised at a farm in the State of Minas Gerais, Brazil. Tracer calves acquired infections during all months of the year, however, highest worm burdens were observed in the rainy season. The following nematode species were recovered from tracer calves: Cooperia punctata, C. spatulata, C. pectinata, Haemonchus similis, H. placei, Oesophagostomum radiatum, Trichostrongylus colubriformis, T. axei, Bunostomum phlebotomum and Trichuris discolor. Cooperia was most prevalent, representing 74.4% of the total of all nematodes recovered. This was followed by Haemonchus 19.2%, Oesophagostomum 4.5%, Trichostrongylus, Trichuris and Bunostomum, represented less than 1% of the total. Thirty Nellore cows with calves were used to evaluate the dynamics of the infections. The cows were the principal source of contamination to pasture and to infection of suckling calves during the pre-weaning period, and probably, also contributed to increased infection of weaned calves at the beginning of the rainy season. Strongyloides was the first infection observed in the calves. The peak egg count was observed in the 2nd month, however, all Strongyloides egg counts became negative in the 7th month. Strongylate nematode egg counts were low in calves from the 3rd month of age onward, and increased gradually until calves were 1 year old. A gradual reduction then occurred until the end of the trial. From the 3rd month onward Cooperia and Haemonchus infective larvae (L3) were most prevalent in fecal cultures. Fecal cultures after the 4th month also resulted in recoveries of Oesophagostomum, Trichostrongylus, and Bunostomum L3. Cooperia was the predominant genus in fecal cultures until the calves were 9 months old. After the calves were 9 and 12 months of age, L3 of Cooperia and Oesophagostomum, respectively decreased in prevalence. Trichostrongylus and Bunostomum L3 were only minimally present in the fecal cultures at this time.

Experimental hybridization between Oesophagostomum dentatum and O. quadrispinulatum in pigs.

To investigate eventual hybridization between two nodular worm species of pigs, Oesophagostomum dentatum and O. quadrispinulatum, we used either mature, adult worms or 10-day-old fourth-stage larvae (L4) as starting material, employing a nonsurgical transplantation technique. Following the transfer of adult worms the ensuing first generation of larvae gave rise to adult worms that were found by morphological examination to be purely O. dentatum. Therefore, we decided to use the immature L4 as starting material. After the transfer of L4 to recipient pigs, fecal cultures were established and the L3 derived from the O. dentatum male O. quadrispinulatum female cross gave rise to adult but infertile worms, which morphologically had the sexual characters of their parent generation, whereas other characteristics were intermediate between the two species. Attempts to reproduce the hybrid worms or the reciprocal cross were unsuccessful, indicating that hybridization between the two species is a rarely occurring phenomenon.