Isolation and characterisation of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR.

In light of the lack of molecular data on the sexual differentiation, maturation and interaction of parasitic nematodes of livestock, the present study investigated sex-specific gene expression in the nodule worm, Oesophagostomum dentatum (Strongylida). Using the technique of RNA arbitrarily-primed polymerase chain reaction (RAP-PCR), 31 expressed sequence tags (ESTs) differentially-displayed between the sexes were cloned. Northern blot analysis proved ten ESTs to be expressed exclusively in males (adults and fourth-stage larvae), while two were expressed solely in female stages. None of the ESTs were expressed in infective third-stage larvae. Sequence analysis and subsequent database searches revealed two male-specific ESTs to have significant similarity to Caenorhabditis elegans (predicted) proteins, a protein containing an EGF-like cysteine motif and a serine/threonine phosphatase. Another two male-specific ESTs had similarity to non-nematode sequences. The two female-specific ESTs had similarity to vitellogenin-5 and endonuclease III (predicted) from C. elegans. The remaining ESTs had no similarity to any nucleic acid or protein sequences contained in the databases. The isolation and characterisation of sex-specific ESTs from O. dentatum provides a unique opportunity for studying the reproductive biology of parasitic nematodes at the molecular level, with a view toward novel approaches for parasite control.

A mutation scanning approach for the identification of hookworm species and analysis of population variation.

To overcome limitations in the morphological identification of different developmental stages of hookworms to species, we have established a polymerase chain reaction-linked single strand conformation polymorphism technique (PCR-SSCP) utilizing the internal transcribed spacers (ITS) of ribosomal (r)DNA. These spacers were specifically chosen because they provide reliable species markers for strongylid nematodes. ITS spacers were amplified by PCR from DNA derived from individual parasites of seven species of hookworm, then denatured and subjected to electrophoresis in a mutation detection enhancement (MDE) (non-denaturing) gel matrix. PCR SSCP analysis showed that the single-strand ITS patterns produced allowed the unequivocal identification of all species. The method also allowed the direct display of sequence variation within some species where multiple individual worms were examined. These findings demonstrate the usefulness of the SSCP approach for hookworm identification, the detection of population variation and the direct display of sequence variation in rDNA.

Parasitic helminths of the pig: factors influencing transmission and infection levels.

The occurrence of parasitic helminth species as well as infection intensities are markedly influenced by the type of swine production system used. The present review focusses mainly on the situation in temperate climate regions. Generally, over the past decades there has been a decrease in the number of worm species and worm loads in domestic pigs due to a gradual change from traditional to modern, intensive production systems. The reasons for some species being apparently more influenced by management changes than others are differences in the basic biological requirements of the pre-infective developmental stages, together with differences in transmission characteristics and immunogenicity of the different worm species. Control methods relevant for the different production systems are discussed. Outdoor rearing and organic pig production may in the future be confronted with serious problems because of particularly favourable conditions for helminth transmission. In addition, in organic farms preventive usage of anthelmintics is not permitted.

The effect of fasting on Ascaris suum and Oesophagostomum spp. in growing pigs.

Experiments were conducted to study the possible influence of fasting on Ascaris suum and Oesophagostomum spp. In growing pigs. Forty young crossbred pigs naturally infected with A. suum and Oesophagostomum spp. were used. In one experiment 10 pigs were fasted and offered water ad libitum for 6 days, in another experiment for 10 days. Subsequently, these pigs, together with 10 non-fasted control pigs per experiment were slaughtered, and worm numbers, worm location, sex, developmental stage and female worm fecundity were determined. Pigs fasted for 10 but not for 6 days had decreased numbers of A. suum and Oesophagostomum spp. at slaughter vs controls, and worms were found in more distal locations in the gastrointestinal tract. Fasting for both 6 and 10 days significantly lowered the fecundity of both worm species.

In vitro characterization of anthelmintic susceptibility of field isolates of the pig nodular worm Oesophagostomum spp., susceptible or resistant to various anthelmintics.

A larval development assay (LDA) and an egg hatch paralysis assay (EHPA) were used to measure the sensitivity to anthelmintics of eggs and larvae of nodular worms (Oesophagostomum spp.) in pigs. The tests were carried out using in vivo defined resistant and susceptible isolates of Oesophagostomum dentatum, O. quadrispinulatum and Oesophagostomum spp. For measurement of pyrantel/morantel and levamisole sensitivity the LDA was found able to distinguish between susceptible or resistant isolates of Oesophagostomum. The EHPA was able to detect levamisole resistance, but the test failed to show differences in response to pyrantel between pyrantel susceptible and resistant lines. The possible routine application of LDA and EHPA in the diagnosis of anthelmintic resistance in Oesophagostomum spp. is discussed.

False-positive Ascaris suum egg counts in pigs.

False-positive Ascaris suum egg counts in pig faeces are frequently observed under both experimental and natural conditions. Data from 12 experiments with A. suum infections in pigs were summarized and showed that the percentage of false-positive faecal samples ranged from 4 to 36%. False-positive egg count values varied greatly between pigs and experiments (range 20-1060 eggs per gram faeces). Indoor experiments with pigs housed groupwise in pens generally produced more and higher false-positive egg counts, which may reflect differences in surface area and hence exposure to infective eggs, compared with pasture experiments. The positive predictive value (the number of pigs diagnosed positive by faecal sample that actually harboured worms) was low for indoor experiments (45%) compared with pasture experiments (89%). Differences in design for indoor experiments, such as floor type and use of bedding material, did not influence the positive predictive value (44-47%). A positive correlation was found (r = 0.56, P < 0.05) between faecal egg counts of true-positive and false-positive pigs that were penned together. The results of this survey strongly support previous suggestions that false-positive A. suum egg counts in pigs are the result of coprophagia in indoor experiments and coprophagia/geophagia in pasture experiments. False-positive A. suum egg counts in pig faeces may vary greatly in prevalence and magnitude, and depend in part on management/housing factors.

Visceral larva migrans: migratory pattern of Toxocara canis in pigs.

The migratory pattern of Toxocara canis was investigated following infection of pigs with 60000 infective eggs. Groups of six pigs were slaughtered at 7, 14 and 28 days after infection (p.i.), and the number of larvae in selected organs and muscles was determined by digestion. A group of uninfected pigs was used as negative controls for blood parameters and weight gain. Toxocara canis migrated well in the pig, although the relative numbers of larvae recovered decreased significantly during the experiment. On day 7 p.i., high numbers of larvae were recovered from the lymph nodes around the small intestine and to some extent also from the lymph nodes around the large intestine, and from the lungs and the liver. On day 14, the majority of larvae were recovered from the lungs and the lymph nodes around the small intestine, and by day 28 p.i. most larvae were found in the lungs. Larvae were recovered from the brain on days 14 and 21, with a maximum on day 14 p.i. No larvae were found in the eyes. Severe pathological changes were observed in the liver and lungs, especially on day 14 p.i.; also, development of granulomas was observed in the kidneys. Finally, a strong specific antibody response towards T. canis L2/L3 ES products was observed from day 14 p.i. until termination of the experiment, and the maximum eosinophil response was observed 14 days p.i. The pig is a useful non-primate model for human visceral larva migrans, since T. canis migrate well and induce a strong immunological response in the pig. However, the importance of the pig as a paratenic host is probably minor, because of the relatively early death of most of the larvae.

In vitro characterization of lines of Oesophagostomum dentatum selected or not selected for resistance to pyrantel, levamisole and ivermectin.

Lines of Oesophagostomum dentatum artificially selected or not selected for resistance to pyrantel, levamisole and ivermectin were used in this study. From the 10th generation of selection eggs were collected from each line and subjected to an in vitro larval development assay (LDA) and an egg hatch assay (EHPA). Significant differences were observed between an unselected line of O. dentatum and the lines selected for resistance to levamisole or pyrantel in both assays. The LDA was more sensitive than EHPA in detecting anthelmintic resistance in O. dentatum. The results obtained from the LDA confirmed side-resistance between levamisole and morantel/pyrantel. The in vitro tests failed to show significant differences between ivermectin-sensitive and resistant lines.

The efficacy of ivermectin against nodular worms of pigs: the response to treatment using three different dose levels against Oesophagostomum dentatum and Oesophagostomum quadrispinulatum.

Anthelmintic efficacies of 3 different doses of ivermectin (IVM) were evaluated in 3 isolates of nodular worms in pigs. An isolate of Oesophagostomum quadrispinulatum (OQ) was recently obtained from a commercial farm where poor efficacy of IVM at the recommended dose (300 micrograms.kg-1 body weight) was detected. On this farm, IVM had been used for treatment of sows twice yearly for 6 years. Two other isolates, an O. dentatum (OD) and a mixed Oesophagostomum dentatum and Oesophagostomum quadrispinulatum isolate (ODQ) were obtained from a farm where anthelmintics had never been used. Efficacies of IVM against adult worms of the OQ-isolate at dose rates of 150, 300 and 600 micrograms.kg-1 body weight ranged from 40.5-78.6%. Efficacies against larval stages (L3 and L4) were superior. Efficacies against the OD-isolate were 88.7, 96.1 and 99.6%, respectively. In the ODQ-isolate the efficacies of IVM against adult stages furnished similar results. In conclusion, the efficacy of IVM against O. dentatum was high but against both isolates of O. quadrispinulatum poorer. This suggests that IVM is intrinsically less effective against O. quadrispinulatum and therefore not indicative of acquisition of anthelmintic resistance in the OQ-isolate.

Pigs become infected after ingestion of livers and lungs from chickens infected with Ascaris of pig origin.

An experimental infection with Ascaris of pig origin showed that Ascaris suum larvae can migrate extra-intestinally in chickens. Furthermore, after feeding piglets with Ascaris infected chicken liver and lungs, it was possible to recover larvae from their lungs. These observations suggest that the chicken could serve as a paratenic host for Ascaris. There is also the possibility for zoonotic transmission if raw chicken livers are consumed by humans.

The influence of high- and low-fibre diets on the activity of piperazine against Oesophagostomum spp. in pigs.

An in vivo study evaluated the effect of diet on the efficacy of piperazine against nodular worms of pigs. Twenty pigs, later allocated into five groups, were each infected (and 37 days later re-infected) with 3000 infective larvae of a mixed isolate of Oesophagostomum dentatum and Oesophagostomum quadrispinulatum. Beginning on day 23 post infection (p.i.), pigs in groups 1 and 2 were fed a low-fibre diet consisting of 70% barley flour and 30% protein concentrate, while pigs in groups 3, 4 and 5 were fed a high-fibre diet consisting of 55% barley flour, 21% oat-husk meal and 24% protein concentrate. On day 42 p.i., pigs in groups 1 and 3 were orally dosed with 200 mg piperazine dihydrochloride (Ascarex D, 53%) per kg bodyweight, the recommended dose, while pigs in group 4 were given 100 mg kg-1. Groups 2 and 5 served as non-treated controls for the respective dietary regimens. Eight days after treatment, the pigs were slaughtered and worms recovered from the caecum and large intestine (divided into five sections) and counted. The mean worm count reduction (WCR) in group 1 (full-dose piperazine with low-fibre diet) was 89.8%, while the high-fibre diet in group 3 increased the WCR to 99.4%. In group 4, where the pigs were fed the high-fibre diet and treated with only 100 mg piperazine kg-1, the WCR was 90.9%, identical to the "low fibre" group 1 treated with twice this piperazine dose. There was a zero efficacy recorded against immature worms in all three treated groups. The high-fibre diet improved the efficacy of piperazine against more pathogenic and generally more tolerant O. quadrispinulatum to 99.2% compared with 84.3% at the low-fibre diet.

Comparison of methods for recovery of Ascaris suum larvae from tissues of mice.

Experiments were undertaken to compare procedures for isolating Ascaris suum from mice. A standardised intragastric procedure for inoculation of A. suum eggs, which had a very low interdose variation (S.D. = 6.2% of mean inoculation dose), was developed. There was no significant difference in the number of larvae recovered from the large intestine 4 h post inoculation (p.i.) by using either Baermannisation of washed intestinal wall or submucosal scrapings. In contrast, a significantly higher recovery of larvae was achieved by incubating the intestines vertically in a cylindrical saline-filled funnel. An agar-gel method was used to recover larvae from mucosal scrapings; however, the number of larvae recovered was the lowest of the methods tried. It was found that a significant increase in larval recovery from the liver at 24 h p.i. resulted when the liver was pressed through a garlic press, rather than by using homogenisation. For lung recovery, the highest recovery of larvae (at day 8 p.i.) was obtained by disintegration of tissue with a pair of scissors and incorporating the tissue into agar-gel. The methods presented in this study may be useful for investigation of the histology, morphology and molecular biology of the early of A. suum.

An in vivo dose-response study of fenbendazole against Oesophagostomum dentatum and Oesophagostomum quadrispinulatum in pigs.

A dose-response study using fenbendazole (FBZ) was carried out in pigs infected with O. dentatum and O. quadrispinulatum to determine the minimum effective dose rate of the drug. Thirty pigs were randomly divided into 6 groups of 5 pigs and infected with 5000 infective larvae each. The animals were re-infected 5 days before treatment (Day 30 after the first infection) with the same number of larvae. On Day 35 the pigs in groups 1-5 were treated with FBZ at the following dose rates: 2.5 mg kg-1 (i.e. 50% of the registered dose level), 1.0 mg kg-1 (20%), 0.25 mg kg-1 (5%), 0.1 mg kg-1 (2%) and 0.05 mg kg-1 (1%), respectively. Pigs in group 6 served as non-treated controls. Seven days after treatment (Day 42 after infection) the pigs were slaughtered, worms recovered from the large intestine and counted. The species and sex of adult worms was determined. A high faecal egg count reduction (FECR) after treatment was observed in groups 1, 2 and 3 (98%, 88% and 91%, respectively), while in groups 4 and 5 the egg counts were not affected by treatment. The mean worm count reduction was high in groups 1, 2 and 3 (100%, 99.9% and 98.6%, respectively), but declined in groups 4 and 5 (77% and 40%, respectively). FBZ showed a high efficacy against immature worms in groups 1 and 2, while in groups 3, 4 and 5 counts were not reduced. Species differentiation revealed a higher effect of FBZ against O. dentatum than against O. quadrispinulatum. Sex differentiation indicated a slightly higher (not significant) efficacy against females than males in both species. This study demonstrated a high efficacy of FBZ against the nodular worms in pigs, even at 5% of the currently registered dose level.

Experimental Oesophagostomum dentatum infection in the pig: worm populations resulting from single infections with three doses of larvae.

This report describes the effect of different dose levels of infection upon worm burdens and development and fecundity of the parasites. Three groups each of 40, 9-week-old, helminth naive pigs were inoculated once with either 2000 (group A), 20,000 (group B), or 200,000 (group C) infective third stage larvae of Oesophagostomum dentatum. Subgroups of 5 pigs from each major group were killed 3, 6, 11, 14, 18, 25, 34 and 47 days post inoculation (p.i.) and the large intestinal worm burdens were determined. Faecal egg counts were determined at frequent intervals after day 13 p.i. There were no overt clinical signs of gastrointestinal helminthosis during the experiment. Faecal egg counts became positive in groups A and B at around day 19 p.i., whereas most pigs in the high dose group C did not have positive egg counts until day 27-33 p.i. and some pigs remained with zero egg counts until the end of the study. Throughout the experiment the worm populations in group C consisted mainly of immature larval stages, while those in groups A and B were predominantly adult stages after days 14-18. Adult worms from the low dose group A were significantly longer than those from group C. At high population densities, stunted development of worms and reduced fecundity among female worms were found. Furthermore, there was a tendency for the distribution of the worms within the intestine to be altered with increasing population size.

The preventive effect of the fungus Duddingtonia flagrans on trichostrongyle infections of lambs on pasture.

Four groups of 8 parasite-naive Dorset-crossbred lambs, 3-4 months old, were turned out on infected pasture on 2 May and allocated to 4 separate paddocks. From May to September, 2 groups received Duddingtonia flagrans (10(6) chlamydospores per kg body weight per lamb per day) mixed in 100 g of barley, while the other 2 groups received barley only. All groups remained set-stocked until slaughter for worm counts on 10 October. In late June, all lambs were treated with fenbendazole due to severe parasitic gastroenteritis in all groups. The faecal egg counts were comparable for the 2 treatments throughout the grazing period. Larval development of Ostertagia/Trichostrongylus spp. in faecal cultures was 1-28% in the fungi-fed groups compared with 60-80% in the untreated groups (P < 0.05). In September, pasture larval counts of Ostertagia/Trichostrongylus were 930 and 4400 L3 kg-1 on paddocks of fungi-fed and untreated groups, respectively. Corresponding figures for Nematodirus spp. were 7200 and 11600 L3 kg-1, respectively. At slaughter, the number of immature Ostertagia spp. was 62% lower in the fungi-fed groups compared with the untreated groups (P < 0.05). Four parasite-free lambs were introduced to each paddock during the period 3-23 October and slaughtered for worm counts after 3 weeks of housing. The total worm burden of tracers on paddocks previously grazed by fungi-fed lambs was reduced 86% (P < 0.05; geometric means) compared with control groups, while significant reductions were also seen in abomasal worm counts (68%; P < 0.05), N. spathiger (98%; P < 0.05) and for N. battus (97%; P < 0.01). It is concluded that dosing sheep with D. flagrans while grazing may limit the build up of pasture contamination in the late grazing season and subsequently limit the intake of larvae in sheep.

Experimental Oesophagostomum dentatum infections in the pig: worm populations resulting from trickle infections with three dose levels of larvae.

This paper describes the worm populations in pigs experimentally infected by trickle infections with different dose levels of the nodular worm, Oesophagostomum dentatum. Four groups each of 20 helminth naïve pigs, 10-12 weeks old, were inoculated with 0 (group 1), 100 (group 2), 1000 (group 3), or 10,000 (group 4) infective larvae twice weekly, and the pigs were killed after 10-13 weeks. No overt clinical signs were observed, and only group 4 had slightly lower food conversion rate (P < 0.05) than the controls. Faecal egg counts revealed that the nodular worms in pigs of groups 2 and 3 had a short prepatent period (3-4 1/2 weeks) and a fairly stable egg output, while the worms in the pigs of group 4 had prepatent periods of 3-10 weeks and low, unstable egg excretion. The mean worm burdens increased with the dose rate (group 2: 929 worms; group 3: 7467 worms; group 4: 19,847 worms), but detailed analyses of the worm populations from 10 pigs from each of the infected groups revealed a clear dose-dependency in worm recovery, percentage adult worms, worm lengths and female fecundity, as all these measures declined significantly with increasing dose level. The adult worms seemed to be shorter and less fertile when they were located posteriorly to their predilection site, and especially in group 4 many stunted infertile adults measuring only 2-5 mm were found in the posterior half of the colon, but there were no indications of worm expulsion. Superimposed on the main experiment was a cohort study in which 4 pigs of group 3 were given a single dose of 1000 pyrantel resistant larvae at day 56 (all other larvae were pyrantel sensitive), treated with 28 mg pyrantel per kg body weight at day 85 and killed at day 90. Appropriate control groups were included. The mean establishment of the cohort was similar to previously uninfected controls, but between-animal variation was much higher in the trickle infected group.

Evaluation of the infectivity of Trichinella spp. for reptiles (Caiman sclerops).

Experimental inoculation with nine well-characterised Trichinella isolates was performed on caimans (Caiman sclerops) to determine their infectivity for reptiles belonging to the family Crocodilidae. As controls, the same larval batches of Trichinella isolates were inoculated into mice and guinea pigs. It was suggested that Trichinella pseudospiralis was more likely to infect reptiles than encapsulating species, but whereas all Trichinella species established in mice and guinea pigs, the caimans remained negative. The finding that caimans could not be experimentally infected contrasts with a recent report on infections in farmed crocodiles (Crocodylus niloticus).

Necator americanus (Nematoda: Ancylostomatidae) from Africa and Malaysia have different ITS-2 rDNA sequences.

The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.

The effect of experimental trichostrongyle infections of housed young calves on the subsequent course of natural infection on pasture.

The present studies were designed to investigate whether experimental, mixed trichostrongyle infections of stabled calves prior to their first grazing season could confer sufficient immunity to significantly reduce egg excretion after turnout, and thereby prevent loss-producing infections later on. The study comprised four groups, each of seven calves. During spring, two of the groups received two different dose levels of infective larvae twice weekly, and one group received larger larval doses at monthly intervals. One group served as non-experimentally exposed controls. In May all groups grazed separately on similarly contaminated plots. All experimental groups had reduced egg counts, and herbage infectivity of their plots was significantly lower than that of the controls. These findings were reflected in serum pepsinogen levels and in specific serum antibody responses. Additionally, clinical disease was only observed in the control group animals.

Schistosoma japonicum in the pig: the host-parasite relationship as influenced by the intensity and duration of experimental infection.

Parasitologic, clinicopathologic, and pathologic aspects of Schistosoma japonicum infections of varying durations and intensities were studied in growing pigs injected intramuscularly with a dose of either 0, 100, 500, or 2,000 cercariae and killed at 4, 11, 17, or 24 weeks postinfection (PI). The number of viable worm pairs decreased significantly in the high dose group after 11 weeks PI but not in the lower dose groups; however, a stable population of immature worms persisted throughout the study in all dose groups. Liver egg counts also tended to decrease in the high dose group after 11 weeks but not in the other groups. Fecal egg excretion began at six weeks PI, was highest at eight weeks PI with a pronounced peak occurring only in the high dose group, and then decreased to low levels by 14 weeks PI in all groups. Egg counts from the feces as well as the liver correlated strongly with worm pair numbers during the acute phase of infection. The only clinicopathologic abnormality observed was an increase in circulating eosinophils corresponding to cercarial dose in all infected pigs by week six with peak counts occurring between six and eight weeks PI. The pigs exhibited no clinical signs of disease aside from diarrhea at the onset of patency. However, lesions were present throughout the large intestine of all infected pigs from 11 weeks PI, gradually decreasing with time. Severe liver fibrosis occurred in the 500 and 2,000 dose groups mainly at 11 weeks PI and then decreased in severity. In the liver, but not in the intestine, the severity of lesions at all time points was proportional to the cercarial dose given. The results indicate that after several weeks of patency, pigs with high intensities of S. japonicum infection are able to effectively eliminate the majority of adult worms while maintaining a stable population of immature schistosomes.