Temperature dependent embryonic development of Trichuris suis eggs in a medicinal raw material.

The therapeutic potential of infective pig whipworm eggs, Trichuris suis ova (TSO), is currently tested in several clinical trials on immune-mediated diseases. This paper studied the embryonic development of TSO in a medicinal raw product, where the parasite eggs were suspended in sulphuric acid (pH1). Unembryonated T. suis egg batches were stored at 5, 10, 15, 20, 25, 30, and 40°C (±1°C) and examined at 2, 4, 8, and 14 weeks. Subsequently, sub-batches from each temperature were allowed to embryonate for additional 14 weeks at 25°C, and selected samples were tested for infectivity in Göttingen minipigs. Both male and female pigs were used to evaluate eventual gender specific infectivity. Storage at 30°C up to 14 weeks and subsequent embryonation for 14 weeks at 25°C did not significantly reduce the overall larval establishment in minipigs, as compared to storage at 5°C and subsequent embryonation at 25°C. As marked impairment of egg development was observed during storage at 40°C, a second set of unembryonated egg batches were incubated at 30, 32, 34, 36, 38, and 40°C (±1°C) for 1-8 weeks. The development of the eggs was repeatedly examined by manual light microscopy, multispectral analysis (OvaSpec), and an egg hatching assay prior to the final testing in minipigs (Trial 1). These methods showed that the development started earlier at higher temperatures, but the long-term storage at higher temperature affected the egg development. The present study further documents tolerance of the TSO to storage at temperature 5-15°C, at which temperature development of larvae is not initiated.

In vitro hatching of Trichuris suis eggs.

Eggs of the pig whipworm, Trichuris suis ova (TSO), are currently tested in human clinical trials for their potential immunomodulatory capacity. The biological potency of TSO (egg viability and infectivity) is traditionally assessed in Göttingen minipigs as the establishment of intestinal larvae after inoculation with a known number of eggs. To minimize testing in animal models, development of an in vitro egg hatching assay is proposed as a reliable, cost-effective, and a faster alternative to test the egg viability. The present study aimed to investigate the influence of different chemical, physical, and biological factors on egg hatching. Thus, in a series of experiments and in different combinations, the eggs were stimulated with glass beads, artificial gastric juice, bile salt and trypsin solution, fermentation gut medium, or stimulated with mucosal scrapings from the ileum and the large intestine of the infected and uninfected Göttingen minipig. Mechanical stimulation with glass beads presented a simple and reproducible method for egg hatching. However, incubation of eggs with mucosal scrapings from the ileum, caecum, and colon for 24 h at 38 °C significantly increased hatching.

The jejunal cellular responses in chickens infected with a single dose of Ascaridia galli eggs.

This histopathological study was carried out in order to investigate the cellular response in the jejunum to Ascaridia galli during the first 7 weeks of infection. Fourty-two ISA Brown chickens (7 weeks old) were infected orally with 500 embryonated A. galli eggs each while 28 chickens were left as uninfected controls. Six infected and four control chickens were necropsied at each time point 3, 7, 10, 14, 21, 28 and 42 days post-infection (dpi). Samples for histopathology were taken from three sites of the jejunoileum. Significantly higher eosinophil counts were seen in infected chickens compared to uninfected at 3, 7, 10, 14 and 28 dpi (P < 0.01). In both groups, the initial number of mast cells was high, but this high level of mast cells remained for a longer period in the infected group compared to the control group. Significantly higher counts were thus found in the infected group at 21 (P < 0.001), 28 (P < 0.01) and 42 dpi (P < 0.05). A. galli infection induced changes in the mucosal thickness as reduced villi length at 7, 10, 14, 21 and 28 dpi and in the degree of general cellular infiltration in the lamina propria of the mucosal layer. No adult worms were seen during the experiment; therefore, A. galli larvae have elicited a moderate cellular response in the lamina propria, mainly consisting of eosinophils in the early phase and later of mast cells.

Dose-dependent establishment of Trichuris suis larvae in Göttingen minipigs.

Embryonated eggs of the pig whipworm Trichuris suis (TSOee) constitute the active pharmaceutical ingredient (API) in a medicinal product explored in human clinical trials against several immune-mediated diseases. The measurement of TSO biological potency (hatchability and infectivity) is a requirement for the assessment of TSO's pharmacological potency in human clinical trials. The present study aims to validate the dose-dependent establishment of T. suis larvae in Göttingen minipigs and eventual clinical implication of a dose range (1000-10,000 TSO). Four groups of 5 minipigs were inoculated with doses of 1000, 2500, 7500, and 10,000 TSOee, respectively, to evaluate a range of concentrations of TSOee in a minipig infectivity model. Unembryonated eggs (TSOue) were added to keep the total egg number in the inoculum constant at 10,000 eggs. Two groups received 2500 and 7500 TSOee per pig without the addition of TSOue as controls. The intestinal larval establishment at 21 days post inoculation (dpi) demonstrated a clear positive linear dose-response relationship between numbers of inoculated TSOee and recovered larvae. There was a low level of variation in larval counts in all study groups. Thus, the infectivity model in minipigs within the tested dose range offers a reliable, sensitive and accurate assay for testing biological potency of TSO.

OvaSpec - A vision-based instrument for assessing concentration and developmental stage of Trichuris suis parasite egg suspensions.

BACKGROUND: OvaSpec is a new, fully automated, vision-based instrument for assessing the quantity (concentration) and quality (embryonation percentage) of Trichuris suis parasite eggs in liquid suspension. The eggs constitute the active pharmaceutical ingredient in a medicinal drug for the treatment of immune-mediated diseases such as Crohn׳s disease, ulcerative colitis, and multiple sclerosis. METHODS: This paper describes the development of an automated microscopy technology, including methodological challenges and design decisions of relevance for the future development of comparable vision-based instruments. Morphological properties are used to distinguish eggs from impurities and two features of the egg contents under brightfield and darkfield illumination are used in a statistical classification to distinguish eggs with undifferentiated contents (non-embryonated eggs) from eggs with fully developed larvae inside (embryonated eggs). RESULTS: For assessment of the instrument׳s performance, six egg suspensions of varying quality were used to generate a dataset of unseen images. Subsequently, annotation of the detected eggs and impurities revealed a high agreement with the manual, image-based assessments for both concentration and embryonation percentage (both error rates <1.0%). Similarly, a strong correlation was demonstrated in a final, blinded comparison with traditional microscopic assessments performed by an experienced laboratory technician. CONCLUSIONS: The present study demonstrates the applicability of computer vision in the production, analysis, and quality control of T. suis eggs used as an active pharmaceutical ingredient for the treatment of autoimmune diseases.

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.

Population dynamics of Ascaridia galli following single infection in young chickens.

The population dynamics of Ascaridia galli was studied in 70 ISA Brown layer pullets, 42 of them were each experimentally infected with 500 embryonated A. galli eggs and 28 chickens were kept as uninfected controls. Six chickens from the infected group and 4 from the control group were necropsied at 3, 7, 10, 14, 21, 28 and 42 days post-infection (d.p.i.). The mean worm recovery varied from 11-20% of the infection dose with the highest recovery at 3 d.p.i. and the lowest at 21 and 42 d.p.i. (P < 0·05). More larvae were recovered from the intestinal wall than from the content (P < 0·0001) and intestinal content larvae were longer than those from the wall (mean length 1·6 and 1 mm, respectively, P < 0·0001). Although larvae were growing over time, a population of small-sized larvae (length < 1 mm) was recovered at all d.p.i. During the first week of infection most of the larvae were located in the anterior half of the jejunoileum but they moved posteriorly with the age of infection. Thus, a subpopulation of larvae mainly in the lumen grew with time while another subpopulation remained small and associated with the mucosa. During the infection both subpopulations moved to a more posterior localization in the gastrointestinal (GI) tract.

Viability of Ascaris suum eggs in stored raw and separated liquid slurry.

Separation of pig slurry into solid and liquid fractions is gaining importance as a way to manage increasing volumes of slurry. In contrast to solid manure and slurry, little is known about pathogen survival in separated liquid slurry. The viability of Ascaris suum eggs, a conservative indicator of fecal pollution, and its association with ammonia was investigated in separated liquid slurry in comparison with raw slurry. For this purpose nylon bags with 6000 eggs each were placed in 1 litre bottles containing one of the two fractions for 308 days at 5 °C or 25 °C. Initial analysis of helminth eggs in the separated liquid slurry revealed 47 Ascaris eggs per gramme. At 25 °C, egg viability declined to zero with a similar trend in both raw slurry and the separated liquid slurry by day 308, a time when at 5 °C 88% and 42% of the eggs were still viable in separated liquid slurry and raw slurry, respectively. The poorer survival at 25 °C was correlated with high ammonia contents in the range of 7.9-22.4 mM in raw slurry and 7.3-23.2 mM in liquid slurry compared to 3.2-9.5 mM in raw slurry and 2.6-9.5 mM in liquid slurry stored at 5 °C. The study demonstrates that at 5 °C, A. suum eggs have a higher viability in separated liquid slurry as compared to raw slurry. The hygiene aspect of this needs to be further investigated when separated liquid slurry is used to fertilize pastures or crops.

Survival of weed seeds and animal parasites as affected by anaerobic digestion at meso- and thermophilic conditions.

Anaerobic digestion of residual materials from animals and crops offers an opportunity to simultaneously produce bioenergy and plant fertilizers at single farms and in farm communities where input substrate materials and resulting digested residues are shared among member farms. A surplus benefit from this practice may be the suppressing of propagules from harmful biological pests like weeds and animal pathogens (e.g. parasites). In the present work, batch experiments were performed, where survival of seeds of seven species of weeds and non-embryonated eggs of the large roundworm of pigs, Ascaris suum, was assessed under conditions similar to biogas plants managed at meso- (37°C) and thermophilic (55°C) conditions. Cattle manure was used as digestion substrate and experimental units were sampled destructively over time. Regarding weed seeds, the effect of thermophilic conditions (55°C) was very clear as complete mortality, irrespective of weed species, was reached after less than 2 days. At mesophilic conditions, seeds of Avena fatua, Sinapsis arvensis, Solidago canadensis had completely lost germination ability, while Brassica napus, Fallopia convolvulus and Amzinckia micrantha still maintained low levels (~1%) of germination ability after 1 week. Chenopodium album was the only weed species which survived 1 week at substantial levels (7%) although after 11 d germination ability was totally lost. Similarly, at 55°C, no Ascaris eggs survived more than 3h of incubation. Incubation at 37°C did not affect egg survival during the first 48 h and it took up to 10 days before total elimination was reached. In general, anaerobic digestion in biogas plants seems an efficient way (thermophilic more efficient than mesophilic) to treat organic farm wastes in a way that suppresses animal parasites and weeds so that the digestates can be applied without risking spread of these pests.

Ascaridia galli in chickens: intestinal localization and comparison of methods to isolate the larvae within the first week of infection.

This study was conducted to observe the localization and to compare methods for isolation of minute Ascaridia galli larvae in chicken intestine. Firstly, six 7-week-old layer pullets were orally infected with 2,000 embryonated A. galli eggs and necropsied either at 3, 5 or 7 days post infection (dpi). More than 95 % of the recovered larvae were obtained from the anterior half of the jejunoileum, suggesting this part as the initial predilection site for A. galli larvae. Secondly, the intestinal wall of one layer pullet infected with 20,000 A. galli eggs 3 days earlier was digested in pepsin-HCl for 90 min. The initial 10 min of digestion released 51 % of the totally recovered larvae and the last 30 min of continuous digestion yielded only 5 %. This indicates that the majority of larvae were located superficially in the intestinal mucosa. Thirdly, 48 7-week-old layer pullets were infected with 500 A. galli eggs and necropsied at 3 dpi to compare three different larval isolation methods from the intestinal wall, viz., EDTA incubation, agar-gel incubation and pepsin-HCl digestion, resulting in mean percentages of the recovered larvae: 14.4, 18.2 and 20.0 %, respectively (P = 0.15). As conclusion, we recommended Pepsin-HCl digestion as the method of choice for larval recovery from the intestinal wall in future population dynamics study due to high efficiency and quick and simple detection. The agar-gel method was considered to be a prerequisite for molecular and immunological investigations as the larvae were more active and fully intact.

Localization of Ascaridia galli larvae in the jejunum of chickens 3 days post infection.

The normal habitat of the parasitic stages of Ascaridia galli is in the small intestine of poultry but the exact localization is poorly understood. Therefore, a histological study was conducted in order to localize the larvae during the early phase of infection. Six layer pullets seven-week old were infected orally with 20,000 embryonated A. galli eggs each, whereas four chickens were left as un-infected controls. At necropsy 3 days after infection the first half of jejunum/ileum was divided into two equally sized sections (J1 and J2). After taking samples for histology from the middle of J1 and J2 and the junction between these determined JX, the two sections were subjected to parasitological examination. A higher number of A. galli larvae were recovered from section J2 than J1 and the majority of larvae were recovered from the most profound layers. Based on histology 144 larvae were identified and their location was noted. The highest number of larvae was observed in the JX sample as compared to J1 and J2 (P<0.001). Most of them were located in the profound crypt zone of the mucosa (51%) as compared to the other zones (P<0.05). The number of larvae was higher in the lumen (63%) compared to the epithelium (32%) and lamina propria (5%) (P<0.001). A significantly higher number of eosinophils were found in lamina propria of the infected group compared to the control group (P<0.001). This experiment clearly showed that only few larvae had penetrated the epithelium and were positioned in the lamina propria at 3 days post infection. It was far more common that the larvae were localized within the epithelium or in the lumen of the crypts. It is therefore suggested that at least in this early phase "mucosal phase" is a more appropriate term to be used for the A. galli larval localization as compared to the term "histotrophic phase" currently used in many textbooks.

Symptoms after ingestion of pig whipworm Trichuris suis eggs in a randomized placebo-controlled double-blind clinical trial.

UNLABELLED: Symptoms after human infection with the helminth Trichuris suis have not previously been described. Exposure to helminths has been suggested as immune therapy against allergy and autoimmune diseases. We randomized adults with allergic rhinitis to ingest a dose of 2500 T. suis eggs or placebo every 21 days for 168 days (total 8 doses) in a double-blind clinical trial. In a previous publication, we reported a lack of efficacy and a high prevalence of adverse gastrointestinal reactions. The aim of the present study was to present a detailed description of the adverse event data and post-hoc analyses of gastrointestinal reactions. Adverse events and severity (mild, moderate, severe) were recorded daily by subjects, classified by organ using MedDRA 10.0, and event rates compared between subjects on T. suis treatment vs. subjects on placebo. T. suis-specific serum IgG antibodies were measured by a fluoroenzymeimmunoassay (Phadia ApS). During 163 days complete follow-up, subjects ingesting T. suis eggs (N = 49) had a three to 19-fold higher rate of events (median duration, 2 days) with gastrointestinal reactions (moderate to severe flatulence, diarrhea, and upper abdominal pain) compared with placebo subjects (N = 47). The highest incidence of affected subjects was seen from the first few days and until day 42 (3(rd) dose): 63% vs. 29% for placebo; day 163: 76% vs. 49% for placebo. Seroprevalences increased concurrently in the T. suis group: Day 59, 50%; day 90, 91%; day 170, 93%. The combined duration of episodes with onset before day 42 was ≤ 14 days in 80% of affected subjects. Age, gender, total IgE, and recent intestinal symptoms at baseline did not predict gastrointestinal side effects. In conclusion, during the first 2 months, repeated ingestions of 2500 T. suis eggs caused frequent gastrointestinal reactions lasting up to 14 days, whereas 4 months further treatment mainly provoked a subclinical stimulation. TRIAL REGISTRATION: University hospital Medical Information Network trial registry Reg. no. R000001298, Trial ID UMIN000001070.

Prevalence of gastrointestinal nematodes in growing pigs in Kabale District in Uganda.

During the last 30 years, pig production in Uganda and neighbouring counties has increased markedly. Pigs are mainly kept as a source of income for small-scale farmers; however, the pig production is subject to several constraints, one of them being worm infections. A study was carried out in rural communities in Kabale District in the South Western part of Uganda in September and October 2007 in order to estimate the prevalence of gastrointestinal nematode parasites in pigs based on coprological examination. Fifty-six households were randomly selected and visited. Housing system and deworming history were recorded. Faeces was sampled from rectum of one to five pigs (age, 3-12 months) per household. A total of 106 pigs were examined coprologically of which 91% excreted nematode eggs. The following prevalences of nematode eggs were recorded: strongyles (89%), Ascaris suum (40%), Trichuris suis (17%) and spiruroid eggs (48%). On household level, rearing pigs on slatted floors in pens significantly reduced the faecal egg excretion of strongyle eggs with almost 80% (p=0.010) and a significant interaction between floor type and anthelmintic treatment was found for spiruroids (p=0.037). Fifteen T. suis egg positive pigs were selected for post-mortem examination of the gastrointestinal tract. The post-mortem examinations revealed that 93% pigs were infected with Oesophagostomum spp. (worm burden, min-max 10-2,180), 73% with A. suum (1-36), 67% with T. suis (6-58), and 20% with Hyostrongylus rubidus (worms not quantified). In general, nematode infections were widespread and polyparasitism common in pigs in Uganda. However, worm burdens were moderate which may be related to recent deworming or to the practice of rearing pigs on slatted floors in wooden elevated pens.

Trichuris suis ova therapy for allergic rhinitis: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Parasitic helminth infections can protect against allergic airway inflammation in experimental models and have been associated with a reduced risk of atopy and a reduced course of asthma in some observational studies. Although no clinical evidence exists to support the use of helminth therapy for allergic disease, the helminth Trichuris suis has demonstrated efficacy in treatment of inflammatory bowel disease. OBJECTIVE: To determine efficacy of helminth therapy for allergic rhinitis. METHODS: We conducted a double-blind, placebo-controlled, parallel group trial in which 100 subjects age 18 to 65 years with grass pollen-induced allergic rhinitis were randomly assigned to ingest a total of 8 doses with 2500 live T suis ova or placebo with an interval of 21 days. The primary outcome was a change in mean daily total symptom score for runny, itchy, sneezing nose (maximum change, 9.0) or in percentage of well days during the grass pollen season. RESULTS: Treatment with T suis ova (N = 49) compared with placebo (N = 47) caused transient diarrhea peaking at day 41 in 33% of participants (placebo, 2%), and increased eosinophil counts (P < .001) and T suis-specific IgE (P < .05), IgG (P < .001), IgG(4) (P < .003), and IgA (P < .001), whereas there was no significant change in symptom scores (0.0; 95% CI, -0.5 to 0.4; P = .87), well days (3%; 95% CI, -9% to 14%; P = .63), total histamine (P = .44), grass-specific IgE (P = .76), or diameter of wheal reaction on skin prick testing with grass (P = .85) or 9 other allergens. CONCLUSION: Repeated treatment with the helminth T suis induced a substantial clinical and immunologic response as evidence of infection, but had no therapeutic effect on allergic rhinitis.

Population dynamics of Ascaris suum in trickle-infected pigs.

The population dynamics of Ascaris suum was studied by long-term exposure of pigs to infective eggs. The pigs were experimentally inoculated with 25 A. suum eggs/kg/day, and 7, 8, and 8 pigs were necropsied at weeks 4, 8, and 14 postinoculation (PI), respectively. Despite the fact that the pigs were continuously reinfected, dramatic reductions in numbers of liver lesions (white spots) and migrating lung larvae were observed as a function of time. However, even at the end of the study, a few larvae were able to complete migration, but these larvae seemed unable to mature in the small intestine. Thus, the adult worm population seemed to consist of worms from the first part of the exposure period. The noticeable decrease in number of white spots suggests that the level of exposure is not reflected in the number of white spots in the late phase of a continuous infection. The serum levels of A. suum L3-specific IgG1 and IgA were significantly elevated by week 4 PI, after which the antibody levels declined. The population dynamics and parasite regulating mechanisms are discussed for A. suum in pigs as well as for the closely related species A. lumbricoides in humans.

Ascaris suum infection negatively affects the response to a Mycoplasma hyopneumoniae vaccination and subsequent challenge infection in pigs.

Since their first introduction more than a century ago, vaccines have become one of the most cost-effective tools to prevent and manage infectious diseases in human and animal populations. It is vital to understand the possible mechanisms that may impair optimal vaccine efficacy. The hypothesis posed in this study was that a concurrent Ascaris suum infection of pigs vaccinated with a Mycoplasma hyopneumoniae (Mh) vaccine would modulate the protective immune response to a subsequent challenge infection. Four groups of pigs were either (1) untreated (group C), (2) vaccinated against Mh 3 weeks after the start of the study (group V), (3) given a trickle infection with A. suum throughout the study (group A), or (4) given a trickle infection with A. suum and vaccinated against Mh (group AV). All pigs were subsequently inoculated with live Mh bacteria 4 weeks after the Mh vaccination and necropsied after another 4 weeks. All pigs in group V sero-converted 3 weeks after vaccination (100%), as opposed to only 33% of group AV pigs that were Mh-vaccinated and given A. suum. At the end of the study, only 78% of pigs in group AV had sero-converted. Pigs in group AV had a higher mean percentage of lung pathology and the variation was significantly higher in these pigs compared to pigs in group V. The pattern of gene expression in the lungs and draining lymph nodes indicated a local Th2-skewed response induced by A. suum. Our study indicated that A. suum significantly compromised the effect of Mh vaccination. The impact of reduced vaccine efficacy caused by a common gastrointestinal helminth emphasises the importance of parasite control. More focus should be put into this area of research to outline the practical consequences of this interaction, and to be able to predict, prevent and correct negative interactions.

Albendazole and mebendazole have low efficacy against Trichuristrichiura in school-age children in Kabale District, Uganda.

Three groups of Trichuris trichiura-infected school-age children were treated with one dose 400mg albendazole, 100mg mebendazole twice daily for 3 d, or 100mg mebendazole twice daily for 5 d. The albendazole study investigated cure and egg reduction rates and found that only 5 of 66 infected children were egg-negative 7 d post-treatment, giving a cure rate of 8% and a geometric mean egg reduction rate of 89%. However, at day 14 post-treatment, all children were again egg-positive with significantly higher egg counts than at day 7 (P<0.001). The two mebendazole studies aimed for the recovery of adult T. trichiura worms. After the 3 d course of mebendazole treatment, only four worms were recovered on days 3-5 after start of treatment from 2 of 34 infected children. With the 5 d course of mebendazole treatment, 10 of 21 infected children expelled a total of 27 worms. In the last case the first worm appeared on day 4 post-treatment, and the highest number of worms was recovered when the study ended at day 7. In conclusion, even with the longest treatment regimen and collecting stool samples over seven consecutive days, only very few worms were recovered. The results of this study suggest that alternative drugs and/or alternative regimens in current control programmes against T. trichiura need renewed attention.

Multiplex PCR on single unembryonated Ascaris (roundworm) eggs.

A sensitive and inexpensive method for DNA isolation and amplification by polymerase chain reaction (PCR) from single unembryonated Ascaris sp. eggs is described. The resistant shell of single eggs was crushed mechanically and PCR applied to the crude egg contents without any further purification steps. The ITS1 region of the rDNA and three regions of the mtDNA could be successfully amplified. Using two primer sets, it was possible to amplify the rDNA and mtDNA simultaneously in one single reaction. The ability to perform PCR on single unembryonated eggs may result in better and more precise species identification of eggs recovered from faecal material, environmental samples and possibly archaeological samples. In addition, single egg PCR makes it possible to perform population genetic studies without having to recover adult worms by deworming or autopsy.

A novel technique for identification of Ascaris suum cohorts in pigs.

The objective of the present study was to develop a fast, cheap and reliable technique for identifying different cohorts of the swine parasite, Ascaris suum. A polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP) technique on mt-DNA was used to identify unique haplotypes of four gravid A. suum females on agarose gels after eggs were recovered from each of the worms. Each of four pigs was inoculated with 2000 embryonated eggs originating from one of the four identified Ascaris haplotypes, respectively. Ascaris larvae were isolated from the small intestine at day 14 post-infection using an agar technique. Single larvae from each pig were transferred to 96-well PCR plates and a simple DNA extraction using a worm lysis buffer was carried out and followed by the PCR-RFLP analysis. More than 100 larvae from each of the four pigs were analysed and all were found to have the same haplotype as the parental female. We conclude that unique haplotypes of female A. suum and offspring can be identified by means of PCR-RFLP on mt-DNA and suggest that this method can be used in future research on Ascaris population biology using cohorts with distinct mt-DNA profile.

Survival of Isospora suis oocysts under controlled environmental conditions.

Isospora suis is a coccidian parasite infecting piglets soon after birth. While the gross epidemiology of I. suis is well known, little knowledge exists on the ecology of the oocysts. To study the development and survival of oocysts of I. suis under controlled laboratory conditions, known numbers of oocysts ( approximately 200 in each of 4 replicates) were exposed to all combinations of 4 relative humidities (53-100% RH) and 3 temperatures (20 degrees , 25 degrees , 30 degrees C). Determination of viability was based on morphological and fluorescent properties of the oocyst as well as on the permeability of the oocyst wall characterized by inclusion/exclusion of the fluorescent dye propidium iodide. The viability of the oocysts was studied over time by fluorescence and light microscopy until <5% of the oocysts were considered to be viable. The sporulation rate increased with temperature, however, the infective sporocyst stage was reached within 24h at all temperatures, while RH did not seem to affect sporulation. Results show a rapid reduction in viable oocysts exposed to high temperatures (25 degrees C and 30 degrees C) in combination with low relative humidities (53% RH and 62% RH), at which conditions oocysts died within 24h. Viability was higher when oocysts were exposed to higher relative humidities (75% RH and 100% RH) as well as a lower temperature (20 degrees C). However, even at 75% RH the oocysts died within 24-60 h at 30 degrees C to 20 degrees C, respectively, while the most favourable condition appeared to be 100% RH and 25 degrees C at which condition the percentage of viable oocysts decreased from 100% to 17% in 96 h. The results indicate that it may be possible to reduce the infection pressure of I. suis in modern sow herds by changing the environmental conditions and/or the management within the farrowing pens, and thereby increase animal welfare without relying on the use of routine medication.