The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans.

O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by ß-elimination or endo-ß-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or ß-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galß3-(±Galß4)Galß4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

Oesophagostomum dentatum: potential as a model for genomic studies of strongylid nematodes, with biotechnological prospects.

There are substantial gaps in the knowledge of the molecular processes of development and reproduction in parasitic nematodes, despite the fact that understanding such processes could lead to novel ways of treating and controlling parasitic diseases, through blocking or disrupting key biological pathways. Biotechnological advances through large-scale sequencing projects, approaches for the analysis of differential gene and protein expression and functional genomics (e.g., double-stranded RNA interference) now provide opportunities to investigate the molecular basis of developmental processes in some parasitic nematodes. The porcine nodule worm, Oesophagostomum dentatum (order Strongylida), may provide a platform for testing the function of genes from this and related nematodes, given that this species can be grown and maintained in culture in vitro for periods longer than other nematodes of the same order. In this article, we review relevant biological, biochemical and molecular biological and genomic information about O. dentatum and propose that the O. dentatum - pig system provides an attractive model for exploring molecular developmental and reproductive processes in strongylid nematodes, leading toward new intervention methods and biotechnological outcomes.

Studies on the interaction between Salmonella enterica ser. Typhimurium and intestinal helminths in pigs.

Concomitant infections with helminths and bacteria may affect the course and the resulting disease outcome of the individual infections. Salmonella, Oesophagostomum, Trichuris and Ascaris coexist naturally in pig herds in Denmark, and possible interactions were studied. Pigs in one experiment were trickle infected with low or moderate dose levels of Oesophagostomum spp. and challenge infected with S. Typhimurium. In another experiment, pigs were inoculated with S. Typhimurium followed by a challenge exposure to either Oesophagostomum, Trichuris or Ascaris. Enhancement of the Salmonella infection was not demonstrated in either experiment. The helminth effect on the pigs was modest and may explain the lack of influence on the Salmonella infection. A previous experiment with a larger Oesophagostomum infection level resulted in enhancement of the S. Typhimurium infection. A dose dependency of the interaction is therefore suggested. However, the relatively high worm burdens in the present study suggest that infection with these common pig helminths does generally not influence the course of concurrent S. Typhimurium infections under natural conditions.

Products of fourth-stage larvae of Oesophagostomum dentatum induce proliferation in naïve porcine mononuclear cells.

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune-modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum-infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third- (L3) and fourth-stage larvae (L4) of O. dentatum. Numbers of vital cells and blast-transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L4 (but not L3) significantly increased the numbers of vital poMNC in vitro (up to 187%). In addition, L4 products (homogenates and supernates, 0.1-10 microg/ml) but not those of L3 induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum-infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen-induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L3 and indicate that the parasite alters its set immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.

Ultrasonographic detection and assessment of preclinical oesophagostomum bifurcum-induced colonic pathology.

In northern Ghana and Togo, Oesophagostomum bifurcum infects an estimated 250,000 people, as determined by cultures of stool samples. The juvenile stages of the helminth develop within colonic wall nodules, causing Dapaong tumor or multinodular disease, at the rate of 1 case per week at Nalerigu Hospital in Ghana. Our aim was to discover whether suspected colonic-wall pathology is ultrasonographically visible in asymptomatic individuals living in the area where O. bifurcum is endemic. A total of 464 persons from 3 villages, ranging from highly infected to noninfected, were examined with ultrasonography. Anechogenic colonic lesions with posterior wall enhancement were observed in 71 (54.2%) of 131 and 57 (24.5%) of 233 persons from the villages of endemicity, and no lesions were seen in persons from the village outside the area of endemicity. We describe the lesions noted in this study as nodules caused by O. bifurcum, on the basis of their association at a population level with prevalence of larvae in stools, their expected ultrasonographic appearance and distribution (on the basis of our surgical experience with oesophagostomiasis), and the lack of a convincing differential diagnosis.

Experimental Oesophagostomum bifurcum in monkeys.

OESOPHAGOSTOMUM BIFURCUM: larvae, cultured from human stools collected in northern Ghana, were used to establish experimental infections in monkeys. A patent infection was established in a rhesus monkey (Macaca mulatta) and this infection was used to generate larvae to inoculate additional monkeys. In all, 17 animals were inoculated. Thirteen of 15 animals developed antibodies to the infection between 19 and 62 days post inoculation (PI); two animals had a positive response before inoculation. Four of ten animals developed patent infections between 88 and 134 days and passed eggs in the faeces. Egg shedding was consistent in only one animal, but at low levels of one or two eggs per 2 mg direct smear, and extended over a 400 day period. In the other three animals, egg shedding was sporadic and of only 2-4 weeks duration. In seven animals necropsied between 19 and 22 days PI, one to 17 early fourth-stage larvae were recovered from nodules in the bowel wall; in an eighth animal examined at 314 days, six immature adult worms (early fifth stage) were recovered from nodules in the bowel wall. The morphological features and growth of these recovered larvae are described. Three animals were inoculated with larvae that had been dried for one week at 28 degrees C; two animals began shedding eggs at 128 and 134 days PI, respectively. The present results suggest that the parasite obtained from humans is poorly adapted to lower primate hosts, and supports the concept that Oesophagostomum bifurcum found in humans and monkeys in the same geographical region of northern Ghana and Togo are distinct and that the infections in humans are not likely to represent zoonotic infections acquired from monkeys.

Assessment of antibody-independent cellular cytotoxicity (AICC) of porcine neutrophilic granulocytes by quantitative flow cytometry. Lack of modulation by larval products of Oesophagostomum dentatum.

After infection of pigs by the larvae of Oesophagostomum dentatum, granulomas are formed around the third-stage larvae in the submucosa of the gut which contain a considerable number of neutrophils. This has no obvious impact on the larvae, which develop to fourth-stage larvae within these granulomas. We therefore asked, whether the products of O. dentatum larvae modulate the functional capacity of porcine neutrophils. The antibody-independent cellular cytotoxicity (AICC) was chosen as a model system. This assay was developed for the pig and quantified using flow cytometry. Bovine lymphoblastoid cells (cell line Anna TA1) served as targets. The measurement of cytotoxicity was based on the determination of absolute numbers of vital target cells. This procedure proved to be reliable and required no additional labelling of target and/or effector cells. Porcine neutrophils, when stimulated with phorbol 12-myristate 13-acetate (PMA; > or = 10 nmol/l), killed target cells at effector: target ratios between 1:1 and 9:1. AICC was not demonstrable after 4 h but could be observed between 16 h and 20 h after in vitro co-culture. Killing of targets required close physical contact between effector and targets, since supernatants of PMA-stimulated polymorphonuclear cells were not able to lyse the target cells. Homogenates of third- and fourth-stage larvae of O. dentatum did not affect the vitality of porcine granulocytes or target cells in vitro, nor did they modulate the AICC capacity of porcine granulocytes.

Parenterally administered gastrointestinal nematode infective larvae viable after more than 15 years in liquid nitrogen.

After cryopreservation for 13.3-15.8 years, the viability of the infective larvae (L3) of Trichostrongylus axei, T. colubriformis, Oesophagostomum columbianum, Haemonchus contortus, Ostertagia circumcincta, T. falculatus, Nematodirus spathiger, Chabertia ovina and Dictyocaulus filaria was assessed in sheep, by being deposited at their predilection sites. D. filaria was, however, an exception, in that the L3 were injected into the jugular vein. The mean development of all the species was 22.8%, but if three species (O. columbianum, C. ovina and D. filaria), that developed poorly are disregarded, then the mean development was 33.4%, similar to previous tests after shorter periods of cryopreservation. The L3 of some of the species appeared sluggish when examined 10-15 min after being thawed, and in the case of H. contortus practically all the larvae of the original batch tested in the previous trials of the series appeared dead when thawed for use in the present trial, and were replaced by another batch of L3 of the same species. When re-examined after about 8 h, however, a high percentage of the L3 of the original batch appeared to have become revitalised, and their viability was tested in a trial reported elsewhere. The intestinal cells of the majority of the L3 of N. spathiger, O. circumcincta and C. ovina were vesiculated when they were thawed. Nevertheless, the degree of development of the former two species was of the highest in the trial, and it can be concluded that this phenomenon does not necessarily impede the viability of larvae.

Development and infectivity of eggs and larvae derived from pigs trickle-infected with Oesophagostomum dentatum at different dose levels.

We examined the impact of different Oesophagostomum dentatum dose levels and durations of infection on the development and infectivity of the following generation. Pigs were trickle-infected with 200, 2,000 or 20,000 L3/week over 20 weeks. Egg hatch assays were performed at monthly intervals; however, no consistent differences were found between any of the dose groups in the development of eggs into first-stage larvae. To compare larval infectivity, larvae were derived from faecal cultures set up from the low- and the high-dose groups in the early and the late part of the experiment, and were inoculated into helminth-free pigs (5,000 L3/pig). Worm establishments were significantly higher (P<0.05) in the group of pigs receiving larvae derived early in the experiment from the low-dose group compared with the two groups receiving larvae from high-dose groups, thus indicating an adverse effect of high doses of trickle infection on the later infectivity of L3 larvae derived from excreted eggs.

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.

Development of the free-living stages of Hyostrongylus rubidus and Oesophagostomum spp. at different temperatures and humidities.

Faeces containing a mixture of Hyostrongylus rubidus and Oesophagostomum spp. eggs were mixed with vermiculite and water and set up at combinations of different relative humidities (65.5%, 79.5% 90.0%, and 100%) and temperatures (5 degrees C, 10 degrees C, 15 degrees C, 20 degrees C, and 25 degrees C) in order to study the rate of egg hatching and larval development. The study established that the development from egg to infective larva showed similar patterns for the two parasites. Optimum development and survival was in the temperature range from 15 degrees C to 20 degrees C and at humidities from 79.5 to 95.5%.

[Pathogenic role of O. venulosum in lambs]. / Vurkhu patogennata rolia na O. venulosum pri agneta.

Studied was the effect of an experimental infection of lambs with 1000 and 4000 larvae of Oesophagostomum venulosum on the activity of some more important serum enzymes, the level of sulfhydryl groups, and the level of sugar. It was found that the activity of glutamic acid oxal-acetate transaminase and lactic acid dehydrogenase rose, and that of alkaline phosphatase dropped. At 4000 larvae the level of glutamic acid pyruvate transaminase rose. The concentration of blood sugar dropped. There was also a drop of the SH groups. It was best manifested from the first to the third week following infection. In the fourth week the level of the total and residual SH groups showed a trend toward coming back to normal, however, the initial values were not reached.