Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum

During its development from free-living infectious third-stage larvae to the adult worms in the large intestines of pigs, Oesophagostomum dentatum experiences several environmental changes. Differences in protein patterns can reflect such changes. Somatic and ES antigens and glycoproteins of pre-parasitic, histotropic and intestinal stages were compared by single-dimension SDS–PAGE and stage-specific proteins were defined. Furthermore, fourth-stage larvae derived from different sources—in-vitro cultivation and intestinal contents—were compared and also found to be different. It is hypothesised that O. dentatum reacts to environmental stimuli by differential expression of specific proteins as a possible mode of adaptation to the host.     

Prostaglandin D(2) synthesis in Oesophagostomum dentatum is mediated by cytosolic glutathione S-transferase

Glutathione S-transferases (GSTs) of Oesophagostomum dentatum possess considerable similarity to synthetic prostaglandin D synthase (PGDS), and therefore their ability to convert prostaglandin (PG) H₂ to PGD₂in vitro was investigated with a commercial Prostaglandin D Synthase Inhibitor Screening Assay Kit. Fractioned homogenates of O. dentatum third-stage larvae only displayed cytosolic but not microsomal GST. Both total larval homogenate and isolated GST could metabolise PGH₂ to PGD₂, which could be inhibited by the GST inhibitor sulfobromophthalein (SBP) in a dose-dependent manner, whereas reactions to the specific PGDS inhibitor HQL-79 were not dose-dependent. Inhibition of larval development by SBP in vitro was abolished by the addition of PGD₂ but not by PGH₂, supporting the assumption that GST acts as PGDS and is important for nematode development. Since motility and viability of O. dentatum larvae are reduced in vitro by various inhibitors of eicosanoid metabolism, enzymes of this pathway, including GST, constitute putative intervention targets.     

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 (B_max) 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 B_max 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.     

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum

BackgroundThe porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.MethodsDifferent enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.ResultsWhereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered ‘male’, while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc_2–4PC_2–4 motifs not observed in our previous studies on other nematodes.ConclusionThe interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.General significanceUnusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.     

Differentiation of two Oesophagostomum spp. from pigs, O. dentatum and O. quadrispinulatum, by computer-assisted image analysis of fourth-stage larvae

A method was developed to differentiate between fourth-stage larvae (L4) of two species of porcine nodular worms, Oesophagostomum dentatum and O. quadrispinulatum, by computer-assisted analysis of digitised microphotographs of L4 grown in vitro for various time periods and of L4 ex vivo. The overall lengths of the larvae and the lengths of the oesophagus as well as parameters describing the shape of the oesophagus and buccal capsule were measured and a formula based on these parameters was developed that could differentiate between the two species on the basis of the morphometric data. It was demonstrated that morphometry can produce unbiased data which can be employed for the calculation of indices suitable for the differentiation of morphologically different specimens. Computer-based techniques facilitate the processing of the complex data and offer the option for automation of measurements for routine applications.     

Fatty acid patterns of different stages of Oesophagostomum dentatum and Oesophagostomum quadrispinulatum as revealed by gas chromatography

Total methylated fatty acid patterns of various developmental stages (third-stage larvae (L3), L3 and fourth-stage larvae (L4) cultured in vitro, L4 and female and male adults derived from intestinal contents) of the porcine nodular worms Oesophagostomum dentatum and Oesophagostomum quadrispinulatum and their cultivation medium were analysed by gas chromatography using Microbial Identification computer software. Fatty acids ranging from C-12 to C-20 could be separated. For each stage and species, characteristic patterns were found. The most prevalent fatty acids were C-18. The freshly exsheathed larvae contained the greatest variety of fatty acids (including short-chain fatty acids C-12 to C-15) with approximately equal amounts of fatty acids with odd and even chain lengths, whereas more advanced stages consisted of a lower number of fatty acids with mostly even chain lengths >/=C-16. Intestinal stages contained less odd-numbered fatty acids and less branched fatty acids than others. In contrast to intestinal L4, cultivated L4 had high amounts of C-15:0 and C-17:0. Sheathed L3 contained more C-18 than freshly exsheathed ones, and medium incubated for 7 days in the presence of parasites contained C-13 to C-15 and monounsaturated C-16, but less C-18 and C-20:4 than fresh medium or medium incubated without worms. Based on the evaluation of stage- and species-specific fatty acid patterns random samples could be assigned to the correct stage and species. In a dendrogram based on fatty acid patterns the same stages of the two species formed the closest relationships, and the intestinal stages formed a clade distinct from the cultivated larvae and L3. All stages contained considerable relative amounts of arachidonic acid, the main precursor of eicosanoids. The fixed differences between species and stages indicate genetic regulation of fatty acid patterns, while environmental influences are mirrored by differences between cultivated and intestinal stages. Regulation of fatty acid patterns probably plays a role in worm physiology and host-parasite interaction.     

Oesophagostomum dentatum: effect of glutathione S-transferase (GST) inhibitors on GST activity and larval development

Sulphobromophthalein (SBP) inhibits isolated glutathione S-transferase of the porcine nodule worm Oesophagostomum dentatum (Od-GST) and reduces larval development in vitro. In this study possible inhibitory effects of various inhibitors were evaluated in an enzymatic (CDNB) assay with isolated Od-GST and in a larval development assay (LDA). Reversibility was tested in the LDA by removing the inhibitor from culture halfway through the cultivation period. SBP, indomethacin and ethacrynic acid inhibited both enzyme activity and larval development in a dose-dependent and reversible manner. HQL-79 also reduced larval development but had only a minor effect on the isolated enzyme. The phospholipase A₂ inhibitors dexamethasone and hydrocortisone had no major effect. High thermal stability of Od-GST was demonstrated with increasing activity between 4 and 50 °C. Differences between Od-GST and GST of other organisms indicate structural and possibly functional peculiarities and highlight the potential of such enzymes as targets of intervention.     

Oesophagostomum infections in humans

Oesophagostomum spp are normally found as nematode parasites of ruminants, pig and monkeys. Occasionally humans are involved. In the past decade it became clear that, in some parts of Africa, humans are adequate final hosts. In those areas, prevalences of infection are high and morbidity is significant. The presence of lumen-dwelling adult worms, which do not seem to cause a great deal of pathology, can be demonstrated through coproculture. The presence of immature worms, encapsulated in nodules and responsible for pathology, on the other hand, is more difficult to confirm. It is not known what factors limit the distribution of endemic human oesophagostomiasis to a small focus in West Africa. The relationship between the 'helminthomas' described a long time ago in Uganda and the human Oesophagostomum infections in West Africa is unclear and it remains a mystery how humans get infected so effectively by ingesting L3 larvae. In this overview, Ton Polderman and Coby Blotkamp give an account of what is known and what is still to be elucidated in human Oesophagostomum infections.     

Immunization of lambs against Oesophagostomum columbianum, using irradiated third stage larvae

The effect of gamma irradiation at doses of 40- and 50 kR on the development of third stage larvae of Oesophagostomum columbianum and the protection conferred by these irradiated larvae against the nematode, was studied in 6-8 week old male Kashmir Merino lambs. At 40- and 50 kR doses, the third stage larvae failed to develop to the adult stage in the intestine. Though single vaccination with 2000, 50 kR irradiated larvae failed to protect the animals against the infection, vaccination with the same number of 40 kR irradiated larvae conferred a partial protection. The presence of adult worms of O. columbianum in sites outside the intestine of 6-8 week old lambs have been demonstrated for the first time.     

Cytosolic glutathione S-transferases of Oesophagostomum dentatum

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

Oesophagostomum columbianum: gamma-irradiated third-stage larvae for immunization in lambs

40-kR gamma-irradiated third-stage larvae of Oesophagostomum columbianum were used for the immunization of Kashmir Merino lambs. Male lambs (aged from 8 to 12 weeks) were immunized in two separate experiments by two doses of irradiated larvae, given 21 days apart, and subsequently challenged with normal larvae. Judging by the establishment of worms resulting from the challenge infections in the immunized and control groups of lambs in the two experiments, a high degree of immunity was shown to develop in young lambs vaccinated with 500, followed 21 days later with 2000, 40-kR irradiated larvae. Lambs from the immunized groups showed more nodules in the intestine, a high percentage of which were positive for histotrophic stages of O. columbianum, than did controls. The importance of this finding in relation to the possible use of a vaccine for the immuno-prophylaxis of oesophagostomiasis in sheep and other animals is discussed.     

Polymerase chain reaction detection of a novel human KIT (mast/stem cell growth factor receptor) gene polymorphism by single-strand conformation polymorphism analysis or by SmaI or BstNI cleavage

We describe a common single-base polymorphism of the KIT gene that alters both SmaI and BstNI restriction sites, but is most easily detected as a single-strand conformation polymorphism (SSCP) using the polymerase chain reaction (PCR).     

Molecular separation of Oesophagostomum stephanostomum and Oesophagostomum bifurcum (Nematoda: Strongyloidea) from non-human primates.

The ITS-2 sequences for adult specimens of Oesophagostomum stephanostomum from the common chimpanzee and Oesophagostomum bifurcum from the Mona monkey were determined. For both species, the length and GC content of the ITS-2 sequences were 216 bp and 43%, respectively. While there was no unequivocal sequence difference among individual worms representing each of the two species, five (2.3%) interspecific nucleotide differences were detected. These differences were associated with the presence of unique restriction sites in the ITS-2 sequence of 0. stephanostomum for multiple endonucleases of diagnostic value for the differentiation of the two taxa by restriction analysis. Pairwise comparisons of the ITS-2 sequences of O. stephanostomum and O. bifurcum with published ITS-2 sequences for five different congeners indicated that these species from the subgenus Conoweberia are closely related, in accordance with previous morphological studies.     

Oesophagostomum dentatum: expression patterns of enzymes involved in eicosanoid production

Cytosolic and membrane-bound proteins of various stages of Oesophagostomum dentatum, the nodular worm of pigs, were investigated for the presence of lipoxygenases (LOX) and cyclooxygenases (COX) using polyclonal and monoclonal antibodies. Putative 12-LOX and 15-LOX, but not 5-LOX, were detected in both fractions of all developmental stages in the expected size range of 75 kDa, with an isoelectric point of 6.0-6.5. The protein could be precipitated with 50% ammonium sulfate, as described for mammalian LOX. An antibody directed against both COX isoforms and one against mammalian COX-2 detected proteins of approximately 70 kDa with an isoelectric point of 6.0-6.5 in the membrane-bound fractions of third-stage larvae and adults, but not in the fourth-stage larvae. Anti-COX-1 or more specific anti-COX-2 antibodies failed to detect proteins. The constitutive LOX expression supports the assumption that the metabolites of this enzyme previously detected in O. dentatum serve intrinsic functions, while the production of anti-inflammatory COX-products in the invasive and luminal stages of the parasite implies a possible role in host-parasite interactions.     

Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: characterization of the complete mitochondrial genome sequences of the two pig nodule worms

In the present study, the complete mitochondrial DNA (mtDNA) sequences of the pig nodule worm Oesophagostomum quadrispinulatum were determined for the first time, and the mt genome of Oesophagostomum dentatum from China was also sequenced for comparative analysis of their gene contents and genome organizations. The mtDNA sequences of O. dentatum China isolate and O. quadrispinulatum were 13,752 and 13,681 bp in size, respectively. Each of the two mt genomes comprises 36 genes, including 12 protein-coding genes, two ribosomal RNA and 22 transfer RNA genes, but lacks the ATP synthetase subunit 8 gene. All genes are transcribed in the same direction and have a nucleotide composition high in A and T. The contents of A+T are 75.79% and 77.52% for the mt genomes of O. dentatum and O. quadrispinulatum, respectively. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (maximum likelihood, maximum parsimony and Bayesian inference), all revealed that O. dentatum and O. quadrispinulatum represent distinct but closely-related species. These data provide novel and useful markers for studying the systematics, population genetics and molecular diagnosis of the two pig nodule worms.     

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.     

Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum

In this study, sequence variation in three mitochondrial DNA regions, namely cytochrome c oxidase subunit (cox1) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), between Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical origins in Mainland China was examined, and their phylogenetic relationships were reconstructed. A partial of the cox1 (pcox1), nad1, and nad4 genes (pnad1 and pnad4) were amplified separately from individual nodule worms by PCR and were subjected to direct sequencing in order to define sequence variations. While the intraspecific sequence variations within each of the two species were 0.3-5.2% for pcox1, 0-4.9% for pnad1, and 0-7.1% for pnad4, the interspecific sequence differences were significantly higher, being 10.7-13.4% for pcox1, 11-14.6% for pnad1, and 14.9-18% for pnad4, respectively. There were a number of nucleotide positions in the pcox1, pnad1, and pnad4 sequences with no apparent intraspecific variation but distinct interspecific differences among those samples of Oesophagostomum spp. examined, which may be used as genetic makers for the identification and differentiation of the Oesophagostomum spp. Phylogenetic analyses using three inference methods, namely Bayesian inference, maximum likelihood, and maximum parsimony based on the combined sequences of pcox1, pnad1, and pnad4 revealed that the O. dentatum and O. quadrispinulatum form monophyletic groups, respectively. These findings demonstrated clearly the usefulness of the three mitochondrial sequences for studying systematics, population genetic structures, and the molecular ecology of Oesophagostomum spp.     

The veterinary importance of nodular worms (Olesophagostomum spp)

The nodular worms (Oesophagostomum spp) are important parasites of food animals and primates. The archaic line that parasitizes pigs appears to be less pathogenic than the parallel evolutionary lines in ruminants and primates. Morphological types of O. dentatum in pigs and an apparent new host record for O. venulosum, which normally affects sheep and wild ruminants, in cattle suggests aggressive recruitment within the genus. The same Oesophagostomum spp occur in humans and in non-human primates but it is not clear which is the natural host.     

Oesophagostomum radiatum in calves: intestinal hemorrhage associated with larval emergence

Intestinal blood loss in calves infected with various numbers of Oesophagostomum radiatum was measured using ⁵¹Cr-labeled erythrocytes, and compared with the subsequent changes in the packed cell volume of jugular blood. Emergence of histotropic fourth-stage larvae from the submucosal cysts was associated with intestinal hemorrhage in all infected calves but only in the calves given a near lethal dose of larvae was the bleeding sufficient to cause anemia. At the lower infection rates the hemorrhage caused by larvae was considerably less than that produced by the ensuing adult population. The proportion of adult worms recovered from the infecting dose diminished as the dose increased.