Distinguishing Oesophagostomum dentatum from Oesophagostomum quadrispinulatum developmental stages by a single-strand conformation polymorphism method

At some life-cycle stages, it is impossible to distinguish between the two species of porcine nodular worm, Oesophagostomum dentatum and Oesophagostomum quadrispinulatum, using morphological features. A PCR-based single-strand conformation polymorphism technique was established to overcome this limitation. The rDNA region spanning the second internal transcribed spacer was amplified by PCR from genomic DNA from morphologically well-defined adult worms. The PCR products were then denatured and subjected to electrophoresis in a non-denaturing gel matrix. Single-strand conformation polymorphism analysis of the products generated characteristic and reproducible patterns for each of the two species and allowed their unequivocal delineation. The single-strand conformation polymorphism was also applied effectively to assess the purity of nine laboratory-maintained cultures of infective third-stage larvae believed to be monospecific for O. dentatum or O. quadrispinulatum. The analysis showed that all six O. dentatum cultures were indeed monospecific, whereas the three cultures believed to be monospecific for O. quadrispinulatum were either a mixture of O. dentatum and O. quadrispinulatum larvae or pure O. dentatum larvae. These findings demonstrated the usefulness of the single-strand conformation polymorphism approach for the routine monitoring of the purity of parasite lines and indicated its value for studies on the population biology of porcine nodular worms.     

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.     

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.     

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.     

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.     

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 profiles of Chlamydia using capillary gas chromatography

Fatty acid profiles of purified elementary bodies of Chlamydia trachomatis (CT) serotypes D, G and L3 were investigated by gas liquid chromatography (GLC) utilizing three fused silica capillary columns of different polarities. CT serotype C and C. psittaci (CP) strain DD34 were investigated using one column only due to the lack of adequate quantities of purified material. Significantly similar fatty acid profiles were observed in the serotypes examined. However, based on the percentage ratio of 13-methyl tetradecanoate (i-15:0) to 12-methyl tetradecanoate (a-15:0), serotypes D and L3, with ratios of 0.18 and 0.19, respectively, could be differentiated from serotypes C and G with ratios of 1.3 and 1.5, respectively. CP demonstrated a ratio of 0.4, thus differentiating it from the CT serotypes examined. Fatty acids i-15:0 and a-15:0 were absent in uninfected McCoy cells. Results were significantly comparable in all three capillary columns. This study suggests that GLC could be used for identification and differentiation of Chlamydia serotypes.This paper was presented, in part, at the 55th Conjoint Meeting on Infectious Diseases (CAC-MID) on November 24, 1987, Ottawa, Canada.     

Characterisation of stage-specific proteins of Oesophagostomum dentatum by preparative isoelectric focusing and lectin blotting

The nodular worm of pigs, Oesophagostomum dentatum, has previously been shown to undergo distinct biochemical changes during its life cycle. This phenomenon was studied in more detail for the early parasitic stages. Differences between infective third-stage larvae (L3), parasitic fourth-stage larvae cultivated in vitro (L4c), and pre-adult larvae recovered from the intestinal contents of pigs (L4p) were compared with respect to their protein and glycoprotein patterns by solubility-based protein fractionation and preparative isoelectric focusing followed by SDS-PAGE or by Western blotting with various lectins. While differences between the L4 were only minor (only three bands were specific for either L4c or L4p), L3 displayed distinctly different protein patterns with four L3-specific and nine L4-specific bands. Concanavalin A bound to a variety of glycoproteins, partly in a stage-specific manner, while Ricinus communis Agglutinin 120, Wheat Germ Agglutinin, Peanut Agglutinin and Soybean Agglutinin bound to fewer, partly stage-specific, molecules.     

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.     

Fatty acids ofSerratia determined by gas chromatography

The fatty acids in whole-cell methanolysates of 19Serratia strains were identified and quantitated by gas liquid chromatography. The major fatty acid components were 3-OH-C14∶0, C16∶0, C16∶1, and C18∶1+2. They contributed 50–80% of the components in each strain. Significant quantities were also contributed by C14∶0. The other components tended to contribute less than 3% each. Principal component analysis of the fatty acid composition data yielded a three-dimensional ordination of strains that roughly reflected the present taxonomic knowledge on the genusSerratia.     

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

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

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.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

Ribosome-mRNA contact sites at different stages of translation initiation as revealed by cross-linking of model mRNAs.

Two model mRNAs, one with and one without the Shine-Dalgarno (SD) sequence, were bound to Escherichia coli 30S ribosomal subunits in the presence and absence of initiation factors and initiator tRNA and then cross-linked by diepoxybutane. The distribution of the cross-linked mRNA among rRNA and ribosomal proteins (r-proteins) and the extent to which individual r-proteins react was found to be affected by the presence or absence of the SD sequence and by the initiation factors and initiator tRNA. The results are consistent with the hypothesis that the position of the 30S-bound mRNA is shifted under the influence of the initiation factors and fMet-tRNA from a stand-by position towards a second site where the decoding of the initiation triplet by the initiator tRNA occurs.     

Fatty acids ofLactobacillus bulgaricus determined by gas chromatography — Mass spectrometry

Fatty acids (in the form of pyrrolidinides) ofLactobacillus bulgaricus were identified by gas chromatography—mass spectrometry using two columns differing in the polarity of the stationary phase. In addition to the described fatty acids (primarily the most abundant vaccenic acid), we detected nine fatty acids that have not yet been described in the genusLactobacillus (all of them minority compounds) and in four of them we determined the position of the double bond. The occurrence of vaccenic acid was related to other sources of positional isomers of octadecenoic acids (parsley seed - petroselmic acid; olive oil — oleic acid).     

A Study on the Transmission of Oesophagostomum dentatum and Hyostrongylus rubidus among Outdoor Reared Pigs in Denmark

This study was carried out to obtain basic information on the transmission of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor reared pigs in Denmark. Eighteen 10 weeks old worm-free pigs were allocated into 3 groups of 6 pigs each. In May, all pigs were turned out on the same parasitologi-cally naive pasture, and after 2 weeks the pigs in groups 2 and 3 were experimentally infected with 10,800 O. dentatum and 8,700 H. rubidus infective larvae, respectively. Pigs in group 1 served as non-infected controls. All pigs were reared together on the experimental pasture for further 134 days until slaughter in October. Strongyle egg counts, differentiation of infective larvae at species level, serum pepsinogen, and herbage larval infectivity were monitored at regular intervals throughout. Both strongyle species established in the originally parasite-free pigs (group 1) and cross infections were established in group 2 and 3. The pigs were exposed to steadily increasing herbage infectivity of both species of strongyles. At the end of the experiment, geometric mean worm burdens of O. dentatum in groups 1,2 and 3 were 1202, 6136 and 1431 respectively, the burden in group 2 being significantly higher (p<0.05) than that of the 2 other groups. The geometric mean worm burdens of H. rubidus in groups 1, 2 and 3 were 4907, 3679 and 5246 respectively, showing no significant differences between groups.     

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.