A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing.

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

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 A(2) 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.

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 quadrispinulatum (Marcone, 1901) Alicata, 1935--a new for Poland parasite of swine]. / Oesophagostomum quadrispinulatum (Marcone, 1901) Alicata, 1935--nowy dla Polski pasoiyt swin.

MATERIAL AND METHODS: Large intestines of five slaughtered sows, aging 10 months to 3 years and originating from different herds of southern Poland, were examined parasitologically according to an agar-gel method. The intestines were uncoiled and divided into three sections: section 1--caecum and the first 0-20% part of colon, section 2-20--60% of large intestine and section 3--60-100% length of gut. Adult worms of Oesophagostomum were differentiated on the basis of species and sex, using the shape of buccal capsule and oesophagus, the length of male spicules, and the distance from vulva to anus as well as from anus to the tip of tail of females. RESULTS: In the case of two sows, in 30% of the contents from the section 1 of large intestines a total of 10 specimens of Oesophagostomum quadrispinulatum (Marcone, 1901) Alicata, 1935 were found. This is a new nematode species in the parasitic fauna of Poland and the present record enlarges its geographical range. The infection with O. quadrispinulatum seems to be more dangerous from that of O. dentatum--the most common porcine nodular worm.

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.

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

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

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

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

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.

Population dynamics of the parasitic stages of Oesophagostomum dentatum in pigs in single and trickle infections.

A mathematical model was developed to describe the dynamics of the parasitic stages of Oesophagostomum dentatum in pigs. An immigration-death model with constant establishment, development and death rates was fitted to L3, L4 and adult worm burdens observed in a single-infection experiment. Female worm length was modelled by a function of worm age and total worm burden, while worm egg production (eggs per gram faeces per female worm) was modelled by a function of worm age and worm length. The model was then used to predict worm burdens observed in a trickle-infection experiment. The predicted worm burdens were much higher than those observed, suggesting that worm death rates were higher during the trickle infection. After increasing worm death rates to fit the observed worm burdens, female worm lengths and egg production in the trickle infection were predicted. At the medium- and high-dose rates, predicted worm lengths and, thus, egg production were lower than observed, while at the low-dose rate predicted egg production was too high. It appeared that in the trickle infections, total worm burden had less influence on observed female worm length and egg production than in the single infections. The results suggest that the demography of O. dentatum in pigs differs between single and trickle infections.

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.

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

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

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.

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

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

The role of pigs as transport hosts of the human helminths Oesophagostomum bifurcum and Necator americanus.

We conducted a study in an endemic area of both Oesophagostomum bifurcum and Necator americanus in northern Ghana to examine the possibility of pigs acting as transport hosts for these two human helminth species, due to the commonly observed coprophagic habits of pigs. Under controlled conditions four parasite-free pigs consumed fresh faeces from people heavily infected with both helminths, and faeces were subsequently collected from the rectum of the pigs from 5 to 50 h post-feeding. Four to five per cent of the O. bifurcum and N. americanus eggs fed to the pigs were viable and retrieved as third-stage larvae after coproculture of the pigs' faeces. We discuss the possible impact of the coprophagic habits of pigs as potential parasite transport hosts during different seasons in this area of West Africa.

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.

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.

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.

A possible influence of diet composition on the establishment of nematodes in the pig.

Two groups of 20 pigs weighing approx. 26 kg were offered either ground barley plus protein supplement (GBP) or commercial pelleted feed (CPF), two diets consisting of nearly the same amounts of carbohydrates, proteins and micronutrients. All pigs were experimentally infected with 600 infective Ascaris suum eggs and 6000 infective Oesophagostomum spp. larvae and followed coprologically for approx. 12 weeks post infection, whereafter they were slaughtered. The number of Oesophagostomum spp. and A. suum eggs was significantly higher in the GBP-fed pigs compared with the CPF-fed pigs (P < 0.0001). The mean worm burden of adult Oesophagostomum spp. was higher in the GBP-pigs than in the CPF-pigs (P < 0.05), while the mean worm burden of adult A. suum in the two groups was not significantly different. The results indicate that two diets, both fulfilling the requirements of modern pig raising, may result in different establishment rates of natural adult gastrointestinal helminths in swine.

Development and distribution of Oesophagostomum columbianum in young lambs after oral or intraruminal infection.

There was no difference in the establishment or rate of development of Oesophagostomum columbianum in young lambs after infective larvae were administered either orally or by injection directly into the rumen. However, the linear distributions in the intestine of encysted third-stage larvae differed according to the route of infection. The distributions of fourth-stage larvae, after migration to the large intestine, did not differ. It is suggested that the distribution of parasitic third-stage O. columbianum is host-dominated, depending on the site of exsheathment and the rate of passage of ingesta, but that of fourth-stage and adult worms involves active site selection by the parasite.

Relative contributions of late dry-season and early rains pasture contaminations with trichostrongyle eggs to the wet-season herbage infestation in eastern Nigeria.

Two worm-free grass paddocks, P1 and P2, were artificially contaminated in March and April-May, respectively, with bovine faeces containing known numbers of trichostrongyle (mainly Cooperia, Haemonchus and Trichostrongylus spp.) eggs in order to determine the relative contributions of late dry-season and early rains pasture contaminations to the wet-season herbage larval infestation in Nsukka, eastern Nigeria. The resulting herbage infestation was assessed by means of larval counts and tracer studies. A sudden rise in herbage infestation occurred simultaneously in both paddocks in late April, this apparently being determined by the onset of the first substantial rainfall of the wet season. Peak infestations in both paddocks also occurred simultaneously in May. The infestation in P1 was much larger, and the larval population persisted longer, than that in P2 and later gave rise to a second smaller peak in June. No L3 were recovered in herbage samples from either of the paddocks after the third week of July. Both paddocks were infective to goats in May-June, while P2 was also infective in July-August. The results suggest that in the Nigerian derived savanna the initial wet-season herbage infestation in pastures grazed by infected cattle during the dry and wet seasons will consist of L3 from late dry-season and early rains pasture contaminations, the former being the major contributor to the infestation. Consequently, pastures contaminated during the late dry season may not be safe for susceptible animals to graze at the start of the succeeding rainy season.