BACKGROUND
parasitic nematodes of livestock cause substantial economic losses due to poor productivity, failure to thrive and deaths  <cit> .
the financial losses associated with these endoparasites are estimated at billions of dollars per annum  <cit> .
lungworms of the genus dictyocaulus  are key parasitic nematodes which cause pathological effects and clinical disease in different ruminant hosts, particularly in young animals  <cit> .
dictyocaulus viviparus, the bovine lungworm, causes a severe and frequently fatal bronchitis  which is of major importance in many countries  <cit> .
severe cases of dictyocaulosis lead to emphysema and pneumonia – heavy infections can cause a mortality rate of >20% among affected cattle  <cit> .
dictyocaulus viviparus has a direct life cycle  <cit> .
the adult stages  live in the bronchi, where the ovoviviparous females produce eggs from which first-stage larvae  usually hatch rapidly whilst in the lung or the intestinal tract.
the l1s are then shed in the faeces of the bovine host.
under favourable environmental conditions, l1s develop through to the infective third-stage larvae  during a period of ~4– <dig> days.
after ingestion by the host, l3s migrate through the gut wall to the mesenteric lymph nodes, moult, and, as fourth-stage larvae , are transported to the lungs.
l4s penetrate the alveoli, moult and then develop into adults.
however, larval stages can remain inhibited in the lungs for up to  <dig> months.
in cattle, the period from ingestion of l3s to reproductive maturity of the adult worms is 3– <dig> weeks.
while there is considerable knowledge of the morphological changes taking place during the life cycle of d. viviparus, very little is understood about the fundamental molecular and biochemical processes underlying the development and survival of this parasite and the parasite-host interplay.
insights into such processes are fundamentally important and could provide a basis for the identification of molecular targets for the rational design of nematocidal compounds, vaccines or/and for diagnosis.
to date, studies of d. viviparus have been limited to individual genes and proteins.
for instance, before the present study ,  <dig> gene sequences,  <dig> protein sequences and  <dig> research articles relating to d. viviparus were available in public databases.
current technological advances in genomics provide exciting opportunities for exploring basic molecular biological and biochemical aspects of d. viviparus and related nematodes.
for instance, expressed sequence tag  data sets facilitate the prediction and categorization of key molecules, particularly those linked to development , sexual differentiation and maturation, based on comparisons with other organisms for which sequence and functional genomic data sets are available.
also, the complete genome sequence of the free-living nematode caenorhabditis elegans and the wealth of information on gene expression and function for this nematode  <cit>  provide a means of evaluating homologues and orthologues  <cit> , since d. viviparus cannot be maintained or propagated effectively in vitro for the functional testing of genes and gene products.
also, the potential of gene silencing techniques  <cit>  provides a prospect for the functional analysis of molecules in this and other parasitic nematodes.
in the present study, we provide a first insight into the transcriptome of the adult stage of d. viviparus via est sequencing and apply a newly established computational platform  <cit>  for the clustering and comparative analyses of the data set against data available for a range of organisms, with an emphasis on the best characterized nematode, c. elegans.
the representative ests from this dataset have been annotated functionally at the gene and protein levels to aid in assigning, in the main, gene ontologies, protein families and biochemical pathways.
such annotation techniques have enabled us to pin-point genes that could be considered in the development of intervention strategies.
the present data provide a foundation for future investigations in areas, such as the stage-, sex- and tissue-specific gene transcription or expression, whole genome sequencing and proteomics of d. viviparus.
RESULTS
general est analysis
of  <dig> clones sequenced, a total of  <dig> quality ests were obtained , achieving a sequencing success of 84% , which is consistent with previous studies  <cit> .
these pre-processed ests ranged from 80– <dig> bp, with a mean of  <dig> bp and a standard deviation  of  <dig> bp.
after clustering, the mean length of the contigs  increased to  <dig>  bp.
the g+c content of the coding sequences was  <dig> %, consistent with other nematodes from clade v  <cit>  and slightly more than c. elegans  and its congener, c. briggsae   <cit> .
under the assumption that the d. viviparus genome codes for ~ <dig>  proteins  <cit> , the est clusters were predicted to represent ~10–15% of the proteins encoded by this genome.
the cluster analysis of the  <dig> ests from adult d. viviparus yielded  <dig> representative ests , of which  <dig>  had open reading frames .
all rests were then subjected to analyses using estexplorer  <cit> , a semi-automated bioinformatics pipeline .
also, we queried rests  against three databases containing protein sequences from different organisms, in order to categorize the molecules from d. viviparus.
data were compared with protein sequences available for  c. elegans ,  parasitic nematodes , and  organisms other than nematodes   <cit> .
three-way comparison of d. viviparus rests with homologues from c. elegans, wormpep and parasitic nematodes have been figuratively presented using simitri  <cit>  .
for this comparison, similarity searches of the  <dig> representative sequences resulted in  <dig>  homologues to c. elegans,  <dig>  to those from other parasitic nematodes ,  <dig>  homologues in organisms other than nematodes, and  <dig> % had no significant similarity to any other organism  for which sequence data are currently available .
the simitri plot  shows that, given the current database contents, the sampled transcriptome from d. viviparus is equally close to available c. elegans and parasitic nematode sequences, compared with non-nematodes.
comparative analyses with c. elegans data sets
the comparative analysis to identify homologues in c. elegans is important because d. viviparus and this free-living nematode are both considered to belong to clade v of the nematoda  <cit> , and because c. elegans also represents the best characterized nematode in many respects, particularly in terms of its genome, genetics, biology, physiology, biochemistry, as well as the localization and functions of molecules  <cit> .
specifically, the comparative analysis  of all rests with c. elegans data  revealed  <dig>  key, well-characterized molecules associated with various biological processes , including development, regulation of biological processes, response to abiotic and biotic stimuli and reproduction.
'non-wildtype' rnai phenotypes in c. elegans  were associated with  <dig>  of these  <dig> molecules .
of the  <dig> c. elegans homologues, the functions for  <dig>  rests could be inferred using gene ontologies   <cit> , with  <dig>  sequences being mapped to key biological pathways .
overall, the functional classification revealed that approximately half of the rests had homologues in c. elegans and parasitic nematodes, one tenth were specific to parasitic nematodes, and one third of the rests did not match any sequence in current databases, possibly representing novel genes.
as multiple ests can be derived from the same gene, it was important to predict how many unique genes were represented by the rests.
mapping the  <dig> d. viviparus rests to c. elegans revealed that, of the  <dig> d. viviparus rests with similarities to  <dig> c. elegans genes, the majority of these  had a one-to-one relationship to their c. elegans homologue.
the remaining  <dig> rests mapped to multiple non-overlapping regions from  <dig> c. elegans genes .
after discounting for fragmentation, we estimated that  <dig> unique d. viviparus genes were identified, with a suggested new gene discovery rate of  <dig> % .
c. elegans  rnai phenotypes can provide some indication of the relevance and functions of orthologous genes in other nematodes, particularly in parasitic nematodes of clade v, for which the complexity of an obligate parasitic life cycle and the lack of an effective  laboratory culture system make high-throughput functional screening impractical  <cit> .
we retrieved c. elegans rnai data representing d. viviparus homologues.
of  <dig> d. viviparus rests,  <dig> had homologues in c. elegans which could be silenced by rnai .
the rnai phenotypes  included adl , age , bmd , dpy , egl , emb , gro , let , lvl , lva  and unc , whereas  <dig> homologues had no observable rnai phenotype in c. elegans.
we also found that 23% of the most abundant  and 22% of the transcripts predicted to represent secreted proteins of d. viviparus  had c. elegans homologues with non-wildtype rnai phenotypes.
comparative analysis with data for other nematodes within clade v
d. viviparus is a member of clade v of the phylum nematoda  <cit> , which comprises members of the orders strongylida, rhabditida and diplogasterida.
we conducted a comparative analysis of all  <dig> rests with data  <cit>  for various nematodes  belonging to clade v , to examine gene conservation within this clade.
the analysis revealed  <dig>  rests to have significant similarity to molecules from the members of clade v. these rests represented house-keeping  as well as nematode-specific  genes.
we could assign go terms to  <dig> of these  <dig> rest sequences, associated with  <dig> different biological processes, such as embryonic development, intracellular protein transport, protein metabolism and responses to abiotic and/or biotic stimuli.
furthermore,  <dig> sequences could be mapped to biological pathways predicted to be associated with ribosomal proteins and the proteasome system, including  <dig> predicted secreted proteins mapping to cysteine proteinases, secreted protein  <dig> precursor and parasite pepsinogen.
abundant transcripts in adult d. viviparus
a high level of representation in a cdna library usually correlates with high transcript abundance in the original biological sample  <cit> , although artefacts of library construction can result in a selection for or against representation of some transcripts.
the d. viviparus clusters were ranked according to the number of contributing ests, and the top  <dig> clusters, which represented  <dig>  of the total number of rests  obtained, were investigated in detail .
a number of clusters had significant alignments to known proteins, the majority of which were house-keeping genes, such as elongation factors, ribosomal proteins, aldolases, kinases, proteases and actins.
some of these genes have been identified in a number of other nematodes, including ancylostoma caninum, ancylostoma ceylanicum, dirofilaria immitis, strongyloides ratti and meloidogyne incognita  <cit> , requiring detailed characterisation to understand their functions in d. viviparus.
we analysed the most abundantly expressed transcripts from d. viviparus and found that  <dig> of the  <dig>  contigs had no significant similarity to any sequence in the non-redundant protein database.
as most of the nematode data are available only as ests and not included in the blast databases, we further compared these  <dig> contigs with sequences against parasite genomes using wu-blast <dig> and blastn against the ncbi 'other-ests' database.
we found that five sequences did not match any sequence in the databases, whereas seven entries had similarity to ests from other nematodes.
the other  <dig>  contigs were assigned functionality based on blastp against the nr database, and all of them had homologues in either non-parasitic  or parasitic nematodes.
a summary of these findings is provided in table  <dig>  most of the homologues were found to be house-keeping or structural genes , and four contigs were represented by molecules specific to nematodes, such as the major sperm proteins.
comparison with cdnas from third-stage larvae of d. viviparus
recently, strube et al.
<cit>  identified and characterized  <dig> cdnas differentially transcribed between experimentally induced hypobiotic and infective third-stage larvae  of d. viviparus using a suppressive-subtractive hybridization  approach.
we compared these  <dig> sequences against our dataset of  <dig> rests, using blastn, to identify whether any of them were represented in the adult stage, with only one match.
the sole sequence common to both datasets was l3ni  <dig> , which matched est d.viviparus_42_a <dig> in the present dataset, being a homologue of the c. elegans hypothetical protein c05d <dig>  .
thus, this est study of adult d. viviparus represents a novel dataset for parasitic nematodes in clade v, representing the metastrongyloidea .
functional classification of rests from adult d. viviparus
we annotated all  <dig> rests systematically using a range of bioinformatic tools .
this annotation included functional classifications of rests using gene ontologies   <cit> , pathway mapping using kegg   <cit> , visualisation of est data comparisons using simitri  <cit>  and analyses of the d. viviparus secretome using signalp  <cit> , tmhmm  <cit>  and psortii  <cit> .
results of these analyses are described in the following two sections
a. gene ontologies
gene ontology  has been used widely to predict gene function and classification.
go provides a dynamic vocabulary and hierarchy that unifies descriptions of biological, cellular and molecular functions across genomes.
we used blast2go  <cit> , a sequence-based tool to assign go terms, extracting them for each blast hit obtained by mapping to extant annotation associations.
we found that  <dig>  of  <dig> rests could be functionally assigned to biological processes , cellular components  and molecular functions .
a summary go representation  of the d. viviparus rests is given in table  <dig>
amongst the most common go categories representing biological processes were: binding , catalytic activity  and structural molecule activity ; development , metabolism , reproduction  and growth .
the largest number of go terms in cellular components was for cell part , membrane-bound organelle  and non-membrane-bound organelle .
a complete listing of go mappings assigned for rests is provided in additional file  <dig>
b. pathway analysis using kegg assignments
biochemical functionality was predicted by mapping all  <dig> rests to pathways, using kobas implemented within estexplorer  <cit> , with an e-value cut-off of  <dig> e- <dig>  enzyme commission  numbers were used to appraise which sequences pertained to a specific pathway.
a total of  <dig>  sequences were mapped to  <dig> kegg pathways, with  <dig> sequences representing metabolic enzymes characterized by unique ec numbers.
the top  <dig>  pathways are shown in table  <dig>
molecules involved in signal transduction mechanisms  and glycolysis/gluconeogenesis  had the highest representation amongst the sequences mapped to kegg pathways.
we also identified  <dig> predicted proteins with potential roles in host-parasite interactions, with  <dig> molecules predicted to be involved in antigen processing and/or presentation, six in t-cell receptor signalling pathway and seven cd molecules.
although, at this stage, the precise role of such molecules in the parasite-host interplay is unclear, they could be involved in manipulating or evading the host's immune response or associated with the parasite's innate immune response.
extensive experimental work would be required to test these proposals.
furthermore, we identified families of proteins representing serine, cysteine and metallo-proteinases as well as proteinase inhibitors .
while these enzymes are inferred to mediate or modulate proteolytic functions, which, in turn, may facilitate tissue migration and other interactions with host cells, the proteinase inhibitors may protect the parasite against digestion by host or endogenous proteinases excreted/secreted in the lung of the mammalian host  <cit> .
a complete listing of the kegg mappings is available as supplementary data .
secretome analysis
an important starting point in the identification of potential novel drug or vaccine candidates in parasites is the prediction of molecules that are secreted or excreted in or around the host- parasite interface  <cit> .
examples of such proteins are the aspartyl protease inhibitor   <cit> , mi-msp- <dig>   <cit>  and the ancylostoma-secreted protein   <cit> .
in the present data set , we identified  <dig> putatively secreted proteins representing a non-redundant catalogue of d. viviparus molecules .
of these,  <dig>  sequences had no significant similarity to any sequence available in current databases, whereas  <dig>  had homologues in nematodes, with  <dig>  c. elegans and/or c. briggsae matches, and  <dig>  homologues in various other parasitic nematodes, including the blood-feeding nematodes ancylostoma ceylanicum, necator americanus and haemonchus contortus.
the secretome analysis  revealed a number of unique features.
firstly, seven of the putative secreted protein entries were homologous to either sperm-specific family members  <cit>  or major sperm proteins , consistent with the mass spectrometric analysis of secreted molecules from d. viviparus  <cit> .
secondly, acetylcholinesterase  has been identified as an important enzyme secreted by adult d. viviparus, thought to be involved in parasite survival in the host and, therefore, being a vaccine candidate  <cit> .
we now report an entry for secreted acetylcholinesterase  in the secretome analysis of the current est dataset , which may also represent a target for further characterization.
CONCLUSIONS
the present study has given us a first glimpse of the transcriptome of the adult stage of the bovine lungworm, d. viviparus, and represents a starting point for studies in a number of different fundamental and applied areas.
we used a comprehensive est analysis pipeline, estexplorer, for this purpose, for functional annotation at the dna and protein levels  <cit> .
from this single study of  <dig> ests, we have identified  <dig> novel sequences , with high confidence, with no known homologue in any other nematode or mammal for which sequence data are presently available in public databases.
these molecules are particularly interesting, as they may represent genes that may be specific to parasitism or to the species.
however, such molecules are very challenging to work on, as their potential functions cannot be predicted using current bioinformatic approaches.
however, there is considerable scope in exploring such molecules in the future, using a combination of genomic and proteomic approaches.
insights into such molecules and/or their interaction with the bovine host could provide opportunities for developing novel intervention approaches.
from a systematic viewpoint, d. viviparus, belongs to the metastrongyloidea  based on nuclear ribosomal dna sequence data  <cit> , as distinct from the trichostrongyloidea , strongyloidea  and ancylostomatoidea , and thus represents, from biological, host-parasite relationship and molecular evolutionary perspectives, a very interesting species for comparative genomic analysis with nematodes from these superfamilies.
therefore, this nematode brings a number of important benefits for future investigations, particularly for genome sequencing and for subsequent comparative evolutionary analyses.
indeed, d. viviparus, among other strongylid nematodes, has recently been selected for whole genome sequencing, to be carried out at the genome sequencing center of washington university in st louis, usa  <cit> .
for d. viviparus, the genomic information from the present study underpins future microarray analyses, focused on exploring the transcriptional profiles among different stages  versus infective versus late larval stages), sexes  and tissues  of the parasite.
such studies, particularly those of the molecules differentially transcribed and expressed during the transition to parasitism, the invasion of the host and hypobiosis, could provide unique insights into such key molecular developmental and reproductive processes.
while there is some controversy regarding the applicability and usefulness of rnai to some parasitic nematodes, such as the strongylida  <cit> , comparative studies of gene-silencing and transgenesis in c. elegans are considered useful for exploring the function and regulation of some relatively conserved parasite genes, provided data are interpreted with caution  <cit> .
this is particularly the case with the continued increase in genome sequence information.
with the future availability of whole genome sequence data for d. viviparus, it will also be possible to carry out meaningful mass spectroscopic analyses of differentially expressed proteins  <cit> , allowing large-scale analysis of proteins from small amounts of parasite material.
such analyses will enable the link to be made between the regulation of transcription and translation and, importantly, in the study of parasites, will allow the analysis of proteins expressed within short time frames within or external to the host animal, or within organs or micro-environments within the parasite  <cit> .
hence, the application of an integrated bioinformatic-genomic-phenomic-proteomic  approach, focusing on developmental processes and mechanisms, could enhance our understanding of the molecular biology of moulting, invasion of and establishment in the host, hypobiosis , and sexual differentiation, maturation and behaviour of d. viviparus.
clearly, progress in such fundamental areas could lead to the development of exciting new ways of treating, controlling or preventing this lungworm and other parasitic nematodes, by blocking or disrupting key biological pathways in them.

 parasitic nematodes of livestock cause substantial economic losses due to poor productivity, failure to thrive and deaths. the financial losses associated with these endoparasites are estimated at billions of dollars per annum. lungworms of the genus dictyocaulus are key parasitic nematodes which cause pathological effects and clinical disease in different ruminant hosts, particularly in young animals. dictyocaulus viviparus, the bovine lungworm, causes a severe and frequently fatal bronchitis which is of major importance in many countries. severe cases of dictyocaulosis lead to emphysema and pneumonia – heavy infections can cause a mortality rate of >20% among affected cattle. dictyocaulus viviparus has a direct life cycle. the adult stages live in the bronchi, where the ovoviviparous females produce eggs from which first-stage larvae usually hatch rapidly whilst in the lung or the intestinal tract. the l1s are then shed in the faeces of the bovine host. under favourable environmental conditions, l1s develop through to the infective third-stage larvae during a period of ~4– days. after ingestion by the host, l3s migrate through the gut wall to the mesenteric lymph nodes, moult, and, as fourth-stage larvae, are transported to the lungs. l4s penetrate the alveoli, moult and then develop into adults. however, larval stages can remain inhibited in the lungs for up to months. in cattle, the period from ingestion of l3s to reproductive maturity of the adult worms is 3– weeks. while there is considerable knowledge of the morphological changes taking place during the life cycle of d. viviparus, very little is understood about the fundamental molecular and biochemical processes underlying the development and survival of this parasite and the parasite-host interplay. insights into such processes are fundamentally important and could provide a basis for the identification of molecular targets for the rational design of nematocidal compounds, vaccines or/and for diagnosis. to date, studies of d. viviparus have been limited to individual genes and proteins. for instance, before the present study, gene sequences, protein sequences and research articles relating to d. viviparus were available in public databases. current technological advances in genomics provide exciting opportunities for exploring basic molecular biological and biochemical aspects of d. viviparus and related nematodes. for instance, expressed sequence tag data sets facilitate the prediction and categorization of key molecules, particularly those linked to development, sexual differentiation and maturation, based on comparisons with other organisms for which sequence and functional genomic data sets are available. also, the complete genome sequence of the free-living nematode caenorhabditis elegans and the wealth of information on gene expression and function for this nematode provide a means of evaluating homologues and orthologues, since d. viviparus cannot be maintained or propagated effectively in vitro for the functional testing of genes and gene products. also, the potential of gene silencing techniques provides a prospect for the functional analysis of molecules in this and other parasitic nematodes. in the present study, we provide a first insight into the transcriptome of the adult stage of d. viviparus via est sequencing and apply a newly established computational platform for the clustering and comparative analyses of the data set against data available for a range of organisms, with an emphasis on the best characterized nematode, c. elegans. the representative ests from this dataset have been annotated functionally at the gene and protein levels to aid in assigning, in the main, gene ontologies, protein families and biochemical pathways. such annotation techniques have enabled us to pin-point genes that could be considered in the development of intervention strategies. the present data provide a foundation for future investigations in areas, such as the stage-, sex- and tissue-specific gene transcription or expression, whole genome sequencing and proteomics of d. viviparus.  general est analysis of clones sequenced, a total of quality ests were obtained, achieving a sequencing success of 84%, which is consistent with previous studies. these pre-processed ests ranged from 80– bp, with a mean of bp and a standard deviation of bp. after clustering, the mean length of the contigs increased to bp. the g+c content of the coding sequences was %, consistent with other nematodes from clade v and slightly more than c. elegans and its congener, c. briggsae. under the assumption that the d. viviparus genome codes for ~ proteins, the est clusters were predicted to represent ~10–15% of the proteins encoded by this genome. the cluster analysis of the ests from adult d. viviparus yielded representative ests, of which had open reading frames. all rests were then subjected to analyses using estexplorer, a semi-automated bioinformatics pipeline. also, we queried rests against three databases containing protein sequences from different organisms, in order to categorize the molecules from d. viviparus. data were compared with protein sequences available for c. elegans, parasitic nematodes, and organisms other than nematodes. three-way comparison of d. viviparus rests with homologues from c. elegans, wormpep and parasitic nematodes have been figuratively presented using simitri. for this comparison, similarity searches of the representative sequences resulted in homologues to c. elegans, to those from other parasitic nematodes, homologues in organisms other than nematodes, and % had no significant similarity to any other organism for which sequence data are currently available. the simitri plot shows that, given the current database contents, the sampled transcriptome from d. viviparus is equally close to available c. elegans and parasitic nematode sequences, compared with non-nematodes. comparative analyses with c. elegans data sets the comparative analysis to identify homologues in c. elegans is important because d. viviparus and this free-living nematode are both considered to belong to clade v of the nematoda, and because c. elegans also represents the best characterized nematode in many respects, particularly in terms of its genome, genetics, biology, physiology, biochemistry, as well as the localization and functions of molecules. specifically, the comparative analysis of all rests with c. elegans data revealed key, well-characterized molecules associated with various biological processes, including development, regulation of biological processes, response to abiotic and biotic stimuli and reproduction.'non-wildtype' rnai phenotypes in c. elegans were associated with of these molecules. of the c. elegans homologues, the functions for rests could be inferred using gene ontologies, with sequences being mapped to key biological pathways. overall, the functional classification revealed that approximately half of the rests had homologues in c. elegans and parasitic nematodes, one tenth were specific to parasitic nematodes, and one third of the rests did not match any sequence in current databases, possibly representing novel genes. as multiple ests can be derived from the same gene, it was important to predict how many unique genes were represented by the rests. mapping the d. viviparus rests to c. elegans revealed that, of the d. viviparus rests with similarities to c. elegans genes, the majority of these had a one-to-one relationship to their c. elegans homologue. the remaining rests mapped to multiple non-overlapping regions from c. elegans genes. after discounting for fragmentation, we estimated that unique d. viviparus genes were identified, with a suggested new gene discovery rate of %. c. elegans rnai phenotypes can provide some indication of the relevance and functions of orthologous genes in other nematodes, particularly in parasitic nematodes of clade v, for which the complexity of an obligate parasitic life cycle and the lack of an effective laboratory culture system make high-throughput functional screening impractical. we retrieved c. elegans rnai data representing d. viviparus homologues. of d. viviparus rests, had homologues in c. elegans which could be silenced by rnai. the rnai phenotypes included adl, age, bmd, dpy, egl, emb, gro, let, lvl, lva and unc, whereas homologues had no observable rnai phenotype in c. elegans. we also found that 23% of the most abundant and 22% of the transcripts predicted to represent secreted proteins of d. viviparus had c. elegans homologues with non-wildtype rnai phenotypes. comparative analysis with data for other nematodes within clade v d. viviparus is a member of clade v of the phylum nematoda, which comprises members of the orders strongylida, rhabditida and diplogasterida. we conducted a comparative analysis of all rests with data for various nematodes belonging to clade v, to examine gene conservation within this clade. the analysis revealed rests to have significant similarity to molecules from the members of clade v. these rests represented house-keeping as well as nematode-specific genes. we could assign go terms to of these rest sequences, associated with different biological processes, such as embryonic development, intracellular protein transport, protein metabolism and responses to abiotic and/or biotic stimuli. furthermore, sequences could be mapped to biological pathways predicted to be associated with ribosomal proteins and the proteasome system, including predicted secreted proteins mapping to cysteine proteinases, secreted protein precursor and parasite pepsinogen. abundant transcripts in adult d. viviparus a high level of representation in a cdna library usually correlates with high transcript abundance in the original biological sample, although artefacts of library construction can result in a selection for or against representation of some transcripts. the d. viviparus clusters were ranked according to the number of contributing ests, and the top clusters, which represented of the total number of rests obtained, were investigated in detail. a number of clusters had significant alignments to known proteins, the majority of which were house-keeping genes, such as elongation factors, ribosomal proteins, aldolases, kinases, proteases and actins. some of these genes have been identified in a number of other nematodes, including ancylostoma caninum, ancylostoma ceylanicum, dirofilaria immitis, strongyloides ratti and meloidogyne incognita, requiring detailed characterisation to understand their functions in d. viviparus. we analysed the most abundantly expressed transcripts from d. viviparus and found that of the contigs had no significant similarity to any sequence in the non-redundant protein database. as most of the nematode data are available only as ests and not included in the blast databases, we further compared these contigs with sequences against parasite genomes using wu-blast and blastn against the ncbi'other-ests' database. we found that five sequences did not match any sequence in the databases, whereas seven entries had similarity to ests from other nematodes. the other contigs were assigned functionality based on blastp against the nr database, and all of them had homologues in either non-parasitic or parasitic nematodes. a summary of these findings is provided in table most of the homologues were found to be house-keeping or structural genes, and four contigs were represented by molecules specific to nematodes, such as the major sperm proteins. comparison with cdnas from third-stage larvae of d. viviparus recently, strube et al. identified and characterized cdnas differentially transcribed between experimentally induced hypobiotic and infective third-stage larvae of d. viviparus using a suppressive-subtractive hybridization approach. we compared these sequences against our dataset of rests, using blastn, to identify whether any of them were represented in the adult stage, with only one match. the sole sequence common to both datasets was l3ni, which matched est d.viviparus_42_a in the present dataset, being a homologue of the c. elegans hypothetical protein c05d. thus, this est study of adult d. viviparus represents a novel dataset for parasitic nematodes in clade v, representing the metastrongyloidea. functional classification of rests from adult d. viviparus we annotated all rests systematically using a range of bioinformatic tools. this annotation included functional classifications of rests using gene ontologies, pathway mapping using kegg, visualisation of est data comparisons using simitri and analyses of the d. viviparus secretome using signalp, tmhmm and psortii. results of these analyses are described in the following two sections a. gene ontologies gene ontology has been used widely to predict gene function and classification. go provides a dynamic vocabulary and hierarchy that unifies descriptions of biological, cellular and molecular functions across genomes. we used blast2go, a sequence-based tool to assign go terms, extracting them for each blast hit obtained by mapping to extant annotation associations. we found that of rests could be functionally assigned to biological processes, cellular components and molecular functions. a summary go representation of the d. viviparus rests is given in table amongst the most common go categories representing biological processes were: binding, catalytic activity and structural molecule activity; development, metabolism, reproduction and growth. the largest number of go terms in cellular components was for cell part, membrane-bound organelle and non-membrane-bound organelle. a complete listing of go mappings assigned for rests is provided in additional file b. pathway analysis using kegg assignments biochemical functionality was predicted by mapping all rests to pathways, using kobas implemented within estexplorer, with an e-value cut-off of e- enzyme commission numbers were used to appraise which sequences pertained to a specific pathway. a total of sequences were mapped to kegg pathways, with sequences representing metabolic enzymes characterized by unique ec numbers. the top pathways are shown in table molecules involved in signal transduction mechanisms and glycolysis/gluconeogenesis had the highest representation amongst the sequences mapped to kegg pathways. we also identified predicted proteins with potential roles in host-parasite interactions, with molecules predicted to be involved in antigen processing and/or presentation, six in t-cell receptor signalling pathway and seven cd molecules. although, at this stage, the precise role of such molecules in the parasite-host interplay is unclear, they could be involved in manipulating or evading the host's immune response or associated with the parasite's innate immune response. extensive experimental work would be required to test these proposals. furthermore, we identified families of proteins representing serine, cysteine and metallo-proteinases as well as proteinase inhibitors. while these enzymes are inferred to mediate or modulate proteolytic functions, which, in turn, may facilitate tissue migration and other interactions with host cells, the proteinase inhibitors may protect the parasite against digestion by host or endogenous proteinases excreted/secreted in the lung of the mammalian host. a complete listing of the kegg mappings is available as supplementary data. secretome analysis an important starting point in the identification of potential novel drug or vaccine candidates in parasites is the prediction of molecules that are secreted or excreted in or around the host- parasite interface. examples of such proteins are the aspartyl protease inhibitor, mi-msp- and the ancylostoma-secreted protein. in the present data set, we identified putatively secreted proteins representing a non-redundant catalogue of d. viviparus molecules. of these, sequences had no significant similarity to any sequence available in current databases, whereas had homologues in nematodes, with c. elegans and/or c. briggsae matches, and homologues in various other parasitic nematodes, including the blood-feeding nematodes ancylostoma ceylanicum, necator americanus and haemonchus contortus. the secretome analysis revealed a number of unique features. firstly, seven of the putative secreted protein entries were homologous to either sperm-specific family members or major sperm proteins, consistent with the mass spectrometric analysis of secreted molecules from d. viviparus. secondly, acetylcholinesterase has been identified as an important enzyme secreted by adult d. viviparus, thought to be involved in parasite survival in the host and, therefore, being a vaccine candidate. we now report an entry for secreted acetylcholinesterase in the secretome analysis of the current est dataset, which may also represent a target for further characterization.  the present study has given us a first glimpse of the transcriptome of the adult stage of the bovine lungworm, d. viviparus, and represents a starting point for studies in a number of different fundamental and applied areas. we used a comprehensive est analysis pipeline, estexplorer, for this purpose, for functional annotation at the dna and protein levels. from this single study of ests, we have identified novel sequences, with high confidence, with no known homologue in any other nematode or mammal for which sequence data are presently available in public databases. these molecules are particularly interesting, as they may represent genes that may be specific to parasitism or to the species. however, such molecules are very challenging to work on, as their potential functions cannot be predicted using current bioinformatic approaches. however, there is considerable scope in exploring such molecules in the future, using a combination of genomic and proteomic approaches. insights into such molecules and/or their interaction with the bovine host could provide opportunities for developing novel intervention approaches. from a systematic viewpoint, d. viviparus, belongs to the metastrongyloidea based on nuclear ribosomal dna sequence data, as distinct from the trichostrongyloidea, strongyloidea and ancylostomatoidea, and thus represents, from biological, host-parasite relationship and molecular evolutionary perspectives, a very interesting species for comparative genomic analysis with nematodes from these superfamilies. therefore, this nematode brings a number of important benefits for future investigations, particularly for genome sequencing and for subsequent comparative evolutionary analyses. indeed, d. viviparus, among other strongylid nematodes, has recently been selected for whole genome sequencing, to be carried out at the genome sequencing center of washington university in st louis, usa. for d. viviparus, the genomic information from the present study underpins future microarray analyses, focused on exploring the transcriptional profiles among different stages versus infective versus late larval stages), sexes and tissues of the parasite. such studies, particularly those of the molecules differentially transcribed and expressed during the transition to parasitism, the invasion of the host and hypobiosis, could provide unique insights into such key molecular developmental and reproductive processes. while there is some controversy regarding the applicability and usefulness of rnai to some parasitic nematodes, such as the strongylida, comparative studies of gene-silencing and transgenesis in c. elegans are considered useful for exploring the function and regulation of some relatively conserved parasite genes, provided data are interpreted with caution. this is particularly the case with the continued increase in genome sequence information. with the future availability of whole genome sequence data for d. viviparus, it will also be possible to carry out meaningful mass spectroscopic analyses of differentially expressed proteins, allowing large-scale analysis of proteins from small amounts of parasite material. such analyses will enable the link to be made between the regulation of transcription and translation and, importantly, in the study of parasites, will allow the analysis of proteins expressed within short time frames within or external to the host animal, or within organs or micro-environments within the parasite. hence, the application of an integrated bioinformatic-genomic-phenomic-proteomic approach, focusing on developmental processes and mechanisms, could enhance our understanding of the molecular biology of moulting, invasion of and establishment in the host, hypobiosis, and sexual differentiation, maturation and behaviour of d. viviparus. clearly, progress in such fundamental areas could lead to the development of exciting new ways of treating, controlling or preventing this lungworm and other parasitic nematodes, by blocking or disrupting key biological pathways in them. 
we could functionally classify sequences using the gene ontologies and established pathway associations to sequences in kyoto encyclopedia of genes and genomes. the bioinformatic analyses of ests data for d. viviparus has elucidated sets of relatively conserved and potentially novel genes. they cause pathological effects and clinical disease in various ruminant hosts, particularly in young animals. in this study, we provide first insights into the transcriptome of the adult stage of d. viviparus through the analysis of expressed sequence tags. of the c. elegans homologues, had observed'non-wildtype' rnai phenotypes, including embryonic lethality, maternal sterility, sterility in progeny, larval arrest and slow growth. the genes discovered in this study should assist research toward a better understanding of the basic molecular biology of d. viviparus, which could lead, in the longer term, to novel intervention strategies. the characterization of the d. viviparus transcriptome also provides a foundation for whole genome sequence analysis and future comparative transcriptomic analyses. lungworms of the genus dictyocaulus are parasitic nematodes of major economic importance. dictyocaulus viviparus, called the bovine lungworm, is a major pathogen of cattle, with severe infections being fatal. using our est analysis pipeline, we estimate that the present dataset of ests is derived from genes based on cluster and comparative genomic analyses of the ests. of the representative ests, had homologues in the free-living nematode c. elegans, in parasitic nematodes, in organisms other than nematodes, and had no significant match to any sequence in the current databases.
 lungworms of the genus dictyocaulus are parasitic nematodes of major economic importance. they cause pathological effects and clinical disease in various ruminant hosts, particularly in young animals. dictyocaulus viviparus, called the bovine lungworm, is a major pathogen of cattle, with severe infections being fatal. in this study, we provide first insights into the transcriptome of the adult stage of d. viviparus through the analysis of expressed sequence tags.  using our est analysis pipeline, we estimate that the present dataset of ests is derived from genes based on cluster and comparative genomic analyses of the ests. of the representative ests, had homologues in the free-living nematode c. elegans, in parasitic nematodes, in organisms other than nematodes, and had no significant match to any sequence in the current databases. of the c. elegans homologues, had observed'non-wildtype' rnai phenotypes, including embryonic lethality, maternal sterility, sterility in progeny, larval arrest and slow growth. we could functionally classify sequences using the gene ontologies and established pathway associations to sequences in kyoto encyclopedia of genes and genomes. in addition, we predicted secreted proteins which could represent potential candidates for developing novel anthelmintics or vaccines.  the bioinformatic analyses of ests data for d. viviparus has elucidated sets of relatively conserved and potentially novel genes. the genes discovered in this study should assist research toward a better understanding of the basic molecular biology of d. viviparus, which could lead, in the longer term, to novel intervention strategies. the characterization of the d. viviparus transcriptome also provides a foundation for whole genome sequence analysis and future comparative transcriptomic analyses. 