BACKGROUND
fasciola hepatica, the liver fluke, is a digenetic trematode helminth, causing highly damaging hepatobiliary disease  in mammalians including economically important ruminants  and humans  <cit> .
fasciolosis results in a significant economic loss in livestock industry worldwide and more cases have been reported in humans in different countries  <cit> .
the disease is currently treated with anti-helminthics , but the observed anti-helminthic drug resistance  <cit>  necessitates more effective strategies for the treatment and/or the prevention of fasciolosis.
for the elucidation of infection mechanisms of the liver fluke and the development of better strategies in dealing with fasciolosis, the most critical step is the identification and characterisation of the genes which are important for the establishment of parasitism.
the transcriptome of f. hepatica was reported in a previous publication where a previous sequencing platform  was employed for the purpose of describing general biological characteristics of the parasite  <cit> .
however, the sequence data from that study is likely to be still further from encapturing the entirety of the transcriptome profile of f. hepatica and the virulence factors of the parasite, particularly those related to host immunomodulation, are worthwhile for additional investigation with current ngs platforms .
the current ngs technologies produce greater transcriptomic data in comparison with the previous sequencing systems and increase the chance of detection of parasite transcripts which are expressed at lower levels  but with significant importance in immune evasion.
one of the most promising approaches for determining the virulence factors of parasitic organisms is in silico comparison of parasites’ transcripts with publicly available data  <cit> .
a recent data resource, the helminth secretome database   <cit> , contains a broad repertoire of excretory/secretory  protein sequences  of various parasitic helminths including trematodes, cestodes and nematodes.
es proteins of endoparasites are, in general, thought to play vital roles in the establishment of infection  <cit> .
additionally, vaccine investigation and online information network , provides gene and protein sequences that are affiliated with infection mechanisms of various micro  and macro  infectious organisms  <cit> .
compared to other resources providing whole genome data of numerous pathogens, both hsd and violin contain filtered data better suiting to glean pathogen-related molecules of infectious organisms.
however, the major caveat of the specialised databases is the presence of insufficiently refined data  that are related with regular physiological events in both parasitic and non-parasitic organisms, but not indeed linked to virulence.
parasitism genes which are not directly involved in virulence, but rather associated with regular physiological mechanisms, could be uncovered by sequence homology with taxonomically similar, free-living  organisms  <cit> .
very recently, a large nucleotide sequence collection from free-living/non-infectious trematodes  of dugesiidae family  has become publicly available at dna data bank of japan .
additionally, a comprehensive data for a well studied free-living model nematode, caenorhabditis elegans, is freely accessible from a regularly updated resource, wormbase .
the data of non-parasitic organisms from current resources have been useful tool to investigate the genes that are under directional selection through the comparative analysis of nucleotide diversity by assessing nonsynonymous/synonymous  substitution rates  <cit> .
it is a well known fact that parasitism related genes important for the evasion of defensive systems of host and the corresponding genes in the host are under constant selective pressure favoring nucleotide subsitutions  <cit> .
for example, the strong influence of directional selection in the evolution of avirulence genes to gain immunomodulatory properties has been clearly reported in multiple studies  <cit> .
furthermore, lineage-specific sub- or neo-functionalisation of genes which are vital for the establishment and maintenance of parasitism could be identified through comparative genomics  <cit> .
apart from the analysis of sequence homology with infectious and non-infectious organisms, the virulence genes associated with modulation of host immune responses  can be identified by the manual inspection of encoded protein motifs in public databases  <cit> .
to date, in vivo and in vitro studies have shown that the liver fluke modulates host immune responses for enhancing its virulence  <cit> ; however, which genes of the parasite imitate the components of host immune system have not yet been elucidated in detail.
the main purpose of this study was to glean virulence and immunomodulatory f. hepatica genes through comparative transcriptome profiling with the transcriptomes of non-parasitic related organisms by focusing on the genes which are evolved in lineage-specific manner, under positive selection and show similar motifs of host immune system genes involved in cytokine signaling.
RESULTS
transcriptome profile of f. hepatica
workflow illustrating the experimental steps of the study is demonstrated in figure  <dig>  from a total of  <dig> , <dig> sequence reads, generated by the sequencing instrument  with paired-end 2x 100 bp reading,  <dig>  contigs  were de novo assembled, of which,  <dig>  transcripts were annotated with blast searches  as described in additional file  <dig>  the obtained base number in this study was approximately  <dig>  times higher than that reported in the previous transcriptome study of f. hepatica  <cit> .
the transcript n <dig> was 522 bp and the length of a total of  <dig>  transcripts was equal or greater than the observed n <dig> length in this work.
in the present study, f. hepatica g + c content was  <dig> %, which was similar to that reported in the previous related studies of f. hepatica   <cit> .
the identified transcript sequences in our study corresponded to a total of  <dig>  unique accession numbers .figure  <dig> experimental design.
the transcriptome study is consisted of wet lab procedures and data analysis processes.
the parasite transcript sequences were analysed in terms of positive selection, cytokine signaling and pathogen database homology.
the parasite sequences showing the signs of positive selection  were termed psrs .
the cytokine signaling relation for the inferred parasite proteins were detected by the protein motif information and the related transcripts were termed csrs .
non-virulence-related transcripts  were determined by the sequence homology of the parasite transcripts with the data of the non-pathogen associated databases  by excluding the non-parasite organism homologous psrs and csrs.
the parasite transcripts showing the exclusive sequence homology with the pathogen-associated databases  , but not being nvts, were termed prds .
the parasite transcripts including psrs, csrs and pdrs were considered virulence-related transcripts .
immunomodulation categorisable vrs  were termed virulence and immunomodulation-related transcripts .
asterisk  indicates that data from other resources  were used for functional categorisation of some psrs and pdrs.
the identity of species was genetically confirmed by the presence of a f. hepatica transcript , in addition to the morphological identification of the isolated parasite.
the amino acid sequence for this protein at the correct frame of the transcript sequence was valine  as in f. hepatica, but not leucine  as in f. gigantica  <cit> , confirming the species specificity of the isolated parasite in this study.
in terms of drug resistance, the isolated parasite in the present study was found to be susceptible to albendazole .
this was extrapolated by the comparative analysis of the translated aminoacid sequences of a f. hepatica transcript  with the drug susceptibility associated amino acid residues  of tubulin beta- <dig> of f. hepatica  <cit> .
nonsynonymous/synonymous substitution rate of f. hepatica transcripts
a total of  <dig>  orthologous pairs  could be subjected to nonsynonymous/synonymous substitution rate analysis .
ka/ks ratio was calculable for a total of  <dig>  transcript pairs .
ka/ks analysis revealed that majority of the analysed f. hepatica transcripts  have ka/ks <  <dig> , hinting a purifiying selection against nonsynonymous changes as expected, a minority of the transcripts  have ka/ks >  <dig>  and a tiny portion of the transcripts  have ka/ks =  <dig> .
f. hepatica transcripts with ka/ks >  <dig> were hereafter called transcripts under positive selection .
only the small percentage of the orthologous transcripts were under positive selection, which confirms the hypothesis that some of the genes are diversing from the former versions to possess virulence capabilities as previously suggested  <cit> .figure  <dig> nonsynonymous/synonymous substitution rate of the liver fluke transcripts.
ka/ks ratio distribution  of a total of  <dig>  dugesiidae or c. elegans orthologous f. hepatica transcripts is shown.
the majority of the transcripts  were found to be associated with negative selection .
a group of transcripts  could be under directional selection .
another group of transcripts  were related to neutral selection.
only a small portion of the transcripts  showed the signs of positive selection  and, of this, a minority  reached statistical significance .
the level of homology of f. hepatica transcripts in cytokine signaling
the detailed analysis of interproscan descriptions for all functionally categorised transcripts revealed that some of the protein motifs , inferred from a group of transcript sequences , suggesting possible involvement of them in cytokine signaling .
sequence homology of f. hepatica transcripts with the specialised secondary databases and virulence-related transcripts of the parasite
approximately half of the total liver fluke transcript number  showed sequence homology with nucleotide or protein sequences of non-parasitic organisms including dugesiidae species and c. elegans  .
the level of sequential homology of f. hepatica transcripts with dugesia sp.
and schmidtea sp.
was slightly higher  than that with the transcripts of c. elegans , but this increased to  <dig> % when considering the parasite transcripts sequentially homologous to any of dugesiidae species .
the parasite transcripts which were similar to the sequences of dugesiidae species or c. elegans  but not showed the signs of positive selection  and/or cytokine signaling relation, a total of  <dig>  liver fluke sequences , were named non-virulence-related transcripts .
based on the transcript number, approximately a quarter of f. hepatica transcriptome  showed homology with the sequences from helminth secretome database , but only a minor part of these transcripts  were out of the category of nvts that these transcripts were named hts .
a smaller percentage of f. hepatica transcriptome showed homology with the data of vaccine investigation and online information network , but only a minority of these transcripts  were not observed in the category of nvts and these transcripts were termed vts .
a number of non-nvts  were found sequentially homologous to both hsd and violin databases .
all hts, vts and hts/vts were called pdr  under pdr subgroup.table  <dig>
sequence homology of the
f. hepatica
transcripts with the secondary databases and virulence-related transcripts of the parasite
a)
f. hepatica
transcripts predicted to be similar to:
number
percentage
data type
analysis/database
c. elegans
b) virulence-related
f. hepatica
transcripts
number
percentage
numerical values for the f. hepatica transcripts which show similarity with nucleotide/protein sequences of the pathogen-associated and non-pathogen-associated databases  and the numbers of the virulence-related f. hepatica transcripts   are listed.
a total of  <dig>  liver fluke transcripts  and an additional  <dig>  transcripts  showed homology with sequences of dugesiidae species and c. elegans, respectively, yielding a total of  <dig>  transcripts   .
overall,  <dig>  liver fluke transcripts   were determined to be non-virulence-related  after excluding the transcripts showing the signs of positive selection   and the relationship with cytokine signaling .
approximately a quarter of the total liver fluke transcripts  showed sequence homology with the hsd data and  <dig> % of the total liver fluke transcripts showed sequential similarity with the violin data.
a total of  <dig>  transcripts  and another set of  <dig> transcripts  were exclusively homologous to the hsd  and violin data , respectively but not identified in the category of nvts .
a small number of vrs  were common for both hts and vts .
in total,  <dig>  vrs  were included in pdr subgroup .
a total of  <dig> vrs showing the signs of positive selection  were observed in psr group alone.
some of vrs  were only identified by csr subgroup.
a number of psrs  were common for pdr subgroup.
three of vrs were detected by both psr and csr subgroups.
only one of vrs was determined by all the subgroups .
overall, a total of  <dig>  transcripts, identified at least by one of the subgroups, were predicted to be virulence-related.
the percentage values indicate the ratio of transcript number to total transcript number .
nvts: non-virulence-related transcripts.
overall,  <dig>  % of all identified f. hepatica transcripts was assumed to be virulence-related transcripts  on the basis of the degree of homology with sequences from publicly available databases; i) pathogen-associated data , ii) observation of the signs of positive selection  and iii) cytokine signaling relation , yielding a total of  <dig>  transcripts  .
a number of the transcripts with ka/ks >  <dig>  were also observed in pdr subgroup .
of the cytokine signaling related transcripts , one transcript showed the signs of positive selection and sequence homology with the hsd database , three transcripts  showed the signs of positive selection without sequential similarity of any pathogen related databases, and the others showed sequence homology with the non-parasitic databases .
a number of csrs  showed non-similarity with any specialised databases used in this study.
protein function profile for whole transcriptome and vr group
the interproscan search for whole transcriptome of the parasite revealed a total of  <dig>  unique accession number indicating protein families, domains or functional sites.
protein sequences inferred from a total of  <dig>  transcripts  were categorised in various functional groups by considering the biological functions of the parasite in previous publications  <cit> .
the majority of the protein sequences  were functionally categorisable with interproscan information and the rest  were classifiable on the basis of the information from the other resources such as gene ontology, uniprot, ncbi or referred publications .
the analysis indicated that the abundant transcripts were mostly involved in nucleic acid binding/transcription  and signaling , and only  <dig> % of all the categorised transcripts  was associated with immunomodulation .
biological functions of the virulence-related transcripts under vr group, and pdr and psr subgroups were found to be mostly related to nucleic acid binding/transcription, signaling and unknown mechanisms, respectively .
interestingly, the relative abundance of the transcripts with unknown mechanisms in psr subgroup  was higher than those in pdr subgroup , suggesting possible novel functions enhancing the virulence of the parasite .
the relative quantity of immunomodulation related transcripts within pdr subgroup  was higher more than twice, compared to those within psr subgroup  , likely hinting the importance of high diversity at lineage/genus level for gaining immunomodulation capability.
the protein function of all the transcripts under csr group was inherently related to immunomodulation.figure  <dig> relative abundances of the liver fluke transcripts in various biological functions based on manual inspection of protein motifs.
the relative abundances  of all the functionally categorised transcripts within whole transcriptome , vr group and its psr and pdr subgroups  in various functions are demonstrated.
nucleic acid binding/transcription and signaling mechanisms were highly pronounced functions of the whole transcriptome, with relative transcript abundances of  <dig> % and  <dig> %, respectively .
this pattern was similar within vr group except the relative transcript abundance of unknown mechanisms was higher within this group , psr subgroup  and pdr subgroup .
the transcript proportions of amino acid/protein metabolism, catalytic activity, energy, neuron system and signaling within psr subgroup were higher  than those within pdr subgroup.
the relative transcript ratios for immunomodulation, ion/haem binding and proteolysis/protein degradation functions were higher  in pdr subgroup in comparison with psr subgroup.
subcellular localisation profile for whole transcriptome and vr group
subcellular localisation signals of the parasite protein sequences were mostly related to cytosol , extracellular space  and nucleus  .
similarly, most of the detected subcellular localisation signals of vrs were associated with the cytosol, extracellular and nucleus parts, in order .
the extracellular localisation signal was more oftenly observed within vr group  and pdr subgroup  and csr subgroup , in comparison to psr subgroup .
further details about the subcellular localisation for the transcripts are shown individually in additional file  <dig> figure  <dig> subcellular localisation signals of the liver fluke transcripts.
the relative amount  of the detected subcellular localisation signals within whole transcriptome , vr group and its subgroups including csr subgroup, pdr subgroup and psr subgroup  are demonstrated.
the relative abundances for the cytosol, extracellular part and nucleus signals were found high levels within the total transcriptome,  <dig> %,  <dig> %,  <dig> %, respectively.
this profile was similarly found within vr.
the relative percentages of the extracellular signal in csr subgroup, pdr subgroup and vr group are higher  than those in psr subgroup.
the abundances of the signals of mitochondria&peroxisome, endoplasmic reticulum and plasma membrane were proportionally slightly higher  in psr subgroup than those within vr group and other subgroups .
virulence and immunomodulation-related transcripts and genes of f. hepatica
of vrs , the immunomodulation categorised pdrs, psrs, and all csrs, a total of seventy-one transcripts, corresponding to  <dig> putative genes, were named virulence and immunomodulation-related transcripts  under vir set .
further details about the sequence characteristics including available interproscan accesion number for virs are shown in additional file  <dig>  the majority of virs were specifically detectable through the level of homology with the pathogen related databases   and cytokine signaling relation   and the minor part of virs, observable in pdr subgroup , csr subgroup  or both , showed the signs of positive selection  .table  <dig>
virulence and immunomodulation-related transcripts of
f.hepatica
transcript no
accession no
description
subgroup
hpi
immunological property
resource
virulence and immunomodulation-related transcripts of f. hepatica  with accession and description of the corresponding sequences, identified by the transcript subgroups, are listed.
the predicted immunomodulatory property function and the possibility for the host-parasite interaction  for virs are shown.
data resources for the categorisation and/or prediction of function are indicated.
most proteins inferred from virs  were predicted to be possibly located in the extracellular space except a vir protein which is possibly located at the cell plasma membrane.
accesion with description was obtained from the blast searches in the ncbi protein database or genedb .
asterisk  indicates potential association with extracellular localisation.
double asterisk  indicates potential association with plasma membrane location.
no asterisk in the hpi column indicates the signs of cytoplasmic locations for the related transcripts except # <dig>  which showed the signs of being located in mitochondria and/or nucleus.
ips: interproscan.
a number of virs  showed sequential similarities with mhc i and an important part of virs indicated the relationship with tgf-β signaling based on sequence homologies with tgf-β, tgf-β receptor or other proteins associated with stimulation or inhibition of tgf-β .
the other sequence homologies were associated with various immunomodulatory molecules including t-cell receptor, toll/interleukin- <dig> receptor, tnf receptor, cluster of differentiations , il- <dig> receptor accessory protein, interleukin-4/interleukin- <dig>  tnf-α, modulators of t-cell function, suppressors of cytokine signaling and of ikbke  <dig>  or molecules involved in other immunomodulation-related mechanisms .
the majority of virs  individually were potentially located at extracellular space  or localised within plasma membrane  , possibly indicating direct interactions of the parasite proteins with host immune system.figure  <dig> distribution of putative virulence and immunomodulation-related orthologous genes by the immunomodulation property.
the number of the virulence and immunomodulation-related transcripts and the corresponding putative orthologous genes based on the predicted immunomodulation property are demonstrated.
the number of the putative orthologous genes showing mhc i homology  was the highest, which was followed by the others showing sequence homologies with tgf-β receptor , t-cell or toll/interleukin- <dig> receptors , and the relation with tgf-β stimulation .
gene ontology categories related to whole transcriptome, vr group/subgroups and vir set
gene ontology categories  for whole transcriptome of the liver fluke, in comparison with vr group, its subgroups  and vir set are shown in table  <dig>  the relative transcript abundances of the cellular  and metabolic processes  were found to be higher, in comparison to other biological processes in the total transcriptome, and this pattern was similar for vr group and its pdr and psr subgroups.
however, the proportions of the transcripts in response to stimulus  and immune system process  were the highest within vir set than those within vr group and its subgroups.
furthermore, vir set has higher sequence proportions in molecular transducer activity  and receptor activity , compared to vr group and its subgroups except csr subgroup.
the relative abundance in the go cellular compartmant category within vir set was skewed to membrane .table  <dig>
gene ontology for the liver fluke transcripts in different classifications using blast2go
gene ontology category
description
transcript classification
csr subgroup
pdr subgroup
psr subgroup
vr group
vir set
wt
biological process
molecular function
cellular component
gene ontology categories  for the whole transcriptome  and vr group with its subgroups  and vir set are shown.
the relative transcript abundances of response to stimulus  and immune system process  were predominant in vir set while the proportional transcript abundances for the cellular process  and metabolic process  were remarkably higher within wt, vr group, pdr and psr subgroups .
the relative transcript abundances of molecular transducer  and receptor activities  appeared to be much higher within vir set  and csr subgroup , compared to the other subgroups, vr group and wt.
the proportional transcript abundance for the membrane  was predominant within vir set  and csr subgroup , but the cellular part   was with the highest transcript ratio within vr group and its pdr and psr subgroups and wt.
numerical values and brackets indicate the total number of transcript sequences and the relative transcript abundance  within each transcript classification, respectively.
biological pathways related to whole transcriptome, vr group/subgroups and vir set
a total of  <dig> different kegg biological pathways  were determined in whole transcriptome of the parasite, in which the purine  and pyrimidine metabolisms  were the biological pathways with higher abundant transcript numbers, where the relative abundances of the other pathways, in transcript number, were less than 5% .
the pathway with the highest transcript number was the purine  metabolism within psr and pdr subgroups; however, the relative transcript abundance for the pyrimidine metabolism  within pdr subgroup was approximately twice than that within psr subgroup .
aminobenzoate degradation , beta-alanine metabolism , glycine, serine and threonine metabolism  were uniquely identified in pdr subgroup while butanoate metabolism  and pentose phosphate pathway  were found specific to psr subgroup among the transcript subgroups .table  <dig>
comparisons of the biological pathways of the liver fluke transcripts in different classifications
biological pathway
transcript classification
csr subgroup
pdr subgroup
psr subgroup
vr group
vir set
wt
biological pathways, identified at least for one of the transcript classifications, are shown.
most transcripts within vr group and its psr and pdr subgroups were related to purine metabolism , similar to the profile of wt.
the relative transcript abundance for the pyrimidine metabolism  within psr subgroup was found approximately half in pdr subgroup.
aminobenzoate degradation , beta-alanine metabolism , glycine, serine and threonine metabolism  were uniquely identified in pdr subgroup among the other subgroups.
butanoate metabolism  and pentose phosphate pathway  were only identified in psr subgroup in comparison with the other subgroups.
no any biological pathway was determined in csr subgroup and vir set.
discussion
in this study, we applied detailed in silico analyses to determine the virulence and immunomodulation-related genes of the liver fluke through a comparative assessment of the transcriptome profile with current ngs technology .
the observed go categories of f. hepatica transcripts in terms of biological processes were mostly concordant with the previous classification  <cit> .
however, some go categories, including signaling , receptor activity  and membrane  were firstly described in the present study.
this indicates overall more comprehensive coverage of total transcriptome profile of this parasite.
beside go analysis approach with blast2go, the manual inspection approach, including analysis of all the detected protein motifs, significantly increased the number of the categorised transcript into various functional terms that were previously used in the related studies  <cit> .
comparative transcriptome profile analysis of the parasite transcriptome with the sequences from non-parasitic organisms  revealed that  <dig> % of the obtained parasite transcripts shared significant homology, and the remaining  were possibly be lineage- or genus-specific.
a small part of the transcriptome  showed the sequence homology with the pathogen related databases but not with the non-parasite related databases.
the important strategy for picking out candidate transcripts with virulence was based on the identification of the transcripts with ka/ks >  <dig> with the assumption of the possible fast evolutionary pattern of parasitism associated genes  <cit> .
this strategy has been successfully employed to identify virulence and immunomodulation-related genes of other parasitic organisms such as plasmodium and theileria species  <cit> .
in our study, as a result of ka/ks analysis of  <dig>  orthologous transcripts, a total of 418 f. hepatica transcripts were found to be with ka/ks >  <dig>  hinting at a faster evolutionary rate because of likely involvement of these genes in the process of parasitism.
more detailed analysis of the transcriptome with the motifs of proteins known to be associated with cytokine signaling was useful for the elucidation of other important genes from f. hepatica.
the similar evolutionary characteristics of previously described virulence genes including cathepsin protease l <dig> and l <dig>  <cit> , cathepsin protease b  <cit> , thioredoxin/peroxiredoxin  <cit> , glutathione s-transferase   <cit> , protein disulfide-isomerase  <cit>  and 14-3- <dig> protein  <cit>  boosts the authenticity of parasitism associated genes identified in this study.
the presence of more transcripts likely encoding immunomodulatory proteins within the pathogen database related subgroup , in comparison to the positive selection related subgroup , suggests a possible role for nucleotide diversity at the lineage/genus for the establishment of parasitism.
because of more frequent extracellular localisation of the transcripts within pdr subgroup and the cytokine signaling related subgroup  relative to psr subgroup, it could be postulated that these gene products could directly interact with host proteins.
pyrimidine metabolism and other aminoacid related metabolisms such as aminobenzoate degradation , beta-alanine metabolism , glycine, serine and threonine metabolism , specific to pdr subgroup, could hint a possible lineage/genus specific nucleotide diversification.
through the comparative transcriptome analysis of the liver fluke sequences, a set of virulence-related transcripts  were found likely to possess immunomodulatory functional characteristics.
to our knowledge, all the virulence and immunomodulatory genes of f. hepatica identified hitherto have not been reported, with the exception of two putative genes  which are related to cd <dig>  <cit> .
the proportion of transcripts with receptor activity  was higher in vir set, in comparison with vr group and its pdr and psr subgroups, possibly denoting that virs are coevolved with host proteins due to direct interactions.
the skewed proportionality in abundance of membrane  in vir set supports this further.
majority of virs and their corresponding genes showed sequence similarity with host immune receptors  or cytokines  that these host molecules are known to be involved in cd4+ t-helper cell differentiation and regulation of the subsequent immune responses  <cit> .
the identification of the transcripts sequentially similar to tgf-β cytokine , tgf-β receptor  or tgf-β antagonists  could be important for controlling tgf-β cytokine levels.
additionally, the other genes that share sequence similarity with somatomedin b and thrombospondin  , hypothetical proteins containing thrombospondin  domain , proposed to stimulate tgf-β expression  <cit> , or bone morphogenetic protein antagonist noggin proteins, known to inhibit of the effects of tgf-β   <cit> , could play roles in regulating the activities of tgf-β.
our results showed that sequence homology with receptor like genes was not limited to tgf-β receptor, some other parasite genes were found to have sequential similarities with toll/interleukin- <dig> receptor , tnf receptor  or il- <dig> receptor accessory protein  that all of which are known to be involved in controlling proinflammatory responses  <cit> .
putative parasite genes  with sequence homologies to mhc class i receptor had the highest proportion of all identified parasitism genes.
the predicted protein sequences of the related transcripts  cover the region of alpha  <dig> and  <dig> domains, but not of alpha  <dig> domain of mhc class  <dig> molecule.
we speculate that the parasite peptides may be presented to host immune cells  by alpha  <dig> and  <dig> domains, but the cytotoxic effects of immune cells could be potentially blocked because of the absence of alpha  <dig> domain.
this proposed mechanism may result in suppression of subsequent pro-inflammatory responses including related cd4+ t-cell differentiation  and cytotoxic cell killing mechanisms, known to be harmful to the liver fluke  <cit> .
another interesting finding was the presence of a number of putative genes with t-receptor homology for a number of putative genes .
the imitation of t-cell receptor by the parasite may interefere with the presentation of the parasite’s antigen to t-cells and subsequent cellular and humoral responses.
taken together with the observed homologies related to t-cell receptor and the proteins of tgf-β signaling accentuates the importance of the stimulation of th <dig> type responses for the success of parasitism as suggested by another study conducted for trichuris suis  <cit> .
some of the parasite transcripts encoding interleukin-4/interleukin- <dig> conserved site   or showing sequential similarity with tnf-α  cytokine may possibly act in driving immune responses to th <dig> and th <dig>  respectively.
the other transcript  encoding a protein motif , known to induce the expression of il- <dig> cytokine , could be important in balancing the th <dig> and th <dig> responses  <cit> .
the identification of a transcript sharing sequence similarity with cd <dig>   was another noteworthy finding.
cd <dig> is a membrane protein of suppressed t-reg cells and associated with negative regulation of t-reg associated cytokine signaling and t-cell activation   <cit> .
we suggest that f. hepatica transcripts which coevolved with cd <dig> could be important for the regulation of t-reg associated responses.
the other cluster of differentiation  similarity was found to be associated with cd <dig> , stimulating various regulatory factors in b and t lymphocytes through binding molecules such as cd <dig> and cd <dig>   <cit> .
two of f. hepatica transcripts, # <dig> and # <dig>  were found to share high level of sequential similarities with macrophage inhibition factor and ctg4a , respectively.
both proteins are known to be involved in the activation of macrophages during inflammation  <cit> .
in relation to this, three genes, #42935/# <dig>  # <dig>  # <dig>  were associated with binding proline   <cit>  which is secreted by th <dig> type immune response-related alternatively activated macrophages promoting the development of fibrosis  <cit> .
taken together, the parasite proteins appear to be involved in the regulation of the macrophage activation and control of the development of excessive fibrotic tissue, in concordance with observations in clinical studies  <cit> .
there are other identified transcripts which showed sequential similarities with a neutrophil protein   <cit> , molecules known to suppressors of cytokine signaling   <cit>  and of ikbke <dig>   <cit>  or modulators of t-cell   <cit> .
these genes could be other components of the immunomodulatory mechanism induced by the parasite, but which specific immune responses they stimulate can not be predicted with the available data.
CONCLUSIONS
by comparative analysis of total transcriptome of f. hepatica with publicly open databases, a number of putative genes , which are potentially critical for virulence through immunomodulation or associated mechanisms and firstly described in this study.
the majority of these genes appeared to be lineage- or genus-specific, suggesting a modus operandi through the enhancement of sequential diversity for genes encoding proteins which are likely to be at the frontline for the establishment of parasitism.
in addition, the nucleotide diversity stemming from positive selectional pressure was found to be associated with cytokine signaling mechanisms by relying on the observed homologies with known genes such as toll/interleukin- <dig> receptor, tgf-β receptor, cd <dig> and a s. mansoni orthologous protein containing thrombospondin  domain .
a significant percentage of the transcripts have a remarkable level of sequential similarity with host immune receptors and cytokines, which are known to be part of array of immunological responses through cd4+ t-helper differentiation, indicating modulation of host immune system via controlling cellular responses associated with the t-helper heterogeneity .
in conclusion, the blockage of the effects of aforementioned parasite proteins with rnai or other strategies  would be a good approach in the fight against fasciolosis.
this may even be important in promoting the efficacy of other immunoprophylactic molecules which are experimentally tested in the prevention of fasciolosis.
apart from the dealing with fasciolosis, synthetic versions of the identified virulence and immunomodulatory genes reported in this study could be important to control undesired pro-inflammatory responses, by considering immunoregulatory effects of the parasite and current therapeutic approaches in the treatment of autoimmune defects   <cit> .
studies including the synthesis of the indicated genes in prokaryotic and eukaryotic systems and evaluating their immunological effects in vitro and in vivo are underway.
the present approach can be used for other studies with similar purposes.

 fasciola hepatica, the liver fluke, is a digenetic trematode helminth, causing highly damaging hepatobiliary disease in mammalians including economically important ruminants and humans. fasciolosis results in a significant economic loss in livestock industry worldwide and more cases have been reported in humans in different countries. the disease is currently treated with anti-helminthics, but the observed anti-helminthic drug resistance necessitates more effective strategies for the treatment and/or the prevention of fasciolosis. for the elucidation of infection mechanisms of the liver fluke and the development of better strategies in dealing with fasciolosis, the most critical step is the identification and characterisation of the genes which are important for the establishment of parasitism. the transcriptome of f. hepatica was reported in a previous publication where a previous sequencing platform was employed for the purpose of describing general biological characteristics of the parasite. however, the sequence data from that study is likely to be still further from encapturing the entirety of the transcriptome profile of f. hepatica and the virulence factors of the parasite, particularly those related to host immunomodulation, are worthwhile for additional investigation with current ngs platforms. the current ngs technologies produce greater transcriptomic data in comparison with the previous sequencing systems and increase the chance of detection of parasite transcripts which are expressed at lower levels but with significant importance in immune evasion. one of the most promising approaches for determining the virulence factors of parasitic organisms is in silico comparison of parasites’ transcripts with publicly available data. a recent data resource, the helminth secretome database, contains a broad repertoire of excretory/secretory protein sequences of various parasitic helminths including trematodes, cestodes and nematodes. es proteins of endoparasites are, in general, thought to play vital roles in the establishment of infection. additionally, vaccine investigation and online information network, provides gene and protein sequences that are affiliated with infection mechanisms of various micro and macro infectious organisms. compared to other resources providing whole genome data of numerous pathogens, both hsd and violin contain filtered data better suiting to glean pathogen-related molecules of infectious organisms. however, the major caveat of the specialised databases is the presence of insufficiently refined data that are related with regular physiological events in both parasitic and non-parasitic organisms, but not indeed linked to virulence. parasitism genes which are not directly involved in virulence, but rather associated with regular physiological mechanisms, could be uncovered by sequence homology with taxonomically similar, free-living organisms. very recently, a large nucleotide sequence collection from free-living/non-infectious trematodes of dugesiidae family has become publicly available at dna data bank of japan. additionally, a comprehensive data for a well studied free-living model nematode, caenorhabditis elegans, is freely accessible from a regularly updated resource, wormbase. the data of non-parasitic organisms from current resources have been useful tool to investigate the genes that are under directional selection through the comparative analysis of nucleotide diversity by assessing nonsynonymous/synonymous substitution rates. it is a well known fact that parasitism related genes important for the evasion of defensive systems of host and the corresponding genes in the host are under constant selective pressure favoring nucleotide subsitutions. for example, the strong influence of directional selection in the evolution of avirulence genes to gain immunomodulatory properties has been clearly reported in multiple studies. furthermore, lineage-specific sub- or neo-functionalisation of genes which are vital for the establishment and maintenance of parasitism could be identified through comparative genomics. apart from the analysis of sequence homology with infectious and non-infectious organisms, the virulence genes associated with modulation of host immune responses can be identified by the manual inspection of encoded protein motifs in public databases. to date, in vivo and in vitro studies have shown that the liver fluke modulates host immune responses for enhancing its virulence; however, which genes of the parasite imitate the components of host immune system have not yet been elucidated in detail. the main purpose of this study was to glean virulence and immunomodulatory f. hepatica genes through comparative transcriptome profiling with the transcriptomes of non-parasitic related organisms by focusing on the genes which are evolved in lineage-specific manner, under positive selection and show similar motifs of host immune system genes involved in cytokine signaling.  transcriptome profile of f. hepatica workflow illustrating the experimental steps of the study is demonstrated in figure  from a total of, sequence reads, generated by the sequencing instrument with paired-end 2x 100 bp reading, contigs were de novo assembled, of which, transcripts were annotated with blast searches as described in additional file the obtained base number in this study was approximately times higher than that reported in the previous transcriptome study of f. hepatica. the transcript n was 522 bp and the length of a total of transcripts was equal or greater than the observed n length in this work. in the present study, f. hepatica g + c content was %, which was similar to that reported in the previous related studies of f. hepatica. the identified transcript sequences in our study corresponded to a total of unique accession numbers.figure experimental design. the transcriptome study is consisted of wet lab procedures and data analysis processes. the parasite transcript sequences were analysed in terms of positive selection, cytokine signaling and pathogen database homology. the parasite sequences showing the signs of positive selection were termed psrs. the cytokine signaling relation for the inferred parasite proteins were detected by the protein motif information and the related transcripts were termed csrs. non-virulence-related transcripts were determined by the sequence homology of the parasite transcripts with the data of the non-pathogen associated databases by excluding the non-parasite organism homologous psrs and csrs. the parasite transcripts showing the exclusive sequence homology with the pathogen-associated databases, but not being nvts, were termed prds. the parasite transcripts including psrs, csrs and pdrs were considered virulence-related transcripts. immunomodulation categorisable vrs were termed virulence and immunomodulation-related transcripts. asterisk indicates that data from other resources were used for functional categorisation of some psrs and pdrs. the identity of species was genetically confirmed by the presence of a f. hepatica transcript, in addition to the morphological identification of the isolated parasite. the amino acid sequence for this protein at the correct frame of the transcript sequence was valine as in f. hepatica, but not leucine as in f. gigantica, confirming the species specificity of the isolated parasite in this study. in terms of drug resistance, the isolated parasite in the present study was found to be susceptible to albendazole. this was extrapolated by the comparative analysis of the translated aminoacid sequences of a f. hepatica transcript with the drug susceptibility associated amino acid residues of tubulin beta- of f. hepatica. nonsynonymous/synonymous substitution rate of f. hepatica transcripts a total of orthologous pairs could be subjected to nonsynonymous/synonymous substitution rate analysis. ka/ks ratio was calculable for a total of transcript pairs. ka/ks analysis revealed that majority of the analysed f. hepatica transcripts have ka/ks < , hinting a purifiying selection against nonsynonymous changes as expected, a minority of the transcripts have ka/ks >  and a tiny portion of the transcripts have ka/ks = . f. hepatica transcripts with ka/ks >  were hereafter called transcripts under positive selection. only the small percentage of the orthologous transcripts were under positive selection, which confirms the hypothesis that some of the genes are diversing from the former versions to possess virulence capabilities as previously suggested.figure nonsynonymous/synonymous substitution rate of the liver fluke transcripts. ka/ks ratio distribution of a total of dugesiidae or c. elegans orthologous f. hepatica transcripts is shown. the majority of the transcripts were found to be associated with negative selection. a group of transcripts could be under directional selection. another group of transcripts were related to neutral selection. only a small portion of the transcripts showed the signs of positive selection and, of this, a minority reached statistical significance. the level of homology of f. hepatica transcripts in cytokine signaling the detailed analysis of interproscan descriptions for all functionally categorised transcripts revealed that some of the protein motifs, inferred from a group of transcript sequences, suggesting possible involvement of them in cytokine signaling. sequence homology of f. hepatica transcripts with the specialised secondary databases and virulence-related transcripts of the parasite approximately half of the total liver fluke transcript number showed sequence homology with nucleotide or protein sequences of non-parasitic organisms including dugesiidae species and c. elegans. the level of sequential homology of f. hepatica transcripts with dugesia sp. and schmidtea sp. was slightly higher than that with the transcripts of c. elegans, but this increased to % when considering the parasite transcripts sequentially homologous to any of dugesiidae species. the parasite transcripts which were similar to the sequences of dugesiidae species or c. elegans but not showed the signs of positive selection and/or cytokine signaling relation, a total of liver fluke sequences, were named non-virulence-related transcripts. based on the transcript number, approximately a quarter of f. hepatica transcriptome showed homology with the sequences from helminth secretome database, but only a minor part of these transcripts were out of the category of nvts that these transcripts were named hts. a smaller percentage of f. hepatica transcriptome showed homology with the data of vaccine investigation and online information network, but only a minority of these transcripts were not observed in the category of nvts and these transcripts were termed vts. a number of non-nvts were found sequentially homologous to both hsd and violin databases. all hts, vts and hts/vts were called pdr under pdr subgroup.table sequence homology of the f. hepatica transcripts with the secondary databases and virulence-related transcripts of the parasite a) f. hepatica transcripts predicted to be similar to: number percentage data type analysis/database c. elegans b) virulence-related f. hepatica transcripts number percentage numerical values for the f. hepatica transcripts which show similarity with nucleotide/protein sequences of the pathogen-associated and non-pathogen-associated databases and the numbers of the virulence-related f. hepatica transcripts are listed. a total of liver fluke transcripts and an additional transcripts showed homology with sequences of dugesiidae species and c. elegans, respectively, yielding a total of transcripts. overall, liver fluke transcripts were determined to be non-virulence-related after excluding the transcripts showing the signs of positive selection and the relationship with cytokine signaling. approximately a quarter of the total liver fluke transcripts showed sequence homology with the hsd data and % of the total liver fluke transcripts showed sequential similarity with the violin data. a total of transcripts and another set of transcripts were exclusively homologous to the hsd and violin data, respectively but not identified in the category of nvts. a small number of vrs were common for both hts and vts. in total, vrs were included in pdr subgroup. a total of vrs showing the signs of positive selection were observed in psr group alone. some of vrs were only identified by csr subgroup. a number of psrs were common for pdr subgroup. three of vrs were detected by both psr and csr subgroups. only one of vrs was determined by all the subgroups. overall, a total of transcripts, identified at least by one of the subgroups, were predicted to be virulence-related. the percentage values indicate the ratio of transcript number to total transcript number. nvts: non-virulence-related transcripts. overall, % of all identified f. hepatica transcripts was assumed to be virulence-related transcripts on the basis of the degree of homology with sequences from publicly available databases; i) pathogen-associated data, ii) observation of the signs of positive selection and iii) cytokine signaling relation, yielding a total of transcripts. a number of the transcripts with ka/ks >  were also observed in pdr subgroup. of the cytokine signaling related transcripts, one transcript showed the signs of positive selection and sequence homology with the hsd database, three transcripts showed the signs of positive selection without sequential similarity of any pathogen related databases, and the others showed sequence homology with the non-parasitic databases. a number of csrs showed non-similarity with any specialised databases used in this study. protein function profile for whole transcriptome and vr group the interproscan search for whole transcriptome of the parasite revealed a total of unique accession number indicating protein families, domains or functional sites. protein sequences inferred from a total of transcripts were categorised in various functional groups by considering the biological functions of the parasite in previous publications. the majority of the protein sequences were functionally categorisable with interproscan information and the rest were classifiable on the basis of the information from the other resources such as gene ontology, uniprot, ncbi or referred publications. the analysis indicated that the abundant transcripts were mostly involved in nucleic acid binding/transcription and signaling, and only % of all the categorised transcripts was associated with immunomodulation. biological functions of the virulence-related transcripts under vr group, and pdr and psr subgroups were found to be mostly related to nucleic acid binding/transcription, signaling and unknown mechanisms, respectively. interestingly, the relative abundance of the transcripts with unknown mechanisms in psr subgroup was higher than those in pdr subgroup, suggesting possible novel functions enhancing the virulence of the parasite. the relative quantity of immunomodulation related transcripts within pdr subgroup was higher more than twice, compared to those within psr subgroup, likely hinting the importance of high diversity at lineage/genus level for gaining immunomodulation capability. the protein function of all the transcripts under csr group was inherently related to immunomodulation.figure relative abundances of the liver fluke transcripts in various biological functions based on manual inspection of protein motifs. the relative abundances of all the functionally categorised transcripts within whole transcriptome, vr group and its psr and pdr subgroups in various functions are demonstrated. nucleic acid binding/transcription and signaling mechanisms were highly pronounced functions of the whole transcriptome, with relative transcript abundances of % and %, respectively. this pattern was similar within vr group except the relative transcript abundance of unknown mechanisms was higher within this group, psr subgroup and pdr subgroup. the transcript proportions of amino acid/protein metabolism, catalytic activity, energy, neuron system and signaling within psr subgroup were higher than those within pdr subgroup. the relative transcript ratios for immunomodulation, ion/haem binding and proteolysis/protein degradation functions were higher in pdr subgroup in comparison with psr subgroup. subcellular localisation profile for whole transcriptome and vr group subcellular localisation signals of the parasite protein sequences were mostly related to cytosol, extracellular space and nucleus. similarly, most of the detected subcellular localisation signals of vrs were associated with the cytosol, extracellular and nucleus parts, in order. the extracellular localisation signal was more oftenly observed within vr group and pdr subgroup and csr subgroup, in comparison to psr subgroup. further details about the subcellular localisation for the transcripts are shown individually in additional file figure subcellular localisation signals of the liver fluke transcripts. the relative amount of the detected subcellular localisation signals within whole transcriptome, vr group and its subgroups including csr subgroup, pdr subgroup and psr subgroup are demonstrated. the relative abundances for the cytosol, extracellular part and nucleus signals were found high levels within the total transcriptome, %, %, %, respectively. this profile was similarly found within vr. the relative percentages of the extracellular signal in csr subgroup, pdr subgroup and vr group are higher than those in psr subgroup. the abundances of the signals of mitochondria&peroxisome, endoplasmic reticulum and plasma membrane were proportionally slightly higher in psr subgroup than those within vr group and other subgroups. virulence and immunomodulation-related transcripts and genes of f. hepatica of vrs, the immunomodulation categorised pdrs, psrs, and all csrs, a total of seventy-one transcripts, corresponding to putative genes, were named virulence and immunomodulation-related transcripts under vir set. further details about the sequence characteristics including available interproscan accesion number for virs are shown in additional file the majority of virs were specifically detectable through the level of homology with the pathogen related databases and cytokine signaling relation and the minor part of virs, observable in pdr subgroup, csr subgroup or both, showed the signs of positive selection.table virulence and immunomodulation-related transcripts of f.hepatica transcript no accession no description subgroup hpi immunological property resource virulence and immunomodulation-related transcripts of f. hepatica with accession and description of the corresponding sequences, identified by the transcript subgroups, are listed. the predicted immunomodulatory property function and the possibility for the host-parasite interaction for virs are shown. data resources for the categorisation and/or prediction of function are indicated. most proteins inferred from virs were predicted to be possibly located in the extracellular space except a vir protein which is possibly located at the cell plasma membrane. accesion with description was obtained from the blast searches in the ncbi protein database or genedb. asterisk indicates potential association with extracellular localisation. double asterisk indicates potential association with plasma membrane location. no asterisk in the hpi column indicates the signs of cytoplasmic locations for the related transcripts except # which showed the signs of being located in mitochondria and/or nucleus. ips: interproscan. a number of virs showed sequential similarities with mhc i and an important part of virs indicated the relationship with tgf-β signaling based on sequence homologies with tgf-β, tgf-β receptor or other proteins associated with stimulation or inhibition of tgf-β. the other sequence homologies were associated with various immunomodulatory molecules including t-cell receptor, toll/interleukin- receptor, tnf receptor, cluster of differentiations, il- receptor accessory protein, interleukin-4/interleukin- tnf-α, modulators of t-cell function, suppressors of cytokine signaling and of ikbke or molecules involved in other immunomodulation-related mechanisms. the majority of virs individually were potentially located at extracellular space or localised within plasma membrane, possibly indicating direct interactions of the parasite proteins with host immune system.figure distribution of putative virulence and immunomodulation-related orthologous genes by the immunomodulation property. the number of the virulence and immunomodulation-related transcripts and the corresponding putative orthologous genes based on the predicted immunomodulation property are demonstrated. the number of the putative orthologous genes showing mhc i homology was the highest, which was followed by the others showing sequence homologies with tgf-β receptor, t-cell or toll/interleukin- receptors, and the relation with tgf-β stimulation. gene ontology categories related to whole transcriptome, vr group/subgroups and vir set gene ontology categories for whole transcriptome of the liver fluke, in comparison with vr group, its subgroups and vir set are shown in table  the relative transcript abundances of the cellular and metabolic processes were found to be higher, in comparison to other biological processes in the total transcriptome, and this pattern was similar for vr group and its pdr and psr subgroups. however, the proportions of the transcripts in response to stimulus and immune system process were the highest within vir set than those within vr group and its subgroups. furthermore, vir set has higher sequence proportions in molecular transducer activity and receptor activity, compared to vr group and its subgroups except csr subgroup. the relative abundance in the go cellular compartmant category within vir set was skewed to membrane.table gene ontology for the liver fluke transcripts in different classifications using blast2go gene ontology category description transcript classification csr subgroup pdr subgroup psr subgroup vr group vir set wt biological process molecular function cellular component gene ontology categories for the whole transcriptome and vr group with its subgroups and vir set are shown. the relative transcript abundances of response to stimulus and immune system process were predominant in vir set while the proportional transcript abundances for the cellular process and metabolic process were remarkably higher within wt, vr group, pdr and psr subgroups. the relative transcript abundances of molecular transducer and receptor activities appeared to be much higher within vir set and csr subgroup, compared to the other subgroups, vr group and wt. the proportional transcript abundance for the membrane was predominant within vir set and csr subgroup, but the cellular part was with the highest transcript ratio within vr group and its pdr and psr subgroups and wt. numerical values and brackets indicate the total number of transcript sequences and the relative transcript abundance within each transcript classification, respectively. biological pathways related to whole transcriptome, vr group/subgroups and vir set a total of different kegg biological pathways were determined in whole transcriptome of the parasite, in which the purine and pyrimidine metabolisms were the biological pathways with higher abundant transcript numbers, where the relative abundances of the other pathways, in transcript number, were less than 5%. the pathway with the highest transcript number was the purine metabolism within psr and pdr subgroups; however, the relative transcript abundance for the pyrimidine metabolism within pdr subgroup was approximately twice than that within psr subgroup. aminobenzoate degradation, beta-alanine metabolism, glycine, serine and threonine metabolism were uniquely identified in pdr subgroup while butanoate metabolism and pentose phosphate pathway were found specific to psr subgroup among the transcript subgroups.table comparisons of the biological pathways of the liver fluke transcripts in different classifications biological pathway transcript classification csr subgroup pdr subgroup psr subgroup vr group vir set wt biological pathways, identified at least for one of the transcript classifications, are shown. most transcripts within vr group and its psr and pdr subgroups were related to purine metabolism, similar to the profile of wt. the relative transcript abundance for the pyrimidine metabolism within psr subgroup was found approximately half in pdr subgroup. aminobenzoate degradation, beta-alanine metabolism, glycine, serine and threonine metabolism were uniquely identified in pdr subgroup among the other subgroups. butanoate metabolism and pentose phosphate pathway were only identified in psr subgroup in comparison with the other subgroups. no any biological pathway was determined in csr subgroup and vir set. discussion in this study, we applied detailed in silico analyses to determine the virulence and immunomodulation-related genes of the liver fluke through a comparative assessment of the transcriptome profile with current ngs technology. the observed go categories of f. hepatica transcripts in terms of biological processes were mostly concordant with the previous classification. however, some go categories, including signaling, receptor activity and membrane were firstly described in the present study. this indicates overall more comprehensive coverage of total transcriptome profile of this parasite. beside go analysis approach with blast2go, the manual inspection approach, including analysis of all the detected protein motifs, significantly increased the number of the categorised transcript into various functional terms that were previously used in the related studies. comparative transcriptome profile analysis of the parasite transcriptome with the sequences from non-parasitic organisms revealed that % of the obtained parasite transcripts shared significant homology, and the remaining were possibly be lineage- or genus-specific. a small part of the transcriptome showed the sequence homology with the pathogen related databases but not with the non-parasite related databases. the important strategy for picking out candidate transcripts with virulence was based on the identification of the transcripts with ka/ks >  with the assumption of the possible fast evolutionary pattern of parasitism associated genes. this strategy has been successfully employed to identify virulence and immunomodulation-related genes of other parasitic organisms such as plasmodium and theileria species. in our study, as a result of ka/ks analysis of orthologous transcripts, a total of 418 f. hepatica transcripts were found to be with ka/ks >  hinting at a faster evolutionary rate because of likely involvement of these genes in the process of parasitism. more detailed analysis of the transcriptome with the motifs of proteins known to be associated with cytokine signaling was useful for the elucidation of other important genes from f. hepatica. the similar evolutionary characteristics of previously described virulence genes including cathepsin protease l and l, cathepsin protease b, thioredoxin/peroxiredoxin, glutathione s-transferase, protein disulfide-isomerase and 14-3- protein boosts the authenticity of parasitism associated genes identified in this study. the presence of more transcripts likely encoding immunomodulatory proteins within the pathogen database related subgroup, in comparison to the positive selection related subgroup, suggests a possible role for nucleotide diversity at the lineage/genus for the establishment of parasitism. because of more frequent extracellular localisation of the transcripts within pdr subgroup and the cytokine signaling related subgroup relative to psr subgroup, it could be postulated that these gene products could directly interact with host proteins. pyrimidine metabolism and other aminoacid related metabolisms such as aminobenzoate degradation, beta-alanine metabolism, glycine, serine and threonine metabolism, specific to pdr subgroup, could hint a possible lineage/genus specific nucleotide diversification. through the comparative transcriptome analysis of the liver fluke sequences, a set of virulence-related transcripts were found likely to possess immunomodulatory functional characteristics. to our knowledge, all the virulence and immunomodulatory genes of f. hepatica identified hitherto have not been reported, with the exception of two putative genes which are related to cd. the proportion of transcripts with receptor activity was higher in vir set, in comparison with vr group and its pdr and psr subgroups, possibly denoting that virs are coevolved with host proteins due to direct interactions. the skewed proportionality in abundance of membrane in vir set supports this further. majority of virs and their corresponding genes showed sequence similarity with host immune receptors or cytokines that these host molecules are known to be involved in cd4+ t-helper cell differentiation and regulation of the subsequent immune responses. the identification of the transcripts sequentially similar to tgf-β cytokine, tgf-β receptor or tgf-β antagonists could be important for controlling tgf-β cytokine levels. additionally, the other genes that share sequence similarity with somatomedin b and thrombospondin, hypothetical proteins containing thrombospondin domain, proposed to stimulate tgf-β expression, or bone morphogenetic protein antagonist noggin proteins, known to inhibit of the effects of tgf-β, could play roles in regulating the activities of tgf-β. our results showed that sequence homology with receptor like genes was not limited to tgf-β receptor, some other parasite genes were found to have sequential similarities with toll/interleukin- receptor, tnf receptor or il- receptor accessory protein that all of which are known to be involved in controlling proinflammatory responses. putative parasite genes with sequence homologies to mhc class i receptor had the highest proportion of all identified parasitism genes. the predicted protein sequences of the related transcripts cover the region of alpha and domains, but not of alpha domain of mhc class molecule. we speculate that the parasite peptides may be presented to host immune cells by alpha and domains, but the cytotoxic effects of immune cells could be potentially blocked because of the absence of alpha domain. this proposed mechanism may result in suppression of subsequent pro-inflammatory responses including related cd4+ t-cell differentiation and cytotoxic cell killing mechanisms, known to be harmful to the liver fluke. another interesting finding was the presence of a number of putative genes with t-receptor homology for a number of putative genes. the imitation of t-cell receptor by the parasite may interefere with the presentation of the parasite’s antigen to t-cells and subsequent cellular and humoral responses. taken together with the observed homologies related to t-cell receptor and the proteins of tgf-β signaling accentuates the importance of the stimulation of th type responses for the success of parasitism as suggested by another study conducted for trichuris suis. some of the parasite transcripts encoding interleukin-4/interleukin- conserved site or showing sequential similarity with tnf-α cytokine may possibly act in driving immune responses to th and th respectively. the other transcript encoding a protein motif, known to induce the expression of il- cytokine, could be important in balancing the th and th responses. the identification of a transcript sharing sequence similarity with cd was another noteworthy finding. cd is a membrane protein of suppressed t-reg cells and associated with negative regulation of t-reg associated cytokine signaling and t-cell activation. we suggest that f. hepatica transcripts which coevolved with cd could be important for the regulation of t-reg associated responses. the other cluster of differentiation similarity was found to be associated with cd, stimulating various regulatory factors in b and t lymphocytes through binding molecules such as cd and cd. two of f. hepatica transcripts, # and # were found to share high level of sequential similarities with macrophage inhibition factor and ctg4a, respectively. both proteins are known to be involved in the activation of macrophages during inflammation. in relation to this, three genes, #42935/# # # were associated with binding proline which is secreted by th type immune response-related alternatively activated macrophages promoting the development of fibrosis. taken together, the parasite proteins appear to be involved in the regulation of the macrophage activation and control of the development of excessive fibrotic tissue, in concordance with observations in clinical studies. there are other identified transcripts which showed sequential similarities with a neutrophil protein, molecules known to suppressors of cytokine signaling and of ikbke or modulators of t-cell. these genes could be other components of the immunomodulatory mechanism induced by the parasite, but which specific immune responses they stimulate can not be predicted with the available data.  by comparative analysis of total transcriptome of f. hepatica with publicly open databases, a number of putative genes, which are potentially critical for virulence through immunomodulation or associated mechanisms and firstly described in this study. the majority of these genes appeared to be lineage- or genus-specific, suggesting a modus operandi through the enhancement of sequential diversity for genes encoding proteins which are likely to be at the frontline for the establishment of parasitism. in addition, the nucleotide diversity stemming from positive selectional pressure was found to be associated with cytokine signaling mechanisms by relying on the observed homologies with known genes such as toll/interleukin- receptor, tgf-β receptor, cd and a s. mansoni orthologous protein containing thrombospondin domain. a significant percentage of the transcripts have a remarkable level of sequential similarity with host immune receptors and cytokines, which are known to be part of array of immunological responses through cd4+ t-helper differentiation, indicating modulation of host immune system via controlling cellular responses associated with the t-helper heterogeneity. in conclusion, the blockage of the effects of aforementioned parasite proteins with rnai or other strategies would be a good approach in the fight against fasciolosis. this may even be important in promoting the efficacy of other immunoprophylactic molecules which are experimentally tested in the prevention of fasciolosis. apart from the dealing with fasciolosis, synthetic versions of the identified virulence and immunomodulatory genes reported in this study could be important to control undesired pro-inflammatory responses, by considering immunoregulatory effects of the parasite and current therapeutic approaches in the treatment of autoimmune defects. studies including the synthesis of the indicated genes in prokaryotic and eukaryotic systems and evaluating their immunological effects in vitro and in vivo are underway. the present approach can be used for other studies with similar purposes. 
the identification of f. hepatica genes involved in the parasite’s virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. of these, a group of the virulence-related genes, not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors, cytokines, cluster of differentiations or molecules associated with other immunomodulatory mechanisms. in brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution and cytokine signaling relation were conducted. a number of the parasite transcripts were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. some of the genes appeared to be under positive selection, imitating proteins associated with cytokine signaling. in this study, to identify the parasite’s putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult f. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. with a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of f. hepatica, which are firstly described here, could be employed for the development of better strategies in the battle against both zoonotically and economically important disease, fasciolosis. ngs of the whole transcriptome resulted in, sequence reads, yielding a total of transcripts. fasciola hepaticawhole transcriptometrematoderna-seqvirulenceimmunomodulationevasionissue-copyright-statement© the author 2015
 fasciola hepatica causes chronic liver disease, fasciolosis, leading to significant losses in the livestock economy and concerns for human health in many countries. the identification of f. hepatica genes involved in the parasite’s virulence through modulation of host immune system is utmost important to comprehend evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. in this study, to identify the parasite’s putative virulence genes which are associated with host immunomodulation, we explored whole transcriptome of an adult f. hepatica using current transcriptome profiling approaches integrated with detailed in silico analyses. in brief, the comparison of the parasite transcripts with the specialised public databases containing sequence data of non-parasitic organisms or of numerous pathogens and investigation of the sequences in terms of nucleotide evolution and cytokine signaling relation were conducted.  ngs of the whole transcriptome resulted in, sequence reads, yielding a total of transcripts. a number of the parasite transcripts were predicted to be virulence-related on the basis of the exclusive homology with the pathogen-associated data, positive selection or relationship with cytokine signaling. of these, a group of the virulence-related genes, not previously described, were found likely to be associated with immunomodulation based on in silico functional categorisation, showing significant sequence similarities with various immune receptors, cytokines, cluster of differentiations or molecules associated with other immunomodulatory mechanisms. some of the genes appeared to be under positive selection, imitating proteins associated with cytokine signaling.  with a comparative transcriptome profiling approach, we have identified a number of potential immunomodulator genes of f. hepatica, which are firstly described here, could be employed for the development of better strategies in the battle against both zoonotically and economically important disease, fasciolosis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fasciola hepaticawhole transcriptometrematoderna-seqvirulenceimmunomodulationevasionissue-copyright-statement© the author 2015 