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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Vet Parasitol. Author manuscript; available in PMC May 26, 2010.
Published in final edited form as:
PMCID: PMC2677129
NIHMSID: NIHMS99006

The transcriptomes of the cattle parasitic nematode Ostertagia ostartagi

Abstract

Ostertagia ostertagi is a gastrointestinal parasitic nematode that affects cattle and leads to a loss of production. In this study, we present the first large-scale genomic survey of Ostertagia ostertagi by the analysis of expressed transcripts from three stages of the parasite: third-stage larvae, fourth-stage larvae and adult worms. Using an in silico approach, 2,284 genes were identified from over 7,000 expressed sequence tags and abundant transcripts were analyzed and characterized by their functional profile. Of the 2,284 genes, 66% had similarity to other known or predicted genes while the rest were novel and potentially represent genes specific to the species and/or stages. Furthermore, a subset of the novel proteins were structurally annotated and assigned putative function based on orthologs in C. elegans and corresponding RNA interference phenotypes. Hence, over 70% of the genes were annotated using protein sequences, domains and pathway databases. Differentially expressed transcripts from the two larval stages and their functional profiles were also studied leading to a more detailed understanding of the parasite’s life-cycle. The identified transcripts are a valuable resource for genomic studies of O. ostertagi and can facilitate the design of control strategies and vaccine programs.

Keywords: Cattle, Parasite, Nematode, Transcripts, ESTs, Ostertagia ostertagi, Comparative genomics

1. Introduction

Ostertagiosis is caused by the nematode Ostertagia ostertagi, a stomach parasite of cattle found predominantly in temperate regions of the world. Ostertagia belongs to the order Strongylida, which includes numerous livestock parasites and hookworms (Ancylostoma and Necator species), and is part of clade V along with the extensively characterized free-living nematode Caenorhabditis elegans (Blaxter et al., 1998). Ostertagia ostertagi eggs are passed within the host from the female worm, winding up in dung where they hatch to the first larval stage (L1), and then develop to the second larval stage (L2). The infective third larval stage (L3) has a protective layer that enables it to survive long periods in the environment and also enables mobility for migration and dissemination on pasture. After host ingestion, the L3 loose their protective sheath and burrow into the gastric glands of the abomasum where they develop to fourth stage larvae (L4). The L4 can proceed directly to adult worms at 3 - 4 weeks post infection or enter seasonal arrested development for up to 6 months (diapause). The mechanism of this arrest is unknown. In C. elegans, arrest as a non-feeding dauer larvae (L3d) is triggered in the L1/early L2 by adverse conditions for survival such as lack of food, overcrowding or environmental extremes (Riddle and Albert, 1997). It is not yet known whether L4 arrest in a trichostrongyle like O. ostertagi has any ancestral connection or shared mechanisms with the C. elegans L3d.

Type I ostertagiosis occurs when the adult worm proceeds directly through the larval stages without any inhibition. Type II ostertagiosis occurs when the L4 larvae resume development after a period of seasonal arrest. Surprisingly, seasonal arrest is regional where some L4 become developmentally inhibited in the spring and summer, and in less temperate regions of the world they become inhibited in the fall and winter. Both types of ostertagiosis cause damage to the host abomasum and are accompanied by weight loss and severe diarrhea that can lead to a loss of productivity and increased morbidity and mortality. Infections with the entire class of gastrointestinal (GI) nematodes cost the cattle producers hundreds of millions of dollars annually.

Anthelmintic drugs are typically used to treat ostertagiosis. Although evidence for resistance to these drugs has yet to surface in O. ostertagi, it is nonetheless on the increase among this broad group of GI nematodes including Haemonchus, Cooperia, and Trichostrongylus (Wolstenholme et al., 2004). Ostertagia ostertagi infection does not rapidly confer natural immunity to re-infection. Other modes of intervention such as vaccination with Ostertagia polyprotein products (Vercauteren et al., 2004) and cysteine proteinase {Geldhof, 2002 #2370} have been investigated for preventing infections though with equivocal results. This study has generated the first extensive genomics resource for O. ostertagi. The information offers insights into mechanisms associated with the host-parasite interface and also the parasite’s life cycle which may accelerate progress towards identification of effective targets and strategies for parasite control.

2. Material and methods

2.1. Ostertagia ostertagi material, RNA extraction, and library construction

Third stage exsheathed larvae were collected from 14-day fecal-sphagnum moss cultures of O. ostertagi eggs. The larvae were recovered by overnight passage on a Baermann apparatus, and then cleaned by passage through a 20-micron nylon mesh. The larvae were then subjected to a treatment with 1.25% chlorox to induce excystation. The larvae were washed with 5 changes of PBS and then pelleted and snap frozen in liquid nitrogen.

Late L4 were recovered 10 days after infection of young calves with O. ostertagi L3. The calves were killed and the abomasa (gastric glands) were recovered and washed extensively and then placed in a Baermann apparatus containing warm PBS for approximately 4 hours. The L4 that migrated from the tissues were washed extensively, pelleted and snap frozen in liquid nitrogen.

Pulverization was performed using an Alloy Tool Steel Set (Fisher Scientific Interbnational). Total RNA of L3 and L4 was prepared using TRIzol reagent (GibcoBRL, Life Technologies). Two types of cDNA libraries were constructed from each stage: SMART based (Mitreva et al., 2005) and SL1-1 based (Mitreva et al., 2004a).

2.2. EST sequencing and clustering

EST processing and clustering were performed as described (McCarter et al., 2003; Mitreva et al., 2004c). Submissions have been deposited to GenBank, and information for clone requests is available at www.nematode.net (Martin et al., in press). One hundred and five EST were defined as chimeras and excluded from the analysis. Later examination of the O. ostertagi culture used as a source of the L3 sample identified a minority population of Cooperia oncophora worms, a very closely related trichostrongylid intestinal nematode that frequently co-infects hosts along with O. ostertagi. The O. ostertagi culture used for the L4 samples was found not to have any C. oncophora worms. To determine the extent of C. oncophora ESTs in the O. ostertagi sample in silico, all L3- and L4-biased and –specific clusters from the sample were screened against 857 C. oncophora and 186 O. ostertagi sequences retrieved from NCBI (June 2008) using WU-BLAST. As orthologs might not possibly exist in the NCBI database for both species, we also analyzed the subset of the L3-enriched genes that had homology to both species (with E-value better than 1e-05). We identified two genes (with 99 and 2 ESTs respectively) out of nine that were more homologous to C. oncophora than O. ostertagi and the remaining genes showed stronger identity with O. ostertagi. This analysis was repeated with L4-enriched contigs to evaluate the incidence of C. oncophora. From the L4 set, five genes showed sequence identity with C. oncophora and O. ostertagi and only one of them had stronger matches to the C. onchophora homologs. In both analysis (L3 and L4), whenever the hits were better to C. oncophora the subject was different units of the cytochrome oxidase, and the second best hit was to O. ostertagi. The O. ostertagi hit was with higher % identity but shorter alignment which resulted in a better e-value for the C. onchophora hit. This analysis indicates that we can not confirm that C. oncophora transcripts are present in the O. ostertagi L3 sample and if they are the presence is not substantial, and furthermore, given preliminary evidence of genetic and biological similarities among these trichostrongyles, it is unlikely that small amounts of contamination would skew the overall results. Furthermore, in case of the overlapping clusters comprised of ESTs from multiple stages, this concern is eliminated as contamination was not an issue with the L4 and the adult sample (Fig. 1).

Fig. 1
Distribution of clusters based on life stages of origin. Total number of clusters is 2,284, majority of clusters are represented by only one stage, though further dept of sampling will likely increase representation by multiple stages.

Expressed Sequence tags (EST) (6557) from L3, L4, and the adult stage were submitted to GenBank. Additional ESTs (485) were downloaded from GenBank on 01/03/2006. NemaGene clustering was performed on ESTs from the L3, L4 and adult worms. This clustering groups all ESTs into contigs representing transcripts with potential isoforms. Contigs that are mostly identical are then grouped into unique clusters (McCarter et al., 2003). Rarely, clusters may also include highly related gene family members. After quality control and screening, 7,042 ESTs were organized into contigs which were translated using prot4EST (Wasmuth and Blaxter, 2004). There were 4,605 contigs with valid translations that clustered into 2,284 putative genes.

2.3. Structural and functional annotation, and comparative analysis using bioinformatics approaches

Transmembrane and signal peptide predictions

Phobius (Kall et al., 2004), a combined SP and TM prediction method based on hidden Markov modeling was used with default settings. Each query sequence was further annotated as SP-only, TM-only or TM with SP, For EST clusters, Phobius annotation was obtained for each contig and then summarized at the NemaGene cluster level (www.nematode.net).

Interpro and Gene Ontology mappings

Default parameters for InterProScan v16.1 were used to search against InterPro database. Raw InterProScan results for the translated EST contigs were summarized at the EST cluster level. Gene Ontology annotations were obtained from the InterProScan using the -goterms flag as a parameter. Gene ontologies were further assigned and displayed graphically by AmiGO utilizing default parameters. Complete GO mappings for the all the genes can be accessed at http://nematode.net/cgi-bin/amigo/go_ostertagia_ostertagi/go.cgi. Go term enrichment was obtained after processing the GO occurrences through the FUNC server (Prufer et al., 2007).

KEGG pathway mappings (Kanehisa and Goto, 2006)

WU-BLAST matches of the O. ostertagi against KEGG database version 46.0 was used for pathway mapping with a filter of 1e-05. Table 3 and supplemental on line ST3 lists mappings for the top scoring and C. elegans hits to the KEGG enzymes that meet the E-value filter. For hypergeometric comparisons of L3-specific and L4-specific genes, all hits meeting the E-value cut-off were used. KEGG associations are available at http://www.nematode.net/KEGGscan/cgi-bin/KEGGscan_hit_distribution.cgi?species_selection=Ostertagia%20ostertagi.

Table 3
KEGG metabolic pathways mapped by O. ostertagi cDNA clusters and corresponding mappings in C. elegans.

3. Results and discussion

3.1. Characteristics of the generated transcripts and the identified genes

We generated cDNA libraries from three stages of O. ostertagi. A total of 7,042 ESTs (Table 1) that passed our sequencing and quality control screening were clustered using an internal pipeline (McCarter et al., 2003) to reduce redundancy, and were grouped into 4,611 contigs that were translated into amino acid sequences. Valid translations were obtained for 4,605 contigs and these clustered into 2,284 genes. Gene estimation from ESTs can sometimes result in over-representation because the short transcript length can lead to non-overlapping clusters which are part of a single gene (i.e. fragmentation). Gene fragmentation was estimated at 2.1% by comparison to C. elegans as a reference. The distribution of EST per cluster size shows that the largest cluster has 971 ESTs and less than 3% of clusters have more than 10 ESTs. Sixty-six percent of the clusters contain only one EST. This distribution reflects the abundance of transcripts from the stage-specific expression of the three libraries that were sampled. Highly expressed transcripts are indicative of the expression levels of the stages being sampled and can also represent important functional elements.

Table 1
Ostertagia ostertagi cDNA libraries and properties.

Clusters that contain transcripts from all three libraries represent transcripts expressed in all the stages of the parasite and are likely involved in core biological processes. The distribution of the clusters based on library composition is illustrated in Fig. 1. Less than 1% of all clusters contain transcripts from all stages and 12% (264) share transcripts among both larval stages. Such low numbers of shared clusters have also been reported in other EST projects (e.g. (Mitreva et al., 2004b; Thompson, 2005). Proteins enriched in these two specific life-cycle stages are discussed below.

3.2. Functional classification

Vast amounts of genomic information are available on public databases and offer a reliable way to identify similarity and/or homology to previously sequenced species. To functionally characterize the O. ostertagi genes, three phylogenetic databases were built. The first database includes three Caenorhabditis species (C. elegans, C. briggsae and C. remanei); the second database contains all nematode species other than Caenorhabditis; and the last database is the non-redundant protein database from Genbank excluding any nematode sequences (databases built: May 2007). A similarity search of the 2,284 genes using WU-BLAST (Gish, 1996-2002) versus these three phylogenetic databases as subject (with e-values better than 1e-05) indicates that 66% (1,497/2,284) of the O. ostertagi genes have sequence homologs among known and predicted proteins from other species. Over 70% of these genes matched all three databases (Fig. 2). In addition, the O. ostertagi gene clusters were compared to the draft genome of Haemonchus contortus (downloaded on February 10, 2009from http://www.sanger.ac.uk/Projects/H_contortus/), clade V Strongylida nematode that infects goats and sheep. This comparison identified 67% (1,529/2,284) O. ostertagi genes that had homologies with e-values better than 1e-05, of which 148 were homologies not shared with the three phylogenetic databases presented in Fig. 2. The average identity between these genes was 70%, with one third having % identity greater than 80 (data not shown). In all probability, these shared gene products are widely conserved among metazoans and associated with core biological processes. Five percent of Ostertagia genes with identity only to genes from Caenorhabditis species are potential nematode Clade V lineage-specific genes whereas the 639 O. ostertagi genes that did not show identity with any database are likely Trichostrongyloidea or Ostertagia-specific sequences.

Fig. 2
Distribution of O. ostertagi cluster BLAST matches by database. Databases used were: Caenorhabditis spp: C. elegans, C. briggsae, C. remanei; Other nematodes, all GenBank nucleotide data from nematodes except Caenorhabditis species; Non-nematodes, NR ...

Comparison to C. elegans showed that 52% (1,187/2,284) of the O. ostertagi genes have well-conserved putative orthologs in C. elegans. These orthologs were determined based on the C. elegans and O. ostertagi gene pairs that have highest sequence similarity to each other in reciprocal WU-BLAST search (with e-values better than 1e-05). Gene groups that had a high degree of conservation (with e-value better than 1e-100) included ribosomal proteins, collagen proteins, cytochrome subunits, zinc metalloproteases and serine/threonine kinases (Supplemental Information, ST1). RNA interference (RNAi) has been reported in O. ostertagi in a few genes but the knockdown effects have not been reliable (Visser et al., 2006). However, extrapolation from the C. elegans orthologs/homologs with observed RNAi phenotypes can be very informative for functional analysis. Of orthologous genes, 46% have observable C. elegans RNAi phenotypes. Further, when phenotypes were observed, 82% exhibited severe effects including embryonic, larval, or adult lethal, sterile adults or progeny, and larval or adult growth arrest (Supplemental Information, ST2).

The InterPro (Apweiler et al., 2001) database was searched to identify similarities to annotated protein domains and 1,172 genes shared sequence similarity with 1,035 unique Interpro domains (Supplemental Information, ST4). Well-represented domains include collagen triple helix repeat, nematode cuticle collagen, EGF-like region and transthyretin-like (Table 2). Among the sequences evaluated, 884 genes have Gene Ontology (The Gene Ontology, 2008) mappings and the three GO categories, biological process (670 genes), cellular component (451 genes) and molecular function (764 genes). Data constituting these mappings are shown in Fig. 3.

Fig. 3
Percentage representation of Gene Ontology (GO) categories for O. ostertagi clusters: A. Biological processes; B. Cellular component; C. Molecular function.
Table 2
The 25 most abundant InterPro domains by O. ostertagi cDNA clusters and corresponding mappings in C. elegans.

Unique clusters (605) mapped to 143 biochemical pathways grouped in 11 KEGG categories (Kanehisa and Goto, 2006)(Table 3). A complete listing of the pathways is available as supplementary material on line Supplemental Information ST3) and graphical representations of the KEGG mappings are available at www.nematode.net (Martin et al., in press). This viewer provides associations to specific enzyme commission (EC) numbers, KEGG Orthology identifiers, significance of the similarity and the O. ostertagi genes associated with the enzyme. Using this web tool, it is possible to make comparisons of the mappings between O. ostertagi and another nematode species for any enzyme in the metabolic pathways. This visualization can shed insight into the differences in parasitism, adaptations to the environment, etc. between the 32 partial nematode genomes available currently. Comparisons with the C. elegans KEGG viewer provides a mechanism to associate RNAi phenotype information assigned to the C. elegans genes associated with the pathways. Potential chokepoints in these metabolic pathways can be determined and this enables the identification of genes of interest based on their function.

Most nematode vaccine targets are excretory/secretory (ES) products or intestinal antigens that are membrane-bound. Excretory-secretory protein products are often involved in host-parasite interactions or have a role in parasite development and survival within the host. As such, they present suitable targets for alternative intervention strategies (S. E. Newton, 2003). However, it should be noted that prediction of signal peptides defines cellular events and not parasitological events. Therefore, a “secretory signal” characterizes the possibility that the protein in question will be secreted from the cell and not necessarily from the parasite and into the host. Comparing the hidden markov models (HMM) of different sequenced regions, we identified 254 O. ostertagi genes that have signal peptides for secretion, 355 genes with transmembrane domains and 44 genes with both features. Of the genes with signal peptides, 43% had associations to 122 Interpro domains and 49% of the TM to 187 domains (Supplemental Information, Table ST5).

3.3. Stage-enriched expression

3.3.1. Stage-biased expression

Ostertagia L3- and L4-biased or –specific clusters were identified, functionally characterized and compared to provide insight into adaptation and enrichment in each stage. The stage-biased clusters, comprised entirely of one of the two stages, resulted in 870 L3- and 955 L4-biased genes. Using a hypergeometric distribution analysis on KEGG metabolic pathways, we identified the pathways enriched in each of the stages. Fatty acid metabolism, metabolism of xenobiotics by cytochrome P450, C21-steroid hormone metabolism, linoleic acid metabolism, arachidonic acid metabolism, galactose metabolism and pentose and glucuronate interconversions are among the most enriched pathways among genes biased to L3. Pathways enriched in L4 stage include oxidative phosporylation and ubiquinone biosynthesis. Fatty acids have a role in locomotion and infection of host of L3 larvae in other nematodes (Medica and Sukhdeo, 1997) and xenobiotic metabolism in nematodes has relevance for anthelminthic drug applications (Kotze et al., 2006). The enriched pathways from both stages are listed in Supplemental Information (ST6).

Analysis of GO terms using the FUNC web server (Prufer et al., 2007) with this dataset show that in the L3 stage, oxidoreductase activity, signal transduction, binding, aromatic compound biosynthesis and GTP cyclohydrolase I activity are significantly over-represented. Genes involved in the degradation of fatty-acids and aromatic compounds show up-regulation in the C. elegans dauer stage (Burnell et al., 2005). Phosphate transport, structural constituents of the cuticle, serine-type peptidase activity, intracellular part and extracellular region are enriched in the L4 genes. A list of over-represented GO terms is presented in Supplemental Information (ST7). Enzymes belonging to the enriched KEGG pathways and functional domains of these GO terms reveal functional profiles that illustrate stage-specific adaptation and therefore, can serve as potential focus points for designing control studies.

3.3.2. Stage-specific expression

The stage-specific clusters were identified based on stage of origin and published statistical methodology (Audic and Claverie, 1997). While some of these will not remain stage-specific once more sequencing is performed, at present we were able to identify 36 possible L3-specific genes (comprised of 1,527 ESTs) and 71 L4-specific genes (1,428 ESTs) (significance threshold of 0.05). Investigating the differences in function between these two sets of genes provided valuable insight into the parasite’s life-cycle and can help identifying putative targets for designing effective vaccines and drugs targeting specific stages.

WU-BLAST analysis of the 36 L3-specific clusters shows that the majority of them have similarity to known or hypothetical proteins (Table 4). The most well represented functional category is the GTP-cyclohydrolase enzyme. This transcript, represented by 971 ESTs, putatively encodes GTP-cyclohydrolase I (EC 3.5.4.1.6) enzyme which is part of the KEGG folate biosynthesis pathway. This enzyme has previously been studied using RT-PCR and shown to be up-regulated in the L3 compared to other stages and its expression is localized to muscle and intestinal cells. (Moore et al., 2000). Gene products involved in electron transport were also identified in this dataset. To date, three genes have been identified as belonging to the cytochrome oxidase subunit protein superfamily and we have identified two of these subunits which participate in electron transport and possess transmembrane domains. Another cluster was homologous to the 14-3-3b protein from Meloidugyne incognita (Jaubert et al., 2004) and this regulatory protein has been shown to be important in the development of other parasites.

Table 4
Transcripts significantly enriched in L3 larval.

An L3-specific gene has significant alignment to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This enzyme (EC 1.2.1.9) acts as a catalyst during glycolysis. Onchocerca volvulus is a filarial parasite where the L3 is the infective stage (via black fly bite into the circulation of the human host). The GAPDH protein in O. volvulus, which is also up-regulated during the L3 stage, shares sequence identity with the antigens that have been reported to protect against O. volvulus infections (Erttmann et al., 2005). Methyl Malonyl CoA Mutase, UDP-GlucuronosylTransferase, NADH hydrogenase subunit and Peptidylglycine--hydroxylating monooxygenase are some of the other prominent functional categories.

Using the Gene ontology (GO) as an alternative functional classification of the 36 genes, identified 21 genes that could be assigned GO mapping. Of the 19 that had an assignment in biological process, 15 were classified as cellular process (GO:0009987). By molecular function, of the 19 genes that had an assignment, 15 were classified as catalytic activity (GO:0003824) and by cellular component, of the nine genes that had an assignment, four were classified as intracellular part (GO:0044424). Among the genes expressed in the L3, seven do not share homology with other known protein sequences and are novel. Of these, five genes have putative signal peptides or transmembrane domains. As ES products have been implicated in maintaining a positive environment for the parasite, and are intriguing from vaccine development point of view, these ES warrant further investigation.

A total of 71 genes were identified to be L4-enriched and 58 of these genes have significant alignment to confirmed or hypothetical proteins from other nematodes (Table 5). The gene group enriched with the most number of ESTs (305) in L4-specific genes is the collagen protein group. The nematode cuticle is made up of collagen proteins that are known to be differently-expressed by stage (Elling et al., 2007; Mitreva et al., 2004c). The cuticle of parasitic nematodes has an important role as an interface between parasite and host and therefore, these genes could potentially provide targets for intervention strategies. The other abundant functional groups are ribosomal proteins, SXC1 protein and the 17 kDa ES antigen protein. Among the genes enriched in the L4 stage, ten do not share sequence identity with other known protein sequences and are novel. Of these, one gene has signal peptides or transmembrane domain predictions.

Table 5
Transcripts significantly enriched in L4 larval.

Of the 71 L4 genes, 40 were assigned GO terms. Of 33 that had an assignment in biological process, 19 were involved with cellular metabolic process (GO:0044237). By molecular function, of the 35 that had an assignment, 20 were assigned to structural molecular activity (GO:0005198) and by cellular component, of the 31 that had an assignment, the most abundant was intracellular part (GO:0044424), which constituted 26 of the assignments.

4. Conclusion

This study is the first large-scale survey of the larval and adult O. ostertagi transcribed genomes. The generation and analysis of the over 2,300 genes presented here is a valuable addition to resources for the study of parasitic worms, laying a foundation for further comparative studies on biology, parasitism and evolution of nematodes. The O. ostertagi EST sequencing data have been submitted to public databases and the functional annotations are available online (www.nematode.net)(Martin et al., 2009). This information is therefore accessible as a resource to researchers working on parasitic nematodes for studies, including but not limited to microarrays, RT-PCR, RNA interference screens and proteomic experiments. Such studies will aid in the identification of genes involved in host recognition, infection, migration and immune evasion as well as the characterization of targets for vaccines and drugs.

Fig. 4Fig. 4
Cluster composition of stage-specific clusters: A. EST distribution in L3-specific transcripts (for better visualization the biggest cluster is excluded); B. EST distribution in L4-specific clusters.

Supplementary Material

Acknowledgments

Ostertagia ostertagi EST sequencing at Washington University was supported by NIH-NIAID grant AI 46593 to MM. The authors would like to thank Claire Murphy and Mike Dante for technical assistance.

Footnotes

Accession numbers Nucleotide sequence data reported in this paper and sequenced at the Washington University’s Genome Center are available in the GenBank, EMBL and DDBJ databases. The accession numbers for the Ostertagia ostertagi ESTs are 19379209-19381350, 12652777-21653605, 21265177-21265790, 20130441-20133438.

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