Project description:Purpose: The goal of this study was to track fluctuations in gene expression across all defined life cycle stages of the cattle lungworm Dictyocaulus viviparus. Methods: Batches of worms from a Hannover, Germany strain were harvested in duplicate as eggs, L1, L2, L3, L4, hypobiotic L5, mixed-sex L5, male L5, female L5, mature males and mature females. RNA was isolated, reverse transcribed and sequenced on the Illumina platform. Finally, reads were mapped to the D. viviparus draft genome assembly and read counts associated with each feature were used to estimate gene expression levels and predict differential gene expression at various transitional points across the life cycle. Results: Principal component analyses indicated that major gene expression shifts occur upon hatching, infection of the bovid host, and sexual maturation.
Project description:Entomopathogenic nematodes (EPNs) are unique parasitic nematodes due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. Although it has been widely believed that EPNs rely on their bacterial partners for killing insect hosts, compelling evidence from previous studies challenges this model. We developed an improved method of activating millions of Steinernema carpocapsae infective juveniles (IJs) in vitro to harvest excreted/secreted (ES) proteins for bioactivity tests and proteomics analysis. We found that a low dose of the ES proteins from early activated nematodes is lethal to Drosophila melanogaster adults within 2-6 hours. We analyzed the protein composition of this venom using mass spectrometry and identified 472 proteins. Many of these venom proteins share high homology with those of vertebrate-parasitic nematodes. Among many different families of proteins found in the venom, proteases and protease inhibitors are especially abundant. Some toxin-related proteins such as Shk domain-containing proteins were also detected. We further analyzed the transcriptomes of individual non-activated IJs and nematodes that were activated in vitro and in vivo, which revealed a dramatic shift in gene expression during IJ activation. By comparing the whole transcriptomes and the genes encoding venom proteins between the in vitro and in vivo activated nematodes, we confirmed that the in vitro activation is a good approximation of the in vivo process. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of ES products.
Project description:BACKGROUND: Lungworms of the genus Dictyocaulus (family Dictyocaulidae) are parasitic nematodes of major economic importance. They cause pathological effects and clinical disease in various ruminant hosts, particularly in young animals. Dictyocaulus viviparus, called the bovine lungworm, is a major pathogen of cattle, with severe infections being fatal. In this study, we provide first insights into the transcriptome of the adult stage of D. viviparus through the analysis of expressed sequence tags (ESTs). RESULTS: Using our EST analysis pipeline, we estimate that the present dataset of 4436 ESTs is derived from 2258 genes based on cluster and comparative genomic analyses of the ESTs. Of the 2258 representative ESTs, 1159 (51.3%) had homologues in the free-living nematode C. elegans, 1174 (51.9%) in parasitic nematodes, 827 (36.6%) in organisms other than nematodes, and 863 (38%) had no significant match to any sequence in the current databases. Of the C. elegans homologues, 569 had observed 'non-wildtype' RNAi phenotypes, including embryonic lethality, maternal sterility, sterility in progeny, larval arrest and slow growth. We could functionally classify 776 (35%) sequences using the Gene Ontologies (GO) and established pathway associations to 696 (31%) sequences in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, we predicted 85 secreted proteins which could represent potential candidates for developing novel anthelmintics or vaccines. CONCLUSION: The bioinformatic analyses of ESTs data for D. viviparus has elucidated sets of relatively conserved and potentially novel genes. The genes discovered in this study should assist research toward a better understanding of the basic molecular biology of D. viviparus, which could lead, in the longer term, to novel intervention strategies. The characterization of the D. viviparus transcriptome also provides a foundation for whole genome sequence analysis and future comparative transcriptomic analyses.
Project description:The bovine lungworm, Dictyocaulus viviparus (order Strongylida), is an important parasite of livestock that causes substantial economic and production losses worldwide. Here we report the draft genome, variome, and developmental transcriptome of D. viviparus. The genome (161?Mb) is smaller than those of related bursate nematodes and encodes fewer proteins (14,171 total). In the first genome-wide assessment of genomic variation in any parasitic nematode, we found a high degree of sequence variability in proteins predicted to be involved host-parasite interactions. Next, we used extensive RNA sequence data to track gene transcription across the life cycle of D. viviparus, and identified genes that might be important in nematode development and parasitism. Finally, we predicted genes that could be vital in host-parasite interactions, genes that could serve as drug targets, and putative RNAi effectors with a view to developing functional genomic tools. This extensive, well-curated dataset should provide a basis for developing new anthelmintics, vaccines, and improved diagnostic tests and serve as a platform for future investigations of drug resistance and epidemiology of the bovine lungworm and related nematodes.
Project description:Current diagnostic methods for detecting the presence or absence of Dictyocaulus viviparus in dairy herds, are insensitive when based on detection of antibody levels in bulk tank milk (BTM). Here we present a novel technique to confirm the presence of the parasite based on a pooled-milk sample from 10 randomly selected first - lactation heifers (FLH). This study was run in two parts. First, a longitudinal study was performed to look at infection dynamics in milk samples across the grazing season using a prototype ELISA developed by Svanova (Boehringer-Ingelheim, Uppsala). We identified that mean ODR values in milk samples from FLH was significantly higher than that for older cows (0.13 versus 0.07 respectively, p < 0.001) suggesting that samples from the FLH cohort should be pooled to produce the test. Second, the pooled - milk test was evaluated on a cross-sectional survey of UK dairy herds (n = 25 grazing and n = 25 zero-grazing herds) to evaluate test performance under field conditions. The optical density ratio (ODR) cut-off value for our pooled-milk test using 10 FLH milk samples was optimal at a value of 0.16. Pooling 10 FLH samples created a sensitivity and specificity of 66.7% and 95.5% respectively. In comparison, whole-herd BTM samples had a maximum sensitivity of 37.5% and specificity of 63.6% at an ODR cut-off of 0.18. The area under the curve according to receiver-operative-characteristic (ROC) analysis was high for the 10-heifer test (0.87) but poor for the whole herd BTM testing (0.45). This study provides a more sensitive diagnostic test strategy for the screening of D.viviparus in dairy herds. Testing herds at the end of a grazing season would facilitate the planning of effective control measures, such as the use of the lungworm vaccination or strategic deworming, for the following grazing season. This may prove to be a useful test strategy for the diagnosis of a variety of parasitic diseases of livestock.
Project description:Parasitic nematodes cause diseases of major economic importance in animals. Key representatives are species of Dictyocaulus (=lungworms), which cause bronchitis (=dictyocaulosis, commonly known as "husk") and have a major adverse impact on the health of livestock. In spite of their economic importance, very little is known about the immunomolecular biology of these parasites. Here, we conducted a comprehensive investigation of the adult transcriptome of Dictyocaulus filaria of small ruminants and compared it with that of Dictyocaulus viviparus of bovids. We then identified a subset of highly transcribed molecules inferred to be linked to host-parasite interactions, including cathepsin B peptidases, fatty-acid and/or retinol-binding proteins, ?-galactoside-binding galectins, secreted protein 6 precursors, macrophage migration inhibitory factors, glutathione peroxidases, a transthyretin-like protein and a type 2-like cystatin. We then studied homologues of D. filaria type 2-like cystatin encoded in D. viviparus and 24 other nematodes representing seven distinct taxonomic orders, with a particular focus on their proposed role in immunomodulation and/or metabolism. Taken together, the present study provides new insights into nematode-host interactions. The findings lay the foundation for future experimental studies and could have implications for designing new interventions against lungworms and other parasitic nematodes. The future characterisation of the genomes of Dictyocaulus spp. should underpin these endeavours.
Project description:The lungworm, Dictyocaulus viviparus, causes parasitic bronchitis in cattle, and is responsible for substantial economic losses in temperate regions of the world. Here, we undertake the first large-scale exploration of available transcriptomic data for this lungworm, examine differences in transcription between different stages/both genders and identify and prioritize essential molecules linked to fundamental metabolic pathways, which could represent novel drug targets. Approximately 3 million expressed sequence tags (ESTs), generated by 454 sequencing from third-stage larvae (L3s) as well as adult females and males of D. viviparus, were assembled and annotated. The assembly of these sequences yielded ~61,000 contigs, of which relatively large proportions encoded collagens (4.3%), ubiquitins (2.1%) and serine/threonine protein kinases (1.9%). Subtractive analysis in silico identified 6928 nucleotide sequences as being uniquely transcribed in L3, and 5203 and 7889 transcripts as being exclusive to the adult female and male, respectively. Most peptides predicted from the conceptual translations were nucleoplasmins (L3), serine/threonine protein kinases (female) and major sperm proteins (male). Additional analyses allowed the prediction of three drug target candidates, whose Caenorhabditis elegans homologues were linked to a lethal RNA interference phenotype. This detailed exploration, combined with future transcriptomic sequencing of all developmental stages of D. viviparus, will facilitate future investigations of the molecular biology of this parasitic nematode as well as genomic sequencing. These advances will underpin the discovery of new drug and/or vaccine targets, focused on biotechnological outcomes.