The vicuna (Vicugna vicugna) as a natural host of Dictyocaulus filaria in Peru.

Lungworm infection, or verminous pneumonia, is a parasitic disease that causes serious problems in small and large ruminants. Despite the fact that nematodes of the genus Dictyocaulus in cattle and sheep are the main cause of this disease, there are few studies on the natural infections of South American camelids. For this reason, this study aims to report the natural infection by Dictyocaulus filaria in vicunas (Vicugna vicugna) for the first time. During a shearing season (chaku) in Cuzco, Peru, two accidentally killed adult vicunas were submitted to the IVITA-Marangani research center in Cuzco for their respective necropsies. The tracheas of both vicunas had numerous nematodes, as seen during the necropsy. The nematodes were collected in 70% ethanol and were morphologically identified as D. filaria. Likewise, the DNA of six nematodes was extracted, and the ITS2 region and the 28S rRNA gene were amplified and sequenced. The nucleotide sequences of both genetic markers were up to 100% identical with previously reported D. filaria DNA sequences found in the goat yearlings from Turkey, sheep from Iran, Turkey, and India, and the argali from Uzbekistan, which confirmed the morphological diagnosis. This finding represents the first molecular confirmation of a natural D. filaria infection in a South American camelid. It will be necessary to carry out future studies to know the current situation of verminous pneumonia in domestic and wild South American camelids and to know the negative effects of the disease on them.

Distribution of large lungworms (Nematoda: Dictyocaulidae) in free-roaming populations of red deer Cervus elaphus (L.) with the description of Dictyocaulus skrjabini n. sp.

Lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in domestic and wild ungulates. This study investigates the distribution, morphology and genetic diversity of D. cervi and a new lungworm species, Dictyocaulus skrjabini n. sp. infecting red deer Cervus elaphus, fallow deer Dama dama and moose Alces alces in Poland and Sweden. The study was conducted on 167 red deer from Poland and on the DNA of lungworms derived from 7 fallow deer, 4 red deer and 2 moose collected in Sweden. The prevalence of D. cervi and D. skrjabini n. sp. in dissected red deer in Poland was 31.1% and 7.2%, respectively. Moreover, D. skrjabini n. sp. was confirmed molecularly in 7 isolates of fallow deer lungworms and 1 isolate of red deer lungworms from Sweden. Dictyocaulus skrjabini n. sp. was established based on combination of their distinct molecular and morphological features; these included the length of cephalic vesicle, buccal capsule (BC), buccal capsule wall (BCW), distance from anterior extremity to the nerve ring, the width of head, oesophagus, cephalic vesicle, BC and BCW, as well as the dimensions of reproductive organs of male and female. Additionally, molecular analyses revealed 0.9% nucleotide sequence divergence for 1,605 bp SSU rDNA, and 16.5­17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7­19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2­18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

Morphological and molecular description of Dictyocaulus xanthopygus sp. nov. (Nematoda: Trichostrongyloidea) from the Manchurian wapiti Cervus elaphus xanthopygus.

Dictyocaulus xanthopygus sp. nov. (Nematoda: Trichostrongyloidea) was isolated from the lungs of the Manchurian wapiti in Primorsky kray, Russia. The newly described species exhibits morphological characteristics of Dictyocaulus but is distinct from congeneric species based on morphological (lengths of body and esophagus, distances from the anterior end to nerve ring and to excretory pore, the thickness of the buccal capsule, etc.) and molecular features. High levels of genetic divergence as well as Bayesian phylogenetic analyses based on 18S rRNA nuclear and cox1 mitochondrial genes supported the independence of Dictyocaulus xanthopygus sp. nov. Secondary structures of helix 39 of 18S rRNA were identical, while ES9 adjacent to the helix has a unique conformation for newly described worms. Energy-efficient conformational rearrangements of rRNA secondary structures can be applicable in studies on the pathogenesis, epidemiology, taxonomy and evolutionary biology of parasites. Additionally, bracketed dichotomous keys to six valid species of Dictyocaulus were prepared.

Molecular characterization of Dictyocaulus nematodes in wild red deer Cervus elaphus in two areas of the Italian Alps.

Nematodes of the genus Dictyocaulus are the causative agents of parasitic bronchitis and pneumonia in several domestic and wild ungulates. Various species have been described in wild cervids, as the case of Dictyocaulus cervi in red deer, recently described as a separate species from Dictyocaulus eckerti. In Italy, information on dictyocaulosis in wildlife is limited and often outdated. In this work, 250 red deer were examined for the presence of Dictyocaulus spp. in two areas of the Italian Alps (n = 104 from Valle d'Aosta, n = 146 from Stelvio National Park), and the retrieved lungworms were molecularly characterized. Lungworms were identified in 23 and 32 animals from Valle d'Aosta and Stelvio National Park, respectively. The nematodes, morphologically identified as D. cervi, were characterized molecularly (18S rDNA, ITS2, and coxI). Consistently, almost all specimens were found to be phylogenetically related to D. cervi. Three individuals, detected from both study sites and assigned to an undescribed Dictyocaulus sp., clustered with Dictyocaulus specimens isolated from red deer and fallow deer in previous studies. Within each of D. cervi and the undescribed Dictyocaulus sp., the newly isolated nematodes phylogenetically clustered based on their geographical origin. This study revealed the presence of D. cervi in Italian red deer, and an undetermined Dictyocaulus sp. that should be more deeply investigated. The results suggest that further analyses should be focused on population genetics of cervids and their lungworms to assess how they evolved, or co-evolved, throughout time and space and to assess the potential of transmission towards farmed animals.

DICTYOCAULUS CERVI-LIKE LUNGWORM INFECTION IN A ROCKY MOUNTAIN ELK (CERVUS CANADENSIS NELSONI) FROM WYOMING, USA.

Dictyocaulus spp. infections are common in North American cervids, with Dictyocaulus viviparus described as most common. A Rocky Mountain elk (Cervus canadensis nelsoni) was found dead in Wyoming, US with significant bronchitis and pneumonia. In the bronchi and trachea, numerous large nematodes were found and grossly identified as Dictyocaulus spp. lungworms. Macroscopic alterations, such as distended interlobular septa and edema with foam and mucus observed on cut surface and in trachea and bronchi, were consistent with those commonly described in D. viviparus infections. Female lungworms were identified to Dictyocaulus spp. level via morphologic examination and molecular analyses based on mitochondrial cyclooxygenase 1 and 18S ribosomal RNA genes. A phylogenetic analysis was conducted employing the maximum likelihood method. Based on both morphologic and genetic assays, the isolated lungworms were most likely a strain of Dictyocaulus cervi. Within the female adult worms, free first stage larvae were observed besides worm eggs, which had not been described for Dictyocaulus spp. Phylogenetic analysis revealed that our parasites clustered closely with D. cervi, forming a subclade with that species within a larger clade that includes Dictyocaulus eckerti. While the elk tested positive for chronic wasting disease, it is assumed that significant pathology in the present case was caused directly by infection with the D. cervi-like lungworm, not previously described in North America.

An assessment of the use of cox1 and cox3 mitochondrial genetic markers for the identification of Dictyocaulus spp. (Nematoda: Trichostrongyloidea) in wild ruminants.

Lungworms of the genus Dictyocaulus Railliet and Henry, 1907 (Nematoda: Trichostrongyloidea) are the causative agents of parasitic bronchitis (dictyocaulosis, husk) of various ungulate hosts, including domestic and wild ruminants. Correct diagnosis of lungworm species and a better understanding of the transmission patterns of Dictyocaulus spp. are crucial in minimising the risk of its cross transmission between wildlife and livestock, and for the control of dictyocaulosis. The study was conducted on large lungworms collected from European bison, roe deer and red deer. The study resulted in 14 sequences of the partial cox1 region of Dictyocaulus spp. and 10 novel DNA sequences of partial cox3 region, including the first available mt cox3 sequence, of the roe deer lungworm (D. capreolus). The European bison was infected with bison genotype of D. viviparus, whereas red deer and roe deer were infected with D. cervi and D. capreolus respectively. The current study revealed that the cox3 nucleotide sequences of D. capreolus and D. viviparus were 100% homologous to each other. Our findings indicate that the mt cox3 gene does not serve as an efficient mt marker for systematic, population genetic or molecular epidemiological studies of Dictyocaulus lungworms.

Interrelationships of Dictyocaulus spp. in Wild Ruminants with Morphological Description of Dictyocaulus cervi n. sp. (Nematoda: Trichostrongyloidea) from Red Deer, Cervus elaphus.

Lungworms from the genus Dictyocaulus cause parasitic bronchitis (dictyocaulosis) characterized by coughing and severe lung pathology in both domestic and wild ruminants. In this study we investigated the interrelationships of Dictyocaulus spp. from European bison (Bison bonasus L.), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) by nucleotide sequence analysis spanning the 18S RNA gene (small subunit [SSU]) and internal transcribed spacer 2 (ITS2) regions of the ribosomal gene array as well as the mitochondrial cytochrome c oxidase subunit 1 (cox1). Molecular analyses of sequence data obtained partly with novel primers from between 10 and 50 specimens from each host were carried out. Bayesian inference analysis revealed that each host species was infected with different genotypes. Analysis of cox1 sequence data showed a diverse genetic background and high evolutionary potential of Dictyocaulus taxa. Data from lungworms of European bison revealed a distinct genotype of Dictyocaulus viviparus, whereas Dictyocaulus capreolus was only found in roe deer. In contrast, red deer were infected with a taxon with unique SSU, ITS2, and cox1 sequences. These results indicate the occurrence of a novel genotype from red deer, which differs significantly from the National Center for Biotechnology Information reference sequence of Dictyocaulus eckerti. The molecular evidence was consistent with a morphological study with description and imaging of Dictyocaulus cervi n. sp. recovered from red deer. Dictyocaulus cervi n. sp. can be distinguished from D. eckerti on the basis of the absence of cervical papillae, the occurrence of a single ring of 4 symmetrical submedian cephalic papillae, length of the tail in females, morphometry of the female reproductive system, and measurements of gubernacula in males. In conclusion, our findings further strengthen the idea that the genetic complexity and diversity among Dictyocaulus lungworms infecting wildlife ruminants is larger than previously believed and warrants further investigation.

Genetic diversity and population genetics of large lungworms (Dictyocaulus, Nematoda) in wild deer in Hungary.

Dictyocaulus nematode worms live as parasites in the lower airways of ungulates and can cause significant disease in both wild and farmed hosts. This study represents the first population genetic analysis of large lungworms in wildlife. Specifically, we quantify genetic variation in Dictyocaulus lungworms from wild deer (red deer, fallow deer and roe deer) in Hungary, based on mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data, using population genetic and phylogenetic analyses. The studied Dictyocaulus taxa display considerable genetic diversity. At least one cryptic species and a new parasite-host relationship are revealed by our molecular study. Population genetic analyses for Dictyocaulus eckerti revealed high gene flow amongst weakly structured spatial populations that utilise the three host deer species considered here. Our results suggest that D. eckerti is a widespread generalist parasite in ungulates, with a diverse genetic backround and high evolutionary potential. In contrast, evidence of cryptic genetic structure at regional geographic scales was observed for Dictyocaulus capreolus, which infects just one host species, suggesting it is a specialist within the studied area. D. capreolus displayed lower genetic diversity overall, with only moderate gene flow compared to the closely related D. eckerti. We suggest that the differing vagility and dispersal behaviour of hosts are important contributing factors to the population structure of lungworms, and possibly other nematode parasites with single-host life cycles. Our findings are of relevance for the management of lungworms in deer farms and wild deer populations.

Dictyocaulus viviparus genome, variome and transcriptome elucidate lungworm biology and support future intervention.

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.

Development of a multiplex PCR for identification of Dictyocaulus lungworms in domestic and wild ruminants.

Dictyocaulus lungworms are the causative agents of parasitic bronchitis (dictyocaulosis) characterised by coughing and severe lung pathology in domestic and wild ruminants. The objective of this study was to design a simple molecular test that could detect of lungworm DNA from both adult and larval lungworms and could distinguish between the most common Dictyocaulus species found in cattle and in some species of wild ruminants. A multiplex PCR test with four novel primers targeting species-specific regions of the second internal transcribed spacer (ITS2) was designed based on our own sequence data as well as on available sequence information in GenBank. After PCR amplification of lungworms from European bison (Bison bonasus), cattle (Bos taurus), moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), products were analysed with gel electrophoresis. This resulted in three specific bands of different size depending on the species analysed. Dictyocaulus viviparus collected from cattle or European bison resulted in a ca. 560 bp band, D. capreolus collected from roe deer produced a band ca. 400 bp and the longest DNA band (ca. 660 bp) was obtained with DNA from Dictyocaulus sp. collected from red deer and moose. Dictyocaulus eckerti bands with expected size of 714 bp were not observed in our study. The multiplex method produced consistent results with samples from both Sweden and Poland and overcame the limitations of traditional techniques based on differences in morphological features of parasites at different life stages.

Palaeosymbiosis revealed by genomic fossils of Wolbachia in a strongyloidean nematode.

Wolbachia are common endosymbionts of terrestrial arthropods, and are also found in nematodes: the animal-parasitic filaria, and the plant-parasite Radopholus similis. Lateral transfer of Wolbachia DNA to the host genome is common. We generated a draft genome sequence for the strongyloidean nematode parasite Dictyocaulus viviparus, the cattle lungworm. In the assembly, we identified nearly 1 Mb of sequence with similarity to Wolbachia. The fragments were unlikely to derive from a live Wolbachia infection: most were short, and the genes were disabled through inactivating mutations. Many fragments were co-assembled with definitively nematode-derived sequence. We found limited evidence of expression of the Wolbachia-derived genes. The D. viviparus Wolbachia genes were most similar to filarial strains and strains from the host-promiscuous clade F. We conclude that D. viviparus was infected by Wolbachia in the past, and that clade F-like symbionts may have been the source of filarial Wolbachia infections.

Analysis of the transcriptome of adult Dictyocaulus filaria and comparison with Dictyocaulus viviparus, with a focus on molecules involved in host-parasite interactions.

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, ß-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.

Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU).

Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

Assessment of the genetic relationship between Dictyocaulus species from Bos taurus and Cervus elaphus using complete mitochondrial genomic datasets.

BACKGROUND: Dictyocaulus species are strongylid nematodes of major veterinary significance in ruminants, such as cattle and cervids, and cause serious bronchitis or pneumonia (dictyocaulosis or "husk"). There has been ongoing controversy surrounding the validity of some Dictyocaulus species and their host specificity. Here, we sequenced and characterized the mitochondrial (mt) genomes of Dictyocaulus viviparus (from Bos taurus) with Dictyocaulus sp. cf. eckerti from red deer (Cervus elaphus), used mt datasets to assess the genetic relationship between these and related parasites, and predicted markers for future population genetic or molecular epidemiological studies. METHODS: The mt genomes were amplified from single adult males of D. viviparus and Dictyocaulus sp. cf. eckerti (from red deer) by long-PCR, sequenced using 454-technology and annotated using bioinformatic tools. Amino acid sequences inferred from individual genes of each of the two mt genomes were compared, concatenated and subjected to phylogenetic analysis using Bayesian inference (BI), also employing data for other strongylids for comparative purposes. RESULTS: The circular mt genomes were 13,310 bp (D. viviparus) and 13,296 bp (Dictyocaulus sp. cf. eckerti) in size, and each contained 12 protein-encoding, 22 transfer RNA and 2 ribosomal RNA genes, consistent with other strongylid nematodes sequenced to date. Sliding window analysis identified genes with high or low levels of nucleotide diversity between the mt genomes. At the predicted mt proteomic level, there was an overall sequence difference of 34.5% between D. viviparus and Dictyocaulus sp. cf. eckerti, and amino acid sequence variation within each species was usually much lower than differences between species. Phylogenetic analysis of the concatenated amino acid sequence data for all 12 mt proteins showed that both D. viviparus and Dictyocaulus sp. cf. eckerti were closely related, and grouped to the exclusion of selected members of the superfamilies Metastrongyloidea, Trichostrongyloidea, Ancylostomatoidea and Strongyloidea. CONCLUSIONS: Consistent with previous findings for nuclear ribosomal DNA sequence data, the present analyses indicate that Dictyocaulus sp. cf. eckerti (red deer) and D. viviparus are separate species. Barcodes in the two mt genomes and proteomes should serve as markers for future studies of the population genetics and/or epidemiology of these and related species of Dictyocaulus.

Genes of the bovine lungworm Dictyocaulus viviparus associated with transition from pasture to parasitism.

Genes necessary to enable nematode parasitic life after free-living larval life are of substantial interest to understand parasitism. We investigated transcriptional changes during transition to parasitism in the bovine lungworm Dictyocaulus viviparus, one of the most important parasites in cattle farming due to substantial economic losses. Upregulated transcripts in either free-living, developmentally arrested L3 or parasitic immature L5 were identified by suppression subtractive hybridization (SSH) followed by differential screening and subsequent virtual Northern blot verification. From 400 sequenced clones of parasitic L5, 372 (93.0%) upregulated high quality ESTs were obtained clustering into 30 contigs and 38 singletons. Most conceptual translated peptides were SCP/TAPS "family" members also known as pathogenesis-related protein (PRP) superfamily (28.5% of total ESTs), cysteine proteases (24.5%), and H-gal-GP orthologues (9.9%). These proteins are predicted to play key roles in fundamental biological processes such as nutrition and development but also parasite-host interactions and immune defense mechanisms. Increased energy requirement of the rapidly developing L5 lungworm stage was obvious in a proportion of 12.2% upregulated ESTs being components of the respiratory chain. From the developmentally arrested L3 stage sequencing of 200 clones resulted in 195 high quality ESTs (97.0%) clustering into 7 contigs and 3 singletons only. Besides a hypothetical protein (70.1% of total ESTs) most transcripts encoded the cleavage stimulation factor subunit 2 (17.5%), which is a component of the poly(A(+)) machinery and found to be involved in gene silencing. Obtained data provide the basis for future fundamental research into genes associated with parasitic lifestyle but also applied research like vaccine and/or drug development.

In vitro studies on the sexual maturation of the bovine lungworm Dictyocaulus viviparus during the development of preadult larvae to adult worms.

The bovine lungworm Dictyocaulus viviparus is one of the most important parasites in grazing cattle. However, not much is known about morphology and molecular aspects of sexual maturation occurring during development of preadult larvae (L5) to adults. Since studies in the pulmonary compartments are infeasible, an in vitro cultivation method was established. The study was conducted with L5 during in vitro cultivation, assessing longitudinal growth and sexual maturation. Best results were achieved with RPMI-1640 medium with L-glutamine, 50% fetal bovine serum, amphotericin B (0.25 mg/ml), penicillin (10,000 U/ml), and streptomycin (10 mg/ml) at 39°C and 5% atmospheric CO2. During cultivation, individuals grew from an average length of 4.64 to 9.88 mm independent of their density per setup. Regarding sexual maturation, female individuals started to lay eggs, whereas the testes of male individuals were filled with spermatozoa. Consequently, adult female and adult male worms developed. However, no copulation was observable and eggs did not embryonate. Development was further investigated by quantitative real-time PCR transcriptional analysis of major sperm protein (msp) and vitellogenin (vit) representing male and female sexual development, respectively. Male msp transcription peaked after 5 days of cultivation [corresponding to 20 days post infection (dpi)] and decreased gradually afterwards. Female vit transcription showed the highest rate after 15 days of cultivation (30 dpi), however it never reached the transcription rate in female adults isolated from the host. All in all, the present study gives not only insights into morphological differentiation but provides data lightening molecular aspects of sexual maturation in D. viviparus.

Transcriptional differences between hypobiotic and non-hypobiotic preadult larvae of the bovine lungworm Dictyocaulus viviparus.

The survival of the bovine lungworm Dictyocaulus viviparus, one of the most important parasites in cattle, inside the host is ensured by arrested development during adverse environmental conditions, commonly referred to as hypobiosis. In the present study, a subtractive hybridization approach was used to compare the transcription profiles of hypobiotic and non-hypobiotic larvae (L5hyp and L5, respectively). Thereby, 75 L5hyp-enriched and 58 L5-enriched representative ESTs (rESTs) were identified. Subsequent sequence similarity search revealed that 28 L5hyp-rESTs and 11 L5-rESTs were homologous to known transcripts, whereas 47 L5hyp-rESTs and 47 L5-rESTs showed no homologies with published sequences, thus possibly representing parasitic or even Dictyocaulus-specific genes. The differential transcripts were predicted to be involved in nucleic acid synthesis, DNA binding, metabolic pathways and signal transduction. Overall, data presented in this paper provide a first basis for further characterization and analysis of genes driving normal as well as arrested (hypobiotic) parasite development.

GTP-cyclohydrolase and development in Teladorsagia circumcincta and Dictyocaulus viviparus (Nematoda: Strongylida).

GTP-Cyclohydrolase (GTP-CH) is necessary for the production of tetrahydrobiopterin, a required cofactor for the three aromatic amino acid hydroxylases and nitric oxide synthases. The gene encoding GTP-CH is transcribed at high levels in infective third larval stages of a number of parasitic trichostrongylid nematodes. We explore the potential role of GTP-CH within the processes of nematode development and environmentally-induced hypobiosis. For two species of parasitic nematode that are of major economic and welfare importance to livestock in temperate regions, Teladorsagia circumcincta and Dictyocaulus viviparus, we have demonstrated that each of the pre-parasitic larval stages transcribe high mean levels of cat-4 (the gene encoding GTP-CH). Using quantitative real-time polymerase chain reaction analysis and two different isolates of D. viviparus, only one of which is capable of entering hypobiosis, we have shown that there were only minor differences between these isolates in mean cat-4 transcript levels, both during the parasitic stages and during the earlier environmental life cycle stages (L(1)-L(3)). Taken together, these data indicate that, although both species of nematode produce high levels of cat-4 transcript in pre-parasitic larval stages, GTP-CH levels are unlikely to be involved in the induction of parasite hypobiosis. Alternative roles for GTP-CH in larval development are discussed.

Deep insights into Dictyocaulus viviparus transcriptomes provides unique prospects for new drug targets and disease intervention.

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.

Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus.

The bovine lungworm Dictyocaulus viviparus is of major economic importance in cattle farming in the temperate zones. The invertebrate protein paramyosin is one of the main components of muscle thick filaments but can also exhibit immunomodulatory functions. It represents a promising vaccine candidate in parasitic helminths. In this study, D. viviparus paramyosin (DvPmy) was characterized on the transcriptional as well as genomic level. The identified genomic sequence comprises 19 introns compared to only 10 introns in the Caenorhabditis elegans orthologue. Quantitative real time PCR transcriptional analysis revealed paramyosin transcription throughout the whole parasite's life cycle with the highest transcription rate in the agile moving first-stage larvae and the lowest in motionless hypobiosis induced third stage larvae. Recombinantly expressed DvPmy was found to bind collagen and IgG. Thereby the present study is the first showing that nematode paramyosin has the capability for immunomodulation and thus may be involved in host immune defence.