Biological function of Dictyocaulus viviparus asparaginyl peptidase legumain-1 and its suitability as a vaccine target.

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35-39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

Fatty acid composition of free-living and parasitic stages of the bovine lungworm Dictyocaulus viviparus.

The development of parasitic nematodes proceeds via multiple stages, often implicating the necessity to adapt to different environments. Especially the transition from free-living to parasitic stages is accompanied by a significant change in the environmental conditions. To shed light on possible adaptations to these transitions, the fatty acid composition of different developmental stages of the bovine lungworm Dictyocaulus viviparus was investigated. Fatty acids of D. viviparus eggs, the free-living first, second and third larval stage (L1-L3) as well as the parasitic preadult stage and adult male and female worms residing in the lungs of infected hosts were quantified by gas chromatography after transesterification to their fatty acid methyl esters. The fatty acid content and diversity were higher in parasitic stages compared to those of free-living larvae. The most prevalent fatty acids in both parasitic and free-living stages were stearic (C18:0), palmitic (C16:0), palmitoleic (C16:1) and caprylic acid (C8:0). A variety of (poly-)unsaturated FAs was found in the parasitic stages and in the eggs, which was similar to the variety of FAs found in bovine surfactant. This finding indicates that parasitic stages of D. viviparus take up FAs from their environment. While eggs contained the highest concentration of fatty acids, a decrease was observed from eggs to L1 and further from L2 to L3. The lowest concentration was found in 38-days-old L3, which suggests that FAs serve as an energy reserve for the free-living, non-feeding larval stages. The free-living larvae contained mainly saturated fatty acids and only traces of unsaturated fatty acids, which is in contrast to the phospholipid saturation hypothesis of cold tolerance. Instead, a trade-off between desiccation stress and temperature adaptation may favour a higher amount of saturated FAs in the free-living larval stages. Further studies explicitly examining the FA composition of the different classes of lipids are necessary to better describe the adaptative responses of the FA metabolism to different environmental conditions.

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.

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.

Genetic regulation of arrested development in nematodes: are age-1 and daf-gene orthologs present in Dictyocaulus viviparus?

In opposite to the free-living soil nematode Caenorhabditis elegans, the genetic regulation of hypobiosis or inhibited or arrested development in parasitic nematodes is completely unknown. In C. elegans, the daf-genes or the age-1 gene are of major importance in signaling pathways regulating arrested development. To investigate if orthologs of these genes are present in the bovine lungworm Dictyocaulus viviparus, a PCR analysis with gene-specific primer combinations was performed. No orthologs of the age-1 or daf-genes could be identified in D. viviparus. The possible differences in the role of the daf-genes concerning arrested development in parasitic and free-living nematodes will be discussed.

Nematoda: aerobic respiratory pathways of adult parasitic species.

Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.