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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.

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.

An outbreak of severe parasitic pneumonia caused by Dictyocaulus viviparus was diagnosed in adult dairy cows in the municipality of Arabutã, Southern Brazil. The total morbidity in the herd was 71.9%, and the morbidity amongst adult lactating cattle was 100%. The main clinical signs observed were dyspnea, tachypnea, nasal discharge, decreased milk production, and cough. A necropsy was conducted on one animal in order to establish the diagnosis. The herd had been treated previously with levamisole; however, clinical signs persisted and became worse. After treatment with eprinomectin the severity of clinical signs decreased, and the respiratory condition subsequently disappeared. It is believed that the high morbidity presented in this outbreak is related to epidemiological factors, such as increased rainfall in 2014 and 2015, associated with low immunity of the herd. This is the first report of dictyocaulosis in adult dairy cattle in Brazil. Furthermore, it describes an outbreak presenting very high morbidity.

Specimens of Dictyocaulus spp. were extracted from the respiratory tracts of 3 ruminant hosts including roe deer (Capreolus capreolus), red deer (Cervus elaphus), and chamois (Rupicapra rupicapra) from wild populations in the province of León, northwestern Spain. The near-complete nuclear small-subunit ribosomal RNA gene, and 2 regions of the large-subunit ribosomal RNA gene, were amplified by PCR and sequenced. The SSU rDNA gene sequences indicated a high level of similarity between the isolate from C. elaphus and the published sequences for Dictyocaulus eckerti. SSU rDNA gene sequences were identical in the isolates from C. capreolus and R. rupicapra, and both corresponded to published sequences for D. capreolus. The LSU rDNA gene sequences differed in isolates from the latter 2 hosts, indicating the possible presence of an undescribed Dictyocaulus sp. in R. rupicapra. These results showed that the LSU rDNA gene sequences are useful indicators of genetic and species diversity in species of Dictyocaulus.

The efficacy of eprinomectin on Dictyocaulus filaria and Cystocaulus ocreatus in naturally infected sheep was evaluated in the present study. In total, 30 infected sheep were randomly divided into two groups: treated (n = 15) and untreated (n = 15). A single pour-on dose of eprinomectin (0.5 mg/kg) was administered to the treated group. No medication was used in the untreated group. Faecal larval counts were performed on pre-treatment (day 0) and post-treatment (days 7, 14, 21 and 42) days. Eprinomectin was found to be 100% effective against D. filaria on day 7 post-treatment when compared with the untreated group and it maintained this effect on days 14, 21 and 42. However, the decrease in faecal larval count of C. ocreatus was found to be 86, 86 and 91%, on days 14, 21 and 42, respectively.

Mitochondrial DNA (mtDNA) sequence data and amplified fragment length polymorphism (AFLP) patterns were compared for the lungworm Dictyocaulus viviparus, a nematode parasite of cattle. Eight individual D. viviparus samples from each of 8 herds in Sweden and 1 laboratory isolate were analysed, with the aim of describing the diversity and genetic structure in populations using different genetic markers on exactly the same DNA samplesNucleotide sequence data reported in this paper have been submitted to GenBank under the Accession numbers DQ299539-DQ299826.. There was qualitative agreement between the whole-genome AFLP data and the mtDNA sequence data, both indicating relatively strong genetic differentiation among the Swedish farms. However, the AFLP data detected much more genetic variation than did the mtDNA data, even after allowing for the different inheritance patterns of the markers, and indicated that there was much less differentiation among the populations. The mtDNA data therefore seemed to be more informative about the most recent history of the parasite populations, as the general patterns were less obscured by detailed inter-relationships among individual worms. The 4 mtDNA genes sequenced (1542 bp) showed consistent patterns, although there was more genetic variation in the protein-coding genes than in the structural RNA genes. Furthermore, there appeared to be at least 3 distinct genetic groups of D. viviparus infecting Swedish cattle, 1 of which was predominant and showed considerable differentiation between farms, but not necessarily within farms. Second, the 2 smaller genetic groups occurred on farms where the predominant group also occurred, suggesting that these farms have had multiple introductions of D. viviparus.

Dictyocaulus capreolus n. sp. recovered from roe deer, Capreolus capreolus and moose, Alces alces in Sweden is described and figured. Morphological studies revealed the new species to be closest to D. eckerti and D. africanus on the basis of mouth shape, all three species having an elongate mouth opening. The other species of the genus, including D. viviparus, all have a circular to oval mouth opening. Dictyocaulus capreolus n. sp. can be distinguished from D. eckerti and D. africanus on the basis of the morphology of the buccal capsule and the bursa. These morphological studies support earlier evidence of the presence of a new species of Dictyocaulus in roe deer and moose that could be distinguished from D. eckerti and D. viviparus using either a PCR-linked hybridization assay or image analysis software to study the dimensions of the buccal capsule.

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.

The existence of bronchopulmonary nematodes in German roe deer Capreolus capreolus is well documented, with two types of lung parasites that have been described previously: Dictyocaulus capreolus and Varestrongylus capreoli. However, little is known about the impact of these parasites on their host animal or which parameters influence outbreak and intensity of infection. The aim of this study was to obtain new information on the relevance of factors such as season, environmental conditions or age, sex, and body mass of the infected roe deer. To obtain our results, the respiratory tracts of 762 roe deer from south‐eastern Germany were examined. In the sample, 42.5% of roe deer were infested with V. capreoli and 14.0% with D. capreolus, and 51.3% of animals were completely free of lung parasites. Testing for influencing variables, our regression models found both sex and age of the roe deer to statistically influence infestation, with male sex and younger age correlating with both stronger infestation and higher infestation rates. Accordingly, in male animals the infestation rates with V. capreoli and D. capreolus (45.1% and 20.1%, respectively) were higher than in females (39.4% and 8.0%, respectively). The overall infestation rate of juvenile animals was remarkably higher (73%) than those of sub‐adults (38.3%) or adults (28.4%). Across all age groups, infested animals showed lower body weights compared to non‐infested animals. According to our multiple linear regression model, roe deer infected with D. capreolus on average weighed 0.65 kg less than their healthy counterparts, and in the case of V. capreoli 0.72 kg less on average. While the burden on the well‐being of infested animals can only be assumed, these concrete figures (reduced body weight in infested compared to healthy animals) demonstrate the economic damage lung parasites cause to meat harvesting from bagged roe deer.