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Bovine lungworms (Dictyocaulus viviparus) are nematodes which cause a respiratory disease known as verminous bronchitis or pneumonia. In this paper, we describe 20 outbreaks of bovine dictyocaulosis recorded between 2000 and 2023 in Central Argentina. Outbreaks occurred more frequently during the autumn-winter season (from April to August) and affected cattle under 1 year old in beef production systems. An average morbidity and mortality of 26.22 % and 8.44 % were registered, respectively. The main clinical signs observed were respiratory distress (coughing, tachypnea, dyspnea, and nasal discharge), weight loss, weakness, decubitus, and diarrhea. Necropsies were performed in thirty-one calves, heifers, and steers. Gross findings included diffuse interstitial or multifocal pneumonia, with marbled appearance intermingling atelectasis and red-gray firm areas of consolidation, and subpleural and interlobular emphysema and edema. Microscopically, lungs were characterized by abundant edema and mixed intra-alveolar multifocal to coalescent infiltrate. Frequently, adult worms and/or larvae were spotted in the bronchi or alveoli, respectively. Some cases exhibited proliferation of type 2 pneumocytes and hyaline membranes covering the alveolar septa. Co-infections with gastrointestinal nematodes were frequently found in fecal samples. Given the perspective of anthelmintic resistance and future changes in environmental conditions due to climate change, integrated parasitic control strategies are mandatory and should be tailored to each production system. The information gathered in this research provides an overview of lungworm infections in livestock production systems from Central Argentina and could be useful for surveilling, monitoring and designing strategic interventions for the control of this important parasitic disease in the region., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest Not applicable., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2023. The Author(s).)

Outbreaks of cattle lungworm disease (Dictyocaulus viviparus) are explosive and costly. The unpredictability of the disease often encourages farmers to apply blanket anthelmintic treatments to the herd, which impede the acquisition of immunity, increase the risk of drug resistance, and interfere with efforts to reduce anthelmintic use against ubiquitous gastrointestinal nematodes. Improving our understanding of the factors which lead to a high risk of infection with lungworm, (including climatic pressure), would support a more targeted management. We present GLOWORM-FL-DV, the first mathematical model of the free-living stages of D. viviparus. The ecology of D. viviparus is unique compared with other strongylid nematodes due to its relationship with Pilobilus spp. fungi, which enhance the transmission potential. The role of the fungi was therefore incorporated into the model framework, informed by laboratory observations of Pilobolus spp. development and sporulation. The thermal niche of D. viviparus was characterised based on published and laboratory observations. Mortality of parasitic larvae increased significantly below 0 o C, and larval development occurred above 1.4 o C, whereas the estimated minimum temperature for migration via Pilobolus spp. was 8.8 o C. Model predictions were compared with antibody levels in bulk milk tank samples collected at two-weekly intervals from eight dairy herds across Great Britain over two grazing seasons. The model predicted high levels of larval abundance on pasture 46 days (38-52 days) before a rise in antibody levels and 22-26 days before the onset of clinical signs. The model assesses the impact of climate and weather on lungworm larval availability at pasture and provides a framework for the development of a risk forecasting system. This could help to focus vigilance for clinical signs at high-risk times and facilitate the targeted use of anthelmintics to prevent outbreaks, in support of sustainable parasite control., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The lungworm Dictyocaulus viviparus has an important role in cattle health and productivity worldwide, since infections can lead to substantial economic losses. Despite its importance, few studies investigating the epidemiological aspects of infection by this parasite have been conducted. The aim of this study was to report the occurrence of lungworm infection in beef cattle herds reared in an area of livestock production in the northeastern region of Brazil. From September 2020 to August 2021, monthly fecal samples (n = 493) were collected from 46 beef cattle. Among all the animals assessed, lungworm larvae were detected in 8.7% (4/46). None of them presented any clinical sign suggestive of infection by lungworm parasites. Twenty larvae were retrieved, with the minimum number (n = 1) detected in October and December, and the maximum number (n = 13) in November. These presented a mean length of 363 μm (± 28.65 μm) and mean width of 19 μm (± 1.03 μm), and were morphologically similar to Dictyocaulus sp.. This study reports the occurrence of this parasite in this livestock production area. Lastly, local veterinarians need to be aware of inclusion of this parasite in the differential diagnosis of other respiratory infections in beef cattle.

Dictyocaulus viviparus, the causative agent of bovine parasitic bronchitis, is an important parasite of dairy cattle. Infections can lead to substantial economic losses, due to mortality, reduced weight gain and milk production and treatment costs. There have been relatively few studies investigating herd management risk factors for infections with D. viviparus and lungworm-associated production losses. The aims of this study were (1) to assess the impact of (sub)clinical lungworm infections on productivity in dairy cows and, (2) to identify or confirm risk factors, related to herd management, for infections in grazing dairy cattle. Using a recombinant Major Sperm Protein (MSP)-based ELISA, the presence of D. viviparus antibodies in bulk tank milk (BTM) samples was evaluated on 717 and 634 farms at two-week intervals during two grazing seasons (2018 and 2019). Associations between milk antibody levels and production data (mean milk yield in kg/cow/day, percentage of fat and protein) were assessed, as well as associations with putative risk factors in the herd management, gathered through a questionnaire survey. In both years, there was a substantial, but non-significant, difference in the annual mean milk yield on farms with at least one BTM sample above the cut-off of 0.41 ODR, compared with the mean milk yield on farms that stayed under this threshold on each sampling day (-0.17 and -0.70 kg milk/cow/day in 2018 and 2019, respectively). In 2019, this association was stronger, and significant, when the cut-off was exceeded in at least two consecutive BTM samples (-1.74 kg milk/cow/day). BTM results were also significantly negatively associated with the closest milk production data during the two-weekly BTM sampling intervals in 2019. A single or two consecutive positive tests were used in the risk factor analysis as a proxy for lungworm-associated milk yield losses. Purchase of new animals (Odds Ratio (OR) = 2.68) and the proportion of the first grazing season covered by preventive anthelmintic treatment (OR up to 3.88, depending on proportion) were positively associated with lungworm-associated milk yield losses, while mowing at least 50 % of the pastures (OR = 0.57) was negatively associated with lungworm-associated milk yield losses. Our results suggest that the ELISA holds promise to identify herds with significant production losses due to lungworm infections, under the condition that BTM sampling is done repeatedly during the grazing season. Based on the confirmed risk factors, adjustments of the farm management could perhaps mitigate these losses., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© Wildlife Disease Association 2021.)

The lungworm Dictyocaulus viviparus can have a major impact on bovine health and productivity. Enzyme-Linked Immunosorbent Assays (ELISA), based on a recombinant Major Sperm Protein (MSP), have been developed to detect D. viviparus-specific antibodies in bulk tank milk (BTM). The objectives of this study are to assess the association between BTM optical density ratio's (ODR) and farmer-reported lungworm outbreaks based on the clinical sign "coughing" throughout the grazing season and to compare the sensitivity and specificity of two ELISAs under field conditions. The Hannover MSP-ELISA and the prototype Svanova MSP-ELISA were used for the detection of D. viviparus antibodies in BTM samples on 717 dairy farms during the 2018 grazing season. Assuming all herds to be truly lungworm infected, the results show that the Svanova ELISA had a lower sensitivity (40-65%) and specificity (75-90%) for the detection of D. viviparus infections in BTM compared to the Hannover ELISA, which had a sensitivity of 42% and 74% and specificity of 100% and 98% at a cut-off of 0.41 ODR and 0.25 ODR, respectively. Therefore, analyses of the associations between milk antibody levels and farmer-reported outbreaks during the 2018 and 2019 grazing season were assessed using the Hannover ELISA, on 717 and 634 farms, respectively. A positive association was found between a farmer-reported outbreak and having at least two consecutive positive BTM ODR's at a cut-off of 0.41 in 2018 (Odds Ratio (OR) = 5.5) and 2019 (OR = 2.8). Furthermore, there was a significant association between a farmer-reported outbreak and having a positive BTM ODR in August (OR 2018 = 4.4; OR 2019 = 2.8) and October (OR 2018 = 3.7; OR 2019 = 1.8). On the farms with a farmer-reported outbreak and positive BTM samples, over half (2018 = 77%; 2019 = 57%) of the positive ODR's were situated before the outbreak and 47% (2018) and 42% (2019) within 12 weeks before the outbreak. In conclusion, there is a positive association between farmer-reported outbreaks and the occurrence of a positive BTM sample at the cut-off of 0.41 ODR using the Hannover ELISA., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The large lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in various ungulate hosts, including red deer. Recently, the red deer-derived lungworm D. cervi was described and separated from D. eckerti. Little is known of the transmission patterns, epidemiology, geographical distribution and pathogenicity of D. cervi. Histological examinations were performed on 22 formalin-fixed lung tissue samples of hunted red deer. Exclusively, D. cervi adults were derived from 15 red deer and confirmed molecularly (GenBank accession: MH183394). Dictyocaulus cervi infection was associated with various degrees of lung pathology, including interstitial pneumonia, bronchitis and bronchiolitis with an influx of eosinophils, lymphocytes, plasma cells and macrophages; massive hyperplasia of lymphoid follicles within bronchiolar tissue, and hyperplasia of the bronchial and bronchiolar epithelium. Furthermore, emphysema, atelectasis and lung tissue congestion were noted. Interestingly, interstitial and subpleural fibrosis was seen in adult Dictyocaulus-negative samples, suggesting either a prepatent phase of Dictyocaulus infection or infection/coinfection with protostrongylid nematodes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Background: Infections with the bovine lungworm Dictyocaulus viviparus might lead to reduced milk production and detrimental impacts on milk quality resulting in considerable economic losses in dairy farming., Methods: In the presented field study, 1988 faecal samples were collected from 1166 Black and White dairy cows allocated in 17 small and medium-sized German grassland farms. Faecal samples were collected in summer and autumn 2015 to assess D. viviparus larvae excretion. Test-day records were used to estimate the association between patent D. viviparus infections in individual cows and the milk production parameters milk yield, milk protein and milk fat content by using linear mixed models. Bulk tank milk (BTM) samples from each farm and individual milk samples from those cows which were excreting larvae in summer were collected in autumn. In addition, occurrence of the clinical symptom "coughing" was noted in individual cows during autumn sampling to determine its association with patent lungworm infections., Results: Patent D. viviparus infections were found on 23.5% (4/17) of farms with a prevalence at the individual cow level of 0.9% (9/960) in summer and 3.4% (35/1028) in autumn. No BTM sample exceeded the BTM ELISA cut-off value of 0.410 optical density ratio (ODR), the mean value was 0.168 ODR. Only one individual milk sample exceeded the individual milk ELISA cut-off value of 0.573 ODR (mean value of 0.302 ODR). A patent D. viviparus infection status was associated with a lower average daily milk yield of 1.62 kg/cow/day (P = 0.0406). No significant association was found with milk protein or fat content representing milk quality parameters. Coughing was observed in 5.9% (61/1028) of cows. Of the coughing cows, only 4.9% (3/61) had a patent lungworm infection. Fisher's exact test showed no significant difference between infected and non-infected coughing cows., Conclusions: Farmers and veterinarians should be aware that patent lungworm (re)infections in dairy cows reduce milk yield, despite the absence of clinical signs. Furthermore, if dairy cows present with coughing, other differential diagnoses need to be considered in addition to dictyocaulosis.

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.

The aim of this study was to evaluate the influence of dictyocaulosis (mild or severe) on enzymes of NTPDase, 5'-nucleotidase, and adenosine deaminase (ADA) of dairy cows naturally infected by Dictyocaulus viviparus. Blood and faeces were collected from 22 dairy cows of the same farm to evaluate NTPDase (ATP and ADP substrate), 5'-nucleotidase, and ADA activities on days 0 (pre-treatment) and 10 (post-treatment). Seric activities of NTPDase (ATP substrate), 5'-nucleotidase, and ADA were lower (P<0.05) in D. viviparus infected animals compared to uninfected cows. The number of D. viviparus larvae per gram of faeces varied among the animals, and they showed different degrees of severity according to respiratory clinical signs of the disease (cough and nasal discharge). Later, these cows were divided into two groups: those with mild (n=10) and severe (n=12) disease. Cows with severe disease showed higher NTPDase activity (ATP substrate) than those with mild disease (P≤0.05). The opposite occurred with NTPDase (ADP substrate), 5'-nucleotidase, and ADA in cows with severe disease, that is, the enzymatic activity of these seric enzymes significantly decreased (P≤0.05) compared to animals with mild disease. Infected animals showed reduced NTPDase activity (ATP and ADP substrate) after treatment. No enzymatic changes were observed for 5'-nucleotidase, and ADA pre- and post-treatment (P>0.05). Based on these results, we conclude that dictyocaulosis alters NTPDase, 5'-nucleotidase, and ADA activities of cow naturally infected by the parasite, in consequence the enzymes act as inflammatory markers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lungworm infection is caused by a Dictyocaulus filaria nematode parasitizing the bronchi and bronchioles of sheep and goats. Various anthelmintics, including albendazole, levamisole, fenbendazole, ivermectins, and others, are used to treat the animals. The aim of this investigation was to study the impact of lungworm infestation on the biochemical parameters of animals during combination treatment with albendazole and T- and B-activin. Experiments were carried out in 20 uninfected mongrel lambs aged 4-5 months. Infectious D.filaria larvae were given with water to 15 lambs once orally at a dose of 1000 larvae per head. 5 uninfected lambs served as a control group. The time course of changes in serum bio- chemical parameters was studied in animals. Treatment with Albena in combination with T- and B-activin in lambs ex- perimentally infested with lungworm was found to restore their biochemical reactivity. After sheep treatment with Albena alone, biochemical parameters were noted to tend to normalize, but their normal full recovery did not take place.

The objective of this study was to conduct a comprehensive field survey using a Dictyocaulus viviparus major sperm protein ELISA on bulk tank milk samples from Belgian dairy herds to gain insights in: (1) the sensitivity (Se) and specificity (Sp) of the test under field conditions; (2) the value of the test to predict a future clinical lungworm outbreak; (3) its associations with milk production parameters and (4) its associations with herd management factors. A total of 1248 herds were sampled, with samplings occurring in the middle ("August") and towards the end ("October") of the grazing season. A completed questionnaire on potential risk factors and potentially lungworm-induced clinical signs was obtained from 587 farms and milk production records could be obtained from 343 herds. The median (25th-75th percentile) D. viviparus antibody level (ODR) was 0.25 (0.19-0.31) in "August" and 0.24 (0.19-0.32) in "October". At a threshold of 0.41 ODR, the Se and Sp were estimated using mixture models at 50 and 99%, respectively. At the same threshold, the positive and negative predictive value of the ELISA applied in "August" on the occurrence of farmer-reported lungworm symptoms in the period August-November were 65% and 69%, respectively. D. viviparus antibody levels were significantly higher in the north vs. the south of the country, in large herds and in herds that did not mow pastures or that frequently purchased new animals. An increase in the ELISA result of "August" over the interquartile range was associated with a drop in the annual average milk yield, milk protein% and milk fat% of -0.50kgcow -1 day -1 , 0.02 and 0.02, respectively. The relationships between the ELISA results in "October" and milk production parameters were also negative, but lower and non- or only marginally significant. We conclude that the bulk tank milk ELISA has a low value to predict lungworm disease on an individual farm based on a fixed sampling date in the middle of the grazing season. On the other hand, the test has been potential to detect subclinical production impacts and study risk factors through epidemiological surveys., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

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.

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.

Background: A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs., Methods: The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI)., Results: Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement., Conclusions: A versatile bead-based assay using fluorescence detection (xMAP technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques.

Infections with Dictyocaulus viviparus and Ostertagia ostertagi nematode parasites are of importance to bovine health and production in temperate areas across the world. Losses due to these parasites in dairy herds can be considerable due to decreased milk productivity and fertility. However, information on current epidemiological patterns in Irish dairy herds is limited. Bulk milk samples were collected from a total of 319 dairy farms across the Republic of Ireland. The D. viviparus samples were tested with an ELISA based on recombinant major sperm protein, while the O. ostertagi samples were tested with an ELISA based on crude saline extract, whole worm O. ostertagi antigen. Management data were collected from the farms using a questionnaire. Logistic regression was used to find significant associations between the presence of antibodies against D. viviparus and O. ostertagi and management factors. The overall prevalence of D. viviparus infection was 62.8%, while over 98% of herds had antibodies to O. ostertagi at the specified cut-off. Both D. viviparus and O. ostertagi antibodies were highest in November, which could be explained by the accumulated uptake of larvae through the grazing season. In herds of farmers that dosed their in-calf heifers with anthelmintics were significantly more likely to be positive for antibodies against D. viviparus infection. This study highlights that both D. viviparus and O. ostertagi infections are widespread in dairy herds in Ireland throughout the grazing season., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The lungworm Dictyocaulus viviparus, causing parasitic bronchitis in cattle, induces a temporary protective immunity that prevents clinical disease. A radiation-attenuated larvae based vaccine is commercially available in a few European countries, but has the disadvantages of a live vaccine. As a recombinant subunit vaccine would overcome these disadvantages, the parasite's muscle protein paramyosin (PMY) was tested as a recombinant vaccine antigen., Methods: D. viviparus-PMY was recombinantly expressed in Escherichia coli as a glutathione-S-transferase (GST)-fused protein. Emulsified in adjuvant Saponin Quil A, the protein was given intramuscularly into calves. Two independent recombinant PMY (rPMY) vaccination trials with negative control groups (first trial: adjuvant only; second trial: non-fused GST) as well as an additional positive control group in the second trial, using the Bovilis Dictol live vaccine to verify vaccination results, were performed. To determine the vaccination success, shedding of larvae as well as worm burden and worm sizes were analyzed. Additionally, ELISA-based determination of development of immunglobulins IgM, IgA, IgE, IgG as well as the subclasses IgG1 and IgG2 was performed. To analyze PMY localization in the bovine lungworm, immunohistochemical staining of adult worms was carried out., Results: Immunohistochemical staining revealed that PMY is part of the bovine lungworm's pharyngeal and body wall muscles. Vaccination with rPMY resulted in 47% [geometric mean: 67%] and 57% (geometric mean: 71%) reduction of larvae shedding in the first and second vaccination trial, respectively. Worm burden was reduced by 54% (geometric mean: 86%) and 31% (geometric mean: 68%), respectively, and worms of rPMY-vaccinated cattle were significantly shorter in both trials. Furthermore, ELISAs showed a clear antibody response towards rPMY with exception of IgE for which titers could not be detected. After challenge infection, rPMY antibodies were only exceptionally elevated among study animals indicating PMY to be a hidden antigen., Conclusions: Even though vaccination with the attenuated live vaccine was with 94% (geometric mean: 95%) reduction in larvae shedding and 93% (geometric mean: 94%) reduction in worm burden superior to rPMY vaccination, results using the latter are promising and show the potential for further development of a recombinant PMY-based vaccine against the bovine lungworm.

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.

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, β-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., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study was to monitor local cytokine responses to Dictyocaulus viviparus in calves during primary infection and re-infection. Bronchoalveolar lavage fluid (BALF) was collected weekly from experimentally infected calves and interleukin-2 (IL-2), IL-4, IL-5, IL-10 and IFN-γ mRNA expression was quantified in BALF cells. The major finding was a prominent transient increase in IL-4 mRNA expression, compared with that of uninfected calves, observed in BALF cells collected 2-3 weeks post-primary D. viviparus infection. At 2 weeks post-infection, macroscopic worms were also first observed in BALF. Calves re-infected after 10 weeks were partially immune which was evident at slaughter 5 weeks post-infection as a lower worm burden than in previously naïve calves infected at the same time. IL-4 mRNA expression in BALF cells 2 weeks post-re-infection was increased compared with that of uninfected animals but not as high as that of primarily infected calves. BALF cell expression of the other cytokines tested for was not as clearly effected by the D. viviparus infection. It seems likely that the strong IL-4 response observed during primary infection reflects an innate response to the worms that may initiate an ensuing Th2 response, which confers protective immunity., (© 2013 John Wiley & Sons Ltd.)

In November 2008, a total of 19,910 bulk tank milk (BTM) samples were obtained from dairy farms from all over Germany, corresponding to about 20% of all German dairy herds, and analysed for antibodies against the bovine lungworm Dictyocaulus viviparus by use of the recombinant MSP-ELISA. A total number of 3,397 (17.1%; n = 19,910) BTM samples tested seropositive. The prevalences in individual German federal states varied between 0.0% and 31.2% positive herds. A geospatial map was drawn to show the distribution of seropositive and seronegative herds per postal code area. ELISA results were further analysed for associations with land-use and climate data. Bivariate statistical analysis was used to identify potential spatial risk factors for dictyocaulosis. Statistically significant positive associations were found between lungworm seropositive herds and the proportion of water bodies and grassed area per postal code area. Variables that showed a statistically significant association with a positive BTM test were included in a logistic regression model, which was further refined by controlled stepwise selection of variables. The low Pseudo R(2) values (0.08 for the full model and 0.06 for the final model) and further evaluation of the model by ROC analysis indicate that additional, unrecorded factors (e.g. management factors) or random effects may substantially contribute to lungworm infections in dairy cows. Veterinarians should include lungworms in the differential diagnosis of respiratory disease in dairy cattle, particularly those at pasture. Monitoring of herds through BTM screening for antibodies can help farmers and veterinarians plan and implement appropriate control measures.

Objective: To determine the prevalence, identify the species involved and assess possible risk factors of lungworm infection in small ruminant slaughtered in abattoir of Mashhad in the northeast of Iran from October 2010-August 2011., Methods: Faecal and post mortem examination were conducted on 350 and 2 500 animals, respectively., Results: The overall prevalence of lungworm infection was 10.85% and 3.80% in coproscopic and post mortem examination respectively, and this difference was found to be significant. Higher prevalence of lungworm infection was recorded in post mortem examination in sheep (4.1%) than in goats (0.5%) (P< 0.05). The proportion of infection with Dictyocaulus filaria, Protostrongylus rufescens and mixed infection were 3.7%, 0.1% and 0.2% in sheep while in goats, the infection was reported with Dictyocaulus filaria (0.5%) only. The seasonal dynamics of lungworm infection showed that prevalence was highest in winter (7.8%) with a remarkable decline during the dry time (summer) (0.8%) which the difference was significant (P<0.001). The animals of less than one year old showed greater infection in post mortem examination than older animals significantly (P<0.001). Also, the infection rate between male and female animals showed significant difference (P<0.05) with prevalence rate of 4.6% and 2.0%, respectively., Conclusions: Due to its impact on production, emphasis should be given for the control and prevention of lungworm infection in this area., (Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

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.

A one-year field study analysing lungworm seropositivity by use of the MSP-ELISA was performed (1) to investigate the antibody dynamics in individual milk samples following field (re-)infections of dairy cows with the bovine lungworm Dictyocaulus viviparus, (2) to investigate the correlation between individual and bulk tank milk (BTM) antibody titres and (3) to review the current individual as well as BTM cut-off value, which was extrapolated from dilution experiments (Fiedor et al., 2009). Over a one-year period individual and BTM samples were collected monthly on 15 dairy farms. Following a critical review of previous cut-off values, individual and BTM samples were subjected to different cut-off thresholds. Following Receiver-Operating-Characteristics (ROC) analysis, individual milk samples were assessed with the cut-off value 0.573, previously shown to be associated with each 100% sensitivity and specificity. In addition, the present study enabled BTM cut-off adjustment based on field data. To ensure reliable detection of herds with an in-herd prevalence of ≥20% the BTM cut-off was lowered from 0.493 to 0.410, corresponding to 100% sensitivity and 97.32% specificity. Regression analysis showed that the percentage of seropositive animals related to the corresponding BTM ODR correlated moderately (r=0.581, P<0.001), whereas a strong correlation (r=0.764, P<0.001) was found between mean individual and BTM ODR per herd and sampling month. Seasonal antibody pattern became obvious in a single-peaked antibody curve in late summer/early autumn for individual milk whilst BTM showed a two-peaked distribution with an additional spring peak besides the late summer/early autumn peak. This leads to the conclusion that the BTM-ELISA could be a useful tool to detect and control pasture contamination in the spring, following sexual maturation of hypobiotic lungworm larvae harboured by clinically asymptomatic carrier animals. In addition to the knowledge gained on antibody patterns in dairy herds and the relationship of individual and BTM, the present study enabled sensitivity and specificity calculations for the obsolete BTM cut-off value 0.493 to be performed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9-99.7%) and 98.1% (CI: 95.3-99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3-4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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.

The FLOTAC flotation technique has been introduced as a new diagnostic tool to detect parasitic elements from faeces. Samples from naturally infected young deer were used for counting Dictyocaulus larvae and strongylid eggs. The FLOTAC technique, using 11 different flotation solutions with specific gravities (sg) between 1.20 and 1.45, was compared with the Baermann technique and the saturated sodium chloride (sg 1.20)-based McMaster method. In addition, a comparison was made between the FLOTAC technique with magnesium sulphate (sg 1.28) and the Baermann technique for larval recovery from faeces that were examined on the day of collection or after 7 days storage at 4 degrees C. On the whole egg counts between the FLOTAC using different flotation solutions and the McMaster were unremarkable. In contrast, variations of larval counts were detected between different flotation solutions as well as with the Baermann technique. Most flotation solutions with a specific gravity of 1.20 floated significantly fewer lungworm larvae (p < 0.05) compared to flotation solutions with a higher specific gravity. Magnesium sulphate (sg 1.28) consistently produced the highest mean larval counts in all conducted experiments. Larval counts using magnesium sulphate (sg 1.28) were higher than with the Baermann technique both on the day of collection and after 7 days. Overall, the use of magnesium sulphate (sg 1.28) with FLOTAC for larval counts resulted in higher counts than the Baermann recovery technique and was the better choice of those flotation solutions examined. Furthermore, magnesium sulphate (sg 1.28) was also reliable for strongylid egg detection with the FLOTAC apparatus.

In the postmortem examination of 54 adult donkeys slaughtered for the carnivore animals in Kafr El-Sheikh Zoological Garden, Dictyocaulus arnfieldi (Cobbold, 1884) Railliet and Henry, 1907 was demonstrated in their lungs. The biological data of the different developmental stages were recorded and discussed, where the period required for maturation, hatching and development of 2nd and 3rd stage larvae was 84 hours, general discussion was given.

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.

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 Le6n, 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.

Case Description: The owner of a herd of 74 Holstein-Friesian cattle reported decreased milk production, weight loss, and coughing among lactating cows. Owner-initiated antimicrobial treatment was unsuccessful; 1 lactating cow died, and 50% of the lactating cows had clinical signs of respiratory distress, such as tachypnea and coughing., Clinical Findings: On the basis of history, physical examination findings, and fecal examination results, affected animals were determined to have Dictyocaulus viviparus (lungworm) infestation. The disease history suggested that the herd contained cows with subclinical patent lungworm infestations; after introduction of susceptible heifers, the pastures had become heavily infested with D viviparus and clinical problems subsequently developed in both newly introduced and resident cows., Treatment and Outcome: Affected and unaffected heifers and adult cows were treated with a pour-on formulation of eprinomectin (0.5 mg/kg [0.23 mg/lb]). One animal died, but 2 weeks after treatment, clinical signs among affected cattle were markedly improved. Ten weeks after treatment, milk production improved from 23 kg/cow/d (51 lb/cow/d) to 28 kg/cow/d (62 lb/cow/d)., Clinical Relevance: The outbreak provides additional evidence that dictyocaulosis is becoming more common among adult dairy cattle, rather than almost exclusively affecting young stock. This may be attributable to anthelmintic use and management practices on dairy farms. Combined with anecdotal reports of an increase in the incidence of dictyocaulosis among adult cattle in North America, D viviparus infestation should be included as a differential diagnosis for decreased milk production, weight loss, and coughing among adult dairy cattle.

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.

This paper presents investigation of lungworm disease outbreaks that is based on retrospective examination of cases recorded between 1994 and 2000 on a government beef cattle breeding centre in the state of Pahang, peninsular Malaysia. The breed of cattle on the centre was Nelore and the mean population over a 7-year period (from 1994 to 2000) was 1612. All animals were allowed to graze on pasture and mixed grazing was practiced on the farm. The routine de-worming programme was performed using levamisole and ivermectin from 1994 to 1998 and abamectin in 1999 and 2000 on 1 to 3-month-old calves and an annual dose given to the adult cattle. Nelore was introduced into the farm in 1991, three years before the first outbreak from Brazil where Dictyocaulus viviparus infection had been reported. No lungworm infection had been observed in the farm prior to the animal introduction. Within the 7-year period, 36 fatalities occurred and the annual mortality rate due to lungworm infection was 0.31%. The highest rate was recorded in 1997. Among the total 36 deaths, about 75% of deaths occurred in calves aged between 6 months and 12 months, 67% were males and 33% were female cattle. The highest number of deaths (19%) occurred in the month of November. In conclusion, D. viviparus infection may have been introduced into a tropical climate along with consignments of cattle from lungworm endemic areas resulting in fatal disease outbreaks for a few years following the animal's initial introduction.

Calves with naturally acquired Dictyocaulus viviparus infection mount an effective immune response. In the search for protection-inducing antigens, we found that several D. viviparus third-stage larval (L3) and adult ES products carry N-glycans. Deglycosylation of the worm antigens using PNGase F resulted in reduced IgA, IgE, IgG1 and IgG2 (but not IgM) reactivities in sera of primary infected animals, suggesting that the carbohydrate moieties contained immunodominant epitopes. Challenge infection resulted in increased specific serum antibody levels against ES and L3 in the re-infected and challenge control groups. Testing of sera by enzyme-linked immunosorbent assay (ELISA) demonstrated a significant increase in IgG1 and IgE (but not IgA or IgG2) reactivity against the deglycosylated antigens in the re-infected group compared with the challenge control group. Sera from calves vaccinated with irradiated larvae showed a strong anti-N-glycan response, but no booster response against the protein backbone after challenge infection, consistent with the absence of a memory response. Together, our results suggest that D. viviparus proteins carry immunodominant N-glycan moieties that elicit a strong but short-lived immune response during infection and after vaccination, whereas the protein backbones effectively induce a memory response which results in a long-lasting, potentially protective immune response in re-infected, but not in vaccinated calves.

A cross-sectional study of lungworm infection was carried out with the aim of determining the prevalence of lungworm infection of small ruminants and identifying the species of the respiratory helminthes circulating in six districts of northeastern Ethiopia: Debresina, Legambo, Habru, Kalu, Chaffa-Dawi and Artumana-Fersejelle. Faecal and postmortem examination were conducted from 1162 and 104 animals, respectively. An overall infection rate of 53.6% and 66.3% was found by faecal and postmortem examinations, respectively. Significant difference (p<0.05) was found between areas of different altitude with an infection rate of 30.4%, 32.5% and 71.3% at low, medium and high altitude areas, respectively. Prevalence on the different months was insignificant (p>0.05). The prevalence of Dictyocaulus filaria and Muellerius capillaris infection showed a significant difference (p<0.05) between young, adult and old age groups. The prevalence of D. filaria decreases and that of M. capillaris increase with increasing age of the animal. Animals under relatively good management system have been found less affected with significant difference (p>0.05) than their counterparts at relatively poor management system. The infection rate between male and female animals showed significant difference (p<0.05) with prevalence rate of 44.4% and 59.3%, respectively. A significant difference between sheep and goats was also noted with infection rate of 24.4% and 50.7%, respectively. Monthly worm burden of D. filaria infection showed significant difference (p>0.05) while it was insignificant in case of M. capillaris. Due to its impact on production, emphasis should be given for the control and prevention of lungworm infection in highland areas.

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 samples. 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.

The effect of targeted selective anthelmintic treatment on the seroprevalence of the lungworm Dictyocaulus viviparus in cattle was investigated. The study was commenced on an organic dairy enterprise in Sweden in November 1998 after the observation of an outbreak dictyocaulosis in the herd, and then continued for almost 3 years. The first year sampling was conducted on a monthly basis and then biannually with the exception of between August and November 2000 when sampling was performed monthly following a second outbreak of dictyocaulosis. Throughout the study, blood samples were examined for specific IgG(1) levels from all animals in the herd that had been grazing for more than 3 months. At the first sampling occasion, 13% out of the 90 blood samples were seropositive. One month later, after targeted selective treatment with eprinomectin (Eprinex), Merial), the whole herd was seronegative. Seroprevalence then gradually increased and 1 year later it returned to levels similar to those observed at the start of the study. At turnout in April 2000, seroprevalence was 1.3% but it then rapidly increased to 28% and 30% in August and September, respectively. This increase was mainly due to an increase in FSG animals of which many were coughing. Consequently, all seropositive animals were injected with ivermectin (Ivomec), Merial) at 0.05 mg/kg body weight in late August 2000. Although all animals recovered, seroprevalence was only reduced to 12% 1 month later. The differences in seroprevalence after both of these anthelmintic treatments were probably attributed to the timing. The first deworming with eprinomectin was conducted in November when the infection already was transient, whereas ivermectin in connection with the second outbreak was injected in a more acute phase of the infection cycle. Infection levels in 2001 were low with seroprevalences of 2.3% and 5.6% in May and September, respectively. These results show that dictyocaulosis in Sweden can be effectively controlled by the use of macrocyclic lactones. However, the infection was not eradicated from the herd despite close monitoring of the seroprevalence and targeted selective treatment of every seropositive animal on two occasions.

Monitoring the health of wildlife populations is important for understanding and controlling the risk of infections to livestock, humans and/or other wildlife. In this paper, we analyse the results of surveys of parasites and non-specific signs of diseases carried out on organs from 638 red and 107 sika deer culled in four regions of Scotland between 1991 and 1997. Infections of the lung by Elaphostrongylus spp. were significantly greater in red than sika deer. Older animals were more heavily infected with Elaphostrongylus spp. and Sarcocystis spp., and infections with Sarcocystis spp. tended to be heavier in more recent years. The results suggest that a combination of key indicator parasite species and non-specific signs of disease may be useful for monitoring the health of wildlife populations at a national scale. However, they also demonstrate that such monitoring needs to be long-term, carried out according to standard protocols and at an appropriate resolution to enable integration with data on other potentially influential environmental factors.

Mononuclear cell populations in the lungs of calves infected with Dictyocaulus viviparus were studied during primary infection and reinfection in order to identify cells involved in development of protective immunity to parasitic bronchitis. Three groups of calves were either inoculated with 500 third-stage larvae at both weeks 0 and 10 (n = 6), inoculated only at week 10 (n = 6), or remained uninfected (n = 3). The animals were monitored weekly by collection of bronchoalveolar lavage fluid (BALF), blood and faeces. Among mononuclear BALF-cell populations, the gamma/delta TCR-expressing cells showed a pronounced transient increase in proportion as well as in relative cell size 2 weeks post primary infection, whereas CD4-, CD8-, Ig- and CD14-expressing cells showed no significant differences related to the infection. The increase in gamma/delta TCR-expressing cells coincided with significantly increased proportions of eosinophils and recovery of adult worms in BALF. After reinfection, gamma/delta TCR-expressing cells increased again, but not until week 3 post inoculation, whereas eosinophils were increased by week 2 and reached higher levels than after primary infection. After reinfection, establishment of D. viviparus was less successful than after primary infection. In conclusion, these results indicate a role for gamma/delta TCR-expressing lymphocytes in the pathogenesis of D. viviparus infection.

The high degree of immunity induced by the bovine lungworm, Dictyocaulus viviparus, makes it an ideal model in which to study nematode-induced protective immune responses. Here, cytokine responses were measured over the course of an experimental infection of D. viviparus. Local cytokine messenger RNA (mRNA) transcripts were measured in lung parenchyma, tracheal rings and draining lymph nodes using semi-quantitative reverse transcriptase-polymerase chain reaction. Responses were measured in animals necropsied at 15, 22 and 43 days post-infection (DPI). The responses elicited at these time points were compared with cytokine levels observed in uninfected animals. Interleukin (IL)-4, IL-5, IL-10, IL-12p35, IL-13 and interferon gamma (IFNgamma) mRNA levels were measured in duplicate at each site. By 42 DPI, very few parasites were recovered, either from faeces or lungs. Transcripts of all cytokines increased in the lung parenchyma, tracheal rings and caudal mesenteric lymph nodes by 15 DPI. The response was rapid and peaked during the time of larval migration through the lungs. By 42 DPI, expression levels of most cytokines were reduced to levels similar to, or below, base line values measured in uninfected animals. Highest levels of IL-10, IL-12p35, IL-13 and IFNgamma transcript were measured in the bronchial lymph nodes of uninfected animals. IgG1 levels were negatively correlated with expression levels of all cytokines. The results demonstrate that a mixed cytokine response occurs over the course of a primary infection during which the parasites were eliminated by day 43 DPI. These results agree with those obtained for other helminths in cattle and challenge the hypothesis that polarised Th2 responses are essential for protection against nematodes in this species. These observations are important in the development of recombinant vaccines, particularly when considering adjuvant choice.

A serological survey was carried out to determine the prevalence and geographical distribution of Dictyocaulus viviparus in calves after their first grazing season in Sweden. A total of 754 animals from 76 randomly selected herds in seven geographical regions were examined between September 24 and December 19, 2001. To get an indication about the geographical distribution of the infection 41 herds with beef-suckler calves were investigated. On each farm, blood was collected from 8 to 10 animals after an average of 26 +/- 24 days post-housing to determine specific IgG1 levels against a possible lungworm sperm antigen that is highly specific against patent infections of D. viviparus. We also investigated the seroprevalence of lungworm infection in relation to cattle management. In one region additional samples were analysed from 35 herds either with: (a) beef-suckling calves that were dewormed at housing, (b) untreated organically raised dairy calves, and finally from conventionally raised dairy calves either, (c) with or, (d) without a prophylactic anthelmintic treatment programme against gastrointestinal parasites on pasture. A questionnaire was used to obtain information about herd size and management, including measures to control nematode parasites on the farm. A total of 86 (11.8%) out of 754 animals had antibodies against D. viviparus, and at least one infected individual was detected in 30 (39.5%) of the 76 herds examined. Lungworm infected animals were found throughout the country and there was no significant differences between regions, although in southern and southwestern Sweden 70.0% of the herds were infected. Furthermore, there were no major differences in the seroprevalence in relation to management. Between 40.0 and 44.4% of the herds were infected irrespective of management, with the exception of calves from organic herds where no seropositive samples were found (0%). This result is in contrast to previous findings of lungworms in Sweden, and indicates that the parasite status on organic farms is diverse.

We have examined the population genetic structure in a collection of nine isolates of the parasitic lungworm Dictyocaulus viviparus. Eight of the isolates were sampled from cattle in geographically separated farms throughout south-central Sweden, and one isolate was a laboratory strain that has been maintained in experimentally infected calves for almost four decades. A total of 72 worms were examined, with eight individual worms from the same individual host representing each isolate. The genetic variation as revealed by amplified fragment length polymorphism analysis using four selective primer combinations was high. Depending on the primer combination a total of 66-79 restriction fragments were amplified, with 26-44 peaks of similar complexity from each of the isolates. The heterozygosity within populations was relatively small, as were the population mutation and immigration rates, which seemed to be in neutral equilibrium. The genetic diversity was therefore reasonably well structured in the field; and the laboratory isolate was quite distinct from the field samples. There was no relationship between the patterns of genetic diversity and the geographical proximity of the farms. The estimates of heterozygosity were much larger and more consistent than those previously estimated for this nematode species using mitochondrial sequencing, and the genetic structuring was thus much less pronounced and the gene flow greater. We attribute these differences in estimation to the broader sampling of loci available using amplified fragment length polymorphism markers, which may therefore constitute a superior technique for the study of patterns of lungworm diversity. Furthermore, the data estimating gene flow for D. viviparus was less than previously reported for closely related species in North America. This might be related to different rates of movements of infected hosts. It seems likely that lungworm infections are rather persistent on different farms, and the sudden outbreaks of disease that can be observed with host movements are most likely to be related to the introduction of susceptible stock.