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

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 In Italy, information on dictyocaulosis in wildlife is limited and often outdated.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

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

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

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

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

[(IMPACT OF LUNGWORM INFESTATION ON THE BIOCHEMICAL PARAMETERS OF ANIMALS DURING COMBINATION TREATMENT WITH ALBENDAZOLE AND T- AND B-ACTIVIN)].

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.

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

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

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

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

Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves.

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.

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

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

Dictyocaulosis in cattle: Retrospective analysis of 20 outbreaks in Central Argentina.

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.

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

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

Predicting the unpredictable? A climate-based model of the timing of peak pasture infectivity for Dictyocaulus viviparus.

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 0oC, and larval development occurred above 1.4oC, whereas the estimated minimum temperature for migration via Pilobolus spp. was 8.8oC. 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.

Larvoscopic study on Dictyocaulus sp. in the faeces of beef cattle in northeastern Brazil.

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.

Efficacy of eprinomectin pour-on treatment in sheep naturally infected with Dictyocaulus filaria and Cystocaulus ocreatus.

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.

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

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

Risk factors for lungworm-associated milk yield losses in grazing dairy 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.

Comparison of the FLOTAC technique with the McMaster method and the Baermann technique to determine counts of Dictyocaulus eckerti L1 and strongylid eggs in faeces of red deer (Cervus elaphus).

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.

Association between Dictyocaulus viviparus bulk tank milk antibody levels and farmer-reported lungworm outbreaks.

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.

Pathological lesions in the lungs of red deer Cervus elaphus (L.) induced by a newly-described Dictyocaulus cervi (Nematoda: Trichostrongyloidea).

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.

The effect of patent Dictyocaulus viviparus (re)infections on individual milk yield and milk quality in pastured dairy cows and correlation with clinical signs.

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.

A transcriptomic analysis of the adult stage of the bovine lungworm, Dictyocaulus viviparus.

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.