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.

The first finding of Dictyocaulus cervi and Dictyocaulus skrjabini (Nematoda) in feral fallow deer (Dama dama) in Australia.

Feral deer are widespread throughout Australia with the capacity to impact livestock production via transmission of parasites. Samples of Dama dama (fallow deer), Rusa unicolor (sambar deer), Cervus elaphus (red deer) and an unidentified deer were sourced from various locations in south-eastern Australia for examination for parasites. Adult nematodes were collected from the lungs of all deer species across four separate geographical locations. The nematodes were identified as species of Dictyocaulus through both morphological and molecular means. Species identification based on morphological features was difficult, with many measurements from described species overlapping. Molecular analyses targeting three markers, namely 18S rRNA, ITS2, and cox1 revealed the presence of two distinct species: Dictyocaulus cervi and Dictyocaulus skrjabini. These are the first genetically confirmed reports of species of Dictyocaulus in feral deer in Australia, and although cross-transmission of species of Dictyocaulus with livestock has not yet been reported, it cannot be completely discounted without further research.

Opening a can of lungworms: Molecular characterization of Dictyocaulus (Nematoda: Dictyocaulidae) infecting North American bison (Bison bison).

Dictyocaulus is a globally distributed genus of lungworms of domestic and wild ungulates. Dictyocaulus adults inhabit the bronchi, frequently causing subclinical and clinical disease, and that impacts animal health and production. North American bison (Bison bison) and cattle (Bos taurus) share various parasitic nematode species, particularly in areas where co-grazing occurs. The current assumption is that North American bison share the lungworm D. viviparus with cattle, but this has not been confirmed on a molecular basis. The aim of this study was to molecularly characterize Dictyocaulus lungworm isolates from North American plains bison (Bison bison bison). Fecal samples were collected from 5 wild conservation bison herds located in Iowa, North Dakota, Oklahoma, Colorado, and Montana in 2019 and 2020, and from ranched and feedlot bison from 2 herds in Oklahoma and Texas. First-stage lungworm larvae (L1) were isolated via Baermann technique. Genomic DNA was extracted from L1s of up to 3 samples per herd and followed by PCR and sequencing targeting the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA and the partial cytochrome oxidase c subunit 1 (cox1) of mitochondrial DNA. Phylogenetic analyses were performed in MEGA X 10.1. Sequences of North American plains bison Dictyocaulus belong to a single, uncharacterized species, clustering in well-supported clades (100% and 100% bootstrap support for ITS2 and cox1, respectively), differing from D. viviparus of cattle in North America and Europe, and European bison (Bison bonasus). Our results contradict previous assumptions regarding parasite identity, highlighting the need for characterization of this species through morphological and molecular methods, elucidating its biology and host range, and potential impact on host health. Further investigation into the biodiversity of Dictyocaulus species infecting bovids and cervids in North America is warranted.

First report of a newly-described lungworm, Dictyocaulus cervi (Nematoda: Trichostrongyloidea), in moose (Alces alces) in central Europe.

Lungworms from the genus Dictyocaulus are the causative agents of verminous pneumonia in domestic and wild ungulates. Recently, in 2017, a new species was isolated from red deer and described as Dictyocaulus cervi; however, little is known about its epidemiology and pathogenicity in other cervids. The aim of our study was to determine the extent of infection with Dictyocaulus nematodes in the moose population in Poland. Parasitological necropsies were performed in 18 moose and 249 faecal samples were analysed. A combination of multiplex PCR and analysis of the partial SSU, cox1 and cyt B regions revealed the presence of D. cervi infection in two of the necropsied moose. Histopathological examinations revealed changes, including multiple cross sections of larvae of nematodes in alveoli, massive pulmonary fibrosis, mononuclear cell infiltration and diffuse alveolar damage in the lungs of four animals. The lesions were more pronounced when adult Dictyocaulus nematodes were present in the bronchi and bronchioles. Some of the observed pathological changes could be attributed to co-infection by nematodes of the Protostrongylidae, whose larvae were found in all four animals with lung pathologies. In the faeces, Dictyocaulus sp. larvae only occurred together with Protostrongylidae larvae; in addition, higher numbers of Protostrongylidae larvae were excreted in the faeces of animals with dictyocaulosis. The present study is the first report of the presence of D. cervi in moose, and demonstrates the value of multiplex PCR in the identification of Dictyocaulus nematodes. Our findings indicate that co-infections with multiple species of lung nematodes in moose may be commonplace, and this should be considered as a factor aggravating the course of parasitosis.

A novel pooled milk test strategy for the herd level diagnosis of Dictyocaulus viviparus.

Current diagnostic methods for detecting the presence or absence of Dictyocaulus viviparus in dairy herds, are insensitive when based on detection of antibody levels in bulk tank milk (BTM). Here we present a novel technique to confirm the presence of the parasite based on a pooled-milk sample from 10 randomly selected first - lactation heifers (FLH). This study was run in two parts. First, a longitudinal study was performed to look at infection dynamics in milk samples across the grazing season using a prototype ELISA developed by Svanova (Boehringer-Ingelheim, Uppsala). We identified that mean ODR values in milk samples from FLH was significantly higher than that for older cows (0.13 versus 0.07 respectively, p < 0.001) suggesting that samples from the FLH cohort should be pooled to produce the test. Second, the pooled - milk test was evaluated on a cross-sectional survey of UK dairy herds (n = 25 grazing and n = 25 zero-grazing herds) to evaluate test performance under field conditions. The optical density ratio (ODR) cut-off value for our pooled-milk test using 10 FLH milk samples was optimal at a value of 0.16. Pooling 10 FLH samples created a sensitivity and specificity of 66.7% and 95.5% respectively. In comparison, whole-herd BTM samples had a maximum sensitivity of 37.5% and specificity of 63.6% at an ODR cut-off of 0.18. The area under the curve according to receiver-operative-characteristic (ROC) analysis was high for the 10-heifer test (0.87) but poor for the whole herd BTM testing (0.45). This study provides a more sensitive diagnostic test strategy for the screening of D.viviparus in dairy herds. Testing herds at the end of a grazing season would facilitate the planning of effective control measures, such as the use of the lungworm vaccination or strategic deworming, for the following grazing season. This may prove to be a useful test strategy for the diagnosis of a variety of parasitic diseases of livestock.

A novel pooled milk test strategy for the herd level diagnosis of Dictyocaulus viviparus.

Current diagnostic methods for detecting the presence or absence of Dictyocaulus viviparus in dairy herds, are insensitive when based on detection of antibody levels in bulk tank milk (BTM). Here we present a novel technique to confirm the presence of the parasite based on a pooled-milk sample from 10 randomly selected first - lactation heifers (FLH). This study was run in two parts. First, a longitudinal study was performed to look at infection dynamics in milk samples across the grazing season using a prototype ELISA developed by Svanova (Boehringer-Ingelheim, Uppsala). We identified that mean ODR values in milk samples from FLH was significantly higher than that for older cows (0.13 versus 0.07 respectively, p < 0.001) suggesting that samples from the FLH cohort should be pooled to produce the test. Second, the pooled - milk test was evaluated on a cross-sectional survey of UK dairy herds (n = 25 grazing and n = 25 zero-grazing herds) to evaluate test performance under field conditions. The optical density ratio (ODR) cut-off value for our pooled-milk test using 10 FLH milk samples was optimal at a value of 0.16. Pooling 10 FLH samples created a sensitivity and specificity of 66.7% and 95.5% respectively. In comparison, whole-herd BTM samples had a maximum sensitivity of 37.5% and specificity of 63.6% at an ODR cut-off of 0.18. The area under the curve according to receiver-operative-characteristic (ROC) analysis was high for the 10-heifer test (0.87) but poor for the whole herd BTM testing (0.45). This study provides a more sensitive diagnostic test strategy for the screening of D.viviparus in dairy herds. Testing herds at the end of a grazing season would facilitate the planning of effective control measures, such as the use of the lungworm vaccination or strategic deworming, for the following grazing season. This may prove to be a useful test strategy for the diagnosis of a variety of parasitic diseases of livestock.

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.

Ectonucleotidase and adenosine deaminase as inflammatory marker in dairy cows naturally infected by Dictyocaulus viviparus.

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.

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

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