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

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

Antibodies against Dictyocaulus viviparus major sperm protein in bulk tank milk: Association with clinical appearance, herd management and milk production.

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-1day-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.

Vaccination of calves with yeast- and bacterial-expressed paramyosin from the bovine lungworm Dictyocaulus viviparus.

Previously, vaccination of cattle with Escherichia coli-expressed bovine lungworm paramyosin (EcPMY) adjuvanted with Quil A resulted in considerable reduction in worm burden and larvae shedding (Strube et al., 2015). To further evaluate the protective potential of PMY, cattle vaccination trials were performed using either E. coli- (EcPMY) or Pichia pastoris-expressed PMY (PpPMY) with different adjuvants (Matrix-Q(™) or Quil A). Combinations EcPMY+Matrix-Q(™) (trial 1), PpPMY+Matrix-Q(™) (trial 2) and PpPMY+Quil A (trial 3) were tested against challenge infections with 2000 Dictyocaulus viviparus larvae. Even though GM worm burden and larvae shedding was lower in almost all vaccinated groups, there were high variations between individuals hampering significant differences. However, in all vaccinated groups, lungworms were significantly shorter compared with those in controls. In vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant (r)PMY revealed no significant proliferation following vaccinations or challenge infection. All vaccinated cattle showed a significant rise in specific antibodies, particularly IgG and its subclass IgG1, and detected the native lungworm PMY in immunoblots starting 2 weeks after the first vaccination. The use of a different rPMY-adjuvant combination or combined vaccination with additional recombinant antigens might be a promising future approach towards a new vaccine against lungworms in cattle.

Vaccination with recombinant paramyosin against the bovine lungworm Dictyocaulus viviparus considerably reduces worm burden and larvae shedding.

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.

Parasite control practices on pasture-based dairy farms in the Republic of Ireland.

Dictyocaulus viviparus, Ostertagia ostertagi (nematode parasites), and Fasciola hepatica (trematode parasite) result in productivity losses on dairy farms and impact on animal health through clinical and sub-clinical disease. Parasite control in livestock systems is largely based on the use of chemoprophylactic agents (anthelmintics), grazing management, or a combination of both. The objective of this study was to document current parasite control measures employed by Irish dairy farmers in a predominantly pasture-based livestock system. A questionnaire survey of 312 geographically representative farmers was completed in 2009 with a follow up survey completed in 2011. Statistical analysis highlighted significant differences in chemoprophylactic usage between 2009 and 2011. In particular, an increase in the use of albendazole for both trematode (19% in 2009 to 36% in 2011) and nematode (30% in 2009 to 58% in 2011) control was observed. This was most likely due to flukicide restrictions introduced in the Republic of Ireland in 2010 for dairy animals. Logistic regression highlighted regional differences in chemoprophylactic use. Farmers in southern parts of Ireland, an area with good quality soil, less rainfall, and a higher density of dairy farms than other regions, were approximately half as likely to dose for F. hepatica and were more likely (OR>2.0) to use albendazole for both nematode and fluke control. Approximately 30% of respondents who used a chemoprophylactic treatment for nematodes, used a product which was 'unsuitable for purpose' (e.g. ivermectin for the treatment of F. hepatica), highlighting the need for increased awareness, continuing research, and regionally targeted education tools regarding optimal parasite control.

Differential N-glycan- and protein-directed immune responses in Dictyocaulus viviparus-infected and vaccinated calves.

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.

Preventive vaccination of lactating and pregnant heifers against lungworm: safety and protection in three dairy herds.

A study of the safety of a vaccine against lungworm was carried out with pregnant and lactating heifers from three dairy herds with a previous history of lungworm outbreaks in adult cows. Half of the heifers were vaccinated while the other half were not. A slight temporary cough following the vaccination was only observed in one herd. No adverse effects on pregnancy or milk production were seen. All heifers were serologically and coprologically examined before the first, before and after the second immunization, 3 months after introduction to pasture and at the end of the grazing season. Serological and faecal examination of the dairy cows before introduction into pasture confirmed the presence of at least one Dictyocaulus viviparus carrier in each herd. Lungworm infection occurred in all herds during the grazing season, most prominently in the herd with the highest number of heifers. In this herd, mild coughing associated with the lungworm infection was noticed, especially in the non vaccinated heifers. No other signs or symptoms were observed. It is concluded that a vaccine against D. viviparus can be used safely in heifers, before they are introduced into the adult herd, and that this vaccine can be used as a preventive measure against lungworm outbreaks in adult cattle.

The application of mass spectrometry to identify immunogenic components of excretory/secretory products from adult Dictyocaulus viviparus.

Proteomics has come to the forefront in the post-genomic era. The ability to compare and identify proteins expressed in a particular cell type under specific physiological or pathological states requires a range of technologies, including separation of complex protein or peptide mixtures, densitometry-based or isotope-coded methods for comparison of multiple proteomes, and mass spectrometric methods for identification of individual low abundance proteins. Although an emergent technology, thus far, proteomics has provided new perspectives on many problems in biomedical science. In parasitology, proteomics has been used to answer specific biological questions relating to survival and development, and also to identify candidates for vaccines. Here, we describe an ongoing research programme in which proteomics is being used to identify potential vaccine candidates for the bovine lungworm, Dictyocaulus viviparus. This work is focusing on antibody responses to the adult parasite excretory/secretory (ES) products, with selection of candidate antigens based on differential screening with serum from immune versus non-immune animals to simplify the proteome and the ensuing analytical challenges. Thus far, we have identified seven candidate proteins using this strategy. Of these, one protein showed significant identity to a previously cloned gene from D. viviparus, whilst the other six proteins have shown no significant identities. Isolation of further peptide sequences is now warranted to facilitate cloning of the genes encoding these antigens.

Efficacy trial of an irradiated cattle lungworm vaccine in red deer (Cervus elaphus).

Lungworm (Dictyocaulus sp.) is the parasite of most concern to the New Zealand deer industry. Although lungworm can be controlled by anthelmintics there is an increasing concern over excessive drenching programmes and reliance on chemicals for parasite control. A live irradiated larval vaccine developed for cattle has been available in Europe for the past 40 years but has never been evaluated in red deer in New Zealand. Four groups of red deer and two of cattle were hand reared from birth in parasite-free conditions. The cattle acted as a control group to ensure that the vaccine was still efficacious on arrival in New Zealand. Two groups of deer were vaccinated, and all four groups were challenged with either D. viviparus or deer origin Dictyocaulus, tentatively identified as D. eckerti. The vaccine provided excellent protection to cattle under New Zealand conditions, there was no larval output in the vaccinated cattle and no adults were found in their lungs at necropsy. In red deer, patency was delayed in the vaccinated groups regardless of challenge species and larval output was lower but was not prevented. Adult lungworms were found in the lungs of all deer at necropsy but fewer were recorded in the vaccinated deer. Although Huskvac provided a degree of protection for red deer it was not effective enough to recommend its use.

[Lungworm disease in dairy cattle: symptoms, diagnosis, and pathogenesis on the basis of four case reports]. / Longwormproblemen bij melkkoeien: symptomatologie, diagnostiek en pathogenese aan de hand van een viertal cases.

Clinical lungworm disease appears to occur frequently in Dutch dairy herds. Because the clinical diagnosis is difficult to make in adult cattle, the clinical diagnosis, laboratory diagnosis, differential diagnosis, therapy, and prevention are discussed in this article. In addition, four cases of lungworm disease in adult cattle are presented. The main clinical complaints were coughing, decreased milk production, and weight loss. Several lactating cows died in one herd. The disease history of four herds revealed that introduction of susceptible cows or heifers to herds with cows with subclinical patent lungworm infections had resulted in a pasture infection, leading to clinical problems in both the newly introduced and 'resident' cows of the herd. Further history analysis of the fourth herd revealed that re-introduction of lungworm infection by newly purchased cows in a lungworm free herd resulted in clinical lungworm problems in adult and young animals. The fourth case led to the conclusion that lungworm infection must have been re-introduced by cows purchased from another farm.

Dictyocaulus viviparus: re-emerging or never been away?

For over 40 years a highly effective vaccine against the bovine lungworm has been commercially available. The use of it successfully reduced the number of outbreaks in calves. However, the past decade has seen a dramatic increase in lungworm outbreaks in adult cows in the UK. This might indicate that Dictyocaulus viviparus is re-emerging as a significant parasite in the dairy cattle industry. Much is still unknown, and here the most important aspects requiring urgent attention are put into perspective.

Treatment vs non-treatment of helminth infections in cattle: defining the threshold.

Helminth infections are an important cause of lost productivity in livestock world-wide, often necessitating anthelmintic treatment. However, a large part of the anthelmintics are used indiscriminately because the parasite levels are too low to justify treatment or because the treatments are not correctly programmed, resulting in undertreatment or overtreatment. The objective of this paper is to discuss possible thresholds for anthelmintic treatment of some of the more important helminth infections in livestock, i.e. gastrointestinal nematodes, lungworms and liver fluke, to promote a more appropriate use of anthelmintics. When possible, a distinction is made between therapeutic thresholds, production-based thresholds and preventive thresholds. A "therapeutic threshold" is intended to identify (an) animal(s) with parasite levels that necessitate immediate treatment. The therapeutic threshold is basically the diagnosis of clinical disease, and can be determined relatively easily for the parasites discussed in this paper. A "production-based threshold" is intended to measure the effects of sub-clinical parasitism on productivity parameters, such as weight gain and milk yield, before clinical disease occurs. Finally, a "preventive threshold" is meant to predict future infection levels, to enable the application of appropriate control measures.

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products.

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freund's incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.

Vaccine development and diagnostics of Dictyocaulus viviparus.

Parasitic bronchitis is a serious disease of cattle and is caused by the nematode, Dictyocaulus viviparus. For over 30 years, a radiation-attenuated larval vaccine has been used for prevention of this disease. This vaccine has been used with considerable success in the UK and parts of Western Europe, however, it has several disadvantages. It has a short shelf-life and the vaccine has to be produced annually necessitating the use of donor calves. Following vaccination, calves must receive further boosting from natural challenge to maintain protective immunity. Sales of the irradiated larval vaccine have decreased dramatically since the 1970s. This is thought to be due to increased reliance of farmers on anthelmintic programmes to control lungworm infection. It is possible that, under certain circumstances, these programmes do not allow sufficient parasite exposure to stimulate protective immunity to further Dictyocaulus challenge. This is borne out by the recent documented increase in the number of outbreaks of parasitic bronchitis in the UK. A stable vaccine against D. viviparus that is capable of stimulating a more prolonged immunity would be beneficial. Recent research has been directed at identification and isolation of components thought to be involved in parasite survival in the host and examination of their potential as vaccine candidates. One of these components is acetylcholinesterase (AChE), an enzyme secreted by adult worms. This review describes the development of the secreted AChE as a vaccine candidate, as well as documenting recent developments in the immunodiagnosis of D. viviparus.

Cross-sectional serological survey on gastrointestinal and lung nematode infections in first and second-year replacement stock in the netherlands: relation with management practices and use of anthelmintics.

A cross-sectional survey was carried out on 86 farms randomly distributed in The Netherlands. After housing following the first and the second grazing season (FGS and SGS) serum samples were collected to determine IgG levels against Cooperia oncophora and Dictyocaulus viviparus, and the pepsinogen content. A questionnaire was used to inquire on grazing management practices and the use of anthelmintic drugs. On 80.7 and 60.2% of the farms FGS and SGS animals, respectively, were treated at least once with an anthelmintic drug. The percentage for the SGS animals indicates that the use of anthelmintic drugs in those animals has increased enormously over the last 10-15 years. Generally, parasitic nematode control in the FGS is good on most farms, but it can be characterised as being overprotective. There is a tendency that if anthelmintic drugs are used in the FGS they also are used more often in the SGS. On 12 farms (14%), no anthelmintic drugs were given in the FGS and the SGS. These farms did not differ from the others with respect to management practices in any obvious way. The serological results were in general very low, indicating low levels of exposure to gastrointestinal nematode infection in both FGS and SGS animals. This was not surprising in view of the good to high level of nematode control practices reported by the farmers. Although not statistically significant, a consistent result was that serological results for the SGS animals were more often positive or on average higher on those farms where FGS parasite control tended to be excessive. For D. viviparus, a prevalence rate of 41% positive farms was found. Following comparison with previous data, it is speculated that lungworm (sero-)prevalence in replacement stock may be declining as a result of continuing high levels of parasite control in replacement stock. It is concluded that the results confirm previous surveys, lending support to the conclusion that parasitic nematode control on Dutch dairy farms, certainly in FGS calves, is good but tends to be overprotective.

The efficacy of two isolates of the nematode-trapping fungus Duddingtonia flagrans against Dictyocaulus viviparus larvae in faeces.

A series of experiments was carried out to examine the effects of two different isolates of the nematode-trapping fungus Duddingtonia flagrans to reduce the number of free-living larvae of the bovine lungworm, Dictyocaulus viviparus. A laboratory dose-titration assay showed that isolates CI3 and Troll A of D. flagrans significantly reduced (P < 0.05 to P < 0.001) the number of infective D. viviparus larvae in cultures at dose-levels of 6250 and 12,500 chlamydospores/g of faeces. The larval reduction capacity was significantly higher for Troll A compared to CI3 when lungworm larvae were mixed in faecal cultures with eggs of Cooperia oncophora or Ostertagia ostertagi and treated with 6250 chlamydospores/g of faeces. Both fungal isolates showed a stronger effect on gastrointestinal larvae than on lungworm larvae. Two plot trials conducted in 1996 and 1997 involved deposition of artificial faecal pats containing free-living stages of D. viviparus and C. oncophora on grass plots. Herbage around the pats was collected at regular intervals and infective larvae recovered, counted and identified. These experiments showed that both D. flagrans isolates reduced the number of gastrointestinal as well as lungworm larvae in faecal pats. During both plot trials, the transmission of C. oncophora larvae, but not D. viviparus, from faecal pats to the surrounding herbage was clearly affected by climatic conditions. After collection of faecal pats from the grass plots one month after deposition, the wet and dry weight of pats as well as organic matter content were determined. No differences were found between the fungus-treated and non-treated control pats. This indicated that the rate of degradation of faeces was not affected by the addition of the fungus.

Strategic control of gastrointestinal nematode and lungworm infections with eprinomectin at turnout and eight weeks later.

Two groups of 10 first-year grazing cattle were either left untreated as controls (group 1) or treated with eprinomectin as a pour-on application at turnout and eight weeks later (group 2). The control group developed a mild infection with gastrointestinal nematodes and lungworms during the season, whereas the treated animals remained healthy. The interval between the treatments allowed the establishment of adult worms, but the egg counts remained negligible. The total number of eggs shed by the treated cattle during the grazing season was significantly smaller than by the controls.

[Prevention of gastrointestinal and lungworm infections in alpine calves: use of doramectin pour-on in before and after the alpine grazing season]. / Prophylaxe des Magen-Darm- und Lungenwurmbefalls bei gealpten Jungrindern: Einsatz von Doramectin pour-on bei Alpauftrieb und Alpabtrieb.

The effect of doramectin pour-on (Dectomax 0.5% pour-on, Pfizer) against gastrointestinal nematodes (GIN) and lungworms was investigated in calves grazed on alpine pastures in Switzerland. Doramectin pour-on (0.5 mg/kg body weight) was administered topically to calves (n = 57, Group D) before and after the alpine grazing season (mid-June to end of September) when the calves were kept on altitudes between 1800 and 2600 m together with 20 untreated calves (Group K). Calves of both groups were grazed together during the alpine season and were subsequently moved to prealpine and home pastures where they were kept until stabling (end of November). Faecal examinations, performed 4 to 5 weeks after anthelmintic treatment of Group D calves showed a marked reduction of the trichostrongyle egg output, which was also low in the untreated calves. Low numbers of Dictyocaulus-larvae were excreted by the calves at turnout to the alpine pastures. At the end of September the mean excretion of Dictyocaulus-larvae had increased markedly in both groups. After treatment of Group D calves the larval output remained negative until stabling. In contrast, the larval excretion of the untreated calves (Group K) reached highest levels at the end of October and 4 calves exhibited clinical signs of dictyocaulosis. The results indicate that the strategic use of doramectin pour-on in June and in late September provided good protection against severe infections with D. viviparus. The infection level with GIN confirmed that these parasites are of minor relevance under the extensive grazing conditions on high alpine pastures.