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.

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.

Dictyocaulus viviparus genome, variome and transcriptome elucidate lungworm biology and support future intervention.

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.

Transcriptional differences between hypobiotic and non-hypobiotic preadult larvae of the bovine lungworm Dictyocaulus viviparus.

The survival of the bovine lungworm Dictyocaulus viviparus, one of the most important parasites in cattle, inside the host is ensured by arrested development during adverse environmental conditions, commonly referred to as hypobiosis. In the present study, a subtractive hybridization approach was used to compare the transcription profiles of hypobiotic and non-hypobiotic larvae (L5hyp and L5, respectively). Thereby, 75 L5hyp-enriched and 58 L5-enriched representative ESTs (rESTs) were identified. Subsequent sequence similarity search revealed that 28 L5hyp-rESTs and 11 L5-rESTs were homologous to known transcripts, whereas 47 L5hyp-rESTs and 47 L5-rESTs showed no homologies with published sequences, thus possibly representing parasitic or even Dictyocaulus-specific genes. The differential transcripts were predicted to be involved in nucleic acid synthesis, DNA binding, metabolic pathways and signal transduction. Overall, data presented in this paper provide a first basis for further characterization and analysis of genes driving normal as well as arrested (hypobiotic) parasite development.

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.

GTP-cyclohydrolase and development in Teladorsagia circumcincta and Dictyocaulus viviparus (Nematoda: Strongylida).

GTP-Cyclohydrolase (GTP-CH) is necessary for the production of tetrahydrobiopterin, a required cofactor for the three aromatic amino acid hydroxylases and nitric oxide synthases. The gene encoding GTP-CH is transcribed at high levels in infective third larval stages of a number of parasitic trichostrongylid nematodes. We explore the potential role of GTP-CH within the processes of nematode development and environmentally-induced hypobiosis. For two species of parasitic nematode that are of major economic and welfare importance to livestock in temperate regions, Teladorsagia circumcincta and Dictyocaulus viviparus, we have demonstrated that each of the pre-parasitic larval stages transcribe high mean levels of cat-4 (the gene encoding GTP-CH). Using quantitative real-time polymerase chain reaction analysis and two different isolates of D. viviparus, only one of which is capable of entering hypobiosis, we have shown that there were only minor differences between these isolates in mean cat-4 transcript levels, both during the parasitic stages and during the earlier environmental life cycle stages (L(1)-L(3)). Taken together, these data indicate that, although both species of nematode produce high levels of cat-4 transcript in pre-parasitic larval stages, GTP-CH levels are unlikely to be involved in the induction of parasite hypobiosis. Alternative roles for GTP-CH in larval development are discussed.

Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus.

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.

Evaluation of reference genes for quantitative real-time PCR to investigate protein disulfide isomerase transcription pattern in the bovine lungworm Dictyocaulus viviparus.

Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. For Dictyocaulus viviparus, the housekeeping genes beta-tubulin, beta-actin, elongation factor 1alpha (ef-1alpha), glyceraldehyde-3-phosphatase dehydrogenase (gapdh), and 60S ribosomal protein L37a (60S rpL37a) were characterized and evaluated. Evaluation using the geNorm software revealed ef-1alpha and beta-tubulin as the most suitable reference genes, whereas the coefficient of variation approach resulted in ef-1alpha and 60S rpL37a as transcripts with the least variation among 12 developmental lungworm stages. The critical influence of reference genes on qPCR data analysis, with the possible consequence of erroneous, misleading results due to inappropriate reference genes used for data normalization, is shown for protein disulfide isomerase 2 (pdi-2) transcription patterns. Proper normalization of pdi-2 transcription using ef-1alpha and beta-tubulin as reference genes resulted in a more than 7-fold enriched pdi-2 transcription in L1 compared to that in eggs, and a dramatic decrease in L3. Following an increase in the L5 stage there is again a decrease of pdi-2 transcription in adult lungworms. These fluctuations in the transcription levels reflect the requirement of cuticule collagen during bovine lungworm development.

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.

Genetic regulation of arrested development in nematodes: are age-1 and daf-gene orthologs present in Dictyocaulus viviparus?

In opposite to the free-living soil nematode Caenorhabditis elegans, the genetic regulation of hypobiosis or inhibited or arrested development in parasitic nematodes is completely unknown. In C. elegans, the daf-genes or the age-1 gene are of major importance in signaling pathways regulating arrested development. To investigate if orthologs of these genes are present in the bovine lungworm Dictyocaulus viviparus, a PCR analysis with gene-specific primer combinations was performed. No orthologs of the age-1 or daf-genes could be identified in D. viviparus. The possible differences in the role of the daf-genes concerning arrested development in parasitic and free-living nematodes will be discussed.

Differential gene expression in hypobiosis-induced and non-induced third-stage larvae of the bovine lungworm Dictyocaulus viviparus.

Hypobiosis is of particular importance in overwintering of the bovine lungworm Dictyocaulus viviparus. However, in parasitic nematodes there is no information available on the genetic mechanisms of hypobiosis. Suppression subtractive hybridisation was performed to identify upregulated transcripts of hypobiosis-induced and non-induced third-stage D. viviparus larvae, respectively. Subtracted libraries containing 105 clones of the hypobiosis-induced and 104 clones of the non-induced larvae were generated. By differential screening and Southern dot blot, 26 clones of the hypobiosis-induced and 22 clones of the non-induced larvae were confirmed to be differentially expressed. Sequencing of rapid amplification of cDNA ends (RACE) and spliced-leader-1 PCR products was performed to further characterise selection of the differentially regulated gene transcripts. The genes encoding an N-methyltransferase and a superoxide dismutase were upregulated in the hypobiosis-induced and non-induced larvae, respectively. The expression patterns of these genes were validated by quantitative real-time PCR. This revealed differential gene expression, particularly for the N-methyltransferase.

Lungworms of ruminants.

Several species of nematodes parasitize the respiratory tract of ruminants. Disease due to infection with these parasites occurs less frequently in North America than in other parts of the world but can be locally and regionally important. The life cycles, epidemiology, pathology, diagnosis and treatment of Dictyocaulus viviparus, Dictyocaulus filaria, and Muellerius capillaris are summarized in this article.

Suppressive treatment of abamectin against Dictyocaulus viviparus and the occurrence of resistance in first-grazing-season calves.

Dictyocaulus viviparus cause severe lung infections and are endemic in some areas of temperate and tropical countries. Treatment is based on strategic nematode control programs using potent compounds with no reports of drug failure. Macrocyclic lactones (MLs) are available at different concentrations and combinations and have being used heavily by producers. The objective of this work was to determine the efficacy of the MLs ivermectin, moxidectin, doramectin, and abamectin, and the combination ivermectin plus abamectin in naturally infected calves (n=70). Initial infection was determined by necropsy of tracer animals. Faecal larval counts determined that none of the compounds used was able to eliminate D. viviparus for up to 28 days after treatment. The "Area Nova" strain was isolated for future work.

The health of wild red and sika deer in Scotland: an analysis of key endoparasites and recommendations for monitoring disease.

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.

Effects of condensed tannins and crude sesquiterpene lactones extracted from chicory on the motility of larvae of deer lungworm and gastrointestinal nematodes.

The objective of this study was to determine the effects of condensed tannins (CT) and an extract containing crude sesquiterpene lactones (CSL) from chicory (Cichorium intybus) on the motility of the first-(L1) and third-stage (L3) larvae of deer lungworm Dictyocaulus viviparus and the L3 larvae of gastrointestinal nematodes in vitro, using the larval migration inhibition (LMI) assay. The CT and CSL had a profound effect on the motility of the larvae displayed by their ability to inhibit larval passage through nylon mesh sieves. Incubation of lungworm L1 larvae in rumen fluid (collected from deer fed pasture) containing 100, 400 and 1000 microg CT/ml, inhibited 12, 28 and 41% of the larvae from passing through the sieves, respectively, while the incubation of L3 larvae with rumen fluid (pH 6.6) containing the same concentrations inhibited 26, 37 and 67% of L3 larvae from passing through the sieves, respectively. Gastrointestinal larvae seem more susceptible to CT than lungworm larvae especially at higher concentrations. CT inhibited 27, 56 and 73% of gastrointestinal larvae from passing through the sieves when used at a concentration of 100, 400 and 1000 microg/ml, respectively. CT were more effective (P<0.001) at reducing the motility of lungworm L1 and L3 larvae when added to the rumen fluid than when added to the abomasal fluid (pH 3.0). Addition of 2 microg polyethylene glycol/microg CT eliminated the inhibitory effect of CT against L1 and L3 larvae especially during incubation in rumen fluid, confirming the effect as due to CT. The CSL extract also showed similar inhibitory activity against L1 and L3 lungworm and L3 gastrointestinal larvae in both fluids, indicating that this extract was not affected by the pH of the fluid, and was more effective against L3 than L1 lungworm larvae. Condensed tannins appeared to be more effective than CSL at inactivating L1 and L3 lungworm and L3 gastrointestinal larvae in rumen fluid, but CSL were particularly effective against L3 lungworm larvae in abomasal fluid. Activity of these secondary compounds explains the reduced parasite problem of young deer grazing chicory.

The effect of treatment with eprinomectin on lungworms at early patency on the development of immunity in young cattle.

An experiment was carried out to study the effect of topical application of eprinomectin at early patency on the build up of infection and development of protection against Dictyocaulus viviparus in young cattle. Three groups of six calves were used and parasitological and blood variables were monitored at weekly intervals throughout the trial. At the start of the experiment calves in groups A and B were experimentally inoculated with 100 D. viviparus infective third-stage larvae (L3) for five consecutive days, whereas calves in group C served as uninfected controls. The calves in group A were each treated with eprinomectin (0.5mg/kg bodyweight) in a pour-on formulation at early patency at day 24 post the first inoculation, whereas the calves in groups B and C were left untreated. Seven weeks following anthelmintic treatment all groups were challenged with 1500 L3. Another 4 weeks later the animals were sacrificed and established worms in the lungs were counted. Moderate transient signs of lungworm disease occurred both in groups A and B. However, group B calves were found to be about 8 times more resistant than those in group A, whereas the naive infection controls in Group C was found to be about 35 times more susceptible to infection. Also the ELISA values showed that the course of infection was different between experimental groups. The eosinophil counts prior to and at the time of slaughter indicate that immunity was involved in the protection and the response was correlated with previous exposure and worm load. Weight gains differed significantly, but only between groups A and C and between groups B and C that on an average were approximately 13kg heavier at the termination of the experiment. It was concluded that eprinomectin was effective against established adult lungworms. However, the untreated calves (group B) developed a more marked resistance to lungworms compared to those that were subjected to anthelmintic treatment at early patency (group A). On the other hand, the cumulative number of excreted larvae was on an average 43 times higher in group B as compared to group A. Consequently, infected calves that remain out on pasture should be treated. This will restrain transmission of the parasite despite the fact that immunity is deteriorated.

Efficacy of moxidectin 1% injectable and 0.2% oral drench against natural infection by Dictyocaulus filaria in sheep.

Two separate trials (I and II) with 34 and 32 Churra ewes, respectively, and distributed into two groups, have been carried out to evaluate the efficacy of two different formulations of moxidectin at a dose rate of 0.2mg/kg body weight (b.w.) against natural infection by Dictyocaulus filaria in sheep. Trial I was designed to evaluate a 1% moxidectin injectable formulation, whereas in trial II a 0.2% moxidectin oral drench formulation was used. The efficacy was measured on the basis of the reduction of the faecal larval counts and of adult worm recoveries at slaughter. In each trial, a group of animals was treated on day 0 with moxidectin 1% injectable or moxidectin 0.2% oral drench and the other group acted as untreated control. When the faecal larval counts was compared within the treated groups, the efficacy was over 95% until day +13, and 100% at the remainder of the sampling dates after the application of injectable moxidectin, whereas in trial II, the larvae per gram (lpg) of faeces increased until the first sampling time post treatment (p.t.), day +6, and zero counts were recorded for all animals by the following days. On the basis of adult worm recoveries at necropsy, the efficacy of the treatment was 100% in both trials, however, adult worms were detected at slaughter for all control sheep. These results indicate that moxidectin 1% injectable and moxidectin 0.2% oral drench, administered at 0.2mg/kg b.w., were 100% effective against D. filaria infection in sheep. No adverse reactions to the treatments were observed in the animals.

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.

Immunity in calves against Dictyocaulus viviparus following a low primary infection.

Thirty Holstein Friesian heifer calves, 3 months of age, were used to evaluate whether a low primary infection with Dictyocaulus viviparus would result in immunity against challenge infection 5 or 12 weeks later. Fifteen calves were experimentally infected with 30 larvae at day 0, while the other calves remained as uninfected controls. On day thirty-five 6 primary infected (G1) and 6 non-infected calves (G2) were challenged with 2000 larvae. These groups were necropsied on day 70. On day 84 the remaining 9 primary infected (G3) and 9 non-infected (G4) calves were similarly challenged and necropsied on day 119. Respiratory signs developed in most calves from approximately 2 weeks after challenge, but signs were more severe in G2 and G4 than in G1 and G3. The low primary infection resulted in significantly reduced faecal larval excretion (> 80%) and worm burdens (> 70%). In the primary infected groups the proportion of female worms in the worm burden was increased, suggesting that part of the developed immunity affected survival of the worms after their establishment in the host. Worm lengths and worm fecundity were also significantly reduced. All observed effects were stronger following challenge on day 84 than on day 35, but the effect of the duration of a primary infection (contrast between G1 and G3) was only statistically significant in the case of mean faecal larval excretion on the day of necropsy (P < 0.05) and fecundity (P < 0.05).

Effect of condensed tannins extracted from four forages on the viability of the larvae of deer lungworms and gastrointestinal nematodes.

The inhibitory activity of condensed tannins extracted from four forage legume plants were evaluated by using a larval migration inhibition assay. The first (L1) and third (L3) stages of deer lungworm (Dictyocaulus viviparus), and the third stage (L3) of deer gastrointestinal nematodes were incubated with tannins extracted from Lotus pedunculatus, Lotus corniculatus, sulla (Hedysarum coronarium) and sainfoin (Onobrychus viciifolia). The tannins extracted from all the forages had inhibitory activity as measured by their ability to paralyse the larvae and inhibit them from passing through sieves. At the highest concentration used (1200 microg/ml) the tannins extracted from sainfoin had the highest activity against ensheathed L1 lungworm larvae (58 per cent), followed by L. pedunculatus (45 per cent), sulla (42 per cent) and L. comiculatus (35 per cent) when the larvae were incubated at 37 degrees C. The same trend, but with lower activities, was observed when the larvae were incubated at 22 degrees C. Anthelmintic activity against L3 lungworm larvae was evaluated by measuring the death rate of ensheathed L3 larvae after incubation with condensed tannins for two, 24 and 48 hours at room temperature (22 degrees C). The death rate was significantly higher (P<0.001) after 48 hours incubation than after two hours or 24 hours, and significantly higher (P<0.001) after 24 hours than after two hours incubation. Condensed tannins from sainfoin had the highest inhibitory activity followed by L. pedunculatus, sulla and L. comiculatus. The tannins from sainfoin also had the highest activity against L3 larvae of gastrointestinal nematodes, followed by L. pedunculatus, sulla and L. comiculatus. Exsheathed larvae of gastrointestinal nematodes were significantly more susceptible to the action of the tannins than ensheathed larvae.