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.

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.

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.

Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.

We report a European wide assessment of the economic burden of gastrointestinal nematodes, Fasciola hepatica (common liver fluke) and Dictyocaulus viviparus (bovine lungworm) infections to the ruminant livestock industry. The economic impact of these parasitic helminth infections was estimated by a deterministic spreadsheet model as a function of the proportion of the ruminant population exposed to grazing, the infection frequency and intensity, the effect of the infection on animal productivity and mortality and anthelmintic treatment costs. In addition, we estimated the costs of anthelmintic resistant nematode infections and collected information on public research budgets addressing helminth infections in ruminant livestock. The epidemiologic and economic input data were collected from international databases and via expert opinion of the Working Group members of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). In order to reflect the effects of uncertainty in the input data, low and high cost estimates were obtained by varying uncertain input data arbitrarily in both directions by 20 %. The combined annual cost [low estimate-high estimate] of the three helminth infections in 18 participating countries was estimated at € 1.8 billion [€ 1.0-2.7 billion]. Eighty-one percent of this cost was due to lost production and 19 % was attributed to treatment costs. The cost of gastrointestinal nematode infections with resistance against macrocyclic lactones was estimated to be € 38 million [€ 11-87 million] annually. The annual estimated costs of helminth infections per sector were € 941 million [€ 488 - 1442 million] in dairy cattle, € 423 million [€ 205-663 million] in beef cattle, € 151million [€ 90-213 million] in dairy sheep, € 206 million [€ 132-248 million] in meat sheep and € 86 million [€ 67-107 million] in dairy goats. Important data gaps were present in all phases of the calculations which lead to large uncertainties around the estimates. Accessibility of more granular animal population datasets at EU level, deeper knowledge of the effects of infection on production, levels of infection and livestock grazing exposure across Europe would make the largest contribution to improved burden assessments. The known current public investment in research on helminth control was 0.15 % of the estimated annual costs for the considered parasitic diseases. Our data suggest that the costs of enzootic helminth infections which usually occur at high prevalence annually in ruminants, are similar or higher than reported costs of epizootic diseases. Our data can support decision making in research and policy to mitigate the negative impacts of helminth infections and anthelmintic resistance in Europe, and provide a baseline against which to measure future changes.

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.

Apparent prevalence of and risk factors for infection with Ostertagia ostertagi, Fasciola hepatica and Dictyocaulus viviparus in Swiss dairy herds.

Infections with helminth parasites can negatively affect performance of dairy cows. Knowledge on infection intensity, spatial distributions and risk factors are key to develop targeted treatment strategies. Canada and most EU countries have conducted large investigations, but respective data for Switzerland were missing. We now performed a bulk tank milk serosurvey for Ostertagia ostertagi, Fasciola hepatica, and Dictyocaulus viviparus on a total of 1036 voluntarily participating dairy herds that were sampled at confinement periods, i.e. in winter 2014/15 or 2015/16, respectively. All samples were analyzed with commercial ELISAs for antibodies (AB) against O. ostertagi and F. hepatica, and those of the first sampling period additionally with an in-house ELISA for AB against D. viviparus. Testing for the latter parasite was not done in the second year of the study, as the sampling period might have missed infections due to the short lived nature of specific antibodies. The possible influence of geographic, climatic, and farm management variables on AB levels were assessed for each parasite using scanning cluster and multiple regression analysis. Overall seroprevalence for O. ostertagi was 95.5% (95% C.I.: 94.0-96.6), with a mean optical density ratio (ODR) of 0.83, for F. hepatica 41.3% (95% C.I.: 38.3-44.4), and for D. viviparus 2.9% (95% C.I.: 1.6-4.7). There were no significant differences between the two sampling periods. For all parasites, significant geographic clusters of higher AB levels could be established. Furthermore, AB levels against all three parasites were positively correlated with each other, indicating either cross-reactions or co-infections. For O. ostertagi, herd size and percentage of pasture in the ration were positively correlated with AB levels. For F. hepatica, altitude above sea level (a.s.l.) positively, and milk production per cow and year was negatively correlated with AB levels. This work provides baseline data for further studies performing in-depth risk factor analysis and investigating management as well as targeted treatment options to control the parasites.

Dynamics of infections with gastrointestinal parasites and Dictyocaulus viviparus in dairy and beef cattle from Costa Rica.

A longitudinal survey was carried out to determine and describe the prevalence and intensity of gastrointestinal parasite infections and Dictyocaulus viviparus in a dairy and a beef cattle farm of two different ecological zones in Costa Rica. The influence of anthelmintic treatment, age and meteorological factors (rainfall, minimum and maximum temperatures) on gastrointestinal nematodes and D. viviparus counts was determined. Calves were subjected to monthly sampling of feces and blood between April 2002 and March 2003. Coprological techniques were used to detect gastrointestinal helminthes, protozoan and D. viviparus. Blood samples were analyzed for antibodies to D. viviparus by ELISA. The most prevalent gastrointestinal parasites detected on both farms (dairy cattle, A; beef cattle, B) were Eimeria spp. (94.7%, 93.7%), Strongylidae (75.0%, 81.4%), Buxtonella sulcata (38.0%, 21.6%) and Strongyloides papillosus (29.8%, 31.7%), whereas Moniezia benedeni (4.8%, 9.1%), Trichuris spp. (7.3%, 13.2%), Toxocara vitulorum (0.0%, 1.8%) and Entamoeba bovis (2.5%, 1.1%) were less prevalent. Mean fecal egg counts (FEC) showed highest values of Strongylidae in April, May and July (>335.3 eggs/g feces) on farm A, and April, May and August (>304.3 eggs/g feces) on farm B. S. papillosus presented low FEC throughout the year on farm A, on farm B the highest values were obtained in April (303.0 eggs/g feces). Trichuris spp. presented maximum FEC values in May (328.6 eggs/g feces) on farm A and in June (157.5 eggs/g feces) on farm B. Treatment and age had significant influence on infection intensity of Strongylidae (farms A and B), S. papillosus (farms A and B) and Trichuris spp. (farm A). Rainfall had significant effect on S. papillosus (farms A and B) and Trichuris spp. (farm B). Maximum temperature showed significant effect on S. papillosus (farm A) and Trichuris spp. (farms A and B). Minimum temperature had significant influence on Strongylidae (farm A), S. papillosus (farms A and B) and Trichuris spp. (farm B). Haemonchus spp. (57%, 66%) and Cooperia spp. (30.0%, 30.7%) were the most prevalent genera identified by coproculture on both farms, in contrast, Trichostrongylus spp. and Oesophagostomum spp. were less frequent. Patent lungworm infections were low on both farms (10.8%, 1.8%). On farm A, high prevalence of antibodies against D. viviparus was determined only at the beginning of the study, in contrast, on farm B the seroprevalence fluctuated throughout the year. Treatment, age and maximum temperature had significant effect on D. viviparus counts on farm A, but not on farm B.

Interrelationships of Dictyocaulus spp. in Wild Ruminants with Morphological Description of Dictyocaulus cervi n. sp. (Nematoda: Trichostrongyloidea) from Red Deer, Cervus elaphus.

Lungworms from the genus Dictyocaulus cause parasitic bronchitis (dictyocaulosis) characterized by coughing and severe lung pathology in both domestic and wild ruminants. In this study we investigated the interrelationships of Dictyocaulus spp. from European bison (Bison bonasus L.), roe deer (Capreolus capreolus), and red deer (Cervus elaphus) by nucleotide sequence analysis spanning the 18S RNA gene (small subunit [SSU]) and internal transcribed spacer 2 (ITS2) regions of the ribosomal gene array as well as the mitochondrial cytochrome c oxidase subunit 1 (cox1). Molecular analyses of sequence data obtained partly with novel primers from between 10 and 50 specimens from each host were carried out. Bayesian inference analysis revealed that each host species was infected with different genotypes. Analysis of cox1 sequence data showed a diverse genetic background and high evolutionary potential of Dictyocaulus taxa. Data from lungworms of European bison revealed a distinct genotype of Dictyocaulus viviparus, whereas Dictyocaulus capreolus was only found in roe deer. In contrast, red deer were infected with a taxon with unique SSU, ITS2, and cox1 sequences. These results indicate the occurrence of a novel genotype from red deer, which differs significantly from the National Center for Biotechnology Information reference sequence of Dictyocaulus eckerti. The molecular evidence was consistent with a morphological study with description and imaging of Dictyocaulus cervi n. sp. recovered from red deer. Dictyocaulus cervi n. sp. can be distinguished from D. eckerti on the basis of the absence of cervical papillae, the occurrence of a single ring of 4 symmetrical submedian cephalic papillae, length of the tail in females, morphometry of the female reproductive system, and measurements of gubernacula in males. In conclusion, our findings further strengthen the idea that the genetic complexity and diversity among Dictyocaulus lungworms infecting wildlife ruminants is larger than previously believed and warrants further investigation.

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 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.

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 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.

The spermatogenesis of Dictyocaulus filaria (Nematoda, Trichostrongyloidea).

A cytological study was carried out, using male Dictyocaulus filaria, that revealed the diploid number of chromosomes was 2n = 11 and the sex determining mechanism was XO. The behaviour of the chromosomes in the different stages of meiosis was also investigated. Cross, open ring and rod bivalents were observed in diakinesis. The chromosomes appeared to be acrocentric since they acquired a radial disposition in Metaphase-II. The chiasma frequency was 1 and the nucleolus-organizing region was located at the ends of the chromosomes.

Viability and pattern of emergence of Dictyocaulus viviparus larvae in sporangia of the fungus Pilobolus kleinii.

Recoveries of third stage Dicytocaulus viviparus larvae (L3) from Pilobolus species sporangia ranged from 23 per cent at 21 days to 3 per cent after 90 days for sporangia attached to polythene discs positioned on pasture. There was a continuous release of L3 for up to 16 days from sporangia which were placed under conditions simulating those occurring on pasture.

Bile agar migration test: a novel method for testing the viability of Dictyocaulus viviparus larvae in lungworm vaccine.

A bile agar migration (BAM) test was used to determine the viability of Dictyocaulus viviparus larvae in lungworm vaccines. Percentage migration values for 10 batches of vaccine varied from 8.8 to 33.2 per cent compared with 99 per cent of unirradiated larvae tested under similar conditions. On the other hand a conventional viability count (CVC), based on subjective assessment of larval shape, gave figures of 95.1 to 98.7 per cent for the 10 vaccine batches. The BAM test may therefore have potential for providing a more discriminatory assessment of vaccine quality than CVC.

Pilobolus species and rapid translation of Dictyocaulus viviparus from cattle faeces.

In a series of five laboratory experiments observations were made on the role of Pilobolus in the translation of infective lungworm larvae from faeces of cattle. The results indicated that a substantial proportion of the lungworm larvae present in the faeces may be translated from the faeces by this fungus within eight days at a temperature of 15 degrees C. No clear relationship was observed between the numbers of Pilobolus and the translation of lungworm larvae. Further a longitudinal study on the occurrence of Pilobolus on faecal pats of grazing calves showed that between the beginning of July and the middle of September peak emergence of sporangia generally occurred within one week and most sporangia emerged within three weeks. From faecal pats which had been deposited at the end of September and the middle of October emergence of sporangia was lower and mainly occurred after two to four weeks.

Cell adherence to larvae of Dictyocaulus viviparus in vitro.

Bovine eosinophils survived for up to 48 hours in vitro in a medium of undiluted bovine serum and became adherent to the surface of Dictyocaulus viviparus larvae earlier than other cells found in peripheral blood. Cell adherence was associated with a heat labile factor in normal bovine serum and a heat stable factor in hyperimmune serum. A factor associated with leucocytes in vitro appeared to cause larval immobility. Cells from calves treated with levamisole behaved identically to those from other sources. It is suggested that eosinophils are an important element in host defence against D viviparus infection.

Effect of sub-lethal doses of albendazole on the fecundity of Dictyocaulus viviparus.

During an evaluation of the efficacy of prolonged low-dose albendazole therapy on nematode infections in calves, it was observed that a dose-rate of 2 mg kg-1 given daily for 10 days was highly effective at expelling Dictyocaulus viviparus, but smaller doses suppressed the faecal output of larvae without killing the lungworms. A more detailed study with four experimentally infected calves given 1 mg kg-1 day-1 for 10 days showed that faecal output of larvae ceased after six to eight days but resumed four to six days after the withdrawal of the drug. The albendazole-induced sterility was therefore reversible. A subsidiary study showed that the strain of parasite used was fully susceptible to albendazole.

Overwintering of larvae of the cattle lungworm (Dictyocaulus viviparus) on pasture in Kentucky.

Seventeen dairy calves, raised lungworm-free, were used in an experiment to determine whether lungworm (Dictyocaulus viviparus) larvae survived on a pasture in central Kentucky during the winter of 1979/1980. On Apr 22, 1980, the calves were placed on pasture. Three calves were put on pasture A, on which no cattle had grazed for over 20 years. Fourteen calves were put on pasture B, which had been vacant since Dec 12, 1979 (132 days before beginning of experiment), when several lungworm-infected calves had been removed from the pasture. Fecal samples from the 17 calves were examined for lungworm larvae at weekly intervals for about 6 weeks; lungworm larvae were not found in any of the 3 calves on pasture A, but were found in 12 of 14 (86%) calves on pasture B. One calf from pasture A and 3 calves from pasture B were euthanatized 37 days after being placed on pasture; lungworms were recovered from 1 calf from pasture B.

Cryopreservation of the infective larvae of the common nematodes of ruminants.

Exsheathed infective larvae (L 3) of 19 species of nematodes were tested for infectivity in either sheep or cattle after they had been frozen in 0,9% NaCl solution, stored for a relatively short time in the gas phase of liquid nitrogen and subsequently thawed. In addition, 13 of these species were tested after similar storage for up to 18 months. In sheep, Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum columbianum were viable after 2 years of cryopreservation, a mean of greater than 90% of the L 3 being alive when thawed after this period. Similar results were obtained with Chabertia ovina L 3 after 18 months and with Marshallagia marshalli, Trichostrongylus falculatus and Dictyocaulus filaria, after a short period of freezing. On the other hand, Gaigeria pachyscelis and Strongyloides papillosus survived freezing for up to 7 months but neither was viable at the end of this period, nor was exsheathed G. pachyscelis viable without freezing. Most of these infestations were established by inoculating the infective larvae into the abomasum and/or duodenum. M. marshalli, T. falculatus and C. ovina also proved infective after oral dosing. D. filaria, the only other species tested by this route, was not infective when dosed per os after thawing. The infective larvae of the bovine nematodes, Haemonchus placei, Ostertagia ostertagi, Nematodirus helvetianus, Oesophagostomum radiatum, Cooperia pectinata and Cooperia punctata survived freezing for a mean of 26 months, greater than 90% being alive on thawing, but infectivity was generally lower than with the same genera in sheep. Even when not frozen, exsheathed Bunostomum phlebotomum was non-infective. When Cooperia spp. after thawing were tested for infectivity by the oral route, more worms developed in one calf infested orally than in another infested by inoculation into the duodenum. Ova of H. contortus, M. marshalli, O. circumcincta, T. colubriformis, T. falculatus, N. spathiger, C. ovina, H. placei, O. ostertagi, Cooperia spp. and N. helvetianus were recovered from the faeces of animals infested with cryopreserved L 3. No ova of O. columbianum or O. radiatum were recovered from faeces, because differential larval counts were performed before they were patent. Nevertheless, gravid females were obtained post-mortem. Frozen L 3 of N. helvetianus were used to re-establish a pure strain in calves, 2,3 million ova being recovered from infestations with 10 670 L 3 frozen for 26 months. The infectivity of the progeny of frozen L 3 was tested with M. marshalli and C. ovina. In both instances infectivity was high and the worms which developed also produced ova, thus completing the cycle. This appears to be the first report of infective larvae of parasitic nematodes retaining their infectivity after being frozen in liquid nitrogen (gas phase) for longer than 2 years. This is also apparently the first time that M. marshalli T. colubriformis, T. falculatus, T. axei, N. spathiger, C...