Potential role of hares in the spread of liver fluke in the Netherlands.

Hares (Lepus europeanus) sharing pasture with cattle from six locations in the Netherlands were examined for the presence of liver fluke (Fasciola hepatica) and shown to have prevalences of infection ranging from 0 to 41%. The mitochondrial haplotypes of liver flukes present in the hare populations were determined and compared with those found in cattle from a farm where triclabendazole resistance has been reported. Phylogenetic analysis indicated that the flukes present in the hares belonged to the same clades as those present in the cattle. A consideration of the life cycle of the liver fluke and the seasonal breeding pattern and ecology of hares supports the suggestion that hares may act as a refugia for liver fluke and as a vector for the spread of drug-resistant genotypes.

Anthelmintic effects of phytogenic feed additives in Ascaris suum inoculated pigs.

Two experiments were performed to determine the anthelmintic effect of some phytogenic feed additives on a mild infection of Ascaris suum in growing and finishing pigs. Usually, an infection of A. suum is controlled by using conventional synthetic drugs. Organic farmers, however, prefer a non-pharmaceutical approach to worm control. Therefore, phytotherapy could be an appropriate alternative. In the first experiment, a commercial available organic starter diet was supplemented with 3% of a herb mixture, adding 1% Thymus vulgaris, 1% Melissa officinalis and 1% Echinacea purpurea to the diet, or with 4% of a herb mixture, thereby adding the mentioned herbs plus 1% Camellia sinensis (black tea). A negative control group (no treatment) and a positive control group (treatment with conventional synthetic drug flubendazole) were included. In the second experiment, the anthelmintic properties against A. suum of three individual herbs, Carica papaya, Peumus boldus and Artemisia vulgaris, each in a dose of 1%, were tested. Pigs were infected with 1000 infective worm eggs each. Each experiment was performed with 32 individually housed growing pigs (8 replicates/treatment), which were monitored for 67 days. It was hypothesized that the herbs would block the cycles of the larvae, thereby preventing the development of adult worms. Therefore, phytogenic feed additives were not supplied during the whole experimental period, but only from the start until D39. Pigs were inoculated with infective worm eggs during five consecutive days (D17-D21). At D67 all pigs were dissected, whereafter livers were checked for the presence of white spots. Also numbers of worms in the small intestine were counted. In experiment 1, the numbers of worm-infected pigs were similar for both the herb supplemented (groups 3 and 4) and the unsupplemented (group 1) treatments (5-6 pigs of 8), while the treatment with flubendazole (group 2) resulted in 0 infected pigs. In experiment 2, herb addition (groups 2-4) did not significantly reduce the number of worm-infected pigs compared to the negative control (group 1). It can be concluded that the tested herb mixtures and individual herbs in the diets of growing and finishing pigs did not decrease the number of pigs which were infected with A. suum, although the herb mixture without black tea and also boldo leaf slightly (P<0.10) reduced the number of worms in the intestinal tract. The tested herb mixtures and individual herbs did not affect the performance of the pigs.

A comparison of the efficacy of two ivermectin formulations against larval and adult Ascaris suum and Oesophagostomum dentatum in experimentally infected pigs.

A study was conducted to evaluate and compare the efficacy of two injectable formulations of ivermectin (IVM-1 and IVM-2) at a dose rate of 0.3 mg/kg bodyweight versus placebo in the treatment and control of larval and adult stages of Ascaris suum and Oesophagostomum spp. in experimentally infected pigs. Seventy helminth free pigs were allocated on a liveweight basis to 7 groups each comprising 10 pigs (A-G). Group A served as an untreated control group. Groups B and C were used to investigate the efficacy of both formulations against adult stages of A. suum and Oesophagostomum spp., Groups D and E for efficacy against larval stages of A. suum and Groups F and G for efficacy against larval stages of Oesophagostomum spp. Pigs of groups A, B, C, D and E were infected on Day-0 with 1000 infective A. suum eggs each. Infective larvae of Oesophagostomum spp. (10,000/pig) were given on Day-0 to pigs of Groups F and G and on Day-21 to pigs of Groups A, B and C. Treatment was given to pigs of Group A (saline as placebo) on Day-7 and -28, IVM-1 to pigs of Group F on Day-7, pigs of Group D on Day-14 and pigs of Group B on Day-49. IVM-2 was given to pigs of Group G on Day-7, Group E on Day-28 and Group C on Day-49. Pigs of Groups F and G were sacrificed on Day-28, pigs of Groups A, D and E on Day-49 and pigs of Groups B and C on Day-56. Post mortem worm counts showed the following efficacies: (IVM-1) against larval A. suum 100%, against adult A. suum 94.4%, against larval Oesophagostomum spp. 52.0% and against adult Oesophagostomum spp. 83.0%. (IVM-2) against larval A. suum 100%, against adult A. suum 90.3%, against larval Oesophagostomum spp. 94.0% and against adult Oesophagostomum spp. 94.7%.

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease.

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.

An experimental study on triclabendazole resistance of Fasciola hepatica in sheep.

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted. The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.

Resistance of Fasciola hepatica against triclabendazole in cattle and sheep in The netherlands.

In the winter of 1998/1999, sheep on a farm in the province of North Holland, The Netherlands, died from subacute and chronic liver fluke disease despite four previous treatments with triclabendazole (TCBZ). Faecal examinations of sheep and cattle on the farm showed high number of liver fluke eggs. In a randomised clinical trial, the fluke egg output was monitored weekly for 3 weeks in sheep which were treated with TCBZ or with closantel; in dairy cows treated with TCBZ or with clorsulon; and in heifers treated with TCBZ or clorsulon. The results showed a significant reduction of 99.7, 98.1 and 99.2%, respectively, in fluke egg output at 21 days in all non-TCBZ treated animals. TCBZ treatment produced percentage decreases of 15.3, 4.3 and 36.6%, respectively. These results are highly indicative of the presence of TCBZ-resistant Fasciola hepatica in sheep and cattle on this farm.

Nematode parasites of adult dairy cattle in the Netherlands.

Abomasa, blood samples and faecal samples for examination of nematode infections were collected from 125 dairy cows during the period November 1997-October 1998. Of these, 12 had no grazing history and were, therefore, excluded from this study. From the remaining 113, 88.5% had nematode eggs in the faeces. Larval identification of the positive cultures showed that Ostertagia spp. larvae were most frequent (97%), followed by Trichostrongylus spp. (29%), Oesophagostomum spp. (23%), Cooperia punctata (20%), Cooperia oncophora (4%), Haemonchus contortus (2%) and Bunostomum phlebotomum (1%). The geometric mean EPG was 2.4. Two cows excreted larvae of Dictyocaulus viviparus (0.1 and 0.6 LPG resp.). Worms were found in the abomasa of 108 cows (96%). In all these abomasa Ostertagia spp. was present (100%). Trichostrongylus axei was found in 47 abomasa (43.5%) and two cows (2%) were infected with Capillaria bovis. The geometric mean of the total abomasal worm counts was 1743 and of Ostertagia spp. alone 1615. Almost all male worms were Ostertagia ostertagi, only occasionally Skrjabinagia lyrata10,000) total worm burden. Ostertagia specific antibodies were highest in late summer and autumn and lowest in spring and early summer. The same pattern, although not so pronounced, was observed for the serum pepsinogen values. No clear seasonal pattern was found for the Cooperia specific antibodies. Antibodies against D. viviparus were detected in seven cows (6%).

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.

A novel ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level.

We describe an ex vivo rat infection model to study protective immunity against Fasciola hepatica at the gut level. An exact number of newly excysted juveniles (NEJs) was injected into a gut segment with an intact blood supply and which was still attached to a live anaesthetized rat. NEJs that penetrated the gut wall during the following 6 h were recovered from a beaker filled with medium and were counted under a microscope. This infection model was validated and enabled us to exactly quantify the infection dose whilst at the same time exactly quantifying the number of NEJs penetrating the gut wall. The mean sum of NEJs that migrated through the gut wall into the beaker (peritoneal fraction), plus NEJs that remained in the gut wall and the gut lumen was 87% of the infective dose (+/-3.6% SEM; n=18). The function of the ex vivo segments was well-preserved, as demonstrated by only minor leakage of an inert liquid marker. The ex vivo model enabled us to measure protection against F. hepatica at the gut level. In naive rats 52% (+/-2.4% SEM; n=40) of the injected NEJs penetrated the gut wall, whereas in previously infected rats only 12% (+/-1.8% SEM; n=40) were able to do so, irrespective of the infection dose. Thus, when rats were orally primed, the migration of NEJs through the gut wall was 77% less than the migration in naive rats. We conclude that the ex vivo model should be valuable in studies of the induction and expression of protective immunity against F. hepatica in the intestine, and will aid in development and optimization of vaccines.

Protection of Fasciola hepatica in the gut mucosa of immune rats is associated with infiltrates of eosinophils, IgG1 and IgG2a antibodies around the parasites.

We investigated the immune effector mechanisms that underlie protection against F. hepatica in the gut wall of immune rats, using (immuno)histochemistry. In the lamina propria of immune Wistar rats, four weeks after oral infection, frequencies of IgE-positive cells, eosinophils and mucosal mast cells were significantly increased, compared with naïve rats. These factors represent the traditional effector mechanisms against helminths. No significant differences were detected between the two groups in frequencies of IgM-, IgG2a-, IgG1- and IgA- positive cells, CD4- and CD8-positive cells, NK cells, macrophages, neutrophils or goblet cells. Upon challenge of immune rats with F. hepatica in an ex vivo gut segment, NEJs that migrated through the (sub)mucosa were coated with IgG1 and IgG2a antibodies and surrounded by eosinophils. No IgE or IgA antibodies were detected on the parasites. The onset of these immune effector responses, two h after challenge, was related to the expression of protection. These results suggest that NEJs are killed by an eosinophil-mediated cytotoxic response involving IgG antibodies. These antibodies were not produced in the intestine, but infiltrated the gut upon challenge. The observed immune effector responses were not restricted to the site where the primary infection is located, namely the small intestine, but were also detected in the large intestine. The presence of the protective immune mechanisms in two other rat strains demonstrates the pivotal importance of these responses, irrespective the genetic background of the host.

Effect of three sustained-release devices on parasitic bronchitis in first year calves.

The effect of three intraruminal sustained-release devices (SRD) against Dictyocaulus viviparus infection was tested in five groups of six calves. Group 1 served as untreated controls, and groups 2, 3 and 4 were dosed with a levamisole SRD, a fenbendazole SRD, and an ivermectin SRD, respectively. Group 5 was vaccinated against lungworm and received a levamisole SRD. The calves were turned out on May 28 and the devices given seven days later. All the calves received trickle infections with a total of 200 lungworm larvae between 9 and 34 days after turnout. They were housed on October 28, challenged with 5000 lungworm larvae and slaughtered three weeks later. No clinical signs of parasitic bronchitis were observed during the study. The treated groups gained significantly more weight (P < 0.05) than the controls, but did not differ among themselves. Larvae were first detected in the faeces of the control group between 25 and 32 days after the first infection, and had a group mean of 21 larvae per gram (lpg) after 60 to 80 days, after which the lpg gradually decreased. In group 2, larvae were detected near the end of the grazing season and never exceeded a group mean of 1.5 lpg. In group 3, a very low larval output was observed after housing (group mean 0.1 lpg). Groups 4 and 5 never became patent. The results of an ELISA followed the pattern of larval output; optical densities above the cut-off value were recorded in groups 1, 2 and 3. On the basis of worm recoveries after challenge, group 1 was immune. Group 4 had significantly more lungworms than group 2. There were no significant differences in worm numbers between groups 2, 3 and 5, but the worms in group 5 were retarded in growth (P < 0.05).

Studies on the survival of Ascaris suum eggs under laboratory and simulated field conditions.

A series of four experiments was carried out to study the survival of Ascaris suum eggs: in a pig slurry unit on a farm, in the laboratory under anaerobic conditions and different relative humidities (rH), and under simulated field conditions. Survival of eggs in the pig slurry unit was 20% after four weeks and 0% after 16 weeks. Anaerobic conditions had only a minor influence on survival: after 12 weeks, more than 80% of the eggs could still develop. At high relative humidities (100% and 75%), survival was respectively 96% and 62% after 12 weeks. At 47.5% rH, survival dropped to 0% after 10 weeks and at 7.5% rH in 8 weeks. A. suum eggs kept in pig slurry under dry and sunny outdoor conditions survived for 2-4 weeks, whereas under wet and shady conditions, 90% of the eggs were still viable after 8 weeks.

A survey of anthelmintic resistance in nematodes of sheep in The Netherlands.

The prevalence of anthelmintic resistance in nematodes of sheep was surveyed in 1994 on 70 farms in the Netherlands. An in vitro egg hatch assay, faecal egg count reduction (FECR) 14 days after treatment, and larval cultures were used as methods of investigation. Oxfendazole was tested on 69, ivermectin on 51, and levamisole on 36 farms. The median effective dose (ED50) of thiabendazole could be determined on 64 farms. On 60 farms (94%) the ED50 value was > or = 0.12 microgram ml-1, which is indicative of the presence of benzimidazole (BZ) resistance. On two farms egg output was too low to do a FECR test. Based on the results of the FECR test, BZ resistance was present on 56 farms (84%), on 2 farms there was a suspicion of resistance and on 9 farms no resistance could be found. No clear indications were found for the presence of resistance against ivermectin or levamisole. BZ resistance was demonstrated in Haemonchus contortus, Cooperia curticei, Ostertagia spp. and/or Trichostrongylus spp. No resistance was observed in species from the genus Nematodirus, Chabertia ovina and/or Oesophagostomum spp.

Rhodoquinone is synthesized de novo by Fasciola hepatica.

Most adult parasitic helminths have an anaerobic energy metabolism in which fumarate is reduced to succinate by fumarate reductase. Rhodoquinone (RQ) is an essential component of the electron transport associated with this fumarate reduction, whereas ubiquinone (UQ) is used in the aerobic energy metabolism of parasites. Not known yet, however, is the RQ and UQ composition during the entire life cycle nor the origin of RQ in parasitic helminths. This report demonstrates the essential function of RQ in anaerobic energy metabolism during the entire life cycle of Fasciola hepatica, as the amount of RQ present reflected the importance of fumarate reduction in various stages. We also studied the origin of RQ, as earlier studies on the protozoan Euglena gracilis suggested that RQ is synthesized from UQ. Therefore, in parasitic helminths RQ might be synthesized by modification of UQ obtained from the host. However, we demonstrated that in F. hepatica adults RQ was not produced by modification of UQ obtained from the host but that RQ was synthesized de novo, as (i) the chain-length of the quinones of F. hepatica adults was not related to the chain length of the quinone of the host, (ii) despite many attempts we could never detect any in vitro conversion of UQ9 into RQ9 or into UQ10, neither by intact adult flukes nor by homogenates of F. hepatica adults and (iii) F. hepatica adults used mevalonate as precursor for the synthesis of RQ. We also showed that the rate of quinone synthesis in F. hepatica adults was comparable to that in the free-living nematode Caenorhabditis elegans. These results prompted the suggestion that RQ is synthesized via a pathway nearly identical to that of UQ biosynthesis: possibly only the last reaction differs.

Rhodoquinone and complex II of the electron transport chain in anaerobically functioning eukaryotes.

Many anaerobically functioning eukaryotes have an anaerobic energy metabolism in which fumarate is reduced to succinate. This reduction of fumarate is the opposite reaction to succinate oxidation catalyzed by succinate-ubiquinone oxidoreductase, complex II of the aerobic respiratory chain. Prokaryotes are known to contain two distinct enzyme complexes and distinct quinones, menaquinone and ubiquinone (Q), for the reduction of fumarate and the oxidation of succinate, respectively. Parasitic helminths are also known to contain two different quinones, Q and rhodoquinone (RQ). This report demonstrates that RQ was present in all examined eukaryotes that reduce fumarate during anoxia, not only in parasitic helminths, but also in freshwater snails, mussels, lugworms, and oysters. It was shown that the measured RQ/Q ratio correlated with the importance of fumarate reduction in vivo. This is the first demonstration of the role of RQ in eukaryotes, other than parasitic helminths. Furthermore, throughout the development of the liver fluke Fasciola hepatica, a strong correlation was found between the quinone composition and the type of metabolism: the amount of Q was correlated with the use of the aerobic respiratory chain, and the amount of RQ with the use of fumarate reduction. It can be concluded that RQ is an essential component for fumarate reduction in eukaryotes, in contrast to prokaryotes, which use menaquinone in this process. Analyses of enzyme kinetics, as well as the known differences in primary structures of prokaryotic and eukaryotic complexes that reduce fumarate, support the idea that fumarate-reducing eukaryotes possess an enzyme complex for the reduction of fumarate, structurally related to the succinate dehydrogenase-type complex II, but with the functional characteristics of the prokaryotic fumarate reductases.

[Anthelmintic resistance in sheep nematodes in The Netherlands]. / Anthelmintica-resistentie bij nematoden van het schaap in Nederland.

The prevalence of anthelmintic resistant nematodes in sheep was investigated in 1994 with a faecal egg count reduction test (FECRT) and an egg hatch assay (EHA) on 71 farms in the Netherlands. A benzimidazole (oxfendazole) was tested on 70 farms, ivermectin on 51 farms, and levamisole on 35 farms. Based on the FECRT benzimidazole resistance was present on 56 farms, 2 were suspected, on 9 no resistance was observed, while 3 farms could not be tested due to a too low egg output before treatment. No clear indications for resistance to ivermectin and levamisole were found. Benzimidazole resistance was demonstrated in Haemonchus contortus, Cooperia curticei, Ostertagia spp. and/or Trichostrongylus spp. No resistance was seen in species from the genus Nematodirus, Chabertia ovina and/or Oesophagostomum spp. De results of the EHA confirmed the results of the FECRT. On 59 farms the ED50 value was > 0.12 microgram ml-1 thiabendazole.

The reappearance of eggs in faeces of horses after treatment with ivermectin.

The reappearance of nematode eggs in faeces after ivermectin treatment was studied in 104 horses on 10 farms during the stabling period. Faecal samples were taken at weekly intervals. Sampling was discontinued when the mean egg output per farm was > 10% of the pre-treatment egg output. This point was reached after 63 days, when the mean output of eggs had decreased to 70.3%. Before treatment, 95.9% of the cultured larvae were of the cyathostome type, the others belonged to Gyalocephalus capitatus, Strongylus vulgaris, S. edentatus, Oesophagodontus/Poteriostomum spp., Triodontophorus spp. and Trichostrongylus axei. After treatment, the cultures nearly always produced 100% cyathostome larvae, although occasionally low numbers (< 1%) of larvae of large strongyles (mainly S. edentatus, rarely S. vulgaris and Oesophagodontus spp.) and T. axei were seen.

An epidemiological study of Fasciola hepatica in The Netherlands.

Transmission of F. hepatica under natural conditions was analysed in a three year programme. The variables used were the indirect haemagglutination (IHA) technique, worm establishment in tracer lambs and the population dynamics, infection rate and shedding pattern of Lymnaea truncatula. It is concluded that fluke eggs, infected snails and metacercariae on herbage can survive the winter in the Netherlands. Metacercarial availability was positively correlated to the amount of rainfall in the grazing period. The role developed eggs that survive the winter is important, because this results in earlier infections in the herd. The use of the serological diagnosis method IHA is important to detect F. hepatica infection in an early stage. Use of cellophane paper on floats is a useful method for determining the shedding pattern of cercariae from L. truncatula. It is concluded that collection of metacercariae on cellophane floats, inventarization of L. truncatula and its infection level are useful tools for the prediction of liverfluke infections.