Pathophysiological studies on Dicrocoelium dendriticum infection in sheep

Red cell kinetics and plasma protein metabolism were investigated in two experiments using 20 adult sheep naturally infected with Dicrocoelium dendriticum, but free of other liver and gastrointestinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium burdens were injected with 51Cr-labelled red cells and 125I-labelled albumin, the results indicated that there were no significant differences in the turnover rate of labelled red cells or albumin between any of the groups. In the second experiment, two groups of sheep with low and high worm burdens were studied using the same radioisotope tracers; in addition, 59Fe-citrate was used to assess red cell iron incorporation rates in the two groups. Although the red cell half-lives of the infected sheep were just significantly longer, both were within normal limits and the difference was attributed to random variation within the two small groups of sheep. No significant differences were found in the other parameters. It was concluded that burdens of up to 4000 D. dendriticum do not cause significant blood or plasma protein loss in sheep.     

Hepatogenous photosensitisation in Scottish sheep casued by Dicrocoelium dendriticum

Dicrocoeliosis was identified as the probable predisposing cause of weight loss and hepatogenous photosensitisation affecting half of a group of 14month-old ewe lambs on a farm on the Inner Hebridean island of Coll. Oral dosing of the ewe lambs with 15mg/kg albendazole only achieved a 79.2% reduction in Dicrocoelium dendriticum egg count, 21day post treatment. Thus, this report of dicrocoeliosis differs from the disease that occurs elsewhere, both in the severity and nature of its clinical signs, and in the response of the parasite to anthelmintic drug treatment. These differences could indicate the existence of a genetically divergent D. dendriticum population, which may have evolved within the unique and isolated biotope afforded by the machair on the island of Coll. Better understanding of these factors is a prerequisite for effective and sustainable disease control.     

An evaluation of the efficacy of netobimin against Dicrocoelium dendriticum in sheep

A trial was carried out to assess the efficacy of a nitrophenylguanidine compound, netobimin against Dicrocoelium dendriticum in naturally infected sheep. At a dose rate of 20 mg/kg bodyweight administered orally the drug was highly effective, producing a mean reduction of 98.9 per cent in the fluke burdens of treated animals compared with untreated controls. No side effects were observed in the treated sheep.     

The inability of tapeworm Hymenolepis diminuta and fluke Dicrocoelium dendriticum to metabolize praziquantel

Biotransformation enzymes can, to a certain extent, protect parasitic worms against the toxic effects of anthelmintics and can contribute to drug-resistance development. The objective of our work was (1) to find and identify phase I and II metabolites of the anthelmintic praziquantel (PZQ) formed by the lancet fluke (Dicrocoelium dendriticum) and the rat tapeworm (Hymenolepis diminuta) and (2) to compare PZQ metabolites in helminths with PZQ biotransformation in rat as host species. Ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used for this purpose. During in vitro incubations, mitochondria-like and microsomes-like fractions (prepared from homogenates of adult worms or from rat liver homogenate) were incubated with 10 and 100 μM PZQ. Liquid/liquid extraction was used for samples during in vitro experiments. In the ex vivo study, living D. dendriticum and H. diminuta adults were incubated in RPMI-1640 medium in the presence of 50 nM or 100 nM PZQ for 24h. After incubation, the worms were removed from the medium and homogenized. Homogenates of worms, medium from the incubation of worms or rat hepatocytes and rat urine (collected during 24h after oral PZQ administration) were separately extracted using solid-phase extraction. The results showed that both D. dendriticum and H. diminuta enzymatic systems are not able to metabolize PZQ. On the other hand, thirty one different phase I and four phase II PZQ metabolites were detected in rat samples using UHPLC/MS/MS analyses. These results show that our experimental helminths, as the members of tapeworm and fluke groups of parasites, are not able to deactivate PZQ, and that the biotransformation enzymes of the studied helminths do not contribute to PZQ-resistance.     

The transport of albendazole and albendazole sulphoxide in the lancet fluke (Dicrocoelium dendriticum)

Albendazole (ABZ) is one of the most important benzimidazole compounds possessing high activity against the lancet fluke, Dicrocoelium dendriticum. ABZ sulphoxide (ABZ.SO) is the main molecule present in the bloodstream of an ABZ-treated host. The aim of this study was to characterise the pattern of ex vivo uptake of ABZ and ABZ.SO by lancet flukes and the export of both anthelmintics from these parasites. Transport of these anthelmintics in both living and dead flukes was compared. The adult flukes were collected from mouflons (Ovis musimon) which had been infected naturally. Results showed that more lipophilic ABZ was imported to a higher extent than ABZ.SO, and that significantly higher concentrations of ABZ were detected within living flukes as compared to dead ones. The same pattern was revealed in the study of ABZ and ABZ.SO export from the flukes' bodies. In addition to passive diffusion, active ABZ uptake and active efflux of ABZ and ABZ.SO in D. dendriticum could be assumed.     

Phase I biotransformation of albendazole in lancet fluke (Dicrocoelium dendriticum)

Dicroceliosis, a lancet fluke infection, is a frequent parasitosis of small ruminants and the anthelmintic drug albendazole (ABZ) is effective in control of this parasitosis. The aim of our project was to study the metabolism of ABZ and ABZ sulphoxide (ABZ.SO) in lancet fluke. Both invitro (subcellular fractions of fluke homogenates) and exvivo experiments (adult flukes cultivated in medium) were performed for this purpose. ABZ was metabolised invitro by lancet fluke NADPH-dependent enzymes by two oxidative steps (sulphoxidation and sulphonation). The apparent kinetic parameters of these reactions have been determined. In the exvivo experiments, only ABZ sulphoxidation was observed. The stereospecificity in ABZ sulphoxidation invitro was slight, with preferential formation of (+)-ABZ.SO enantiomer. In contrast (−)-ABZ.SO formation predominated in exvivo experiments. Sulphoreduction of ABZ.SO occurred neither invivo nor exvivo. The detection of ABZ oxidative metabolites indicates the presence of drug metabolising oxidases in lancet fluke.     

Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy)

An indirect enzyme-linked immunosorbent assay (ELISA) with Dicrocoelium dendriticum excretory/secretory antigens was used to evaluate the presence of serum antibodies against the trematode in 738 sheep randomly chosen in Sardinia (Italy). Coprological sedimentation was used to discover egg-output. Seropositivity was detected in 86.2% tested sheep, whereas faecal prevalence was 6.7%; all that were faecal-positive also were ELISA-positive.     

Isoenzyme patterns of phosphatases and esterases in Fasciola hepatica and Dicrocoelium dendriticum

A study was made of alkaline and acid phosphatase isoenzymes and non-specific esterases in homogenates of the trematodes Fasciola hepatica and Dicrocoelium dendriticum obtained from infected tissues of Capra hircus and Ovis aries using horizontal polyacrylamide gel electrophoresis.

The esterase patterns in F. hepatica and D. dendriticum were different. Homogenate of F. hepaticafrom both hosts gave four enzyme bands. For D. dendriticum, however, while homogenates of parasites from C. hircus also gave four bands, those of O. aries gave only three bands.

Acid and alkaline phosphatases in homogenates of both parasites showed three enzyme bands, but there was a host species difference betwen the enzyme patterns of specimens collected from C. hircus versus O. aries.     

甘肃临夏绵羊双腔吸虫的感染与鉴定

为调查甘肃省临夏回族聚集地中绵羊双腔吸虫的自然感染情况,有效地指导防治工作,选取了具有代表性的屠宰场进行调查。首先经过形态学观察,对疑似病原进行分离,再通过PCR方法及DNA测序来鉴定。形态鉴定结果表明,在350只绵羊中共发现8只绵羊感染了双腔吸虫,分子生物学鉴定结果表明,这8只绵羊感染的双腔吸虫均为矛形双腔吸虫。     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

Effectiveness of cambendazole (Bonlam paste) in sheep invaded by tramatodes (Dicrocoelium dendriticum)]

Tests were performed to examine the effectiveness of a new form of cambendazole (Bonlam paste, produced by MSD, USA) in sheep, naturally invaded by the trematodes Dicrocoelium dendriticum. Single administration of 25 mg per 1 kg live weight induced a rapid decrease in the number of the excreted eggs of D. dendriticum, as counted during ovoscopic examination seven, ten, and fifteen days after administration of the chemical. The intenseffectiveness of the preparation, determined post mortem, was 95.1%. Despite this, several dicrocoelia with normal motility and with no changes on the cuticle or internal organs were found in all the animals dissected. The animals tolerated the treatment with no signs of side-effects.     

Prevalence of Fasciola hepatica and Dicrocoelium dendriticum in the cow: inspection in an east Switzerland abattoir

On the basis of meat inspection in an abattoir in the eastern part of Switzerland, the incidence of liverflukes in cattle was recorded weekly during one year. The prevalence was compared with surveys from the seventies and nineties. The average incidence of Dicrocoelium dendriticum infection did not change. Although the prevalence of Fasciola hepatica apparently slightly decreased, an actual decrease of liverflukes could not be demonstrated.     

Relationship between egg output and parasitic burden in lambs experimentally infected with different doses of Dicrocoelium dendriticum (Digenea)

The relationship between egg elimination and parasitic burden was studied in two groups of 12 lambs experimentally infected with 1000 and 3000 Dicrocoelium dendriticum metacercariae, respectively. Half the animals in each group were slaughtered 2 months post-infection (p.i.) and the other half 6 months p.i. In order to detect and follow elimination of D. dendriticum eggs by the lambs, faeces samples collection started one and a half months p.i. and continued fortnightly until the end of the experiment. Egg elimination was first detected between days 49 and 79 p.i. (mean = 59 +/- 1.6 SE). Mean eggs per gram (epg) was higher in the lambs infected with 3000 metacercariae (347.2 +/- 42.4 epg) than in those infected with 1000 (194.8 +/- 14.4), although no significant differences were detected between both groups using the Student 't' test. Egg elimination was higher in the faeces samples taken in the afternoon (mean = 357.8 +/- 47.6 epg) than in those from the morning (mean = 215.7 +/- 21.3). The percentage of metacercariae which became established as worms was higher in the animals dosed with 1000 metacercariae (21.6%) than in those infected with 3000 (16.3%). The number of worms recovered on necropsy of each animal varied between 30 and 2063 (mean = 346.6 +/- 80.5) and their length between 2.6 and 7.1 mm (mean = 5.2 +/- 0.1). The mean number of parasites for lambs infected with 3000 metacercariae (489.3 +/- 163.1) was higher than that obtained from those dosed with 1000 (215.7 +/- 41.4), although more worms were collected in some cases from the lambs infected with the latter dose than the former. In general there was an increase in the number of epg eliminated as days p.i. and parasitic burden increased. A positive relationship was observed via the correlation coefficient between the number of epg eliminated by each of the lambs throughout the experiment and that of worms recovered. This relationship was more intense on considering only the number of epg eliminated between days 120 and 180 p.i.     

Assessment of climatic and orographic conditions on the infection by Calicophoron daubneyi and Dicrocoelium dendriticum in grazing beef cattle (NW Spain)

From February 2003 to March 2004, 1148 faecal samples from autochthonous Rubia Gallega cattle breed were collected in 170 farms from Lugo (NW Spain), in order to find out the prevalence and intensity of egg-excretion by Calicophoron daubneyi and Dicrocoelium dendriticum, and the possible influence of external factors as annual mean temperature and rainfall, altitude and mean slope. Twenty-six percent (95% CI 19%, 33%) and the 18% (12, 24) of the farms were infected with rumen and lancet flukes. C. daubneyi egg-output (Me=24) was found in 13% (11, 15) of the individual samples. Six percent (5, 7) of the animals in the study had low D. dendriticum eggs (Me=8). By estimating the odds ratio values it was shown that the highest probability of infection by C. daubneyi was in pastures with a mean slope of less than 13% (OR=1.9) and situated under 600 m (OR=1.6). Annual mean rainfall and temperature were not identified as risk factors for the infection with rumen flukes. Mountainous pastures with a mean slope of higher than 25% (OR=5.8) and situated over 600 m (OR=24.6) where precipitation was high (>1000 mm; OR=7) and temperature low (<11 degrees C; 2.8) had involved the highest risk of infection by D. dendriticum. Because of the prevalences found in this study, employment of suitable management practices with strategic treatments with efficacious anthelmintics are needed to reduce the presence of both trematodes and to increase the health status of grazing beef cattle.