Dicrocoelium Dentriticum in Explanted Liver: Report of an Unusual Finding.

Dicrocoelium dentriticum, a member of trematode type helminths, is a liver parasite of ruminants. Humans are infected accidentally by ingestion of intermediate host, through infected ants via eating of raw vegetables or drinking of contaminated water. Infection is often asymptomatic or results in subtle symptoms; therefore, infections are usually unrecognized. However, it can produce chronic cholangitis and swelling or adenomatous proliferation in the bile ducts and lead to abdominal pain, diarrhea, fatigue, jaundice, and other symptoms. We report a 49-year-old female patient with end-stage hepatic cirrhosis from viral hepatitis B and D coinfection who underwent liver transplant. Shortly after transplant, she developed symptoms suggesting an obstructed biliary duct. Liver needle biopsy was done 24 hours after transplant to rule out rejection. Biopsy of her explanted liver was also examined pathologically. Microscopic examination of the liver needle biopsy ruled out rejection. Prepared sections of explanted liver revealed a helminth in the common bile duct. Morphologic reconstruction of helminth by microscopic findings and consultation with an expert parasitologist supported the diagnosis of Dicrocoelium dentriticum.

Efficacy and safety of oral praziquantel against Dicrocoelium dendriticum in llamas.

Dicrocoelium dendriticum can cause severe pathological changes of the liver and bile system in camelids, and therapeutic options for treatment are limited. To address this problem, the efficacy of two different dose rates of praziquantel was investigated in llamas suffering from natural D. dendriticum infections. 53 llamas were examined under field conditions on two occasions: before and two weeks after treatment. At the beginning of the study, the animals were weighed, randomly allocated to one of the treatment groups (n=21 each) or the control group (n=11) and dosed orally using a praziquantel-containing paste (250 mg/ml) at a dose of either 25 mg (group 1) or 50 mg (group 2) per kg of body weight. Criteria for efficacy were faecal egg count reduction (FECR) and extensity effect. Animals treated with 25 mg/kg of body weight showed a FECR of 85%. Therapy with 50 mg/kg led to a FECR of 91%. Almost twice the number of animals of group 1 (33%) still shed eggs two weeks after treatment compared with group 2. The results of this study indicate that 50 mg/kg oral praziquantel is required for efficacious dosing and that this dose rate is safe in llamas and thus is recommended for the treatment of camelids naturally infected with D. dendriticum.

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.

In vitro oxidative metabolism of xenobiotics in the lancet fluke (Dicrocoelium dendriticum) and the effects of albendazole and albendazole sulphoxide ex vivo.

Dicrocoeliosis, a parasitic infection caused by Dicrocoelium dendriticum (lancet fluke), is often treated by the anthelmintic drug albendazole (ABZ). In the lancet fluke, ABZ metabolism via enzymatic sulphoxidation was found, but no information about ABZ oxidases has been available. The aim of our project was to find out which enzyme of the lancet fluke is responsible for ABZ sulphoxidation, as well as to assay the activities of oxidation enzymes. We also studied whether ex vivo 24-h exposures of flukes to ABZ or its sulphoxide (ABZ.SO) would alter ABZ sulphoxidation rate and the activities of tested enzymes. In subcellular fractions from flukes, marked activities of peroxidase (Px), glutathione Px (GPx), catalase (CAT), superoxide dismutase, and thioredoxin glutathione reductase were found. Using specific inhibitors, the participation of flavine monooxygenases in ABZ-oxidation was found. The ex vivo exposition of flukes to ABZ or ABZ.SO did not change the rate of ABZ sulphoxidation in vitro, but the ex vivo exposure of flukes to anthelmintics increased Px, CAT, and GPx activity. The modulation of these enzyme activities after ABZ or ABZ.SO exposition may be characteristic of the parasite’s protective mechanism against oxidative stress caused by drug treatment.

Liquid chromatography/mass spectrometric identification of benzimidazole anthelminthics metabolites formed ex vivo by Dicrocoelium dendriticum.

With further use of chemical agents in the control of parasitic infections, an increased number of drug resistance occurrences to antiparasitic drugs has been reported. Induction of enzymes responsible for detoxification of given drugs can contribute to drug resistance development in a parasitic organism. The identification of formed metabolites allows the characterization of the enzymes participating in biotransformation and possibly in drug resistance development. The objective of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole, flubendazole and mebendazole formed in ex vivo incubations by the parasitic helminth Dicrocoelium dendriticum, a parasite of ruminants and other grazing animals, using liquid chromatography/mass spectrometric (LC/MS) techniques. In the ex vivo study, approximately 50 living D. dendriticum adults were incubated in 5 mL RPMI-1640 medium in the presence of 10.0 micromol L(-1) benzimidazole drug (5% CO(2), 38 degrees C) for 24 h. The bodies of the parasite were then removed from the medium. After homogenization of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid-phase extraction. The extracts were analyzed using LC/MS with electrospray ionization. The results showed that D. dendriticum enzymatic systems are capable of phase I oxidation and reduction as well as phase II conjugation reactions. Detected phase I metabolites comprised albendazole sulfoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methyl derivatives of both reduced flubendazole and reduced mebendazole were observed.

Parasiticidal effects of peroxynitrite on ovine liver flukes in vitro.

Peroxynitrite (ONOO-) is a cytotoxic anion, produced by interaction between nitric oxide and superoxide in vivo in some inflammatory cells. This study investigated its effects on Fasciola hepatica and Dicrocoelium dendriticum isolated from ovine livers and kept in bile at room temperature. Peroxynitrite was synthesized using a quenched flow reactor and assayed spectrophotometrically. It was applied at different concentrations (10(-3.5) to 10(-2.3 M)) to the flukes kept in bile. The viability of the peroxynitrite-treated flukes was compared with a control group (n=5-7 per group). Control F. hepatica and D. dendriticum lived for 226+/-11and 208+/-14min, respectively. Life times were decreased by peroxynitrite at all concentrations used (P<0.001). At the highest concentration of peroxynitrite, F. hepatica and D. dendriticum lived only for 6.1+/-0.4 and 4.1+/-4.1+/-0.2min, respectively. Correlation between peroxynitrite concentration and parasite viability was significant in the case of F. hepatica (r= -0.842; P= 0.0035). A single application of peroxynitrite can decrease the life span of ovine liver flukes. A failure in the activation of hepatic macrophages in infected animals may lead to a decreased production of free radicals and, thus, peroxynitrite. Such a failure is likely to deprive the body of a defence tool against multicellular parasites.

Effect of treatment with netobimin on milk production of sheep.

In the Basque country lambing takes place during winter, followed by milking until late spring or summer, so it was considered that this would be the most profitable period for deworming, when there was an increased production pressure on ewes with depressed immune status owing to the peri-parturient relaxation of immunity. The drug employed was netobimin, and the trial was carried out in 22 commercial flocks, in each of which ewes were allocated to one of three similar groups. One group was left as a non-treated control (T0), the second was dosed 15 days before parturition (T1), and the remaining group dosed both at 15 days before and 15 days after lambing (T2). Mean total milk production in the T2 group increased by 8.8 and 6.3% in the second and third month post-partum, respectively, compared with that of the controls (T0), while over the standard lactation period of 120 days the T2 group showed a significant (8.9%) increase in production compared with the controls. The T1 group did not significantly differ from T0. The market value of the increase in milk production was calculated to be approximately 700% of the cost of treatment with netobimin.

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.

Light and electron microscopic studies on the effect of praziquantel on Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica (Trematoda) in vitro.

The fine structure of the tegument of three trematode species, Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica, was studied by means of light scanning (SEM) and transmission electron microscopy (TEM) after in vitro exposure to 0, 1, 10, and 100 micrograms/ml of the anthelmintic praziquantel for 5, 15, 30, and 60 min. In S. mansoni and D. dendriticum the resulting vacuolization of the tegument was confined to numerous small areas scattered all over the surface of the parasites and this finally led to the disruption of the apical tegumental layer. No changes were found in the tegument of F. hepatica after treatment with praziquantel.

[Effectiveness of cambendazole (Bonlam paste) in sheep invaded by tramatodes (Dicrocoelium dendriticum)]. / Efektívnost cambendazolu (Bonlam pasta) u oviec invadovaných trematódmi Dicrocoelium dentriticum.

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.