Pharmacological and morphological characteristics of the muscular system of the giant liver fluke (Fascioloides magna - Bassi 1875).

Motility is required for feeding, reproduction and maintenance of the fluke in the host's liver. According to that, the neuromuscular system can be an attractive drugable target for chemotherapy. Musculature of the Fascioloides magna is organized into three layers, an outer circular layer, beneath this layer the longitudinal layer, and third, the oblique, or diagonal layer underlies the longitudinal layer. In our study, the administration of atropine or caffeine did not cause classic muscle contractions of F. magna muscle strips. However, the Electrical Field Stimulation (EFS) induced stable and repeatable contractions, which enabled us to examine their sensitivity to the various substances. Acetylcholine (ACh) (300 µM and 1 mM), caused only a slight relaxation, without affecting the amplitude of spontaneous contractions or the amplitude of contractions induced by EFS. Contrary to that, atropine (100 µM) caused a significant increase in the basal tone and an increase of EFS-induced contractions. If acetylcholine is an inhibitory neurotransmitter in trematodes, the described effects of atropine are achieved by the blockade of inhibitory neurotransmission. On the other hand, with respect to the process of excitation-contraction coupling, the plant alkaloid ryanodine (30 µM) significantly reduced the basal tone, as well as EFS-induced contractions of F. magna muscle strips. Ryanodine inhibited the potentiating effect of atropine on the basal tone and contractions caused by EFS, which indicates that the contractile effect of atropine is dependent on Ca(++) release from intracellular stores. Caffeine (500 µM) caused relaxation of fluke muscle strips and at the same time significantly enhanced the EFS-induced contractions. Both effects of caffeine can be explained by entry of extracellular Ca(++) into muscle cells. The muscle contractility of F. magna depends both on the entry of extracellular calcium, and calcium release from intracellular stores, which are under the control of RyRs. Our results also suggest that antitrematodal drugs could potentially be developed from substances with selective anti-cholinergic activity.

Effect of Lysimachia ramosa (Primulaceae) on helminth parasites: motility, mortality and scanning electron microscopic observations on surface topography.

The alcoholic extract of Lysimachia ramosa Wall (Primulaceae) was tested in vitro against helminth parasites, Fasciolopsis buski and Ascaris suum, from porcine hosts and Raillietina echinobothrida from domestic fowl. The live adult parasites, collected from a freshly autopsied host, were exposed to different concentrations (5-50mg) of the test plant extract in physiological phosphate-buffered saline (PBS) having 0.1% dimethyl sulphoxide (DMSO) at 37+/-1 degrees C. The treated parasites revealed complete inactivation and flaccid paralysis that was followed by death at varying periods of time. A dose-dependent loss of motility and mortality was observed in all the treated parasites. Scanning electron microscopic observations revealed conspicuous deformity of the surface architecture in all the parasites exposed to the test plant extract. The general tegument in F. buski showed shrinkage and loss of scale-like spines; proglottides all along the strobilar length in R. echinobothrida appeared shrunken and deformed and the cuticular surface of A. suum appeared disorganised, having lost transverse striations. The botanicals of the test plant seem to be effective against all the three types of helminth parasites.

Experimental infection of liver flukes, Fasciola hepatica and Fascioloides magna, in Bison (Bison bison).

This experimental study was conducted to evaluate the susceptibility of American bison (Bison bison) to liver flukes, Fascioloides magna and Fasciola hepatica. Six bison were each experimentally inoculated with 600 metacercariae of Fascioloides magna, and three were later treated with triclabendazole suspension at 40 mg/kg of body weight. Four additional bison were each experimentally inoculated with 600 metacercariae of Fasciola hepatica. Five control bison were placebo controls. Two controls and all inoculated bison were euthanized 10 mo (Fascioloides magna) and 7 mo (Fasciola hepatica) after inoculation. None of the control bison or the bison inoculated with Fascioloides magna had flukes or lesions characteristic of fluke infection at necropsy. All four bison inoculated with Fasciola hepatica had characteristic liver fluke lesions at necropsy, and three of four bison contained four, 103, and 111 adult flukes, respectively. Fluke eggs were detected in feces of all Fasciola hepatica-inoculated bison during the experiment, but not from the Fascioloides magna-infected bison or control bison. Clinical signs of infection were not observed during the experiment, but hemoglobin and packed cell volumes were lower in the Fasciola hepatica bison when compared to controls, and eosinophil levels were increased. Triclabendazole at 40 mg/kg of body weight appeared to be safe in bison because no toxic reactions were observed. Results from this study indicated bison are susceptible to infection with Fasciola hepatica and are efficient definitive hosts. Because no Fascioloides magna were recovered, bison may have a decreased susceptibility or innate resistance to Fascioloides magna infection, which may account for a lack of reported infections in this host.

Pharmacokinetic behavior in sheep and cattle of 5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1H-benzimidazole, a new fasciolicide agent.

The physicochemical properties, pK(a), Log P and solubility of compound alpha, (5-chloro-2-(methylthio)-6-(1-naphthyloxy)-1H-benzimidazole), a new fasciolicide agent, were characterized using conventional methods. Also, its pharmacokinetics was evaluated in sheep and cattle. In both species an oral dose of 12 mg/kg was administered. Blood samples were collected during 144 h and analyzed by using an HPLC assay. Results showed that compound alpha is a weak base with a pK(a) value of 2.87 and log P of 1.44. The solubility was very low in aqueous solvents. Pharmacokinetic studies showed that in both species compound alpha could not be detected at any sampling time. The mean half-life (t(1/2)) values of alpha sulphoxide in sheep and cattle were 19.86 and 29.87 h, while the half-life values of alpha sulphone were 19.43 and 46.32 h respectively. C(max) values of alpha sulphoxide did not differ between species while alpha sulphone values were higher in cattle. Plasma protein binding of alpha sulphoxide was between 82% and 86%. These results, combined with the previous efficacy studies, suggest that compound alpha could be a promising fasciolicide agent.

Ultrastructural observations on Fasciolopsis buski and its alterations caused by shoot extract of Alpinia nigra.

The ultrastructural alterations in the tegument of Fasciolopsis buski in response to incubation in the alcoholic extract of Alpinia nigra were evaluated using transmission electron microscopy. The body tegument of the trematode is composed of an external syncytial layer, musculature, and an inner layer containing tegumental cells. The syncytium comprises various organelles like mitochondria, lysosomes, and tegumentary bodies of the type 2 kind with rare sighting of the type 1. Severe distortion and disorganization of the tegument was revealed in the parasite exposed to the A. nigra extract in the current study. The extent of vacuolization was such that vacuoles proceeded down to the basal lamina causing the syncytium to separate from the tegument at different places. There was depletion of parenchyma material and loss of connecting tubules running down from the syncytium to the tegumental cells causing the cells to be deprived of any proper boundaries.

Anthelmintic efficacy of genistein, the active principle of Flemingia vestita (Fabaceae): alterations in the free amino acid pool and ammonia levels in the fluke, Fasciolopsis buski.

The crude root-peel extract of Flemingia vestita, its active principle genistein and the reference flukicide oxyclozanide were tested against Fasciolopsis buski, the giant intestinal trematode. The amino acid composition of F. buski was demonstrated using HPLC and it was observed that the free amino acid (FAA) pool of the control worm consisted of aspartate, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, lysine, histidine, arginine, phosphoserine, taurine, citrulline, ornithine, beta-alanine, and gamma-amino butyric acid (GABA). Of the amino acids detected valine was found to be the maximum in quantitative analysis. In qualitative analysis the FAA pool of the parasites under various treatments remained same as that of the control; however, quantitatively the level of various FAAs in the parasite was significantly affected. The treated parasites showed a marked decrease in the levels of arginine, ornithine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, serine, threonine, and taurine following treatment with 20 mg/ml of crude peel extract, 0.5 mg/ml of genistein and 20 mg/ml of the reference drug, though an increase in the levels of glutamic acid, glutamine, phosphoserine, citrulline and GABA was noticeable. Enhanced levels of GABA and citrulline under the influence of genistein may be implicated in alterations of nitric oxide release and consequent neurological change (e.g. paralysis) in the parasite. Ammonia in the tissue homogenate as well as in the incubation medium showed a quantitative increase compared to the controls after treatment with the various test materials. The ammonia level increased by 40.7%, 66.4% and 18.16% in treatments with F. vestita, genistein and oxyclozanide, respectively, at the mentioned dosages. The changes in the levels of the amino acids and nitrogen components post treatment suggest that the amino acid metabolism in the parasite may have been altered under the influence of the test materials.

Anthelmintic efficacy of Flemingia vestita: genistein-induced effect on the activity of nitric oxide synthase and nitric oxide in the trematode parasite, Fasciolopsis buski.

The root-tuber peel of Flemingia vestita, an indigenous leguminous plant of Meghalaya (Northeast India), has usage in local traditional medicine as curative against worm infections. The peel and its active component, genistein, have been shown to cause flaccid paralysis, deformity of tegumental architecture and alterations in the activity of several enzymes in platyhelminth parasites. To investigate further the mode of action and anthelmintic efficacy of the plant-derived components, the crude peel extract of F. vestita and genistein were tested, hitherto for the first time, in respect of the unique neuronal messenger nitric oxide (NO) and the enzyme nitric oxide synthase (NOS) in Fasciolopsis buski, the large intestinal fluke of swine and human host. NADPH-diaphorase histochemical staining (a selective marker for NOS in neuronal tissues), which was demonstrable in the neuronal cell bodies in the cerebral ganglia, the brain commissure, the main nerve cords and in the innervation of the pharynx, ventral sucker, terminal genitalia and genital parenchyma of the parasite, showed a stronger activity in the treated worms. In biochemical analysis also, the NOS activity showed a significant increase in the parasites treated with the test materials and reference drug, compared to the untreated controls. The increase in NOS activity in the treated parasites can be attributed to an inducing effect of the plant-derived components.

Effect of root-tuber extract of Flemingia vestita, a leguminous plant, on Artyfechinostomum sufrartyfex and Fasciolopsis buski: a scanning electron microscopy study.

The tegumental surface of Artyfechinostomum sufrartyfex as viewed under the scanning electron microscope revealed the presence of double rows of spines in the collar. The dorsal surface (6-8 rows) and the ventral surface are provided with posteriorly directed spines. The normal body surface of Fasciolopsis buski shows posteriorly directed scales throughout the ventral surface; the dorsal surface is free of any scales but has domed, coarsely distributed papillae. When treated in vitro with ethanol root-tuber extract of Flemingia vestita, an indigenous medicinal plant in Meghalaya, India, at a concentration of 5, 10, and 20 mg/ml phosphate-buffered saline (PBS), A. sufrartyfex became paralyzed within 1.1-1.4, 0.8-1.0, and 0.3-0.5 h, respectively. Following similar treatment, F. buski took 3.0-3.6, 1.5-2.0, and 0.6-0.8 h, respectively, to reach a paralytic state. Oxyclozanide B.P. was used as the reference drug and paralyzed the worm, taking slightly less time than the crude extract for both species of flukes. Stereoscanning observations on the tegumental surface of treated (20 mg extract/ml PBS) A. sufrartyfex revealed sloughing off of most of the spines or their deformation as well as wrinkles and rupture of the general tegument. Severe tegumental alterations and deformities were also displayed by F. buski exposed to 20 mg extract/ml PBS.

Efficacy of triclabendazole against natural infections of Fascioloides magna in wapiti.

Fourteen free-ranging adult wapiti (Cervus elaphus nelsoni) were captured in Banff National Park, Alberta (Canada) and held in captivity near Edmonton. A 24% suspension of triclabendazole at doses of 30 to 100 mg/kg body weight was drenched into the rumen of eight females and four males. Two male wapiti were used as untreated controls. Animals were killed and examined at 4 (n = 3), 6 (n = 4), or 8 (n = 4) wk after treatment. Efficacy was 90% against immature Fascioloides magna collected 4 wk after treatment and 98% against adult flukes collected 4, 6 or 8 wk after treatment. All 32 flukes recovered from control wapiti were active and apparently healthy. Treatment at 50 to 60 mg/kg is recommended against F. magna in wapiti. A protocol for treating infected wapiti is outlined.

Efficacy of triclabendazole against experimentally induced Fascioloides magna infections in sheep.

Efficacy of oral administration of 20 mg of triclabendazole/kg of body weight was evaluated against 12-week Fascioloides magna infections in 12 sheep, each inoculated orally with 250 viable metacercariae. From 6 sheep treated with triclabendazole, 1 immature F magna was recovered, whereas 116 F magna with a mean length of 19 +/- 6.5 mm were recovered from 6 untreated control sheep. Efficacy of triclabendazole was 99.14%. Signs of toxicosis or illness were not observed in the sheep.

Evaluation of clorsulon against immature Fascioloides magna in cattle and sheep.

Efficacy of clorsulon was evaluated against infection with immature Fascioloides magna in 24 cattle and 12 sheep. Infections were induced by oral administration of 600 metacercariae/host. In cattle, clorsulon at dosages of 7 and 21 mg/kg of body weight was 65 and 100% effective against 8-week-old flukes, and 20 and 74% effective against 16-week-old flukes, respectively. In sheep, clorsulon at a dosage of 21 mg/kg was 92% effective against 8-week-old flukes. Significantly (P less than 0.05) more F magna were recovered from untreated sheep than from untreated cattle.

Effects of praziquantel on human intestinal flukes (Fasciolopsis buski and Heterophyes heterophyes).

Human intestinal flukes (Fasciolopsis buski and Heterophyes heterophyes) were treated with praziquantel in vivo or in vitro and then studied by transmission (TEM) and scanning (SEM) electron microscopy. F. buski worms recovered from patients, which had been cured by oral treatment of 1 X 15, 1 X 25 or 1 X 40 mg praziquantel/kg body weight, showed typical contraction and alterations together with vacuolization and bleb formation of their tegument. H. heterophyes worms recovered from an experimentally infected dog were incubated in media containing 1 microgram praziquantel/ml for 5-60 min. Their tegument showed lesions the extent and duration of which increased with exposure time, finally leading to death. The damage observed in vitro was very similar to that found in F. buski recovered from cured patients. The cure rate was very high when using 1 X 15 mg praziquantel/kg.

Efficacy of albendazole against Fascioloides magna in sheep.

Sheep experimentally infected with Fascioloides magna were used in a controlled study to determine the flukicide activity of different doses of albendazole (methyl [5(propylthio)-1H-benzimidazol-2-yl] carbamate). Sheep, about 4 months old, were inoculated with 100 metacercariae of F magna and treated with the various doses of albendazole 10 weeks later. At 6 weeks after treatment, the sheep were necropsied, flukes were recovered and measured, and the pathologic changes were recorded. The sheep were given different doses of albendazole (5, 7.5 [single dose or double dose], 10, and 15 mg/kg) and were compared with both nontreated inoculated and nontreated noninoculated controls. There were up to 70% fewer worms recovered in the group given 2 doses of 7.5 mg/kg than there were in the nontreated controls. There was also a significant reduction in flukes recovered in the groups given the 7.5, 10, and 15 mg/kg doses. Because a single F magna fluke has the potential to kill a sheep, it is imperative to consider the number of sheep with no flukes after treatment. According to that criterion, 50% of the sheep were protected at dose rates of 7.5 (single dose, double dose), 10, and 15 mg/kg of body weight.

Field trial on the treatment of fasciolopsiasis with praziquantel.

Eight-five of 816 (10.7%) students attending a primary school in Central Thailand were examined and found infected with Fasciolopsis buski. All of students ate fresh water lily stems and most ate other fresh water plants including caltrop, water cress and morning glory. The 85 students were given praziquantel in randomized single doses of 15, 25 or 40 mg/kg body weight. Side effects were mild and transient and consisted of headache, dizziness, nausea, sleepiness, abdominal discomfort, anorexia, diarrhea, epigastric pain, vomiting and lassitude. Those receiving the highest dosages had more side effects than students in the other 2 groups. Large blisters were observed on the tegument of F. buski passed in feces and this was believed to be caused by the drug. The authors recommend a single dose of praziquantel in a dosage of 15 mg/kg of body weight for the treatment of parasitosis.

The effect of five fasciolicides on malate dehydrogenase activity and mortality of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum.

The effect of oxyclozanide, hexachlorophene, nitroxynil, rafoxanide and diamphenethide on malate dehydrogenase activity of homogenates of Fasciola gigantica, Fasciolopsis buski and Paramphistomum explanatum was investigated. The ratio of oxaloacetate reduction to malate oxidation in homogenates of Fasciola gigantica, Fasciolopsis buski and P. explanatum was 4.5:1, 3.6:1 and 5.2:1 respectively. Oxyclozanide and rafoxanide at 10(-3) M inhibited enzyme activity by 100% in homogenates from all three species while hexachlorophene at 10(-3) M also caused 100% inhibition in homogenates from Fasciola gagantica and P. explanatum but only 65% of malate oxidation in Fasciolopsis buski homogenates. Nitroxynil at 10(-3) M produced 60% inhibition in F. buski homogenates yet had little effect at this concentration on preparations from the other species. Little inhibition was seen with diamphenethide, even at high concentrations. Rapid death of Fasicola gigantica and P. explanatum resulted in vitro when 10(-3) M oxyclozanide, hexachlorophene, nitroxynil or rafoxanide, were added to the incubation medium. Fasciolopsis buski was killed by 10(-3) M oxyclozanide but at this concentration the remaining compounds only caused reduced activity. Assay of malate dehydrogenase following drug treatment in vitro failed to show any appreciable reduction in enzyme activity in Fasciola gigantica and P. explanatum but oxyclozanide and hexachlorophene produced inhibition in Fasciolopsis buski. The mode of action of these compounds is discussed.

Effects of six fasciolicides against Fascioloides magna In white-tailed deer.

Thirty-three white-tailed deer (Odocoileus virginianus) of various ages, both sexes, and in good physical condition were captured for anthelmintic evaluation of six compounds against the large American liver fluke, Fascioloides magna. Based on fluke mortality, hexachlorophene administered at the rate of 12 to 26 mg/kg of body weight was lethal to 5 of 10 mature flukes in seven deer. Nitroxynil at 11 to 24 mg/kg inhibited egg production, but did not kill mature flukes in eight deer. Rafoxanide at 12 to 25 mg/kg killed 6 of 8 (75%) immature flukes in eight deer, but was not effective against 17 mature flukes. Clioxanide at 16 to 38 mg/kg, diamphenethide at 255 to 280 mg/kg, and hexachloroethane at 463 to 629 mg/kg were not effective against F. magna in four, two and four deer, respectively. There was no indication that treatment with fasciolicides at the higher dose rates was more efficacious than at the lower dose rates. Detection of fluke eggs in the feces was a reliable method for diagnosing the presence of mature F. magna in deer prior to treatment, but was not reliable for measuring fasciolicidal activity of all compounds tested.