Inhibition of triclabendazole metabolism in vitro by ketoconazole increases disruption to the tegument of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP 450) enzyme pathway was inhibited using ketoconazole (KTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 system was inhibited by a 2-h pre-incubation in ketoconazole (40 μM), then incubated for a further 22 h in NCTC medium containing either KTZ, KTZ + nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM), KTZ + NADPH + TCBZ (15 μg/ml), or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO; 15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ–resistant isolate. However, co-incubation with KTZ + TCBZ, but more particularly KTZ + TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own: in the syncytium, for example, there was severe swelling of the basal infolds and their associated mucopolysaccharide masses, accompanied by an accumulation of secretory bodies just below the apex. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis, production, and transport of secretory bodies were badly disrupted. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.     

Potentiation of triclabendazole sulphoxide-induced tegumental disruption by methimazole in a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 μM), then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); MTZ+NADPH+TCBZ (15 μg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 μg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than triclabedazole-resistant isolate. However, co-incubation with MTZ+TCBZ, but more particularly MTZ+TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own, with severe swelling of the basal infolds and mucopolysaccharide masses in the syncytium, accompanied by a reduction in numbers of secretory bodies. The synthesis and production of secretory bodies in the tegumental cells was severely affected as well. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes, and this process may play a role in the development of drug resistance.     

Enhancement of the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica using the metabolic inhibitor ketoconazole

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered by using the metabolic inhibitor, ketoconazole (KTZ) to inhibit the cytochrome P450 (CYP 450) system within Fasciola hepatica. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 enzyme system was inhibited by a 2 h pre-incubation in KTZ (40 μM). Flukes were then incubated for a further 22 h in NCTC medium containing either KTZ; KTZ + nicotinamide adenine dinucleotide phosphate (NADPH; 1 nM); KTZ + NADPH + TCBZ (15 μg/ml); or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO;15 μg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible isolate than the TCBZ-resistant isolate. However, co-incubation with KTZ and TCBZ/TCBZ.SO led to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own, with greater swelling and blebbing of the tegument and even the loss of the apical plasma membrane in places. With the Cullompton isolate, there was limited potentiation of drug action in combination with KTZ, and only with TCBZ.SO. The results support the concept of altered drug metabolism within TCBZ-resistant isolates and indicate that this process may play a role in the development of drug resistance.     

Fasciola hepatica: disruption of the vitelline cells in vitro by the sulphoxide metabolite of triclabendazole

 The effects of the active sulphoxide metabolite of the fasciolicide triclabendazole (Fasinex, Ciba-Geigy) on the vitelline cells of Fasciola hepatica were determined in vitro by transmission electron microscopy using both intact flukes and tissue-slice material. At a triclabendazole concentration of 15 μg/ml the vitelline cells of intact flukes showed ultrastructural changes only after prolonged incubation periods (12–24 h). The changes observed were a swelling of the granular endoplasmic reticulum (GER) cisternae with decreased ribosomal covering in the intermediate-type cells and condensation of chromatin and disappearance of the nucleolus in the nucleus of the stem cell. Similar changes were evident more quickly (by 6 h) in whole flukes treated at the higher concentration of 50 μg/ml. The shell globule clusters were loosely packed in the intermediate type-2 cells, and the number of intermediate type-1 cells declined with more prolonged incubation. Disruption of the nurse-cell cytoplasm was also observed from 12 h onwards. After only 6 h incubation of tissue-slice material at 50 μg/ml, intermediate type-1 cells were absent, shell globule clusters in mature cells were loosely packed and the nurse-cell cytoplasm was badly disrupted. By 12 h the vitelline cells were vacuolated and grossly abnormal. The results are discussed in relation to postulated actions of triclabendazole against the microtubule component of the cytoskeleton and against protein synthesis in the fluke. Received: 15 August 1995 / Accepted: 24 October 1995     

Fasciola hepatica: disruption of spermatogenesis by the fasciolicide compound alpha

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the spermatogenic cells in the testis were examined by histology and transmission electron microscopy. Disruption to the testes became increasingly severe over time. The testis tubules shrank in size, became vacuolated, and contained fewer cells. Identification of cell types became difficult, and apoptotic eosinophilic bodies were the predominant feature at 72 h p.t. Changes to the spermatogonia were evident at 24 h p.t., the cells containing swollen and electron-lucent mitochondria. The proportion of tertiary spermatogonia increased at 48 h p.t., and they showed signs of autophagy. Multinucleate spermatogonia were a feature of drug treatment at this time point, and they contained autophagic vacuoles. By 72 h p.t., it was difficult to identify primary and secondary spermatogonia, and there were no recognisable clusters of tertiary spermatogonia. Most spermatogonial cells were multinucleate and in the process of breaking down. With regard to the primary spermatocytes, fragmentation of the cytophore was observed at 24 h p.t. Intact rosettes were rare after 48 h treatment; collections of cells were seen, but were not organised into clusters. By 72 h p.t., no spermatocyte cells could be recognised. The results indicate that spermatogenesis was severely affected by compound alpha.     

Efficacy of nitroxynil against Fasciola hepatica resistant to triclabendazole in a naturally infected sheep flock

The aim of this study was to compare the efficacy of triclabendazole (TCBZ) and nitroxynil against a TCBZ-resistant Fasciola hepatica strain in a naturally infected sheep flock. The efficacies were measured by the faecal egg count reduction test. The level of F. hepatica antigens was tested in faeces; and haematological indices such as total proteins (TP), albumin, hepatic enzymes and total IgG were also studied. The results confirmed the resistance of F. hepatica against TCBZ in the flock with an efficacy during the first month post-treatment between 59.4% and 73.8%. In the nitroxynil group, the efficacy during the same period ranged between 81.3% and 86%, likely because the efficacy of this drug against 7- to 9-week-old immature stages is only 50–90%. Anemia was showed in all groups and white blood cells were always higher than the reference range. The values of TP and albumin were within normal range in most of the sheep, and an increase in hepatic enzymes confirmed the liver damage. Regarding total IgG, some negative correlations were found with egg excretion, and in relation to the level of antigens in faeces, these ones decreased immediately after treatment. We conclude that nitroxynil could be an alternative in case of TCBZ resistance.     

Fasciola hepatica: morphological effects of a combination of triclabendazole and clorsulon against mature fluke

A study has been carried out to investigate the morphological effects of half-strength triclabendazole (TCBZ), half-strength clorsulon, and a combination of these two drugs against mature Fasciola hepatica. The Cullompton TCBZ-susceptible isolate was used for these experiments. Flukes were incubated for 24 h in vitro in TCBZ sulphoxide (7.5 μg/ml), clorsulon (5 μg/ml), or a combination of the two drugs. For the in vivo experiment, rats were dosed with TCBZ (6.25 mg/kg body weight), clorsulon (5 mg/kg body weight), or a combination of the two drugs and flukes recovered after 48 h. Surface changes to the flukes were assessed by scanning electron microscopy. Treatment with the combination of drugs produced greater disruption to the flukes than the individual drugs at half-strength, both in vivo and in vitro. Disruption to the tegument of the flukes induced by the individual drugs at half-strength was relatively minor and less than that caused by the drugs at full-strength. The results suggest that there are additive effects between TCBZ and clorsulon, which may be indicative of synergy: the use of drug combinations would be of value in the treatment of triclabendazole-resistant fluke.     

Fasciola hepatica: ultrastructural effects of a combination of triclabendazole and clorsulon against mature fluke

A study has been carried out to investigate the ultrastructural effects of triclabendazole (TCBZ) at half-normal concentration, clorsulon at half-normal concentration, and a combination of these two drugs against mature Fasciola hepatica. The Cullompton TCBZ-susceptible isolate was used for these experiments. Flukes were incubated for 24 h in vitro in TCBZ sulphoxide (7.5 μg/ml), clorsulon (5 μg/ml), or a combination of the two drugs. For the in vivo experiment, rats were dosed with TCBZ (5 mg/kg body weight), clorsulon (5 mg/kg body weight), or a combination of the two drugs, and flukes recovered after 48 h. Fine structural changes within the tegumental syncytium and tegumental cells were assessed by transmission electron microscopy. Treatment with the combination of drugs produced greater disruption to the flukes than the individual drugs at half-normal concentrations, both in vivo and in vitro; also than TCBZ.SO at normal concentration in vitro. The changes observed aid in the understanding of the gross changes to the tegumental surface described previously (Meaney M, Allister J, McKinstry B, McLaughlin K, Brennan GP, Forbes AB, Fairweather I. Parasitol Res 99:609–621, 2006). The results indicate that there are additive effects between TCBZ and clorsulon and suggest that the use of drug combinations would be of value in the treatment of TCBZ-resistant fluke.     

Fasciola hepatica: the effect of the sodium inophore monensin on the adult tegument

The effect on the tegument of adultFasciola hepatica of incubation in the sodium ionophore monensin, the Na⁺/K⁺-ATPase inhibitor ouabain and ouabain pretreatment followed by monensin has been determined in vitro by scanning and transmission electron microscopy (SEM, TEM). With monensin incubation alone (1×10^–6 M), a flattening of the tegument with some loss of spines on the ventral surface is evident from 0.5 h onwards. Internally, the subtegumental musculature becomes grossly swollen, although there is no swelling of the infoldings of the basal plasma membrane of the tegument, even after 24 h incubation. Ouabain incubation (1×10^–3 M) induces folding of the apical surface of the tegument from 0.5 h onwards, and this is accompanied by the formation of bleds and microvilli. Brief (0.5 h) exposure to ouabain (1×10^–3 M) followed by monensin treatment (1×10^–4 M, 3 h) leads to gross vacuolation of the tegument, but this is not due to swelling of the basal infoldings. The other main feature of ouabain-pretreated flukes is the projection of basal lamina-like material into the tegumental syncytium. Monensin treatment alone (1×10^–6 M) results in the Golgi complexes of the tegumental cells becoming very diffuse from 1.5 h onwards, and relatively few secretory bodies are present in the cytoplasm. After 0.5 h incubation in ouabain (1×10^–3 M), the Golgi complexes of the tegumental cells are indistinct, although numerous secretory bodies are still present. The classical monensin-induced swelling of the Golgi cisternae is observed in the tegumental cells only when monensin treatment (1×10^–4 M, 3 h) was preceded by brief (0.5 h) exposure to ouabain (1×10^–3 M). The results are discussed in relation to the postulated osmoregulatory role of the tegument and the role of sodium pumps in membrane function in the fluke.     

Fasciola hepatica: Tegumental surface changes in adult and juvenile flukes following treatment in vitro with the sulphoxide metabolite of triclabendazole (Fasinex)

The effects of the novel benzimidazole, triclabendazole (Fasinex, Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental surface ofFasciola hepatica has been examined in vitro. The tegument of adult flukes incubated in TCBZ-SX (50 g/ml) appeared swollen and blebbed after only 6 h. In addition, progressive spine loss at the oral cone was evident following 12 h treatment. After 24 h, the tegumental syncytium and spines had completely sloughed away, leaving an exposed basal lamina and empty spine sockets. Juvenile flukes (3 weeks old) also demonstrated tegumental alterations after treatment with TCBZ-SX (20 g/ml). The syncytium became extremely roughened and corrugated on both dorsal and ventral surfaces after only 3 h. Following 6- and 9-h incubations, there were many deep furrows, which were especially pronounced on the ventral surface, and by 18 h, the juvenile tegument was severely disrupted, especially on the ventral surface. In all cases, the effects were more marked than in the previous incubation periods. The results confirm the potent activity of triclabendazole againstF. hepatica and suggest that the tegument of adult and juvenile flukes may be a target organ for this important fasciolicide.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

In vivo and in vitro inhibition of hepatic microsomal drug metabolism by ketoconazole.

Ketoconazole (KC), a broad spectrum antifungal drug, has been recognized recently as a cause of hepatic injury. The mechanism of the adverse reaction remains unclear: a metabolic idiosincrasy has been suggested. However as a substituted imidazole, KC might be expected to interfere with the hepatic microsomal mixed function oxidases. Ethylmorphine N-demethylase (E-DM) and aniline hydroxylase (A-OH) activities were determined in rat liver microsomes in the presence of increasing amounts of KC. Both were inhibited in an exponential fashion. The E-DM inhibition was almost complete at concentrations greater than 250 microM and was of the mixed type. A much weaker effect was observed for A-OH. A significant inhibition of E-DM was also observed when KC was administered in vivo to rats either orally for 7 days at the dose of 100 mg/kg/day (P less than 0.02) or intraperitoneally for 4 days at the dose of 50 or 100 mg/kg day (P less than 0.01 or P less than 0.001 respectively). A-OH activity was significantly reduced (P less than 0.01) only after ip administration of 100 mg/kg/day of the drug for 4 days. Neither the amount of cytochrome P-450 nor NADPH cytochrome c reductase activity were affected at the doses considered. These data show that KC interferes with hepatic oxidative drug metabolism and suggest that this mechanism might be involved in the unwanted side effects of therapy with KC.     

The energy metabolism of Fasciola hepatica during its development in the final host

Mature liver flukes, Fasciola hepatica, of different ages were isolated from the bile ducts of experimentally infected rats. Their energy metabolism was studied during aerobic incubation with [6-14C]glucose. The results showed that the aerobic potentials of the parenchymal liver flukes are not lost immediately after arrival in the bile ducts, but in a later phase. During the development of the newly excysted juvenile into the mature adult the major part of ATP production in aerobic incubations is successively contributed by three different pathways of glucose breakdown. The Krebs cycle, which is by far the main energy-yielding pathway of the juvenile fluke, is gradually replaced by aerobic acetate formation and, finally, by the anaerobic dismutation reactions of the adult liver fluke. This observed decrease in Krebs-cycle activity per mg protein is not the result of a decrease in activity per individual fluke. The Krebs-cycle activity per fluke actually increases enormously during its whole development. This indicates that the aerobic potential of adult F. hepatica is not just a remnant of earlier aerobic stages but that classical, mammalian type mitochondria are produced during the entire development of the fluke. Calculations are presented which demonstrate that the Krebs-cycle activity of the developing F. hepatica is directly proportional to the surface area of the fluke. This supports our view that Krebs-cycle activity is limited by the diffusion of oxygen and can only occur in the outer layer of the liver fluke during its entire development in the final host.     

Human infection by Fasciola hepatica in Venezuela: report of a geriatric case

A new case of human infection due to Fasciola hepatica is reported in the Venezuelan parasitological literature. The patient is an 81-year-old female asymptomatic, with an eosinophilia of 21% and critrosedimentation rate of 26 mm/h and was found during a routine check up. These values were normal at the time of treatment (4 months later), as were several tests of hepatic function, blood chemistry and peripheral blood haematological values. The number of eggs of the parasite were between 90 and 130/g of facees. The patient was successfully treated with the human formulation of trielabendazole at 2 single doses of 10 mg/kg, each separated by 24 h. The same laboratory tests mentioned above did not show modifications, except for a slight increase in the cosinophil counts at 2 and 11 days and erythrosedimentation rate at 11 days post-treatment. Coproparasitological observations carried out at 12, 18, 60 and 120 days post-treatment were negative. No clinical symptoms were registered up to 2 months after treatment. The patient most probably acquired the infection at home through the ingestion of commercially available lettuce from an endemic area of bovine fascioliasis, very distant from home, and between 4 and 7 months previous to treatment. We stress the need to investigate F. hepatica infections which he been neglected, probably because of limited knowledge by health workers, lack of specific symptoms and absence of more sensitive diagnostic procedures.     

Fasciola hepatica: Disruption of spermatogenesis by the microfilament inhibitor cytochalasin B

The distribution of actin filaments in the spermatogenic cells ofFasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100 g/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.     

Fasciola hepatica : in vitro production of daughter rediae and cercariae from first- and second-generation rediae

Fasciola hepatica mother rediae belonging to the first generation and the first cohort of the second generation were extracted from their snail intermediate host (Lymnaea truncatula) and maintained in vitro for 16 days to determine the quantity of daughter rediae and cercariae produced by each generation. L-15 Leibovitz medium with a saline fraction adjusted to that of the L.?truncatula hemolymph was used for experiments. A total of 35 daughter rediae exited from the 21 mother rediae of the first and second generations, which were 14–32 days old at the onset of culture. Microscope examination of the mother rediae demonstrated the presence of numerous redial embryos and differentiating daughter rediae, whereas no cercaria was seen within their body. A total of 49 cercariae exited from the 17 mother rediae of the first and second generations, which were 29–50 days old at the onset of culture. No differentiating daughter redia was seen in these last mother rediae. The productivity of the first or second generation (first cohort) was nearly identical in terms of cercariae.     

Infection of Lymnaea cubensis by Fasciola hepatica in a high altitude region in Venezuela

Lymnaea cubensis is the vector in a region of distome infection in Trujillo State at more than 1000 m altitude where almost all bovines are infected. A quarter of the molluscs were infected with a mean number of 20 rediae per infected molluc each redia capable of producing 16 cercariae. Prevalence of infection increases with size of molluse and is highest in those 4 to 5 mm long. However, Lymnaea as small as 3 mm produce cercariae and molluscicide treatments should be begun as soon as forms of this size appear.     

Fasciola hepatica: an unusual case of adaptation to a Moroccan population of Galba truncatula

Parent snails, F1, and F2, originating from a Moroccan population of Galba truncatula, were experimentally subjected to sympatric miracidia of Fasciola hepatica at 20°C to determine the mode of adaptation of this parasite to the local intermediate host via the study of redial burdens and by following cercarial shedding. In spite of an increase in the global prevalences of infections, the frequencies recorded for immature or mature infections in dissected snails did not show any significant variations from parents to F2. If the redial burdens in mature infections were mainly composed of first-generation rediae in parent snails, the numbers of rediae belonging to subsequent generations significantly increased in the F1 and F2 generations. In mature infections, the lengths of first-generation rediae and their contents significantly decreased from parent snails to F2. Conversely, in the first redial cohort of the second generation (R2a rediae), only the intraredial contents increased significantly with snail generation. In mature infections, daughter redia production was ensured by all mother rediae of the first generation in parent snails, whereas it was progressively effected by the first mother redia in the F1 and F2 infections. The numbers of cercariae shed by snails increased significantly from parents to F2. If the cercariae produced by first-generation rediae decreased with snail generation, an inverse finding was noted in the case of R2a rediae. We suggest that snails impose a bottleneck on the development of the first mother redia, so that other mother rediae of the first generation react with greater growth (until 3.9 mm in parent snails), an important daughter redia production and a high cercarial production. As the first mother redia remained alive until day 49 post-exposure but without growth after week 2, this mode of development must be considered as a new developmental pattern of redial generations, and it is useful to determine its frequency in snails, as it has never been found in naturally or experimentally infected snails in central France.     

Fasciola hepatica: coprological diagnosis in comparison to the worm burden in sheep and cattle

Bulls artificially infected with a varying number of metacercariae of Fasciola hepatica were kept under uniform, standardized laboratory conditions. 6 months after the infection the daily range of variation and the distribution of the Fasciola eggs in the faeces were determined 3 times a day (morning, noon and afternoon) over two test periods, each of 5 days. This test, carried out in over 800 individual faeces samples, showed that the excretion of Fasciola eggs over a period of several days fluctuates considerably within one animal and within one infection group; the excretion of Fasciola eggs over one day varies widely at different times in each animal and also in each infection group; the distribution of Fasciola eggs in the faeces is always irregular within one day and also over several days in all animals. The variations demonstrated here must be taken into consideration when assessing the results of coproscopical diagnosis because studies of the faeces carried out on a single occasion can lead to completely incorrect conclusions being drawn.     

Fasciola hepatica: tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide

The effect of the deacetylated (amine) metabolite of diamphenethide (10 g/ml) on the tegumental surface of Fasciola hepatica over a 24 h period in vitro has been determined by scanning electron microscopy. Blebbing begins around the oral sucker after 3 h and then passes backwards along the body, reaching the ventral sucker and midbody by 6 h, and finally the posterior end of the body (by 12 h). Initially, the blebs are small, the tegument surrounding the spines is swollen and the tegument generally has a smooth, swollen appearance. This submerges the spines below the body surface. At higher magnification the surface is seen to bear microvillous-like projections in addition to the blebs and surface pitting is deeper than normal. Later on, the blebs increase in size and burst, causing lesions and loss of spines. Lesions begin to appear on the oral cone and ventral sucker after 6 h, in the midbody by 12 h and on the dorsal surface of the posterior region after 24 h. By this time the damage is extensive: around the oral and ventral suckers, and over large areas of the oral cone and midbody region the tegument has been stripped off to expose the basal lamina beneath. The dorsal surface of the fluke is consistently more severely affected than the ventral surface.