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Liver flukes include Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis spp., Fascioloides magna, Gigantocotyle explanatum and Dicrocoelium spp. The two main species, F. hepatica and F. gigantica, are major parasites of livestock and infections result in huge economic losses. As with C. sinensis, Opisthorchis spp. and Dicrocoelium spp., they affect millions of people worldwide, causing severe health problems. Collectively, the group is referred to as the Food-Borne Trematodes and their true significance is now being more widely recognised. However, reports of resistance to triclabendazole (TCBZ), the most widely used anti-Fasciola drug, and to other current drugs are increasing. This is a worrying scenario. In this review, progress in understanding the mechanism(s) of resistance to TCBZ is discussed, focusing on tubulin mutations, altered drug uptake and changes in drug metabolism. There is much interest in the development of new drugs and drug combinations, the re-purposing of non-flukicidal drugs, and the development of new drug formulations and delivery systems; all this work will be reviewed. Sound farm management practices also need to be put in place, with effective treatment programmes, so that drugs can be used wisely and their efficacy conserved as much as is possible. This depends on reliable advice being given by veterinarians and other advisors. Accurate diagnosis and identification of drug-resistant fluke populations is central to effective control: to determine the actual extent of the problem and to determine how well or otherwise a treatment has worked; for research on establishing the mechanism of resistance (and identifying molecular markers of resistance); for informing treatment options; and for testing the efficacy of new drug candidates. Several diagnostic methods are available, but there are no recommended guidelines or standardised protocols in place and this is an issue that needs to be addressed., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cytochemical staining techniques were carried out en bloc with in vitro excysted and gut-penetrated Fasciola gigantica larvae in order to visualise the glycocalyx of the tegument, a structure which comprises the parasite component of the host-parasite interface, yet is incompletely preserved by conventional fixation and preparation techniques for electron microscopy. Positive reactivity with ruthenium red and periodic acid-thiocarbohydrazine-osmium (PATCO) techniques revealed that the glycocalyx is polyanionic and carbohydrate-rich throughout its depth. It comprises a trilaminate arrangement, with a thin dense zone and fibrillar layer closely apposed to the outer aspect of the apical plasma membrane, invested by an irregular thick mucopolysaccharide capsule. The latter, not recorded in adult flukes, may represent a specific adaptation to facilitate invasion in the face of host immunity, and may also protect the parasite surface from the action of host- and parasite-derived proteases. Early in the invasion of a naïve host, the glycocalyx may be partly responsible for triggering the responses of innate immunity, while later in infection, or when an anamnestic response is initiated in an immunocompetent host, the antibodies and activated lymphocytes of specific acquired immunity are invoked to interact with the parasite surface. The cytochemical properties of the glycocalyx, together with its potential for dynamic turnover due to exocytosis of the T0 tegumental secretory bodies, are likely to aid neutralisation of potentially damaging immune effectors and ensure their removal from the vicinity of the parasite by sloughing in complex with glycocalyx components., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 μM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 μM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 μM and those of the in vivo assays., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Using in vitro procedures to prepare newly excysted metacercariae and gut-penetrated juvenile Fasciola gigantica, the ultrastructural features of the tegumental syncytium and perikarya of these ephemeral stages in the host-invasion process were compared. The T0-type tegumental cells in newly excysted metacercariae are packed with stored T0 granules which, following transport to the surface membrane of the syncytium, discharge by exocytosis to maintain the glycocalyx. The T0 cells become depleted of T0 granules during the penetration process, shrink in size, and initiate autophagy in the cytoplasm to facilitate metamorphosis from a storage function to active biosynthesis. The novel products appear to include lysosomes which contribute to the autophagosomes, and T1 granules, necessary for maintenance of the glycocalyx and immunoprotection, as the invasion process continues into the host liver. Residual bodies, the end-products of autophagy, are eliminated from the apical membrane of the tegumental syncytium into the host-parasite interface. There they may present a transient source of parasite-derived molecules, including lysosomal cathepsin-type proteases, with potential for interaction with the host's immune system, and so might be exploited as targets for vaccinal and immunomodulatory studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A questionnaire to obtain information on tapeworm control practices was sent to 252 sheep farmers in Northern Ireland (NI) in 2012. Replies were received from 228 flock owners. Most farmers considered that tapeworm infections had less impact on productivity than gastrointestinal nematodes, flukes and ectoparasites. The majority of respondents (61.8%) did not treat for tapeworms. Of those that did, the average number of treatments given per year was 2.3, with some owners treating up to 6 times a year. The highest percentages of treatments were given over the period May-July. Benzimidazole compounds were the predominant class of drugs used (48.2%), followed by macrocyclic lactones (MLs) (31.2%). Levamisole, oxyclozanide, closantel and Monepantel were also used; together with MLs, their combined use accounted for 51.9% of all treatments given, and represents inappropriate product choice. Diagnostic data for tapeworm infections in NI over the period 2007-2014 was retrieved from the database held by the Veterinary Sciences Division at Stormont. Positive diagnoses remained low throughout this period: the highest recorded figure was 3.1%, in 2007. Despite there being little-to-no justification for treating sheep for M. expansa on the basis of any likely benefit to the health or production of the animals, many farmers in NI do treat for tapeworm and often with ineffective products. This is of concern, in that it could lead to the inadvertent development of anthelmintic resistance in nematode and trematode parasites., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The ultrastructure of the ovary of Fasciola hepatica collected from field-infected sheep, was compared with that of flukes from laboratory-infected rats harbouring the Oberon or the Cullompton fluke isolate. At the periphery of the ovarian tubules, in all flukes, interstitial tissue was identified that appears to provide physical support and facilitate the metabolism of the germinal-line cells. Oogonia undergo mitotic division to maintain the cell population and to produce oocytes. Early oocytes feature conspicuous synaptonemal complexes in the nucleoplasm, and these become less evident as the oocytes grow in size, move towards the core of the ovarian tubule, and synthesise osmiophilic bodies. The latter may represent cortical granules, and serve to block polyspermy. The identity of the synaptonemal complexes was confirmed by immunocytochemical labelling of synaptonemal proteins. The occurrence of synaptonemal complexes in the oocytes of all fluke types examined indicates that pairing of bivalent chromosomes, with the potential for genetic recombination and chiasmata formation, is a feature of the triploid aspermic parthenogenetic Cullompton flukes, as well as of the wild-type out-breeding field-derived and Oberon isolate flukes. In oocytes within shelled eggs in the proximal uterus of all flukes, condensed chromosomes align at meiotic metaphase plates. Following the reduction division, two equal pronuclei appear in each oocyte in the distal uterus. On the basis of these observations, a mechanism of facultative parthenogenesis for F. hepatica is proposed that accommodates the survival and clonal expansion of triploid aspermic isolates., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Reports of resistance to triclabendazole (TCBZ) among fluke populations have increased in recent years. Allied to this, there has been a rise in the prevalence of the disease, which has been linked to climate change. Results from questionnaire surveys conducted in Northern Ireland (NI) in 2005 (covering the years 1999-2004) and 2011 (covering the years 2008-2011) have provided an opportunity to examine the extent to which fluke control practices have changed over a prolonged time-frame, in light of these changes. A number of differences were highlighted. There was a significant shift away from the use of TCBZ over time, with it being replaced largely by closantel. The timing of treatments had moved earlier in the year, perhaps in response to climate change (and an altered pattern of disease). In relation to the frequency of drug treatments, there were no major changes in the overall pattern of drug treatments between the two survey points, although on both occasions approximately one-third of flock owners gave more than 3 treatments per year to ewes. In lowland areas in 2011, flock owners were rotating drug classes more often (each year and at each treatment) than in 2005, whereas in upland areas, flock owners were rotating less often and more were not rotating at all. Between 2005 and 2011, the percentage of flock owners giving quarantine treatments to bought-in stock had halved, to a very low level (approximately 10%). Using data from a complementary TCBZ resistance survey (Hanna et al., 2015), it has been shown that the way in which data are selected and which efficacy formula is applied can influence the calculation of drug efficiency and impact on diagnosis of resistance., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lambs infected with the Cullompton isolate of Fasciola hepatica were treated orally or subcutaneously with 10mg/kg of closantel at 16 weeks post-infection. Adult flukes were recovered from the liver of individual animals at 12h, 24h, or 36h post-treatment. The flukes were processed for histological analysis. In general, degenerative changes in the reproductive and somatic tissues were progressive, and were most marked in flukes exposed to closantel in vivo for 36h. However, flukes from a 12h subcutaneously-treated lamb showed marked deterioration of the testis, possibly because a portion of the dose has been delivered intravenously. Fewer intact eggs were seen in the uterus of flukes exposed to closantel for longer times (whether administered subcutaneously or orally to the host). The most conspicuous closantel-induced effect in flukes from treated hosts was progressive damage to the tegumental syncytium. While the flukes from 24h-treated hosts showed relatively minor damage to limited areas of the syncytium, towards the posterior end, the flukes from 36h-treated hosts (and flukes from the lamb that putatively received intravenous dosage) had lost large areas of the surface syncytium from the posterior end and dorsal surface, although the syncytium over the anterior end and the anterior ventral surface was largely spared. In areas where the syncytium had sloughed, the underlying structures such as the vitelline follicles, gut profiles and testis profiles, showed marked degeneration and breakdown. Other changes included cell depletion and early stage apoptosis in the testis, ovary and vitelline follicles. This study establishes a model for histological changes in closantel-sensitive F. hepatica exposed to closantel in vivo. Histopathological studies could be complementary to the efficacy controlled test for for closantel resistance in fluke populations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Chronic fasciolosis is often diagnosed by faecal egg counting (FEC), following concentration of the eggs in the sample by a zinc sulphate floatation method. However, concentration by a sedimentation technique gives improved sensitivity. Interpretation of FEC results for fasciolosis is complicated by factors such as the long pre-patent period and irregular egg shedding. Thus, FEC reduction tests (FECRT), when used alone, are not completely reliable for diagnosis of anthelmintic susceptibility or resistance in local fluke populations, especially when parasite burdens are small. A Fasciola hepatica coproantigen ELISA test has been introduced which more accurately reflects the presence of flukes in the host bile ducts in late pre-patent infections, and absence of flukes following successful chemotherapeutic intervention. The aim of the present study was to elucidate the specificity of the F. hepatica coproantigen ELISA technique, particularly regarding potential cross-reactivity with rumen fluke (paramphistome), gastrointestinal nematode and coccidian infections. The method involved parallel testing of a large battery of faecal samples from field-infected cattle and sheep using floatation and sedimentation FECs and coproantigen analysis. No evidence was found for significant false positivity in the F. hepatica coproantigen ELISA due to paramphistome, coccidian and/or gastrointestinal nematode co-infections. With sedimentation FECs less than 10 F. hepatica eggs per gram (epg), the likelihood of a positive coproantigen result for the sample progressively decreased. Diagnosis of fasciolosis should be based on consideration of both FEC and coproantigen ELISA findings, to ensure optimum sensitivity for pre-patent and low-level infections., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An in vivo study in the laboratory rat model has been carried out to monitor changes to the female reproductive system in adult Fasciola hepatica following treatment with the artemisinins, artemether and artesunate. Rats infected with the triclabendazole (TCBZ)-resistant Sligo isolate were dosed orally with artemether at a concentration of 200mg/kg and flukes recovered at 24, 48 and 72 h post-treatment (pt). Rats infected with the TCBZ-resistant Oberon isolate were dosed orally with artesunate at a concentration of 200mg/kg and flukes recovered 24, 48, 72 and 96 h pt. The flukes were processed for histological and transmission electron microscope (TEM) examination of the uterus, Mehlis' gland, ovary and vitellaria. After treatment with artemether, egg production had become abnormal by 72 h pt, with free vitelline cells and masses of shell protein material within the uterus; spermatozoa were absent. The Mehlis' gland and ovary retained a normal morphology over the 3-day period. A change in the cell population in the vitelline follicles was seen at 48 h pt, with a decline in the number of immature cells. This became more marked by 72 h and the follicles became progressively vacuolated over the 3-day period. At the TEM level, there were changes in the immature vitelline cells at 24h pt, as evidenced by a decrease in shell protein production and the presence of lipid droplets and abnormal mitochondria. Spaces in the follicles separated the cells from each other. The changes became progressively more severe with time, so that, by 72 h pt, the follicles were very disrupted, containing cells in the advanced stages of apoptotic breakdown. In extreme cases, the follicles were scarcely recognisable and had become filled with cellular debris. Fine structural changes to the vitelline cells induced by artesunate treatment were similar to those described for artemether, but generally occurred more quickly and were greater; this was particularly true of the swelling of the ger cisternae. Overall, the results have shown that artemisinin treatment has a severe impact on egg production by TCBZ-resistant flukes, an effect that is mediated by disruption of the vitelline cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In order to investigate the incidence and distribution of adult fluke resistance to the fasciolicide tricalbendazole (TCBZ) amongst populations of Fasciola hepatica in sheep flocks in Northern Ireland (NI), individual rectal faeces samples were collected from 3 groups of 20 sheep, before (pre-dose), and 21 days after (post-dose) treatment of the animals with TCBZ, nitroxynil or closantel, on each of 13 well-managed sheep farms distributed across the province. The efficacy of each flukicide was determined for each farm, using faecal egg count reduction (FECRT) and F. hepatica coproantigen ELISA testing. In certain flocks, 2 sheep with high pre-dose faecal egg counts (FEC) were killed 3 days and 21 days respectively after TCBZ treatment, and the histology of the fluke reproductive organs was compared with that of flukes from untreated sheep, and from sheep treated with nitroxynil or closantel 2 days prior to death, using haematoxylin and eosin (H&E) staining and an in situ hybridisation method (TdT-mediated dUDP nick end labelling [TUNEL]) to demonstrate apoptosis. Results from FECRT revealed that in all flocks with a high fluke burden, TCBZ was ineffective in treating chronic fasciolosis, and this finding was generally supported by the results of the coproantigen reduction test (CRT). The histology of reproductive organs of flukes from TCBZ-treated sheep in these flocks was normal, when compared with untreated flukes, and this, together with the FECRT and CRT findings, indicated a likely diagnosis of TCBZ resistance in all the flocks with a high fluke burden. In contrast, nitroxynil and closantel were found to be fully effective against TCBZ-resistant flukes in each of the flocks bearing a high chronic fluke burden. All of the flocks with a high fluke burden and TCBZ resistance were managed on lowland in the South and East of NI. Upland flocks, in the North and West, had low fluke burdens, or were clear of infection; and FECs were too low to allow valid resistance testing. The study highlights the high level of penetration of TCBZ resistance throughout F. hepatica populations in areas of intensively managed sheep production with a high level of fluke challenge. Further, it emphasises the importance of pre-emptive chemotherapeutic action against chronic fasciolosis, using flukicides effective against the egg-producing adult flukes to minimise pasture contamination for the next season's lamb crop. This study also exemplifies the use of several complementary methods (FECRT; CRT; fluke histology; comparative anthelmintic efficacy testing) for confirmation of a diagnosis of fluke drug resistance., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by the inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R-VPL]. In the first experiment, flukes were initially incubated for 2 h in R-VPL (100 μM), then incubated for a further 22 h in R-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg/ml, or 0.1327 μM). For controls, flukes were incubated for 24 h in R-VPL and TCBZ.SO on their own. In a second experiment, flukes were removed from the incubation media following cessation of movement. In the third experiment, Sligo flukes were incubated in lower concentrations of R-VPL (10 μM) and TCBZ.SO (15 μg/ml, or 0.0398 μM). Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R-VPL alone had minimal effect on either isolate. After treatment with TCBZ.SO alone, there was greater surface disruption to the Cullompton than Sligo isolate. However, combined treatment of R-VPL+TCBZ.SO led to more severe surface changes to the Sligo isolate than with TCBZ.SO on its own; this potentiation of drug activity was not seen with the Cullompton isolate. The phenomenon was evident at both concentrations of TCBZ.SO. Inclusion of R-VPL in the incubation medium also reduced the time taken for the flukes to become inactive; again, this effect was more distinct with the Sligo isolate. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The prevalence of anthelmintic resistance in Northern Ireland sheep flocks was evaluated between July and October 2011. Sampling kits were sent to 172 flock owners and returns were received from 91. Within this survey population, 27 flock owners used benzimidazole products, 10 used levamisole products, 15 used avermectin products, 26 used milbemycin products and 4 flock owners used the amino acetonitrile derivative, Monepantel. The remaining 9 flock owners used combination drenches (broad spectrum wormer plus fasciolicide). However, 15 sets of samples were ineligible for faecal egg count reduction testing due to either too low an egg count or insufficient faecal volume. Treatment efficacy below 95%, indicating significant resistance, was detected in 81% (n=24) of flocks tested for benzimidazole resistance; in 14% (n=1) of flocks tested for levamisole resistance; and in 50% (n=7) and 62% (n=13) of flocks tested for avermectin and milbemycin resistance, respectively. Monepantel resistance was absent in all (n=3) flocks tested. Combination products (broad spectrum nematocide plus flukicide) containing levamisole were entirely effective, while treatment efficacy below 95% was detected in 60% (n=3) of flocks where the nematocide in the combination product was a benzimidazole. Where parasite identification based on coproculture was completed, Trichostrongylus was the dominant genus detected in all cases post-treatment, indicating the occurrence of anthelmintic-resistant Trichostrongylus spp. populations. Benzimidazole efficacy was highest in treating Trichostrongylus spp. (51%) and lowest when treating Teladorsagia spp. Levamisole was 100% effective in treating Cooperia, but ineffective (0%) in treating Trichostrongylus spp. Avermectin efficacy was highest when treating Haemonchus contortus (100%) and Teladorsagia spp. (73%), with a marginally lower efficacy against Trichostrongylus spp. (71%). Moxidectin efficacy was 33% against Trichostrongylus spp., 68% against Teladorsagia spp., 97% against Cooperia spp. and 100% against Haemonchus contortus infections., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Reports of anthelmintic resistance to multiple drugs in individual parasite species, and in multiple parasite species across virtually all livestock hosts, are increasingly common. A working group of UK researchers and practitioners devised a set of guidelines in 2003 (Sustainable Control of Parasites in Sheep, 'SCOPS') aimed at maintaining anthelmintic efficacy on farms. Over the years that followed, these guidelines were promoted through meetings, promotional literature and the agricultural press. Results from questionnaires conducted in Northern Ireland (NI) in 2005 (covering 1999-2004) and 2011 (covering 2008-2011) have provided an opportunity to examine the extent to which these campaigns have influenced parasite control on sheep farms. The percentage of flocks at risk of under-dosing through inaccurate weight estimation in NI has increased by 15.9% since 2005. The number of flocks at risk of under-dosing through non-calibrated equipment has increased by 14.3% since 2005. The size of the in refugia population may have potentially doubled, as indicated by an increased compliance with the recommendation (wherever possible) to leave a portion of the flock untreated. However, whether this is indeed the case cannot be explicitly determined without a measure of the impact of various factors, including host immunity, environment/climate, previous anthelmintic treatment and the species of parasite present., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A questionnaire to obtain information on nematode control practices and sheep management was sent to over 1000 farmers in Northern Ireland. Replies were received from 305 flock owners, and data from 252 of them were analysed. Farms were divided into lowland and upland areas. Sizes of pasture and stocking rates on lowland and upland farms were 59.5 hectares, 6.99 sheep/hectare and 62.9 hectares and 10.01 sheep/hectare, respectively. Mean drenching rates for lambs and adults were 2.33 and 2.44, respectively, in lowland flocks and 2.73 and 2.71, respectively, in upland flocks. Between 2008 and 2011, the most frequently identified compounds in use were benzimidazoles and moxidectin in lowland flocks, and benzimidazoles and avermectins in upland flocks. Over the same period the most frequently identified commercial formulations were Tramazole(®), Panacur(®) and Allverm(®) (white drench), Levacide(®) (yellow drench), Oramec(®) (clear drench; avermectin), Cydectin(®) (clear drench; moxidectin) and Monepantel(®) (orange drench). Most respondents (56.35%) treated their lambs at weaning and the most common time to treat ewes was identified to be pre-mating (67.86% of respondents). The results of the questionnaire survey revealed that lowland annual drench frequency was 2.33 and 2.44 in lambs and ewes, respectively, although drench frequencies were higher in upland flocks: 2.73 and 2.71 for lambs and ewes, respectively. Annual drench rotation was practiced by 43.96% of flock owners, but whether this was true rotation or pseudo-rotation (i.e., substitution of one anthelmintic product by another product belonging to the same chemical group of anthelmintics) could not be explicitly determined., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1-4days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have determined the mitochondrial genotype of liver fluke present in Bison (Bison bonasus) from the herd maintained in the Bialowieza National Park in order to determine the origin of the infection. Our results demonstrated that the infrapopulations present in the bison were genetically diverse and were likely to have been derived from the population present in local cattle. From a consideration of the genetic structure of the liver fluke infrapopulations we conclude that the provision of hay at feeding stations may be implicated in the transmission of this parasite to the bison. This information may be of relevance to the successful management of the herd., (Copyright © 2012 Elsevier B.V. All rights reserved.)

While the influence of temperature and moisture on the free-living stages of gastrointestinal nematodes have been described in detail, and evidence for global climate change is mounting, there have been only a few attempts to relate altered incidence or seasonal patterns of disease to climate change. Studies of this type have been completed for England Scotland and Wales, but not for Northern Ireland (NI). Here we present an analysis of veterinary diagnostic data that relates three categories of gastrointestinal nematode infection in sheep to historical meteorological data for NI. The infections are: trichostrongylosis/teladorsagiosis (Teladorsagia/Trichostrongylus), strongyloidosis and nematodirosis. This study aims to provide a baseline for future climate change analyses and to provide basic information for the development of nematode control programmes. After identifying and evaluating possible sources of bias, climate change was found to be the most likely explanation for the observed patterns of change in parasite epidemiology, although other hypotheses could not be refuted. Seasonal rates of diagnosis showed a uniform year-round distribution for Teladorsagia and Trichostrongylus infections, suggesting consistent levels of larval survival throughout the year and extension of the traditionally expected seasonal transmission windows. Nematodirosis showed a higher level of autumn than Spring infection, suggesting that suitable conditions for egg and larval development occurred after the Spring infection period. Differences between regions within the Province were shown for strongyloidosis, with peaks of infection falling in the period September-November. For all three-infection categories (trichostrongylosis/teladorsagiosis, strongyloidosis and nematodirosis), significant differences in the rates of diagnosis, and in the seasonality of disease, were identified between regions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wild-type flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lambs infected with the Cullompton triclabendazole (TCBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3h, 24h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24h, demonstrating that this process is affected very rapidly pt., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10mg/kg live weight and ketoconazole at a dosage of 10mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ+inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica., (© 2011 Elsevier B.V. All rights reserved.)

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The liver fluke remains an economically significant parasite of livestock and is emerging as an important zoonotic infection of humans. The incidence of the disease has increased in the last few years, as a possible consequence of changes to the World's climate. Future predictions suggest that this trend is likely to continue. Allied to the changing pattern of disease, reports of resistance to triclabendazole (TCBZ) have appeared in the literature, although they do not all represent genuine cases of resistance. Nevertheless, any reports of resistance are a concern, because triclabendazole is the only drug that has high activity against the migratory and damaging juvenile stages of infection. How to deal with the twin problems (of increasing incidence and drug resistance) is the overall theme of the session on "Trematodes: Fasciola hepatica epidemiology and control" and of this review to introduce the session. Greater knowledge of fluke epidemiology and population genetics will highlight those regions where surveillance is most required and indicate how quickly resistant populations of fluke may arise. Models of disease risk are becoming increasingly sophisticated and precise, with more refined data analysis programmes and Geographic Information Systems (GIS) data. Recent improvements have been made in our understanding of the action of triclabendazole and the ways in which flukes have become resistant to it. While microtubules are the most likely target for drug action, tubulin mutations do not seem to be involved in the resistance mechanism. Rather, upregulation of drug uptake and metabolism processes appear to be more important and the data relating to them will be discussed. The information may help in the design of new treatment strategies or pinpoint potential molecular markers for monitoring fluke populations. Advances in the identification of novel targets for drugs and vaccines will be made by the various "-omics" technologies that are now being applied to Fasciola. A major area of concern in the current control of fasciolosis is the lack of reliable tests for the diagnosis of drug (TCBZ) resistance. This has led to inaccurate reports of resistance, which is hindering successful disease management, as farmers may be encouraged to switch to less effective drugs. Progress with the development of a number of new diagnostic tests will be reviewed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study, F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An in vivo study in the laboratory rat model was carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from combination treatment of triclabendazole (TCBZ) and the metabolic inhibitor, ketoconazole (KTZ). Rats were infected with the TCBZ-resistant Oberon isolate of F. hepatica and divided into 3 groups at 12 weeks post-infection. The first group was dosed orally with TCBZ at a dosage of 10mg/kg and KTZ at a dosage of 10mg/kg. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.). A second group of rats was treated with TCBZ alone (10mg/kg) and sacrificed at 96 h p.t. The third group acted as untreated controls. Surface changes were monitored by scanning electron microscopy (SEM). In flukes from the TCBZ+KTZ-treated group, the results showed a progressive and time-dependent increase in the level of disruption to the tegumental syncytium. Swelling, furrowing, blebbing and sloughing of the syncytium increased with time p.t. Another feature seen was a thick layer of tegumental shedding in some fluke samples at different times p.t. By comparison, flukes treated with TCBZ alone remained unaffected. The results demonstrated that the Oberon isolate is only sensitive to drug action in the presence of ketoconazole, indicating that combining triclabendazole with a metabolic inhibitor could be used to preserve the effectiveness of the drug against TCBZ-resistant populations of F. hepatica., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles, Mehlis' gland and ovary. Over the 4-day post-treatment period, there was a progressive reduction in the number of oogonia and oocytes in the ovary and evidence of apoptosis. Vacuolation and a decrease in the number of Mehlis' gland cells were observed from 48 h pt onwards and disruption of the normal role of the gland in egg formation was evident. The vitelline follicles showed a gradual decrease in size and became vacuolated; the population structure in each follicle changed to be one consisting mainly of mature cells and the production of shell protein material declined. The follicle became disorganised as the cells broke down and released their contents into the lumen of the follicle. While the uterus appeared to contain eggs at 48 h pt in whole-mount specimens, no properly-formed eggs were observed in histological sections. By 96 h pt, the uterus was completely devoid of eggs. Overall, egg production was seen to be severely affected by TCBZ treatment and flukes were incapable of producing normal eggs within 2 days of treatment. The implications of this in terms of the epidemiology of the disease are discussed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The objective of the present study was to analyse the reproductive viability (using histopathologic studies) of Fasciola hepatica from cattle artificially infected and treated subcutaneously with a new experimental formulation of triclabendazole (8mg/kg b.w.). The results of the efficacy controlled test, which only takes into account the presence of live adult flukes, indicated that, whilst in the control group (n=7) 533 live specimens were recovered, in the test groups (doses of 8 and 12mg/kg b.w.) only 195 and 47 adults were recovered, respectively. These numbers indicate efficacies of 69% and 95.6%, respectively. It was observed in that dose of 8mg/kg b.w. some specimens remained viable, but they were infertile, which severely compromises the biological cycle of the trematode. In the testis tubules of flukes treated with the low dose of TCBZ (8mg/kg), very few cells were present and the vitelline follicles were markedly reduced in size and each follicle contained very few cells. This would have direct implications for the pathogenesis of the parasitosis since the remaining parasites would produce little clinical-productive manifestations, would stimulate the immune response and would find it difficult to establish future re-infestations/re-infections. Consequently, these observations will also prompt a review of certain methodological and interpretative aspects related to efficacy tests, where the only discriminative factor is the reduction of the adult parasite load. On one hand, histopathological studies could be complementary to the efficacy controlled test for TCBZ or other BZD formulations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A sheep trial was performed to evaluate two diagnostic assays, a faecal egg count reduction test (FECRT) and a coproantigen reduction test (CRT), for the diagnosis of resistance of Fasciola hepatica to triclabendazole (TCBZ). The FECRT defines successful TCBZ treatment as a 95% or greater reduction in fluke faecal egg counts (FECs) at 14 days post-treatment (dpt). The CRT defines effective TCBZ treatment as faeces negative for Fasciola coproantigens at 14dpt, as measured by the commercial BIO K201 coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium). Forty-nine indoor-reared sheep were split into four trial groups and each sheep was infected with 200 metacercariae of 1 of 4 F. hepatica isolates, previously described as susceptible (Cullompton and Fairhurst) and resistant (Leon and Oberon) to TCBZ action, respectively. TCBZ treatment was administered at 12 weeks post-infection (wpi) to one sub-group in each infected sheep group, and these sheep were culled at 4 weeks post-treatment (wpt). Untreated sheep sub-groups, were culled at a parallel time-point, that is, at 16wpi. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period, sub-sampled and examined by a standardised egg sedimentation protocol and by the BIO K201 ELISA. Results supported the use of both the FECRT and the CRT for the diagnosis of resistance of F. hepatica to TCBZ. In addition, the study confirmed the TCBZ susceptibility of the Cullompton and Fairhurst F. hepatica isolates and the TCBZ resistance of the Oberon F. hepatica isolate. However, the Leon F. hepatica isolate was found to be susceptible, rather than resistant, to TCBZ action., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A survey of literature on experimental infections with the liver fluke, Fasciola hepatica published between 2005 and 2009 has revealed a general lack of information on where fluke material (i.e. metacercariae) was sourced from. Even less information was given on the drug status of the fluke isolate used, which is a particular concern for those studies that involved anthelmintics. In these two respects, information on the liver fluke lags far behind that for nematodes, where such information is given almost as a matter of course. Of additional concern is that, at times, information about the source and drug history of fluke isolates was incorrect. The overall aim of the review is to demonstrate why it is important to provide as much information as possible on what fluke material is being used. It also attempts to correct some of the errors in the literature and gather together what information is available about the provenance of those isolates that have been used in recent experimental studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Eight indoor-reared cross-bred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48 h, 72 h and 96 h post-treatment (pt). Two control sheep were euthanized alongside the 96 h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps liver and, if present, from the gall bladder and they were processed for transmission electron microscopy (TEM). Disruption to the ultrastructure of the tegument became increasingly severe over time pt. Flukes recovered at 48 h pt showed widespread blebbing of the apical plasma membrane and swelling of the mucopolysaccharide masses surrounding the basal infolds. There was evidence of reduced secretory activity in the tegumental cells and spacing between the cells. Sloughing of the tegumental syncytium was observed at 72 h pt. The subtegumental musculature, parenchyma and tegumental cells were severely disrupted. At 96 h pt, all of the flukes were totally devoid of tegument. Disruption to the subtegumental tissue and somatic musculature was severe, and was so extreme in some specimens that the tegumental cells were barely discernible. Disruption to the gastrodermis was also progressive, though not as severe as disruption to the tegument. There was a general decline of secretory activity with time pt. Autophagic activity was apparent from 48 h pt and became more widespread with increasing time, culminating in breakdown of the gastrodermal cell cytoplasm. The mitochondria were swollen and electron-lucent and the cisternae of the granular endoplasmic reticulum were dilated and fragmented from 72 h pt., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. At 12 weeks post-infection, sheep were dosed with 10mg/kg triclabendazole. Two sheep per time period were euthanized at 48h, 72h and 96h post-treatment. Two control sheep were euthanized alongside the 96h triclabendazole-treated sheep. Flukes were recovered from each of the sheeps' liver and, if present, from the gall bladder, and they were processed for scanning electron microscopy (SEM). Flukes recovered 48h post-treatment were active. Disruption to the tegument took the form of swelling, widespread blebbing and some loss of the tegument covering the spines. By 72h post-treatment, all flukes recovered were dead and a number were recovered from the gall bladder. Typically, the posterior end of the flukes was elongated and in this region the tegumental syncytium had sloughed away. Tegumental loss was more widespread on flukes recovered from the gall bladder. At 96h post-treatment, only one fluke was recovered from the liver and three from the gall bladder. All the flukes were dead and they were totally devoid of tegumental syncytium.

The binding of drugs to plasma proteins--especially serum albumin--is an important factor in controlling the availability and distribution of these drugs. In this study we have investigated the binding of two drugs commonly used to treat liver fluke infections, albendazole (ABZ) and triclabendazole (TCBZ), and their sulphoxide metabolites to bovine serum albumin (BSA). Both ABZ and TCBZ caused shifts in the mobility of BSA in native gel electrophoresis. No such changes were observed with the sulphoxides under identical conditions. The drugs, and their sulphoxides, caused quenching of the intrinsic tryptophan fluorescence of BSA, indicating association between the drugs and this protein. Quantification of this quenching suggested a 5-10-fold reduction in affinity of the sulphoxides compared to the parent compounds. These results are discussed in respect to previous work on the pharmacodynamics of these fasciolicides and will inform the design of novel anthelmintics., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15mg/kg). The lambs were euthanised 48, 72 and 96h after TCBZ treatment, or 24, 48 and 72h after Cpd alpha treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanized 24h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24h post-treatment onwards. The development of these lesions may be related to the known anti-tubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd alpha is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings.

The benzimidazole compounds albendazole (ABZ) and triclabendazole (TCBZ) are both effective against Fasciola hepatica, although ABZ is only effective against adult flukes. Additionally, ABZ is a broad-spectrum nematodicidal compound with well-known ovicidal activity. However, no data on the ovicidal effect of TCBZ against F. hepatica eggs are available. The work reported here evaluated the comparative ovicidal effect of ABZ, TCBZ and their sulphoxide metabolites on F. hepatica eggs recovered from bile of sheep artificially infected with either a TCBZ-susceptible (Cullompton) or a TCBZ-resistant (Sligo) isolate of F. hepatica. Additionally, the effects of different non-flukicidal methylcarbamate benzimidazole compounds on the hatching of F. hepatica eggs were evaluated. Eggs (500 eggs/mL, n=4) were incubated for 12h either with TCBZ, TCBZ sulphoxide (TCBZ.SO), ABZ (5, 10 and 20 nmol/mL) or without drug (untreated control) (Experiment 1). Additionally, the effect of TCBZ and TCBZ.SO (5 nmol/mL) on egg hatchability was examined after a long (15 days) drug exposure (Experiment 2). Furthermore, the ovicidal effect of ABZ and ABZ.SO at different concentrations (5, 1, 0.5, 0.1 and 0.05 nmol/mL) (Experiment 3), and the effect of fenbendazole (FBZ), oxfendazole (OFZ), mebendazole (MBZ), flubendazole (FLBZ) (5 nmol/mL) and reduced-FLBZ (R-FLBZ) (2 microg/mL) on fluke eggs, were evaluated after a 12-h exposure (Experiment 4). Egg hatch was assessed by direct microscopic observation after incubation at 25 degrees C for 15 days. TCBZ and TCBZ.SO did not affect egg hatch after a 12-h incubation. A similar result was obtained after a much longer drug exposure (15 days) (Experiment 1 and 2). However, a significant (P<0.05) inhibition of egg hatch was observed in ABZ- and ABZ.SO-incubated eggs (Experiments 1 and 3). Additionally, the non-flukicidal compounds (Experiment 4) affected egg hatchability, particularly FLBZ and R-FLBZ. In conclusion, ABZ and ABZ.SO had a clear inhibitory effect on egg development of F. hepatica. However, the most extensively used flukicidal compound, TCBZ, and its main sulphoxide metabolite, did not affect egg hatch, even in TCBZ-susceptible flukes.

Seventy indoor-reared sheep were divided into 10 groups to test the efficacy of the experimental fasciolicide, compound alpha (15mg/kg) against triclabendazole (TCBZ)-resistant and TCBZ-susceptible F. hepatica infections. Activity against the Sligo TCBZ-resistant isolate was tested at three time points post-infection (p.i.): 3 days, 4 weeks and 12 weeks (Groups 1-3, respectively). A parallel trial was carried out using TCBZ (10mg/kg) (Groups 5-7): this provided a direct comparison between the efficacies of the two drugs. Group 4 served as an untreated Sligo control. Groups 8 and 9 were setup to test the efficacy of TCBZ and compound alpha against 12-week-old and 4-week-old TCBZ-susceptible, Cullompton infections, respectively. Group 10 served as an untreated Cullompton control. Sheep were sacrificed at 16 weeks p.i. and efficacies were determined. All remaining flukes were collected and measured, before being processed for whole-mount staining to assess the condition of their reproductive structures (testis, vitellaria, ovary and uterus). A second study was carried out to test the activity of compound alpha (15mg/kg) against mature 12-week-old TCBZ-susceptible F. hepatica infections in sheep. Eighteen sheep were divided into two groups, A and B. Group A was treated and Group B served as an untreated control group. Efficacy was determined by reduction in faecal egg counts. The results showed that, whilst compound alpha was very active against adult TCBZ-susceptible flukes, producing a 100% reduction in faecal egg counts, it only caused a 62.5% reduction in fluke burden against juvenile flukes. Moreover, compound alpha was not effective against any stage of infection with TCBZ-resistant F. hepatica in sheep. Data from the trial also revealed biological differences between the two isolates. Thus, Sligo flukes were smaller in size and produced fewer eggs than the Cullompton flukes and their cysts were less infective to sheep. However, they reached the bile ducts more quickly and their eggs appeared in the faeces >2 weeks earlier.

A study has been carried out to determine the relative activity of triclabendazole (TCBZ) and its sulphoxide (TCBZSO) and sulphone (TCBZSO(2)) metabolites against the adult stage of the liver fluke, Fasciola hepatica. Flukes were incubated for 24h in vitro in 15mug/ml of each of the compounds and prepared for scanning and transmission electron microscopy. All three compounds induced changes to the surface morphology of the fluke, the changes comprising swelling and blebbing to a greater or lesser extent in different regions of the fluke. TCBZSO(2) was more disruptive anteriorly and TCBZSO posteriorly. Internal ultrastructural changes were evident following incubation with each of the compounds, with an order of severity TCBZSO(2)>TCBZSO>TCBZ. Swelling of the basal infolds and mitochondria were observed in the tegumental syncytium. In the tegumental cell bodies, there was a reduction in the number of secretory bodies, disruption of the Golgi complexes and swelling of the mitochondria. Severe flooding of the internal tissues was observed with TCBZSO(2) and, to a lesser extent, with TCBZSO and TCBZ. The results demonstrate that both TCBZ and TCBZSO(2) are capable of disrupting the fluke in vitro and are not the inactive compounds they were assumed to be previously. They may well contribute to drug action in vivo as well, indicating that drug action is due to the additive effects of several metabolites, rather than being due to a single active metabolite, namely, TCBZSO.

A total of 8 calves approximately 6 months old and 22 lambs of similar age were infected with metacercariae of Fasciola hepatica of various laboratory-maintained isolates including: Cullompton (sensitive to triclabendazole) and Sligo, Oberon and Leon (reported as resistant to triclabendazole). Ten to 16 weeks after infection, flukes were harvested from these experimental animals and the histology of the testis tissue was examined in a representative sample of flukes from each population. Adult wild-type flukes were also collected from 5 chronically infected cattle and 7 chronically infected sheep identified at post-mortem inspection. The testis tissue of these flukes was compared with that of the various laboratory-maintained isolates. Whilst the testes of the wild-type, Oberon and Leon flukes displayed all the usual cell types associated with spermatogenesis in Fasciola hepatica (spermatogonia, spermatocytes, spermatids and mature sperm), the Cullompton flukes from both cattle and sheep showed arrested spermatogenesis, with no stages later than primary spermatocytes represented in the testis profiles. The presence of numerous eosinophilic apoptotic bodies and nuclear fragments suggested that meiotic division was anomalous and incomplete. In contrast to the wild-type flukes, no mature spermatozoa were present in the testes or amongst the shelled eggs in the uterus. A high proportion of the eggs collected from these flukes hatched to release normal-appearing miracidia after an appropriate incubation period, as indeed was the case with all isolates examined and the wild-type flukes. It is concluded that the eggs of Cullompton flukes are capable of development without fertilization, i.e. are parthenogenetic. The implications of this for rapid evolution of resistant clones following an anthelmintic selection event are discussed. Amongst the Sligo flukes examined, two subtypes were recognised, namely, those flukes with all stages of spermatogenesis and mature spermatozoa present in the testes (type 1), and those flukes with all stages of spermatogenesis up to spermatids present, but no maturing spermatozoa in the testes (type 2). Each sheep infected with the Sligo isolate had both type 1 (approximately 60%) and type 2 (approximately 40%) flukes present in the population. Spermatozoa were found amongst the eggs in the uterus in 64% of flukes and this did not necessarily reflect the occurrence of spermatozoa in the testis profiles of particular flukes, suggesting that cross-fertilization had occurred. The apparent disruption of meiosis in the spermatocytes of the Cullompton flukes is consistent with reports that Cullompton flukes are triploid (3n=30), whereas the Sligo and wild-type flukes are diploid (2n=20). In the Sligo flukes the populations are apparently genetically heterogenous, with a proportion of the flukes unable to produce fully formed spermatozoa perhaps because of a failure in spermiogenesis involving elongation of the nucleus during morphogenesis.

Eight indoor-reared crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection with 10 mg/kg triclabendazole. Two treated sheep were euthanized at 48 h, 72 h and 96 h post-treatment with triclabendazole. Two control sheep were euthanized alongside the 48 h triclabendazole-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for scanning electron microscopy (SEM). Flukes were still active 48 h post-treatment and displayed limited morphological disruption. There was some blebbing and sloughing of the tegument around the oral sucker. In several of the specimens, an extra layer had been deposited on the fluke surface, giving it a flattened appearance. At 72 h post-treatment, only one fluke remained alive and the disruption varied in degree. In the majority of flukes, there was severe swelling of the tegument, accompanied by isolated areas of flattening along the lateral margins of the flukes and in the tail region. Limited areas of sloughing occurred in the tail region. In more seriously affected specimens, the syncytium had been stripped away to reveal the basal lamina and some deeper lesions were also observed. By 96 h post-treatment, all the flukes were dead and were grossly disrupted. They were totally devoid of tegument and deep lesions exposed the internal tissues of the fluke.

Eight indoor-reared, crossbred sheep with no pre-exposure to Fasciola hepatica were infected, by oral gavage, with 200 metacercarial cysts of the triclabendazole-susceptible, Cullompton isolate of F. hepatica. Anthelmintic dosing occurred at 4 weeks post-infection using 15mg/kg compound alpha. Two treated sheep per time period were euthanized at 24h, 48h and 72h post-treatment with compound alpha. The two sheep from the control group were euthanized alongside the 24h alpha-treated sheep. Juvenile flukes were recovered from each of the sheeps' liver and processed for examination by electron microscopy. The surface morphology of the flukes' tegument was assessed using scanning electron microscopy (SEM). The ultrastructure of the tegumental syncytium and underlying tegumental cells and connections and somatic musculature were investigated using transmission electron microscopy (TEM). Both the SEM and TEM results revealed a level of disruption that increased with time, culminating at 72h with extensive tegumental loss and substantial degeneration of the cell bodies. The effects of compound alpha on the surface morphology were not particularly apparent until 48h post-treatment, when disruption included swelling and blebbing of the tegument. At 72h post-treatment, SEM revealed loss of the entire syncytial layer over large areas of the flukes. In the areas where the syncytium was lost and the basal lamina exposed, lesions of varying sizes had developed, revealing underlying tissues. Though minor forms of disruption to the ultrastructure of the syncytium were observed using TEM 24h post-treatment, it was at 48h post-treatment that substantial stress responses occurred. They included the presence of autophagic vacuoles and 'open' bodies at the apex of the syncytium and swelling of the basal infolds. The mitochondria within the syncytium and tegumental cells became progressively more disrupted over the three time periods and, by 72h post-treatment, they were frequently distorted and swollen in appearance, and contained severely swollen cristae. By 72h, the number of secretory bodies, particularly T1 bodies, had become significantly depleted in their respective cell bodies, cytoplasmic processes and in the tegumental syncytium. Both the circular and longitudinal muscle bundles were severely disrupted 72h post-treatment. They frequently contained a reduced number of muscle fibres and, in more severe instances, there was an absence of fibres altogether.

Triclabendazole (TCBZ) has been the drug of choice to treat liver fluke infections in livestock for >20 years, due to its high activity against both adult and juvenile flukes. More recently, it has been used successfully to treat human cases of fascioliasis. Resistance to TCBZ first appeared in the field in Australia in the mid-1990s. Since then, resistance has been reported from a number of countries throughout Europe: Ireland, Scotland, Wales, Spain and The Netherlands. The heavy reliance on a single drug puts treatment strategies for fascioliasis at risk. Should resistance develop further, the prospect is an alarming one. This review will present an overview of progress in understanding the mechanism of resistance to TCBZ, examining possible changes in the target molecule, in drug influx/efflux mechanisms and in the metabolism of TCBZ by the fluke. The review will also consider ways to deal with resistance, covering drug-oriented options such as: the use of alternative drugs, drug combinations and the search for new compounds.