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Application of 'omics' technology, and advances in in vitro methods for studying the growth of Fasciola hepatica, have highlighted the central role of migrating neoblasts in driving forward development and differentiation towards the adult-like form. Neoblast populations present molecular heterogeneity, morphological variation and changes associated with recruitment of these stem cells into their final tissue locations. However, terminal differentiation towards function, has received much less attention than has been the case for the free-living Platyhelminths. An actively replicating neoblast population, comprising cells with heterochromatic nuclei consistent with regulation of gene expression, has been identified in the parenchyma of juvenile Fasciola gigantica migrating in the liver of experimentally infected mice. In some of these cells, early cytoplasmic differentiation towards myocyte function was noted. Neoblasts have also been identified close to, and incorporated in, the subtegumental zone, the gastrodermis and the excretory ducts. In these locations, progressive morphological differentiation towards terminal function has been described. This includes the appearance of specific progenitors of type-1, type-2 and type-3 tegumental cells, the latter possibly contributing to tegumental spine development. 'Cryptic' surface molecular differentiation is postulated to account for recognition and 'docking' of migrating neoblasts with their final site for terminal differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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.)

An in vivo study in the laboratory rat model has been carried out to monitor changes to the tegument and gut of adult Fasciola hepatica following treatment with myrrh ('Mirazid'). Rats infected with the triclabendazole-resistant Dutch isolate were dosed orally with Mirazid at a concentration of 250 mg/kg and flukes recovered 2, 3 and 7 days post-treatment (pt). The flukes were processed for examination by scanning and transmission electron microscopy. A variety of changes to the external surface were observed, culminating in the sloughing of the tegumental syncytium. Internal changes to the syncytium and tegumental cell bodies were more severe and were evident from 2 days pt onwards. Swelling of the basal infolds (leading to flooding of the surface layer) and a decline in secretory body production were the major changes seen. The gastrodermal cells were less severely affected than the tegument, pointing to a trans-tegumental route of uptake for Mirazid by the fluke. Some loss of muscle fibres in the main somatic muscle layers was observed, which may be correlated with the decline in movement of flukes seen at recovery.

An in vivo study in the laboratory rat model has been carried out to monitor changes to the spermatogenic cells in the testis tubules of 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 200 mg/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 200 mg/kg and flukes recovered 24, 48, 72 and 96 h pt. The flukes were processed for histological and transmission electron microscope (TEM) examination. Changes to the spermatogenic cells were evident at 24 h pt with artemether. The spermatogonial and spermatocyte cells contained abnormal mitochondria, there were fewer spermatids and spermatozoa in the tubules than normal, and a number of cells showed signs of apoptosis. There was a further decline in cell numbers at 48 h pt and the organization of the spermatocyte and spermatid rosettes was atypical. Sperm formation had become abnormal and those spermatozoa present possessed only a single axoneme. By 72 h pt, the testis tubules were vacuolated and filled with abnormal cells and cell debris. Only spermatogonial cells could be identified and there was widespread evidence of apoptosis in the cells. Distinct cellular changes following artesunate treatment did not become apparent until 48 h pt. The changes seen were similar to those described for artemether, but were generally less severe at matching time-periods. The fine structural changes occurring in the spermatogenic cells were compared to those observed in other cell types and fluke tissues and the overall information was collated to identify the cellular targets for artemisinin action and to establish the time-line for drug action.

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.)

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 inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was 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 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. 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.

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.)

The liver flukes, Fasciola hepatica and Fasciola gigantica, are considered to be sister species and between them present a major threat worldwide to livestock production. In this study sequence data have been employed from informative regions of the nuclear and mitochondrial genomes of over 200 morphologically F. hepatica-like or F. gigantica-like flukes from Europe, sub-Saharan Africa and South Asia to assess genetic diversity. Evidence is presented for the existence of four well-separated clades: African gigantica-like flukes, Indian gigantica-like flukes, European hepatica-like flukes and African high-altitude hepatica-like flukes. Application of the Biological Species Concept to trematodes is problematic; however, the degree of separation between these groups was sufficient for them to be considered as distinct species using the four times rule for speciation., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

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.

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.)

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.

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 P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mum). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); PB+NADPH+TCBZ (15 microg/ml); or PB+NADPH+TCBZ.SO (15 microg/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 than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. 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.

Uptake of triclabendazole by the liver fluke, Fasciola hepatica has been studied by experiments designed to block either oral uptake of drug, by use of ligatures, or trans-tegumental diffusion, by allowing the drug to bind to an excess of bovine serum albumin (BSA) in the medium. Changes to the tegumental system, musculature and gut were assessed using transmission electron microscopy. Flukes were incubated in vitro for 24 h in TCBZ.SO (15 microg/ml). Disruption to the tegument and muscle was similar in ligatured and non-ligatured flukes, suggesting that closing the oral route did not affect drug uptake. The ultrastructure of the gastrodermal cells remained unchanged. Non-ligatured flukes were also incubated for 24 h in vitro in TCBZ.SO (15 microg/ml) in the presence of red blood cells (RBCs). Oral uptake of blood was demonstrated, but gut ultrastructure remained normal, whereas the tegument was severely disrupted. In separate experiments, ligatured and non-ligatured flukes were incubated in TCBZ.SO (15 microg/ml) in the presence of BSA (30 mg/ml) for 24 h in vitro. There was a marked decrease in the degree of tegumental disruption observed compared with TCBZ.SO action alone; again, the gut remained normal. The findings support previous morphological and pharmacological studies indicating that trans-tegumental uptake of triclabendazole predominates in the liver fluke.

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.

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks postinfection. Adult flukes were recovered from the bile ducts at 24, 48, and 72 h post-treatment (p.t.). Changes to the surface morphology of the flukes were assessed using scanning electron microscopy (SEM). Flukes were still active at 24 h p.t. and displayed limited areas of disruption, which were restricted to the oral cone region. At 48 h p.t., a reduced level of motility was observed in approximately 50% of the flukes recovered. Swelling of the tegument was more widespread and was accompanied by blebbing and partial loss of the tegumental covering of the spines. By 72 h p.t., the reduction in motility was greater, and approximately one quarter of the flukes recovered were inactive. In the majority of the flukes examined, the midbody region was marked by a discoloration of the flukes' tissues. This was seen to be due to the loss of the tegumental syncytium. Sloughing extended into the tail region in some specimens and, in the more badly-affected specimens, the basal lamina was breached to expose the underlying musculature. Elsewhere on the body, the tegument that remained was relatively normal, although areas of swelling and blebbing were present. Overall, the results provided information on the time-scale of changes to the surface morphology of the fluke that underpin the efficacy of compound alpha.

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.

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.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

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-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 is the drug of choice against Fasciola hepatica infections in humans and animals. However, parasite resistance against triclabendazole is spreading in the veterinary field, and there are no drugs of comparable activity currently available for the treatment and control of fascioliasis. We investigated the efficacy of single oral doses of artemether and OZ78 against adult triclabendazole-resistant F. hepatica harboured in rats, and compared the results with triclabendazole administered at two different doses. Single oral doses of 100 mg/kg OZ78 and 200 mg/kg artemether resulted in worm burden reductions of 100%. Whereas a single 10 mg/kg dose of triclabendazole achieved a worm burden reduction of only 4.0%, a five-fold higher dose yielded a significant worm burden reduction of 60.9%. However, the lower dose of triclabendazole administered to rats harbouring a triclabendazole-sensitive F. hepatica isolate resulted in a worm burden reduction of 95.3%. Our findings confirm that artemether and OZ78 possess good fasciocidal properties, even against a triclabendazole-resistant F. hepatica isolate, and hence these drugs might become useful in areas where triclabendazole resistance is common.

Male Sprague-Dawley rats were dosed orally with nitroxynil at a concentration of 40 mg/kg, and adult Fasciola hepatica were recovered after 24, 48 and 72 h. Fine structural changes to the tegument and gut were monitored by transmission electron microscopy. Flukes were also incubated for 24 h in vitro in nitroxynil at a concentration of 100 microg/ml. Following treatment in vivo, there was an accumulation and accelerated release of secretory bodies at the apex of the tegumental syncytium. Some swelling of the mucopolysaccharide masses surrounding the basal infolds was evident after 48 and 72 h. There was an initial accumulation of T1 secretory bodies at the base of the syncytium, but this decreased at 72 h, coinciding with a decline in their production in the tegumental cells. The mitochondria were consistently swollen in the tegumental cells. At 72 h, large vacuolations were observed between the muscle layers and there was flooding around the underlying tissues. Some tegumental cells were seen to be degenerating and beginning to disintegrate. After 24 h treatment in vitro, the basal infolds were swollen and the crystalline structure of the spines was disrupted. Flooding of the internal tissues was evident and, in the tegumental cells, Golgi complexes and secretory bodies were absent. The mitochondria in the tegumental cells were swollen. In the gastrodermal cells, changes were evident at the earliest time period in vivo. The lamellae were disrupted, few secretory bodies were present, the mitochondria and cisternae of granular endoplasmic reticulum (ger) were swollen and there was an increased number of secretory bodies. These changes became progressively more severe with time. Similar changes were evident following treatment in vitro; vesiculation of the ger was also seen. The results indicate that oral uptake is the predominant route of entry of nitroxynil into the fluke.

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.

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 microg/ml), clorsulon (5 microg/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.

The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

In this study, the susceptibility of two isolates of Fasciola hepatica--the Fairhurst and Oberon isolates--to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-week-old flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 microg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.

Male Sprague-Dawley rats were dosed orally with nitroxynil at a concentration of 40 mg/kg and adult Fasciola hepatica recovered after 24 h, 48 h and 72 h. Surface changes to the flukes were monitored by means of SEM. After the 24 h treatment, extensive swelling and blebbing of the tegument was observed on both surfaces, although the dorsal anterior region was more severely affected than either the posterior dorsal region or entire ventral surface. At high magnification, microvillus-like projections were evident, giving the surface a roughened appearance. After 48 h, the changes evident at 24 h had become more severe and some tegumental loss had occurred in the oral region of the fluke. Surface disruption was particularly evident along the lateral margins of the fluke in this region. In some specimens a single large swelling was present in the dorsal midbody region. The swelling was a more typical feature of flukes recovered. After 72 h, tegumental loss was more widespread, occurring over the oral cone and anterior midbody on the dorsal surface. Overall the dorsal surface was consistently more severely affected than the ventral surface, and the anterior region of the fluke was more disrupted than the posterior region. After 24 h in vitro incubation, the oral cone and midbody exhibited considerable spine loss and swelling. Overall, the dorsal surface was more disrupted than the ventral surface and the anterior region of the fluke was more disrupted than the posterior region. Regional differences in the response of the fluke to nitroxynil will be compared to previously published data with other fasciolicides. The results indicate that the tegument is an important target for nitroxynil action. Disruption of this, the fluke's main line of defence, would allow the drug access to other internal tissues, leading to more widespread damage.

The ultrastructural changes in Fasciola hepatica induced by the fasciolicide clorsulon were assessed using scanning electron microscopy. At 8 and 44 weeks post-infection, male Sprague-Dawley rats infected with F. hepaticawere dosed orally with clorsulon at a concentration of 12.5 mg/kg and mature flukes recovered from the bile duct after 24 h, 48 h, and 72 h in both experiments. An in vitro incubation was also set up using mature fluke (8 weeks old) incubated with clorsulon for 24 h at a concentration of 10 microg/ml. After 24 h in vivo, the young-mature flukes (8 weeks old) showed significant disruption to the tegumental surface, particularly in the anterior mid-body region, where a distinct band of swelling and blebbing was evident. The band began just behind the ventral sucker and ran posteriorly along both margins. The apical cone region of the fluke was characterised by swelling and blebbing of the surface between the spines. Similar changes were evident after 48 h in vivo, but the disruption was more severe and the mid-body band had spread posteriorly. In approximately half of the specimens recovered after 72 h in vivo, widespread disruption had occurred, with sloughing of the apical membrane or the entire syncytium, over almost all of the oral cone and anterior mid-body. For all time periods, the anterior half of the fluke was more severely affected than the posterior half. No differences were seen between the dorsal and ventral surfaces. Old-mature flukes (44 weeks old) showed regionally similar, but more severe and widespread disruption than that seen in the young-mature flukes. The onset of surface changes occurred more quickly in old-mature flukes as well. Eight-week-old flukes which had been incubated for 24 h in vitro showed surprisingly little disruption, but this may be due to the method by which the drug is taken up by the fluke.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.

The development of the vitellaria of Fasciola hepatica within the liver of its rat host was studied by means of whole-mount stained preparations and transmission electron microscopy, together with light and electron immunocytochemistry using an antibody to vitelline protein B, an eggshell precursor protein synthesized by F. hepatica. No vitelline cells could be identified in flukes recovered from the liver parenchyma, by any of the methods used. In contrast, follicles were present in flukes at the earliest time of recovery from the bile duct, namely, 5 weeks 3 days post-infection. The vitellaria in these flukes formed a row of small follicles on either side of the body. Development of the follicles was rapid: by 6 weeks 3 days, the vitellaria resembled those in the adult fluke and eggs were present in the uterus. Immunolabelling was confined to the shell protein globules in the vitelline cells, confirming the packaging of the eggshell protein within the shell globule clusters.

A commercial coproantigen ELISA test for fasciolosis, based on the use of MM3 monoclonal antibody for antigen capture, was investigated for possible cross-reactivity with Paramphistomum cervi, a trematode that commonly infects cattle and sheep grazing in fluke-infested pasture in Ireland. Histological sections of adult and immature Fasciola hepatica and P cervi were incubated with MM3 monoclonal antibody, and its binding to tissue-localised coproantigen was subsequently visualised by immunocytochemistry. In a related study, the soluble antigenic fractions derived from homogenates of P cervi adults and Taenia hydatigena metacestodes were tested for cross-reactivity with MM3 monoclonal antibody in an antigen-capture ELISA, using known F hepatica-positive and F hepatica-negative ovine faecal samples as natural controls. It was found that, while intense immunocytochemical labelling was located over the gastrodermis and gut contents of adult and immature F hepatica, sections of adult and immature P cervi were unlabelled. In the ELISA tests, the soluble fractions of F hepatica reacted strongly with MM3 monoclonal antibody, but those of P cervi and T hydatigena gave negative results. These findings support the specificity of the coproantigen ELISA test for fasciolosis in areas where paramphistomosis and cysticercosis are liable to occur singly or as coinfections with F hepatica. [ABSTRACT FROM AUTHOR]