Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Foodborne trematodes (FBT) of public health significance include liver flukes (Clonorchis sinensis, Opisthorchis viverrini, O. felineus, Fasciola hepatica and F. gigantica), lung flukes (Paragonimus westermani and several other Paragonimus spp.) and intestinal flukes, which include heterophyids (Metagonimus yokogawai, Heterophyes nocens and Haplorchis taichui), echinostomes (Echinostoma revolutum, Isthmiophora hortensis, Echinochasmus japonicus and Artyfechinostomum malayanum) and miscellaneous species, including Fasciolopsis buski and Gymnophalloides seoi. These trematode infections are distributed worldwide but occur most commonly in Asia. The global burden of FBT diseases has been estimated at about 80 million, however, this seems to be a considerable underestimate. Their life cycle involves a molluscan first intermediate host, and a second intermediate host, including freshwater fish, crustaceans, aquatic vegetables and freshwater or brackish water gastropods and bivalves. The mode of human infection is the consumption of the second intermediate host under raw or improperly cooked conditions. The major pathogenesis of C. sinensis and Opisthorchis spp. infection includes inflammation of the bile duct which leads to cholangitis and cholecystitis, and in a substantial number of patients, serious complications, such as liver cirrhosis and cholangiocarcinoma, may develop. In lung fluke infections, cough, bloody sputum and bronchiectasis are the most common clinical manifestations. However, lung flukes often migrate to extrapulmonary sites, including the brain, spinal cord, skin, subcutaneous tissues and abdominal organs. Intestinal flukes can induce inflammation in the intestinal mucosa, and they may at times undergo extraintestinal migration, in particular, in immunocompromised patients. In order to control FBT infections, eating foods after proper cooking is strongly recommended.

Fasciola hepatica is a trematode parasite that affects mammals, including humans. In Brazil, fascioliasis, a disease caused by the parasite, is of great importance. The disorder affects the welfare of the Brazilian population through impairing the agricultural production of cattle, where the disease causes weight loss as a result of liver damage. This study aimed to evaluate the genetic diversity of F. hepatica throughout Southern Brazil to determine its geographic origin and estimate the colonization route of the parasite. To accomplish these aims, flukes were collected from slaughterhouses in three endemic areas of Rio Grande do Sul and Paraná states. DNA was isolated using the phenol–chloroform protocol from single flukes and two mitochondrial genes, cytochrome oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 1 (Nad1), were amplified and sequenced. Ten haplotypes of COI were found from 75 isolated parasites and the total haplotype and nucleotide diversity observed were 0.475 and 0.002, respectively. Using the Nad1 gene, we found 24 haplotypes from 79 samples, resulting in haplotype and nucleotide diversity values of 0.756 and 0.004, respectively. An analysis of molecular variance showed that 57.4% and 77.5% of variation was within populations (FST), while 9.0 and 36.8% of variation was among groups (FCT) when considering COI and Nad1 genes, respectively. For COI, the fixation index values of 0.425 and 0.368 were obtained for FST and FCT, respectively, while analysis of Nad1 0.225 and 0.089 index values were obtained for FST and FCT, respectively. We have determined that F. hepatica found in the two distinct areas originated from several geographical regions, since we found haplotypes that were shared with at least three different continents. These data are in accordance with the recent colonization of Brazil, and the recent import of cattle from South American, European and, possibly, some African countries. The observed FST and FCT values for COI and Nad1 genes of F. hepatica may be a result of limited movement of animals within states and support the lack of geographical structure of the parasite in Brazil, which are in agreement with the observed cattle production systems in this region.

Fascioliasis is a condition caused by the trematode Fasciola hepatica. In this paper, the spatial distribution of F. hepatica in bovines in Brazil was modelled using a decision tree approach and a logistic regression, combined with a geographic information system (GIS) query. In the decision tree and the logistic model, isothermality had the strongest influence on disease prevalence. Also, the 50-year average precipitation in the warmest quarter of the year was included as a risk factor, having a negative influence on the parasite prevalence. The risk maps developed using both techniques, showed a predicted higher prevalence mainly in the South of Brazil. The prediction performance seemed to be high, but both techniques failed to reach a high accuracy in predicting the medium and high prevalence classes to the entire country. The GIS query map, based on the range of isothermality, minimum temperature of coldest month, precipitation of warmest quarter of the year, altitude and the average dailyland surface temperature, showed a possibility of presence of F. hepatica in a very large area. The risk maps produced using these methods can be used to focus activities of animal and public health programmes, even on non-evaluated F. hepatica areas.

FhCaBP2 is a Fasciola hepatica protein which belongs to a family of helminth calcium-binding proteins which combine an N-terminal domain containing two EF-hand motifs and a C-terminal dynein light chain-like (DLC-like) domain. Its predicted structure showed two globular domains joined by a flexible linker. Recombinant FhCaBP2 interacted reversibly with calcium and manganese ions, but not with magnesium, barium, strontium, copper (II), colbalt (II), iron (II), nickel, lead or potassium ions. Cadmium (II) ions appeared to bind non-site-specifically and destabilize the protein. Interaction with either calcium or magnesium ions results in a conformational change in which the protein's surface becomes more hydrophobic. The EF-hand domain alone was able to interact with calcium and manganese ions; the DLC-like domain was not. Alteration of a residue (Asp-58 to Ala) in the second EF-hand motif in this domain abolished ion-binding activity. This suggests that the second EF-hand is the one responsible for ion-binding. FhCaBP2 homodimerizes and the extent of dimerization was not affected by calcium ions or by the aspartate to alanine substitution in the second EF-hand. The isolated EF-hand and DLC-like domains are both capable of homodimerization. FhCaBP2 interacted with the calmodulin antagonists trifluoperazine, chlorpromazine, thiamylal and W7. Interestingly, while chlorpromazine and thiamylal interacted with the EF-hand domain (as expected), trifluoperazine and W7 bound to the DLC-like domain. Overall, FhCaBP2 has distinct biochemical properties compared with other members of this protein family from Fasciola hepatica, a fact which supports the hypothesis that these proteins have different physiological roles.

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.

A study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10−4m) on its own, TCBZ.SO (15 μg mL−1) alone, a combination of R(+)-VPL (1×10−4m) plus TCBZ.SO (15 μg mL−1), TCBZ.SO (50 μg mL−1) on its own, or a combination of TCBZ.SO (50 μg mL−1) plus R(+)-VPL (1×10−4m). They were also incubated in TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive; and in TCBZ.SO (50 μg mL−1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 μg mL−1) alone or in combination with R(+)-VPL (1×10−4m) until they became inactive, or with TCBZ.SO (50 μg mL−1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

A total of 20 749 bulk tank milk (BTM) samples was collected in November 2008 from all over Germany, corresponding to 20·9% of all German dairy herds. The BTM samples were analysed for antibodies against Fasciola hepatica using the excretory–secretory (ES) ELISA. A geospatial map was drawn to show herd prevalences per postal code area. Various spatial risk factors were tested for potential statistical associations with the ELISA results in logistic regression supported by a geographical information system (GIS). The mean seroprevalence was 23·6% and prevalences in different German federal states varied between 2·6% and 38·4%. GIS analysis revealed statistically significant positive associations between the proportion of grassed area and water bodies per postal code area and positive BTM ELISA results. This can be explained by the biology of the intermediate host, the amphibious snail Galba (Lymnea) truncatula and the pasture-borne nature of fasciolosis. The full logistic regression model had a Pseudo-R2 of 22%, while the final model obtained by controlled stepwise model building revealed a Pseudo-R2 of 14%, indicating that additional, unrecorded factors and random effects contributed substantially to the occurrence of positive ELISA results. Considering the high seroprevalences in some areas and the economic impact of fasciolosis, farmers and veterinarians should be strongly advised to implement effective liver fluke control programmes.

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of 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 PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ.

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 μm). 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 μg/ml); or PB+NADPH+TCBZ.SO (15 μg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible 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.

Adult Fasciola hepatica were incubated for 48 h in vitro in the synthetic peroxide, OZ78 at a concentration of 100 μg/ml and then prepared for scanning and transmission electron microscopy. There was limited disruption to the external fluke surface, with only slight swelling and blebbing of the interspinal tegument in the midbody and ventral tail regions. By contrast, significant disruption was observed to the ultrastructure of the tegument and subtegumental tissues. There was severe swelling of the basal infolds in the tegumental syncytium and the flooding spread internally to affect the subtegumental tissues. In the tegumental system, there was swelling of the cisternae of granular endoplasmic reticulum and of the mitochondria, with the latter showing signs of breaking down. Autophagic vacuoles and lipid droplets were present and the synthesis of tegumental secretory bodies was much reduced. The gastrodermal cells were severely affected, with swelling and degeneration of the mitochondria and the presence of autophagic vacuoles and lipid droplets. The granular endoplasmic reticulum was swollen and vesiculated and the cells contained few secretory bodies. Both the vitelline and testis follicles showed evidence of extensive cellular disruption and degeneration. This study confirms previous data indicating the potential flukicidal activity of OZ78.

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 haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

The effects of the novel benzimidazole, triclabendazole (TCBZ) (‘Fasinex’, Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental ultrastructure of Fasciola hepatica were determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. At a concentration of 15 µ/ml, the tegument of the whole adult fluke showed ultrastructural changes only after prolonged time-periods, with vacuolation at the base of the syncytium and accumulation of T2 secretory bodies in the tegumental cells. At a concentration of 50 µ/ml, with both whole flukes and tissue-slices, the tegument appeared extremely abnormal with accumulation of secretory bodies towards the base of the syncytium. With longer incubation times, the tegument was completely sloughed away and the tegumental cells became synthetically inactive. The tegument of the 3-week-old juvenile became progressively convoluted at the apex, while in the basal regions there was severe vacuolation. In the tegumental cells, there were accumulations of T1 secretory bodies. These results confirm TCBZ as a potent fasciolicide, being very effective in disrupting the fluke tegument. They may go some way to explain the mode of action of this important fasciolicide.

A dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

The effect of the microtubule inhibitors coichicine (1×10−3 M) and tubulozole-C(1×10−6 M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of T1 secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type I cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, ‘yolk’ globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 μm). Flukes were then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); MTZ+NADPH+TCBZ (15 μg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 μg/ml). 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 surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance.

In East Africa, Fasciola gigantica is generally the causative agent of fasciolosis but there have been reports of F. hepatica in cattle from highland regions of Kenya, Ethiopia, Uganda and Zaire. The topography of the Southern Highlands of Tanzania provides an environment where the climatic conditions exist for the sustenance of lymnaeid species capable of supporting both Fasciola hepatica and F. gigantica. Theoretically this would allow interaction between fasciolid species and the possible creation of hybrids. In this report we present molecular data confirming the existence of the snail, Lymnaea truncatula, at high altitude on the Kitulo Plateau of the Southern Highlands, Tanzania, along with morphometric and molecular data confirming the presence of F. hepatica in the corresponding area. At lower altitudes, where climatic conditions were unfavourable for the existence of L. truncatula, the presence of its sister species L. natalensis was confirmed by molecular data along with its preferred fasciolid parasite, F. gigantica. Analysis based on a 618 bp sequence of the 28S rRNA gene did not reveal the presence of hybrid fasciolids in our fluke samples.

A morphological study has been carried out to determine the effect of the active sulphoxide metabolite of the benzimidazole anthelmintic, albendazole (ABZ-SO) on the adult liver fluke, Fasciola hepatica. Whole flukes were treated with ABZ-SO for 12 and 24 h at a concentration of 10 μg/ml. The changes in response to drug treatment were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). No surface changes were apparent following 12 h ABZ-SO treatment, but localized blebbing was observed after 24 h, which became more extensive towards the posterior region of both surfaces. TEM of sections from the posterior midbody region revealed that ABZ-SO caused the accumulation of secretory bodies in the tegumental cells and in their cytoplasmic connections and, after 24 h, just above the basal plasma membrane. Localized blebbing of the apical membrane also occurred. The morphology of the Golgi complexes within the tegumental cells began to change after 12 h treatment with ABZ-SO and, by 24 h, few complexes were observed. A distinct increase in tubulin immunoreactivity occurred after 12 h treatment, but this decreased after 24 h. The results obtained are consistent with those expected for microtubule inhibition. They are discussed in relation to the action of established microtubule inhibitors, as well as the benzimidazole derivative, triclabendazole.