Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.     

Clonorchicidal properties of the synthetic trioxolane OZ78

Because the synthetic trioxolane OZ78 is active against the liver fluke Fasciola hepatica, we were motivated to investigate the in vivo and in vitro activity against another liver fluke, namely Clonorchis sinensis. Rats infected with C. sinensis for 2 and 5 wk were treated orally with single doses of OZ78 (75, 150, or 300 mg/kg). Worm burden reductions were assessed against untreated control rats. Scanning electron microscopy (SEM) was used to observe adult C. sinensis after recovery from rats 1-3 days posttreatment with a single 300 mg/kg oral dose of OZ78 and after in vitro exposure to concentrations of 1, 10, and 100 microg/ml of OZ78. A single 300 mg/kg oral dose of OZ78 resulted in worm burden reductions of 78.5% and 98.5% against juvenile and adult C. sinensis, respectively. SEM observations revealed tegumental surface alterations, including blebbing and sloughing. OZ78 emerges as a new compound with a broad spectrum of activity against major foodborne trematode infections.     

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

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

Anthelmintic activity of triclabendazole

Triclabendazole was tested in vitro and in vivo against a range of helminths. Although in vitro activity was found against Hymenolepis diminuta (0.5 microgram/ml), Fasciola hepatica (2.5 micrograms/ml), Taenia crassiceps and Schistosoma mansoni (50 micrograms/ml), in vivo activity was only found against F. hepatica, a single oral dose of 40 mg/kg killing 99% of adult flukes in the rat. This spectrum of activity suggests a mechanism of action unlike that of other benzimidazole anthelmintics.     

Liver fluke vaccines in ruminants: strategies,progress and future opportunities

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30–54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.     

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3)

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.     

Decrease of Metagonimus yokogawai Endemicity along the Tamjin River Basin

The Tamjin River which flows from Jangheung-gun via Gangjin-gun to the South Sea was reported to be a highly endemic area of Metagonimus yokogawai infection in 1977 and 1985. However, there were no recent studies demonstrating how much change occurred in the endemicity, in terms of prevalence and worm burden, of metagonimiasis in this river basin. Thus, a small-scale epidemiological survey was carried out on some residents along the Tamjin River basin in order to determine the current status of M. yokogawai infection. A total of 48 fecal samples were collected and examined by the Kato-Katz thick smear and formalin-ether sedimentation techniques. The egg positive rate of all helminths was 50.0%, and that of M. yokogawai was 37.5%, followed by C. sinensis 22.9% and G. seoi 4.2%. To obtain the adult flukes of M. yokogawai, 6 egg positive cases were treated with praziquantel 10 mg/kg in a single dose and purged with magnesium sulfate. A total of 5,225 adult flukes (average 871 specimens per person) of M. yokogawai were collected from their diarrheic stools. Compared with the data reported in 1977 and 1985, the individual worm burdens appeared to have decreased remarkably, although the prevalence did not decrease at all. It is suggested that the endemicity of M. yokogawai infection along the Tamjin River has been reduced. To confirm this suggestion, the status of infection in snail and fish intermediate hosts should be investigated.     

The effects of rafoxanide and nitroxynil on the survival, growth and morphology of Fasciola hepatica in rabbits.

Two experiments were carried out to assess the effects of nitroxynil (10 mg/kg p.o.) and rafoxanide (6.7 mg/kg p.o.) against 2,4, 6 or 8 weeks old F. hepatica in rabbits. The results show that the efficacy and incidence of stunted and abnormal surviving flukes were directly related to the age of infection at treatment. Rafoxanide was the more potent compound against immature forms. The testes were the most consistently abnormal reproductive organs in flukes surviving treatment with both drugs and spermatogenesis per se was disrupted.     

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.     

Seasonal dynamics of Fasciola hepatica burdens in grazing Timahdit sheep in Morocco

Seasonal transmission of Fasciola hepatica was observed in sentinel sheep and the dynamics of the snail intermediate host, Lymnaea truncatula, was followed over a 3-year study period in the Middle-Atlas mountains in Morocco. High fluke burdens were recorded in both lambs and ewes in the fall and winter, suggesting that transmission occurred in late spring. Fluke burdens ranged from one to 302 in ewes and from one to 345 in lambs. Infections with 200 or more flukes were always fatal. A unique feature of this study was the annual cyclical fluctuation of the fluke burdens. Burdens reached maximum levels during the winter and then declined to low numbers by late spring and summer. This suggested self-regulation which may be dependent on breed resistance or may be related to forage factors, including lack of forage (nutritional stress). Snail populations were cyclical and correlated with fluke transmission as observed in the sentinel sheep. The weather was observed to affect the snail populations which in turn limited fluke transmission.     

Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.     

High prevalence of liver and intestinal fluke infections among residents of Savannakhet Province in Laos

The prevalence of liver and intestinal fluke infections was surveyed on residents of Savannakhet Province, Laos. Fecal specimens were collected from a total of 981 residents in 4 Mekong riverside villages and examined by the Kato-Katz thick smear technique. The results revealed that the overall helminth egg positive rate was 84.2%, and the positive rate for small trematode eggs, including Opisthorchis viverrini, heterophyids, or lecithodendriids, was 67.1%. To obtain adult flukes, 38 small trematode egg positive cases were treated with a 20-30 mg/kg single dose of praziquantel and purged. Diarrheic stools were then collected from 29 people and searched for helminth parasites using stereomicroscopes. Mixed infections with O. viverrini and 6 kinds of intestinal flukes were found, namely, Haplorchis taichui, Haplorchis pumilio, Haplorchis yokogawai, Prosthodendrium molenkampi, Phaneropsolus bonnei, and echinostomes. The total number of flukes collected was 7,693 specimens (av. no. per treated person; 265.3). The most common species was O. viverrini, followed by H. taichui, P. molenkampi, echinostomes, H. pumilio, P. bonnei, and H. yokogawai. The results indicate that foodborne liver and intestinal fluke infections are prevalent among residents of Savannakhet Province, Laos.     

In vitro and in vivo studies on the bioactivity of a ginger (Zingiber officinale) extract towards adult schistosomes and their egg production

The bioactivity of an ethyl acetate extract of ginger (Zingiber officinale) towards Schistosoma mansoni adult pairs, both cultured in vitro and in vivo in laboratory mice, was investigated by monitoring worm mortality and fecundity. In vitro, a concentration of 200 mg l(-1) of extract killed almost all worms within 24 h. Male worms seemed more susceptible than female under these conditions. Cumulative egg output of surviving worm pairs in vitro was considerably reduced when exposed to the extract. For example, after 4 days of exposure to 50 mg l(-1), cumulative egg output was only 0.38 eggs per worm pair compared with 36.35 for untreated worms. In vivo efficacy of the extract was tested by oral and subcutaneous delivery of 150 mg kg(-1) followed by assessment of worm survival and fecundity. Neither delivery route produced any significant reduction in worm numbers compared with untreated controls. Worm fecundity was assessed in vivo by cumulative egg counts per liver at 55 days post infection with mice treated subcutaneously. Such infections showed egg levels in the liver of about 2000 eggs per worm pair in 55 days, in both treated and control mice, with no significant difference between the two groups. To ensure that density-dependent effects did not confound this analysis, a separate experiment demonstrated no such influence on egg output per worm pair, at intensities between 1 and 23 worms per mouse.     

Epidemiological Survey on the Infection of Intestinal Flukes in Residents of Muan-gun, Jeollanam-do, the Republic of Korea

Infection status of intestinal flukes was investigated in residents of Muan-gun, Jeollanam-do, the Republic of Korea. Total 1,257 fecal samples of residents were examined by formalin-ether sedimentation technique and Kato-Katz thick smear method. Helminth eggs were detected from 95 (7.6%) residents, and eggs of heterophyid flukes and Clonorchis sinensis were found from 62 (4.9%) and 40 (3.2%) cases, respectively. The larger heterophyid eggs, somewhat dark-brown in color and 37.7 × 21.5 µm in average size, and found in 32 (2.6%) out of 62 egg positive cases of heterophyid flukes. To confirm the adult flukes, we performed worm recovery from 12 cases after praziquantel treatment and purgation with MgSO₄. A total of 1,281 adult flukes, assigned to 7 species, were recovered from 9 cooperative cases. Heterophyes nocens (total 981 specimens) was collected from 9 cases, Stictodora fuscata (80) from 7, Gymnophalloides seoi (75) from 5, Pygidiopsis summa (140) from 3, Stellantchasmus falcatus (3) from 2, and Stictodora lari and Acanthotrema felis (each 1 worm) from 1 case each. The intrauterine eggs of S. fuscata collected from the recovered worm were identical with the larger heterophyid eggs detected in the stool examination. By the present study, it was confirmed that A. felis is a new intestinal fluke infecting humans, and residents in Muan-gun, Jeollanam-do are infected with variable species of intestinal trematodes.     

The consequences of short-term grazing of bioactive forages on established adult and incoming larvae populations of Teladorsagia circumcincta in lambs

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.     

Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica

Polyethylene glycol-precipitated immune complexes (PIC) from the sera of 5 calves with Fasciola hepatica worm burdens ranging between 27 and 70 flukes were examined for parasite antigen content at 2, 4, 6, 8, 10, and 16 wk postinfection (PI) by the enzyme-linked immunosorbent assay (ELISA). Three assays were devised using an affinity-processed rabbit antibody to worm excretory/secretory (FhES) antigens. The PIC plate assay detected parasite antigen by adherence of anti-FhES antibody to PIC incubated overnight on ELISA plates, and tests were visualized using anti-rabbit peroxidase-linked antibody. The serum complex and PIC capture assay utilized the anti-FhES immunoglobulin as an antigen capture antibody linked to the solid phase. The attached complexes were then detected by the adhering bovine antibody, either soluble complexes in serum or as PIC. All assays showed circulating immune complex (CIC) values elevated at 6-8 wk PI, which generally coincided with increased host circulating antibody to FhES antigens. The greatest detection rate for all of the immune complex (IC) detection assays occurred with the PIC capture assay. It detected antigen in almost 90% of sera tested at 6 and 8 wk PI. Both the serum complex and PIC capture assay detected greatest amounts of CIC in those animals with the largest worm burdens, whereas the PIC plate assay showed no such trend. This study shows that F. hepatica antigen detection in CIC can be used to aid immunologic diagnosis of fascioliasis.     

Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts.

1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.     

Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78

The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24-72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 microg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug.     

Hyperendemicity of Haplorchis taichui Infection among Riparian People in Saravane and Champasak Province,Lao PDR

In this study, we found that Haplorchis taichui, a heterophyid intestinal fluke, is highly prevalent, with heavy worm loads, among riparian people in Saravane and Champasak province, Lao PDR. Fecal specimens were collected from 1,460 people (717 men and 743 women) in 12 riparian (Mekong river) districts and were examined by the Kato-Katz fecal smear technique. The overall helminth egg positive rate was 78.8% and 66.4% in Saravane and Champasak province, respectively. The positive rate for small trematode eggs (STE), which included H. taichui and other heterophyids, Opisthorchis viverrini, and lecithodendriids, was 69.9% and 46.3% in Saravane and Champasak province, respectively. To obtain adult flukes, 30 STE-positive people were treated with 40 mg/kg praziquantel and then purged. Whole diarrheic stools were collected 4-5 times for each person and searched for fluke specimens using a stereomicroscope. Mixed infections with various species of trematodes (H. taichui, Haplorchis pumilio, O. viverrini, Prosthodendrium molenkampi, Centrocestus formosanus, and Echinochasmus japonicus) and a species of cestode (Taenia saginata) were found. However, the worm load was exceptionally high for H. taichui compared with other trematode species, with an average of 21,565 and 12,079 specimens per infected person in Saravane and Champasak province, respectively, followed by H. pumilio (41.9 and 22.5, respectively) and O. viverrini (9.4 and 1.5, respectively). These results show that diverse species of intestinal and liver flukes are prevalent among riparian people in Saravane and Champasak province, Lao PDR, with H. taichui being the exceptionally dominant species.     

Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether

The tegumental changes in adult Fasciola hepatica induced by artemether and artesunate were assessed utilizing scanning electron microscopy (SEM). F. hepatica were incubated with artemether and artesunate for 48h at a concentration of 10microg/ml in the absence or presence of haemin. For the latter experiment both, a triclabendazole-resistant and sensitive F. hepatica isolate were used. For the in vivo studies rats were treated with single 200mg/kg oral doses of artemether and artesunate and flukes recovered from the bile ducts after 24-96h. SEM analysis of the flukes incubated in the presence of the drugs without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing, blebbing and eruptions, particularly in the ventral and dorsal mid-body and tail region, was evident. No difference in the extent of damage could be observed between artemether and artesunate and between the triclabendazole-resistant and non-resistant flukes. After 24h in vivo disruption of the tegument was evident in the artemether-treated flukes, and the damage increased in severity 48-72h post-treatment. Sloughing, swelling and extensive furrowing of the tegument was observed in several flukes, in particular in the tail region and the ventral apical cone region. In the artesunate treatment, tegumental damage was evident after 72h, but seemed slightly less pronounced when compared to the artemether-treated specimens examined at the same time point. Concluding our experiments confirm that artemether and artesunate are potent fasciocidal drugs and the tegument of adult F. hepatica appears to be a target for the action of these drugs.