The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica.

Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

Ovicidal in vitro activity of the fixed oil of Helianthus annus L. and the essential oil of Cuminum cyminum L. against Fasciola hepatica (Linnaeus, 1758).

The fascioliasis is a parasitic disease of importance in veterinary medicine and public health. For this parasitosis, the treatment by synthetic fasciolicides is used and due to their intense use although they have been shown less effective because of the establishment of resistant Fasciola hepatica population to these drugs, with a global concern. The use of derived products of plants with biological activity has been shown promising in the control of parasites. In this context, we evaluated the chemical composition and action of ovicidal in vitro fixed oil of Helianthus annuus L. (FOH) and essential oil of Cuminum cyminum L. (EOC), as well as their combination (FOH + EOC) of F. hepatica. In the assay in vitro of F. hepatica were submitted to different concentrations of oils, such as FOH (2.3 mg/mL + 0,017 mg/mL); EOC (2.07 mg/mL + 0,004 mg/mL) and the combination of (1.15 mg/mL + 1.03 mg/mL to 0,0085 mg/mL + 0,008 mg/mL) as well as a positive control of thiabendazole (0.025 mg/mL) and a negative control with distilled water and tween. The identification of the majority chemical compounds was performed by gas chromatography. The -cell viability of the oils was tested in MDBK cellular line by the MTT method. The majority compounds in the FOH were the linoleic (53.6%) and oleic (35.85%) unsaturated fatty acids, and the majority phytochemicals compounds in the EOC were the Cumaldehyde (26.8%) and the 2-Caren 10-al (22.17%). The EOC and the combination presented effectiveness of 99% (±1) and of 94% (±1) in the concentration of 0.03 mg/mL and 0.035 mg/mL+0.03 mg/mL, respectively, and the FOH was insufficiently active as ovicidal. The cell viability at this concentration of EOC was 93%. From the results above we could infer that the EOC is promising as a new alternative for the fascioliasis control.

Pilot Evaluation of Two Fasciola hepatica Biomarkers for Supporting Triclabendazole (TCBZ) Efficacy Diagnostics.

Fasciola hepatica, the causative agent of fasciolosis, is a global threat to public health, animal welfare, agricultural productivity, and food security. In the ongoing absence of a commercial vaccine, independent emergences of anthelmintic-resistant parasite populations worldwide are threatening the sustainability of the few flukicides presently available, and particularly triclabendazole (TCBZ) as the drug of choice. Consequently, prognoses for future fasciolosis control and sustained TCBZ application necessitate improvements in diagnostic tools to identify anthelmintic efficacy. Previously, we have shown that proteomic fingerprinting of F. hepatica excretory/secretory (ES) products offered new biomarkers associated with in vitro TCBZ-sulfoxide (SO) recovery or death. In the current paper, two of these biomarkers (calreticulin (CRT) and triose phosphate isomerase (TPI)) were recombinantly expressed and evaluated to measure TCBZ efficacy via a novel approach to decipher fluke molecular phenotypes independently of molecular parasite resistance mechanism(s), which are still not fully characterised or understood. Our findings confirmed the immunoreactivity and diagnostic potential of the present target antigens by sera from TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) F. hepatica experimentally infected sheep.

Efficacy of purified fractions of Artemisia ludoviciana Nutt. mexicana and ultraestructural damage to newly excysted juveniles of Fasciola hepatica in vitro.

The study aimed to evaluate the fasciolicidal efficacy of extracts and fractions of Artemisia ludoviciana and identify the active substance. Extracts from A. ludoviciana were obtained by using hexane, ethyl acetate and methanol. To test the extracts, newly excysted juveniles of Fasciola hepatica were artificially obtained. The extracts were tested at concentrations of 125, 250, 375 and 500 mg/L. In each test run, an untreated control group and control wells containing triclabendazole sulfoxide were also included. The flukes were examined at 24, 48 and 72 h after treatment. Ethyl acetate extract (ALEAE) showed 100 % efficacy at 48 h of exposure (P < 0.05). Then, this extract was fractionated by column chromatography (CC), and the obtained fractions were evaluated in vitro as previously mentioned. The results indicated that fraction 3 yielded 100 % efficacy at 48 h (P < 0.05). Subsequently, the purification of fraction 3 was performed. New fractions were obtained (A-L), with sub-fraction "J" exhibiting 100 % efficacy at 24 h (P < 0.05). These sub-fractions were submitted to phytochemical analysis, demonstrated the presence of sesquiterpene lactones. Likewise, were analyzed by HPLC/MS/DAD, and the results showed that artemisinin was the main compound. In addition, flukes treated were examined by scanning electron microscopy (SEM) showing areas of inflammation throughout the tegument.

Selective flukicide treatment of non-lactating cows and the corresponding production impact of Fasciola hepatica in dairy herds in Sweden.

A control strategy against Fasciola hepatica infection based on selective treatment of non-lactating animals was evaluated in four Swedish dairy herds. The study was conducted over the course of two consecutive seasons in moderately to highly F. hepatica infected herds with robotic milking, where heifers and dry cows received an oral drench with albendazole (10 mg/kg) during three visits in January, February and March in both 2017 and 2018. This resulted in an anthelmintic coverage between 38 % and 58 % of the animals. Furthermore, on each visit, the infection status of all dewormed animals along with 15 randomly selected milking cows were monitored by detection of F. hepatica coproantigens. Individual milk samples were also collected quarterly from the whole herds for measurements of individual antibody levels against the parasite using milk ELISA. In addition, individual data on milk yield and quality were collected on a monthly basis between 2016 and 2018. To further study the impact of the infection on milk production, truly F. hepatica positive and negative cows in the first lactation were identified based on the results from coproantigen and milk ELISA assays. Total F. hepatica coproantigen prevalence in the herds varied between 28 % and 85 % in the first year, and between 27 % and 68 % in the second year of the study. We found that two years of treatments resulted in a significant decrease of coproantigen-positivity especially on the two most heavily infected farms. These results were confirmed by a similar drop in within-herd prevalences obtained by milk ELISA results. The infection had a significant negative impact on milk yields in untreated F. hepatica positive cows. No consistent long-term effect was observed at the herd level probably due to the influx of animals infected before puberty and/or adult animals that were re-infected at dry-off. This is the first study of the effects of F. hepatica infection on milk yield and quality in dairy herds in Sweden.

Providing information about triclabendazole resistance status influences farmers to change liver fluke control practices.

BACKGROUND: Reports of disease and production losses associated with Fasciola hepatica, the common liver fluke, have increased in recent years. Resistance to triclabendazole, one of the principal veterinary medicines used to prevent losses, has been reported and is now considered widespread in fluke endemic regions of the UK. METHODS: Thirteen farmers participated in a trial in 2013 and the triclabendazole resistance status was obtained for each farm. Based on these results, a knowledge exchange programme on fluke control was delivered to nearly 100 farmers in the region. In this follow-up study, 11 farmers involved in the original trial, participated in semistructured in-depth qualitative interviews in July 2017. RESULTS: Overall, participants identified benefits from participating in the 2013 trial, gaining information about triclabendazole resistance on their farms and knowledge about fluke control. The information on their farm's resistance status was a driver for changing their liver fluke control programmes. Factors such as habitual and repetitive behaviours, grazing restrictions due to agri-environmental schemes, economic pressures and climate change were identified that could impede or prevent the adoption of new control strategies. CONCLUSIONS: The study highlights the significance of resistance to triclabendazole and the impact of knowledge exchange programmes in changing liver fluke control practices.

Efficiency comparison of experimental fosfatriclaben with three commercial fasciolicides in experimentally infected sheep.

In this work, we compare and evaluate the efficiency of fosfatriclaben with three commercial fasciolicides in experimentally infected sheep. Fosfatriclaben is a novel prodrug derived from triclabendazole; it is highly water-soluble with excellent aqueous stability at pH 7, properties that make it ideal for developing intramuscular pharmaceutical compositions in the form of solutions. In order to compare, 30 mixed breed sheep, previously diagnosed negative to fluke eggs, were infected with 200 metacercariae of Fasciola hepatica, twice. Five groups of six animals/each were formed for treatments. Group 1 (G1) was treated with closantel 5% injectable at 5 mg/kg subcutaneously, G2 with clorsulon at 2 mg/kg subcutaneously, G3 with triclabendazole at 12 mg/kg per os, G4 with fosfatriclaben at 6 mg/kg intramuscularly (dose equivalent to triclabendazole content), and G5 remained as the non-treated control. On day 110, fecal samples were examined to determine the percentage of egg reduction after treatment, and sheep were humanely euthanized. The livers were collected, the flukes were extracted, measured, and counted. Efficiency in egg reduction was of 86.8, 90.5, 98.4, and 97.3% for closantel, clorsulon, triclabendazole, and fosfatriclaben, respectively, and efficiency against flukes was of 96.2, 91.9, 99.4, and 95.7%, respectively. No statistical differences were found between treatments. It is concluded that fosfatriclaben at 6 mg/kg intramuscularly presented a high fasciolicide efficiency, similar to the best commercial fasciolicides, having advantage over its predecessor since it uses half of the dose required by triclabendazole to remove flukes in sheep under study.

Liver Fluke Vaccine Assessment in Cattle.

Liver fluke Fasciola hepatica remains an important agent of foodborne trematode disease producing great economic losses due to its negative effect on productivity of grazing livestock in temperate areas. The prevailing control strategies based on antihelminthic drugs are not long term sustainable due to widespread resistance. Hence, vaccination appears as an attractive option to pursue for parasite eradication.

Testing Albendazole Resistance in Fasciola hepatica.

The egg development test is a useful in vitro tool to detect albendazole (ABZ) resistance in Fasciola hepatica. ABZ is the only flukicidal compound with ovicidal activity. The described test is based on the ABZ capacity to affect parasite egg development and hatching in susceptible parasites, while this effect is lost in ABZ-resistant liver fluke isolates. Among many advantages, it is noted that the diagnostic test can be performed on eggs isolated from fecal samples (sheep and cattle), avoiding the sacrifice of animals necessary in controlled efficacy trials. The egg development test described here is a simple, inexpensive, and accessible method, previously employed for diagnosis of ABZ resistance in F. hepatica.

Drug Targets: Screening for Small Molecules that Inhibit Fasciola hepatica Enzymes.

The in vitro screening of small molecules for enzymatic inhibition provides an efficient means of finding new compounds for developing drug candidates. This strategy has the advantage of being rapid and inexpensive to perform. Enzymes are suitable targets for screening when simple methods to obtain them and measure their activities are available and there is evidence of their essential role in the parasite's life cycle. Here, we describe the screening of small molecules as inhibitors of two Fasciola hepatica enzyme targets (cathepsin L and triose phosphate isomerase), an initial step to find new potential compounds for drug development strategies.

Drug resistance in liver flukes.

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

Novel and selective inactivators of Triosephosphate isomerase with anti-trematode activity.

Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health.

Determination of the prevalence and intensity of Fasciola hepatica infection in dairy cattle from six irrigation regions of Victoria, South-eastern Australia, further identifying significant triclabendazole resistance on three properties.

Fasciola hepatica (liver fluke) is a widespread parasite infection of livestock in Victoria, South-eastern Australia, where high rainfall and a mild climate is suitable for the main intermediate host Austropeplea tomentosa. The aims of this study were to quantify the prevalence and intensity of F. hepatica in dairy cattle in the irrigated dairy regions of Victoria and determine if triclabendazole resistance was present in infected herds. Cattle in 83 herds from the following six irrigation regions were tested for F. hepatica: Macalister Irrigation District (MID), Upper Murray (UM), Murray Valley (MV), Central Goulburn (CG), Torrumbarry (TIA) and Loddon Valley (LV). Twenty cattle from each herd were tested using the F. hepatica faecal egg count (FEC) as well as the coproantigen ELISA (cELISA). The mean individual animal true prevalence of F. hepatica across all regions was 39 % (95 % credible interval [CrI] 27%-51%) by FEC and 39 % (95 % CrI 27%-50%) by cELISA with the highest true prevalence (75-80 %) found in the MID. Our results show that 46 % of the herds that took part in this study were likely to experience fluke-associated production losses, based on observations that herd productivity is impaired when the true within-herd prevalence is > 25 %. Using the FEC and cELISA reduction tests, triclabendazole resistance was assessed on 3 herds in total (2 from the 83 in the study; and 1 separate herd that did not take part in the prevalence study) and resistance was confirmed in all 3 herds. This study has confirmed that F. hepatica is endemic in several dairy regions in Victoria: triclabendazole resistance may be contributing to the high prevalence in some herds. From our analysis, we estimate that the state-wide economic loss associated with fasciolosis is in the order of AUD 129 million (range AUD 38-193 million) per year or about AUD 50,000 (range AUD 15,000-75,000) per herd per year.

Exogenous Iron Increases Fasciocidal Activity and Hepatocellular Toxicity of the Synthetic Endoperoxides OZ78 and MT04.

The synthetic peroxides OZ78 and MT04 recently emerged as fasciocidal drug candidates. However, the effect of iron on fasciocidal activity and hepatocellular toxicity of these compounds is unknown. We investigated the in vitro fasciocidal activity and hepatocellular toxicity of OZ78 and MT04 in absence and presence of Fe(II)chloride and hemin, and conducted a toxicological study in mice. Studies were performed in comparison with the antimalarial artesunate (AS), a semisynthetic peroxide. Fasciocidal effects of OZ78 and MT04 were confirmed and enhanced by Fe2+ or hemin. In HepG2 cells, AS reduced cellular ATP and impaired membrane integrity concentration-dependently. In comparison, OZ78 or MT04 were not toxic at 100 µM and reduced the cellular ATP by 13% and 19%, respectively, but were not membrane-toxic at 500 µM. The addition of Fe2+ or hemin increased the toxicity of OZ78 and MT04 significantly. AS inhibited complex I, II, and IV of the mitochondrial electron transport chain, and MT04 impaired complex I and II, whereas OZ78 was not toxic. All three compounds increased cellular reactive oxygen species (ROS) concentration-dependently, with a further increase by Fe2+ or hemin. Mice treated orally with up to 800 mg OZ78, or MT04 showed no relevant hepatotoxicity. In conclusion, we confirmed fasciocidal activity of OZ78 and MT04, which was increased by Fe2+ or hemin. OZ78 and MT04 were toxic to HepG2 cells, which was explained by mitochondrial damage associated with ROS generation in the presence of iron. No relevant hepatotoxicity was observed in mice in vivo, possibly due to limited exposure and/or high antioxidative hepatic capacity.

Chemotactic migration of newly excysted juvenile Clonorchis sinensis is suppressed by neuro-antagonists.

The metacercariae of the Clonorchis sinensis liver fluke excyst in the duodenum of mammalian hosts, and the newly excysted juveniles (CsNEJs) migrate along the bile duct via bile chemotaxis. Cholic acid is a major component of bile that induces this migration. We investigated the neuronal control of chemotactic behavior of CsNEJs toward cholic acid. The migration of CsNEJs was strongly inhibited at sub-micromolar concentration by dopamine D1 (LE-300 and SKF-83566), D2 (spiramide, nemonapride, and sulpiride), and D3 (GR-103691 and NGB-2904) receptor antagonists, as well as a dopamine reuptake inhibitor (BTCP). Neuropeptides, FMRFamide, peptide YY, and neuropeptide Y were also potent inhibitors of chemotaxis. Meanwhile, serotonergic, glutamatergic, and cholinergic inhibitors did not affect chemotaxis, with the exception of fluoxetine and CNQX. Confocal immunofluorescence analysis indicated that dopaminergic and cholinergic neurons were colocalized in the somatic muscle tissues of adult C. sinensis. Our findings suggest that dopaminergic neurons and neuropeptides play a major role in the chemotactic migration of CsNEJs to bile, and their inhibitors or modulators could be utilized to prevent their migration from the bile duct.

The egg hatch test: A useful tool for albendazole resistance diagnosis in Fasciola hepatica.

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

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives.

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices.

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes. Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (ß-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.

Lack of efficacy of triclabendazole against Fasciola hepatica is present on sheep farms in three regions of England, and Wales.

The liver fluke Fasciola hepatica is a parasitic trematode that has a major impact on livestock production and human health. Control of F hepatica is difficult and relies on anthelmintics, particularly triclabendazole, due to its efficacy against both adult and juvenile stages of the parasite. Emergence of triclabendazole-resistant F hepatica populations has been reported in a number of countries, including the UK, but the overall prevalence and distribution of triclabendazole resistance is unknown. In this study, the authors established the presence of reduced efficacy of triclabendazole in sheep flocks in England and Wales, using a validated composite faecal egg count reduction test. Seventy-four sheep farms were sampled from Wales, southwest, northwest and northeast England between Autumn 2013 and Spring 2015. F hepatica eggs were detected in samples from 42/74 farms. Evidence of a lack of efficacy of triclabendazole was detected on 21/26 farms on which the faecal egg count reduction test was completed, with faecal egg count reductions ranging from 89 per cent to 0per cent. Regression analysis suggested that both prevalence of F hepatica and lack of efficacy of triclabendazole were spatially correlated, with higher faecal egg counts and lower percentage reductions on farms located in the northwest of England, and Wales. Overall, the results show that reduced efficacy of triclabendazole is present across England and Wales, with a complete lack of therapeutic efficacy observed on 9/26 farms.

Microscopic alterations in Fasciola hepatica from sheep treated with albendazole / Alterações microscópicas em Fasciola hepatica de ovelhas tratadas com albendazol

Abstract Currently, albendazole is one of the most commonly used drugs because of its affordability. The objective was to evaluate the histopathology of Fasciola hepatica specimens. For this, the efficacy test was performed on sheep treated with albendazole at the dose recommended for F. hepatica, in which the helminths recovered at necropsy were counted and separated for histology. Spermatogenic cells from parasites recovered from treated and control sheep were examined by microscopy. The fecal egg-count reduction test revealed 97.06% efficacy of albendazole in the treatment of F. hepatica. Changes in testicular tubule cells started 48 hours after treatment and became evident within 72 hours, at which point it became difficult to identify cell types. Primary and secondary spermatogonia became increasingly rare and intercellular vacuolization was more evident. Signs of apoptosis, with pycnotic nuclei and evidence of keriorrexia were observed at all times. Cell debris was identified 96 hours after treatment. The results indicated that parasitic spermatogenesis was severely affected by albendazole and demonstrated the importance of the use of histopathology for the diagnosis of therapeutic efficacy in field strains.

Resumo Na atualidade, o albendazol é uma das drogas mais usadas devido à sua acessibilidade econômica. O objetivo foi avaliar a histopatologia dos espécimes de Fasciola hepatica. Para isso, foi realizado o teste de eficácia em ovinos tratados com albendazol na dose recomendada para Fasciola hepatica, no qual os helmintos recuperados em necropsia foram contabilizados e separados para histologia. As células espermatogênicas de parasitas recuperados de ovinos tratados e controle foram examinadas por microscopia. O teste de redução de ovos por grama de fezes revelou 97,06% de eficácia do albendazol no tratamento de F. hepatica. As alterações nas células dos túbulos testiculares iniciaram-se 48 horas após o tratamento e tornaram-se evidentes em 72 horas, altura em que tornou-se difícil identificar os tipos de células. As espermatogônias primárias e secundárias tornaram-se cada vez mais raras e a vacuolização intercelular foi mais evidente. Sinais de apoptose, com núcleos picnóticos e evidência de cariorrexia foram observados em todos os momentos. Os detritos celulares foram identificados 96 horas após o tratamento. Os resultados indicaram que a espermatogênese parasitária foi severamente afetada pelo albendazol e demonstrou a importância do uso da histopatologia para o diagnóstico de eficácia terapêutica em cepas de campo.