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ABSTRACT Background Fascioliasis represents one of the most significant parasitic and foodborne zoonotic diseases in the world. Resistance to currently deployed human and veterinary flukicides is a growing health problem. Zinc oxide nanoparticles (ZnO‐NPs) have developed enormous importance in nanomedicine. The current study aims to evaluate the flukicide activity of ZnO‐NPs on the adult stages of Fasciola hepatica, in vitro assay. Methods Mature flukes of F. hepatica were collected from the biliary tracts of cattle liver from the main slaughterhouses in Aswan Governorate, Egypt. The co‐precipitation process was utilized to create ZnO‐NPs. The adult active flukes of F. hepatica (n = 6) were transferred in Petri dishes (triplicates) containing different concentrations of ZnO‐NPs (0.05, 0.10 and 0.12 mg/mL), with simultaneous maintenance of control in only PBS. The treated and control groups of flukes were maintained inside an incubator at 37°C ± 1°C. Mortality and paralysis of the flukes were observed. The adult flukes were picked up for scanning electron microscopy (SEM) examination from the control medium, and those exposed to the highest effective concentration of ZnO‐NPs. ZnO‐NPs showed dose‐dependent flukicide efficacy against the adult flukes. Results At 0.12 mg/mL concentration of ZnO‐NPs, the mortality of flukes was attained early in 4.05 ± 0.033 h. The photomicrographs obtained by SEM of the flukes revealed that ZnO‐NPs (0.12 mg/mL) had a potent effect on the integument surface and genital system of F. hepatica. Most of the tegument was damaged with complete erosion and loss of the spines and papillae. Furthermore, a severe rupture in the genital system causes the uterus to burst and releases eggs through the genital pore. Conclusions Hence, it could be concluded that ZnO‐NPs performed effective anthelmintic activity against the adult stages of F. hepatica, in vitro. The results of the present study recommend the use of ZnO‐NPs as new preparations with anthelmintic activity.

Pharmacological treatment remains essential to control fasciolosis in areas where infection is endemic. However, there are major constraints to treating food-producing animals. Of particular concern is the lack of flukicides for treating early Fasciola infections in ruminant livestock in some countries. In addition, the information provided in package leaflets, particularly regarding withdrawal periods, is often incomplete, confusing, and/or contradictory. International regulatory bodies should harmonize the use of flukicides in livestock in favor of fairer, safer international trade. In addition, monitoring the efficacy of fasciolicides on farms is also essential to minimize the spread of drug-resistant populations of Fasciola. The current situation regarding flukicide formulations in the European Union and other, non-European countries is analyzed in this review paper. Chemotherapy remains the main tool for controlling fasciolosis in endemic areas. However, treatment of ruminant livestock, the main reservoir of the disease, faces severe technical and legal challenges. The scarce number of flukicide drugs is particularly challenging for some productive sectors. Therapeutic failures have been reported for all available flukicides, but the causes have rarely been identified. This situation is a matter of concern and should be considered a research priority. The information provided in package leaflets about the conditions of use of flukicides is not always as detailed and accurate as it should be. Moreover, the efficacy of treatments is not usually monitored. There are unexplained significant differences between countries, and even within some countries, regarding the withdrawal periods established for veterinary medicines with very similar pharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12–16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65–77%) and 97% (95% CI: 95–98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12–16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle.

G protein-coupled receptors (GPCRs) are established drug targets. Despite their considerable appeal as targets for next-generation anthelmintics, poor understanding of their diversity and function in parasitic helminths has thwarted progress towards GPCR-targeted anti-parasite drugs. This study facilitates GPCR research in the liver fluke, Fasciola hepatica , by generating the first profile of GPCRs from the F. hepatica genome. Our dataset describes 147 high confidence GPCRs, representing the largest cohort of GPCRs, and the largest set of in silico ligand-receptor predictions, yet reported in any parasitic helminth. All GPCRs fall within the established GRAFS nomenclature; comprising three glutamate, 135 rhodopsin, two adhesion, five frizzled, one smoothened, and one secretin GPCR. Stringent annotation pipelines identified 18 highly diverged rhodopsins in F. hepatica that maintained core rhodopsin signatures, but lacked significant similarity with non-flatworm sequences, providing a new sub-group of potential flukicide targets. These facilitated identification of a larger cohort of 76 related sequences from available flatworm genomes, representing new members of existing groups (PROF1/Srfb, Rho-L, Rho-R, Srfa, Srfc) of flatworm-specific rhodopsins. These receptors imply flatworm specific GPCR functions, and/or co-evolution with unique flatworm ligands, and could facilitate the development of exquisitely selective anthelmintics. Ligand binding domain sequence conservation relative to deorphanised rhodopsins enabled high confidence ligand-receptor matching of seventeen receptors activated by acetylcholine, neuropeptide F/Y, octopamine or serotonin. RNA-Seq analyses showed expression of 101 GPCRs across various developmental stages, with the majority expressed most highly in the pathogenic intra-mammalian juvenile parasites. These data identify a broad complement of GPCRs in F. hepatica , including rhodopsins likely to have key functions in neuromuscular control and sensory perception, as well as frizzled and adhesion/secretin families implicated, in other species, in growth, development and reproduction. This catalogue of liver fluke GPCRs provides a platform for new avenues into our understanding of flatworm biology and anthelmintic discovery. [ABSTRACT FROM AUTHOR]

Completion of the F. hepatica lifecycle is dependent on suitable climatic conditions for development of immature stages of the parasite, and its snail intermediate host. Few investigations have been conducted regarding temporal variations in F. hepatica status in Irish dairy herds. The current study aimed to conduct a longitudinal study examining annual and seasonal trends in bulk milk seropositivity over six years, while also investigating associations with soil temperature, rainfall and flukicide treatment. Monthly bulk milk samples (BTM) were submitted by 28 herds between March 2009 and December 2014. In all, 1337 samples were analysed using a Cathepsin L1 ELISA. Soil temperature, rainfall and management data were obtained for general estimating equation and regression analyses. A general decrease in milk seropositivity was observed over the six year study period and was associated with an increased likelihood of treating for liver fluke (OR range = 2.73–6.96). Annual and seasonal analyses of rainfall and F. hepatica BTM status yielded conflicting results. Higher annual rainfall (>1150 mm) yielded a lower likelihood of being BTM positive than annual rainfall of <1000 mm (OR = 0.47; P = 0.036). This was most likely due to farmers being more proactive in treating for F. hepatica in wetter years, although a ‘wash effect’ by high rainfall of the free living stages and snails cannot be ruled out. Higher seasonal rainfall (>120 mm), however, was associated with increased ELISA S/P% values (Coefficient = 9.63S/P%; P = 0.001). Soil temperature was not found to influence F. hepatica to the same extent as rainfall and may reflect the lack of severe temperature fluctuations in Ireland. Flukicides active against both immature and mature F. hepatica were approximately half as likely to record a positive F. hepatica herd BTM status than a flukicide active against only the mature stage of the parasite (OR ≅ 0.45; P < 0.01). This study highlights the importance of examining both annual and seasonal F. hepatica data, which can vary significantly. Additionally, it highlights the progress that can be achieved in fluke control by application of a continuous BTM monitoring program. [ABSTRACT FROM AUTHOR]

Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12–16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65–77%) and 97% (95% CI: 95–98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12–16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle. [ABSTRACT FROM AUTHOR]

Liver fluke is the most important trematode parasite of cattle in the UK, and is endemic. Incidence and geographic range of the parasite has increased rapidly in the last 10 years. Coupled with a complex life cycle and welfare and farm profitability impacts, there are several considerations the clinician must take into account when dealing with an outbreak, or designing a control programme. This article discusses liver fluke in cattle in the United Kingdom. As well as a parasitological summary, clinical signs, diagnosis, treatment and control options are discussed. [ABSTRACT FROM AUTHOR]

Triclabendazole (TCB) is a flukicide used in the treatment of liver fluke in cattle; however, its use is currently prohibited in lactating dairy cows. In this study, following administration of 10% Fasinex (triclabendazole, Novartis Animal Health UK Ltd., Camberley, UK) the milk of 6 animals was used to manufacture dairy products, to ascertain if TCB residues in milk migrate into dairy products. The detection limit of the ultra-high-performance liquid chromatography-tandem mass spectrometry method used was 0.67 µg/kg. The highest concentrations of TCB residue measured, within the individual cow milk yield, was 1,529 ± 244 µg/kg (n = 6), on d 2 posttreatment. Days 2 and 23 posttreatment represented high and low residue concentrations, respectively. At each of these 2 time points, the milk was pooled into 2 independent aliquots and refrigerated. Milk products, including cheese, butter, and skim milk powder were manufactured using pasteurized and unpasteurized milk from each aliquot. The results for high residue milks demonstrated that TCB residues concentrated in the cheese by a factor of 5 (5,372 vs. 918 µg/kg for cheese vs. milk) compared with the starting milk. Residue concentrations are the sum of TCB and its metabolites, expressed as keto-TCB. Residues were concentrated in the butter by a factor of 9 (9,177 vs. 1,082 µg/kg for butter vs. milk) compared with the starting milk. For milk, which was separated to skim milk and cream fractions, the residues were concentrated in the cream. Once skim milk powder was manufactured from the skim milk fraction, the residue in powder was concentrated 15-fold compared with the starting skim milk (7,252 vs. 423 µg/kg for powder vs. skim milk), despite the high temperature (185°C) required during powder manufacture. For products manufactured from milk with low residue concentrations at d 23 posttreatment, TCB residues were detected in butter, cheese, and skim milk powder, even though there was no detectable residue in the milk used to manufacture these products. Triclabendazole residues were concentrated in some milk products (despite manufacturing treatments), exceeding residue levels in the starting milk and, depending on the storage conditions, may be relatively stable over time. [ABSTRACT FROM AUTHOR]

Rafoxanide is an effective treatment for the control of fluke infections in animals, but it is currently not permitted for treating animals whose milk is intended for human consumption. In this study, the persistence of rafoxanide residues in milk, and their migration to dairy products, was investigated following the treatment of six lactating dairy cows with Curafluke 10% oral drench. The highest concentration of rafoxanide residues detected in the individual cows milk ranged from 249 to 627 μg kg−1and occurred at 2–3 days post-treatment. At 2 and 23 days post-treatment (representing high and low residue concentrations) the milk was pooled into two independent aliquots, each containing the full day’s milk produced by three cows. Milk products were made from pasteurised and unpasteurised milk. Pasteurisation appeared to have little impact on the stability of the residues. Rafoxanide concentrated sixfold in the cheese (week 0) compared to the starting milk (2070 vs. 349 μg kg−1) but was four times lower in whey (75 μg kg−1). Rafoxanide residues were up to 14 times higher in butter (week 0) than in the starting milk (5468 vs. 376 μg kg−1). Residues were found to further concentrate in butter and cheese at longer storage and ripening times, respectively. Skim-milk powder was manufactured from skim milk, and residues were 10-fold higher than in the starting skim milk (5468 vs. 376 μg kg−1) despite the 185°C temperature required for the process. Rafoxanide residues were stable in this skim-milk powder when stored at ambient temperature for at least 1 year. Results showed that detectable rafoxanide residues were excreted in milk for 47 days, and concentrated in the fat-based products. The analytical ranges of the UHPLC-MS/MS method used were 1.0–200 μg kg−1(milk and whey) and 10–2000 μg kg−1(other dairy products). [ABSTRACT FROM PUBLISHER]