Search

Fasciolosis is a food-borne anthropozoonotic disease caused by Fasciola spp. that affects multiple hosts, including ruminants and humans. In vitro testing of anthelmintics is of interest to establish the drug's activity without the need for time-consuming and expensive in vivo assays. This study was set to establish a discriminatory dose (DD) by running a dose-titration in vitro experiment (egg hatch test, EHT) of albendazole sulfoxide (ABZ.SO) and nitroxynil (NTX) on eggs of a field strain of Fasciola hepatica. Eggs were recovered from adult parasites isolated from intact bovine livers obtained from a single farm in Paraná, Brazil (FhPar2022 strain) with no ABZ or NTX treatment history. Two hundred eggs were exposed to 18 and 14 concentrations of ABZ.SO and NTX, respectively, for 12h and incubated for 16 days. Egg development and integrity were determined every other day, establishing an index of morphological modification of the different phases. A concentration-dependent effect was observed for egg development in both compounds. ABZ.SO solutions prevent egg hatch, except for the two lowest concentrations. We observed no egg hatch at 6.250-100.0 μmol L -1 for NTX. NTX had an inhibition concentration of 50% (IC 50 ) of 0.043 μmol L -1 with a correlation coefficient of (R 2 ) 0.961. ABZ.SO had an IC 50 of 0.00099 μmol L -1 with a low R 2 of 0.417. Morphological damage was also associated with the increasing concentration of both drugs. Moreover, it was noted that most eggs that reached the eye spot type could hatch, except at 0.39 and 3.12 μmol L -1 of NTX. In ABZ.SO, hatching occurred only at 0.00038, 0.0007, and 0.0015 μmol L-1 concentrations. The obtained DDs of 0.043 μmol L -1 for NTX and 0.00099 μmol L -1 for ABZ.SO can be used to monitor efficacy in field isolates., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Desiree Vera Pontarolo reports financial support was provided by Coordination of Higher Education Personnel Improvement. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis. We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34-169 min and low intrinsic clearance values (<13 μL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules., Competing Interests: Declaration of competing interest None, (Copyright © 2023. Published by Elsevier Inc.)

Fasciola hepatica, the liver trematode, infects ruminants and causes economic loss. Because parasites are developing resistance to commercial drugs, the negative effects of parasitism are increasing. In this study, we aimed to evaluate the efficacy of cumin (Cuminum cyminum) essential oil against F. hepatica eggs and adults. The eggs were incubated with eight concentrations of the essential oil (0.031125-4.15 mg/mL), and viable eggs were counted after 14 days and classified as embryonated or non-embryonated. Adult flukes were incubated in Roswell Park Memorial Institute medium to ensure their viability and then incubated in essential oil. They were observed for 24 h after treatment. The adults were assessed with the two lowest effective oil concentrations used in the ovicidal test. Three controls were used for both tests: nitroxynil, a negative control, and Tween®80. After incubation in oil, the adult specimens were processed for histological analysis and stained with hematoxylin-eosin. In addition, the oil was tested for cytotoxicity using Madin-Darby bovine kidney cells to assess any possible effect on them. The oil was effective in ovicidal and adulticidal inhibition of the trematode, with statistically significant results. All concentrations assessed in the ovicidal test were 100% effective. The adult test was effective within 15 h and inactivated all the specimens at the highest concentration evaluated (0.06225 mg/mL). Histological analysis showed that cumin essential oil resulted in marked areas of vacuolization. The spines showed no structural changes but were surrounded by microvesicles. These findings indicated that cumin oil could be a potential compound in the control of fasciolosis., Competing Interests: Declaration of competing interest The authors declare there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This study investigated the prevalence of bovine liver condemnation due to Fasciola hepatica in abattoirs and its influence on livestock productivity. It also explored risk factors such as season, breed, age and sex in the states of Puebla and Veracruz, Mexico. A total of 5281 cattle livers were inspected in two abattoirs. The overall prevalence of the parasite in cattle throughout 2020 was 24.9% (1313 out of 5281). The highest prevalence was found in Veracruz (28.5%; tropical climate), followed by Puebla (18.4%; temperate climate). The prevalence of fluke infection during the wet season was 19.0% and 25.8% in the states of Puebla and Veracruz, respectively; during the dry season, this was 18.0% and 30.2% respectively. The annual loss caused by condemnation of the liver for both abattoirs was estimated to be US$7502. Using multivariate logistic regression analysis, two models were constructed for potential association with the prevalence of condemned livers, using season, breed, age and sex as risk factors. The results indicate that season, age and sex were significantly associated with an increased risk of parasitic infection. Cattle age was the variable most strongly associated with F. hepatica infection, with the highest prevalence in cattle over three years of age as compared to younger animals, both in the states of Veracruz with a tropical climate (OR 6.443; 95% CI: 4.487-9.251) and in Puebla with a temperate climate (OR 2.854; 95% CI: 1.531-5.321). The results indicate that different factors were significantly associated with the prevalence of condemned livers, and it highlights the importance of continuous monitoring programmes for F. hepatica infection and preventing economic losses in cattle production., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Fasciola hepatica, a worldwide-distributed liver fluke, is one of the causative agents of fasciolosis, a zoonotic disease that affects livestock and humans. In livestock, fasciolosis causes huge economic losses worldwide, reducing animal fertility, milk production, weight gain and condemnation of livers. In spite of the availability of drugs, such as triclabendazole (TCZ), for the treatment of fasciolosis, they do not necessarily prevent liver damage or parasite reinfection and can eventually increase parasite resistance. The aim of this research was to relate the hepatic function, haematological parameters, leukocyte counts in circulation and parasite egg shedding during F. hepatica acute and chronic phases of infection in cattle as well as to determine how these parameters change with TCZ-treatment of chronically infected cattle. Our results show that increased levels of serum aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) were detected in early stages of the experimental infection. Moreover, high circulating eosinophil count and plateletcrit levels were correlated with fluke number in livers from infected cattle. On the other hand, although TCZ-treatment in the chronic phase of infection reduced parasite burden and damage in the liver, it was not able to completely avoid them. In conclusion, our work sheds light into the physiopathological mechanisms induced during fluke infection in cattle, revealing the complexity of the host response to the infection, together with the effects of TCZ-treatment in chronically infected animals., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMɑ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host., Competing Interests: Declaration of competing interest Authors have no conflicts of interest., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fasciolosis is a zoonotic world widely distributed disease caused by the liver fluke Fasciola hepatica, which affects animals and occasionally humans. On the other hand, natural iron oxide particles like magnetite are commonly found in soils where they participate in a wide range of environmental processes like organic matter decomposition, the adsorption of ions and molecules, and chemical reactions that involve the participation of soil living microorganisms. Since Fasciola eggs become soil components after being released with the infected animal faeces, this study focused on the characterization of the natural interaction between natural sub-micrometric magnetite particles and F. hepatica eggs. Our results indicate that particle binding to the F. hepatica egg depends on the particle size and it is also related to the exposed surface area since any condition that favors particle agglomeration leads to the reduction of the particle-eggshell binding intensity. Interestingly, this binding was avoided when proteins or phosphate were incorporated to the incubation solution, but not after formaldehyde fixation of eggs. Finally, when eggs were exposed to an external magnet after being incubated with magnetite particles, they were attracted to it without particles being detached, indicating a strong type of bonding between them. Therefore, the results presented here give new insights in order to improve the possibility of harvesting F. hepatica eggs by using magnetic materials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fasciolosis is an important plant borne trematode zoonosis in ruminants caused by the Fasciola hepatica and Fasciola gigentica, It is classified as a neglected tropical disease and found in more than 50 countries especially where sheep and cattle are reared. Fasciolosis is a serious animal health problems in many rural and urban areas of world, causing significant financial losses due to decrease in production and viscera condemnation in animals. Accurate diagnosis of fasciolosis is always remained a challenging task for the field practitioners. There is no comprehensive summary on the occurrence and distribution of the infection at international level. Therefore, we intended to provide a complete overview on the prevalence and epidemiology of fasciolosis in farm animals from a global prospective. It includes to map the global distribution of fasciolosis in different areas of the world to identify the endemic regions which may be a source of potential disease outbreak. The financial liability related to fasciolosis on the livestock production has also been addressed. For this purpose, the published data during 2000-2015 (15 years) on fasciolosis was reviewed and collected by electronic literature search of four databases including Google, PubMed, Science Direct, and Web of Science. Data presented are contemplated to enhance our current understanding of the parasite's geographical distribution, host range, and economic losses. Information provided would be useful for the application of more effective control strategies against fasciolosis in different geo-economics regions of the world., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Fasciola hepatica is a well-known helminth parasite, with significant economic and public health importance all over the world. It has been known since more than 630 years ago and a considerable research work has been carried out on the life cycle of this important parasite. In the hepatic phase of the life cycle of F. hepatica, it is assumed that the young flukes, after about 6-7 weeks of migration in the liver parenchyma, enter into the bile ducts of the definitive hosts and become sexually mature. Even though the secretion of cysteine peptidases including cathepsin L and B proteases by F. hepatica may justify this opinion, because of several scientific reasons and based on the experimental studies conducted in different animals (reviewed in this article), the entry of parasites into the bile ducts, after their migration in the liver parenchyma seems to be doubtful. However, considering all the facts relating to the hepatic and biliary phases of the life cycle of F. hepatica, two alternative ideas are suggested: 1) some of the migrating juvenile flukes may enter into the bile ducts immediately after reaching the liver parenchyma while they are still very small, or 2) when newly excysted juvenile flukes are penetrating into the intestinal wall to reach the liver through the abdominal cavity, a number of these flukes may enter into the choleduct and reach the hepatic bile ducts, where they mature. According to the previously performed natural and experimental studies in different animals and human beings, the supporting and opposing evidences for the current opinion as well as the evidences that might justify the two new ideas are reviewed and discussed briefly. In conclusion, our present knowledge about the time and quality of the entry of F. hepaticas into the bile ducts, seems to be insufficient, therefore, there are still some dark corners and unknown aspects in this field that should be clarified., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Anthelmintic resistance in livestock parasites is currently a worldwide problem. Fasciola hepatica is a cosmopolitan parasite which causes considerable loss in sheep and cattle production systems all over the world. Chemotherapy is currently the main tool available for its control. The intensive use of triclabendazole, the drug of choice for more than 20 years, has resulted in the development of resistant strains. The therapeutic options are adulticides such as closantel (salicylanilide anthelmintic that binds extensively to plasma albumin) to treat chronic fascioliasis in sheep, and cattle. In the present work, an Egg Hatch Assay (EHA) and morphometric studies were used to evaluate in vivo the ovicidal activity and morphology F. hepatica eggs, recovered from closantel treated sheep collected at different time intervals post treatment. Statistically significant differences (p < 0.0001) were observed in egg morphometry between the control and the treated groups in all the parameters studied. Eggs recovered from treated animals tend to be narrower and longer. Significant differences were found in the embryonation and hatching of eggs between 36 h post treatment (32, 5%) vs. approximately 85% in control, 12 h and 24 h post treatment. Our results confirm that closantel affects in vivo the normal development of the eggs. As one of the first effects, this drug affects the performance of the trematode's reproductive physiology. Even though closantel treated animals may still eliminate eggs in the first days post treatment, these are not viable., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Molecular techniques were used to identify Fasciola species collected from Chiang Mai Thailand. Morphometrically, 65 stained and 45 fresh worms collected from cattle suggested the possible occurrence of both F. gigantica and F. hepatica. Twenty-two worms comprising 15 from cattle and 7 from human patients, were identified subsequently based on three genetic markers: mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2). All of them presented the F. gigantica type in maternally inherited mitochondrial sequences (nad1 and cox1), with six types in each sequence (FgNDI-CM1 to FgNDI-CM6 and FgCOI-CM1 to FgCOI-CM6, respectively). Remarkably, the predominant nad1 type, FgNDI-CM6, was identical to that of aspermic Fasciola sp. formerly reported from Thailand, Japan, Korea, China, Vietnam, and Myanmar. ITS2 sequences were analyzed successfully in 20 worms. Fifteen worms showed the F. gigantica type and five (including one worm from a patient) had mixed ITS2 sequences of both F. gigantica and F. hepatica in the same worms, with additional heterogeneity within both ITS2 types. This study revealed the intermediate form of Fasciola coexisting with F. gigantica for the first time in Thailand.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fascioliasis is a food-borne parasitic disease caused by the trematode species Fasciola hepatica, distributed worldwide, and Fasciola gigantica, restricted to given regions of Africa and Asia. This disease in humans shows an increasing importance, which relies on its recent widespread emergence related to climate and global changes and also on its pathogenicity in the invasive, biliary, and advanced chronic phases in the human endemic areas, mainly of developing countries. In spite of the large neurological affection capacity of Fasciola, this important pathogenic aspect of the disease has been pronouncedly overlooked in the past decades and has not even appear within the numerous reviews on the parasitic diseases of the central nervous system. The aim of this wide retrospective review is an in-depth analysis of the characteristics of neurological and ocular fascioliasis caused by these two fasciolid species. The terms of neurofascioliasis and ophthalmofascioliasis are restricted to cases in which the direct affection of the central nervous system or the eye by a migrant ectopic fasciolid fluke is demonstrated by an aetiological diagnosis of recovered flukes after surgery or spontaneous moving-out of the fluke through the orbit. Cases in which the ectopic fluke is not recovered and the symptoms cannot be explained by an indirect affection at distance may also be included in these terms. Neurofascioliasis and ophthalmofascioliasis cases are reviewed and discussed. With regard to fascioliasis infection giving an indirect rise to neurological affection, the distribution and frequency of cases are analysed according to geography, sex, and age. Minor symptoms and major manifestations are discussed. Three main types of cases are distinguished depending on the characteristics of their manifestations: genuine neurological, meningeal, and psychiatric or neuropsychic. The impressive symptoms and signs appearing in each type of these cases are included. Brain examination techniques and neuroimaging useful for the diagnosis of neurological cases are exposed. Within fascioliasis infection indirectly causing ocular manifestations, case distribution and frequency are similarly analysed. A short analysis is devoted to clarify the first reports of a human eye infection. The affection of related and close organs is discussed by differentiating between cases of the dorsal spine, pulmonary manifestations, heart and vessel affection, findings in blood vessels, skin and dermatologic reactions, cases of ectopic mature flukes, and upper body locations. The clinical complexity of the puzzling polymorphisms, the disconcerting multifocality of the manifestations, and their changes along the evolution of the disease in the same patient, as well as the differences between the clinical pictures shown by different patients, are highlighted. The many syndromes involved are enumerated. The pathogenic and physiological mechanisms underlying neurofascioliasis and ophthalmofascioliasis caused by ectopic flukes and the physiopathogenic processes indirectly affecting the central nervous system and causing genuine neurological, meningeal, psychiatric, and ocular manifestations are discussed. The diagnosis of neurological and ophthalmologic fascioliasis is analysed in depth, including clinical and paraclinical diagnosis, eosinophilia in the blood and cerebrospinal fluid, differential diagnosis from other parasitic infections such as helminthiases and myiases, an update of human fascioliasis diagnosis, and fluke and/or fluke egg recovery by surgery. Diagnostic analyses with faecal and blood samples for fascioliasis patients are updated. Therapy for patients with major neurological manifestations includes both antiparasitic treatments and anti-inflammatory therapeutics. Prognosis in fascioliasis patients with neurological manifestations is discussed, with emphasis on sequelae and fatal cases, and the care of patients with ophthalmologic manifestations is added. Conclusions indicate that neurological cases are overlooked in human fascioliasis endemic areas and also in developing countries in general. In remote zones, rural health centres and small hospitals in or near the human endemic areas do not dispose of the appropriate equipments for neurological analyses. Moreover, physicians may not be aware about the potential relationship between liver fluke infection and neurological implications, and such cases may therefore remain misdiagnosed, even in developed countries. Priority should henceforth be given to the consideration of neurological and ocular affection in human endemic areas, and efforts should be implemented to assess their characteristics and frequency. Their impact should also be considered when estimating the global burden of fascioliasis., (© 2014 Elsevier Ltd All rights reserved.)

The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n=66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus, Turkmenistan, USA, Tunisia and Algeria, indicating that this is the main haplotype involved in the spread of F. hepatica throughout all continents., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Isothermal microcalorimetry (IMC) is an analytical tool that continuously measures the heat flow generated by chemical, physical or biological processes. We have demonstrated that IMC is a useful tool to analyze drug effects on helminths, including adult Fasciola hepatica. Here, we used IMC to examine the activity of triclabendazole and its metabolites triclabendazole sulphone and triclabendazole sulphoxide on juvenile and adult F. hepatica. Worms (one adult or 2-3 juveniles) were placed in 4 or 20 ml glass ampoules containing RPMI 1640 and the test compound (25-100 μg/ml) and the heat flow and motility of worms was examined with TAM48 and TAMIII isothermal microcalorimetry instruments. IMC was found to be precisely document drug effects on juvenile F. hepatica and confirmed the pronounced effect of the benzimidazole derivatives on the motor activity of F. hepatica. Juvenile F. hepatica incubated with 100 μg/ml triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements 8.3, 35 and 6h post-incubation (all p<0.001). The metabolic heat of triclabendazole sulphoxide treated worms (100 μg/ml) was reduced by 50% and 76% 24 and 120 h post-incubation, respectively. Limitations of calorimetric measurements were observed using adult F. hepatica as only three worms could be measured simultaneously and also control worms showed a considerable decrease in heat flow. Adult F. hepatica exposed to triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements after 31 (p=0.009), 49 (p>0.05) and 88 (p>0.05)h. In conclusion, IMC is useful to document drug effects on juvenile F. hepatica and since rapid technological developments in this field are occurring IMC might also hold promise to study adult F. hepatica in the near future., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n=60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene. Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fasciolosis caused by Fasciola spp. (Platyhelminthes: Trematoda: Digenea) is considered as the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. In the endemic regions of the North of Iran, Fasciola hepatica and Fasciola gigantica have been previously characterized on the basis of morphometric differences, but the use of molecular markers is necessary to distinguish exactly between species and intermediate forms. Samples from buffaloes and goats from different localities of northern Iran were identified morphologically and then genetically characterized by sequences of the first (ITS-1) and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA). Comparison of the ITS of the northern Iranian samples with sequences of Fasciola spp. from GenBank showed that the examined specimens had sequences identical to those of the most frequent haplotypes of F. hepatica (n=25, 48.1%) and F. gigantica (n=20, 38.45%), which differed from each other in different variable nucleotide positions of ITS region sequences, and their intermediate forms (n=7, 13.45%), which had nucleotides overlapped between the two Fasciola species in all the positions. The ITS sequences from populations of Fasciola isolates in buffaloes and goats had experienced introgression/hybridization as previously reported in isolates from other ruminants and humans. Based on ITS-1 and ITS-2 sequences, flukes are scattered in pure F. hepatica, F. gigantica and intermediate Fasciola clades, revealing that multiple genotypes of Fasciola are able to infect goats and buffaloes in North of Iran. Furthermore, the phylogenetic trees based upon the ITS-1 and ITS-2 sequences showed a close relationship of the Iranian samples with isolates of F. hepatica and F. gigantica from different localities of Africa and Asia. In the present study, the intergenic transcribed spacers ITS-1 and ITS-2 showed to be reliable approaches for the genetic differentiation of Fasciola spp., providing bases for further studies on F. hepatica, F. gigantica and their intermediate forms in the endemic areas in Asia., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We compared the sedimentation and FLOTAC techniques for the detection and quantification of Fasciola hepatica eggs in faecal samples obtained from 120 experimentally-infected rats before intervention, and in 42 rats after drug administration. Additionally, the average time for a single test was determined. A single FLOTAC showed a higher sensitivity (92.6%) than 2, 4 and 8 sedimentation readings (63.0-85.2%) for detecting F. hepatica eggs in rat faeces post-treatment. On average, it took 21 min to prepare and examine a single FLOTAC, whereas 114 min were needed for the sedimentation method including the reading of 8 slides. In both treated and untreated rats, the sedimentation method resulted in higher mean faecal egg counts (FECs) than FLOTAC (P<0.05). In view of the high sensitivity and efficiency, the FLOTAC technique holds promise for experimental work in the F. hepatica-rat model. Additional research is needed to determine the reasons for the observed differences in FECs., (Copyright 2010 Elsevier Inc. All rights reserved.)

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes., (Copyright 2009 Elsevier Inc. All rights reserved.)

Ribosomal RNA sequences (361 or 362bp) of the second internal transcribed spacer 2 (ITS-2) and a portion of mitochondrial cox1 (423bp) for Fasciola spp. obtained from specimens collected in indigenous and hybrid goats and sheep in Vietnam were characterized for genotypic status and hybridization/introgression. Alignment of 48 ITS-2 sequences (also those from goats and sheep in this study) indicates that F. gigantica and F. hepatica differ typically from each other at seven sites whereas one of these is a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. The isolates from the mountainous goats in the North of Vietnam (Yen Bai province) showed the ITS-2 composition relatively identical to that of F. hepatica. The ITS-2 sequences from populations of Fasciola isolates in goats had probably experienced introgression/hybridization as reported previously in other ruminants and humans. All Vietnamese goat-of-origin specimens had high pairwise percentage of mitochondrial cox1 sequences to F. gigantica (97-100%), and very low identity to F. hepatica (91-93%), suggesting their maternal linkage to be traced to F. gigantica. The presence of hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the goats/sheep in Vietnam, regardless of indigenous or imported hosts, appears to be the first demonstration from a tropical country.

Fasciola hepatica and Fasciola gigantica are trematode parasites responsible for fasciolosis, a disease of ruminant animals which is also increasingly recognised as a disease in humans. By biochemical and in silico methods, we have cloned and characterised the 70 kDa heat-shock proteins (HSP70s) of F. hepatica and F. gigantica. The nucleotide and protein sequences for HSP70 were found to be 98% and 99% identical between liver fluke species, respectively, and to encode conserved amino acid motifs that are of putative functional importance. Western blot analysis demonstrated that HSP70 proteins were expressed at a higher level in F. gigantica recovered from sheep relative to F. hepatica, but HSP70 was not detected in the excretory-secretory products of these liver fluke samples. Real-time reverse-transcriptase PCR analysis of HSP70 expression in parasites from sheep, but not cattle, showed HSP70 expression to be higher in F. gigantica than F. hepatica. These results suggest that hosts refractory to F. gigantica are associated with higher HSP70 expression by this parasite and that HSP70 expression may represent a biochemical marker of the stress response of F. gigantica.

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.

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.

Fascioliasis is of well-known veterinary importance and an increasing human health problem, with reported cases in the five continents. The causative agents, Fasciola hepatica and Fasciola gigantica, present geographical distributions, which overlap in many regions of Africa and Asia, and in which the differentiation of both species is usually difficult because of the many variations in their morphological characteristics. Moreover, in humans, liver fluke classification cannot be achieved by clinical, pathological, coprological or immunological methods. The differential diagnosis between F. hepatica and F. gigantica infection is very important because of their different transmission and epidemiological characteristics. A simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the common restriction enzymes Ava II and Dra II, is described to distinguish between both fasciolid species. It is based on a 618-bp-long sequence of the 28S rRNA gene recently obtained from liver fluke populations of South America, Europe and Africa. This sequence showed a few nucleotide differences between both fasciolids and no intraspecific variations within each species. This assay provides unambiguous results and may be useful for both individual subject diagnosis and epidemiological surveys of humans and animals in endemic regions of sympatry.

There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infection. A cysteine proteinase complex identified in the regurgitant of adult F. hepatica was examined in this context. The thiol-cathepsin-related proteinases of M(r) 28,000 were purified and tested in vaccine trials of sheep infected with liver fluke. Ten animals were immunised with the purified proteinases and developed antibodies to the cysteine proteinases prior to challenge with F. hepatica metacercariae. Infection appeared to cause a boost in antibody response by Week 4 into infection, and antibody levels were generally sustained throughout infection. The cysteine proteinases are not novel antigens, since low-level antibody titres were also detected in nonimmunised controls by late infection. On completion of the trial, there was no difference in worm burden between the two groups. However, faecal egg counts and therefore worm fecundity were significantly decreased.

Using transmission and scanning electron microscopy, the early migration of juvenile Fasciola hepatica was examined in naive and resistant rats. In naive rats, the migration of flukes to the peritoneal cavity was uneventful. In resistant rats, flukes were rapidly coated with antibody whilst still in the gut lumen and a proportion of the flukes were unable to penetrate the intestinal wall. Those that did penetrate were unharmed as they crossed the gut wall, but on entering the peritoneal cavity they were coated with antibody and host cells including eosinophils, neutrophils, macrophages, and mast cells. Eosinophils were seen degranulating onto the fluke surface, and this appeared to result in the erosion of the tegumental syncytium.

Bile ducts were examined histochemically to compare the effects of proline infusion with Fasciola hepatica implantation in rats. After 3 weeks of infusion or implantation, both proline and F. hepatica produced increases in the luminal perimeter and collagen content of the bile duct. However, the effect of the parasite was significantly greater than that of proline, and the parasite produced significant increases in the bile duct wall. These results corroborate earlier biochemical and histological studies indicating the important role of proline in the enlargement of the bile duct in fascioliasis.