The food-borne trematodes, Opisthorchis viverrini and Clonorchis sinensis, are classified as group 1 biological carcinogens: definitive causes of cancer. By contrast, infections with Fasciola hepatica, also a food-borne trematode of the phylum Platyhelminthes, are not carcinogenic. This review explores the premise that the differential activation of macrophages during infection with these food-borne trematodes is a major determinant of the pathological outcome of infection. Like most helminths, the latter stages of infection with all 3 flukes induce M2 macrophages, a phenotype that mediates the functional repair of tissue damaged by the feeding and migratory activities of the parasites. However, there is a critical difference in how the development of pro-inflammatory M1 macrophages is regulated during infection with these parasites. While the activation of the M1 macrophage phenotype is largely suppressed during the early stages of infection with F. hepatica, M1 macrophages predominate in the bile ducts following infection with O. viverrini and C. sinensis. The anti-microbial factors released by M1 macrophages create an environment conducive to mutagenesis, and hence the initiation of tumour formation. Subsequently, the tissue remodelling processes induced by the M2 macrophages promote the proliferation of mutated cells, and the expansion of cancerous tissue. This review will also explore the interactions between macrophages and parasite-derived signals, and their contributions to the stark differences in the innate immune responses to infection with these parasites.

The parasite Fasciola hepatica is an important zoonotic parasite. The development of an animal model of F. hepatica's life cycle is critical for studying the biological characteristics of the parasite in snails and mammals. Eggs of F. hepatica of bovine origin were cultured, and metacercariae were obtained after infection of Galba pervia snails. The life cycle system of F. hepatica was initiated in 2 different animals by orally infecting rabbits, SD rats and Kunming mice with the metacercariae. The animals' survival after infection, parasite migration in the animals and pathological damage to the liver were observed. We discovered that rabbits died due to acute suppurative hepatitis 60–69 days after infection, and eggs were found in the feces on day 63 of infection. The liver of SD rats showed punctate lesions on day 3 of infection, and further changes occurred as the infection progressed. However, liver repair was observed at week 9. SD rats survived for more than a year after infection and continued the F. hepatica life cycle. The liver lesions in Kunming mice after infection were similar but more severe than those in SD rats. Death was observed on the 31st post-infection day. We discovered that while rabbits, SD rats and Kunming mice can all be used as animal models of F. hepatica, SD rats are more suitable experimental animals in terms of tolerance and pathological response.

Pseudosuccinea columella is considered invasive and has become an important intermediate host of both Fasciola species in many regions of the world. This systematic review assessed the geographical distribution of P. columella, and its implications in the transmission of Fasciola hepatica and Fasciola gigantica, globally. A literature search was conducted on Google Scholar, JSTOR and PubMed databases using Boolean operators in combination with predetermined search terms for thematic analysis. Results show that P. columella has been documented in 22 countries from Europe (3), Africa (8), Oceania (2), North America (3) and South America (6). Furthermore, this snail species has shown to adapt to and inhabit a vast array of freshwater bodies including thermal lakes and ditches with acidic soils. Studies showed that P. columella transmits F. hepatica, with natural and experimental infections documented in sub-Saharan Africa, Europe, South America and North America. Experimental infection studies in Cuba showed the presence of P. columella populations resistant to F. hepatica infection. Furthermore, some populations of this invasive snail collected from F. hepatica endemic locations in Brazil, Venezuela, Australia, South Africa, Colombia and Argentina were found without Fasciola infection. As a result, the role played by this snail in the transmission of Fasciola spp. in these endemic areas is still uncertain. Therefore, further studies to detect natural infections are needed in regions/countries where the snail is deemed invasive to better understand the veterinary and public health importance of this snail species in Fasciola-endemic areas and determine the global dispersion of resistant populations of P. columella.

Rumen fluke (Calicophoron daubneyi) has emerged as a prominent parasite of ruminants in Europe over the past decades. Epidemiological questions remain regarding this observed increase in prevalence as well as the prospect for future paramphistomosis risk. This study aimed to identify factors associated with the temporal−spatial prevalence of rumen fluke as measured by veterinary surveillance in a temperate region using zero-inflated negative binomial mixed modelling. Modelling revealed that summer rainfall, raindays and sunshine hours and mean winter temperature as significant positively associated climate variables for rumen fluke prevalence over space and time (P < 0.05). Rumen fluke prevalence was also higher in counties with higher cattle/sheep densities and was positively associated with rumen fluke case rates in the previous years (P < 0.05). Equivalent models for fasciolosis prevalence revealed no significant association with winter temperature and sunshine hours, (P > 0.05). These results confirm a strong association between rainfall and the prevalence of both fluke species in a temperate environment, likely due to the role of Galba truncatula as their intermediate snail host. It also highlights the potential added importance of winter temperature and sunshine hours in rumen fluke epidemiology when compared to liver fluke.

The objective of this study is to determine the prevalence of Fasciola hepatica infection in cattle slaughterhouses, as well as its association with climatic/environmental factors (derived from satellite data), seasonality and climate regions in two states in Mexico. Condemned livers from slaughtered animals were obtained from three abattoirs in the states of Puebla and Veracruz. The overall prevalence of the parasite in cattle between January and December of 2017 was 20.6% (1407 out of 6834); the highest rate of condemnation was observed in Veracruz (26.3%; tropical climate), and the lowest rate was found in Puebla (15.5%; temperate climate). The seasonal prevalence of fluke infection was 18.6%, 14.8% and 28.4% during the wet season, and 17.1%, 12.4% and 22.8% during the dry season in the three abattoir sites, located in the districts of Zacatlán, Teziutlán and Ciudad Alemán, respectively. Liver condemnations due to bovine fasciolosis were prevalent in the Zacatlán, Teziutlán and Ciudad Alemán districts during summer, autumn and summer, respectively. Using generalized estimating equations analysis, we determined six variables – rainfall (wet/dry), land surface temperature day, land surface temperature night, normalized difference vegetation index, seasonality and climate regions (temperate/tropical) – to be significantly associated with the prevalence of condemned livers. Climate region was the variable most strongly associated with F. hepatica infection (odds ratio (OR) 266.59; 95% confidence interval (CI): 241.90–353.34), followed by wet and dry seasons (OR 25.56; 95% CI: 20.56–55.67).

Environmental DNA (eDNA) surveying has potential to become a powerful tool for sustainable parasite control. As trematode parasites require an intermediate snail host that is often aquatic or amphibious to fulfil their lifecycle, water-based eDNA analyses can be used to screen habitats for the presence of snail hosts and identify trematode infection risk areas. The aim of this study was to identify climatic and environmental factors associated with the detection of Galba truncatula eDNA. Fourteen potential G. truncatula habitats on two farms were surveyed over a 9-month period, with eDNA detected using a filter capture, extraction and PCR protocol with data analysed using a generalized estimation equation. The probability of detecting G. truncatula eDNA increased in habitats where snails were visually detected, as temperature increased, and as water pH decreased (P < 0.05). Rainfall was positively associated with eDNA detection in watercourse habitats on farm A, but negatively associated with eDNA detection in watercourse habitats on farm B (P < 0.001), which may be explained by differences in watercourse gradient. This study is the first to identify factors associated with trematode intermediate snail host eDNA detection. These factors should be considered in standardized protocols to evaluate the results of future eDNA surveys.

Earlier reports revealed oxysterol metabolites of Opisthorchis spp. liver fluke origin conjugated with DNA bases, suggesting that the generation of these DNA-adducts may underlie the mutagenicity and carcinogenicity of the infection with these food-borne pathogens. Here, we employed liquid chromatography-mass spectrometry to investigate, compare and contrast spectrograms of soluble extracts from Fasciola hepatica adult worms from bile ducts of cattle with those from O. viverrini and O.felineus from experimentally infected hamsters. F. hepatica and Opisthorchis spp. shared common compounds including oxysterol-like metabolites, bile acids and DNA-adducts, but the spectrometric profiles of F. hepatica included far fewer compounds than Opisthorchis species. These findings support the postulate that parasitic oxysterol-like metabolites could be related to carcinogenesis associated to infection and they point to a molecular basis for the differences among major groups of liver flukes concerning infection-induced malignancy.

Fascioliasis is a zoonotic disease caused by liver flukes transmitted by freshwater lymnaeid snails. Donkey and horse reservoir roles have been highlighted in human endemic areas. Liver fluke infection in mules has received very limited research. Their role in disease transmission, epidemiological importance and Fasciola hepatica pathogenicity are studied for the first time. Prevalence was 39.5% in 81 mules from Aconcagua, and 24.4% in 127 from Uspallata, in high-altitude areas of Mendoza province, Argentina. A mean amount of 101,242 eggs/mule/day is estimated. Lymnaeids from Uspallata proved to belong to ribosomal DNA internal transcribed spacer (ITS) markers ITS-1 and ITS-2 combined haplotype 3C of Galba truncatula. These lymnaeids were experimentally susceptible to infection by egg miracidia from mules. Infectivity, number of cercariae/snail and shedding period fit the enhanced F. hepatica/G. truncatula transmission pattern at very high altitude. This indicates that the mule is able to maintain the F. hepatica cycle independently. Individual burdens of 20 and 97 flukes were found. Mule infection susceptibility is intermediate between donkey and horse, although closer to the latter. Anatomo-pathology and histopathology indicate that massive infection may cause mule death. Haematological value decreases of red blood cells, haemoglobin, leucocytes and lymphocytes indicate anaemia and strong immunosuppression. Strongly increased biochemical marker values indicate liver function alterations. The mule probably played a role in the past exchanges with Chile and Bolivia through Mendoza province. Evidence suggests that mules could contribute to the spread of both F. hepatica and G. truncatula to human fascioliasis-endemic areas in these countries.

It is generally assumed that the digenean human liver fluke, Fasciola hepatica, gained entry to South America during the 15th century upon arrival of Europeans and their livestock. Nonetheless in Patagonia, Argentina, digenean eggs similar to F. hepatica have been observed in deer coprolites dating back to 2300 years B.P. The main objective of our present study was to identify and characterize these eggs using an ancient DNA (aDNA) study. Eggs were isolated and used for aDNA extraction, amplification and sequencing of partial regions from the cytochrome c oxidase subunit 1 and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 mitochondrial genes. Also, phylogenetic trees were constructed using Bayesian and maximum likelihood. Our results confirm the presence of F. hepatica in South America from at least 2300 years B.P. This is the first report and the first aDNA study of this trematode in South America prior to the arrival of the European cattle in the 15th century. The present work contributes to the study of phylogenetic and palaeobiogeographical aspects of F. hepatica and its settlement across America.

Fasciolosis is a zoonotic parasitic disease that seriously endangers the development of animal husbandry and human health. In order to develop a rapid serological diagnostic method for fasciolosis in ruminants, the CatL1D and CatB4 genes of Fasciola hepatica were amplified by reverse transcription polymerase chain reaction (PCR) and cloned, respectively, and then the CatL-B fusion gene (MeCatL-B) was constructed by gene splicing by overlap extension PCR technique. The recombinant rCatL1D, rCatB4 and rMeCatL-B proteins were then prepared by prokaryotic expression, respectively, and the recombinant protein with high specificity and sensitivity was screened via indirect enzyme-linked immunosorbent assay. Using the selected recombinant protein rCatL1D as a diagnostic antigen, we developed a colloidal gold immunochromatographic assay (CGIA) for detecting F. hepatica-specific antibodies, and 426 serum samples of slaughtered sheep were used to evaluate the sensitivity and specificity of F. hepatica CGIA assay. The results showed that the sensitivity and specificity of rCatL1D protein (100%, 96.67%) were higher than those of rCatB4 (94.29%, 80%) and rMeCatL-B (91.43%, 90%). Compared with the gold standard post-mortem inspection, the specificity and sensitivity of the CGIA method was 100% and 97%, respectively, and the consistency rate between these two methods was 99.3%. These results confirmed that the CGIA method based on rCatL1D protein could be a promising approach for rapid diagnosis of sheep fasciolosis because of its high sensitivity and specificity.

A systematic review was conducted focusing on the distribution of Fasciola species and their snail intermediate hosts (IHs) in East and Southern Africa. The reviewed literature showed that both Fasciola hepatica and F. gigantica are present in East and Southern Africa, and infect a wide range of domestic and wild ruminants. Fasciola gigantica was reported in six East African and five Southern African countries, where Radix natalensis (found in low altitudes) was reported to be the main IH. Fasciola hepatica was reported in Tanzania and Ethiopia (East Africa), and in South Africa and Zimbabwe (Southern Africa), where Galba truncatula (found in high altitudes) was documented as the IH in all countries except in Zimbabwe. Both Fasciola species were documented in Tanzania, Ethiopia, Zimbabwe and South Africa. An overlap of the two was observed in areas with an intermediate altitude in Ethiopia and South Africa, where Pseudosuccinea columella was widespread and assumed to transmit both species. Pseudosuccinea columella has been reported in South Africa and Namibia, and proven to transmit F. gigantica in South Africa; its role in Namibia in the transmission of Fasciola species has not been reported. Other lymnaeid species such as R. rubiginosa were reported in South Africa, and R. auricularia in South Africa and Botswana; their role in the transmission of Fasciola species has not been proven. Future studies should aim to determine the role of P. columella in the geographical spread of the two species in East and Southern African countries.

The main intermediate host of Fasciola gigantica in sub-Saharan Africa is Lymnaea (Radix) natalensis. Lymnaea (Pseudosuccinea) columella is capable of transmitting both F. gigantica and F. hepatica and has been reported to be present in South Africa. To date, no natural infection with F. gigantica has been reported despite the wide distribution of the snail. The aim of this study was to confirm whether L. (P.) columella was transmitting F. gigantica and/or F. hepatica in selected locations of KwaZulu-Natal and Eastern Cape provinces of South Africa. Lymnaea (Pseudosuccinea) columella snails were collected from two locations in two provinces of South Africa and screened for cercariae shedding. This was followed by humanely sacrificing the screened snails, and whole tissue of each individual snail was homogenized and amplified using primers designed to amplify the ITS-1 region of Fasciola spp. No cercariae were shed from the screened snails and molecular analysis showed that snails from the two locations were infected with F. gigantica. This study confirms natural infection of L. (P.) columella with F. gigantica in South Africa, where F. gigantica and F. hepatica have already been reported to coexist. Although L. (P.) columella is able to transmit the two species, surprisingly no infection with F. hepatica was detected from the screened snails. The natural intermediate host of F. gigantica in southern Africa, including South Africa, is Lymnaea (Radix) natalensis and comparative studies are needed to determine the competence of the two snail species in the transmission of F. gigantica.

Fasciola hepatica is a helminth parasite that causes huge economic losses to the livestock industry worldwide. Fasciolosis is an emerging foodborne zoonotic disease that affects both humans and grazing animals. This study investigated the associations between climatic/environmental factors (derived from satellite data) and management factors affecting the spatial distribution of this liver fluke in cattle herds across different climate zones in three Mexican states. A bulk-tank milk (BTM) IgG1 enzyme-linked immunosorbent assay (ELISA) test was used to detect F. hepatica infection levels of 717 cattle herds between January and April 2015. Management data were collected from the farms by questionnaire. The parasite's overall herd prevalence and mean optical density ratio (ODR) were 62.76% and 0.67, respectively. The presence of clustered F. hepatica infections was studied using the spatial scan statistic. Three marked clusters in the spatial distribution of the parasite were observed. Logistic regression was used to test three models of potential statistical association from the ELISA results using climatic, environmental and management variables. The final model based on climatic/environmental and management variables included the following factors: rainfall, elevation, proportion of grazed grass in the diet, contact with other herds, herd size, parasite control use and education level as significant predictors. Geostatistical kriging was applied to generate a risk map for the presence of parasites in dairy herds in Mexico. In conclusion, the spatial distribution of F. hepatica in Mexican cattle herds is influenced by multifactorial effects and should be considered in developing regionally adapted control measures.

Although the rumen fluke, Calicophoron daubneyi is now very common and widespread throughout Western Europe, reports of clinical cases are still rare. This study explores the epidemiological background to a severe rumen fluke outbreak in 6-month-old heifers on a dairy farm in Ireland. Sequence analysis of the cytochrome oxidase subunit 1 (Cox1) gene of the rumen fluke metacercariae on pasture failed to identify predominant, possibly pathogenic subtypes. However, estimates of metacercarial load indicated that the animals were exposed to a daily dose of about 5334 C. daubneyi metacercariae for a period of 3 weeks resulting in the build-up of very large numbers of immature worms in the small intestine. It is hypothesized that specific environmental conditions may favour this parasite over its competitor, the liver fluke, Fasciola hepatica, possibly by allowing it to emerge earlier. The possibility that C. daubneyi may be better adapted to the Irish climate than F. hepatica together with the fact that selective treatment against F. hepatica effectively frees the niche for C. daubneyi, may result in the gradual replacement of F. hepatica by C. daubneyi.

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion–secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Fasciola hepatica has both zoonotic importance and high economic impact in livestock worldwide. After ingestion by the definitive host, the Newly Excysted Juveniles (NEJ) penetrate the intestine before reaching the peritoneal cavity. The role of some NEJ-derived proteins in invasion has been documented, but the role of NEJ glycans or lectin-binding receptors during initial infection in the gut is still unknown. To address these questions, the migration of NEJ through rat intestine was recorded at 30 min intervals up to 150 min by two ex vivo methods. Firstly, jejunal sheets were challenged with NEJ incubated with biotinylated lectins. Secondly, untreated NEJ were incubated with distal jejunum pre-treated with lectins. Both Concanavalin A (ConA) and Galanthus nivalis (GNL), which recognize mannose-type N-glycans, significantly inhibited NEJ migration across the jejunum. Most of the lectins bound to the tegument and oral sucker of the NEJ, but only ConA and GNL maintained this interaction over 150 min. None of the lectins examined significantly reduced NEJ migration when pre-incubated with jejunal sheets, suggesting that host glycans might not be essential for initial binding/recognition of the gut by NEJ. Agents capable of blocking mannose-type N-glycans on the NEJ tegument may have potential for disrupting infection.

Reports of Calicophoron daubneyi infecting livestock in Europe have increased substantially over the past decade; however, there has not been an estimate of its farm level prevalence and associated risk factors in the UK. Here, the prevalence of C. daubneyi across 100 participating Welsh farms was recorded, with climate, environmental and management factors attained for each farm and used to create logistic regression models explaining its prevalence. Sixty-one per cent of farms studied were positive for C. daubneyi, with herd-level prevalence for cattle (59%) significantly higher compared with flock-level prevalence for sheep (42%, P = 0·029). Co-infection between C. daubneyi and Fasciola hepatica was observed on 46% of farms; however, a significant negative correlation was recorded in the intensity of infection between each parasite within cattle herds (rho = −0·358, P = 0·007). Final models showed sunshine hours, herd size, treatment regularity against F. hepatica, the presence of streams and bog habitats, and Ollerenshaw index values as significant positive predictors for C. daubneyi (P < 0·05). The results raise intriguing questions regarding C. daubneyi epidemiology, potential competition with F. hepatica and the role of climate change in C. daubneyi establishment and its future within the UK.

Fasciola hepatica is a parasitic trematode that causes serious losses to livestock producers, and also zoonotic disease. The limitations of chemotherapy for the control of fasciolosis have led to significant interest in the development of vaccines to protect cattle and sheep from infection. However, relatively few studies have concentrated on the mechanisms of invasion of the gut by newly excysted juvenile liver flukes (NEJ) and the host response triggered by this event. The aim of this work was to develop an in vitro model to study invasion by NEJ, while also reducing the requirement for challenge infections of experimental animals. Fasciola hepatica metacercariae were excysted in vitro and placed into compartments containing rat distal jejunal sheets. Variations in incubation medium, chamber size and incubation temperature were used to identify optimal conditions for NEJ migration across the gut. Histological examination showed increased migration until 120 min post-incubation. The use of RPMI, without gassing at 39 °C, as the incubation medium was found to be optimal, with 40·5% of NEJ migrating after 150 min. This study describes a readily-reproducible method for studying the migration of F. hepatica NEJ within the definitive host. It will be useful for identifying potential drug and vaccine targets.

Diploid (2n = 2x = 20) and triploid (2n = 3x = 30) Fasciola hepatica have been reported in the UK, and in Asia diploid, triploid and mixoploid (2x/3x) Fasciola spp. exist but there is little information to indicate how common triploidy is, particularly in UK fluke. Here the ploidy of 565 adult F. hepatica from 66 naturally infected British sheep and 150 adult F. hepatica from 35 naturally infected British cattle was determined. All 715 of these parasites were diploid, based on observation of 10 bivalent chromosomes and sperm (n = 335) or, since triploids are aspermic, sperm alone (n = 380). This constitutes the first extensive analysis of the ploidy of F. hepatica field isolates from Great Britain and shows that most F. hepatica isolated from cattle and sheep are diploid and have the capacity to sexually reproduce. These data suggest that triploidy, and by extension parthenogenesis, is rare or non-existent in wild British F. hepatica populations. Given that F. hepatica is the only species of Fasciola present in Britain our results indicate that the parasite is predominantly diploid in areas where F. hepatica exists in isolation and suggests that triploidy may only originate in natural populations where co-infection of F. hepatica and its sister species Fasciola gigantica commonly occurs.

Inter-simple sequence repeats markers were used to determinate the genetic variability of Fasciola hepatica populations recovered from sheep and cattle from Spain (Sp1, Sp2, Sp3 and Sp4), UK (Eng), Ireland (Ir) and Mexico (Mex). Twenty five primers were tested but only five produced 39 reproducible bands, being 71·79% polymorphic bands. This percentage ranged from 10·26% in Sp4 to 48·72% in Sp1, and per host between 28·21 and 48·72% in sheep and between 10·26 and 38·46% in cattle. This relatively low range of genetic diversity within populations, with a mean of 34·40%, implies that a large proportion of variation resided among populations. The population differentiation (Gst = 0·547) indicated that 54·7% of variation is due to differences between populations and 45·3% due to differences within population. The Nei's distance ranged between 0·091 and 0·230 in sheep and between 0·150 and 0·337 in cattle. The genetic relationships between populations and individuals were shown by a UPGMA dendrogram and a principal coordinate analysis; both grouped all populations separately from Sp4, a population of from the Midwest of Spain with the lowest level of diversity. Small genetic distances were observed between Eng and Ir, on the one hand, and Sp1, Sp2, Sp3, from the Northwest of Spain, together with Mex, on the other.