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

The liver fluke, Fasciola hepatica, is one of the major parasite threats to livestock industries world-wide. In sheep and cattle, F. hepatica infection is commonly diagnosed using a range of methods. Aside from conventional coprological and serological diagnostic methods, there are also several molecular methods available based on the detection of liver fluke DNA in faeces. In this study, the outcomes of faecal egg count (FEC), serology and coproantigen ELISA (cELISA) were compared with the performance of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) in diagnosis of F. hepatica from naturally infected cattle and sheep. A total of 64 individual faecal and serum samples were collected from four sheep and beef cattle herds with previous histories of F. hepatica infection. FEC and coproantigen levels were measured in faecal samples and anti-F.hepatica antibody levels were measured in serum samples. DNA samples isolated from faeces were examined both by PCR and LAMP, targeting the internal transcribed spacer 2 (ITS2) region of the F. hepatica genome. Results showed that F. hepatica eggs were present in 28 animals, while coproantigen and specific anti-F. hepatica antibodies were detected in 36 and 53 animals, respectively. Only 3 and 6 samples were positive by PCR and LAMP, respectively. To calculate method specificity and sensitivity, a combination of FEC and cELISA was selected as the composite reference standard (CRS). When compared to the CRS, PCR had a sensitivity of 10.7% and specificity of 100%, whereas LAMP had a sensitivity and specificity of 17.9% and 97.2%, respectively. PCR and LAMP in this field study were highly specific, but both had poor sensitivity compared with FEC and cELISA. Potential reasons for PCR and LAMP failure were inadequate amounts of amplifiable F. hepatica DNA, possibly due to the choice of DNA extraction procedure, amount of faecal material processed, as well as different faeces consistency and composition between different animal species.

Anthelmintic resistance (AR) to Fasciola hepatica is emerging worldwide. Recently, AR to the adulticide compound albendazole (ABZ) was shown in Argentina and Spain. In Sweden, ABZ treatment failure against F. hepatica was first reported in sheep in 2012. The present study tested the efficacy of ABZ and triclabendazole (TCBZ) in sheep naturally infected with F. hepatica using a combination of three different diagnostic methods: faecal egg counts (FEC), coproantigen ELISA (cELISA) and Fasciola egg hatch test (FEHT). Two deworming trials, in November 2014 and January 2015, were performed on two sheep farms (farms A and B) in south-western Sweden. Except ABZ in November, treatment with ABZ or TCBZ achieved sufficient efficacy (97–100%) against adult F. hepatica on farm A. In contrast, ABZ treatment failed in the sheep flock on farm B, despite low initial faecal egg output. On farm B, ABZ efficacy based on FEC was 67% (95% CI: 35–84) and four of eight ewes tested were coproantigen-positive 21 days post-treatment. Ovicidal activity of ABZ against Fasciola eggs in isolates from both farms and one additional bovine isolate were tested by FEHT to exclude the presence of juvenile flukes and other factors such as dosing failure and poor quality of drug product. Irrespective of drug trial, data from FEHT showed significantly lower ovicidal activity of ABZ for the ovine farm B isolate than for the isolate from farm A. This confirms that the low efficacy of ABZ in sheep flock B was associated with ABZ resistance. Overall, the usefulness of three complementary methods for detection of ABZ resistance in the field was demonstrated., Graphical abstract, Highlights • Efficacy of albendazole and triclabendazole against Fasciola hepatica was tested in sheep. • Coproantigen ELISA, faecal egg counts and in vitro Fasciola egg hatch test were used. • Albendazole resistance in F. hepatica was confirmed in one sheep flock. • Fasciola egg hatch test is a useful tool for detection of albendazole resistance.

Transmission patterns of Fasciola hepatica were investigated on beef cattle (n = 3) and sheep (n = 3) farms in Sweden between 2011 and 2012. The dynamics of fluke infection, particularly estimated time of infection, were screened each grazing season by ELISA detection of antibodies in lambs (n = 94) and first grazing season calves (n = 61). Colostral transfer of F. hepatica antibodies from seropositive ewes was detected in sheep up to 11 weeks of age. In sheep, the estimated time of infection differed significantly between herds and years. Typical 'winter infection' was observed on two sheep farms in 2012, but the most prevalent transmission pattern was found to be 'summer infection', characterised by infection of animals in late summer by F. hepatica originating from overwintered and/or spring-excreted eggs. In contrast, beef calves were infected mainly in September-October ('summer infection'). Furthermore, lymnaeid and succineid snails were collected on the pastures used by these animals both in spring and in the autumn each year. In total, 1726, 588, 138, 130, 93 and 42 specimens of Galba truncatula, Lymnaea palustris, Lymnaea glabra, Lymnaea fuscus, Radix peregra and Succinea putris, respectively, were collected and identified. These were subsequently examined for the presence of F. hepatica DNA by species-specific PCR and the findings compared against mean monthly rainfall and temperature data for each farm. The main intermediate host of the liver fluke was G. truncatula, with a prevalence range of F. hepatica infection from 0% to 82%. Only 1 out of 42 terrestrial S. putris tested positive for F. hepatica, casting doubt on the role of this species in transmission of F. hepatica in Sweden. In conclusion, two main peak periods of infection were observed: May-June (from overwintered infected snails = 'winter infection') and August-September (from metacercariae developed and produced by snails during summer = 'summer infection'). The occurrence and frequency of 'winter infection' were dependent on local environmental factors such as snail habitat availability or grazing behaviour of animals, rather than on climatic factors.

Infection with Fascioloides magna (Digenea) causes serious damage to liver tissue in definitive hosts represented by ruminants, especially cervids. The distribution of F. magna includes the indigenous areas in North America, and the areas to which F. magna was introduced-Central Europe, Southeast Europe, and Italy. The North American intermediate host of F. magna, the freshwater snail Pseudosuccinea columella (Lymnaeidae), is an invasive species recorded in South America, the Caribbean, Africa, Australia, and west and Southeast Europe. In Europe, Galba truncatula is the snail serving for transmission, but P. columella has potential to become here a new intermediate host of F. magna. Little is known about interactions between F. magna and P. columella. In this study, the susceptibility of P. columella (Oregon, USA) to the infection by a single miracidium of the Czech strain of F. magna and the influence of F. magna on snail fecundity, shell height, and survival were evaluated. The data show that the Oregon strain of P. columella is a highly suitable host for the Czech strain of F. magna, with the infection rate of 74 %. In addition, a negative effect on survival rate of infected snails was recorded only in the late phase of infection. The infection was accompanied by a major reduction in egg mass production and by a decrease in the number of eggs per egg mass. The shell height of infected snails did not significantly differ from that in unexposed controls.

This study investigated the humoral immune responses of goats experimentally infected with Fascioloides magna and Fasciola hepatica to F. magna excretory/secretory products (FmESP) or F. hepatica excretory/secretory products (FhESP), respectively. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum antibody responses and for possible discrimination of F. magna and F. hepatica infections in goats. Comparison of ESPs of both flukes and evaluation of ESP antigenicity was also studied applying immunoblotting techniques. In all infected goats, antibody level was significantly increased (against negative control) since 2 weeks post infection (WPI). However, the dynamics of antibodies varied between F. magna and F. hepatica groups during the course of the infection. The cross-reaction of antibodies developed against F. magna and F. hepatica with ESP proteins was recorded by ELISA. The species-specific proteins 40, 120 kDa from FmESP and 80, 160 kDa from FhESP (with no antibody cross-reaction) were detected by two dimensional electrophoresis and immunoblot as the potential immunodiagnostic markers. Our results suggest that F. magna and F. hepatica infection could be distinguished by common immunological techniques based on species-specific antigen-antibodies interaction.

Experimental infections of Galba sp. (origin, Colombia) with allopatric isolates of Fasciola hepatica from France or Fascioloides magna from the Czech Republic were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of either parasite. In snails exposed to F. hepatica, 7 of 400 snails harboured several rediae and only two snails contained a small number of free cercariae on day 50 post-exposure. In contrast, the intensity of F. magna infection in Galba sp. progressively increased from the F1 to F5 generations. Spontaneous cercarial shedding of F. magna occurred in 7 of 100 Galba sp. belonging to the F5 generation and the number of shed cercariae did not differ significantly from that noted in control Galba truncatula of French origin. Galba sp. from Colombia can be added to the list of potential intermediate hosts for F. magna.

The geographic distribution of Fasciola hepatica infection in relation to management routines was studied in Swedish dairy herds by testing for F. hepatica antibodies with the enzyme-linked immunosorbent assay (ELISA). In addition, all farmers were sent a questionnaire asking for information about type of production, management routines and historical record of F. hepatica at slaughter. A total of 176 farmers (41%) responded to the questionnaire. A total of 426 bulk tank milk (BTM) samples were randomly selected from the period September to October 2012 representing approximately 10% of all herds in Sweden. The overall seroprevalence was 25% (n = 107; 95% confidence interval = 21-29%) with a concentration of herds located in south-western Sweden. Among the seropositive herds, 31 (29%) had antibody levels indicating production loss. There were no significant differences in seropositivity between organic and conventional herds or due to pasture management routines. The length of grazing period, which increased the risk for heifers, was found to be the most influential factor. A discrepancy was noted between reported F. hepatica presence at meat inspection and herds that were seropositive based on BTM-ELISA results. Although the largest proportion of seropositive BTM samples (80%) came from herds where liver fluke presence had been observed at meat inspection after slaughter, seropositive BTM samples were also diagnosed in five herds (17%) with no remarks at slaughter. In conclusion, F. hepatica is a common parasite in Swedish dairy herds and the month of heifer turn-out and the grazing period length were the most influential factors observed.

Experimental infections of two different populations of Lymnaea fuscus in France and Sweden, with a Czech isolate of Fascioloides magna were carried out to determine if this lymnaeid species enables parasite larval development. Species identification of both snail populations was performed using the morphology of the copulatory organ, and also confirmed by sequencing of the internal transcribed spacer 2 (ITS2) region of the snail genomic rDNA. Only juvenile snails measuring less than 3mm (1-3 weeks of age) were successfully infected (the viable cercariae were recorded) and infection prevalence decreased with age, as documented by increased shell height. In both French and Swedish L. fuscus populations, prevalence ranged between 1.1% and 58.8%. The mean number of metacercariae obtained from cercariae-shedding snails was 13.7 (±11.4), while the total cercarial production noted in snails dissected at day 85 post-exposure was 147.5 (±56.6). Compared to uninfected control snails, we observed reduced growth of infected snails. Despite age-related resistance of snail to the parasite, and limited cercarial production in these experimentally infected snails, F. magna was still able to complete larval development in L. fuscus.

Albendazole (ABZ) has been used for control of ovine fasciolosis in Sweden for several decades. However, increasing prevalence of Fasciola hepatica in Sweden requires attention to diagnostic tools and current control strategies. The coproantigen reduction test (CRT) using commercial Bio-X K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) was recently suggested as a novel method for diagnosis of patent F. hepatica infection. The aim of this study was to compare the efficacy of albendazole and triclabendazole (TCBZ) treatment against F. hepatica in naturally infected sheep in south-western Sweden by CRT, and also to evaluate the usefulness of this test as a diagnostic marker for anthelmintic efficacy following treatment with ABZ. Three weeks after housing, 24 serologically positive ewes on a commercial farm in Sweden were randomly allocated into three groups, which were either dewormed with 5mg/kg ABZ, 10mg/ml TCBZ or left untreated. Twenty-six days after initial administration of these anthelmintics, all of the sheep in the ABZ group and untreated control groups were treated with 10mg/kg TCBZ. While TCBZ caused elimination of coproantigen and eggs in faeces 7 days after application, ABZ treatment failed completely. Neither anthelmintic resistance, underdosing, nor lack of efficacy due to the presence of immature flukes can be out ruled as possible causes. Despite some deviations in conformity between coproantigen levels and presence of fluke eggs, the CRT was a useful tool for measuring treatment efficacies.

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short ( 500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20°C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6–8.3% instead of 23.6–25.9%), the total number of free rediae (10.7–17.9 per snail instead of 26.3–34.7), and that of free cercariae (112.8–136.9 per snail instead of 177.8–248.5). Despite a greater number of free rediae at day 75 p.e. (36.2–45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.

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., Graphical abstract, Highlights • Closantel efficacy against Fasciola hepatica was tested in Swedish cattle. • Closantel treatment failure was observed in two of the three beef cattle herds. • Potential causes of closantel failure are discussed. • Standardised guidelines for evaluation of flukicide efficacy are needed.