Radiopaque Nanorobots as Magnetically Navigable Contrast Agents for Localized In Vivo Imaging of the Gastrointestinal Tract.

Magnetic nanorobots offer wireless navigation capability in hard-to-reach areas of the human body for targeted therapy and diagnosis. Though in vivo imaging is required for guidance of the magnetic nanorobots toward the target areas, most of the imaging techniques are inadequate to reveal the potential locomotion routes. This work proposes the use of radiopaque magnetic nanorobots along with microcomputed tomography (microCT) for localized in vivo imaging applications. The nanorobots consist of a contrast agent, barium sulfate (BaSO4 ), magnetized by the decoration of magnetite (Fe3 O4 ) particles. The magnetic features lead to actuation under rotating magnetic fields and enable precise navigation in a microfluidic channel used to simulate confined spaces of the body. In this channel, the intrinsic radiopacity of the nanorobots also provides the possibility to reveal the internal structures by X-ray contrast. Furthermore, in vitro analysis indicates nontoxicity of the nanorobots. In vivo experiments demonstrate localization of the nanorobots in a specific part of the gastrointestinal (GI) tract upon the influence of the magnetic field, indicating the efficient control even in the presence of natural peristaltic movements. The nanorobots reported here highlight that smart nanorobotic contrast agents can improve the current imaging-based diagnosis techniques by providing untethered controllability in vivo.

The first case of periorbital human dirofilariasis in the Czech Republic.

Dirofilaria repens and Dirofilaria immitis are the most common filarial species affecting humans in Europe. Dirofilaria repens causes subcutaneous or ocular infection, whereas D. immitis is responsible mainly for the pulmonary form. In this report, we present the first human case of periorbital dirofilariasis in the Czech Republic. A 58-year-old woman suffered from an eyelid oedema, redness and pain in the left eye. After excising the parasite from her eyelid, all clinical symptoms disappeared. Based on the morphology and cytochrome oxidase I sequencing, the parasite was identified as D. repens. Histology revealed that the excised worm was female with absent microfilariae in uteri. With respect to the length of the incubation period and the sequence identity with a known Czech isolate, we concluded that D. repens was most likely of autochthonous origin.

A novel approach to imaging engorged ticks: Micro-CT scanning of Ixodes ricinus fed on blood enriched with gold nanoparticles.

Micro-computed tomography (micro-CT) is an exceptional imaging modality which is limited in visualizing soft biological tissues that need pre-examination contrasting steps, which can cause serious deformation to sizeable specimens like engorged ticks. The aim of this study was to develop a new technique to bypass these limitations and allow the imaging of fed ticks in their natural state. To accomplish this, adult Ixodes ricinus females were allowed to engorge in vitro on blood supplemented with PEGylated gold nanoparticles (PEG-AuNPs). In total, 73/120 females divided into 6 groups engorged on blood enriched with 0.07-2.16 mg PEG-AuNPs per ml of blood. No toxic effect was observed for any of the tested groups compared to the control group, in which 12/20 females engorged on clear blood. The ticks were scanned on a Bruker micro-CT SkyScan 1276. The mean radiodensity of the examined ticks exceeded 0 Hounsfield Units only in the case of the two groups with the highest concentration. The best contrast was observed in ticks engorged on blood with the highest tested concentration of 2.16 mg/mL PEG-AuNPs. In these ticks, the midgut and rectal sac were clearly visible. Also, the midgut lumen volume was computed from segmented image data. The reduction in midgut volume was documented during the egg development process. According to this pilot study, micro-CT of ticks engorged on blood supplemented with contrasting agents in vitro may reveal additional information regarding the engorged ticks' anatomy.

Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies.

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

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

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

Small animal in vivo imaging of parasitic infections: A systematic review.

Non-invasive small animal in vivo imaging is an essential tool in a broad variety of biomedical sciences and enables continuous monitoring of disease progression in order to develop and improve diagnostic, therapeutic and preventive measures. Imaging parasites non-invasively in live animals allows efficient parasite distribution evaluation in the host organism and objective evaluation of parasitic diseases' burden and progression in individual animals. The aim of this systematic review was to summarize recent trends in small animal in vivo imaging and compare and discuss imaging of single-cell and multicellular eukaryotic parasites. A literature survey was performed using Web of Science and PubMed databases in research articles published between 1990 and 2018. The inclusion criteria were using any imaging method to visualize a range of protozoan and helminth parasites in laboratory animals in vivo. A total of 92 studies met our inclusion criteria. Protozoans and helminths were imaged in 88% and 12% of 92 studies, respectively. The most common parasite genus studied was the protozoan Plasmodium followed by Trypanosoma and Leishmania. The most frequent imaging method was bioluminescence. Among the helminths, Schistosoma and Echinococcus were the most studied organisms. In vivo imaging is applicable in both protozoans and helminths. In helminths, however, the use of in vivo imaging methods is limited to some extent. Imaging parasites in small animal models is a powerful tool in preclinical research aiming to develop novel therapeutic and preventive strategies for parasitic diseases of interest both in human and veterinary medicine.

Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic.

Candidatus Neoehrlichia mikurensis, the causative agent of tick-borne "neoehrlichiosis" has recently been reported in humans, mammals and ticks in Europe. The aim of this study was to map the distribution of this bacterium in questing ticks in the Czech Republic. A total of 13,325 Ixodes ricinus including 445 larvae, 5270 nymphs and 7610 adults were collected from vegetation by flagging in 140 Czech towns and villages from every region of the Czech Republic. The ticks were pooled into 2665 groups of 5 individuals respecting life stage or sex and tested for the presence of Ca. Neoehrlichia mikurensis by conventional PCR targeting of the groEL gene. The bacterium was detected in 533/2665 pools and 125/140 areas screened, showing an overall estimated prevalence of 4.4 % in ticks of all life stages. Phylogenetic analysis revealed only small genetic diversity among the strains found. Two pools of questing larvae tested positive, suggesting transovarial transmission. According to this study, Ca. Neoehrlichia mikurensis is another tick-borne pathogen widespread in I. ricinus ticks in the Czech Republic.

Three-dimensional reconstruction of the feeding apparatus of the tick Ixodes ricinus (Acari: Ixodidae): a new insight into the mechanism of blood-feeding.

The different components of the mouthparts of hard ticks (Ixodidae) enable these parasites to penetrate host skin, secrete saliva, embed, and suck blood. Moreover, the tick's mouthparts represent a key route for saliva-assisted pathogen transmission as well as pathogen acquisition from blood meal during the tick feeding process. Much has been learned about the basic anatomy of the tick's mouthparts and in the broad outlines of how they function in previous studies. However, the precise mechanics of these functions are little understood. Here, we propose for the first time an animated model of the orchestration of the tick mouthparts and associated structures during blood meal acquisition and salivation. These two actions are known to alternate during tick engorgement. Specifically, our attention has been paid to the mechanism underlining the blood meal uptake into the pharynx through the mouth  and how ticks prevent mixing the uptaken blood with secreted saliva. We animated function of muscles attached to the salivarium and their possible opening /closing of the salivarium, with a plausible explanation of the movement of saliva within the salivarium and massive outpouring of saliva.

Seroprevalence of Fasciola hepatica in cattle in Estonia.

Fasciolosis, an infectious disease caused by the liver fluke Fasciola hepatica, affects grazing cattle world-wide. Liver fluke F. hepatica is prevalent and well-documented in cattle in many European countries, but for the Baltic countries such information is limited. This study investigated the seroprevalence and distribution of F. hepatica in cattle in Estonia. A total of 2461 individual serum samples from 218 farms distributed throughout all 15 Estonian counties, collected between February 2012 and March 2013, were tested for specific anti-F. hepatica antibodies using an in-house enzyme-linked immunosorbent assay (ELISA). In total, 144 individual animals tested seropositive, yielding an animal-level seroprevalence of 5.9% (95% CI 5.0-6.9). The herd-level seroprevalence was 28.4% (95% CI 22.8-34.7) and the herds with at least one seropositive animal were located in 13 of the 15 counties. Of the 62 F. hepatica-positive herds, 14 (6.4%) had an in-herd seroprevalence higher than 25%. With respect to production type, the herd-level seroprevalence was 20.2%, 35.6%, and 36.4% in dairy, mixed, and beef herds, respectively. Animals from the two large islands had higher odds of testing F. hepatica-seropositive than animals from the mainland. Animals from mixed and beef herds had higher odds of testing F. hepatica-seropositive than animals from dairy herds. Mixed and beef herds, and herds with more than 100 cattle, had higher odds of having at least one seropositive animal. This study provided the first serological evidence of the presence and distribution of F. hepatica in cattle herds in Estonia.

Comparison of molecular and conventional methods for the diagnosis of Fasciola hepatica infection in the field.

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.

Assessment of flukicide efficacy against Fasciola hepatica in sheep in Sweden in the absence of a standardised test.

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.

Effect of Fascioloides magna (Digenea) on fecundity, shell height, and survival rate of Pseudosuccinea columella (Lymnaeidae).

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.

First report of closantel treatment failure against Fasciola hepatica in cattle.

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.

Impact of meteorological and environmental factors on the spatial distribution of Fasciola hepatica in beef cattle herds in Sweden.

BACKGROUND: Fasciola hepatica is a parasite with a significant impact on ruminant livestock production. Previous studies in north-west Europe have described its geographical distribution and determined potential predictors of fasciolosis using geographical information system (GIS) and regression modelling. In Sweden, however, information about the distribution of fasciolosis is limited. This study examined the geographical distribution of F. hepatica and identified high-risk areas for beef cattle in Sweden and sought to characterise potential predictors. Beef cattle serum samples were collected during winter 2006-2007 from 2135 herds which were examined for F. hepatica antibodies by enzyme-linked immunosorbent assay (ELISA). Fasciolosis distribution maps were created using GIS based on postcode location of seropositive herds. Spatial scan analysis (SaTScan) was performed to determine high-risk areas. Using datasets on animal density, temperature, precipitation and Corine land cover data, including soil type and soil mineral concentrations in Sweden, bivariate and multiple logistic regression analyses were carried out in R software to reveal potential predictors of F. hepatica infection. RESULTS: Overall herd seroprevalence of F. hepatica in beef cattle was 9.8 % (95 % CI: 8.6-11.1). An irregular spatial distribution of F. hepatica, with two main clusters, was observed in south-west Sweden. The most northerly occurrence of F. hepatica in the world was documented. The final model explained 15.8 % of the variation in F. hepatica distribution in study herds. Absence of coniferous forest was the variable with the highest predictive value. Precipitation in July-September, Dystric Cambisol, Dystric Regosol, and P and Cu concentrations in soil were other negative predictors. Beef cattle herd density, Dystric Leptosol and Fe concentration were positive predictors. CONCLUSIONS: The spatial distribution of F. hepatica in Swedish beef cattle herds is influenced by multi-factorial effects. Interestingly, absence of coniferous forest, herd density, specific soil type and concentration of some soil minerals are more important predictors than climate factors.

Distribution of Fasciola hepatica in Swedish dairy cattle and associations with pasture management factors.

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.

Lymnaea cubensis, an experimental intermediate host for Fascioloides magna.

Single-miracidium infections of Lymnaea cubensis (Pfeiffer) from Guadeloupe with the giant liver fluke Fascioloides magna (Bassi, 1875) (Digenea) were carried out during five successive snail generations to determine if this lymnaeid might sustain complete larval development of the parasite. Controls were constituted by a French population of Galba truncatula (Miller) (a single generation) infected according to the same protocol. It was recorded that prevalence and intensity of F. magna infection in L. cubensis progressively increased from F1 to F5 generations. Cercarial shedding of F. magna was noted only within F5 generation of L. cubensis. However, most measured parameters of infection in this species were significantly lower than those noted for G. truncatula and most L. cubensis died after a single shedding wave. Despite this, L. cubensis can be added to the list of potential intermediate hosts of F. magna.

Transmission patterns of Fasciola hepatica to ruminants in Sweden.

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.

Lymnaea palustris and Lymnaea fuscus are potential but uncommon intermediate hosts of Fasciola hepatica in Sweden.

BACKGROUND: Lymnaea palustris and L. fuscus are members of the European stagnicolines (Gastropoda: Lymnaeidae). The role of stagnicolines in transmission of Fasciola hepatica has been often proposed. To assess the possible relationship between these two stagnicolines and F. hepatica in Sweden, field monitoring in parallel with experimental infections of L. palustris and L. fuscus were conducted. METHODS: Stagnicoline snails were collected and identified on pastures grazed by either sheep or cattle on four farms suffering from fasciolosis in Sweden during 2011-2012. Field-collected L. palustris and L. fuscus were examined for F. hepatica DNA by PCR. In the laboratory, different age groups of L. palustris, L. fuscus and G. truncatula were each exposed to two F. hepatica miracidia and main infection characteristics were obtained. RESULTS: One field-collected L. palustris (out of n = 668) contained F. hepatica as determined by PCR. On the other hand, stagnicolines artificially exposed to F. hepatica miracidia resulted in successful infection with fully differentiated cercariae, but only in juvenile snails (size, 1-2 mm at exposure) and with a prevalence of 51% and 13% in L. palustris and L. fuscus, respectively. In contrast, 90% of juvenile (size, 1-2 mm) and 92% of preadult G. truncatula (size, ≥ 2-4 mm), respectively, were successfully infected. Delayed, reduced and/or no spontaneous cercarial shedding was observed in the two stagnicolines when compared to G. truncatula. However, at snail dissection most cercariae from L. fuscus and L. palustris were able to encyst similarly to those from G. truncatula. CONCLUSION: Both L. fuscus and L. palustris can sustain larval development of F. hepatica but with an apparent level of age resistance. The finding of a single F. hepatica positive specimen of L. palustris, together with infection characteristics from the experimental infection, suggest that L. palustris is a more suitable snail vector of F. hepatica than L. fuscus. The reduced growth observed in both stagnicolines was contrary to the 'parasitic gigantism' theory. Overall, it seems that the epidemiological role of L. palustris in transmission of F. hepatica in Sweden is likely to be much lower than for G. truncatula.

Lymnaea fuscus (Pfeiffer, 1821) as a potential intermediate host of Fascioloides magna in Europe.

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.

The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep.

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.