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1 página.- Trabajo presentado al 24th Annual Meeting of the Spanish Society of Veterinary Pathology(León, Spain, Sep 5-8, 2012), The importance of changes caused by the rumen and reticulum trematode parasite Calicophoron daubneyi (Digenea: Paramphistomidae) is unclear. Most authors suggest that the adult paramphistomes are relatively warmless...

Diarrhea is the main health problem caused by human-related microsporidia, and waterborne transmission is one of the main risk factors for intestinal diseases. Recent studies suggest the involvement of water in the epidemiology of human microsporidiosis. However, studies related to the presence of microsporidia in different types of waters from countries where human microsporidiosis has been described are still scarce. Thirty-eight water samples from 8 drinking water treatment plants (DWTPs), 8 wastewater treatment plants (WWTPs) and 6 recreational river areas (RRAs) from Galicia (NW Spain) have been analyzed. One hundred liters of water from DWTPs and 50 L of water from WWTPs and RRAs were filtered to recover parasites, using the IDEXX Filta-Max® system. Microsporidian spores were identified by Weber’s stain and positive samples were analyzed by PCR, using specific primers for Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem . Microsporidia spores were identified by staining protocols in eight samples (21.0%): 2 from DWTPs, 5 from WWTPs, and 1 from an RRA. In the RRA sample, the microsporidia were identified as E. intestinalis . To the best of our knowledge, this is the first report of human-pathogenic microsporidia in water samples from DWTPs, WWTPs and RRAs in Spain. These observations add further evidence to support that new and appropriate control and regulations for drinking, wastewater, and recreational waters should be established to avoid health risks from this pathogen.

A comprehensive field study was carried out with the following objectives: (a) to assess the usefulness of individual and bulk tank milk analysis for determining Neospora caninum serostatus in individual cows and herds, and (b) to study the associations between N. caninum infection status (based on milk testing), and several productive and reproductive parameters in the animals. Antibodies were detected with a commercially available ELISA test (Bio K 192/5). Analysis of paired serum and milk samples from 1134 lactating cows on 38 farms revealed that 97.6% of the ELISA results were coincident, irrespective of whether serum or milk samples were used. Moreover, multiple linear regression analysis revealed that 86.0% of the variations in ELISA values in milk were due to variations in the serum. The measurement of antibodies in bulk tank milk was a good estimator of the herd level status of N. caninum infection, and enabled detection of infection in 94.7% herds with ≥10.0% seropositive cows and/or in all herds with >4% highly seropositive cows. The odds ratio for abortion in seropositive animals was 9.1 times higher than in seronegative animals. The infection serostatus was also a significant risk factor, as the odds ratio for abortion was even higher (12.0 times) in cows categorized as highly seropositive. ELISA values for the bulk milk from 387 randomly selected herds were negatively associated with average milk production. Moreover, milk production losses mainly occurred on farms categorized as highly positive (i.e. herds with ≥20.0% seropositive cows).

Faecal samples from 224 roe deer (Capreolus capreolus) and 381 wild boars (Sus scrofa) shot during the 2008–2009 hunting season (August–January) in Galicia (NW Spain) were examined to determine the presence and intensity of infection by Cryptosporidium and Giardia. Analysis of a single sample from each of the roe deer revealed that the prevalence of cryptosporidiosis and giardiosis was 1.3% and 5.3% respectively. The prevalence of Giardia infection was significantly higher in juvenile female roe deer than in adult females, but no other significant differences were found in relation to age and sex. In wild boars, the prevalence of cryptosporidiosis and giardiosis was 7.6% and 1.3% respectively. The prevalence of Cryptosporidium infection was significantly higher in juvenile male wild boars than in adult males, but no other significant differences were found in relation to age or sex. In both groups of wild animals, the number of Cryptosporidium oocysts per gram of faeces (OPG) ranged from 5 to 200 and the number of Giardia cysts per gram of faeces (CPG) was between 5 and 47; there were no significant differences between the two groups with respect to number of infections. This is the first large study of Cryptosporidium and Giardia in roe deer and wild boars in hunting areas in Spain and the results demonstrate a low, but widespread prevalence of Cryptosporidium and Giardia in these animals.

Protein electrophoresis was used to study allozyme variation in Fasciola hepatica collected from three locations in Galicia (NW Spain), an area where fascioliasis is endemic. Eleven of 16 loci showed variation in at least one population and 7 loci were polymorphic in all populations studied. Five of these markers showed expected heterozygosities ranging from 0.137 to 0.569. The Nei's unbiased genetic diversity within populations ranged from 0.146 to 0.168. Genotypic frequencies were consistent with panmixia in 25 of 28 cases. Only 2 loci showed a significant deficit of heterozygotes. Genetic distances between populations were small (Da=0.003-0.010). These results suggest high levels of genetic variability and low population structure. This study shows that several of the markers developed are useful for study the population genetic structure of the parasite, which is essential to investigate the evolution of drug resistance that has recently emerged in populations of the study area. © 2010 Elsevier B.V.

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%-99.9%), 100% (95% CI: 98.6%-100%) and 0.997 (95% CI: 0.987-1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R(2)=0.84; p

In this study, we evaluate the MM3-COPRO method for detection of Fasciola coproantigens in human fecal samples, and the usefulness of a new preservative/diluent, CoproGuard, developed for preservation of Fasciola coproantigens. The MM3-COPRO assay was evaluated with 213 samples from healthy patients, 30 Fasciola positive fecal samples (according to the Kato-Katz method), and 83 samples from patients with other parasitic infections. All Fasciola positive specimens were detected with the MM3-COPRO assay (100% sensitivity) and there was no cross-reactivity with other common parasites present in the clinical specimens analyzed (100% specificity). The use of CoproGuard enhanced coproantigen extraction without affecting the detection limit of the assay, and the antigenicity of Fasciola coproantigens in fecal samples stored at 37 degrees C was retained throughout the entire observation period (120 days). We concluded that the MM3-COPRO ELISA combined with the use of CoproGuard may be a very useful tool for the diagnosis of human fascioliasis.

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4-7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3-6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33-66 adults), while it was for F. gigantica infection (burden of 17-69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.

Fasciola hepatica infection is an important cause of lost productivity in livestock worldwide. Effective control of fasciolosis is difficult, especially in milking cows, which can only be treated during dry periods, a control strategy that has not been yet evaluated. In this cross-sectional study, we investigated the effect of the type of flukicide treatment on the prevalence and intensity of infection in dairy cattle from Galicia, an area where fasciolosis is endemic and which is also the main milk-producing region in Spain. Faecal samples were taken from 5188 dairy cows on 275 randomly selected farms for measurement of the concentration of F. hepatica coproantigens by a monoclonal antibody based immunoassay (MM3-COPRO ELISA). On the same day as the sampling, each farm owner/manager was questioned about the types of treatment used on the farm. Three groups of farms were considered according to the fasciolicide treatment: (A) flukicides were not used, (B) an anthelmintic effective against mature stages of flukes was used (albendazole or netobimin) and (C) a fasciolicide effective against immature and mature stages was used (triclabendazole: TCBZ). Results indicated that 16.0% (832/5188) cows from 61.1% (168/275) herds were infected by F. hepatica. The mean coproantigen concentration in infected herds was 13.0ng/ml (range 0.9-112.6ng/ml). The highest individual concentration recorded was 496.6ng/ml. Herd and within-herd prevalences of F. hepatica were similar in all three groups, but surprisingly, individual prevalence and antigen concentration were higher in Group C (p0.05). The percentage of farms with within-herd prevalences25% was very high in all three groups, and no significant differences were observed. In contrast, the percentage of herds with mean antigen concentrations20ng/ml was significantly lower (p0.05) in Groups A and B (14.4% and 14.9%, respectively) than in Group C (50.0%). The proportion of herds that exceeded both limits (25% for prevalence and/or 20ng/ml for coproantigen concentration) was also significantly higher (p0.05) in Group C than in untreated animals (Group A). The survey showed that most dairy farmers are unaware of the existence of F. hepatica infection on their farms, and treatments, when given, are administered without prior diagnosis. Treatment with TCBZ administered only at drying off did not show advantages over other measures including no treatment, or treatment with other benzimidazoles. Consequently, TCBZ should only be used to treat individual animals after correct diagnosis of the infection, and correct management measures taken to control re-infection.

The dynamics of antibody production against Neospora caninum during the gestation period was examined in chronically infected dairy cows. Data were obtained from 86 pregnant parous dairy cows, 21 of which had suffered abortion. The cows belonged to two herds in which a diagnosis of N. caninum infection had been previously confirmed in aborted foetuses. Pregnancy diagnosis and blood collection were performed on post-insemination Days 40, 90, 120, 150, 180, 210, and at parturition or until the time of abortion detection. Blood plasma was tested for antibodies against N. caninum using ELISA. The non-aborting cows were divided into two groups according to whether their antibody values in the second half of gestation had increased or not, while aborting cows were classified as those showing an antibody peak before abortion or those not showing a pre-abortion peak. Differences in antibody values throughout pregnancy in each group of non-aborting and aborting cows were analysed by GLM repeated measures of analysis of variance. While 32 non-aborting cows (49%) showed a significant and consistent increase in anti-Neospora antibody values during the second half of gestation, antibody values in the remaining 33 non-aborting cows were practically constant throughout gestation. An antibody peak around abortion was observed in 11 aborting cows (52%), while antibody values in the remaining 10 aborting cows were similar before and at abortion. Seroprevalence fluctuations, defined as seronegative blood samples at some point during the gestation period, were, furthermore, observed in 2 aborting and 11 non-aborting cows. Our results indicate two clearly distinguishable types of humoral immune dynamics throughout gestation: an increased or flat production of antibodies during the second half of gestation in non-aborting animals and before abortion in aborting cows. The observation that some Neospora-infected dams can exhibit negative antibody values at any time during gestation, particularly at parturition or abortion, prompts future studies designed to explore the use of new ELISA strategies at the farm level.

The objective of the present study was to determine the dynamics of infestation of cattle and pasture by gastrointestinal nematodes in a mild humid environment in northwestern Spain. For this, infestation of pasture by free-living stages (L3), dynamics of faecal egg output, blood pepsinogen levels and worm burden in slaughtered animals were quantified. The results showed a high degree of annual variability, which was dependent on weather conditions. The seasons were clearly defined in the study area, with mild humid winters and relatively dry summers registered throughout the years of the study. Infestation of pasture by larvae varied from year to year, peaking during August in the first year, between August and December in the second year, and during October in the third year. The annual variation was mainly due to weather conditions, particularly the amount of rain in summer. The patterns of faecal egg output were similar in the first and third grazing seasons, with maximum levels observed in May/June; however, in the second year, the peak was reached in October. Blood pepsinogen levels increased from pasture turnout (March/April) until the end of the grazing season (November/December), reaching maximum values from August/September onwards. The nematode parasite species identified at necropsy were Ostertagia osteragi, O. lyrata, Cooperia oncophora, C. macmasteri, C. punctata and Trichuris ovis, with O. ostertagi and C. oncophora predominating. In faecal cultures, the following genera were also identified: Haemonchus, Trichostrongylus, Nematodirus, Bunostomum, Oesophagostomum and Strongyloides. There was a significant correlation (r=0.97, P

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

During calving time on an experimental farm, 32 newborn calves were selected at random and monitored for infection with Cryptosporidium parvum for the first 30 days of their lives. The animals were fed pooled colostrum for 2-3 days after birth and housed in individual pens, which were washed daily using a pressure hose. Fecal smears were examined by microscopy after staining with carbol fuschin for visualization of oocysts. Oocyst shedding was scored semiquantitatively according to the average number of oocysts in 20 randomly selected fields at 1000x magnification. All the animals acquired the infection before 18 days of age. The period of maximum risk was between 9 and 12 days; 50% of the animals were infected by 9.4 days of age. It was found that the earlier the animals acquired the infection, the longer the patent period. Oocyst shedding, which did not always begin with the onset of diarrhea, lasted between 8 and 23 days (mean 12.4+/-3.3 days). Furthermore, fecal samples from 32 periparturient cows (within +/-7 days of giving birth) were filtered, concentrated and examined for oocysts using a fluorescent monoclonal antibody test, which revealed that six of the cows, although asymptomatic, were excreting C. parvum oocysts.

The effectiveness of beta-cyclodextrin, excipient used in pharmaceutical industry, in the treatment of natural infection by Cryptosporidium parvum in suckling calves, was evaluated. Administration of the drug at a dose of 500 mg/kg body weight for 3 consecutive days from birth (prophylactically) or following confirmation of the infection (therapeutically) decreased the severity of diarrhoea and shortened the duration of oocyst shedding.

The monoclonal antibody ES78 was used in a sandwich immunosorbent assay (Sandwich ELISA) for the detection of antigens in sera and faeces in the course of Fasciola hepatica infection in 10 experimentally infected sheep. All infected sheep had circulating antigens in the first week post-infection (WPI). Antigenemia was detectable until WPI 3 in four infected sheep, WPI 4 in five infected sheep and in only one sheep by WPI 5. The detection of coproantigens (Fa(g)) was possible in five infected sheep at WPI-4, in four sheep at WPI-5 and in one sheep only at WPI-6. This technique was compared to an indirect ELISA for the detection of antibodies using excretory secretory antigens of F. hepatica. A significant correlation was found between Fa(g) and egg output and also with adult worm numbers. Our method demonstrated that the diagnosis of active fasciolosis in sheep is possible during all periods of infection.

1 pagina.- Trabajo presentado al 24th Annual Meeting of the Sapnish Society of Veterinary Pathology(Leon, Spain, Sep 5-8, 2012)

Background Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing. Methodology/Principal Findings We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples. Conclusions/Significance The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required., Author Summary Fasciolosis is an important plant-borne trematode zoonosis. This disease is of both clinical and veterinary relevance and, according to the WHO, is considered a re-emerging disease that is spreading around the world. Fasciolosis has a serious impact on health because of the large size of the parasite and the effects of the parasite in down-regulating the host immune response. Human fasciolosis can be distinguished by an acute phase, in which the parasite migrates through different tissues, and a chronic phase in which it invades the bile ducts. Here we describe the development of a rapid, simple and inexpensive immunochromatographic diagnostic method, based on the use of a recombinant cathepsin L1 protein, which performs better than other more complex indirect methods, providing similar specificity and higher sensitivity. The simplicity of the method represents a great advantage for the intervention systems applied in different endemic areas by WHO, such as passive case finding (e.g. Vietnam) and selective treatment (e.g. Egypt). Because of its characteristics, the system can be applied to both phases of the disease, and in holo, meso and hyperendemic areas where point-of-care testing is required.

Infections caused by human parasites (HPs) affect the poorest 500 million people worldwide but chemotherapy has become expensive, toxic, and/or less effective due to drug resistance. On the other hand, many 3D structures in Protein Data Bank (PDB) remain without function annotation. We need theoretical models to quickly predict biologically relevant Parasite Self Proteins (PSP), which are expressed differentially in a given parasite and are dissimilar to proteins expressed in other parasites and have a high probability to become new vaccines (unique sequence) or drug targets (unique 3D structure). We present herein a model for PSPs in eight different HPs (Ascaris, Entamoeba, Fasciola, Giardia, Leishmania, Plasmodium, Trypanosoma, and Toxoplasma) with 90% accuracy for 15 341 training and validation cases. The model combines protein residue networks, Markov Chain Models (MCM) and Artificial Neural Networks (ANN). The input parameters are the spectral moments of the Markov transition matrix for electrostatic interactions associated with the protein residue complex network calculated with the MARCH-INSIDE software. We implemented this model in a new web-server called MISS-Prot (MARCH-INSIDE Scores for Self-Proteins). MISS-Prot was programmed using PHP/HTML/Python and MARCH-INSIDE routines and is freely available at: http://miaja.tic.udc.es/Bio-AIMS/MISS-Prot.php. This server is easy to use by non-experts in Bioinformatics who can carry out automatic online upload and prediction with 3D structures deposited at PDB (mode 1). We can also study outcomes of Peptide Mass Fingerprinting (PMFs) and MS/MS for query proteins with unknown 3D structures (mode 2). We illustrated the use of MISS-Prot in experimental and/or theoretical studies of peptides from Fasciola hepatica cathepsin proteases or present on 10 Anisakis simplex allergens (Ani s 1 to Ani s 10). In doing so, we combined electrophoresis (1DE), MALDI-TOF Mass Spectroscopy, and MASCOT to seek sequences, Molecular Mechanics + Molecular Dynamics (MM/MD) to generate 3D structures and MISS-Prot to predict PSP scores. MISS-Prot also allows the prediction of PSP proteins in 16 additional species including parasite hosts, fungi pathogens, disease transmission vectors, and biotechnologically relevant organisms.

The antigen-specific IgG subclass response may be a convenient indicator of the underlying nature of T helper cell regulation. The aim of the present study was to identify possible differences in Neospora caninum-specific total plasma IgG, IgG1 and IgG2 antibody levels in purebreed and crossbreed pregnancies throughout gestation in beef and dairy cattle chronically infected with N. caninum. Comparisons were also made between aborting and non-aborting dams. The population examined comprised 96 pregnant parous cows seropositive for N. caninum. Plasma antibodies were determined on Days 90, 120, 150, 180 and 210 of gestation or until abortion. Of the 96 pregnancies examined, 12 ended in abortion. None of the 14 Holstein-Friesian (HF) cows inseminated with HF semen (HF-HF group) aborted, whereas 6 (11.0%) of the 54 HF cows inseminated with Limousin semen (HF-L group) and 6 (21.4%) of the 28 Rubia Gallega (RG) beef cows inseminated with RG semen (RG-RG group) aborted. In the 84 non-aborting cows, a significant positive effect of gestation day was observed on total IgG, IgG1 and IgG2 antibodies levels (P

[Background] Paramphistomosis caused by Calicophoron daubneyi and fasciolosis caused by Fasciola hepatica are common parasitic diseases of livestock animals. Transmission of the diseases depends on the presence of intermediate hosts, i.e. freshwater gastropods such as lymnaeids. We carried out a 2-year-long study of the dynamics of the snail population acting as the intermediate host for these parasites, considering the population structure in terms of size/age and infection status. In addition, we determined the kinetics of trematode egg excretion in grazing cows. Generalized Additive Models (GAMs) were used to analyze the associations between different response variables and snail size, sampling month and weather-related variables., [Results] Of the molluscan species examined, Galba truncatula, Radix peregra, Anisus (Anisus) leucostoma and Pisidium casertanum (n = 2802), only G. truncatula was infected with C. daubneyi or F. hepatica, at prevalence rates of 8.2% and 4.4% respectively. The probability of infection with C. daubneyi or F. hepatica was linearly related to snail size, although in different ways (negative for C. daubneyi and positive for F. hepatica). The total snail population increased in winter, when specimens of all size classes were found. Infected snails were more abundant during spring-autumn. Mature cercariae of both parasites were found in most seasons. In the statistical models, the sampling month accounted for a high percentage (71.9–78.2%) of the observed variability in snail abundance. The inclusion of climatic variables in the models moderately increased the percentage of deviance explained (77.7–91.9%). Excretion of C. daubneyi eggs in cow faeces was always higher than that of F. hepatica eggs., [Conclusions] Particular care should be taken to prevent pastures and the surrounding environment being contaminated with parasite eggs during winter-spring, when the number of snails susceptible to miracidial infections is maximal. This is therefore the optimal time for treating grazing animals. Nevertheless, control of trematodosis based only on chemotherapy is difficult in an area such as the study area, where environmental factors favour the regular appearance of snail populations harbouring mature cercariae., The present study was financially supported by the Ministerio de Economía y Competitividad (AGL2011-30563-C03-03). The funders did not have any role in the study design, data collection and analyses, decision to publish or preparation of the manuscript.