Delineating distinct heme-scavenging and -binding functions of domains in MF6p/helminth defense molecule (HDM) proteins from parasitic flatworms.

MF6p/FhHDM-1 is a small protein secreted by the parasitic flatworm (trematode) Fasciola hepatica that belongs to a broad family of heme-binding proteins (MF6p/helminth defense molecules (HDMs)). MF6p/HDMs are of interest for understanding heme homeostasis in trematodes and as potential targets for the development of new flukicides. Moreover, interest in these molecules has also increased because of their immunomodulatory properties. Here we have extended our previous findings on the mechanism of MF6p/HDM-heme interactions and mapped the protein regions required for heme binding and for other biological functions. Our data revealed that MF6p/FhHDM-1 forms high-molecular-weight complexes when associated with heme and that these complexes are reorganized by a stacking procedure to form fibril-like and granular nanostructures. Furthermore, we showed that MF6p/FhHDM-1 is a transitory heme-binding protein as protein·heme complexes can be disrupted by contact with an apoprotein (e.g. apomyoglobin) with higher affinity for heme. We also demonstrated that (i) the heme-binding region is located in the MF6p/FhHDM-1 C-terminal moiety, which also inhibits the peroxidase-like activity of heme, and (ii) MF6p/HDMs from other trematodes, such as Opisthorchis viverrini and Paragonimus westermani, also bind heme. Finally, we observed that the N-terminal, but not the C-terminal, moiety of MF6p/HDMs has a predicted structural analogy with cell-penetrating peptides and that both the entire protein and the peptide corresponding to the N-terminal moiety of MF6p/FhHDM-1 interact in vitro with cell membranes in hemin-preconditioned erythrocytes. Our findings suggest that MF6p/HDMs can transport heme in trematodes and thereby shield the parasite from the harmful effects of heme.

Endogenous transplacental transmission of Neospora caninum during successive pregnancies across three generations of naturally infected sheep.

Endogenous transplacental transmission, which occurs during pregnancy as the result of reactivation of a latent infection in the dam, is the main mechanism of propagation of Neospora caninum within cattle herds. However, the importance of this propagation mechanism has not yet been evaluated in relation to ovine neosporosis. In this study, involving three generations of ewes naturally infected by N. caninum, we demonstrated that endogenous transplacental transmission may also be highly efficient in the ovine host since transmission of infection occurred in 96.6% of gestations and the congenital infection rate ranged between 66.7 and 93%. Nevertheless, parasite burdens decreased gradually in consecutive generations. Reactivation of latent infections had a strong impact on the pregnancy outcome, with high mortality rates recorded in the offspring of the two first generations of ewes (21.4-46.1%). Histological examination of the brain revealed that all aborted foetuses had characteristic lesions of neosporosis (necrotic glial foci) and a few parasite cysts, whereas most stillborn and newborn lambs that died shortly after birth had non-specific lesions (mild glial foci without necrosis) and parasite cysts were more frequent. Microsatellite analysis revealed scarce genetic variability in the N. caninum population, in accordance with a scenario in which infections were of a single origin and were exclusively maintained by clonal propagation through endogenous transplacental transmission.

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis.

Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic methods, typically ELISA (with either native or recombinant antigens), are often used for early diagnosis. The use of native antigens, as in the MM3-SERO ELISA (commercialized as BIO K 211, BIO-X Diagnostics), continues to be beneficial in terms of sensitivity and specificity; however, there is interest in developing ELISA tests based on recombinant antigens to avoid the need to culture parasites. Of the antigens secreted by adult flukes, recombinant procathepsin L1 (rFhpCL1) is the most commonly tested in ELISA to date. However, although adult flukes produce three different clades of CLs (FhCL1, FhCL2, and FhCL5), to our knowledge, the diagnostic value of recombinant FhCL2 and FhCL5 has not yet been investigated. In the present study, we developed and tested three indirect ELISAs using rFhpCL1, rFhpCL2, and rFhpCL5 and evaluated their recognition by sera from sheep and cattle naturally infected with F. hepatica. Although the overall antibody response to these three rFhpCLs was similar, some animals displayed preferential recognition for particular rFhpCLs. Moreover, for cattle sera, the highest sensitivity was obtained using rFhpCL2 (97%), being equal for both rFhpCL1 and rFhpCL5 (87.9%), after adjusting cut-offs for maximum specificity. By contrast, for sheep sera, the sensitivity was 100% for the three rFhpCLs. Finally, the presence of truncated and/or partially unfolded molecules in antigen preparations is postulated as a possible source of cross-reactivity.

The MF6p/FhHDM-1 major antigen secreted by the trematode parasite Fasciola hepatica is a heme-binding protein.

Blood-feeding parasites have developed biochemical mechanisms to control heme intake and detoxification. Here we show that a major antigen secreted by Fasciola hepatica, previously reported as MF6p, of unknown function (gb|CCA61804.1), and as FhHDM-1, considered to be a helminth defense molecule belonging to the family of cathelicidin-like proteins (gb|ADZ24001.1), is in fact a heme-binding protein. The heme-binding nature of the MF6p/FhHDM-1 protein was revealed in two independent experiments: (i) immunopurification of the secreted protein·heme complexes with mAb MF6 and subsequent analysis by C8 reversed-phase HPLC and MS/MS spectrometry and (ii) analysis of the binding ability of the synthetic protein to hemin in vitro. By immunohistochemistry analysis, we have observed that MF6p/FhHDM-1 is produced by parenchymal cells and transported to other tissues (e.g. vitellaria and testis). Interestingly, MF6p/FhHDM-1 is absent both in the intestinal cells and in the lumen of cecum, but it can be released through the tegumental surface to the external medium, where it binds to free heme molecules regurgitated by the parasite after hemoglobin digestion. Proteins that are close analogs of the Fasciola MF6p/FhHDM-1 are present in other trematodes, including Clonorchis, Opistorchis, Paragonimus, Schistosoma, and Dicrocoelium. Using UV-visible spectroscopy and immunoprecipitation techniques, we observed that synthetic MF6p/FhHDM-1 binds to hemin with 1:1 stoichiometry and an apparent Kd of 1.14 × 10(-6) M(-1). We also demonstrated that formation of synthetic MF6p/FhHDM-1·hemin complexes inhibited hemin degradation by hydrogen peroxide and hemin peroxidase-like activity in vitro. Our results suggest that MF6p/FhHDM-1 may be involved in heme homeostasis in trematodes.

Neospora caninum infection as a cause of reproductive failure in a sheep flock.

Neospora caninum has been detected only sporadically in cases of ovine abortion, and it has therefore traditionally been considered as an unimportant parasite in small ruminants. This study was carried out with the aim of identifying the pathogen causing serious reproductive problems on a commercial sheep farm. Sera from all rams and ewes tested negative for antibodies against Border disease virus, Schmallenberg virus and Coxiella burnetii, and infections by these agents were therefore ruled out. Nevertheless, seropositivity to N. caninum and/or Toxoplasma gondii was detected, although the seroprevalence was higher in the case of N. caninum. The percentage of lambings and the number of lambs per dam were significantly lower in ewes that were seropositive to N. caninum while no effect on these parameters was detected in ewes that were seropositive to T. gondii. There was also no evidence of infection by T. gondii in the foetal/lamb tissues analyzed by PCR and/or immunohistopathological techniques. On the contrary, the DNA of N. caninum was detected in 13 out of 14 foetuses/lambs descendant from dams seropositive to this parasite. Characteristic lesions caused by N. caninum and/or its antigen were also detected. Genotyping of the N. caninum DNA revealed only two closely related microsatellite multilocus genotypes. The results clearly demonstrate that infection by N. caninum was the cause of the low reproductive performance of this sheep flock.

Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses.

This work studies the influence of Neospora caninum intra-species diversity on abortion outcome, infection dynamics in terms of parasite dissemination and peripheral-local immune responses in pregnant cattle. Animals were intravenously inoculated at day 70 of pregnancy with 107 tachyzoites of two isolates showing marked differences in virulence in vitro and in pregnant mouse models: Nc-Spain7, a high virulence isolate, and Nc-Spain8, a low-to-moderate virulence isolate. After inoculation, pregnancy was monitored, and dams were culled when foetal death was detected. Foetal mortality occurred in all infected heifers between days 24 and 49 post-infection (pi), however, it was detected sooner in Nc-Spain7-infected animals (median day = 34) than those inoculated with Nc-Spain8 (median day = 41) with a trend towards significance (P < 0.11). Similar histological lesions were observed in placentomes and in most of the foetuses from the two infected groups. However, parasites were more frequently detected in the placenta and foetuses by PCR and in the foetal brain by immunohistochemistry in Nc-Spain7-infected animals. Specific antibodies were detected starting at day 13 post-infection in all infected cattle, with higher IgG levels in Nc-Spain7-infected group. IFN-γ and IL-4 profiles also varied between infected groups in PBMC stimulation assays. Infected animals showed significant increases in their cytokine mRNA levels (IFN-γ, IL-4, IL-10, IL-12p40 and TNF-α) in the caruncle at time of foetal death. Differences between the infected groups were also observed for cytokine profiles. These results demonstrate the influence of the N. caninum isolate on foetal death outcome, infection dynamics and immune responses in cattle.

Bayesian hierarchical modelling of the geospatial distribution of fasciolosis in dairy cattle and the impact on production: Application to the main milk-producing region (Galicia) in Spain.

Fasciolosis caused by Fasciola hepatica is a common parasitic infection among cattle in many countries. Although infected adult cows rarely show overt clinical signs, milk production may be impaired. Thus, significant production losses may occur in dairy herds with a high prevalence of fasciolosis. In this study, Bayesian hierarchical modelling was used to estimate the geospatial distribution of dairy cattle fasciolosis and its impact on milk production. The study was conducted in Galicia, the main milk producing region in Spain and a geographically heterogeneous area. The aims were: 1) to model the geospatial distribution of fasciolosis in dairy herds in the study area, 2) to identify clusters of herds with a high prevalence of fasciolosis, and 3) to assess the effect of fasciolosis on milk yield and quality. A large number of dairy cattle farms (n = 4907), of which 1660 provided production records, were surveyed. Fasciola infection status was determined by applying the MM3-SERO ELISA test to bulk tank milk samples. A high probability of infection was predicted in several zones, particularly in the centre, northeast and southeast of Galicia. Conversely, the predicted probability was very low in some parts of the northwest of the region. Infections with high within-herd prevalence (> 25% lactating cows infected) predominated. High within-herd prevalence was associated with loss of milk production (-1.387 kg/cow/ day, on average). No association between Fasciola infection and either milk fat or protein content was observed. This study has generated the first maps of the spatial distribution of the probability of Fasciola infection in dairy cattle herds in Galicia. The maps presented here can be used for reference purposes, enabling the design of better targeted fasciolosis control programmes in the region. Use of Bayesian hierarchical statistical analysis enabled us to ascertain the uncertainty of the predictions and to account for the spatial autocorrelation in the data. It also enabled us to generate maps showing the residual spatial variation in milk production, a topic that may deserve more detailed study.

Increased specificity of Fasciola hepatica excretory-secretory antigens combining negative selection on hydroxyapatite and salt precipitation.

A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.

New benzimidazole derivative compounds with in vitro fasciolicidal properties.

BACKGROUND: Control of the zoonotic food-borne parasite Fasciola hepatica remains a major challenge in humans and livestock. It is estimated that annual economic losses due to fasciolosis can reach US$3.2 billion in agriculture and livestock. Moreover, the wide distribution of drug-resistant parasite populations and the absence of a vaccine threaten sustainable control, reinforcing the need for novel flukicides. METHODS: The present work analyses the flukicidal activity of a total of 70 benzimidazole derivatives on different stages of F. hepatica. With the aim to select the most potent ones, and screenings were first performed on eggs at decreasing concentrations ranging from 50 to 5 µM and then on adult worms at 10 µM. Only the most effective compounds were also evaluated using a resistant isolate of the parasite. RESULTS: After the first screenings at 50 and 10 µM, four hit compounds (BZD31, BZD46, BZD56, and BZD59) were selected and progressed to the next assays. At 5 µM, all hit compounds showed ovicidal activities higher than 71% on the susceptible isolate, but only BZD31 remained considerably active (53%) when they were tested on an albendazol-resistant isolate, even with values superior to the reference drug, albendazole sulfoxide. On the other hand, BZD59 displayed a high motility inhibition when tested on adult worms from an albendazole-resistant isolate after 72 h of incubation. CONCLUSIONS: BZD31 and BZD59 compounds could be promising candidates for the development of fasciolicidal compounds or as starting point for the new synthesis of structure-related compounds.

Field evaluation of the enhanced MM3-COPRO ELISA test for the diagnosis of Fasciola hepatica infection in sheep.

Fasciolosis is a severe zoonosis responsible for major economic losses in livestock. The enhanced MM3-COPRO test (eMM3-COPRO) and the commercial version BIO K 201 (Bio-X Diagnostics, Rochefort, Belgium) are widely used as immunodiagnostic tools for the specific detection of coproantigens released by Fasciola during the late prepatent and patent stages of infection. However, performance of the eMM3-COPRO has never been evaluated under field conditions. To address this gap, a large number of ovine faecal samples, collected in a region where fasciolosis is endemic (Galicia, NW Spain), were analyzed. Two groups of sheep flocks were selected according to the Fasciola infection status: 'Fasciola-free' and 'Fasciola-infected' flocks. 'Fasciola-free' flocks were seronegative flocks with no history of fasciolosis detected by either coproscopy or necropsy in the last 5 years. Faecal samples from these sheep were used to calculate a cut-off value for infection (OD = 0.021). The cut-off was calculated using a bootstrap resampling method that enables estimation of the sampling distribution of the statistical parameters without making assumptions about the underlying data distribution. 'Fasciola-infected' flocks were characterized by high seroprevalence, a history of fasciolosis and periodical treatment with flukicides. Samples from these flocks were used to estimate the diagnostic accuracy of the eMM3-COPRO relative to coproscopy, which although limited by poor sensitivity is the only reference test available for diagnosing fasciolosis in vivo. To overcome this limitation, all animals classified positive by eMM3-COPRO were treated with triclabendazole and then retested. The eMM3-COPRO displayed higher sensitivity than coproscopy, as it detected coproantigens in all samples with positive coproscopy and in 12% of samples with negative coproscopy. The test also proved highly specific as coproantigens disappeared after the treatment. The eMM3-COPRO was less time consuming than coproscopy, particularly when the procedure involved numerous samples, and showed promise as a tool for monitoring flukicide efficacy.

Ovine Neosporosis: The Current Global Situation.

In the past 20 years, Neospora caninum infection in sheep has been reported in at least 31 countries worldwide from all sheep-rearing continents (Europe, Asia, the Americas, Africa, and Oceania), and its role as an abortifacient agent is becoming more evident. Most studies of ovine neosporosis have focused on its epidemiology, based primarily on serological analysis, with only a few studies investigating the actual presence of the parasite by PCR and/or IHC. Individual seroprevalence rates were highly variable between countries, and even between regions within the same country, ranging from 0.0% to 67.4% positive. Furthermore, most of the studies were not directly comparable due to differences in experimental designs, sample sizes, husbandry systems, ecological factors, and serological tests (e.g., IFAT, ELISA, MAT, Western blot). The latter, along with the scarcity of studies on the relevance of N. caninum as an abortifacient agent, may bias the perception of the importance of this disease. This review summarizes the situation of N. caninum infection in sheep using all available published studies describing natural ovine neosporosis. The epidemiology shows that ovine neosporosis is found worldwide, and it poses a relevant risk to the sustainability of sheep flocks.

Current Challenges for Fasciolicide Treatment in Ruminant Livestock.

Pharmacological treatment remains essential to control fasciolosis in areas where infection is endemic. However, there are major constraints to treating food-producing animals. Of particular concern is the lack of flukicides for treating early Fasciola infections in ruminant livestock in some countries. In addition, the information provided in package leaflets, particularly regarding withdrawal periods, is often incomplete, confusing, and/or contradictory. International regulatory bodies should harmonize the use of flukicides in livestock in favor of fairer, safer international trade. In addition, monitoring the efficacy of fasciolicides on farms is also essential to minimize the spread of drug-resistant populations of Fasciola. The current situation regarding flukicide formulations in the European Union and other, non-European countries is analyzed in this review paper.

Isolation and genetic characterization of Neospora caninum from naturally infected sheep.

Neospora caninum is considered one of the main causes of abortion in cattle but can also cause abortion in sheep. There is limited knowledge of the N. caninum population infecting sheep, and only one N. caninum isolate from a pregnant sheep from Japan has been reported. This study describes the in vitro isolation and genetic characterization of two new sheep isolates of N. caninum implicated in ovine reproductive failure. We used IFN-γ-knockout mice inoculated with PCR-positive brain homogenates from two clinically healthy but congenitally infected lambs at 4.5 months of age for parasite isolation. The lambs were born to dams from a sheep farm that had experienced pregnancy failure caused by N. caninum in successive generations. Tachyzoites were microscopically visualized in peritoneal flushes from all inoculated mice and were also observed in MARC-145 cell cultures within one week after inoculation with peritoneal flushes. Two N. caninum isolates, Nc-Spain11 and Nc-Spain12, were obtained from each lamb. The genotyping of the Nc-Spain11 and Nc-Spain12 isolates based on 9 microsatellite markers showed identical multilocus genotype (MLG). Comparison between a previous N. caninum genotype dataset including 80 MLGs from Argentinean, Spanish, Mexican, German and Scottish bovine isolates and the Japanese sheep isolate showed that the Nc-Spain11 and Nc-Spain12 MLG was unique and differed from the other MLGs. eBURST analyses showed that the Nc-Spain11 and Nc-Spain12 MLG was genetically clustered with other bovine MLGs and one ovine MLG, and the nearest genetic relationship was with an MLG from a bovine abortion collected in the same geographical area of Galicia.

Maternal immune response in the placenta of sheep during recrudescence of natural congenital infection of Neospora caninum.

In order to gain further insight into the pathogenesis and transmission of ovine neosporosis, the serological response of 13 naturally infected pregnant sheep was monitored. All sheep were euthanized upon the detection of a sharp increase in the level of specific antibodies against N. caninum in order to study the maternal immune response after the recrudescence of a chronic infection. Ten sheep were euthanized between 84 and 118 days of gestation, whereas the three remaining and three control not infected, pregnant sheep were euthanized at 135 days of gestation after no sharp increase in antibodies was detected. Vertical transmission was confirmed in 11 sheep by detection of N. caninum-DNA in at least one fetus, confirming recrudescence. Not all of fetuses showed pathologic microscopic lesions, however, multifocal non-purulent encephalitis was the main finding. Furthermore, nine out of the 11 vertical transmission positive sheep had lesions in placentomes (mainly multifocal necrotic foci), and the parasite was detected in eight out of 11 placentas by PCR and/or immunohistochemestry. The placentomes from sheep that suffered recrudescence showed an increased number of T lymphocytes CD3+ (CD4/CD8 < 1) and macrophages (MHC-II+), assessed by immunohistochemestry, together with an up-regulation of IFN-γ, IL-10, IL-4, TNFα, IL-2 and IL-18. IL-17 was only upregulated in the three infected sheep that did not have a sharp increase in antibody levels. In the sheep that showed fetal death at the time of euthanasia (n = 3) the placental microscopic lesions were more severe, the inflammatory infiltrate was higher, and the upregulation of cytokines was greater than in those sheep carrying viable fetuses. This study suggests that, similarly to bovine neosporosis, the time of gestation when recrudescence occurs determines the viability of the fetuses and, thus, seems to be related to the severity of lesions and immune response in the placenta. These results suggest that there might be a correlation, either as cause or as a consequence, between protection against vertical transmission of the parasite and a milder maternal serological response together with a high level of transcription of IL-17 in the placenta.

In-plate recapturing of a dual-tagged recombinant Fasciola antigen (FhLAP) by a monoclonal antibody (US9) prevents non-specific binding in ELISA.

Recombinant proteins expressed in E. coli are frequently purified by immobilized metal affinity chromatography (IMAC). By means of this technique, tagged proteins containing a polyhistidine sequence can be obtained up to 95% pure in a single step, but some host proteins also bind with great affinity to metal ions and contaminate the sample. A way to overcome this problem is to include a second tag that is recognized by a preexistent monoclonal antibody (mAb) in the gene encoding the target protein, allowing further purification. With this strategy, the recombinant protein can be directly used as target in capture ELISA using plates sensitized with the corresponding mAb. As a proof of concept, in this study we engineered a Trichinella-derived tag (MTFSVPIS, recognized by mAb US9) into a His-tagged recombinant Fasciola antigen (rFhLAP) to make a new chimeric recombinant protein (rUS9-FhLAP), and tested its specificity in capture and indirect ELISAs with sera from sheep and cattle. FhLAP was selected since it was previously reported to be immunogenic in ruminants and is expressed in soluble form in E. coli, which anticipates a higher contamination by host proteins than proteins expressed in inclusion bodies. Our results showed that a large number of sera from non-infected ruminants (mainly cattle) reacted in indirect ELISA with rUS9-FhLAP after single-step purification by IMAC, but that this reactivity disappeared testing the same antigen in capture ELISA with mAb US9. These results demonstrate that the 6XHis and US9 tags can be combined when double purification of recombinant proteins is required.

Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis.

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<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.

Evaluation of the flukicide treatment policy for dairy cattle in Galicia (NW Spain).

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 (p<0.05). The percentage of farms with within-herd prevalences >25% was very high in all three groups, and no significant differences were observed. In contrast, the percentage of herds with mean antigen concentrations >20ng/ml was significantly lower (p<0.05) 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 (p<0.05) 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.

Activity of an anti-inflammatory drug against cryptosporidiosis in neonatal lambs.

The efficacy of the anti-inflammatory drug Bobel-24 against experimental infection by Cryptosporidium parvum was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 50 or 500 mg/kg of body weight. The prophylactic/therapeutic treatment was started 4 h before inoculation of the lambs with oocysts and was continued for eight consecutive days. The therapeutic treatment was initiated at the onset of diarrhoea, after confirmation of infection, and was continued for six consecutive days. Infection was monitored by daily examination of faecal samples from the first day until 30 days post-inoculation. The criteria considered in evaluating development of the infection and the drug activity were: oocyst shedding, presence of diarrhoea and weight gain at 15 and 30 days post-inoculation. Bobel-24 was effective as a prophylactic/therapeutic treatment at the lowest dose (50 mg/kg of body weight); in the group treated with this dose of drug there was a longer prepatent period, a shorter patent period and a lower intensity of oocyst excretion than in the untreated control group, and the differences were all statistically significant (P<0.05). Moreover, one animal did not excrete oocysts, and two lambs had diarrhoea, for only 1 and 2 days. In the group treated with the higher dose of the drug, the diarrhoea lasted for a significantly shorter period (P<0.05) than in the untreated group.

Presence of Cryptosporidium spp. and Giardia duodenalis through drinking water.

To evaluate the presence of Cryptosporidium spp. and Giardia duodenalis in the influent and final effluent of sixteen drinking water treatment plants located in a hydrographic basin in Galicia (NW Spain) - in which the principal river is recognised as a Site of Community Importance (SCI) - estimate the efficiency of treatment plants in removing these protozoans and determine the species and genotypes of the parasites by means of a molecular assay. All plant samples of influent and final effluent (50-100 l) were examined in the spring, summer, autumn and winter of 2007. A total of 128 samples were analysed by method 1623, developed by US Environmental Protection Agency for isolation and detection of both parasites. To identify the genotypes present the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and b-giardina (G. duodenalis). The mean concentrations of parasites in the influent were 0.0-10.5 Cryptosporidium spp. oocysts per litre and 1.0-12.8 of G. duodenalis cysts per litre. In the final treated effluent, the mean concentration of parasites ranged from 0.0-3.0 oocysts per litre and 0.5-4.0 cysts per litre. The distribution of results by season revealed that in all plants, the highest numbers of (oo)cysts were recorded in spring and summer. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. Cryptosporidium spp. and G. duodenalis were consistently found at high concentrations in drinking water destined for human and animal consumption in the hydrographic basin under study, in Galicia (NW Spain). It is important that drinking water treatment authorities rethink the relevance of contamination levels of both parasites in drinking water and develop adequate countermeasures.

Vaccination of sheep with Quil-A® adjuvant expands the antibody repertoire to the Fasciola MF6p/FhHDM-1 antigen and administered together impair the growth and antigen release of flukes.

Fasciolosis continues to be a major cause of economic losses in the livestock industry and a growing threat to humans. The limited spectrum of effective anthelmintics and the appearance of resistances urge the need for developing an effective vaccine. Most studies have been focused on the use of TH1-polarizing adjuvants and the use of recombinant Fasciola critical molecules and, despite the efforts, no reproducible protections have been achieved. The F. hepatica MF6p/FhHDM-1 protein is a heme-binding protein also reported to have immunomodulatory properties, constituting a promising target for vaccination and/or as target for the development of new flukicides. Thus, in this study, we investigated the effects of the TH1-polarizing adjuvant Quil A® on sheep immune response to MF6p/FhHDM-1, and the vaccine potential of both native and synthetic forms of this protein against ovine fasciolosis. Subcutaneous injection of Quil A® alone, i.e., without co-injecting any antigen, expands the antibody repertoire to MF6p/FhHDM-1 triggered by a subsequent primoinfection with metacercariae. This effect was not observed with aluminum hydroxide, the most frequently adjuvant used in commercial vaccines. On the other hand, vaccination with synthetic MF6p/FhHDM-1 in Quil A® prompted a 2-4-week delay in the antibody response induced in sheep by a challenge experimental infection. Moreover, fluke populations stablished showed stunted growth and low antigen release probably due to reduced metabolic activity. These observations suggest that primary circulating antibodies induced by the immunization had harmful effects on fluke development. Such effects could not be demonstrated to be associated to TH1 immune response linked events (production of IgG2 isotype antibodies and IFN-γ).