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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Fasciola spp: Mapping of the MF6 epitope and antigenic analysis of the MF6p/HDM family of heme-binding proteins.

MF6p/FhHDM-1 is a small cationic heme-binding protein which is recognized by the monoclonal antibody (mAb) MF6, and abundantly present in parenchymal cells and secreted antigens of Fasciola hepatica. Orthologs of this protein (MF6p/HDMs) also exist in other causal agents of important foodborne trematodiasis, such as Clonorchis sinensis, Opisthorchis viverrini and Paragonimus westermani. Considering that MF6p/FhHDM-1 is relevant for heme homeostasis in Fasciola and was reported to have immunomodulatory properties, this protein is expected to be a useful target for vaccination. Thus, in this study we mapped the epitope recognized by mAb MF6 and evaluated its antigenicity in sheep. The sequence of the MF6p/FhHDM-1 ortholog from F. gigantica (MF6p/FgHDM-1) was also reported. By means of ELISA inhibitions with overlapping synthetic peptides, we determined that the epitope recognized by mAb MF6 is located within the C-terminal moiety of MF6p/FhHDM-1, which is the most conserved region of MF6p/HDMs. By immunoblotting analysis of parasite extracts and ELISA inhibitions with synthetic peptides we also determined that mAb MF6 reacted with the same intensity with F. hepatica and F. gigantica, and in decreasing order of intensity with C. sinensis, O.viverrini and P. westermani orthologs. On the contrary, mAb MF6 showed no reactivity against Dicrocoelium dendriticum and Schistosoma mansoni. The study of the recognition of peptides covering different regions of MF6p/FhHDM-1 by sera from immunized sheep revealed that the C-terminal moiety is the most antigenic, thus being of potential interest for vaccination. We also demonstrated that the production of antibodies to MF6p/FhHDM-1 in sheep infected by F. hepatica occurs relatively early and follows the same pattern as those produced against L-cathepsins.

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.

Transmission of Calicophoron daubneyi and Fasciola hepatica in Galicia (Spain): Temporal follow-up in the intermediate and definitive hosts.

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.

Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens.

ELISA-based methods of detecting Fasciola cathepsins in feces are powerful techniques for diagnosing infections by F. hepatica and F. gigantica. In the last decade, the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium) have been recognized as useful tools for detecting early infections by such trematodes and for monitoring the efficacy of anthelmintic treatments in human and animal species, as they provide some advantages over classic fecal egg counts. However, the sensitivity of MM3-COPRO ELISA can sometimes be compromised by the high variability in the concentration of cathepsins in fecal samples throughout the biological cycle of Fasciola (mainly in cattle) and by differences in the between-batch performance of peroxidase-labeled anti-mouse IgG polyclonal antibodies. To prevent such problems, we investigated whether the incorporation of a commercial streptavidin-polymerized horseradish peroxidase conjugate, in order to reveal bound biotinylated monoclonal antibody MM3, can improve the sensitivity of the MM3-COPRO ELISA. We observed that inclusion of this reagent shifted the previous detection limit of the assay from 0.6 ng/mL to 150 pg/mL and that the modified test is able to identify infection in cows harboring only one fluke. Moreover, we demonstrated that maximal OD values can be achieved with short incubations (30 min each step) at RT with shaking, rather than standard incubations, which significantly accelerates the diagnostic procedure. Finally, we did not find a significant correlation between coproantigen concentration and parasite burden in cattle, which may be due to the low parasite burden (1-10 adult flukes) of the animals used in the present study. As the usefulness of the classic MM3-COPRO test for detecting animal and human infections has already been demonstrated, it is expected that the improvements reported in this study will add new insights into the diagnosis and control of fasciolosis.

Higher physiopathogenicity by Fasciola gigantica than by the genetically close F. hepatica: experimental long-term follow-up of biochemical markers.

BACKGROUND: Fascioliasis is caused by Fasciola hepatica and F. gigantica. The latter, always considered secondary in human infection, nowadays appears increasingly involved in Africa and Asia. Unfortunately, little is known about its pathogenicity, mainly due to difficulties in assessing the moment a patient first becomes infected and the differential diagnosis with F. hepatica. METHODS: A long-term, 24-week, experimental study comparing F. hepatica and F. gigantica was made for the first time in the same animal model host, Guirra sheep. Serum biochemical parameters of liver damage, serum electrolytes, protein metabolism, plasma proteins, carbohydrate metabolism, hepatic lipid metabolism and inflammation were analysed on a biweekly basis as morbidity indicators. Serum anti-Fasciola IgG, coproantigen and egg shedding were simultaneously followed up. RESULTS: rDNA and mtDNA sequencing and the morphometric study by computer image analysis system (CIAS) showed that fasciolids used fitted standard species characteristics. Results demonstrated that F. gigantica is more pathogenic, given its bigger size and biomass but not due to genetic differences which are few. Fasciola gigantica shows a delayed development of 1-2 weeks regarding both the biliary phase and the beginning of egg shedding, with respective consequences for biochemical modifications in the acute and chronic periods. CONCLUSIONS: The higher F. gigantica pathogenicity contrasts with previous studies which only reflected the faster development of F. hepatica observed in short-term experiments.

Immunohistochemical study and mRNA cytokine profile of the local immune response in cattle naturally infected with Calicophoron daubneyi.

In order to recognize the local immune response of the definitive host to Calicophoron daubneyi natural infection, an immunohistochemical study was carried out in the reticulum and rumen in 49 naturally infected cattle. The role of cytokines (IL-4 and IL-10 interleukins and IFN-γ) in the activation of specific defence mechanisms was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays to study cytokine mRNA expression. In all infected animals, CD3+ T lymphocytes seemed to be the main element of the inflammatory infiltrate in the reticular and ruminal lamina propria at the point of the parasite adhesion. Intraepithelial globule leukocytes also showed immunolabelling for CD3. Most CD3+ cells also expressed CD4 (T cell helper) antigen although sporadic CD8+-cytotoxic lymphocytes were observed. Local expression of IFN-γ was observed in damaged papillae at the site of parasite attachment and in scattered cells in the lamina propria. B cells (CD79αcy+, CD45+ and IgG+) were found constantly in relation to lymphoid aggregates. MAC387 was expressed in squamous epithelium and in macrophages of the lamina propria of affected papillae. Macrophages in this location also stained positively for CD163 and CD68. Intraepithelial Langerhans cells and macrophages located in the lamina propria showed immunopositivity for MHCII in the affected areas. RT-qPCR analysis confirmed a statistical significant increase of IFN-γ, and IL-10 expression (p<0.01) in the rumen associated with the presence of flukes. These findings suggest a predominant Th1 polarized local immune response with the probable involvement of Th regulatory cells in cattle C. daubneyi natural infection.

Pathological changes in cattle naturally infected by Calicophoron daubneyi adult flukes.

Local host response and parasite distribution were studied in the forestomachs, abomasum, duodenum and regional lymph nodes of cattle suffering from bovine paramphistomosis. The parasites were found attached, by its ventral sucker, to small conical papillae of the rumen and reticulum. Affected papillae, showed morphological changes denoted by very narrow stalks and expanded heads. Histologically, these changes were characterized by epithelial acanthosis-hyperkeratosis of the epithelium. Infiltration of inflammatory cells was often related with the epithelial changes, although it was also found in the duodenal mucosa and submucosa. These cells were arranged as aggregates or follicles but sparse infiltration of eosinophils, globule leukocytes, mast cells or macrophages was also observed in the lamina propria. Tissue damage and inflammatory reaction were more severe in the ruminal atrium, where the largest number of flukes and affected papillae were observed. In contrast, lesions in the ruminal dorsal sac were absent or mild. Statistical correlation between lesion severity and parasite burden was confirmed.

Cryptosporidium spp. and Giardia duodenalis as pathogenic contaminants of water in Galicia, Spain: the need for safe drinking water.

The objectives of this cross-sectional study were to detect the presence of Cryptosporidium spp. and Giardia duodenalis in drinking water treatments plants (DWTPs) in Galicia (NW Spain) and to identify which species and genotype of these pathogenic protozoans are present in the water. Samples of untreated water (surface or ground water sources) and of treated drinking water (in total, 254 samples) were collected from 127 DWTPs and analysed by an immunofluorescence antibody test (IFAT) and by PCR. Considering the untreated water samples, Cryptosporidium spp. were detected in 69 samples (54.3%) by IFAT, and DNA of this parasite was detected in 57 samples (44.8%) by PCR, whereas G. duodenalis was detected in 76 samples (59.8%) by IFAT and in 56 samples (44.0%) by PCR. Considering the treated drinking water samples, Cryptosporidium spp. was detected in 52 samples (40.9%) by IFAT, and the parasite DNA was detected in 51 samples (40.1%) by PCR, whereas G. duodenalis was detected in 58 samples (45.6%) by IFAT and in 43 samples (33.8%) by PCR. The percentage viability of the (oo)cysts ranged between 90.0% and 95.0% in all samples analysed. Cryptosporidium andersoni, C. hominis, C. parvum and assemblages A-I, A-II, E of G. duodenalis were identified. The results indicate that Cryptosporidium spp. and G. duodenalis are widespread in the environment and that DWTPs are largely ineffective in reducing/inactivating these pathogens in drinking water destined for human and animal consumption in Galicia. In conclusion, the findings suggest the need for better monitoring of water quality and identification of sources of contamination.

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.