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 cercariaeThe 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 manuscriptS

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 fasciolosisWe have previously reported how the combined use of mAb MM3 with polyclonal antibodies obtained from rabbit immunized with Fasciola hepatica excretory-antigens led to the development of the in-house MM3-COPRO ELISA and its commercial version BIO K 201 (BIO X Diagnostics, Belgium), which are widely used to detect human and animal infections caused by F. hepatica. After more than a decade in use, both tests have proven to be useful tools for specifically detecting Fasciola infections, although it has also been found that: i) the conditions of use of the commercial test in some field studies did not enable the sensitivity obtained with the in-house test to be reached, and ii) the batches of the secondary reagent (peroxidase-labeled anti-mouse antibodies) currently available for use in the in-house test do not perform the same as previous batches. To solve these problems, we provide data showing that the incorporation of an enhancement system consisting of streptavidin-polymerized horseradish peroxidase conjugate greatly improved the sensitivity of the MM3-COPRO ELISA and enabled reduction of the incubation time. These modifications enabled the detectability of the assay to be 150 pg/mL, thus enabling detection of infection in animals harboring only one flukeThis work was funded by Ministerio de Ciencia e Innovación, Spain, Grants AGL2011- 30563-C02/C03; Xunta de Galicia, Spain, Grant GPC2014/058; and the European Fund for Regional Development (FEDER). VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario, AP2010-5808) and RAOM is recipient of a fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador, BES-2012- 060270)S

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 efficacyS

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.This work was funded by the Ministerio de Ciencia e Innovación, Spain (Grants AGL2011-30563-C03-01, AGL2011-30563-C03-02 and AGL2011-30563-C03-03); Xunta de Galicia, Spain (Grants GPC 2014/058 and ED431B 2017/18); Network of Biomedical Research on Tropical Diseases (RICET), Spain (Grant RD12/0018/0013) and the European Fund for Regional Development (FEDER). VMS holds a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte (Programa de Formación del Profesorado Universitario). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

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.This work was supported by: Ministerio de Economía y Competitividad (Spain) [grant number AGL2011-30563-C03 and AGL2014-57125R], Ministerio de Economía, Industria y Competitividad (INIA, Spain) [grants numbers RTA2017-00010-C02-01 and RTA2017-00010-C02-02] and the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia, Spain) [grant number ED431B 2017/18]. RAOM holds a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (Programa de Formación de Personal Investigador). VMS is supported by a contract under the grant ED431B 2017/18. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

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

[EN] 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 transmissionSIThe present study was supported by the Ministerio de Ciencia, Innovación y Universidades of Spain (INIA project RTA2014-00013

Characterization of a new albendazole resistant Fasciola hepatica isolate

Characterization of a new albendazole resistant Fasciola hepatica isolateMaría Martínez-Valladares11,2, Elora Valderas-García1,2, Verónica Castilla Gómez de Agüero1,2, Marta González-Warleta3, Laura Ceballos4, Juan P. Lirón4, Rodrigo Sanabria5, Cesar Pruzzo5, Luis I. Alvarez4.1Instituto de Ganadería de Montaña (CSIC-Universidad de León), Departamento de Sanidad Animal (Department of Animal Health), León, Spain.2Departamento de Sanidad Animal (Department of Animal Health), Facultad de Veterinaria, University of Leon, Leon, Spain.3Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, AGACAL, Abegondo, A Coruña, Spain.4Laboratorio de Farmacología, Centro de Investigación Veterinaria de Tandil (CIVETAN), UNCPBA-CICPBA-CONICET, Facultad de Ciencias Veterinarias, Tandil, Argentina.5Laboratorio de Parasitología, Facultad de Ciencias Veterinarias, Universidad Nacional de la Plata (UNLP), La Plata, Argentina.The infection by Fasciola hepatica affects mainly ruminants although is present in a wide variety of species including humans. Fasciolosis control is mainly based on triclabendazole administration, the main drug indicated in humans. Albendazole (ABZ) is used against nematode and liver fluke infections in ruminants. The misuse of these drugs has led to the appearance of anthelmintic resistance. In this study, we characterized an ABZ resistant isolate and evaluate the use of a combined treatment to improve treatment efficacy. The isolate was collected from a slaughterhouse in Argentina, maintained under laboratory conditions and identified by means of the egg hatch test (EHT). Using these eggs metacercariae were produced to infect sheep artificially. When flukes reached the adult stage, animals were divided into two groups, one treated with ABZ (7.5 mg/kg bw) and another with a placebo. All sheep were slaughtered at day 14 post-treatment to collect and count the number of flukes. The resistance of ABZ was confirmed with a reduction of 44% of adult flukes in the treated group. At the necropsy, adult flukes from the livers and eggs from the gall bladders, both from sheep treated with the placebo, were collected. Adult flukes were maintained alive in RPMI medium to let them to excrete eggs for 24 hours. EHT was conducted and different results were observed with the two egg sets. The EHT performed with eggs collected directly from gall bladder confirmed the resistant status of the isolate. However, the EHT with eggs recovered from flukes resulted in a susceptible phenotype, showing that only previously laying eggs (in bile) can express the resistant phenotype. After producing more metacercariae from this resistant isolate, we are testing in vivo the efficacy of a combined treatment to improve its efficacy. The results will be presented during the WAAVP meeting.Fil: Martínez Valladares, María. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Valderas García, Elora. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Castilla Gómez de Agüero, Verónica. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: González Warleta, Marta. Universidad de Leon. Facultad de Veterinaria; ArgentinaFil: Ceballos, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Liron, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Sanabria, Rodrigo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Pruzzo, Cesar Ivan. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Alvarez, Luis Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina28th International Conference of tha World Association for the Advancement of Veterinary ParasitologyDublinIrlandaWorld Association for the Advancement of Veterinary ParasitologyUniversity College Dubli

Paramphistomosis bovina en Galicia

Las paramphistomosis son infecciones producidas por trematodos de la familia Paramphistomidae Fischoeder, 1901, que incluye diferentes géneros y especies que parasitan el tracto gastrointestinal de rumiantes. Los animales se infectan al ingerir pastos contaminados con metacercarias producidas tras el enquistamiento de las fases larvarias emitidas por moluscos que actúan como hospedadores intermediarios. En Galicia, donde también se han detectado explotaciones con altas prevalencias de infección, se desconocían aspectos básicos sobre la epidemiología de estas parasitosis. En consecuencia, se ha realizado un amplio estudio en el que se han abordado muchos aspectos

Development and Evaluation of a New Lateral Flow Immunoassay for Serodiagnosis of Human Fasciolosis

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

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.This work was supported in part by Ministerio de Ciencia e Innovación (Spain) Grant AGL2011-30563-C03, Xunta de Galicia (Spain) Grant GPC2014/058, Instituto de Salud Carlos III-Acción Estratégica de Salud Intramural Grant PI14CIII/00076, and the European Fund for Regional Development (FEDER). The authors declare that they have no conflicts of interest with the contents of this article.S