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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.

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 transpla‑ cental transmission may also be highly efcient 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 out‑ come, with high mortality rates recorded in the ofspring of the two frst generations of ewes (21.4–46.1%). Histologi‑ cal 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-specifc 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.

La neosporosis, asociada a la infección por N. caninum, es una enfermedad de gran importancia en el ganado vacuno y que clásicamente se ha considerado poco relevante en la especie ovina aunque, recientes trabajos, hacen que sea necesario reconsiderar su importancia. Un estudio encaminado a valorar la trasmisión vertical de la neosporosis en ovejas infectadas naturalmente reveló que, en ganado ovino, este parásito presenta una transmisión endógena muy eficiente. Además, el seguimiento de estas ovejas demostró que, al igual que ocurre en el ganado vacuno, los animales infectados pueden abortar, o transmitir el parásito a al feto, en sucesivas gestaciones. Es más, aquellas corderas infectadas congénitamente pueden a su vez transmitir la enfermedad a su descendencia. Esta enfermedad se asocia a fallo reproductivo que puede aparecer en forma de aborto o, más frecuentemente, como mortinatos o nacimiento de corderos débiles y muerte perinatal. El examen histológico de los fetos o corderos afectados muestra generalmente una gliosis multifocal leve acompañada de congestión y leucomalacia en grado variable. La presencia de quistes intracelulares parasitarios es frecuente y puede detectarse en tinciones teñidas con HE, aunque el marcaje inmunohistoquímico facilita su identificación. Recientemente, dentro de un estudio encaminado a valorar la frecuencia de abortos por toxoplasmosis, en cinco rebaños se ha diagnosticado neosporosis como principal causa de aborto, estando afectadas tanto razas cárnicas como lecheras y sistemas de producción intensivo y extensivo. Los fetos estudiados presentaban una encefalitis multifocal no purulenta leve y la presencia del parásito se confirmó mediante detección inmunohistoquímica y amplificación de ácidos nucleicos. El estudio serológico de los rebaños afectados mostró que, en el momento de diagnosticarse el aborto, más de un tercio de las ovejas del rebaño estaban infectadas. Los resultados de estos estudios muestran que la neosporosis ovina

The aim of this study was to assess the prevalence of the major helminth infections affecting organic dairy cattle in northern Spain. Milk and faecal samples were obtained from 443 milking cows. Ostertagia ostertargi and Fasciola hepatica exposure was assessed by detection of specific antibodies in milk samples and F. hepatica infection was diagnosed by the detection of coproantigens in faecal samples. Dictyocaulus viviparus and Calicophoron daubneyi infections were diagnosed by conventional coprological techniques. The prevalence of infections caused by F. hepatica was considerable low, but similar to data reported from conventional farming in the same area. The prevalence rate of C. daubneyi infection was higher than previous data mirroring an increase of the prevalence that was also reported in other European countries in recent years. Specific antibodies against O. ostertargi were detected in all herds and the median levels of antibodies, determined by ELISA, exceeded the thresholds indicating milk production losses. The prevalence of D. viviparus was almost negligible. For each parasite, an ordinal logistic-regression analysis was used to assess the risk of infection by taking into account the administration of effective anthelmintics and the number of lactations. Treatment of cows with fasciolicides decreased the risk of F. hepatica infection in multiparous cows, whereas treatment with oxiclozanide or albendazol did not decrease the risk of C. daubneyi infection or O. ostertargi exposure, respectively. The study findings demonstrate that helminth infection in organic dairy farming is similar or even lower than previous data reported from conventional farming. Special attention should be paid to the impact of these infections on milk production.

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

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, We 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 fluke

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