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      Rapid Enhanced MM3-COPRO ELISA for Detection of Fasciola Coproantigens

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          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.

          Author Summary

          We have previously reported how the combined use of mAb MM3 with polyclonal antibodies obtained from rabbit immunized with Fasciola hepatica excretory-secretory 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.

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          Most cited references34

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          Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis.

          The capacity of climatic conditions to modulate the extent and intensity of parasitism is well known since long ago. Concerning helminths, among the numerous environmental modifications giving rise to changes in infections, climate variables appear as those showing a greater influence, so that climate change may be expected to have an important impact on the diseases they cause. However, the confirmation of the impact of climate change on helminthiases has been reached very recently. Only shortly before, helminthiases were still noted as infectious diseases scarcely affected by climate change, when compared to diseases caused by microorganisms in general (viruses, bacteriae, protozoans). The aim of the present paper is to review the impact of climate change on helminthiases transmitted by snails, invertebrates which are pronouncedly affected by meteorological factors, by focusing on trematodiases. First, the knowledge on the effects of climate change on trematodiases in general is reviewed, including aspects such as influence of temperature on cercarial output, cercarial production variability in trematode species, influences of magnitude of cercarial production and snail host size, cercarial quality, duration of cercarial production increase and host mortality, influence of latitude, and global-warming-induced impact of trematodes. Secondly, important zoonotic diseases such as fascioliasis, schistosomiasis and cercarial dermatitis are analysed from the point of view of their relationships with meteorological factors. Emphasis is given to data which indicate that climate change influences the characteristics of these trematodiases in concrete areas where these diseases are emerging in recent years. The present review shows that trematodes, similarly as other helminths presenting larval stages living freely in the environment and/or larval stages parasitic in invertebrates easily affected by climate change as arthropods and molluscs as intermediate hosts, may be largely more susceptible to climate change impact than those helminths in whose life cycle such phases are absent or reduced to a minimum. Although helminths also appear to be affected by climate change, their main difference with microparasites lies on the usually longer life cycles of helminths, with longer generation times, slower population growth rates and longer time period needed for the response in the definitive host to become evident. Consequently, after a pronounced climate change in a local area, modifications in helminth populations need more time to be obvious or detectable than modifications in microparasite populations. Similarly, the relation of changes in a helminthiasis with climatic factor changes, as extreme events elapsed relatively long time ago, may be overlooked if not concretely searched for. All indicates that this phenomenon has been the reason for previous analyses to conclude that helminthiases do not constitute priority targets in climate change impact studies.
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            Diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario.

            Before the 1990s, human fascioliasis diagnosis focused on individual patients in hospitals or health centres. Case reports were mainly from developed countries and usually concerned isolated human infection in animal endemic areas. From the mid-1990s onwards, due to the progressive description of human endemic areas and human infection reports in developing countries, but also new knowledge on clinical manifestations and pathology, new situations, hitherto neglected, entered in the global scenario. Human fascioliasis has proved to be pronouncedly more heterogeneous than previously thought, including different transmission patterns and epidemiological situations. Stool and blood techniques, the main tools for diagnosis in humans, have been improved for both patient and survey diagnosis. Present availabilities for human diagnosis are reviewed focusing on advantages and weaknesses, sample management, egg differentiation, qualitative and quantitative diagnosis, antibody and antigen detection, post-treatment monitoring and post-control surveillance. Main conclusions refer to the pronounced difficulties of diagnosing fascioliasis in humans given the different infection phases and parasite migration capacities, clinical heterogeneity, immunological complexity, different epidemiological situations and transmission patterns, the lack of a diagnostic technique covering all needs and situations, and the advisability for a combined use of different techniques, at least including a stool technique and a blood technique.
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              Probiotic Bifidobacterium longum alters gut luminal metabolism through modification of the gut microbial community

              Probiotics are well known as health-promoting agents that modulate intestinal microbiota. However, the molecular mechanisms underlying this effect remain unclear. Using gnotobiotic mice harboring 15 strains of predominant human gut-derived microbiota (HGM), we investigated the effects of Bifidobacterium longum BB536 (BB536-HGM) supplementation on the gut luminal metabolism. Nuclear magnetic resonance (NMR)-based metabolomics showed significantly increased fecal levels of pimelate, a precursor of biotin, and butyrate in the BB536-HGM group. In addition, the bioassay revealed significantly elevated fecal levels of biotin in the BB536-HGM group. Metatranscriptomic analysis of fecal microbiota followed by an in vitro bioassay indicated that the elevated biotin level was due to an alteration in metabolism related to biotin synthesis by Bacteroides caccae in this mouse model. Furthermore, the proportion of Eubacterium rectale, a butyrate producer, was significantly higher in the BB536-HGM group than in the group without B. longum BB536 supplementation. Our findings help to elucidate the molecular basis underlying the effect of B. longum BB536 on the gut luminal metabolism through its interactions with the microbial community.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                20 July 2016
                July 2016
                : 10
                : 7
                : e0004872
                Affiliations
                [1 ]Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
                [2 ]Laboratorio de Parasitología, Centro de Investigaciones Agrarias de Mabegondo, INGACAL, Abegondo, A Coruña, Spain
                McGill University, CANADA
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: VMS FMU. Performed the experiments: VMS RAOM MGW MM FMU. Analyzed the data: VMS RAOM MGW MM FMU. Contributed reagents/materials/analysis tools: MGW MM FMU. Wrote the paper: VMS FMU.

                Article
                PNTD-D-16-00728
                10.1371/journal.pntd.0004872
                4954672
                27438470
                822b82f2-262b-4223-b52f-7b7210bc5e38
                © 2016 Martínez-Sernández et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 April 2016
                : 1 July 2016
                Page count
                Figures: 7, Tables: 0, Pages: 20
                Funding
                Funded by: Ministerio de Ciencia e Innovación (ES)
                Award ID: AGL2011-30563-C03
                Award Recipient :
                Funded by: Xunta de Galicia
                Award ID: GPC2014/058
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003176, Ministerio de Educación, Cultura y Deporte;
                Award ID: AP2010-5808
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003329, Ministerio de Economía y Competitividad;
                Award ID: BES-2012-060270
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004837, Ministerio de Ciencia e Innovación;
                Award ID: AGL2011-30563-C02
                Award Recipient :
                This 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). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Immunologic Techniques
                Immunoassays
                Enzyme-Linked Immunoassays
                Biology and Life Sciences
                Organisms
                Animals
                Invertebrates
                Flatworms
                Trematodes
                Fasciola
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                Organisms
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                Vertebrates
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                Bovines
                Cattle
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                Parasitic Diseases
                Nematode Infections
                Medicine and Health Sciences
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                Research and Analysis Methods
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                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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