{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T22:01:29Z","timestamp":1774908089864,"version":"3.50.1"},"reference-count":51,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2020,7,30]],"date-time":"2020-07-30T00:00:00Z","timestamp":1596067200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000268","name":"Biotechnology and Biological Sciences Research Council","doi-asserted-by":"publisher","award":["102108"],"award-info":[{"award-number":["102108"]}],"id":[{"id":"10.13039\/501100000268","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Welsh European Funding Office","award":["#"],"award-info":[{"award-number":["#"]}]},{"name":"Hybu Cig Cyrmu","award":["#"],"award-info":[{"award-number":["#"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Fasciola hepatica, the causative agent of fasciolosis, is a global threat to public health, animal welfare, agricultural productivity, and food security. In the ongoing absence of a commercial vaccine, independent emergences of anthelmintic-resistant parasite populations worldwide are threatening the sustainability of the few flukicides presently available, and particularly triclabendazole (TCBZ) as the drug of choice. Consequently, prognoses for future fasciolosis control and sustained TCBZ application necessitate improvements in diagnostic tools to identify anthelmintic efficacy. Previously, we have shown that proteomic fingerprinting of F. hepatica excretory\/secretory (ES) products offered new biomarkers associated with in vitro TCBZ-sulfoxide (SO) recovery or death. In the current paper, two of these biomarkers (calreticulin (CRT) and triose phosphate isomerase (TPI)) were recombinantly expressed and evaluated to measure TCBZ efficacy via a novel approach to decipher fluke molecular phenotypes independently of molecular parasite resistance mechanism(s), which are still not fully characterised or understood. Our findings confirmed the immunoreactivity and diagnostic potential of the present target antigens by sera from TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) F. hepatica experimentally infected sheep.<\/jats:p>","DOI":"10.3390\/molecules25153477","type":"journal-article","created":{"date-parts":[[2020,8,3]],"date-time":"2020-08-03T07:45:57Z","timestamp":1596440757000},"page":"3477","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Pilot Evaluation of Two Fasciola hepatica Biomarkers for Supporting Triclabendazole (TCBZ) Efficacy Diagnostics"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8878-2873","authenticated-orcid":false,"given":"Clare F.","family":"Collett","sequence":"first","affiliation":[{"name":"Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0304-4730","authenticated-orcid":false,"given":"Russell M.","family":"Morphew","sequence":"additional","affiliation":[{"name":"Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0985-8818","authenticated-orcid":false,"given":"David","family":"Timson","sequence":"additional","affiliation":[{"name":"School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Helen C.","family":"Phillips","sequence":"additional","affiliation":[{"name":"Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter M.","family":"Brophy","sequence":"additional","affiliation":[{"name":"Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth SY23 3DA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,7,30]]},"reference":[{"key":"ref_1","unstructured":"Food and Agriculture Organization (1994). Diseases of Domestic Animals Caused by Liver Flukes: Epidemiology, Diagnosis and Control of Fasciola, Paramphistome, Dicroceoelium, Eurytrema and Schistosome Infections of Ruminants in Developing Countries, Food and Agriculture Organization."},{"key":"ref_2","unstructured":"Behm, C.A., and Sangster, N.C. (1999). Pathology, pathophysiology and clinical aspects. Fasciolosis, CABI Publishing."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.vetpar.2009.03.024","article-title":"Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis","volume":"163","author":"Valero","year":"2009","journal-title":"Vet. Parasitol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1371\/journal.pone.0016126","article-title":"Predicting Impacts of Climate Change on Fasciola hepatica Risk","volume":"6","author":"Fox","year":"2011","journal-title":"PLoS ONE"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1136\/vr.113.14.315","article-title":"Treatment of immature and mature Fasciola hepatica infections in sheep with triclabendazole","volume":"113","author":"Boray","year":"1983","journal-title":"Vet. Rec."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1016\/j.pt.2016.03.002","article-title":"Current Threat of Triclabendazole Resistance in Fasciola hepatica","volume":"32","author":"Kelley","year":"2016","journal-title":"Trends Parasitol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1136\/vr.105209","article-title":"Lack of efficacy of triclabendazole against Fasciola hepatica is present on sheep farms in three regions of England, and Wales","volume":"184","author":"Kamaludeen","year":"2019","journal-title":"Vet. Rec."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.vetpar.2015.01.004","article-title":"Fasciola hepatica vaccine: We may not be there yet but we\u2019re on the right road","volume":"208","author":"Mulcahy","year":"2015","journal-title":"Vet. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1128","DOI":"10.1002\/1615-9861(200109)1:9<1128::AID-PROT1128>3.0.CO;2-0","article-title":"Proteomic analysis of Fasciola hepatica excretory-Secretory products","volume":"1","author":"Jefferies","year":"2001","journal-title":"Proteomics"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"5876","DOI":"10.1021\/pr300654w","article-title":"Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke fasciola gigantica","volume":"11","author":"Morphew","year":"2012","journal-title":"J. Proteome Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3308","DOI":"10.1021\/acs.jproteome.6b00331","article-title":"Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species","volume":"15","author":"Morphew","year":"2016","journal-title":"J. Proteome Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"306","DOI":"10.4269\/ajtmh.2000.63.306","article-title":"Tc45, a dimorphic Trypanosoma cruzi immunogen with variable chromosomal localization, is calreticulin","volume":"63","author":"Ferreira","year":"2000","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"50645","DOI":"10.1074\/jbc.M309497200","article-title":"Identification of an N-domain Histidine Essential for Chaperone Function in Calreticulin","volume":"278","author":"Guo","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2338","DOI":"10.1074\/jbc.M508302200","article-title":"Identification by mutational analysis of amino acid residues essential in the chaperone function of calreticulin","volume":"281","author":"Martin","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"38612","DOI":"10.1074\/jbc.M110.168294","article-title":"Structural basis of carbohydrate recognition by calreticulin","volume":"285","author":"Kozlov","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2182","DOI":"10.1016\/j.biochi.2013.08.014","article-title":"Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica","volume":"95","author":"Zinsser","year":"2013","journal-title":"Biochimie"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/0969-2126(93)90021-8","article-title":"The crystal structure of an engineered monomeric triosephosphate isomerase, monoTIM: The correct modelling of an eight-Residue loop","volume":"1","author":"Borchert","year":"1993","journal-title":"Structure"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1002\/1097-0134(20010215)42:3<383::AID-PROT80>3.0.CO;2-G","article-title":"Modeling, mutagenesis, and structural studies on the fully conserved phosphate-binding loop (loop 8)of triosephosphate isomerase: Toward a new substrate specificity","volume":"42","author":"Norledge","year":"2001","journal-title":"Proteins Struct. Funct. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1002\/pro.5560050206","article-title":"Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies","volume":"5","author":"Schliebs","year":"1996","journal-title":"Protein Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"9655","DOI":"10.1021\/bi963086a","article-title":"A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability","volume":"36","author":"Schliebs","year":"1997","journal-title":"Biochemistry"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1017\/S0031182010001149","article-title":"Population dynamics of the liver fluke, Fasciola hepatica: The effect of time and spatial separation on the genetic diversity of fluke populations in the Netherlands","volume":"138","author":"Walker","year":"2011","journal-title":"Parasitology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1016\/j.ijpara.2016.09.001","article-title":"Climate-Driven longitudinal trends in pasture-borne helminth infections of dairy cattle","volume":"46","author":"Charlier","year":"2016","journal-title":"Int. J. Parasitol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"278246","DOI":"10.1155\/2009\/278246","article-title":"Diagnosis of Parasitic Diseases: Old and New Approaches","volume":"2009","author":"Ndao","year":"2009","journal-title":"Interdiscip. Perspect. Infect. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Mart\u00ednez-Sern\u00e1ndez, V., Mui\u00f1o, L., Perteguer, M.J., G\u00e1rate, T., Mezo, M., Gonz\u00e1lez-Warleta, M., Muro, A., Correia, d.C.J.M., Romar\u00eds, F., and Ubeira, F.M. (2011). Development and evaluation of a new lateral flow immunoassay for serodiagnosis of human fasciolosis. PLoS Negl. Trop. Dis., 5.","DOI":"10.1371\/journal.pntd.0001376"},{"key":"ref_25","unstructured":"Walsh, T.R., Ainsworth, S., Armstong, S., Walker, A., Hodgkinson, S.H.J., and Williams, D. (2018). Development of A Pen-Side Diagnostic Test for Liver Fluke Infections in Cattle and Sheep. British Cattle Conference, University of Liverpool."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.euprot.2014.02.013","article-title":"In vitro biomarker discovery in the parasitic flatworm Fasciola hepatica for monitoring chemotherapeutic treatment","volume":"3","author":"Morphew","year":"2014","journal-title":"EuPA Open Proteom."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1007\/s00436-010-2085-6","article-title":"Entamoeba histolytica calreticulin: An endoplasmic reticulum protein expressed by trophozoites into experimentally induced amoebic liver abscesses","volume":"108","author":"Meza","year":"2011","journal-title":"Parasitol. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.1016\/j.molimm.2008.10.034","article-title":"Trypanosoma cruzi calreticulin: A possible role in Chagas\u2019 disease autoimmunity","volume":"46","author":"Ribeiro","year":"2009","journal-title":"Mol. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1016\/j.molimm.2003.11.018","article-title":"Role of calreticulin from parasites in its interaction with vertebrate hosts","volume":"40","author":"Ferreira","year":"2004","journal-title":"Mol. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4907","DOI":"10.1021\/acs.jproteome.5b00794","article-title":"Identification of up- and down-Regulated proteins in pemetrexed-Resistant human lung adenocarcinoma: Flavin reductase and calreticulin play key roles in the development of pemetrexed-associated resistance","volume":"14","author":"Chou","year":"2015","journal-title":"J. Proteome Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.1111\/j.1582-4934.2010.01137.x","article-title":"Nitric oxide and P-Glycoprotein modulate the phagocytosis of colon cancer cells","volume":"15","author":"Kopecka","year":"2011","journal-title":"J. Cell. Mol. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1599","DOI":"10.1111\/cas.13061","article-title":"Calreticulin is highly expressed in pancreatic cancer stem-Like cells","volume":"107","author":"Matsukuma","year":"2016","journal-title":"Cancer Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.molbiopara.2010.04.003","article-title":"Proteomic analysis of in vitro newly excysted juveniles from Fasciola hepatica","volume":"172","author":"Valero","year":"2010","journal-title":"Mol. Biochem. Parasitol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4940","DOI":"10.1021\/pr1000785","article-title":"Comparative proteomic analysis of triclabendazole response in the liver fluke fasciola hepatica","volume":"9","author":"Chemale","year":"2010","journal-title":"J. Proteome Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1016\/j.biocel.2004.02.030","article-title":"Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum","volume":"37","author":"Gelebart","year":"2005","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1139\/o98-091","article-title":"Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum","volume":"76","author":"Michalak","year":"1998","journal-title":"Biochem. Cell Biol. Biol. Cell."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2835","DOI":"10.1091\/mbc.12.9.2835","article-title":"Calreticulin, a calcium-binding molecular chaperone, is required for stress response and fertility in Caenorhabditis elegans","volume":"12","author":"Park","year":"2001","journal-title":"Mol. Biol. Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S0074-7696(05)45004-4","article-title":"Cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and ERp57","volume":"245","author":"Bedard","year":"2005","journal-title":"Int. Rev. Cytol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1080\/03602530500205135","article-title":"The endoplasmic reticulum in xenobiotic toxicity","volume":"37","author":"Cribb","year":"2005","journal-title":"Drug Metab. Rev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1111\/j.1742-7843.2004.pto940305.x","article-title":"Cytoprotection Following Endoplasmic Reticulum Stress Protein Induction in Continuous Cell Lines","volume":"94","author":"Bedard","year":"2004","journal-title":"Basic Clin. Pharmacol. Toxicol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/S0014-5793(04)00306-0","article-title":"Identification of enolase as a plasminogen-Binding protein in excretory-Secretory products of Fasciola hepatica","volume":"563","author":"Bernal","year":"2004","journal-title":"FEBS Lett."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1078\/1438-4221-00283","article-title":"Housekeeping enzymes as virulence factors for pathogens","volume":"293","author":"Pancholi","year":"2003","journal-title":"Int. J. Med. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1534\/genetics.108.087551","article-title":"Degradation of functional triose phosphate isomerase protein underlies sugarkill pathology","volume":"179","author":"Seigle","year":"2008","journal-title":"Genetics"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Davis, C.N., Phillips, H., Tomes, J.J., Swain, M.T., Wilkinson, T.J., Brophy, P.M., and Morphew, R.M. (2019). The importance of extracellular vesicle purification for downstream analysis: A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens. PLoS Negl. Trop. Dis., 13.","DOI":"10.1371\/journal.pntd.0007191"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"D442","DOI":"10.1093\/nar\/gky1106","article-title":"The PRIDE database and related tools and resources in 2019: Improving support for quantification data","volume":"47","author":"Csordas","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_47","first-page":"95","article-title":"BioEdit: A user-Friendly biological sequence alignment editor and analysis program for Windows 95\/98\/NT","volume":"41","author":"Hall","year":"1999","journal-title":"Nucleic Acids Symp. Ser."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"W636","DOI":"10.1093\/nar\/gkz268","article-title":"The EMBL-EBI search and sequence analysis tools APIs in 2019","volume":"47","author":"Madeira","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.14202\/vetworld.2017.1189-1193","article-title":"Seroprevalence of Fasciola gigantica infection in bovines using cysteine proteinase dot enzyme-Linked immunosorbent assay","volume":"10","author":"Kumar","year":"2017","journal-title":"Vet. World"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1016\/j.vetpar.2006.09.008","article-title":"Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep. I. Comparative immunological and plasma biochemical changes during early infection","volume":"143","author":"Raadsma","year":"2007","journal-title":"Vet. Parasitol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1042\/bj3440281","article-title":"Calreticulin: One protein, one gene, many functions","volume":"344","author":"Michalak","year":"1999","journal-title":"Biochem. J."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/15\/3477\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:53:05Z","timestamp":1760176385000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/15\/3477"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,30]]},"references-count":51,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2020,8]]}},"alternative-id":["molecules25153477"],"URL":"https:\/\/doi.org\/10.3390\/molecules25153477","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,30]]}}}