Pilot Evaluation of Two Fasciola hepatica Biomarkers for Supporting Triclabendazole (TCBZ) Efficacy Diagnostics

Abstract

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.

Fasciola hepatica Cathepsin L Zymogens: Immuno-Proteomic Evidence for Highly Immunogenic Zymogen-Specific Conformational Epitopes to Support Diagnostics Development

“…For 1-D (1-DE) and 2-D (2-DE) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), protein samples were prepared and electrophoresed as previously described, 45 as specified per lane/gel in this study. Gels destined for direct examination or mass spectrometry were fixed (10% (v/v) acetic acid; 40% (v/v) ethanol), washed (H 2 O, 18 MΩ), and then stained with Coomassie blue (PhastGel Blue R, Amersham Biosciences, U.K.) as per the manufacturer’s instructions and destained in acetic acid (1% (v/v)) as required.…”

“…Whole anti-rFhΔpCL1 sera were diluted as required for each application and incubated with membranes at room temperature for an hour prior to incubation with 1:30,000 diluted anti-rabbit IgG-AP secondary antibodies (A3687, Sigma-Aldrich, U.K.) and detected using the BCIP-NBT system and imaged using a Bio-Rad GS-800 calibrated densitometer (Bio-Rad, U.K.) as previously described. 45 Uncropped images of entire membranes from all western hybridization procedures are provided in the Supporting Information ( Supporting Figure S1 ).…”

“…Gels destined for direct examination or mass spectrometry were fixed (10% (v/v) acetic acid; 40% (v/v) ethanol), washed (H 2 O, 18 MΩ), and then stained with Coomassie blue (PhastGel Blue R, Amersham Biosciences, U.K.) as per the manufacturer’s instructions and destained in acetic acid (1% (v/v)) as required. For liquid chromatography-tandem mass spectrometry (LC-MS 2 ), technical replicate (duplicate) gel pieces were excised, prepared, and analyzed as previously described, , except for the use of a HPLC Prot-ID Chip (Agilent 6550 iFunnel Q-TOF, Agilent Technologies, U.K.). Where necessary for complex protein mixtures, samples were analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, U.K.) coupled to an UltiMate 3000 liquid chromatography tower (Dionex, Thermo Scientific, U.K.) and Zorbax Eclipse Plus reversed-phase C18 column at 30 °C (Agilent Technologies, U.K.) operated as follows.…”

“…Ions were formed by fragmentation by collision-induced dissociation with a collision energy of 35%, and resulting ions were detected in the ion trap in centroid mode. Data files were assessed using the MASCOT MS 2 ions search (Matrix Science) against the GenBank database (v204), with the search parameters set as previously described, , except for the inclusion of error tolerance and exclusion of a decoy search tool. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD030293 (DOI: 10.6019/PXD030293), and details of sample nomenclature are available in the Supporting Information (Supporting Table S1).…”

“…For western hybridization procedures, 1- and 2-DE-separated samples were transferred to nitrocellulose membrane (NCM 0.45 μm; GE Healthcare, U.K.), which was confirmed by Amido Black staining, and membranes were prepared as previously described, with antibodies tested as follows. Whole anti-rFhΔpCL1 sera were diluted as required for each application and incubated with membranes at room temperature for an hour prior to incubation with 1:30,000 diluted anti-rabbit IgG-AP secondary antibodies (A3687, Sigma-Aldrich, U.K.) and detected using the BCIP-NBT system and imaged using a Bio-Rad GS-800 calibrated densitometer (Bio-Rad, U.K.) as previously described . Uncropped images of entire membranes from all western hybridization procedures are provided in the Supporting Information (Supporting Figure S1).…”