Parasitol Res DOI 10.1007/s00436-014-3888-7

ORIGINAL PAPER

Identification and expression of Fasciola gigantica thioredoxin Narin Changklungmoa & Pornanan Kueakhai & Somjai Apisawetakan & Suda Riengrojpitak & Prasert Sobhon & Kulathida Chaithirayanon

Received: 14 January 2014 / Accepted: 26 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5’UTR, 78 bp 3’UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels N. Changklungmoa : S. Riengrojpitak Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand N. Changklungmoa e-mail: [email protected] P. Kueakhai : S. Apisawetakan Faculty of Allied Health Sciences, Burapha University, Long-Hard Bangsaen Rd, Saen Sook Sub-district, Mueang District, Chonburi 20131, Thailand P. Sobhon : K. Chaithirayanon (*) Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand e-mail: [email protected]

in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target. Keywords Fasciola gigantica . Thioredoxin . Cloning . Characteristics . Tissue expression

Introduction Fasciola gigantica is the most common Fasciola spp. infecting ruminants and also humans in tropical regions of Asia and Africa. It causes liver damage, anemia, and death in heavily infected animals, thus causing severe economic loss in sheep, cattle, and goat (Fairweather et al. 1999; Spithill et al. 1999; Torgerson and Claxton 1999). Metacercariae are the infective stage which excyst in the host’s duodenum and penetrate through the intestinal wall and liver parenchyma. Finally, they develop to mature stage in the bile ducts. Hemorrhage and liver damage occurs from the parasite’s migration, during which the parasites are exposed to reactive oxygen species (ROS) generated by the host’s immune cells, including neutrophils, eosinophils, and macrophages (Badwey and Karnovsky 1980; Tagaya et al. 1989; Bertini et al. 1999; Nakamura et al. 2001). However, the parasite can evade the damage resulting from the ROS exposure through its possession of a series of antioxidant proteins and enzymes that played essential roles in cellular redox response, including superoxide dismutase (SOD; (Assady et al. 2011), peroxiredoxin (Prx; Chaithirayanon and Sobhon 2010), thioredoxin glutathione reductase (TGR; Maggioli et al. 2011), and thioredoxin (Trx; Alger et al. 2002). Trx is the essential member of the group that can convert hydrogen peroxide (H2O2) to water.

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Trx is a member of an evolutionarily conserved family of redox-active proteins, containing tryptophan, cysteine, glycine, proline, and cysteine (WCGPC) motif that are found in various organisms ranging from microorganisms to mammals (Powis et al. 1997; Alger et al. 2002; Line et al. 2008). The cysteine pair in the active site can reversibly change between thiol-disulfide form (Collet and Messens 2010), and this protein is maintained in their reduced state by an enzyme, thioredoxin reductase, which transfer electrons from NADPH (Holmgren, 1985). Trx of Fasciola hepatica (FhTrx) has been cloned and characterized by Shoda et al. (1999); Salazar-Calderon et al. (2001), and it is a tegument antigen that is expressed in juvenile and adult parasites with a molecular weight (MW) of 12 kDa. In this study, we had cloned, characterized, and localized Trx of F. gigantica (FgTrx). This information will be used for further studies that will exploit this protein as a drug target, and possibly in the development of a vaccine.

Materials and methods Parasite specimens F. gigantica metacercariae were obtained from experimentally infected snails, Lymnaea ollula (Changklungmoa et al. 2013). Newly excysted juveniles (NEJs) were produced by activating the excystment of metacercariae as described previously in 2006). The 2- and 4-week-old juvenile parasites were collected from the livers of golden Syrian hamsters experimentally infected with F. gigantica metacercariae (Kueakhai et al. 2011). Adult parasites and eggs were collected from the bile ducts and gallbladders of naturally infected cattle killed at a local abattoir in Pathumtanee province, Thailand. They were then washed several times with 0.85 % NaCl and kept for further experiments. Cloning and sequence analyses of cDNA of FgTrx The adult F. gigantica cDNA library (Meemon et al. 2004) was used for amplification of FgTrx gene. The specific primers of Trx gene were designed from the conserved sequence of F. hepatica Trx gene (GenBank accession no. AAV59016), which comprised of a forward primer (5′ TGG TGC GGA CCG TGC 3′) and a reverse primer (5′ GCA CGG TCC GCA CCA 3′). The fragment of FgTrx was cloned by polymerase chain reaction (PCR) using λTriplEx2 sequencing primers and specific primers of Trx gene, and then inserted into the pGEM-T easy vector (Promega, Madison, USA). Finally, the purified plasmid-DNA isolated from a single colony was sequenced by Macrogen Inc (South Korea). The nucleotide and deduced amino acid sequences were analyzed by BLAST (The National Center for Biotechnology

Information, NCBI, http://ww.ncbi.nlm.nih.gov/BLAST/) and SignalP 3.0 (Bendtsen et al. 2004; http://www.cbs.dtu.dk/ services/SignalP/). Alignment of multiple homologous sequences from closely related parasites and host species were carried out by ClustalW (http://ww.ebi.ac.uk/ clustralW/). Expression and purification of the recombinant FgTrx protein (rFgTrx) The full-length FgTrx cDNA was subcloned into the pET-30b vector and transformed into Escherichia coli BL21 (DE3). The rFgTrx with six-histidine tagged fusion protein was expressed at 37 °C, for 3 h by inducing the bacteria with isopropyl-β-D- thiogalactoside (IPTG). The bacterial cells were collected and resuspended in a lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and sonicated with 5 cycles of a 10-s burst per cycle on ice. The lysate was purified using nickel-nitrilotriacetic acid (Ni-NTA) at 4 °C, washed twice with a washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0), then eluted by an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The rFgTrx protein was dialyzed in PBS pH 7.4, and concentrated using Amicon Ultra Centrifugal Devices, 3000 NMWL (Millipore Corporation, MA, USA). The purified rFgTrx was kept for further experiments at −80ºC. Production of rabbit polyclonal antibody against rFgTrx Polyclonal anti-rFgTrx was produced by immunizing a New Zealand white rabbit four times at 2-week interval, subcutaneously with 200 μg of rFgTrx protein mixed with complete Freund’s adjuvant (first immunization) and incomplete Freund’s adjuvant (boosting immunizations). Blood samples were collected every 2 weeks after immunization, serum were kept at −20ºC until used. Experiment on animals was approved by The Animal Care and Use Committee (SCMUACUC), Faculty of Science, Mahidol University, Thailand. Insulin reduction assay The activity of rFgTrx protein was determined by insulin reduction assay (Holmgren 1979). The reaction contained PBS (pH 7.4), 1 mM EDTA, 130 μM insulin (Humulin® R, Eli Lilly & Company, IN, USA), 500 μM dithiothreitol (DTT), and rFgTrx protein, and rEcTrx (Merck KGaA, Darmstadt, Germany) was used as a positive control. The 2-[(1-Methylpropyl) dithio]-1H-imidazole (PX12) (Santa Cruz Biotechnology, CA, USA) was used as a Trx inhibitor. The activity was monitored at OD 650 nm in an automatic Titertek Multiscan spectrophotometer (Flow Laboratories, VA, USA). The experiments were performed in triplicate.

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F.gigantica F.hepatica S.mansoni S.japonicum H.sapiens B.taurus

MR---VLATAADLEKLINENKGRLIVVDFFAQWCGPCRNIAPKVEALAKEIPEV MR---LLRTAADLEKLINENKGRLIVVDFFAQWCGPCRNIAPKVEALAKEIPEV MSKLIELKQDGDLESLLEQHKNKLVVVDFFATWCGPCKTIAPLFKELSEKY-DA MSNVLHIETDDDFDSFLKENKDKLIVVDFFATWCGPCKKIAPAFEALSADR-SA MVK--QIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNV MVK--QIESKYAFQEALNSAGEKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNV * : ::. :. :*:**** * *****: * * .. *: . ..

F.gigantica F.hepatica S.mansoni S.japonicum H.sapiens B.taurus

EFAKVDVDQNEEAAAKYSVTAMPTFVFIKDGKEVDRFSGANETKLRETINKHK EFAKVDVDQNEEAAAKYSVTAMPTFVFIKDGKEVDRFSGANETKLRETITRHK IFVKVDVDKLEETARKYNISAMPTFIAIKNGEKVGDVVGASIAKVEDMIKKFI LYVKVDVDKLEETARKYDVSAMPTFIVIKNGEKVDTVVGASIENVEAAIRKHK IFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV VFLEVDVDDCQDVAAECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELI : :****. ::.* : .:..**** :*.*::*. . **. ::. * .

51 51 53 53 52 52

104 104 106 106 105 105

Fig. 1 Alignment of Trx amino acid sequences from F. gigantica, F. hepatica AAF14217.1, S. mansoni AAL79841.1, S. japonicum AAD52699.1, and Trx of host species including B. taurus AAC83380.1and H. sapiens AAF87085.1. Asterisk indicates identical

residues, colon indicates highly conserved residues, and dot indicates moderately conserved residues. The conserved amino acid sequences in bold letters: “WCGPC” is the active site

Preparation of parasite antigens

Determination of the expression levels of natural FgTrx in developmental stages of F. gigantica by ELISA

F. gigantica whole body antigens (WB), extracts of eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult parasites were homogenized in a buffer containing 10 mM Tris–HCl, 150 mM NaCl, 0.5 % Triton X-100, and 10 mM EDTA, pH 7.4, 10 mM phenylmethanesulfonylfluoride (PMSF), and sonicated for 5 min in an ice bath with 15-s pulses. Excretory-secretory antigens (ES) of adult F. gigantica were obtained by incubating adult parasites in PBS for 3 h at 37 °C. Tegumental antigens (TA) were obtained by extracting the adult parasites with a nonionic detergent (1 % Triton X-100, 0.05 M Tris, 0.01 M EDTA, 0.15 M NaCl, pH 8.0) at 37 °C for 20 min (Changklungmoa et al. 2012). After centrifugation at 10,000g for 1 h at 4 °C, each antigencontaining supernatant was collected and kept at −80 °C until used.

A 96-well plate was coated with 100 μl of 10 μg/ml of WBs of egg, metacercariae, NEJ, 2- and 4-week-old juveniles, adult,

Immunoblot analysis The 5 μg per well of antigen fractions were separated on a 15 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with 5 % skim milk in PBS containing 0.1 % Tween-20 (PBST) at room temperature (RT) for 1 h, then incubated with rabbit anti-rFgTrx serum diluted 1:5,000 with PBS at RT for 1 h. The positive bands were visualized using AP-conjugated goat anti-rabbit IgG (Invitrogen-Life Technologies, Carlsbad, CA, USA) diluted 1:2,000 with PBS at RT for 1 h, and the color developed with nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indodyl phosphate (NBT/BCIP) substrates (Roche, Mannheim, Germany). Finally, the reaction was stopped by adding a buffer containing 10 mM Tris–HCl and 1 mM EDTA.

Fig. 2 Coomassie blue-stained 15 % SDS-PAGE of rFgTrx expression purified by the Ni-NTA affinity chromatography. Lane 1 proteins from noninduced condition, lane 2 whole lysate after induction, and lane 3 purified rFgTrx. The position of purified FgTrx is indicated by asterisk . MW markers are shown in the left side

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adult ES and TA, and a serial dilution of rFgTrx from 10−15 to 10−7 g/ml in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), incubated at 4ºC, overnight. The coated plate was washed three times with PBST (0.05% Tween 20), and nonspecific binding was blocked with 1 % bovine serum albumin (BSA) at RT for 1 h. Then, the coated plate was washed three times with PBST. Rabbit polyclonal antirFgTrx, diluted at 1:2,000 with PBS, was added at 100 μl per well and incubated at RT for 1 h. The plate was washed three times with PBST and incubated with 100 μl per well of Fig. 3 Insulin reduction activity assay of rFgTrx. a Insulin reduction reactions with 4 mM rFgTrx (circle), 2 mM rFgTrx (square), 1 mM rFgTrx (triangle), and in the absence of rFgTrx (diamond), all with 5 mM DTT. b Semiquantitation of rFgTrx activity and rEcTrx activity as a positive control shown in black bars. The recombinant protein with inhibitor (PX12) as negative control shown in gray bars. The reactions were performed in triplicate

HRP-conjugated goat anti-rabbit IgG (SouthernBiotech, Birmingham, USA), diluted at 1:5,000 with PBS, at RT for 1 h. Then, the plate was washed three times with PBST and incubated with 100 μl per well of 3,3′,5,5′Tetramethylbenzidine (TMB) (KPL, Gaithersburg, USA) at RT for 5 min. Finally, enzymatic reaction was stopped by adding 1 N HCl at 100 μl per well. The optical density (OD450) was measured at 450 nm in an automatic Titertek Multiscan spectrophotometer (Flow Laboratories, VA, USA). The experiments were performed in triplicate.

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binding was blocked by incubating in 4 % BSA in PBS for 1 h, after which the sections were incubated in rabbit antirFgTrx diluted at 1:2,000 with PBS at 4 °C, overnight. Excess antibody was washed out with PBST for 15 min three times, and then the sections were incubated in the secondary antibody, AP-conjugated goat anti-rabbit IgG (Southern Biotech, Birmingham, USA), diluted 1:2,000 with PBS, for 1 h at RT, and washed with PBST before incubating in NBT/BCIP (Roche, Mannheim, Germany) in the dark. After optimal color development, the reaction was stopped by adding a buffer containing 10 mM Tris–HCl and 1 mM EDTA. The sections were mounted in 90 % glycerol, and photographed under a light microscope (Nikon, ECLIPSE E600).

Fig. 4 The immunoblot analysis of a native FgTrx protein by probing with rabbit anti- rFgTrx: egg (lane 1), metacercariae (Meta, lane 2), NEJ (lane 3), 2-week-old juvenile (2wk, lane 4), 4-week-old juvenile (4wk, lane 4), adult (AD, lane 5), tegumental antigen (TA, lane 6), excretorysecretory antigens (ES, lane 7). The position of native FgTrx is indicated by asterisk. MW markers are shown in the left side

Localization of FgTrx protein by immunohistochemistry The 2- and 4-week-old juveniles and adult F. gigantica were fixed with 4 % paraformaldehyde in PBS for 4 h at 4 °C, and embedded in paraffin. The sections, cut at 5 μm-thick, were deparaffinized and rehydrated in fresh xylene twice for 5 min each, and then treated in 100, 95, 80, and 70 % ethanol and tap water, twice for 5 min each. The sections were microwaved at 700 W in citrate buffer (10 mM citric acid, pH 6.0) three times for 5 min, and then washed with PBS for 5 min three times, treated with 0.1 % glycine in PBS (w/v) at RT for 30 min, and rinsed with PBS for 5 min with shaking. The nonspecific Fig. 5 The limited of detection of rFgTrx by using rabbit antirFgTrx. The reaction was done in triplicate. Arrow indicates the lowest detectable concentration

Results Molecular cloning and characterization of cDNA encoding FgTrx gene The cDNA sequence encoding FgTrx from an adult F. gigantica cDNA library was amplified by PCR using degenerated primers designed from the conserved sequence of F. hepatica Trx gene. The PCR product with an estimated size of 315 bp was separated in a 2 % agarose gel. The complete nucleotide sequence of FgTrx cDNA comprised of 405 bp with 315 bp in the open reading frame (ORF). This ORF was composed of 104 putative amino acids, starting at ATG and ending at TAG. The predicted peptide exhibited a molecular mass of 11.68 kDa, and an isoelectric point of 5.86. The conserved motif “WCGPC” was detected (Fig. 1). No signal peptide was found in FgTrx gene by using SignalP software. FgTrx protein shares high

Parasitol Res Fig. 6 Semiquantitation of expression levels of the natural FgTrx in eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica as estimated by its reactivity with rabbit antirFgTrx using indirect ELISA. The high level expressions of FgTrx (indicated by OD value) were shown in eggs, 4-week-old juvenile, and adult F. gigantica. The reactions were performed in triplicate

identity with F. hepatica (96 %), Schistosoma mansoni (47 %), and Schistosoma japonicum (50 %).

Estimation of FgTrx expression levels in developmental stages of F. gigantica by ELISA

Expression of rFgTrx

The lowest concentration of rFgTrx protein that was detected by rabbit anti-rFgTrx was at 7.5 fg/ml (Fig. 5). The native FgTrx was expressed in all stages of F. gigantica lifecycle but with continuously increased levels from metacercariae, NEJ, 2- and 4-week-old juveniles, and adult. The eggs also contained very high level of FgTrx (Fig. 6).

The FgTrx cDNA was subcloned into pET-30b vector, and rFgTrx protein coupled with His-tag at the C-terminus was expressed in the transfected E. coli BL21 (DE3) at 37ºC for 3 h. The rFgTrx was purified by Ni-NTA chromatography, and then resolved in a 15 % SDS-PAGE as a single band with molecular weight (MW) of 12 kDa (Fig. 2). Insulin reduction assay The rFgTrx was tested for reducing activity using insulin reduction assay. PBS was used as a negative control indicating the baseline for this study, and positive control employed rEcTrx. For the rFgTrx activity, a concentration-dependent increase in insulin reduction was observed (Fig. 3a). The activity of rFgTrx was inhibited by adding Trx inhibitor (PX12; Fig. 3b). The activity of rFgTrx was similar to that of rEcTrx. Detection of Trx protein during the life cycle By immunoblot analysis, WB, TA, and ES antigens from adult parasites showed one positive band detected by rabbit antirFgTrx at MW 12 kDa. In addition, the native FgTrx protein with the same MW was present in various developmental stages of parasites, including eggs, metacercariae, NEJ, 2and 4-week-old juveniles, and adult parasites (Fig. 4).

Fig. 7 The detection of FgTrx protein in the tissues of 2- and 4-week-old„ juveniles and adult F. gigantica by immunohistichemistry, using rabbit anti-rFgTrx as probe and observed by a light microscope. a Negative control of adult parasite section probed with pre-immune sera. b A low magnification of adult parasite section showing positive signal in the tegument (Tg) and parenchyma (Pa), but not in caecum (Ca). c A medium magnification of adult parasite section showing positive signal in the tegument (Tg) and parenchyma (Pc), but not in caecum (Ca), muscle (Ms), and spine (Sp). d A high magnification of adult F. gigantica section showing positive signal in the testis (Ti). e A high magnification of adult F. gigantica section showing positive signal in the ovary (Ov). f A high magnification of adult F. gigantica section showing positive signal in the vetilline gland (Vi). g A high magnification of adult F. gigantica section showing positive signal in the egg (Eg). h A medium magnification of 4week-old juvenile section showing strong signal in the tegument (Tg), but not in ceacum (Ca) and parenchyma (Pc). i A high magnification of 4-week-old juvenile section showing strong signal in the tegument (Tg). j A medium magnification of 2-week-old juvenile section showing strong signal in the tegument (Tg), but not in caecum (Ca), ventral sucker (Vs), oral sucker (Os), and parenchyma (Pc). k A high magnification of 2-week-old juvenile section showing strong signal in the tegument (Tg)

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Distribution of FgTrx protein in F. gigantica tissues The localization by immunohistochemistry showed immunoreactivity against rabbit anti-rFgTrx in the tegument of 2- and

4-week-old juveniles (Fig. 7h, i, j, and k). In adult parasite, positive signal for FgTrx was detected in the tegument, parenchyma, eggs, testes, and vitelline cells (Fig. 7d, e, f, and g). The caecum and muscle were not stained (Fig. 7c). The

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negative control, using pre-immune serum, did not show any positive signal for FgTrx (Fig. 7a).

Discussion The FgTrx sequence showed high conservation with two cysteines at the active site, indicating that FgTrx gene is a member of thioredoxin family (Powis et al. 1997; Alger et al. 2002). The full sequence of rFgTrx was composed of 315 bp ORF, 12 bp 5’UTR, and 78 bp 3’UTR, and the putative FgTrx peptide was composed of 104 amino acids, with a MW of 11.68 kDa, pI 5.86 with no signal peptide; hence, it was not a secreted protein. Multiple alignments of FgTrx putative amino acid sequences with sequences from other related parasites, including F. hepatica, S. japonicum, and S. mansoni, and from the host species including Homo. sapiens and Bos. taurus, exhibited the same conserved sequence at WCGPC. FgTrx protein shares high identity with F. hepatica (96 %), S. mansoni (47 %), and S. japonicum (50 %). When assayed by insulin reduction activity, the recombinant protein rFgTrx exhibited reductive action similar to the rEcTrx (a positive control), and the activities of both proteins were inhibited by PX12. By using immunoblotting, it was confirmed that the native Trx protein expressed in all developmental stages of F. gigantica consisted of a single band with MW at 12 kDa which is similar to that of F. hepatica, and both of which do not have signal peptides (Salazar-Calderon et al. 2001). Following infection, all stages of F. gigantica were exposed to ROS generated from the host’s immune cells, as well as from their own metabolism and cell proliferation, especially in the reproductive organs (vitelline gland, testis, and ovary). By immunohistochemistry, the FgTrx protein was detected at a fairly high level in the tegument of 2- and 4-week-old juveniles, because the tegument is an organ that is directly exposed to ROS, especially H2O2 from the host’s immune cells that these stages of parasite encountered during their migration. Hence, these juvenile parasites protect themselves by expressing a high level of Trx in the tegument for ROS neutralization. In addition, strong signal of FgTrx protein was detected in the tegument, parenchyma, and reproductive organs of the adult parasite. These indicated that tissue expression of FgTrx depends on the location as well as stages of the parasites, as the 2- and 4-week-old juveniles resided in liver parenchyma, the high level of ROS in this tissue induced a high expression level of FgTrx in the tegument. Whereas, the adult parasites were located mainly in bile ducts and having more metabolic activity and cell proliferation in their various organ systems; thus, FgTrx protein was also highly expressed in the parenchyma and reproductive organs in addition to the tegument in order to neutralize ROS generated locally in these tissues. Trx was also highly expressed in the eggs and vitelline gland because of their high metabolic activity and cell proliferation

in the processes of egg and eggshell productions. Quantitatively, by using ELISA, it was shown that FgTrx expression level was high in eggs, 2- and 4-week-old juveniles, and adult parasites but low in metacercariae and NEJ, perhaps because these stages were exposed to relatively low level of free radicals. High expression level of Trx in eggs was similar to that previously described in S. mansoni (Alger et al. 2002). FgTrx may be a good vaccine candidate to prevent F. gigantica infection, especially at the NEJ and juvenile stages because it was expressed at a high level in the tegument of these stages and help to protect them during the early phase of infection. Furthermore, a vaccine using FgTrx combined with other juvenile proteins that are essential for the migration processes, such as cathepsin B2, B3 (Chantree et al. 2012, 2013), and cathepsin L1H (Sansri et al. 2013) could even be more effective. As well, since FgTrx showed abundance in tegumental antigen, it may be released into the blood circulation of infected animals and, as a result, it may be a good candidate for immunodiagnosis especially for early detection. Acknowledgments Mahidol University.

This research was supported by a grant from

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Identification and expression of Fasciola gigantica thioredoxin.

In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by ...
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