ORIGINAL PAPER

Martın Cancela Æ Carlos Carmona Æ Silvina Rossi

Blas Frangione Æ Fernando Goni Æ Patricia Berasain

Purification, characterization, and immunolocalization of paramyosinfrom the adult stage of Fasciola hepatica

Received: 26 August 2003 / Accepted: 25 November 2003 / Published online: 12 February 2004� Springer-Verlag 2004

Abstract Paramyosin, a vaccine candidate in differenthelminthiases, was purified from the adult liver flukeFasciola hepatica using two different procedures. Thefirst started with a crude extraction of paramyosin inhigh-salt buffer followed by gel filtration chromatogra-phy and two precipitation-solubilization cycles; in thesecond, anion exchange chromatography replaced thegel filtration step. In both cases, the apparent molecularweight of the purified protein determined by sodiumdodecyl sulfate gel electrophoresis under reducing andnon-reducing conditions was 97 kDa and 200 kDa,respectively. The molecular weights were consistent withthe presence of a dimeric protein linked by disulfidebridges. Western blot analysis showed that the dimericand monomeric forms were both recognized by anantiserum raised against the F. hepatica 97 kDa band(a-FhPmy), and by an anti-Schistosoma mansoni pa-ramyosin immune serum. Immunohistochemistry usinga-FhPmy demonstrated the localization of paramyosinwithin the subtegumental muscle and in muscle cellssurrounding the gut of adult parasites. We also observedlabeling of extramuscular structures like testes, surfacelamellae of the gut and the tegument of adult flukes.

Introduction

Fasciola hepatica is the etiological agent of fasciolosis, aworldwide zoonosis that affects mammals. In agricul-turally based countries, the infection of ruminants leadsto important economic losses (Spithill and Dalton 1998),and new epidemiological studies suggest that fasciolosismust be considered as an emerging disease in humans,with an estimated 2.4 million people infected, and morethan 180 million at risk (Mas-Coma et al. 1999). Al-though triclabendazole is an effective drug for treatingliver fluke, the cost of the treatment is high, particularlyfor farmers in developing countries. Moreover, cases ofparasites resistant to the drug have been reported,mainly in Europe (Overend and Bowen 1995; Gaassen-beek et al. 2001). Thus, the need for an alternativetherapy, such as an effective vaccine, seems to beessential for controlling of the disease.

Paramyosin, a fibrillar protein widely distributedamong invertebrates but absent in vertebrates, was de-fined as a potential candidate antigen to develop vac-cines against some helminthiases, i.e. schistosomiasisand filariasis. Several vaccination trials are under way,or have been conducted, using either the native or re-combinant protein. Different levels of response andprotection have been achieved in animals (Pearce et al.1988; Flanigan et al. 1989; Nanduri and Kazura 1989;Ramirez et al. 1996).

In general, most paramyosins have a molecularweight of around 100 kDa and include a heptad repeatarrangement characteristic of a-helical coiled coil struc-tures (Lanar et al. 1986). They are predominantly lo-cated in muscle as a component of the core of thickfilaments (Epstein et al. 1985). However, some authorshave shown the existence of extramuscular isoforms inlarval stages of Schistosoma japonicum (Gobert et al.1997) and S. mansoni (Matsumoto et al. 1988).

In addition, paramyosins from different platyhelm-inths can disturb the classic pathway of complement(Laclette et al. 1992), bind collagen (Laclette et al. 1990),

M. Cancela Æ C. Carmona Æ P. Berasain (&)Dpto. de Biologıa Celular y Molecular, Facultad de Ciencias,Instituto de Higiene, Unidad de Biologıa Parasitaria,Av. A. Navarro 3051, C.P. 11600 Montevideo, UruguayE-mail: pberasai@higiene.edu.uyTel.: +598-2-4801597Fax: +598-2-4873073

S. RossiLaboratorio de Inmunotecnologıa, Catedra de Inmunologıa,Facultades de Quımica y Ciencias, Instituto de Higiene,Montevideo, Uruguay

B. Frangione Æ F. GoniDepartment of Pathology and Psychiatry, New York University,New York, USA

F. GoniCatedra de Inmunologıa, Facultad de Quımica,Universidad de la Republica Oriental del Uruguay, Montevideo,Uruguay

Parasitol Res (2004) 92: 441–448DOI 10.1007/s00436-003-1059-3

and Fc-c molecules (Kalinna and McManus 1993; Lo-ukas et al. 2001). These findings suggest a multifunc-tional role for paramyosin, not only as a structuralcomponent of the contractile apparatus, but also as amolecule capable of interacting with the host immunesystem in order to help parasite survival.

Here, we describe the purification to homogeneity ofF. hepatica paramyosin using newly reported method-ologies. In addition, localization in the tissues of adultflukes was determined immunohistochemically.

Materials and methods

Parasites

Mature F. hepatica were extracted from the bile ducts of bovinelivers obtained at local abattoirs. Liver flukes were transported tothe laboratory in bile at 37�C, and washed for 2 h at 37�C in10 mM phosphate-buffered saline (PBS) pH 7.3. Live adult flukeswere frozen and kept at �80�C until used.

Purification of F. hepatica paramyosin

Step 1. Protein extraction in high salt concentration

The purification procedures are illustrated in Fig. 1. Parasites werehomogenized in a glass tissue grinder by mixing 1 weight volume

(vol) of wet parasite with 2 vol of buffer A: 15 mM sodium phos-phate pH 7.0, 50 mM NaCl, 1 mM ethylenediaminetetracetic acid(EDTA), 2 mM phenylmethanesulfonyl fluoride (PMSF), 10 lM L-trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64),10 lM bestatin, 5 mM DL-dithiothreitol (DTT). The homogeneizedmaterial was sonicated over an ice bath for 3 min with 60 s burstsat 20% power followed by 30 s pauses, using an UltrasonicHomogenizer 4710 (Cole-Palmer Instrument, Ill.). The whole flukehomogenate (WFH) was centrifuged at 48,000 g for 30 min at 4�Cand the pellet (P) obtained was washed twice with 4 vol of buffer Aand centrifuged again at 48,000 g for another 30 min. The pelletwas resuspended in 1 vol of buffer B: 20 mM Tris-HCl pH 8.0containing 0.6 M NaCl, 1 mM DTT, 10 lM E64 and 2 mMPMSF. The mixture was shaken for 10 min at 4�C and the solu-bilized proteins (S1) were separated by centrifugation at 48,000 gfor 30 min at 4�C. The pellet obtained (P1) was subjected to thesame procedure to obtain more solubilized proteins (S2). Aliquotsof both supernatants, S1 and S2, were analyzed on 10% sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Samples of comparable composition were pooled and concentratedfourfold by ultra-filtration on a PM10 membrane (Amicon, Mass.).The concentrated material (S3) was then subjected to gel filtrationor to anion exchange chromatography as described below.

Step 2. Gel filtration or anion exchange chromatography of S3

Gel filtration of the crude paramyosin extract (S3) was performedon a Pharmacia Bio-Pilot system using a Superdex 200 HR16/50column (Pharmacia Biotech, Uppsala, Sweden) equilibrated in20 mM Tris-HCl pH 8.0, 0.6 M NaCl and 1 g/l sodium azide(NaN3). Approximately 10 mg of protein in 2 ml were applied andeluted in the same buffer at a flow rate of 2.5 ml/min. Proteinelution was monitored at 280 nm and 1 ml-fractions were collected.Protein fractions were analyzed by 10% SDS-PAGE and Westernblotting. The fractions that showed a 97 kDa band recognized by arabbit hyper-immune serum raised against S. mansoni paramyosin(a-SmPmy) were pooled and subjected to the final step of purifi-cation. The column was calibrated with the following standards:dextran blue, 2,000 kDa; thyroglobulin, 669 kDa; ferritin,440 kDa; catalase, 232 kDa; aldolase, 158 kDa; albumin, 67 kDa;ovalbumin, 43 kDa; chymotrypsinogen A, 25 kDa; and ribonu-clease A, 13.7 kDa (Pharmacia).

Alternatively, the gel filtration step was substituted for an anionexchange chromatography performed in a Mono Q column(Pharmacia Bio-Pilot System). The column was equilibrated withten column volumes of 50 mM Tris-HCl pH 7.6, 0.1 M NaCl, 1 g/lNaN3 at a flow rate of 2 ml/min. Before applying the S3 to thecolumn, the sample was diluted—not dialyzed—with 50 mM Tris-HCl pH 7.6 to achieve a final concentration of 0.1 M NaCl. Theelution of bound proteins was carried out with a NaCl linear gra-dient ranging from 0.1 to 0.65 M. Fractions of 1 ml were collectedand analyzed by SDS-PAGE and by Western blot.

Step 3. Paramyosin precipitation and extraction cycles

Paramyosin-enriched fractions obtained from either gel filtration orthe anion-exchange chromatography were dialyzed against a lowsalt buffer (LSB) consisting of 10 mM potassium phosphate pH6.0, 0.1 M KCl, 2 mM DTT overnight at 4�C with two changes.The precipitate formed was separated by centrifugation at 20,000 gfor 20 min at 4�C. The pellet (P4) was suspended in 5 ml of a highsalt buffer (HSB) consisting of 10 mM potassium phosphatepH 7.6, 0.6 M KCl, 2 mM DTT and dialyzed against the samebuffer overnight at 4�C with two changes. After centrifugation at20,000 g for 20 min at 4�C, the supernatant (S5) was subjected to anew precipitation-extraction cycle, and the final precipitate(FhPmy) suspended in HSB but without DTT.

Protein concentration was determined by the Bradford method(BioRad, Hercules, Calif.) in microtiter plates and bovine serumalbumin (BSA) was used as the protein standard (Bradford 1976).Fig. 1 Purification scheme for Fasciola hepatica paramyosin

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SDS-PAGE and protein staining

Electrophoresis was performed using the Laemmli system (Lae-mmli 1970). Proteins run under non-reducing and reducing con-ditions (10 mM DTT) were stained using either Coomassie brilliantblue R250 (CBB-R250) (Harlow and Lane 1988) or a silver stainprotocol (Heukeshoven and Dernick 1985). The molecular weightmarkers were the high molecular weight range Sigma M3788(Sigma, St. Louis, Miss., USA), which included myosin of 205 kDaand phosphorylase b, 97 kDa and the prestained kit MW-SDS-Blue (Sigma).

Production of immune sera

A group of ten sheep were immunized with the 97-kDa paramyosincut from an 8%-SDS-PAGE gel. Minced gel containing approxi-mately 100 lg of paramyosin in complete Freund’s adjuvant wasadministered at week 0 and a booster of another 100 lg inincomplete Freund’s adjuvant at week 4. Sera were collected3 weeks after the booster, and aliquots positive for the 97-kDaband were pooled and kept until use.

Immunoblotting

After electrophoresis, proteins were electrotransferred onto a0.22 lm nitrocellulose membrane (Bio Rad) for 90 min at 1.2 mA/cm2 (Kyhse-Andersen 1984) and the membrane was blocked with5% skim milk, 2.5% BSA in PBS-0.1% Tween 20 for 1 h at roomtemperature (RT). The strips were probed either with 1:2000 a-SmPmy or with 1:2000 of the polyclonal sheep serum raised againstF. hepatica paramyosin (a-FhPmy) for 1 h at RT. Bound anti-bodies were detected using anti-rabbit IgG horseradish peroxidaseconjugate (1:3,000) (Kirkegaard and Perry, Md., USA), or an anti-sheep IgG horseradish peroxidase conjugate (Sigma), respectively.The blots were developed by using the 4-chloro-naphtol or 3,3-diaminobenzidine (DAB) system (Sigma)

Determination of FhPmy internal amino acid sequences

We performed hot trypsin digestion of FhPmy as a modification ofthe method of Plaut and Tomasi (1970) for IgM. Briefly, a 0.2 mg/ml solution of paramyosin in HSB was digested at pH 8.1, 60�C for1 h with TPCK-treated trypsin (Worthington Biochemical, N.J.,USA), enzyme/protein ratio, 1:5 (w/w). The reaction was stoppedby acidification with 1 M acetic acid and quickly frozen with aspray of liquid nitrogen after the addition of Laemmli electro-phoresis sample buffer. Samples were run on 12.5% SDS-PAGE,electrotransferred onto PVDF membranes (Immobilon PS, Milli-pore, Belford), and the selected fragments were excised and sub-jected to automated amino acid sequence analysis carried out on a477 A microsequencer (Applied Biosystems Carpinteria, Calif.).The resulting phenylthiohydantoin derivatives were identified usingthe online 120 PTH Analyzer (Applied Biosystems). The sequencesobtained were analyzed by using the BLAST protein program(http://www.ncbi.nlm.nih.gov/BLAST/) searching for nearly exactmatches on short stretches.

Immunohistochemistry

Freshly collected liver flukes were washed in PBS and fixed over-night in 4.5% paraformaldehyde in PBS. Parasites were washed,dehydrated, cleared and embedded in paraffin wax. Tissue sectionswere cut 5 lm thick, deparaffinized, hydrated, soaked in PBS for5 min, and subjected to the antigen retrieval (AR) method. For theAR treatment, the slides were immersed in 50 mM Tris-HCl pH 7.6and autoclaved at 95�C for 10 min (McNicol and Richmond 1998).Endogenous peroxidase was inactivated with 1% H2O2 in

methanol for 20 min at RT. Afterwards, the slides were soaked inPBS-Tween 0.05% (PBS-T) for 5 min and incubated with positive(a-FhPmy) or negative sera (1:200) for 30 min at 37�C. The slideswere washed four times with PBS-T, 5 min each and then incubatedwith goat anti-sheep IgG horseradish peroxidase (HRP) conjugate(dilution 1:500) for 30 min at 37�C. Four washes in PBS-T weredone before color development with a freshly prepared solution of6 mg of DAB in 10 ml of Tris-HCl 50 mM pH 7.6 plus 10 ll of30% H2O2. Mayer’s hematoxylin was used as counterstain. Sec-tions were dehydrated and mounted for light microscopy.

Results

Purification of F. hepatica paramyosin by the gelfiltration method

Figure 2a shows 10% SDS-PAGE analysis of the firststep of purification. After the extraction of paramyosinfrom the pellet (P) both supernatants, S1 and S2, wereenriched in a band of 97 kDa apparent molecularweight. To obtain a successful purification, we found outthat the cysteine proteinase inhibitor E64 must bepresent at a concentration of 5 lM in the extractionbuffer in order to prevent proteolysis of the paramyosin.

Supernatants S1 and S2 were pooled (S3) and sub-jected to gel filtration (Fig. 2b). Eluted proteins wereanalyzed by SDS-PAGE (Fig. 2c) and Western blot(Fig. 2d). Paramyosin was detected mainly in the firstpeak coincident with the void volume, but also as a faintband eluted in the second chromatogram peak (Fig. 2c,lane 9, 12, 15, 28 and 31). According to the calibration ofthe column, the proteins eluted in the void volumeshould have a molecular mass higher than 700 kDa. Thea-SmPmy only recognized a band of 97 kDa apparentmolecular weight (Fig. 2d).

Fractions containing paramyosin from the firstpeak were pooled separately from those obtained fromthe second peak, and both pools were subjected totwo dialysis cycles against LSB and HSB. Paramyosinformed a white precipitate (P4) after dialysis in LSB,which was mostly solubilized after extensive dialysis inHSB (S5). After the second precipitation cycle, weobtained a clean supernatant S6 and a white pellet(FhPmy1 and FhPm2) highly enriched in 97-kDa pa-ramyosin as shown by SDS-PAGE analysis underreducing conditions (Fig. 2e). The purity of the finalpreparation obtained from the second peak was betterthan that obtained from the first peak (Fig. 2e). Thea-SmPmy only cross-reacted with the 97-kDa band inthe purified preparations FhPmy1 and FhPmy2(Fig. 2f).

Purification of F. hepatica paramyosin by the anionicexchange chromatography method

Alternatively, supernatant S3 was subjected to anion-exchange chromatography, the elution profile of whichis shown in Fig. 3a. Analysis of the fractions by SDS-PAGE under reducing conditions and Western blot

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showed that paramyosin eluted at a NaCl concentrationbetween 0.15–0.25 M.

Fractions containing paramyosin were pooled andsubjected to two precipitations and one solubilizationcycle. As shown in Fig. 3b, no contaminants were ob-served in the final preparation. Paramyosin was recog-nized by the a-FhPmy in steps S3, P2, PL1, PL2 andFhPmy (Fig. 3c).

SDS-PAGE analysis under non-reducing conditionsshowed that paramyosin migrated as a band of 200 kDaapparent molecular weight (Fig. 4a, lane 1) recognized

by the a-FhPmy antiserum raised against the 97-kDaband (Fig. 4b, lane 1).

Determination of FhPmy internal amino acid sequences

No direct amino acid sequence could be obtained twicefrom the 97 kDa band that was presumed to be blocked.Subsequently, we tried to obtain internal sequences inorder to confirm the identity of the purified protein. Hottrypsin digestion of FhPmy produced several fragments

Fig. 2a–f Purification ofF. hepatica paramyosin usingthe gel filtration method. a 10%SDS-PAGE analysis of theextraction step of F. hepaticaparamyosin: S (supernatant)and P (pellet) obtained afterfluke homogenization; S1, S2and P2: supernatants and pelletafter P extraction using 20 mMTris-HCl pH 8.0 with 0.6 MNaCl. The 97-kDa paramyosinis present in both S1 and S2(arrow). All samples were rununder reducing conditions andproteins stained withCoomassie BB R-250. MWMolecular weight marker. b Gelfiltration chromatogram of S3.c 10% SDS-PAGE analysisunder reducing conditions ofgel filtration fractions (seeunder b). Proteins werevisualized with the silver stainmethod described in materialsand methods. d Western blotanalysis of the same gelfiltration fractions using thea-SmPmy serum. The arrowindicates the presence ofparamyosin. e 10% SDS-PAGEanalysis under reducingconditions of the fractionsobtained during theprecipitation-solubilizationsteps (silver stain). f Westernblot analysis of fractionsobtained after purification ofparamyosin by the gel filtrationmethod. The a-SmPmy serumand the 4-chloro-naphtol-peroxidase system was used todetect reactivity againstparamyosin. The arrow showsthe presence of the 97 kDaF. hepatica paramyosin in S3(extraction step), FhPmy1(from void volume peak) andFhPmy2 (from first peak)

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in the range of 10–45 kDa (Fig. 3d). The amino acidterminal sequences of the selected fragments (Fig. 3d)showed high homology with other paramyosins (Pmy);the sequence from the 36 kDa band aligned with the S.japonicum Pmy and the S. mansoni Pmy at the E294

residue and with the Opistorchis felineus Pmy at the E275

residue (identity 87%, homology 100%); the 30 kDa

fragment showed homology to the Echinococcus granu-losus Pmy fragment L333TIEIKDLQAE (identity 81%,homology 81%); and the sequence from of the 25 kDaband aligned with the sequence ADELQR (identity100%, homology 100%) present in several paramyosins(S. japonicum, S. japonicum, Taenia solium, Hymenolepisnana and Mytilus galloprovincialis).

Fig. 3a–d Purification of F.hepatica paramyosin by theanion-exchange method.a Elution pattern of S3 proteinapplied on a PharmaciaMonoQ anion exchangecolumn (MonoQ-FPLC). Thestippled zone represents elutionof paramyosin. b 10% SDS.-PAGE under reducingconditions of solubilizedparamyosin (S3), eluted fromMonoQ column (PL1, PL2)and precipitated in LSB(FhPmy). WFH Whole flukehomogenate, S supernatantafter homogenization, P2 pelletafter extraction, PL poolsobtained from MonoQ anionicexchange chromatography,LSB supernatant of LSB. Theproteins were visualized bysilver staining. c Immunoblotanalysis of the same fraction aspart b using the a-FhPmy. Thearrow indicates a-FhPmyrecognition. d Trans blot ofFhPmy fragments obtainedafter hot trypsin digestion.Arrows indicate the sequencedbands and its amino acidcomposition are shown in theone letter code

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Tissue localization of paramyosin

The immunostaining patterns using paraformaldehydefixed sections of adult flukes after AR treatment showeda strong reactivity of the a-FhPmy within the subtegu-mental muscles (Fig. 5a) and the muscles surroundingthe gut (Fig. 5c, d). No reactivity in muscles was evidentin AR untreated sections (data not shown). Conversely,we found a strong labelling in the surface lamellae of thegut (Fig. 5c), tegumental surface (Fig. 5a) and testes(Fig. 5e), either with or without AR treatment.

Discussion

We isolated and characterized a paramyosin from tissuesof adult F. hepatica. The protein was predominantlyfound in the insoluble material left after fluke homoge-nization and had to be solubilized in the presence of ahigh salt concentration—0.6 M KCl or NaCl. As inmany helminths, the protein had an apparent molecularweight of 97 kDa by SDS-PAGE under reducing con-ditions. The amino acid sequence obtained from FhPmydigested fragments confirmed that the purified protein isa paramyosin.

Interestingly, in some steps of the purification inwhich DTT was not included, we observed a dimericform of approximately 200 kDa. This suggests that thedimeric form of F. hepatica paramyosin is most probablydue to an interchain disulfide bridge. In agreement withour results, Bullard et al. (1973) and Vinos et al. (1991)described the formation of oligomers in the absence of b-mercaptoethanol when working with Drosophila mela-nogaster paramyosin. These authors also suggested thatthe paramyosin dimer is stabilized not only by hydro-phobic forces, but also by disulfide bridges.

In a first attempt to purify native paramyosin fromliver flukes, we evaluated the use of two methods pre-viously described for the purification of paramyosinfrom T. solium (Laclette et al. 1990) and Caenorhabditiselegans (Harris and Epstein 1977). In our hands, themethod described by Laclette et al. (1990), based onthe collagen-binding property of T. solium paramyosin,produced a preparation containing not only paramyo-sin but also several other proteins including collagenitself. Moreover, when the Harris and Epstein methodwas used, we obtained a final preparation of paramy-osin with substantial contaminants of high molecularweight.

Therefore, we developed two different methods forthe purification of paramyosin from adult F. hepaticawith the goal of obtaining enough material for use inruminant vaccination (unpublished data). Using the gelfiltration method, we obtained a highly enriched pa-ramyosin preparation with high recovery, 0.3 mg/g ofwet parasite; however, the sample still showed contam-inants of low molecular weight. Conversely, the ion-ex-change method gave a lower yield (0.012 mg/g of wetparasite) but a highly purified preparation.

Nevertheless, the ability of paramyosin to precipitateand to solubilize at low and high salt concentrations,respectively, in the presence of DTT was used to con-centrate the preparations obtained. This ability alsoshowed that the disulfide bridge is necessary to form thedimer, but other forces might be responsible for themultimeric aggregation seen in the gel filtration chro-matography.

Anion-exchange chromatography carried out atpH 7.6 produced better results than those obtained atpH 8.6 (data not shown), suggesting that F. hepaticaparamyosin might have an acidic isoelectric point asdescribed for S. mansoni paramyosin (Schmidt et al.1996) (pI=4.7; P. Berasain unpublished data).

The presence of large amounts of paramyosin in thefirst peak of the gel filtration chromatography indicatedthat it might form large aggregates. These results suggestthat paramyosin had different aggregation states underthe assayed conditions. In C. elegans, the presence ofregularly charged patches on the outer surface of thehelix was thought to be responsible for the interactionsbetween paramyosin molecules (Kagawa et al. 1989), orbetween paramyosin and other proteins like myosin orfilagenins (Liu et al. 1998). It is then clear that the needfor a high salt concentration to solubilize paramyosinfrom tissues reflects the existence of strong protein-protein interactions.

Immunohistochemical studies using paraformalde-hyde fixed sections of adult liver flukes and an ARmethod demonstrated the presence of paramyosinwithin the musculature of F. hepatica. Both the subteg-umental muscles and muscle fibers surrounding the gutreacted with the antibody. However, an attempt tolocalize paramyosin in paraformaldehyde fixed sectionswithout the AR method failed to detect paramyosin inthe muscle. Decreased paramyosin immunolabeling in

Fig. 4a, b Presence of S-S bridges in F. hepatica paramyosin.a 10% SDS-PAGE analysis of samples collected from the stippledzone of Fig. 3a under non-reducing (lane 1) and reducing (lane 2)conditions. b Western blot analysis showing the reactivity ofa-FhPmy serum with the dimer (lane 1) and monomer (lane 2) ofparamyosin

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paraformaldehyde fixed sections without the ARmethod was also reported by Laclette et al. (1995) andGobert et al. (1997) for different parasitic stages ofS. mansoni and S. japonicum, respectively, although thelatter found strong immunolabelling when using adultparasites. Our results show that AR is necessary todetect paramyosin epitopes in the musculature ofF. hepatica.

An interesting finding was the presence of intenselabeling within the surface lamellae of the gut and testes

of adult parasites. Further studies must be carried out toconfirm that the immunostaining seen corresponds withthe actual presence of paramyosin in such extramuscularstructures. We also observed some reactivity within thetegumental surface of the adult parasite. This resultsuggests that paramyosin or paramyosin-like epitopespresent on the surface of the fluke could be interactingwith host components, as previously reported forothers paramyosins (Laclette et al. 1992; Kalinna andMcManus 1993; Loukas et al. 2001).

Fig. 5a–f Immunolocalizationof paramyosin in adult flukes.Sections 5 lm-thick weresubjected to antigen retrievaland incubated with positive(a, c–e) and negative (b, f) sera.mf Muscle fibers, cmf circularmuscle fibers, lmf longitudinalmuscle fibers, g gut, Pparenchyma, s spine, sl surfacelamellae, T tegument, t testes, tstegumental surface, vg vitellineglands. Arrows show thereactivity of the a-FhPmyantiserum

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Western blot analysis using a-FhPmy showed reac-tivity with both the dimeric and the monomeric forms ofF. hepatica paramyosin. This protein was also recog-nized by the a-SmPmy, indicating the existence of con-served epitopes between both proteins. Some studieshave demonstrated the existence of cross reactivity be-tween paramyosin molecules in evolutionarily divergentspecies (Wisnewski and Kresina 1995) and they havefound that immunodominant epitopes present in pa-ramyosin could be essential for the molecule and arethus conserved.

Paramyosin is one of the candidate antigens selectedby the World Health Organization for the developmentof a vaccine against schistosomiasis (Kalinna et al.1997), and previous studies have shown its protectivepotential against this disease (Pearce et al. 1988; Flani-gan et al. 1989; Ramirez et al. 1996) as well as filariasis(Nanduri and Kazura 1989). In our case, the purificationof milligram quantities of paramyosin from F. hepaticahas allowed us to explore its immunoprophylactic po-tential in a sheep model, with promising results(unpublished data).

Acknowledgements We thank Dr. Edward Pearce from CornellUniversity, USA for kindly supplying the anti-SmPmy hyper-im-mune serum. This work was supported by grants from IFS(International Foundation for Science) and SIDA (Swedish Inter-national Development Agency). Patricia Berasain was the recipientof a PEDECIBA doctoral fellowship. The experiments described inthis study comply with the current laws of Uruguay.

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