J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 1 4 8 5 – 1 5 0 3

ava i l ab l e a t www.sc i enced i r ec t . com

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Review

Proteomics of foodborne trematodes

Rafael Toledoa, M. Dolores Bernalb, Antonio Marcillaa,⁎aÁrea de Parasitología, Departament de Biologia Cel.lular i Parasitologia, Universitat de València, Burjassot, Valencia, SpainbDepartament de Bioquímica i Biologia Molecular, Universitat de València, Burjassot, Valencia, Spain

A R T I C L E I N F O

⁎ Corresponding author at: Dept. Biologia CeluBurjassot, Valencia, Spain. Tel.: +34 9635444

E-mail address: Antonio.Marcilla@uv.es (A

1874-3919/$ – see front matter © 2011 Elsevidoi:10.1016/j.jprot.2011.03.029

A B S T R A C T

Article history:Received 28 February 2011Accepted 26 March 2011Available online 1 April 2011

Food-borne trematodiases are among the most neglected tropical diseases, not only interms of research funding, but also in the public media. The Trematoda class containsseveral species identified as the causal agents of these diseases whose biological cycle,geographical distribution and epidemiology have been well characterised. The diagnosis ofthese diseases is based on parasitological techniques and only a limited number of drugs arecurrently available for treatments, most of which are unspecific. Therefore, in-depth studiesto identify new and specific targets for both effective diagnosis and treatments are urgentlyneeded. Currently, little molecular information is available regarding the host–parasiteinteraction. In this regard, proteomic studies have the potential to identify diagnosticbiomarkers for the early detection of the diseases, as well as new vaccine targets. In thisreview, a description of the biology, clinical features and current diagnostic tools of themaingroups of trematodes and the corresponding diseases they cause is followed by a discussionof the available studies using proteomic techniques to identify key parasite proteinsinvolved in the pathogenesis of food-borne trematodiases.

© 2011 Elsevier B.V. All rights reserved.

Keywords:ProteomicsParasitesFood-borne trematodes

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14862. Biology of food-borne trematode infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14863. Fish- and invertebrate-borne trematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1487

3.1. Liver flukes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14873.1.1. Opistorchiasis and clonorchiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14883.1.2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1489

3.2. Intestinal flukes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14903.2.1. Diplostomiasis, gymnophalloidiasis and heterophyasis . . . . . . . . . . . . . . . . . . . . . . . . . . 14903.2.2. Echinostomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14923.2.3. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1493

3.3. Lung flukes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14943.3.1. Paragonimiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14943.3.2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1495

lar y Parasitologia, Facultat de Farmàcia, Universitat de València, Av. V.A. Estellés, s/n, 4610091; fax: +34 963544769.. Marcilla).

er B.V. All rights reserved.

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4. Plant-borne trematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14964.1. Liver flukes (fascioliasis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1496

4.1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14964.1.2. Geographical distribution and epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14964.1.3. Clinical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14964.1.4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1496

4.2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14964.2.1. The transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14974.2.2. The secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14984.2.3. Host–parasite interactions and novel targets for diagnosis and vaccines . . . . . . . . . . . . . . . . . 1498

5. Treatment of food-borne trematodiases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14996. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1499References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1500

1. Introduction

Despite the considerable public health impact and theemerging nature of food-borne trematodiases [1–4], thesediseases are among the most neglected of the so-calledneglected tropical diseases [1,5–9]. It should be noted thatthe neglected tropical diseases are found predominantly in theworld's poorest populations in low-income countries, andwhere these diseases are common, they exacerbate poverty.

Compared with malaria or tuberculosis, most of theneglected tropical diseases are orphans with regard toresearch funding and presence in the press media [9–11].Moreover, funding for improving prevention and control isscarce, and the global strategy for the control of schistosomi-asis and food-borne trematodiases and other parasitic worminfections is morbidity control by chemotherapy. Althoughthere is growing international awareness pertaining to theneglected tropical diseases and new political and financialcommitments to do something against them [12,13], withregard to fluke infections, a number of campaigns have beenstopped, and the diseases are not on the priority list of theWorld Health Organization (WHO).

At present, mainly two drugs are currently available:triclabendazole against fascioliasis and praziquantel againstthe other food-borne trematode infections and schistosomi-asis, with the new drugs tribendimidine and peroxidicderivates (e.g. artemisinins and synthetic trioxolanes) beinginvestigated [14]. There has been little incentive to invest inthe discovery and development of trematodicidal drugs.While public–private partnerships work for some of theneglected tropical diseases since 1999 (e.g. the Drugsfor Neglected Diseases Initiative (DNDi) focusing on humanAfrican trypanosomiasis and leishmaniasis), pioneeringprogrammes for major helminth diseases are underway[14,15]. In this context, studies to identify new and specifictargets for treatment are needed. Moreover, the identificationof parasite-specific proteins could clearly facilitate thedesign of new tools for rapid and cheap diagnosis, which inturn could help in breaking the transmission of the parasite.Last but not least, the identification of potential targets forvaccination seems to be one of the best ways to control theseparasite infections.

Proteomics should help in those goals, but at presentproteomic studies are limited by some problems like massspectrometry sensitivity, which ultimately depends on theamount and quality of material available. Parasite material isin most of the cases limited, and to get enough in vivomaterialsmany laboratories maintain the parasite life cycle inthe laboratory using rodents as definitive hosts [16]. Anotherimportant limitation for proteomic studies is the lack ofsequence data available to correctly identify the peptidesobtained in mass spectrometry assays [16].

In the present reviewwe summarise the key characteristicsof food-borne trematodes, depicting their life cycles, geo-graphical distribution and epidemiology, as well as their mainclinical features and how are these diagnosed. Specialattention is focused on reviewing the proteomic studiesavailable and how these studies could help in identifyingand characterising new targets for treatment and diagnosis.Nonetheless, publications of latest draft genome sequencesfor the related trematodes of the genus Schistosoma [17,18] arenowproviding new insights into the biology of trematodes andoffer an opportunity for identification of potential new targetsfor treatment, diagnosis and vaccines.

2. Biology of food-borne trematode infections

The class Trematoda comprises an important group ofparasitic flatworms of medical and veterinary importanceand contains numerous species that are the causative agentsof human and animal diseases. The digenetic trematodesconstitute the largest group of platyhelminths, and in thisreview we will focus on those digenetic trematodes transmit-ted by food.

The life cycle of digeneans is complex but a common featureis that snails act as first intermediate host. Usually there areseven larval stages (e.g. adult, egg,miracidium, sporocyst, redia,cercaria, and metacercaria) with alternations of asexual andsexual reproductive phases in the molluscan and definitivehost. Moreover, the life cycle of some digeneansmay involve anunencysted stage intermediate between the cercaria andmetacercaria and is referred to as the mesocercaria [1,19,20].Fig. 1 depicts a schematic representation of the life cycles of

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major food-borne trematodes including liver, intestinal andlung flukes.

Typically the life cycle of the major food-borne trematodesinclude two or three different hosts: a vertebrate definitivehost, including humans and domestic animals; an inverte-brate first intermediate host (a mollusc); and, frequently, asecond intermediate host carrying the encysted metacercarialstage (Fig. 1). Eggs are produced by adult worms followingsexual reproduction in the definitive host and are released viafaeces for most of the human food-borne trematodes,although Paragonimus spp. release their eggs mainly in thesputum. The egg hatches and a swimming ciliated larva, themiracidium is released. The miracidium, attracted by chemo-taxis and chemokinesis, penetrates the snail intermediatehost or is ingested by the host (Fig. 1). In some cases, the eggsare directly ingested by the intermediate host and themiracidia hatch in the gastrointestinal tract of the snail.Various snail species act as first intermediate host, most ofwhich are trematode-species specific [21].

Within this host, miracidia develop into sporocysts, but insome cases the miracidia directly give rise to redia. Thegerminal cells within the sporocysts multiply and producenew germinal masses which produce daughter sporocysts orrediae. Development of pathernitae (sporocysts and rediae)follows different patterns depending on the digenean species[20]. Finally, these larval stages produce embryos, whichdevelop into cercariae. The free-swimming cercariae escapefrom the host and either come in contact with a compatible

free swimmingCercaria

Mesocercaria

Metacercaria Metacercaria Metacercaria

Adult

Third intermediate host

Second intermediate host

Second intermediate host or paratenic host

Definitive h

vegetation

3

2

2

2

4, 5

1, 3,6, 7

1, 3,6, 7 4, 5

Fig. 1 – Schematic representation of the life cycle patterns of selec2. Alaria; 3. Echinostoma; 4. Fascioliasis; 5. Paramphistomum; 6. NaModified from Toledo et al. [19].

second intermediate host in which penetrate and encyst (e.g.Clonorchis sinensis, Echinostoma spp. or Opistorchis spp.), orencyst on aquatic vegetation such as watercress, water lotus,water caltrop, water chestnut or water lily (e.g. Fasciola hepaticaor Fasciolopsis buski). Numerous invertebrates and poikilother-mal vertebrates serve as second intermediate host. Severalfish species, crustaceans, snails and tadpoles have beenreported to act as second intermediate host. Human andanimal definitive host become infected when eating raw,pickled or insufficiently cooked second intermediate hostharbouring metacercariae, aquatic vegetation or, even, drink-ing contaminated water [22,23]. After their ingestion, themetacercariae excyst in the gastrointestinal tract, releasing ajuvenile worm which migrates to the target organ. Infectionwith Paragonimus spp. might also occur through the consump-tion of undercooked meat of wild boar, which acts as aparatenic host [24]. The survival of the adult worms in thedefinitive host may vary from days to several years [1].

3. Fish- and invertebrate-borne trematodes

3.1. Liver flukes

Although several food-borne trematodes inhabit the liver ofthe definitive host, only those causing opistorchiasis andclonorchiasis are considered in the present section since theyare transmitted by eating fish.

Egg

embryonated

miracidium

miracidium

sporocyst I

sporocyst II

Redia IRedia II

Cercaria

Metacercaria

ost

First intermediate host

Water

1, 2, 3, 4,5, 7

1,2,

3,4,

5,6

3, 4, 5, 6

6

3, 4, 5, 6

3, 7

1, 2

1 2 3, 4, 5, 7

ingestedhatches

7

3

ted genera of intestinal digenetic trematodes. 1. Diplostomum;nophyetus; 6. Heterophyes.

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3.1.1. Opistorchiasis and clonorchiasis

3.1.1.1. Background. The major fish-borne liver flukes –Opistorchis viverrini, O. felineus and Clonorchis sinensis – haveclose morphological and biological characteristics. Differenti-ation of the species is based on several characters of the adultworms such as the size, shape and position of the testes andthe arrangement of the vitelline glands [25].

The life cycle of Opistorchis spp. and C. sinensis is shown inFig. 2. These species follow a three-host life cycle. Humaninfection follows the consumption of raw or undercooked fishharbouring infective metacercariae. Adult worms inhabit intothe intrahepatic bile duct, but they also can be found in thecommon bile duct, cystic duct and, even, in the gallbladder.C. sinensis may survive up to 26 years in the human host [19].Although there is no direct estimate of the life expectancy ofO. viverrini, it is thought that may survive for approximately10 years [25].

3.1.1.2. Geographical distribution and epidemiology. Liverflukes are important public health problems in many partsof the world and they are endemic in Asia and Eastern Europe[24,25]. C. sinensis is widespread in the People's Republic ofChina (PR China), Korea and North Vietnam, while O. viverriniis endemic in Southeast Asia, including Thailand, Lao People'sDemocratic Republic (PDR), Cambodia and Central Vietnam[25]. Recent reports suggested that about 35 million people areinfected with C. sinensis, with up to 15 million human in-fections in PR China alone and another 8–10 million individ-uals infected with O. viverrini in Thailand and Lao PDR [22,26].

Fig. 2 – Life cycle of Clonorchis spp.Source: http://www.dpd.cdc.gov/dpd

It is estimated that 600 million people are at risk of infection[1]. O. felineus is found in Russia and possibly in Eastern Europe[27].

3.1.1.3. Clinical aspects. Most chronic human opistorchiasisand clonorchiasis cases show few specific signs or symptoms,except an increased frequency of palpable liver [28]. Flatu-lence, fatigue, fever, diarrhoea, rash, oedema, abdominal painand enlargement of the liver may appear in moderateinfections (up to 1000 flukes) [25]. Patients of clonorchiasiswith a very high worm burden (up to 25,000 flukes) might alsoexhibit acute pain in the right upper quadrant [23]. Severeopistarchiasis, which is rare, might cause obstructive jaun-dice, cirrhosis, cholangitis, acalculous cholecystitis or bileperitonitis [1].

Cholangiocarcinoma is the most serious complication ofinfections with O. viverrini and C. sinensis. O. viverrini isclassified as definitely carcinogenic (class 1) and C. sinensisas probable carcinogen (class 2A) [29].

In contrast to infections with O. viverrini and C. sinensis,many patients infected with O. felineus suffer from fever tohepatitis-like symptoms in the acute stage of the infection.Chronic symptoms include obstruction, inflammation andfibrosis of the biliary tract, liver abscesses, pancreatitis andsuppurative cholangitis [24].

3.1.1.4. Diagnosis. The detection of eggs in faeces, bile orduodenal fluids is the gold standard for diagnosis. Parasito-logical examination of faecal samples is widely used forroutine diagnosis. The most frequently employed methods to

x/html/clonorchiasis.htm.

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detect eggs are Kato-Katz thick smear, Stoll's dilution and theformaline ethyl acetate concentration technique [27]. Howev-er, the similarity of the eggs of trematodes can makesometimes the specific diagnosis difficult in some cases.

Immunodiagnosis tests have been developed forOpistorchisand Clonorchis infections and good results have been obtainedusing individual antigens and detecting isotype-specificantibodies [30]. Faecal antigen detection by ELISA also showspromise [31]. Recent attention has been focused on thedetection of DNA of the eggs from faecal samples by PCR-based approaches [30,32]. Further details on methods for thediagnosis of trematode infections can be found in the reviewby Johansen et al. [33].

3.1.2. ProteomicsControl measures for opisthorchiasis and clonorchiasis relymainly on treating infected people with praziquantel, with novaccines or new drugs yet available. In this landscape,fundamental molecular biological investigations are essentialto develop novel diagnostic methods and new treatments.However, to date, almost all the molecular studies of flukeshave focused on human blood flukes (schistosomes) [34].

3.1.2.1. The transcriptome. Despite theirmajor socio-economicimpact, no draft or complete nuclear genomic sequence isavailable for liver flukes, and transcriptomic data are scant(Table 1). A preliminary study described 2387 EST from an adultC. sinensis cDNA library [35]. However, recent application ofmassive sequencing technologies is helping greatly to obtainsequence data from these neglected organisms, improving theidentification of parasite proteins by proteomics. In this context,two recent studies provide a first, deep insight into thetranscriptomes ofO. viverrini and C. sinensis using 454 sequencingfrom cDNA libraries and a semiautomated bioinformaticsplatform to assemble and annotate the datasets [36]. In thesestudies, 50,000 unique sequences were identified and compara-tive analyses revealed thatmost of the sequences (85%)werenew[36], thus expanding current databases and providing a resourcefor research groups investigating molecular aspects of these twotrematodes.

As indicated by Young et al. [36], the transcriptomes ofC. sinensis and O. viverrini provide a sound platform forinvestigating diverse aspects of host–parasite interactionsand the pathogenesis, even assisting in testing hypotheses

Table 1 – Representative proteomic and transcriptomic studies

Species Strategies Materials

Clonorchis sinesis 2DE-MS ESP

Opistorchis viverrini 2DE-MS Whole juvenile and adultOFFGEL MS/MS ESP

Echinostoma caproni 2DE-MS Whole sporocystsLC–MS/MS ESP

Echinostoma friedi 2DE-MS, WB ESPEchinostoma paraenseiParagonimus westermani 2DE-MS ESPFasciola hepatica 2DE-MS ESP

2DE-MS, WB ESP in vivo

regarding to the molecular basis of cholangiocarcinoma. Thecomprehensive transcriptomic data will also improve theclassification of different proteins (somatic, excretory/secre-tory and tegumental) from C. sinensis and O. viverrini identifiedin previous proteomic studies [37,38].

When a comparative analysis of C. sinensis, O. viverrini(Opisthorchiidae), Fasciola hepatica (Fasciolidae), S. japonicumand S. mansoni (Schistosomatidae) transcriptomes was per-formed, it showed that the opisthorchiids and fasciolidsshared the greatest (29–31%) protein sequence homology[36]. The proteins encoded by adult C. sinensis and O. viverrinimapped to metabolic, genetic and environmental data/infor-mation and cellular processing pathways inferred to beconserved amongst eukaryotes, whereas ~90% of predictedproteins did not map to any known pathways [36]. Thisobservation could provide the basis to explore novel biologicalpathways and processes that are unique to these parasites,being even potential drug and/or vaccine targets.

The advances in transcriptomics should assist futurecomparative -omic studies of liver flukes and the diseasesthat they cause. Those studies should focus on the differentialtranscription of genes among the different biological phases ofthe parasite to explore the molecular basis of pathologicalchanges and/or carcinogenesis in humans at different stagesof liver fluke disease. But still, the assembly and annotation ofthe nuclear genomes of C. sinensis and O. viverrini is needed.

3.1.2.2. The secretome. Mulvenna et al. [38] characterised300 parasite proteins from the excretory/secretory products(ESP) of O. viverrini using OFFGEL electrophoresis and multiplereaction monitoring (Table 1). The excretory/secretory prod-ucts included a complex mixture of proteins that have beenassociated with cancer, and also identified a cysteine proteaseinhibitor which may contribute to malignant transformationof inflamed cells [38]. More than 160 tegumental proteinswere also identified using sequential solubilisation of iso-lated teguments, being some of those molecules localised tothe surface membrane of the tegument by biotin labellingand fluorescence microscopy. These included annexins,which are potential immunomodulators, and orthologues ofthe schistosomiasis vaccine antigens Sm29 and tetraspanin-2.Mulvenna et al. [38] suggested novel roles in pathogenesis forthe tegument–host interface since more than ten surfaceproteins had no homologues in the public databases.

of food-borne trematodes.

Reference Transcriptomes Reference

[37] 2387 EST [35]>50,000 unique seq [36]

[52] >50,000 unique seq [36][38][89] Underway Unpublished

[85]358 EST [92]

[109][132] 14,031 EST http://www.sanger.ac.uk[131] 1684 EST(juveniles) [126]

>47,800 unique seq [125]

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Similarly to O. viverrini, proteomic studies of Clonorchissinensis have been focused in the study of both, parasite ESPsand host response [37,39]. Host proteins were examined todetermine the effects of C. sinensis infection on proteinexpression in host bile duct epithelium, examining proteomicprofile changes in a human cholangiocarcinoma cell line(HuCCT1) treated with parasite ESP [39].

As mentioned before, Ju et al. [37] established a 2-Dproteome map of the C. sinensis ESPs (Table 1), and identifieddiagnostic candidates for clonorchiasis through an immune-proteomic approach. They detected cysteine proteases inimmunoblot assays with sera obtained from patients andidentified legumain as a potential antigen for clonorchiasis.Since the numbers of registered genes for C. sinensis inGenbank was very limited at that time, they also generatedan EST database by massive sequencing of a cDNA library,which was constructed from adult C. sinensis [37]. Theseauthors successfully identified 62 protein spots. The proteinsidentified include detoxification enzymes, such as glutathioneS-transferase and thioredoxin peroxidase, myoglobin and anumber of cysteine proteases that are expressed abundantly.

3.1.2.3. Host–parasite interactions and targets for potentialnovel diagnostic tools and vaccines. Several studies havedescribed the use of molecular biological techniques in thesearch for useful serodiagnostic antigens for clonorchiasis[40]. Many recombinant proteins have been evaluated againsthelminth-infected human sera. These include a 7-kDa proteinof excretory–secretory products of adult C. sinensis [41],myoglobin [42], a fatty acid-binding protein (FABP) [43] andglutathione S-transferases (Cs28GST and Cs26GST) [44],among others. The results show moderate to low sensitivitiesand high specificities for serodiagnosis of clonorchiasis whenused as a single antigen, suggesting the use of a cocktail ofantigens or chimeric antigens as a strategy to improve thesensitivity [45]. In this context, recently Li et al. [46] selected 11clones from an adult C. sinensis cDNA library by immunoscre-ening using infected human sera. A mix of antigens wereprepared using recombinant proteins from positive clonesand investigated for antigenicity by immunoblotting againstC. sinensis- and helminth-infected patient sera [46]. A mix ofantigens from two recombinant proteins (Cs28GST andCs26GST) produced 76% sensitivity and 95% specificity, but atriple mix containing Cs26GST, Cs28GST and a vitellineprecursor protein reached 87% sensitivity and maintained95% specificity [46]. Recent immunoproteomic studies identi-fied legumains and cysteine proteases as antigens present inthe C. sinensis ESPs [37].

In relation to Opisthorchis species, the first recombinantproteins with potential use in diagnosis included paramyosinof O. felineus [47], a glycine–tyrosine rich eggshell protein fromO. viverrini [48], and more recently an asparaginyl endopepti-dase (from the legumain family) [49] and GST [50,51].

The first high resolution proteomic study onO. viverriniwaspublished by Boonmee et al. [52] with the aim to betterunderstand parasite biology, pathogenesis/carcinogenesisrelated to this parasite and lead to the identification of newtargets of vaccines and drugs. That study showed a compar-ative analysis using two-dimensional gel electrophoresis tohighlight proteins differentially expressed in the maturation

process from juvenile to adult parasites [52]. Approximately210–240 protein spots were resolved by 2-DE in two ranges ofpI (4.5–5.8 and 6.0–8.0), and at least 35 protein spots weredifferentially expressed in 4 week adult compared to 1 weekjuvenile fluke, corresponding to proteins probably involved insex organ development and egg production [52].

Recent studies by the group of Alex Loukas in Australiahave identified O. viverrini ESP proteins which could be eitherinvolved in contributing to the hepatobiliary abnormalities,including cholangiocarcinogenesis (cathepsin F), or in theestablishment of a tumorigenic environment that mayultimately manifest as cholangiocarcinoma (granulin-likegrowth factor) [53,54].

3.2. Intestinal flukes

3.2.1. Diplostomiasis, gymnophalloidiasis and heterophyasis

3.2.1.1. Background. The family Diplostomidae containsdigeneans from numerous orders of birds and mammals. Ingeneral, species of the Diplostomidae have a 3-host life cycle,though some variations of this pattern can be found. Thecercariae emerge from the snails and they penetrate fish,amphibians, molluscs, and annelids forming metacercariae[55]. In some Diplostomidae, the life cycle is expanded toincorporate four hosts by inclusion of a mesocercaria.Definitive hosts become infected by the ingestion of thesecond intermediate host or the paratenic host harbouringmetacercariae. Eggs typically hatch and penetrate the firstintermediate host [56].

At least members of 3 genera of Diplostomidae (Alaria,Neodiplostomum, and Fibricola) are known to parasitize man. Atthe intestinal level, only N. seoulense and F. cratera parasitizehumans.

The family Gymnophallidae consists of a small group ofdigeneans occurring in the intestine, gall bladder and thebursa Fabricii of birds and also in the intestine of mammals.The taxonomy of the family, including generic classification,is unsatisfactory due to considerable homogeneity of itsmembers, their small body size, and the difficulty in observingthe internal structures of these digeneans. A recent revision ofthe family describes 5 valid genera (Gymnophalloides, Parva-trema, Gymnophallus, Pseudogymnophallus, and Bartolius) [57]. Atypical gymnophallid life cycle involves bivalves as firstintermediate host, and bivalves, polychaetes, gastropods, orbrachiopods as second intermediate hosts. The definitive hostbecomes infected after ingestion of the second intermediatehost harbouring the metacercariae. Within the Gymnophalli-dae, studies on the pathology and immunology of theinfection are available only for one species, Gymnophalloidesseoi [58].

The family Heterophyidae contains small egg-shapedtrematodes with infective metacercariae that are usuallyencysted in fish as a second intermediate host. The definitivehost becomes infected by eating raw or poorly cooked fishharbouringmetacercariae. Heterophyids show little specificitytoward the definitive host and numerous fish-eating mam-mals, including humans, can be infected (Fig. 3). The adultworms live between the villi of the anterior region of the smallintestine and they release fully embryonated eggs into water.

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The eggs are then ingested often by littorine snails (particu-larly Littorina littorea and L. scutulata), and hatch within thesnail's intestine. Intramolluscan development comprise spo-rocyst and redial stages, and cercariae are released into thewater where they typically penetrate shore-fish, such ascunners, gudgeon, and charr, and they encyst on the surfaceof the fish (Fig. 4). Metacercariae may remain viable for years[59]. Although there are a great number of genera within theHeterophyidae, most of the studies in relation to humaninfections of these infections are focused on Metagonimusyokogawai. Its life cycle can be maintained easily in thelaboratory in various experimental hosts, thus facilitatingstudies on heterophyids.

3.2.1.2. Geographical distribution and epidemiology. Humaninfections with Neodiplostomum seoulense have been reviewedrecently [21,60]. A total of 28 human cases have been reportedin the Republic of Korea [60–62]. Chai and Lee [60] estimatedthe total number of human cases to be 1000 in the Republic ofKorea. Nevertheless, there are no available studies on thepathology and immunology of N. seoulense infections inhumans. There are no reported cases of human infectionwith F. cratera, a trematode species indigenous to NorthAmerica. Symptoms exhibited by the volunteer were similarto those described in N. seoulense infections.

Human infections with Gymnophalloides seoi have only beenreported in Korea [58–60]. G. seoi is highly prevalent amongvillagers in the southwestern coastal islands of Korea, wherehalf of the population was infected [63]. People becameinfected by consuming raw oysters [59].

Fig. 3 – Life cycle of Metagonimus sSource: http://www.dpd.cdc.gov/dp

Human infections by heterophyids have been often reported.Chai and Lee [60] have listed 12 species of heterophyids thatparasitizehumans inKoreabelonging to thegenera:Metagonimus,Heterophyes, Stictodora, Heterophyopsis, Pygidiopsis, Stellantchasmus,and Centrocestus. Moreover, members of the genera Haplorchishavebeenalso implicated inhumanheterophyiasis [59]. ChaiandLee [60] and Fried et al. [59] provide excellent coverage of humaninfections by these digeneans. The most prevalent species inhumans are M. yokogawai and H. heterophyes, which are distrib-uted mainly in Asia, Africa and Eastern Europe [59]. Humansbecome infected by eating raw, pickled or poorly cooked fish.

3.2.1.3. Clinical aspects. N. seoulense may cause severeenteritis with abdominal pain, fever, diarrhoea, fullness, andanorexia. The clinical aspects of the human infection withN. seoulense have not been studied in detail.

Most of the data available for Gymnophalloides species isbased on studies using laboratory rodents. Although symp-toms vary in humans from endemic areas, some cases ofsevere gastroenteritis and signs of acute pancreatitis havebeen reported [58,63]. Clinical symptoms of this infectioninclude loose stools, pancreatitis, indigestion, diarrhoea andgastrointestinal discomfort [58,60]. G. seoi infections mayrequire medical attention because of their relationships withpancreatic diseases [58].

Low-grade infections of heterophyids are of no clinicalconsequence, but cases with heavy infections are associatedwith diarrhoea, mucus-rich faeces, abdominal pain, dyspep-sia, anorexia, nausea, and vomiting [59,60,64]. Anaphylacticreactions have also been reported [65]. Occasionally, worm

pp.dx/html/Metagonimiasis.htm.

Fig. 4 – Life cycle of Echinostoma spp.Source: http://www.dpd.cdc.gov/dpdx/html/Echinostomiasis.htm.

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eggs may enter the circulatory system though the crypts ofLieberkühn causing emboli which may be fatal depending onthe affected tissue [64].

Chi et al. [66] have studied the pathology of a human case ofmetagonimiasis. The parasitism was incidentally detected inan intestinal segment that was removed surgically for treatingintestinal perforation related to amalignant histiocytosis. Themain histological lesions were massive lymphoplasmacyticand eosinophilic infiltration in the stroma, erosion of theenterocytes in the areas surrounding the worms, goblet celldepletion, and occasional villous oedema.

3.2.1.4. Diagnosis. The diagnosis of Diplostomas and Gym-nophallidae infections can be done by identification ofcharacteristic eggs in the faeces. The eggs of N. seouli areellipsoid to elliptical, thin shelled, with an inconspicuousoperculum, and frequently asymmetrical [20]. No immuno-logical or molecular methods have been developed.

In addition to the parasitological methods, several immu-nological methods have been developed for the diagnosis ofthe infections with Metagonimus yokogawai and Heterophyestaichui. Using crude extract of metacercariae and adult wormsCho et al. [67] developed an indirect ELISA method to detectspecific IgG in cats infected with M. yokogawai. The resultsdemonstrated that the serological diagnosis of metagonimia-sis is feasible from the first few days of the infection. However,the sensitivity of the method was low in infections with areduced number of worms. Lee et al. [68] used a similarmethod to detect metagonimiasis in humans using metacer-carial crude antigens. However, cross reactivity with othertrematodiaisis such as fascioliasis, schistosomiasis and para-

gonimiasis was detected. Ditrich et al. [69] developed indirectELISA andwestern-blotmethods to detectH. taichui in humansusing cytoplasmic and membranous antigens from adultworms. ELISA analysis showed that cytoplasmic antigensweremore sensitive, but cross-reactions between both specieswere detected. Western-blot analysis exhibited differencesbetween both trematode specieswere observed, enabling theirdifferentiation.

A recent study has evaluated three immunological tech-niques like counter current immunoelectrophoresis (CCIE),intradermal (ID) and indirect fluorescent immunoassay (IFI),for the diagnosis of Heterophyes infection in puppets [70]. Theresults indicate that the ID test could be recommended fordiagnosis of heterophyosis in naturally infected animals, butno application to humans has been reported yet.

3.2.2. Echinostomiasis

3.2.2.1. Background. The family Echinostomatidae containsa rather heterogeneous group of cosmopolitan and hermaph-roditic digeneans that parasitize, as adults, diverse vertebratehosts [61,62,71]. Adult echinostomatids are predominantlyfound in birds, but also parasitize mammals and occasionallyreptiles and fishes. Their typical site of location is the intestinethough species parasitizing other sites also exist [71].

Members of the Echinostomatidae follow a 3-host life cycle(Fig. 4). The first intermediate hosts are aquatic snails in whicha sporocyst, two generations of rediae and cercariae develop.Emerged cercariae infect the second intermediate host, whichmay be several species of snails, clams, frogs and even fishes.The definitive host becomes infected after ingestion of the

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second intermediate host harbouring the encysted metacer-cariae (Fig. 4) [62,71].

Species of Echinostoma have been the most widely used instudies on pathological and immunological aspects of infec-tion. Comparative studies on the development of a singlespecies of Echinostoma in different host species in which thecourse of the infection differs have allowed for the analysis ofsome of the factors that regulate the immunopathology andthe immune response in echinostome infections [71–74].

3.2.2.2. Geographical distribution and epidemiology. Echinos-tomes are commonly found in birds (waterfowls) and mam-mals, and their distribution is ubiquitous. Their specificitytoward the definitive host is usually low and an echinostomespecies is able to infect several species of vertebrate hosts.

The incidence of human echinostomiasis is difficult todetermine with any accuracy because of the lack of epidemi-ological surveys. Most of the data relies on historic surveysand occasional case reports. The distribution of humanechinostomiasis is strongly determined by the dietary habits.Humans become infected when they eat raw or inadequatelycooked food, specially fish, snakes, amphibians, clams andsnails containing encysted echinostome metacercariae [72].Moreover, it has been postulated that humans can also beinfected drinking untreated water containing echinostomecercariae, which could become encysted when exposed to thehuman gastric juice [75].

Although echinostomiasis occurs worldwide, most humaninfections are reported from foci in East and Southeast Asia.Echinostomiasis is relatively rare, yet the foci of transmissionremain endemic mostly due to the local dietary preferences.Most of these endemic foci are localised in China, India,Indonesia, Korea, Malaysia, Philippines, Russia, Taiwan, andThailand [76]. Moreover, occasional cases have also beenreported in other countries. In this context, a very recent studyhas estimated the prevalence of infection with echinostomeflukes ranged from 7.5% to 22.4% in 4 schools surveyed inCambodia [77].

The number and identity of the echinostome speciescausing human echinostomiasis is uncertain in relation tothe absence of systematic surveys and occasional case reports.Moreover, the problematical taxonomy of the group compli-cates further the specific diagnosis of the worms found inhumans [76].

3.2.2.3. Clinical aspects. Major clinical symptoms due toechinostome infection may include abdominal pain, diar-rhoea, easy fatigue, and loss of body weight [20,71,74]. Thesymptoms in echinostomiasis seem to be more severe thanthose observed in other intestinal trematode infections.Human morbidity is due to the prolonged latent phase,symptomatic presentations and similarity of symptoms withother intestinal helminth infections [20,78]. Heavy infectionsare associated with eosinophilia, abdominal pain, waterydiarrhoea, anaemia, oedema, and anorexia [74]. Pathologicaldamage includes catarrhal inflammation, erosion and evenintestinal ulceration [20]. Nevertheless the clinical signs inechinostomiasis are poorly known.

Of particular interest are the endoscopic findings in humanE. hortense infections. Several adult wormswere attached to an

ulcerated mucosal layer in the distal part of the stomach [79].The lesion was accompanied by a stage IIc or stage III of earlygastric cancer and multiple ulcerations and bleeding in thestomach and duodenum. Ulceration and bleeding appeared tobe caused by the worms [79]. Other factors observed byendoscopy are mucosal erosions, ulcerative lesions and signsof chronic gastritis [80].

3.2.2.4. Diagnosis. The diagnosis of human echinostomiasisis usually based on recovery of eggs in faecal examinations.The size of human-infecting echinostome eggs is in the rangeof 0.066–0.149 mm in length and 0.043–0.090 mm in width [72].

Occasionally, human echinostomiasis has been diagnosedby gastroduodenal endoscopy performed in relation to severeepigastric symptoms and ulcerative lesions in the stomachand duodenum [80].

Immunological methods also may be useful for thediagnosis and monitoring of echinostome infections. Severaltechniques have been developed based on antibody detection,seroantigen detection and coproantigen detection [81]. Al-though the use of these methods has provided interestingresults, the information is limited to experimental infectionssince the potential cross-reactivity with other helminths hasnot been evaluated [20,82,83].

The application of molecular methods for the diagnosis ofechinostomes is very limited. Most of the molecular studieshave focused on species identification and systematic, and/orphylogenetic studies [16].

3.2.3. ProteomicsAmong the intestinal flukes mentioned above, the onlypublished studies available on proteomics refer to echinos-tomes. The application of proteomics to echinostome proteinsis very recent and there are still very few publications [16].These reports include the identification of E. trivolvis hemo-zoin by laser desorption mass spectrometry [84] and thecharacterization of ESP proteins from adults of E. friedi [85],and E. caproni [86–88], as well as from E. caproni primarysporocysts [89] (Table 1).

In most of the studies on echinostomes proteins, thematerials analysed have been the echinostome excretory–secretory products (ESP), because these materials constitutethe initial contact with the environment, i.e. the host. In thiscontext, the molecular definition of the parasite proteins willhelp to understand the development of chronic infections, orin contrast, the rejection of the intestinal helminths.

3.2.3.1. The transcriptome. The major limitation in echinos-tomes proteomics is the absence of a full genome project,which should provide enough annotated DNA sequence datato produce deduced protein sequences. Most of the identifi-cations of echinostomes proteins have been possible becausethey were homologous to other trematode proteins [16].However, it is estimated that more 75% of proteins may notpresent sufficient homology to allow for the proper identifi-cation in echinostomes. Moreover, not all the available datafor other species like schistosomes are useful in the searchesusing routine bioinformatic tools, thus further modification ofsuch data sets is needed to facilitate these searches. Further-more, half of the proteins identified in schistosomes from

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their transcriptomes do not have a known function, or areeven species-specific [90,91], thus increasing the difficultieswhen working with other trematodes like echinostomes.

Echinostomes lack of transcriptome projects. Our group iscurrently working to obtain and assemble the transcriptomeof E. caproni (unpublished data). Until now, there was only oneEST collection (from E. paraensei) available which contains 358sequences (Table 1) [92]. In our experience, even afterobtaining good mass spectra, reliable identification cannotbe achieved in more than 90% of the cases. Thus, only a smallpercentage of the molecules analysed are properly identifiedusing the currently available proteomic technology.

3.2.3.2. The secretome. In E. friedi, following a proteomicapproach, we have identified structural proteins like actin,tropomyosin, and paramyosin; glycolytic enzymes like enolase,glyceraldehyde 3P dehydrogenase (GAPDH), and aldolase; detox-ifying enzymes like GSTs, and the stress-related protein Hsp70[133]. Other identified proteins in echinostomes include chaper-ones, proteases, signalling molecules and calcium-binding pro-teins. More recently, we have performed a shot-gun liquidchromatography/tandem mass spectrometry (LC–MS) for theseparation and identification of tryptic peptides from the ESP ofE. caproni adultworms [88]. Database searchwas performedusingMASCOT search engine [93] (http://www.matrixscience.com/home.html) and ProteinPilot software v2.0 (http://www.absciex.com/Products/Software/ProteinPilot-Software, Applied Biosys-tems) [88]. Although 4030 peptides were analysed in that study,significant homologies were found for only 274 (6.8%). A total of39 proteins were identified (16 using MASCOT and 23 usingProteinPilot) [88].

In relation to the identification of echinostomes proteins, wecould identify someof themcombiningmass spectrometrydatawith western-blotting using heterologous (cross-reactive) anti-bodies [16]. Unfortunately, this technology is feasible only forconserved proteins; it is also expensive and can be timeconsuming. However, this approach may be sometimes theonly way to get information about the proteome in trematodes.An example of the applicability of this technology has beenreported for E. friedi, where we have confirmed the identity ofechinostome proteins present in the ESP using commercialantibodies against GST, actin, aldolase, Hsp-70 and enolase, aswell as non-commercial antibodies for GAPDH [85].

In our studies we have detected posttranslational modifi-cations in the protein pattern of echinostomes proteinsincluding tyrosine phosphorylation in response to differentstimulus [94], or glycosylation processes [95]. Recent studieson schistosomes have shown that modifications like tyrosinephosphorylation of parasite proteins may be correlated withthe adaptation to the hosts [96].

We report from our investigations a high representation ofproteins in ESP lacking the classical molecular features ofsecretory proteins, including cytoskeletal proteins, heat-shockproteins, glycolytic and ATP-related enzymes, signal transduc-tion proteins, histones and transcriptional regulators. Interest-ingly, all these proteins have been described as the majorcomponents of exosomes [97–99]. These structures have beendescribed in a wide range of cell lines and organisms, includingparasites like Leishmania spp. [100]. The role of parasiteexosomes is described as being responsible for protein export

and communication with host macrophages [101]. Furtherinvestigations could provide sustention to the existence ofexosomes in helminths.

3.2.3.3. Host–parasite interactions and novel targets for diagnosisand vaccines. As mentioned before no proteomic studiesare available for most of the intestinal trematodes. Initialstudies identifieda16-kDacysteineproteinaseofGymnophalloidesseoi which showed no antigenicity on both enzyme-linkedimmunosorbent assay and immunoblots [102]. Antigens ofMetagonimus and Heterophyes species have been described abovein the diagnosis section.

As mentioned before, immunoproteomic studies withE. caproni adults identified major antigens in ESP detected byimmunoglobulins M, A and G, suggesting their potential ascandidates for diagnosis and eventually vaccination [95],constituting the first report in echinostomes of a combinationof proteomics and serology, which has been denominated“immunome” [91]. Other studies used proteomics to charac-terise the proteins released by E. caproni primary sporocysts inthe intermediate host [89].

Saric et al. [103] recently report the use of mass spectrometryto characterise the metabolomic profile of E. caproni infection inblood, stool, andurineand its potential for discover biomarkers ofthe infection. Important differences between the infected and theuninfected control animals were observed, mainly in urine,selecting it as the biofluid of choice for diagnosis of an infection[103].

We have also used proteomics to identify E. caproni proteinscapable of interacting with host matrix proteins like plasmin-ogen, identifying the enzyme enolase as being one of themostabundant and reactive proteins present [86]. These resultsconfirmprevious observations in other trematodes like F. hepatica[94] or Schistosoma bovis [104].

3.3. Lung flukes

3.3.1. Paragonimiasis

3.3.1.1. Background. There are about 15 species of Paragonimusknown to infect humans. P. westermani is the most commonelsewhere, while P. heterotremus is the etiological agent of humanparagonimiasis in PR China, Lao PDR, Vietnam and Thailand[24,27,105].

Human or other definitive hosts (carnivores) becomeinfected after ingestion of raw or undercooked freshwatercrustacean such as crabs, shrimp or crayfishes (Fig. 5). Themetacercariae excyst in the small intestine and penetratethrough the intestinal wall into the abdominal cavity, prior tomigration through the sub-peritoneal tissues, the muscle, theliver, the diaphragm, and finally enters the lung wherematuration occurs. Adult flukes lay eggs, which are coughedup and ejected by spitting with the sputum or swallowed andpassed in the faeces. After hatch, miracidia invade freshwatersnails and finally the cercariae emerge. Crustacea probablyacquire the infection by consuming cercariae or eatinginfected snails containing the fully developed cercariae (Fig. 5).

3.3.1.2. Geographical distribution and epidemiology. About20 million people are infected with lung flukes [22] and

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estimated 293 million people are at risk of infection [8].Human paragonimiasis occurs in three endemic focal areas:Asia (PR China, Japan, Korea, Lao PDR, Philippines, Vietnam,Taiwan and Thailand), South and Central America (Ecuador,Peru, Costa Rica and Columbia) and Africa (Cameroon, Gambiaand Nigeria) [25]. Moreover, occasional cases in other placeshave been reported [27].

Endemic areas can be identified as the people eat raw,pickled and/semi-cooked freshwater species of crabs, shrimpsand crayfishes. The sources of infection in the endemic areashave been recently reviewed by Sripa et al. [25].

3.3.1.3. Clinical aspects. Pathology induced by Paragonimusspp. is related to the migration of the worms from the gut tothe lungs. Ectopic migrations to aberrant sites including thebrain and subcutaneous sites at the extremities have beenreported [24,106]. The presence of the flukes in the lung causeshaemorrhage, inflammatory reaction and necrosis of lungparenchyma and fibrotic encapsulation. In pulmonary para-gonimiasis, the most noticeable symptom is a chronic coughwith brown and blood streaked pneumonia-like sputum.Pulmonary paragonimiasis can be confused with chronicbronchitis, bronchial asthma or tuberculosis [25,106].

3.3.1.4. Diagnosis. Definitive diagnosis is established by thedetection of eggs of Paragonimus in the sputa and/or faeces byparasitological examination. Supporting methods includechest X-ray and immunological tests, including ELISA and

Fig. 5 – Life cycle of Paragonimus sSource: http://www.dpd.cdc.gov/d

immunoblotting-based tests. A recombinant antigen ofP. westermani egg has been tested as an ELISA antigen offeringhigh levels of sensitivity and specificity [107]. Immunoassaysfor the detection of IgG to excretory/secretory products ofP. heterotremus provide a sensitive and specific method fordiagnosis [108].

3.3.2. ProteomicsSimilarly to other foodborne trematodes, only a proteomicstudy on Paragonimus species has been published so far, withother studies describing proteins used in diagnosis.

3.3.2.1. The secretome. The only proteomic study on Para-gonimus analysed excretory–secretory products of adultP. westermani using 2-DE coupled to MS (Table 1) [109]. Thestudy identified 25 different proteins, some of them highlyrepresented like cysteine proteases. Additionally, three previ-ously unknown cysteine proteases were also identified byMALDI-TOF/TOF MS, being the majority of those proteasesreactive against sera from paragonimiasis patients. Chrono-logical changes in the antibody responses to different pro-teases have also been detected in an experimental model ofcanine paragonimiasis [109].

3.3.2.2. Host–parasite interactions and novel targets for diagnosisand vaccines. Reliable diagnostic tools are needed to re-solve the clinical and parasitological diagnostic problems ofparagonimiasis infection. In particular, the diagnosis of ectopic

pp.pdx/html/paragonimiasis.htm.

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paragonimiasis using parasitological techniques is notpossible, and thus, immunological and serological methodsconstitute an alternative, but crude worm extracts are subjectto cross-reactivity among trematodes [109]. Early studies on theidentification of useful serodiagnostic antigens included theuse of recombinant yolk ferritin [110] and cysteine proteases[111]. Park et al. [111] suggested that a new powerful drug forparagonimiasis could be designed and developed focusing onthe exploration of anti-agents against P.westermani like cysteineproteases.

Dekumyoy et al. [112] have identified three polypeptideswhich strongly reacted with sera from patients of paragoni-miasis in enzyme-linked immunotransfer blot (EITB) (orwestern-blot) assays. Those molecules did not react withsera from patients with other helminth infections, andallowed the distinction between two Paragonimus species,P. westermani and P. heterotremus [112].

More recent studies by Lee et al. [113] have described an ELISAusing a recombinant cysteineproteinase antigenwhich exhibiteda high specificity and sensitivity in infected individuals, with nocross-reactivitywithClonorchis sinensisandMetagonimusyokogawai(along with P. westermani, the three major human trematodeparasites in Korea, where the study was performed).

4. Plant-borne trematodes

There are six plant-borne trematode species known affectinghumans: F. hepatica, F. gigantica, Fasciolopsis buski (Fasciolidae),Gastrodiscoides hominis (Gastrodicidae), Watsonius watsoni andFischoederus elongates (Paramphistomidae). Whereas F. hepaticaand F. gigantica are hepatic, the others are intestinal parasites.In the present section wewill focus on themembers of Fascioladue to their greater impact in human health and the existenceof more proteomic studies from these species.

4.1. Liver flukes (fascioliasis)

4.1.1. BackgroundF. hepatica and F. gigantica are the causative agents of liver flukedisease (fascioliasis) in domestic animals and humans. Bothspecies follow a two-host life cycle (Fig. 6). The eggs, released bythe adults residing in the bile ducts of themammalian host, arecarried into the intestine and are passed in the faeces.Embryonation and hatching occurs in freshwater (Fig. 6). Thefree-swimming miracidia find and penetrate in the molluscanintermediate host where the parasite undergoes a series ofdevelopmental stages (sporocyst–rediae–cercaria). The free-swimming cercariae adhere to and encyst, as metacercariae,on vegetation. Following ingestion of the contaminated vegeta-tion the parasite excysts in the small intestine and juvenileworms penetrate through the gut wall and enter the peritonealcavity (Fig. 6). After 10–12 weeks ofmigration theparasites enterthe bile ducts where they mature and may remain for up to1–2 years in cattle or as long as 20 years in sheep [114].

4.1.2. Geographical distribution and epidemiologyAlthough traditionally considered as a disease of livestock,fascioliasis is now recognised as an important emerging

zoonotic disease of humans. It is estimated that between 2.4and 17 million people are currently infected and 91 million areat risk of infection [8]. Human infections normally occur inareas where animal fascioliasis is endemic. Transmissionoccurs where farming communities regularly share the samewater soured as their animals or consume water-basedvegetation. The main types of aquatic plants are watercress,algae, kjosco and tortora [114]. Drinking untreated water maybe a source of infection due to the presence of free-floatingmetacercarial cysts [115].

To date, themajority of reported human cases of fascioliasisare due to infections with F. hepatica. However, some reportsindicatea rise inhuman infectionsdue to F. gigantica inVietnam,and probably Thailand and Cambodia [115].

The highest prevalence of human fascioliasis is found inthe Altiplan region of Northern Bolivia. Prevalence in thisarea may reach 40% in certain communities [115]. Hyperen-demic human fascioliasis has also been reported in the NileDelta region between Cairo and Alexandria [116], with aprevalence of 19% in some villages. Significant levels ofhuman F. hepatica infections also occur, with regularoutbreaks involving up to 100,000 infections, in severalprovinces of Northern Iran [115]. In Europe, human flukeinfections occur more sporadically, though significant out-breaks of the disease occur in France, Portugal and Spain[115]. Sporadic cases also have been reported in the USA[27].

4.1.3. Clinical aspectsTwo different phases can be distinguished in the fascioliasis:acute fascioliasis, corresponding with the migratory stages ofthe life cycle, and chronic fascioliasis, corresponding with thepresence of the adult worms in the bile ducts [117]. Acutefascioliasis is characterised by fever, abdominal pain, hepato-megaly and other gastrointestinal symptoms result fromdestruction of liver tissues by the migratory flukes. Chronicfascioliasis is often sub-clinical or shows symptoms that areindistinguishable from other hepatic diseases such as cho-langitis, cholecystitis and cholelithiasis. Few human deathshave been reported in relation to fascioliasis [118].

4.1.4. DiagnosisDefinitive diagnosis of human fascioliasis relies on detectionof parasite eggs in the faeces. The thick smear Kato-Katzmethodhas been extensively used since low infectionsmay benot detected [114]. Some progress has been made in PCRtechniques for identification of F. hepatica and F. giganticainfections [119]. Moreover, several ELISAs for the detection ofantibodies against the parasite have been developed [120–123].An accurate serological test using recombinant cathepsin Lhas been developed and it can be applied to blood samplestaken onto filter paper. This method has been validated invarious endemic regions [120].

4.2. Proteomics

Information regarding the proteome of Fasciola spp., F. hepaticaand F. gigantica, and Fasciolopsis buski, as well as their in-teractionswith their host at themolecular level, are essential todevelop specific and effective diagnostic, pharmacological and

Fig. 6 – Life cycle of Fasciola hepatica.Source: http://www.dpd.cdc.gov/dpdx/html/Fascioliasis.htm.

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preventive tools. Surprisingly, despite the great socioeconomicimpact and animal health importance of fascioliasis, little isknownabout theseparasites and their interplaywith thehost atthe molecular level. The lack of comprehensive genomic datasets for Fasciola spp. is limiting themolecular biological researchof these parasites. To date, most molecular biological studiesof flatworms have focused on human blood flukes, and thegenome sequences of Schistosoma mansoni and Schistosomajaponicum have been assembled and published [17,18]. This isin contrast to the situation for species of Fasciola because thenuclear genomic research of fasciolids has been neglected.The Fasciola nucleotide sequences available in GenBank™ arerelatively few and highly redundant, and currently there are14,031 adult F. hepatica expressed sequence tags (ESTs)available from the Wellcome Trust Sanger Centre (http://www.sanger.ac.uk/Projects/F_hepatica/). Only the completenucleotide sequence of themitochondrial (mt) DNAmoleculeof F. hepatica is available [124]. The EST and genomic datasetspresently available for Fasciolopsis buski are far too small toprovide sufficient information for molecular studies of thisparasite.

4.2.1. The transcriptomeUsing the 454 sequencing technology (Gs FLX Titanium,Roche), the first transcriptome of the adult stage of F. hepaticahas been reported [185], opening the door to high-throughputproteomic technologies. The characterization of this tran-scriptome has revealed numerous molecules of biologicalrelevance, based on homology searches against blood flukes

(Schstosoma mansoni and S. japonicum), nematodes (allCaenorhabditis species) and mammals, for which comprehen-sive datasets are available. Someof thesemolecules are inferredto be involved in key biological processes or pathways, such asprotein phosphorylation, proteolysis, carbohydrate metabo-lism, translation and RNA-dependent DNA replication; signaltransduction, microtubule-based movement and proteinpolymerization; cell redox homeostasis, regulation of ADP-ribosylation factor (ARF) and Rab GTPase activity, cellular ironhomeostasis, as well as the regulation of actin filamentpolymerization.

A limited set of ESTs (1684 high quality sequences) from thenewly excysted juveniles (NEJ) has been also reported [126].Considering that only 22 sequences fromNEJ were available inGenbank (15 of them encoding cathepsins) until July 2009, thisrepresents a contribution to the knowledge of the genesexpressed by the invasive stage of F. hepatica. Functionalannotation of predicted proteins showed a general represen-tation of diverse biological functions. Besides proteases andantioxidant enzymes expected to participate in the earlyinteraction with the host, various proteins involved in geneexpression, protein synthesis, cell signalling and mitochon-drial enzymes were identified. More than half of the juvenilecontigs (55.3%) were also found in adult ESTs. On the otherhand, there are several juvenile contigs that they mightrepresent stage specific transcripts since they are absentfrom the adult database. The 22.1% of juvenile contigs likelycorrespond to core eukaryotic functions such as ribosomalproteins and common enzymes.

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4.2.2. The secretomeRecently, the publicly available 14,031 ESTs from theWellcome Trust Sanger Centre have been analysed to predictsecretory proteins [127] using the EST2Secretome, a semi-automated bioinformatics platform [128], the secreted proteindatabase SPD [129], and the manually curated signal peptidedatabase SPDb [130]. Among the 160 predicted F. hepaticasecretory proteins, the major components are proteolyticenzymes including cathepsin L (41.2%), cathepsin B, andasparaginyl endopeptidase cysteine proteases, as well asnovel trypsin-like serine proteases and carboxypeptidases.These data, together with the analysis 1-DE of the secretoryproteins from different developmental stages (i.e. metacercar-iae, newly excysted juveniles, and immature and adult stagesof this parasite), complement previous studies characterisingthe major secretory proteins expressed by adult F. hepaticausing 2-DE [131–133].

The proteomic analyses of the adult F. hepatica proteinssecreted in vivo and in vitro led to the identification of differentproteins. In accordance with the transcriptomic predictions,cathepsin L proteases (FhCL1, FhCL2, and FhCL5) are highlyrepresented in adult fluke secretions. Proteomic analysis ofproteins secreted by infective larvae, immature flukes, andadult F. hepatica showed that these proteases are develop-mentally regulated and changes in their expression correlatewith the migration of the parasite [134].

Besides proteases, the parasites secrete an array of proteinswith antioxidant activities that are also highly regulatedaccording to their migration through host tissues: fatty acid-binding proteins (FaBP1, FaBP2, FaBP3, and Fh15), redoxenzymes (peroxiredoxin and thioredoxin and protein-disul-fide isomerase) and glutathione S-transferases (GSTs). Thesemolecules have been implicated in fluke immune avoidancemechanisms [135,136], and may also protect the parasitesfrom harmful reactive oxygen species released by hostimmune cells. In addition, actin and the glycolytic enzymesenolase and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) have all been identified in vitro but have not beendetected in vivo. Enolase has been implicated in autoimmunediseases as well as in invading host tissues by pathogens [137].Our group identified enolase as plasminogen binding proteinin the excretory–secretory materials of F. hepatica adults [138].Nevertheless, as GST, FABP, enolase, actin, and GAPDH arelocated at the surface or just below the surface of the fluke,they may be released during in vitro culture as the tegument issloughed to evade the host immune system [131].

As it occurs in other helminths, like Echinostoma spp., theanalysis of the identified proteins shows that few of them aretargeted for export using a classic eukaryotic amino-terminalsecretion signal peptide. The identified proteases of F. hepaticapresent a canonical secretion signal and therefore are likely tobe secreted via a classical endoplasmic reticulum/Golgipathway from specialised gastrodermal cells of the gut [139].Nevertheless, each of the aforementioned antioxidant andglycolytic enzymes do not possess a signal sequence forsecretion, likely indicating that they are secreted via non-classical mechanisms via a trans-tegumental route and/or theexistence of a vesicle-based secretion system in theseparasites like exosomes as already suggested in this revision(see above).

4.2.3. Host–parasite interactions and novel targets for diagnosisand vaccinesThe available data sets could contribute to the identification ofmolecular markers for the early diagnosis of disease, andprovide a foundation for the prediction of drug targets.Triclabendazole (TCBZ) remains the drug of choice for treatinginfections of the liver flukes, F. hepatica and F. gigantica inlivestock and has become the main drug used to treat humancases of the disease as well because it is effective againstimmature and adult parasites. The mode(s) of action andbiological target(s) of TCBZ at the molecular level have yet tobe resolved, but proteomic assays via 2-DE revealed proteinsdisplaying altered synthesis patterns and responses bothbetween isolates and under TCBZ exposure [140]. The TCBZresponding proteins were grouped into three categories;structural proteins, energy metabolism proteins, and “stress”response proteins. Two of the TCBZ responding proteins, aglutathione transferase and a fatty acid binding protein, werecloned, produced as recombinant proteins, and both found tobind TCBZ at physiologically relevant concentrations, whichmay indicate a role in TCBZ metabolism and resistance.Nevertheless, the development of resistance to triclabenda-zole (reviewed in [141]) has shown anthelmintics to be anunsustainable means of controlling the disease and hasprovided further incentive for the development of molecularvaccines against these pathogens.

As we have indicated, proteomic approaches present aunique opportunity for detecting protein targets for rationalvaccine design. Since excreted/secreted proteins (ESP) or thosepredicted to be expressed on the cell surface, in general, areexposed to the immune system of the host, they representobvious candidates for the development of anti-parasitevaccines [142]. Cathepsin L proteases have been described aspromising vaccine candidates for F. hepatica. These proteases aresecreted into the host tissues where they play important roles inhost–parasite interactions. Vaccination of cattle and sheep withpurified cathepsins L1 and L2 of F. hepatica afforded a highpercentage protection against establishment of adult worms, aswell as having an anti-fecundity effect by inhibiting theembryonation of eggs [143]. While these studies were performedwithnativeproteins, it hasbeenreported thatyeast (Saccaromycescerevisiae and/or Pichia pastoris), transformed with a full-lengthFheCL1 or FheCL2 cDNA, expressed and secreted the functionalenzyme into the culture medium from which homogeneousenzyme could be obtained by conventional purification tech-niques [144,145]. These experiments demonstrate that liver flukecathepsins could be produced at a commercial level using yeastas the heterologous expression system and therefore representexcellent candidates for thedevelopmentof first generationanti-fluke vaccines. The advantages and disadvantages of eachexpression system have been discussed in detail by Daltonet al. [146]. There are several other molecules that are potentialvaccine candidates including GST, FABPs and Leucine amino-peptidase (LAP) [147,148]. Interestingly, LAPwasalso identifiedasone of the major immunogen molecules in F. hepatica infections[122]. While trials with these antigens have given some veryvariable results, it is still feasible that these molecules could bedeveloped as a basis for commercial vaccines, either alone or incombination with other candidates, if the precise conditions forinducing protection were elucidated.

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5. Treatment of food-borne trematodiases

Praziquantel is the drug of choice for the food-borne trema-todiases, except for fascioliaisis. Praziquantel exhibits a broadspectrum against trematodes and has an excellent safetyprofile [149,150]. Although the exact mechanism of action ofpraziquantel has not been elucidated, it has been postulatedthat a disruption of Ca2+ homestasis occurs as praziquantelinduces a rapid contraction of the trematodes [151]. Alltreatment schedules with prazinquantel are well tolerated,with only few adverse events including abdominal pain,dizziness, headache, nausea and urticaria [149]. The dosesemployed for the different trematode infections are shown inTable 2.

Triclabendazole, a benzimidazole derivative, is used for thetreatment of the fascioliasis and holds promise for thetreatment of paragonimiasis. Currently, triclabendazole isregistered for human use only in Ecuador, Egypt, France andVenezuela and the doses employed are shown in Table 2.Abdominal pain, biliary colic, fever, nausea, vomiting, weak-ness, and liver enlargement have been reported as adversereactions. There are some concerns that triclabendazoleresistance might emerge since it has been reported inveterinary medicine [1]. Bithionol also can be used againstfascioliasis when triclabendazole is not available. Howeverlong treatment schedules of 10 to 15 days are required [149].New drugs which include tribendimidine and peroxidicderivates (e.g. artemisinins and synthetic trioxolanes) arebeing investigated [150].

6. Concluding remarks

Neglected tropical diseases like malaria, HIV/AIDS andtuberculosis receive most of the attention including funding,presence in the press media, and resources for research,meanwhile other neglected diseases like schistosomiasis andfoodborne trematodiases, which are highly endemic, receivemuch less consideration. This is even worse in the case offoodborne trematodiases, where easy control measures canbe implemented, but there are still millions of patients. Newtargets for diagnosis, treatment and vaccination are urgentlyneeded, and in this landscape new technologies should

Table 2 – Habitat, infection sources, and treatment of choice of m

Species Habitat Source of infection

Clonorchis sinesis Liver Freshwater fish PrOpistorchis felineus Liver Freshwater fish PrOpistorchis viverrini Liver Freshwater fish PrParagonimus spp. Lung Freshwater crabs, crayfish, wild boar

meatPr

Echinostomatidae Intestine Freshwater fish, frogs, mussels, snails,tadpoles

Pr

Gymnophalloides seoi Intestine Oysters PrHeterophyidae Intestine Freshwater fish. PrFasciola spp. Liver Freshwater vegetables, contaminated

water.Tw

diminished the distance in research between these trop-ical diseases. In this context, molecular biology tools likethe “-omics” should help greatly. Proteomic-based ap-proaches for the study of host–pathogen interactions arecentral to the understanding of the pathogenesis process andthey have the potential to identify new diagnostic biomarkersfor the early detection of diseases, as well as new vaccinetargets. At present, most of the studies of foodbornetrematodes include few or no proteomic approaches, andwhen performed, they exhibited important problems like thesource of material, complicated biological cycles, as well astechnical problems. However, the main bottleneck in theseproteomic studies is the absence of databases, with only fullgenome sequences available for two schistosome species.The growing availability of sequence information fromdiverse parasites through genomic and transcriptomic pro-jects offers new opportunities for the identification ofextracellular proteins that are secreted or released byhelminth parasites. The recent massive sequencing tech-niques with “more affordable” costs have led to the charac-terization of different transcriptomes from foodbornetrematodes like F. hepatica, O. viverrini and C. sinensis. Thisinformation has clearly increased the chance of identifyingproteins in mass spectrometry assays in those and othertrematodes, but the data from the sequences obtained haverevealed that the homology between these transcriptomes is atmost 40%. Additional sequence data from these and othertrematodes is needed to fulfil identification requirements, andimportantly to check for species-specific targets to be used infuture diagnosis, treatment and vaccination.

Acknowledgments

Dr. Lynne Yenush is acknowledged for critical reading of themanuscript.

This work was supported by the projects CGL2005-0231/BOS and SAF2010-16236 from the Ministerio de Ciencia eInnovación and FEDER (European Union), PROMETEO/2009/081fromConselleria d'Educació, Generalitat Valenciana (Valencia,Spain), PS09/02355 from the Fondo de Investigación Sanitaria(FIS) del Ministerio de Ciencia e Innovación (Madrid, Spain)and FEDER, and UV-AE-10-23739 from the Universitat deValència (Valencia, Spain).

ajor food-borne trematodes and their underlying diseases.

Treatment (dose)

aziquantel (3×25 mg/kg for 2 days or single dose of 40 mg/kg)aziquantel (3×25 mg/kg for 2 days or single dose of 40 mg/kg)aziquantel (3×25 mg/kg for 2 days or single dose of 40 mg/kg)aziquantel (3×25 mg/kg for 2 days)

aziquantel (single dose of 25 mg/kg)

aziquantel (single dose of 10 mg/kg)aziquantel (single dose of 25 mg/kg)riclabendazole (single dose of 10 mg/kg or 20 mg/kg in two split dosesithin 12–24 h)

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