Proteomics of foodborne trematodes

J O U R NA L OF PR O TE O MI CS 7 4 ( 2 01 1 ) 1 4 8 5–1 5 0 3

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www.elsevier.com/locate/jprot

Review

Proteomics of foodborne trematodes Rafael Toledoa , M. Dolores Bernalb , Antonio Marcillaa,⁎ a

Área de Parasitología, Departament de Biologia Cel.lular i Parasitologia, Universitat de València, Burjassot, Valencia, Spain Departament de Bioquímica i Biologia Molecular, Universitat de València, Burjassot, Valencia, Spain

b

AR TIC LE I N FO

ABS TR ACT

Article history:

Food-borne trematodiases are among the most neglected tropical diseases, not only in

Received 28 February 2011

terms of research funding, but also in the public media. The Trematoda class contains

Accepted 26 March 2011

several species identified as the causal agents of these diseases whose biological cycle,

Available online 1 April 2011

geographical distribution and epidemiology have been well characterised. The diagnosis of these diseases is based on parasitological techniques and only a limited number of drugs are

Keywords:

currently available for treatments, most of which are unspecific. Therefore, in-depth studies

Proteomics

to identify new and specific targets for both effective diagnosis and treatments are urgently

Parasites

needed. Currently, little molecular information is available regarding the host–parasite

Food-borne trematodes

interaction. In this regard, proteomic studies have the potential to identify diagnostic biomarkers for the early detection of the diseases, as well as new vaccine targets. In this review, a description of the biology, clinical features and current diagnostic tools of the main groups of trematodes and the corresponding diseases they cause is followed by a discussion of the available studies using proteomic techniques to identify key parasite proteins involved in the pathogenesis of food-borne trematodiases. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . Biology of food-borne trematode infections . . . . . . Fish- and invertebrate-borne trematodes . . . . . . . 3.1. Liver flukes . . . . . . . . . . . . . . . . . . . . 3.1.1. Opistorchiasis and clonorchiasis . . . . 3.1.2. Proteomics . . . . . . . . . . . . . . . . 3.2. Intestinal flukes . . . . . . . . . . . . . . . . . 3.2.1. Diplostomiasis, gymnophalloidiasis and 3.2.2. Echinostomiasis . . . . . . . . . . . . . 3.2.3. Proteomics . . . . . . . . . . . . . . . . 3.3. Lung flukes . . . . . . . . . . . . . . . . . . . . 3.3.1. Paragonimiasis . . . . . . . . . . . . . . 3.3.2. Proteomics . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Dept. Biologia Celular y Parasitologia, Facultat de Farmàcia, Universitat de València, Av. V.A. Estellés, s/n, 46100 Burjassot, Valencia, Spain. Tel.: + 34 963544491; fax: +34 963544769. E-mail address: [email protected] (A. Marcilla). 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.03.029

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4.

Plant-borne trematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Liver flukes (fascioliasis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Geographical distribution and epidemiology . . . . . . . . . . . . . . 4.1.3. Clinical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. The transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. The secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3. Host–parasite interactions and novel targets for diagnosis and vaccines 5. Treatment of food-borne trematodiases . . . . . . . . . . . . . . . . . . . . . . . . 6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.

Introduction

Despite the considerable public health impact and the emerging nature of food-borne trematodiases [1–4], these diseases are among the most neglected of the so-called neglected tropical diseases [1,5–9]. It should be noted that the neglected tropical diseases are found predominantly in the world's poorest populations in low-income countries, and where these diseases are common, they exacerbate poverty. Compared with malaria or tuberculosis, most of the neglected tropical diseases are orphans with regard to research funding and presence in the press media [9–11]. Moreover, funding for improving prevention and control is scarce, and the global strategy for the control of schistosomiasis and food-borne trematodiases and other parasitic worm infections is morbidity control by chemotherapy. Although there is growing international awareness pertaining to the neglected tropical diseases and new political and financial commitments to do something against them [12,13], with regard to fluke infections, a number of campaigns have been stopped, and the diseases are not on the priority list of the World Health Organization (WHO). At present, mainly two drugs are currently available: triclabendazole against fascioliasis and praziquantel against the other food-borne trematode infections and schistosomiasis, with the new drugs tribendimidine and peroxidic derivates (e.g. artemisinins and synthetic trioxolanes) being investigated [14]. There has been little incentive to invest in the discovery and development of trematodicidal drugs. While public–private partnerships work for some of the neglected tropical diseases since 1999 (e.g. the Drugs for Neglected Diseases Initiative (DNDi) focusing on human African trypanosomiasis and leishmaniasis), pioneering programmes for major helminth diseases are underway [14,15]. In this context, studies to identify new and specific targets for treatment are needed. Moreover, the identification of parasite-specific proteins could clearly facilitate the design of new tools for rapid and cheap diagnosis, which in turn could help in breaking the transmission of the parasite. Last but not least, the identification of potential targets for vaccination seems to be one of the best ways to control these parasite infections.

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Proteomics should help in those goals, but at present proteomic studies are limited by some problems like mass spectrometry sensitivity, which ultimately depends on the amount and quality of material available. Parasite material is in most of the cases limited, and to get enough in vivo materials many laboratories maintain the parasite life cycle in the laboratory using rodents as definitive hosts [16]. Another important limitation for proteomic studies is the lack of sequence data available to correctly identify the peptides obtained in mass spectrometry assays [16]. In the present review we summarise the key characteristics of food-borne trematodes, depicting their life cycles, geographical distribution and epidemiology, as well as their main clinical features and how are these diagnosed. Special attention is focused on reviewing the proteomic studies available and how these studies could help in identifying and characterising new targets for treatment and diagnosis. Nonetheless, publications of latest draft genome sequences for the related trematodes of the genus Schistosoma [17,18] are now providing new insights into the biology of trematodes and offer an opportunity for identification of potential new targets for treatment, diagnosis and vaccines.

2.

Biology of food-borne trematode infections

The class Trematoda comprises an important group of parasitic flatworms of medical and veterinary importance and contains numerous species that are the causative agents of human and animal diseases. The digenetic trematodes constitute the largest group of platyhelminths, and in this review we will focus on those digenetic trematodes transmitted by food. The life cycle of digeneans is complex but a common feature is that snails act as first intermediate host. Usually there are seven larval stages (e.g. adult, egg, miracidium, sporocyst, redia, cercaria, and metacercaria) with alternations of asexual and sexual reproductive phases in the molluscan and definitive host. Moreover, the life cycle of some digeneans may involve an unencysted stage intermediate between the cercaria and metacercaria 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 and lung flukes. Typically the life cycle of the major food-borne trematodes include two or three different hosts: a vertebrate definitive host, including humans and domestic animals; an invertebrate first intermediate host (a mollusc); and, frequently, a second intermediate host carrying the encysted metacercarial stage (Fig. 1). Eggs are produced by adult worms following sexual reproduction in the definitive host and are released via faeces for most of the human food-borne trematodes, although Paragonimus spp. release their eggs mainly in the sputum. The egg hatches and a swimming ciliated larva, the miracidium is released. The miracidium, attracted by chemotaxis and chemokinesis, penetrates the snail intermediate host or is ingested by the host (Fig. 1). In some cases, the eggs are directly ingested by the intermediate host and the miracidia hatch in the gastrointestinal tract of the snail. Various snail species act as first intermediate host, most of which are trematode-species specific [21]. Within this host, miracidia develop into sporocysts, but in some cases the miracidia directly give rise to redia. The germinal cells within the sporocysts multiply and produce new germinal masses which produce daughter sporocysts or rediae. Development of pathernitae (sporocysts and rediae) follows different patterns depending on the digenean species [20]. Finally, these larval stages produce embryos, which develop into cercariae. The free-swimming cercariae escape from the host and either come in contact with a compatible

second intermediate host in which penetrate and encyst (e.g. Clonorchis sinensis, Echinostoma spp. or Opistorchis spp.), or encyst on aquatic vegetation such as watercress, water lotus, water caltrop, water chestnut or water lily (e.g. Fasciola hepatica or Fasciolopsis buski). Numerous invertebrates and poikilothermal vertebrates serve as second intermediate host. Several fish species, crustaceans, snails and tadpoles have been reported to act as second intermediate host. Human and animal definitive host become infected when eating raw, pickled or insufficiently cooked second intermediate host harbouring metacercariae, aquatic vegetation or, even, drinking contaminated water [22,23]. After their ingestion, the metacercariae excyst in the gastrointestinal tract, releasing a juvenile worm which migrates to the target organ. Infection with Paragonimus spp. might also occur through the consumption of undercooked meat of wild boar, which acts as a paratenic host [24]. The survival of the adult worms in the definitive 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 of the definitive host, only those causing opistorchiasis and clonorchiasis are considered in the present section since they are transmitted by eating fish.

Definitive host Adult

Egg

Water 2

1, 3, 6, 7

4, 5

embryonated

Metacercaria Third intermediate host

Metacercaria

Metacercaria

Second intermediate host

vegetation

1, 3, 6, 7

miracidium

Metacercaria

4, 5

2

hatches

ingested

1, 2 ,

3, 4 ,

5, 6

3

7

miracidium 1, 2, 3, 4, 5, 7

6

sporocyst I 3

Cercaria

sporocyst II

Cercaria 3, 4, 5, 6

5, 7

free swimming

12 1, 2

, 3, 4

Mesocercaria Second intermediate host or paratenic host

2

3, 7

Redia I Redia II

3, 4, 5, 6

First intermediate host Fig. 1 – Schematic representation of the life cycle patterns of selected genera of intestinal digenetic trematodes. 1. Diplostomum; 2. Alaria; 3. Echinostoma; 4. Fascioliasis; 5. Paramphistomum; 6. Nanophyetus; 6. Heterophyes. Modified from Toledo et al. [19].

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Opistorchiasis and clonorchiasis

3.1.1.1. Background. The major fish-borne liver flukes – Opistorchis viverrini, O. felineus and Clonorchis sinensis – have close morphological and biological characteristics. Differentiation of the species is based on several characters of the adult worms such as the size, shape and position of the testes and the arrangement of the vitelline glands [25]. The life cycle of Opistorchis spp. and C. sinensis is shown in Fig. 2. These species follow a three-host life cycle. Human infection follows the consumption of raw or undercooked fish harbouring infective metacercariae. Adult worms inhabit into the intrahepatic bile duct, but they also can be found in the common 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 of O. viverrini, it is thought that may survive for approximately 10 years [25]. 3.1.1.2. Geographical distribution and epidemiology. Liver flukes are important public health problems in many parts of the world and they are endemic in Asia and Eastern Europe [24,25]. C. sinensis is widespread in the People's Republic of China (PR China), Korea and North Vietnam, while O. viverrini is endemic in Southeast Asia, including Thailand, Lao People's Democratic Republic (PDR), Cambodia and Central Vietnam [25]. Recent reports suggested that about 35 million people are infected with C. sinensis, with up to 15 million human infections in PR China alone and another 8–10 million individuals infected with O. viverrini in Thailand and Lao PDR [22,26].

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 opistorchiasis and clonorchiasis cases show few specific signs or symptoms, except an increased frequency of palpable liver [28]. Flatulence, fatigue, fever, diarrhoea, rash, oedema, abdominal pain and enlargement of the liver may appear in moderate infections (up to 1000 flukes) [25]. Patients of clonorchiasis with a very high worm burden (up to 25,000 flukes) might also exhibit acute pain in the right upper quadrant [23]. Severe opistarchiasis, which is rare, might cause obstructive jaundice, cirrhosis, cholangitis, acalculous cholecystitis or bile peritonitis [1]. Cholangiocarcinoma is the most serious complication of infections with O. viverrini and C. sinensis. O. viverrini is classified as definitely carcinogenic (class 1) and C. sinensis as probable carcinogen (class 2A) [29]. In contrast to infections with O. viverrini and C. sinensis, many patients infected with O. felineus suffer from fever to hepatitis-like symptoms in the acute stage of the infection. Chronic symptoms include obstruction, inflammation and fibrosis of the biliary tract, liver abscesses, pancreatitis and suppurative cholangitis [24]. 3.1.1.4. Diagnosis. The detection of eggs in faeces, bile or duodenal fluids is the gold standard for diagnosis. Parasitological examination of faecal samples is widely used for routine diagnosis. The most frequently employed methods to

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

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detect eggs are Kato-Katz thick smear, Stoll's dilution and the formaline ethyl acetate concentration technique [27]. However, the similarity of the eggs of trematodes can make sometimes the specific diagnosis difficult in some cases. Immunodiagnosis tests have been developed for Opistorchis and Clonorchis infections and good results have been obtained using individual antigens and detecting isotype-specific antibodies [30]. Faecal antigen detection by ELISA also shows promise [31]. Recent attention has been focused on the detection of DNA of the eggs from faecal samples by PCRbased approaches [30,32]. Further details on methods for the diagnosis of trematode infections can be found in the review by Johansen et al. [33].

3.1.2.

Proteomics

Control measures for opisthorchiasis and clonorchiasis rely mainly on treating infected people with praziquantel, with no vaccines or new drugs yet available. In this landscape, fundamental molecular biological investigations are essential to develop novel diagnostic methods and new treatments. However, to date, almost all the molecular studies of flukes have focused on human blood flukes (schistosomes) [34].

3.1.2.1. The transcriptome. Despite their major socio-economic impact, no draft or complete nuclear genomic sequence is available for liver flukes, and transcriptomic data are scant (Table 1). A preliminary study described 2387 EST from an adult C. sinensis cDNA library [35]. However, recent application of massive sequencing technologies is helping greatly to obtain sequence data from these neglected organisms, improving the identification of parasite proteins by proteomics. In this context, two recent studies provide a first, deep insight into the transcriptomes of O. viverrini and C. sinensis using 454 sequencing from cDNA libraries and a semiautomated bioinformatics platform to assemble and annotate the datasets [36]. In these studies, 50,000 unique sequences were identified and comparative analyses revealed that most of the sequences (85%) were new [36], thus expanding current databases and providing a resource for research groups investigating molecular aspects of these two trematodes. As indicated by Young et al. [36], the transcriptomes of C. sinensis and O. viverrini provide a sound platform for investigating diverse aspects of host–parasite interactions and the pathogenesis, even assisting in testing hypotheses

regarding to the molecular basis of cholangiocarcinoma. The comprehensive transcriptomic data will also improve the classification of different proteins (somatic, excretory/secretory and tegumental) from C. sinensis and O. viverrini identified in previous proteomic studies [37,38]. When a comparative analysis of C. sinensis, O. viverrini (Opisthorchiidae), Fasciola hepatica (Fasciolidae), S. japonicum and S. mansoni (Schistosomatidae) transcriptomes was performed, it showed that the opisthorchiids and fasciolids shared the greatest (29–31%) protein sequence homology [36]. The proteins encoded by adult C. sinensis and O. viverrini mapped to metabolic, genetic and environmental data/information and cellular processing pathways inferred to be conserved amongst eukaryotes, whereas ~90% of predicted proteins did not map to any known pathways [36]. This observation could provide the basis to explore novel biological pathways and processes that are unique to these parasites, being even potential drug and/or vaccine targets. The advances in transcriptomics should assist future comparative -omic studies of liver flukes and the diseases that they cause. Those studies should focus on the differential transcription of genes among the different biological phases of the parasite to explore the molecular basis of pathological changes and/or carcinogenesis in humans at different stages of liver fluke disease. But still, the assembly and annotation of the nuclear genomes of C. sinensis and O. viverrini is needed.

3.1.2.2. The secretome.

Mulvenna et al. [38] characterised 300 parasite proteins from the excretory/secretory products (ESP) of O. viverrini using OFFGEL electrophoresis and multiple reaction monitoring (Table 1). The excretory/secretory products included a complex mixture of proteins that have been associated with cancer, and also identified a cysteine protease inhibitor which may contribute to malignant transformation of inflamed cells [38]. More than 160 tegumental proteins were also identified using sequential solubilisation of isolated teguments, being some of those molecules localised to the surface membrane of the tegument by biotin labelling and fluorescence microscopy. These included annexins, which are potential immunomodulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Mulvenna et al. [38] suggested novel roles in pathogenesis for the tegument–host interface since more than ten surface proteins had no homologues in the public databases.

Table 1 – Representative proteomic and transcriptomic studies of food-borne trematodes. Species

Strategies

Materials

Reference

Transcriptomes 2387 EST >50,000 unique seq >50,000 unique seq

[35] [36] [36]

Underway

Unpublished

358 EST

[92]

14,031 EST 1684 EST(juveniles) >47,800 unique seq

http://www.sanger.ac.uk [126] [125]

Clonorchis sinesis

2DE-MS

ESP

[37]

Opistorchis viverrini

2DE-MS OFFGEL MS/MS 2DE-MS LC–MS/MS 2DE-MS, WB

Whole juvenile and adult ESP Whole sporocysts ESP ESP

[52] [38] [89]

2DE-MS 2DE-MS 2DE-MS, WB

ESP ESP ESP in vivo

[109] [132] [131]

Echinostoma caproni Echinostoma friedi Echinostoma paraensei Paragonimus westermani Fasciola hepatica

Reference

[85]

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Similarly to O. viverrini, proteomic studies of Clonorchis sinensis have been focused in the study of both, parasite ESPs and host response [37,39]. Host proteins were examined to determine the effects of C. sinensis infection on protein expression in host bile duct epithelium, examining proteomic profile changes in a human cholangiocarcinoma cell line (HuCCT1) treated with parasite ESP [39]. As mentioned before, Ju et al. [37] established a 2-D proteome map of the C. sinensis ESPs (Table 1), and identified diagnostic candidates for clonorchiasis through an immuneproteomic approach. They detected cysteine proteases in immunoblot assays with sera obtained from patients and identified legumain as a potential antigen for clonorchiasis. Since the numbers of registered genes for C. sinensis in Genbank was very limited at that time, they also generated an EST database by massive sequencing of a cDNA library, which was constructed from adult C. sinensis [37]. These authors successfully identified 62 protein spots. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly.

3.1.2.3. Host–parasite interactions and targets for potential novel diagnostic tools and vaccines. Several studies have described the use of molecular biological techniques in the search for useful serodiagnostic antigens for clonorchiasis [40]. Many recombinant proteins have been evaluated against helminth-infected human sera. These include a 7-kDa protein of excretory–secretory products of adult C. sinensis [41], myoglobin [42], a fatty acid-binding protein (FABP) [43] and glutathione S-transferases (Cs28GST and Cs26GST) [44], among others. The results show moderate to low sensitivities and high specificities for serodiagnosis of clonorchiasis when used as a single antigen, suggesting the use of a cocktail of antigens or chimeric antigens as a strategy to improve the sensitivity [45]. In this context, recently Li et al. [46] selected 11 clones from an adult C. sinensis cDNA library by immunoscreening using infected human sera. A mix of antigens were prepared using recombinant proteins from positive clones and investigated for antigenicity by immunoblotting against C. sinensis- and helminth-infected patient sera [46]. A mix of antigens from two recombinant proteins (Cs28GST and Cs26GST) produced 76% sensitivity and 95% specificity, but a triple mix containing Cs26GST, Cs28GST and a vitelline precursor protein reached 87% sensitivity and maintained 95% specificity [46]. Recent immunoproteomic studies identified legumains and cysteine proteases as antigens present in the C. sinensis ESPs [37]. In relation to Opisthorchis species, the first recombinant proteins with potential use in diagnosis included paramyosin of O. felineus [47], a glycine–tyrosine rich eggshell protein from O. viverrini [48], and more recently an asparaginyl endopeptidase (from the legumain family) [49] and GST [50,51]. The first high resolution proteomic study on O. viverrini was published by Boonmee et al. [52] with the aim to better understand parasite biology, pathogenesis/carcinogenesis related to this parasite and lead to the identification of new targets of vaccines and drugs. That study showed a comparative analysis using two-dimensional gel electrophoresis to highlight proteins differentially expressed in the maturation

process from juvenile to adult parasites [52]. Approximately 210–240 protein spots were resolved by 2-DE in two ranges of pI (4.5–5.8 and 6.0–8.0), and at least 35 protein spots were differentially expressed in 4 week adult compared to 1 week juvenile fluke, corresponding to proteins probably involved in sex organ development and egg production [52]. Recent studies by the group of Alex Loukas in Australia have identified O. viverrini ESP proteins which could be either involved in contributing to the hepatobiliary abnormalities, including cholangiocarcinogenesis (cathepsin F), or in the establishment of a tumorigenic environment that may ultimately manifest as cholangiocarcinoma (granulin-like growth factor) [53,54].

3.2.

Intestinal flukes

3.2.1.

Diplostomiasis, gymnophalloidiasis and heterophyasis

3.2.1.1. Background. The family Diplostomidae contains digeneans from numerous orders of birds and mammals. In general, species of the Diplostomidae have a 3-host life cycle, though some variations of this pattern can be found. The cercariae emerge from the snails and they penetrate fish, amphibians, molluscs, and annelids forming metacercariae [55]. In some Diplostomidae, the life cycle is expanded to incorporate four hosts by inclusion of a mesocercaria. Definitive hosts become infected by the ingestion of the second intermediate host or the paratenic host harbouring metacercariae. Eggs typically hatch and penetrate the first intermediate host [56]. At least members of 3 genera of Diplostomidae (Alaria, Neodiplostomum, and Fibricola) are known to parasitize man. At the intestinal level, only N. seoulense and F. cratera parasitize humans. The family Gymnophallidae consists of a small group of digeneans occurring in the intestine, gall bladder and the bursa 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 its members, their small body size, and the difficulty in observing the internal structures of these digeneans. A recent revision of the family describes 5 valid genera (Gymnophalloides, Parvatrema, Gymnophallus, Pseudogymnophallus, and Bartolius) [57]. A typical gymnophallid life cycle involves bivalves as first intermediate host, and bivalves, polychaetes, gastropods, or brachiopods as second intermediate hosts. The definitive host becomes infected after ingestion of the second intermediate host harbouring the metacercariae. Within the Gymnophallidae, studies on the pathology and immunology of the infection are available only for one species, Gymnophalloides seoi [58]. The family Heterophyidae contains small egg-shaped trematodes with infective metacercariae that are usually encysted in fish as a second intermediate host. The definitive host becomes infected by eating raw or poorly cooked fish harbouring metacercariae. Heterophyids show little specificity toward the definitive host and numerous fish-eating mammals, including humans, can be infected (Fig. 3). The adult worms live between the villi of the anterior region of the small intestine and they release fully embryonated eggs into water.

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The eggs are then ingested often by littorine snails (particularly Littorina littorea and L. scutulata), and hatch within the snail's intestine. Intramolluscan development comprise sporocyst and redial stages, and cercariae are released into the water where they typically penetrate shore-fish, such as cunners, gudgeon, and charr, and they encyst on the surface of the fish (Fig. 4). Metacercariae may remain viable for years [59]. Although there are a great number of genera within the Heterophyidae, most of the studies in relation to human infections of these infections are focused on Metagonimus yokogawai. Its life cycle can be maintained easily in the laboratory in various experimental hosts, thus facilitating studies on heterophyids.

3.2.1.2. Geographical distribution and epidemiology. Human infections with Neodiplostomum seoulense have been reviewed recently [21,60]. A total of 28 human cases have been reported in the Republic of Korea [60–62]. Chai and Lee [60] estimated the total number of human cases to be 1000 in the Republic of Korea. Nevertheless, there are no available studies on the pathology and immunology of N. seoulense infections in humans. There are no reported cases of human infection with F. cratera, a trematode species indigenous to North America. Symptoms exhibited by the volunteer were similar to those described in N. seoulense infections. Human infections with Gymnophalloides seoi have only been reported in Korea [58–60]. G. seoi is highly prevalent among villagers in the southwestern coastal islands of Korea, where half of the population was infected [63]. People became infected by consuming raw oysters [59].

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Human infections by heterophyids have been often reported. Chai and Lee [60] have listed 12 species of heterophyids that parasitize humans in Korea belonging to the genera: Metagonimus, Heterophyes, Stictodora, Heterophyopsis, Pygidiopsis, Stellantchasmus, and Centrocestus. Moreover, members of the genera Haplorchis have been also implicated in human heterophyiasis [59]. Chai and Lee [60] and Fried et al. [59] provide excellent coverage of human infections by these digeneans. The most prevalent species in humans are M. yokogawai and H. heterophyes, which are distributed mainly in Asia, Africa and Eastern Europe [59]. Humans become infected by eating raw, pickled or poorly cooked fish.

3.2.1.3. Clinical aspects. N. seoulense may cause severe enteritis with abdominal pain, fever, diarrhoea, fullness, and anorexia. The clinical aspects of the human infection with N. seoulense have not been studied in detail. Most of the data available for Gymnophalloides species is based on studies using laboratory rodents. Although symptoms vary in humans from endemic areas, some cases of severe gastroenteritis and signs of acute pancreatitis have been reported [58,63]. Clinical symptoms of this infection include loose stools, pancreatitis, indigestion, diarrhoea and gastrointestinal discomfort [58,60]. G. seoi infections may require medical attention because of their relationships with pancreatic diseases [58]. Low-grade infections of heterophyids are of no clinical consequence, but cases with heavy infections are associated with diarrhoea, mucus-rich faeces, abdominal pain, dyspepsia, anorexia, nausea, and vomiting [59,60,64]. Anaphylactic reactions have also been reported [65]. Occasionally, worm

Fig. 3 – Life cycle of Metagonimus spp. Source: http://www.dpd.cdc.gov/dpdx/html/Metagonimiasis.htm.

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

eggs may enter the circulatory system though the crypts of Lieberkühn causing emboli which may be fatal depending on the affected tissue [64]. Chi et al. [66] have studied the pathology of a human case of metagonimiasis. The parasitism was incidentally detected in an intestinal segment that was removed surgically for treating intestinal perforation related to a malignant histiocytosis. The main histological lesions were massive lymphoplasmacytic and eosinophilic infiltration in the stroma, erosion of the enterocytes in the areas surrounding the worms, goblet cell depletion, and occasional villous oedema.

3.2.1.4. Diagnosis.

The diagnosis of Diplostomas and Gymnophallidae infections can be done by identification of characteristic eggs in the faeces. The eggs of N. seouli are ellipsoid to elliptical, thin shelled, with an inconspicuous operculum, and frequently asymmetrical [20]. No immunological or molecular methods have been developed. In addition to the parasitological methods, several immunological methods have been developed for the diagnosis of the infections with Metagonimus yokogawai and Heterophyes taichui. Using crude extract of metacercariae and adult worms Cho et al. [67] developed an indirect ELISA method to detect specific IgG in cats infected with M. yokogawai. The results demonstrated that the serological diagnosis of metagonimiasis is feasible from the first few days of the infection. However, the sensitivity of the method was low in infections with a reduced number of worms. Lee et al. [68] used a similar method to detect metagonimiasis in humans using metacercarial crude antigens. However, cross reactivity with other trematodiaisis such as fascioliasis, schistosomiasis and para-

gonimiasis was detected. Ditrich et al. [69] developed indirect ELISA and western-blot methods to detect H. taichui in humans using cytoplasmic and membranous antigens from adult worms. ELISA analysis showed that cytoplasmic antigens were more sensitive, but cross-reactions between both species were detected. Western-blot analysis exhibited differences between both trematode species were observed, enabling their differentiation. A recent study has evaluated three immunological techniques like counter current immunoelectrophoresis (CCIE), intradermal (ID) and indirect fluorescent immunoassay (IFI), for the diagnosis of Heterophyes infection in puppets [70]. The results indicate that the ID test could be recommended for diagnosis of heterophyosis in naturally infected animals, but no application to humans has been reported yet.

3.2.2.

Echinostomiasis

3.2.2.1. Background. The family Echinostomatidae contains a rather heterogeneous group of cosmopolitan and hermaphroditic digeneans that parasitize, as adults, diverse vertebrate hosts [61,62,71]. Adult echinostomatids are predominantly found in birds, but also parasitize mammals and occasionally reptiles and fishes. Their typical site of location is the intestine though 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 which a sporocyst, two generations of rediae and cercariae develop. Emerged cercariae infect the second intermediate host, which may 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 metacercariae (Fig. 4) [62,71]. Species of Echinostoma have been the most widely used in studies on pathological and immunological aspects of infection. Comparative studies on the development of a single species of Echinostoma in different host species in which the course of the infection differs have allowed for the analysis of some of the factors that regulate the immunopathology and the immune response in echinostome infections [71–74].

3.2.2.2. Geographical distribution and epidemiology. Echinostomes are commonly found in birds (waterfowls) and mammals, and their distribution is ubiquitous. Their specificity toward the definitive host is usually low and an echinostome species is able to infect several species of vertebrate hosts. The incidence of human echinostomiasis is difficult to determine with any accuracy because of the lack of epidemiological surveys. Most of the data relies on historic surveys and occasional case reports. The distribution of human echinostomiasis is strongly determined by the dietary habits. Humans become infected when they eat raw or inadequately cooked food, specially fish, snakes, amphibians, clams and snails containing encysted echinostome metacercariae [72]. Moreover, it has been postulated that humans can also be infected drinking untreated water containing echinostome cercariae, which could become encysted when exposed to the human gastric juice [75]. Although echinostomiasis occurs worldwide, most human infections are reported from foci in East and Southeast Asia. Echinostomiasis is relatively rare, yet the foci of transmission remain endemic mostly due to the local dietary preferences. Most of these endemic foci are localised in China, India, Indonesia, Korea, Malaysia, Philippines, Russia, Taiwan, and Thailand [76]. Moreover, occasional cases have also been reported in other countries. In this context, a very recent study has estimated the prevalence of infection with echinostome flukes ranged from 7.5% to 22.4% in 4 schools surveyed in Cambodia [77]. The number and identity of the echinostome species causing human echinostomiasis is uncertain in relation to the absence of systematic surveys and occasional case reports. Moreover, the problematical taxonomy of the group complicates further the specific diagnosis of the worms found in humans [76]. 3.2.2.3. Clinical aspects. Major clinical symptoms due to echinostome infection may include abdominal pain, diarrhoea, easy fatigue, and loss of body weight [20,71,74]. The symptoms in echinostomiasis seem to be more severe than those observed in other intestinal trematode infections. Human morbidity is due to the prolonged latent phase, symptomatic presentations and similarity of symptoms with other intestinal helminth infections [20,78]. Heavy infections are associated with eosinophilia, abdominal pain, watery diarrhoea, anaemia, oedema, and anorexia [74]. Pathological damage includes catarrhal inflammation, erosion and even intestinal ulceration [20]. Nevertheless the clinical signs in echinostomiasis are poorly known. Of particular interest are the endoscopic findings in human E. hortense infections. Several adult worms were attached to an

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

3.2.2.4. Diagnosis. The diagnosis of human echinostomiasis is usually based on recovery of eggs in faecal examinations. The size of human-infecting echinostome eggs is in the range of 0.066–0.149 mm in length and 0.043–0.090 mm in width [72]. Occasionally, human echinostomiasis has been diagnosed by gastroduodenal endoscopy performed in relation to severe epigastric symptoms and ulcerative lesions in the stomach and duodenum [80]. Immunological methods also may be useful for the diagnosis and monitoring of echinostome infections. Several techniques have been developed based on antibody detection, seroantigen detection and coproantigen detection [81]. Although the use of these methods has provided interesting results, the information is limited to experimental infections since the potential cross-reactivity with other helminths has not been evaluated [20,82,83]. The application of molecular methods for the diagnosis of echinostomes is very limited. Most of the molecular studies have focused on species identification and systematic, and/or phylogenetic studies [16]. 3.2.3.

Proteomics

Among the intestinal flukes mentioned above, the only published studies available on proteomics refer to echinostomes. The application of proteomics to echinostome proteins is very recent and there are still very few publications [16]. These reports include the identification of E. trivolvis hemozoin by laser desorption mass spectrometry [84] and the characterization of ESP proteins from adults of E. friedi [85], and E. caproni [86–88], as well as from E. caproni primary sporocysts [89] (Table 1). In most of the studies on echinostomes proteins, the materials analysed have been the echinostome excretory– secretory products (ESP), because these materials constitute the initial contact with the environment, i.e. the host. In this context, the molecular definition of the parasite proteins will help to understand the development of chronic infections, or in contrast, the rejection of the intestinal helminths.

3.2.3.1. The transcriptome. The major limitation in echinostomes proteomics is the absence of a full genome project, which should provide enough annotated DNA sequence data to produce deduced protein sequences. Most of the identifications of echinostomes proteins have been possible because they were homologous to other trematode proteins [16]. However, it is estimated that more 75% of proteins may not present sufficient homology to allow for the proper identification in echinostomes. Moreover, not all the available data for other species like schistosomes are useful in the searches using routine bioinformatic tools, thus further modification of such data sets is needed to facilitate these searches. Furthermore, half of the proteins identified in schistosomes from

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their transcriptomes do not have a known function, or are even species-specific [90,91], thus increasing the difficulties when working with other trematodes like echinostomes. Echinostomes lack of transcriptome projects. Our group is currently working to obtain and assemble the transcriptome of E. caproni (unpublished data). Until now, there was only one EST collection (from E. paraensei) available which contains 358 sequences (Table 1) [92]. In our experience, even after obtaining good mass spectra, reliable identification cannot be achieved in more than 90% of the cases. Thus, only a small percentage of the molecules analysed are properly identified using the currently available proteomic technology.

3.2.3.2. The secretome.

In E. friedi, following a proteomic approach, we have identified structural proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase (GAPDH), and aldolase; detoxifying enzymes like GSTs, and the stress-related protein Hsp70 [133]. Other identified proteins in echinostomes include chaperones, proteases, signalling molecules and calcium-binding proteins. More recently, we have performed a shot-gun liquid chromatography/tandem mass spectrometry (LC–MS) for the separation and identification of tryptic peptides from the ESP of E. caproni adult worms [88]. Database search was performed using MASCOT search engine [93] (http://www.matrixscience.com/ home.html) and ProteinPilot software v2.0 (http://www.absciex. com/Products/Software/ProteinPilot-Software, Applied Biosystems) [88]. Although 4030 peptides were analysed in that study, significant homologies were found for only 274 (6.8%). A total of 39 proteins were identified (16 using MASCOT and 23 using ProteinPilot) [88]. In relation to the identification of echinostomes proteins, we could identify some of them combining mass spectrometry data with western-blotting using heterologous (cross-reactive) antibodies [16]. Unfortunately, this technology is feasible only for conserved proteins; it is also expensive and can be time consuming. However, this approach may be sometimes the only way to get information about the proteome in trematodes. An example of the applicability of this technology has been reported for E. friedi, where we have confirmed the identity of echinostome proteins present in the ESP using commercial antibodies against GST, actin, aldolase, Hsp-70 and enolase, as well as non-commercial antibodies for GAPDH [85]. In our studies we have detected posttranslational modifications in the protein pattern of echinostomes proteins including tyrosine phosphorylation in response to different stimulus [94], or glycosylation processes [95]. Recent studies on schistosomes have shown that modifications like tyrosine phosphorylation of parasite proteins may be correlated with the adaptation to the hosts [96]. We report from our investigations a high representation of proteins in ESP lacking the classical molecular features of secretory proteins, including cytoskeletal proteins, heat-shock proteins, glycolytic and ATP-related enzymes, signal transduction proteins, histones and transcriptional regulators. Interestingly, all these proteins have been described as the major components of exosomes [97–99]. These structures have been described in a wide range of cell lines and organisms, including parasites like Leishmania spp. [100]. The role of parasite exosomes is described as being responsible for protein export

and communication with host macrophages [101]. Further investigations could provide sustention to the existence of exosomes in helminths.

3.2.3.3. Host–parasite interactions and novel targets for diagnosis and vaccines. As mentioned before no proteomic studies are available for most of the intestinal trematodes. Initial studies identified a 16-kDa cysteine proteinase of Gymnophalloides seoi which showed no antigenicity on both enzyme-linked immunosorbent assay and immunoblots [102]. Antigens of Metagonimus and Heterophyes species have been described above in the diagnosis section. As mentioned before, immunoproteomic studies with E. caproni adults identified major antigens in ESP detected by immunoglobulins M, A and G, suggesting their potential as candidates for diagnosis and eventually vaccination [95], constituting the first report in echinostomes of a combination of proteomics and serology, which has been denominated “immunome” [91]. Other studies used proteomics to characterise the proteins released by E. caproni primary sporocysts in the intermediate host [89]. Saric et al. [103] recently report the use of mass spectrometry to characterise the metabolomic profile of E. caproni infection in blood, stool, and urine and its potential for discover biomarkers of the infection. Important differences between the infected and the uninfected 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 proteins capable of interacting with host matrix proteins like plasminogen, identifying the enzyme enolase as being one of the most abundant and reactive proteins present [86]. These results confirm previous 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 Paragonimus known to infect humans. P. westermani is the most common elsewhere, while P. heterotremus is the etiological agent of human paragonimiasis in PR China, Lao PDR, Vietnam and Thailand [24,27,105]. Human or other definitive hosts (carnivores) become infected after ingestion of raw or undercooked freshwater crustacean such as crabs, shrimp or crayfishes (Fig. 5). The metacercariae excyst in the small intestine and penetrate through the intestinal wall into the abdominal cavity, prior to migration through the sub-peritoneal tissues, the muscle, the liver, the diaphragm, and finally enters the lung where maturation occurs. Adult flukes lay eggs, which are coughed up and ejected by spitting with the sputum or swallowed and passed in the faeces. After hatch, miracidia invade freshwater snails and finally the cercariae emerge. Crustacea probably acquire the infection by consuming cercariae or eating infected snails containing the fully developed cercariae (Fig. 5). 3.3.1.2. Geographical distribution and epidemiology. About 20 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, Gambia and Nigeria) [25]. Moreover, occasional cases in other places have been reported [27]. Endemic areas can be identified as the people eat raw, pickled and/semi-cooked freshwater species of crabs, shrimps and crayfishes. The sources of infection in the endemic areas have been recently reviewed by Sripa et al. [25].

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immunoblotting-based tests. A recombinant antigen of P. westermani egg has been tested as an ELISA antigen offering high levels of sensitivity and specificity [107]. Immunoassays for the detection of IgG to excretory/secretory products of P. heterotremus provide a sensitive and specific method for diagnosis [108].

3.3.2.

Proteomics

Similarly to other foodborne trematodes, only a proteomic study on Paragonimus species has been published so far, with other studies describing proteins used in diagnosis.

3.3.1.3. Clinical aspects. Pathology induced by Paragonimus spp. is related to the migration of the worms from the gut to the lungs. Ectopic migrations to aberrant sites including the brain and subcutaneous sites at the extremities have been reported [24,106]. The presence of the flukes in the lung causes haemorrhage, inflammatory reaction and necrosis of lung parenchyma and fibrotic encapsulation. In pulmonary paragonimiasis, the most noticeable symptom is a chronic cough with brown and blood streaked pneumonia-like sputum. Pulmonary paragonimiasis can be confused with chronic bronchitis, bronchial asthma or tuberculosis [25,106].

3.3.2.1. The secretome. The only proteomic study on Paragonimus analysed excretory–secretory products of adult P. westermani using 2-DE coupled to MS (Table 1) [109]. The study identified 25 different proteins, some of them highly represented like cysteine proteases. Additionally, three previously unknown cysteine proteases were also identified by MALDI-TOF/TOF MS, being the majority of those proteases reactive against sera from paragonimiasis patients. Chronological changes in the antibody responses to different proteases have also been detected in an experimental model of canine paragonimiasis [109].

3.3.1.4. Diagnosis.

3.3.2.2. Host–parasite interactions and novel targets for diagnosis and vaccines. Reliable diagnostic tools are needed to re-

Definitive diagnosis is established by the detection of eggs of Paragonimus in the sputa and/or faeces by parasitological examination. Supporting methods include chest X-ray and immunological tests, including ELISA and

solve the clinical and parasitological diagnostic problems of paragonimiasis infection. In particular, the diagnosis of ectopic

Fig. 5 – Life cycle of Paragonimus spp. Source: http://www.dpd.cdc.gov/dpdx/html/paragonimiasis.htm.

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paragonimiasis using parasitological techniques is not possible, and thus, immunological and serological methods constitute an alternative, but crude worm extracts are subject to cross-reactivity among trematodes [109]. Early studies on the identification of useful serodiagnostic antigens included the use of recombinant yolk ferritin [110] and cysteine proteases [111]. Park et al. [111] suggested that a new powerful drug for paragonimiasis could be designed and developed focusing on the exploration of anti-agents against P. westermani like cysteine proteases. Dekumyoy et al. [112] have identified three polypeptides which strongly reacted with sera from patients of paragonimiasis in enzyme-linked immunotransfer blot (EITB) (or western-blot) assays. Those molecules did not react with sera from patients with other helminth infections, and allowed the distinction between two Paragonimus species, P. westermani and P. heterotremus [112]. More recent studies by Lee et al. [113] have described an ELISA using a recombinant cysteine proteinase antigen which exhibited a high specificity and sensitivity in infected individuals, with no cross-reactivity with Clonorchis sinensis and Metagonimus yokogawai (along with P. westermani, the three major human trematode parasites in Korea, where the study was performed).

4.

Plant-borne trematodes

There are six plant-borne trematode species known affecting humans: F. hepatica, F. gigantica, Fasciolopsis buski (Fasciolidae), Gastrodiscoides hominis (Gastrodicidae), Watsonius watsoni and Fischoederus elongates (Paramphistomidae). Whereas F. hepatica and F. gigantica are hepatic, the others are intestinal parasites. In the present section we will focus on the members of Fasciola due to their greater impact in human health and the existence of more proteomic studies from these species.

4.1.

Liver flukes (fascioliasis)

4.1.1.

Background

F. hepatica and F. gigantica are the causative agents of liver fluke disease (fascioliasis) in domestic animals and humans. Both species follow a two-host life cycle (Fig. 6). The eggs, released by the adults residing in the bile ducts of the mammalian host, are carried into the intestine and are passed in the faeces. Embryonation and hatching occurs in freshwater (Fig. 6). The free-swimming miracidia find and penetrate in the molluscan intermediate host where the parasite undergoes a series of developmental stages (sporocyst–rediae–cercaria). The freeswimming cercariae adhere to and encyst, as metacercariae, on vegetation. Following ingestion of the contaminated vegetation the parasite excysts in the small intestine and juvenile worms penetrate through the gut wall and enter the peritoneal cavity (Fig. 6). After 10–12 weeks of migration the parasites enter the bile ducts where they mature and may remain for up to 1–2 years in cattle or as long as 20 years in sheep [114].

4.1.2.

Geographical distribution and epidemiology

Although traditionally considered as a disease of livestock, fascioliasis is now recognised as an important emerging

zoonotic disease of humans. It is estimated that between 2.4 and 17 million people are currently infected and 91 million are at risk of infection [8]. Human infections normally occur in areas where animal fascioliasis is endemic. Transmission occurs where farming communities regularly share the same water soured as their animals or consume water-based vegetation. The main types of aquatic plants are watercress, algae, kjosco and tortora [114]. Drinking untreated water may be a source of infection due to the presence of free-floating metacercarial cysts [115]. To date, the majority of reported human cases of fascioliasis are due to infections with F. hepatica. However, some reports indicate a rise in human infections due to F. gigantica in Vietnam, and probably Thailand and Cambodia [115]. The highest prevalence of human fascioliasis is found in the Altiplan region of Northern Bolivia. Prevalence in this area may reach 40% in certain communities [115]. Hyperendemic human fascioliasis has also been reported in the Nile Delta region between Cairo and Alexandria [116], with a prevalence of 19% in some villages. Significant levels of human F. hepatica infections also occur, with regular outbreaks involving up to 100,000 infections, in several provinces of Northern Iran [115]. In Europe, human fluke infections occur more sporadically, though significant outbreaks of the disease occur in France, Portugal and Spain [115]. Sporadic cases also have been reported in the USA [27].

4.1.3.

Clinical aspects

Two different phases can be distinguished in the fascioliasis: acute fascioliasis, corresponding with the migratory stages of the life cycle, and chronic fascioliasis, corresponding with the presence of the adult worms in the bile ducts [117]. Acute fascioliasis is characterised by fever, abdominal pain, hepatomegaly and other gastrointestinal symptoms result from destruction of liver tissues by the migratory flukes. Chronic fascioliasis is often sub-clinical or shows symptoms that are indistinguishable from other hepatic diseases such as cholangitis, cholecystitis and cholelithiasis. Few human deaths have been reported in relation to fascioliasis [118].

4.1.4.

Diagnosis

Definitive diagnosis of human fascioliasis relies on detection of parasite eggs in the faeces. The thick smear Kato-Katz method has been extensively used since low infections may be not detected [114]. Some progress has been made in PCR techniques for identification of F. hepatica and F. gigantica infections [119]. Moreover, several ELISAs for the detection of antibodies against the parasite have been developed [120–123]. An accurate serological test using recombinant cathepsin L has been developed and it can be applied to blood samples taken onto filter paper. This method has been validated in various endemic regions [120].

4.2.

Proteomics

Information regarding the proteome of Fasciola spp., F. hepatica and F. gigantica, and Fasciolopsis buski, as well as their interactions with their host at the molecular level, are essential to develop specific and effective diagnostic, pharmacological and

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

preventive tools. Surprisingly, despite the great socioeconomic impact and animal health importance of fascioliasis, little is known about these parasites and their interplay with the host at the molecular level. The lack of comprehensive genomic data sets for Fasciola spp. is limiting the molecular biological research of these parasites. To date, most molecular biological studies of flatworms have focused on human blood flukes, and the genome sequences of Schistosoma mansoni and Schistosoma japonicum have been assembled and published [17,18]. This is in contrast to the situation for species of Fasciola because the nuclear genomic research of fasciolids has been neglected. The Fasciola nucleotide sequences available in GenBank™ are relatively few and highly redundant, and currently there are 14,031 adult F. hepatica expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre (http:// www.sanger.ac.uk/Projects/F_hepatica/). Only the complete nucleotide sequence of the mitochondrial (mt) DNA molecule of F. hepatica is available [124]. The EST and genomic datasets presently available for Fasciolopsis buski are far too small to provide sufficient information for molecular studies of this parasite.

4.2.1.

The transcriptome

Using the 454 sequencing technology (Gs FLX Titanium, Roche), the first transcriptome of the adult stage of F. hepatica has been reported [185], opening the door to high-throughput proteomic technologies. The characterization of this transcriptome has revealed numerous molecules of biological relevance, based on homology searches against blood flukes

(Schstosoma mansoni and S. japonicum), nematodes (all Caenorhabditis species) and mammals, for which comprehensive datasets are available. Some of these molecules are inferred to be involved in key biological processes or pathways, such as protein phosphorylation, proteolysis, carbohydrate metabolism, translation and RNA-dependent DNA replication; signal transduction, microtubule-based movement and protein polymerization; cell redox homeostasis, regulation of ADPribosylation factor (ARF) and Rab GTPase activity, cellular iron homeostasis, as well as the regulation of actin filament polymerization. A limited set of ESTs (1684 high quality sequences) from the newly excysted juveniles (NEJ) has been also reported [126]. Considering that only 22 sequences from NEJ were available in Genbank (15 of them encoding cathepsins) until July 2009, this represents a contribution to the knowledge of the genes expressed by the invasive stage of F. hepatica. Functional annotation of predicted proteins showed a general representation of diverse biological functions. Besides proteases and antioxidant enzymes expected to participate in the early interaction with the host, various proteins involved in gene expression, protein synthesis, cell signalling and mitochondrial enzymes were identified. More than half of the juvenile contigs (55.3%) were also found in adult ESTs. On the other hand, there are several juvenile contigs that they might represent stage specific transcripts since they are absent from the adult database. The 22.1% of juvenile contigs likely correspond to core eukaryotic functions such as ribosomal proteins and common enzymes.

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The secretome

Recently, the publicly available 14,031 ESTs from the Wellcome Trust Sanger Centre have been analysed to predict secretory proteins [127] using the EST2Secretome, a semiautomated bioinformatics platform [128], the secreted protein database SPD [129], and the manually curated signal peptide database SPDb [130]. Among the 160 predicted F. hepatica secretory proteins, the major components are proteolytic enzymes including cathepsin L (41.2%), cathepsin B, and asparaginyl endopeptidase cysteine proteases, as well as novel trypsin-like serine proteases and carboxypeptidases. These data, together with the analysis 1-DE of the secretory proteins from different developmental stages (i.e. metacercariae, newly excysted juveniles, and immature and adult stages of this parasite), complement previous studies characterising the major secretory proteins expressed by adult F. hepatica using 2-DE [131–133]. The proteomic analyses of the adult F. hepatica proteins secreted in vivo and in vitro led to the identification of different proteins. In accordance with the transcriptomic predictions, cathepsin L proteases (FhCL1, FhCL2, and FhCL5) are highly represented in adult fluke secretions. Proteomic analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and changes in their expression correlate with the migration of the parasite [134]. Besides proteases, the parasites secrete an array of proteins with antioxidant activities that are also highly regulated according to their migration through host tissues: fatty acidbinding proteins (FaBP1, FaBP2, FaBP3, and Fh15), redox enzymes (peroxiredoxin and thioredoxin and protein-disulfide isomerase) and glutathione S-transferases (GSTs). These molecules have been implicated in fluke immune avoidance mechanisms [135,136], and may also protect the parasites from harmful reactive oxygen species released by host immune cells. In addition, actin and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have all been identified in vitro but have not been detected in vivo. Enolase has been implicated in autoimmune diseases as well as in invading host tissues by pathogens [137]. Our group identified enolase as plasminogen binding protein in the excretory–secretory materials of F. hepatica adults [138]. Nevertheless, as GST, FABP, enolase, actin, and GAPDH are located at the surface or just below the surface of the fluke, they may be released during in vitro culture as the tegument is sloughed to evade the host immune system [131]. As it occurs in other helminths, like Echinostoma spp., the analysis of the identified proteins shows that few of them are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. The identified proteases of F. hepatica present a canonical secretion signal and therefore are likely to be secreted via a classical endoplasmic reticulum/Golgi pathway from specialised gastrodermal cells of the gut [139]. Nevertheless, each of the aforementioned antioxidant and glycolytic enzymes do not possess a signal sequence for secretion, likely indicating that they are secreted via nonclassical mechanisms via a trans-tegumental route and/or the existence of a vesicle-based secretion system in these parasites like exosomes as already suggested in this revision (see above).

4.2.3. Host–parasite interactions and novel targets for diagnosis and vaccines The available data sets could contribute to the identification of molecular markers for the early diagnosis of disease, and provide a foundation for the prediction of drug targets. Triclabendazole (TCBZ) remains the drug of choice for treating infections of the liver flukes, F. hepatica and F. gigantica in livestock and has become the main drug used to treat human cases of the disease as well because it is effective against immature and adult parasites. The mode(s) of action and biological target(s) of TCBZ at the molecular level have yet to be resolved, but proteomic assays via 2-DE revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ exposure [140]. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and “stress” response proteins. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinant proteins, and both found to bind TCBZ at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance. Nevertheless, the development of resistance to triclabendazole (reviewed in [141]) has shown anthelmintics to be an unsustainable means of controlling the disease and has provided further incentive for the development of molecular vaccines against these pathogens. As we have indicated, proteomic approaches present a unique opportunity for detecting protein targets for rational vaccine design. Since excreted/secreted proteins (ESP) or those predicted to be expressed on the cell surface, in general, are exposed to the immune system of the host, they represent obvious candidates for the development of anti-parasite vaccines [142]. Cathepsin L proteases have been described as promising vaccine candidates for F. hepatica. These proteases are secreted into the host tissues where they play important roles in host–parasite interactions. Vaccination of cattle and sheep with purified cathepsins L1 and L2 of F. hepatica afforded a high percentage protection against establishment of adult worms, as well as having an anti-fecundity effect by inhibiting the embryonation of eggs [143]. While these studies were performed with native proteins, it has been reported that yeast (Saccaromyces cerevisiae and/or Pichia pastoris), transformed with a full-length FheCL1 or FheCL2 cDNA, expressed and secreted the functional enzyme into the culture medium from which homogeneous enzyme could be obtained by conventional purification techniques [144,145]. These experiments demonstrate that liver fluke cathepsins could be produced at a commercial level using yeast as the heterologous expression system and therefore represent excellent candidates for the development of first generation antifluke vaccines. The advantages and disadvantages of each expression system have been discussed in detail by Dalton et al. [146]. There are several other molecules that are potential vaccine candidates including GST, FABPs and Leucine aminopeptidase (LAP) [147,148]. Interestingly, LAP was also identified as one of the major immunogen molecules in F. hepatica infections [122]. While trials with these antigens have given some very variable results, it is still feasible that these molecules could be developed as a basis for commercial vaccines, either alone or in combination with other candidates, if the precise conditions for inducing protection were elucidated.

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Praziquantel is the drug of choice for the food-borne trematodiases, except for fascioliaisis. Praziquantel exhibits a broad spectrum against trematodes and has an excellent safety profile [149,150]. Although the exact mechanism of action of praziquantel has not been elucidated, it has been postulated that a disruption of Ca2+ homestasis occurs as praziquantel induces a rapid contraction of the trematodes [151]. All treatment schedules with prazinquantel are well tolerated, with only few adverse events including abdominal pain, dizziness, headache, nausea and urticaria [149]. The doses employed for the different trematode infections are shown in Table 2. Triclabendazole, a benzimidazole derivative, is used for the treatment of the fascioliasis and holds promise for the treatment of paragonimiasis. Currently, triclabendazole is registered for human use only in Ecuador, Egypt, France and Venezuela and the doses employed are shown in Table 2. Abdominal pain, biliary colic, fever, nausea, vomiting, weakness, and liver enlargement have been reported as adverse reactions. There are some concerns that triclabendazole resistance might emerge since it has been reported in veterinary medicine [1]. Bithionol also can be used against fascioliasis when triclabendazole is not available. However long treatment schedules of 10 to 15 days are required [149]. New drugs which include tribendimidine and peroxidic derivates (e.g. artemisinins and synthetic trioxolanes) are being investigated [150].

diminished the distance in research between these tropical diseases. In this context, molecular biology tools like the “-omics” should help greatly. Proteomic-based approaches for the study of host–pathogen interactions are central to the understanding of the pathogenesis process and they have the potential to identify new diagnostic biomarkers for the early detection of diseases, as well as new vaccine targets. At present, most of the studies of foodborne trematodes include few or no proteomic approaches, and when performed, they exhibited important problems like the source of material, complicated biological cycles, as well as technical problems. However, the main bottleneck in these proteomic studies is the absence of databases, with only full genome sequences available for two schistosome species. The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offers new opportunities for the identification of extracellular proteins that are secreted or released by helminth parasites. The recent massive sequencing techniques with “more affordable” costs have led to the characterization of different transcriptomes from foodborne trematodes like F. hepatica, O. viverrini and C. sinensis. This information has clearly increased the chance of identifying proteins in mass spectrometry assays in those and other trematodes, but the data from the sequences obtained have revealed that the homology between these transcriptomes is at most 40%. Additional sequence data from these and other trematodes is needed to fulfil identification requirements, and importantly to check for species-specific targets to be used in future diagnosis, treatment and vaccination.

6.

Acknowledgments

5.

Treatment of food-borne trematodiases

Concluding remarks

Neglected tropical diseases like malaria, HIV/AIDS and tuberculosis receive most of the attention including funding, presence in the press media, and resources for research, meanwhile other neglected diseases like schistosomiasis and foodborne trematodiases, which are highly endemic, receive much less consideration. This is even worse in the case of foodborne trematodiases, where easy control measures can be implemented, but there are still millions of patients. New targets for diagnosis, treatment and vaccination are urgently needed, and in this landscape new technologies should

Dr. Lynne Yenush is acknowledged for critical reading of the manuscript. This work was supported by the projects CGL2005-0231/ BOS and SAF2010-16236 from the Ministerio de Ciencia e Innovación and FEDER (European Union), PROMETEO/2009/081 from Conselleria 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 de València (Valencia, Spain).

Table 2 – Habitat, infection sources, and treatment of choice of major food-borne trematodes and their underlying diseases. Species Clonorchis sinesis Opistorchis felineus Opistorchis viverrini Paragonimus spp.

Habitat Liver Liver Liver Lung

Source of infection

Freshwater fish Freshwater fish Freshwater fish Freshwater crabs, crayfish, wild boar meat Echinostomatidae Intestine Freshwater fish, frogs, mussels, snails, tadpoles Gymnophalloides seoi Intestine Oysters Heterophyidae Intestine Freshwater fish. Fasciola spp. Liver Freshwater vegetables, contaminated water.

Treatment (dose) Praziquantel Praziquantel Praziquantel Praziquantel

(3 × 25 mg/kg (3 × 25 mg/kg (3 × 25 mg/kg (3 × 25 mg/kg

for for for for

2 days or single dose of 40 mg/kg) 2 days or single dose of 40 mg/kg) 2 days or single dose of 40 mg/kg) 2 days)

Praziquantel (single dose of 25 mg/kg) Praziquantel (single dose of 10 mg/kg) Praziquantel (single dose of 25 mg/kg) Triclabendazole (single dose of 10 mg/kg or 20 mg/kg in two split doses within 12–24 h)

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