S. mansoni is an important model to study schistosomiasis

S. mansoni is the most widely used schistosome model, and its life cycle is kept in many institutions worldwide. Fig. 1 summarizes laboratories in Europe, USA, South America, Asia and Northern Africa, which currently maintain the life cycle of S. mansoni. Several of these laboratories such as the Schistosomiasis Resource Center (Biomedical Research Institute; Bethesda, USA) or the Theodor Bilharz Research Institute, Giza, Egypt also provide schistosome material for research or teaching purposes. Remarkably, our search did not identify any laboratories in sub-Saharan Africa (the area considered to be most severely affected by schistosome-induced morbidity (van der Werf et al. Reference van der Werf, de Vlas, Brooker, Looman, Nagelkerke, Habbema and Engels2003), which maintain the complete laboratory life-cycle of S. mansoni.

Fig. 1.

World map highlighting locations of laboratories, which maintain the life cycles of S. mansoni (circles), F. hepatica (triangles) and E. caproni (squares). Laboratories were identified through contacting experts and literature searches (using PubMed) restricted to the past 5 years.

Schistosoma mansoni: Aggeu Magalhães Institute (FIOCRUZ) (Recife, Brazil), Biomedical Research Institute (Rockville, MD, USA), Butantã Institute, São Paulo (São Paolo, Brazil), Catholic University of Rio Grande do Sul (Porto Alegre, Brazil), CDC Atlanta (Atlanta, GA, USA), Chiang Mai University (Chiang Mai, Thailand), DBL-Centre for Health Research and Development (Copenhagen, Denmark), Federal University of Rio de Janeiro (Rio de Janeiro, Brazil), GIDE – Federal University of Minas Gerais (Belo Horizonte, Brazil), Gonçalo Muniz Institute (FIOCRUZ) (Salvador, Brazil), Institut Pasteur de Lille (Lille, France), Instituto René Rachou-Fiocruz (Belo Horizonte, Brazil), London School of Hygiene and Tropical Medicine (London, UK), Natural History Museum (London, UK), National Research Council (Rome, Italy), Oswaldo Cruz Institute (FIOCRUZ) (Rio de Janeiro, Brazil), Queensland Institute of Medical Research (Brisbane, Australia), Swiss Tropical Institute (Basel, Switzerland), Theodor Bilharz Research Institute (Giza, Egypt), Trinity College (Dublin, Ireland), Tufts University (Boston, MA, USA), Tulane University (New Orleans, LA. USA), Universidad San Pablo-CEU (Madrid, Spain), University Erlangen-Nuremberg (Erlangen-Nuremberg, Germany), University New Mexico (Albuquerque, NM, USA), University of Antwerp (Antwerp, Belgium), University of California San Francisco (San Francisco, USA), University of Georgia (Athens, GA, USA), University of Heidelberg (Heidelberg, Germany), University of Melbourne (Melbourne, Australia), University of Nottingham (Nottingham, UK), University of Occupational and Environmental Health (Kitakyushu, Fukuoka, Japan), University of São Paulo (São Paolo, Brazil), University of São Paulo, Ribeirão Preto (São Paolo, Brazil), University of Texas Health Science Center (San Antonio, TX, USA), University of the Health Sciences (Bethesda, MD, USA), University of York (York, UK). Fasciola hepatica: Baldwin Aquatics (Monmouth, OR, USA), Elizabeth Macarthur Agricultural Institute (Camden, Australia), Facultés de Médecine et de Pharmacie (Limoges, France), Queens University of Belfast (Belfast, Northern Ireland), Ridgeway Research Ltd (Gloucestershire, UK), Veterinary Health Research (Armidale, NSW, Australia), Wageningen University (Wageningen, Netherlands). Echinostoma caproni: Lafayette College (Easton, PA, USA), Swiss Tropical Institute (Basel, Switzerland), Universidad de Valencia (Valencia, Spain), Université de Perpignan (Perpignan, France), University of Alberta (Alberta, Canada), University of California Santa Barbara (Santa Barbara, CA, USA).

Briefly, in the definitive host S. mansoni worms develop from a post-penetration larval stage, the schistosomulum. The female worms produce eggs which leave the body in excreta – faeces in the case of S. mansoni, and on contact of the eggs with fresh water free-swimming miracidia hatch out and infect snails. For laboratory culture miracidia are hatched from S. mansoni eggs taken from experimental animal livers and/or intestines and Biomphalaria glabrata is the intermediate host snail species most commonly used (Fig. 2). Following asexual reproduction in the snails, cercariae are shed from the snails into freshwater (Lewis, Reference Lewis2001).

Fig. 2.

Life cycle of S. mansoni highlighting collection points for in vitro and in vivo chemotherapeutic studies. Maturation of parasite occurs within final host (red arrows).

S. haematobium is much less studied, as it is difficult to grow in rodents (Doenhoff et al. Reference Doenhoff, Hagan, Cioli, Southgate, Pica-Mattoccia, Botros, Coles, Tchuem Tchuente, Mbaye and Engels2009). To my knowledge, the life cycle of S. haematobium is currently maintained only in two laboratories, namely the Theodor Bilharz Research Institute (Giza, Egypt) and the Schistosomiasis Resource Center. ‘Sibling’ species of S. haematobium, such as S. bovis, S. matthei or S. margrebowiei, the life cycles of which can be maintained in laboratory rodents, might serve as models for S. haematobium (Agnew et al. Reference Agnew, Murare, Lucas and Doenhoff1989).

S. japonicum, the third major human schistosome species, is mainly studied in China. Nonetheless several institutions outside China maintain the life cycle and infected snails and mammals are also available on request from the Schistosomiasis Resource Center. Though less frequently used, schistosome species infecting other species of definitive hosts might also prove useful for experimental studies: for example, the bird schistosome Trichobilharzia ocellata reaches oviposition after 9 days already and is not pathogenic. Its life cycle has been described as troublesome (Smyth and Halton, Reference Smyth and Halton1983).

S. mansoni in vitro assay based on schistosomula

Following the establishment of the S. mansoni life cycle in the laboratory, in vitro parasite culture techniques were developed (Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007). Early work with schistosomula was based on the recovery of young worms from the lungs of infected rodents, hence only limited worm material was available (Smyth and Halton, Reference Smyth and Halton1983). The discovery that schistosomula can be obtained by transforming cercariae using simple techniques as centrifugation, repeated aspiration through a syringe needle or chemical stimulation was a breakthrough in the field of schistosome cultivation. Today cercariae are often used as starting material for schistosome in vitro studies as large numbers of schistosomula can be obtained in a cost-effective manner (Table 1, Fig. 2) (Smyth and Halton, Reference Smyth and Halton1983; Abdulla et al. Reference Abdulla, Ruelas, Wolff, Snedecor, Lim, Xu, Renslo, Williams, McKerrow and Caffrey2009; Caffrey et al. Reference Caffrey, Williams, Todd, Nelson, Keiser, Utzinger and Selzer2009). In addition, most importantly, the use of mechanically obtained schistosomula reduces and replaces the use of live animals in accordance with the 3Rs (reduce, replace, refine) of animal protection principles (Broadhead and Bottrill, Reference Broadhead and Bottrill1997). An assay with artificially produced schistosomula might serve as a pre-screen in drug sensitivity assays, however one has to keep in mind that mechanically transformed schistosomula possess distinct differences to schistosomula, which have penetrated the skin (e.g. biochemical differences) or juvenile and adult schistosomes (Brink et al. Reference Brink, McLaren and Smithers1977).

Table 1.

In vitro drug screening: techniques employed, culture conditions and evaluation of juvenile and adult S. mansoni, F. hepatica and E. caproni assays

Several techniques for transforming cercariae and maintaining schistosomula are available (Colley and Wikel, Reference Colley and Wikel1974; Howells et al. Reference Howells, Ramalho-Pinto, Gazzinelli, de Oliveira, Figueiredo and Pellegrino1974; Ramalho-Pinto et al. Reference Ramalho-Pinto, Gazzinelli, Howells, Mota-Santos, Figueiredo and Pellegrino1974; Brink et al. Reference Brink, McLaren and Smithers1977; Smyth and Halton, Reference Smyth and Halton1983; Abdulla et al. Reference Abdulla, Ruelas, Wolff, Snedecor, Lim, Xu, Renslo, Williams, McKerrow and Caffrey2009; Caffrey et al. Reference Caffrey, Williams, Todd, Nelson, Keiser, Utzinger and Selzer2009). In our laboratories, for example, a Vortex transformation method based on Ramalho-Pinto (Ramalho-Pinto et al. Reference Ramalho-Pinto, Gazzinelli, Howells, Mota-Santos, Figueiredo and Pellegrino1974) is used. Briefly, 50 ml of an ice-cold cercarial suspension is centrifuged and the packed cercariae are resuspended and vortexed for 2 min to trigger tail loss. For the isolation of cercarial bodies from tails, an ice-cold Hank's basal salt solution (HBSS) is added to the cercarial suspension and the tail-rich supernatant is decanted (Manneck et al. Reference Manneck, Haggenmüller and Keiser2009). Schistosomula are incubated in Basch medium (Basch, Reference Basch1981) supplemented with serum and antibiotics or other culture media (Smyth and Halton, Reference Smyth and Halton1983). Our own studies have shown that schistosomula survive for at least 96 h in different media (Basch, MEM, DMEM or TC 199) regardless of serum supplementation (author's unpublished observations). Abdulla and colleagues have recently reported that schistosomula survive in Basch medium for several weeks (Abdulla et al. Reference Abdulla, Ruelas, Wolff, Snedecor, Lim, Xu, Renslo, Williams, McKerrow and Caffrey2009). In Fig. 3a schistosomula incubated for 24 h in Basch medium are shown. Fig. 3b depicts a scanning electron microscopic (SEM) image of a newly transformed schistosomulum obtained by Vortex transformation. Following incubation of schistosomula with investigational drugs, changes in worm motor activity, morphological/tegumental alterations and occurrence of death are recorded at different time points. These phenotypic changes might be scored using a viability scale from 0 to 3: (3=totally vital, normally active, no morphological changes, 2=slowed activity, first morphological changes and granularity visible, 1=minimal activity, severe morphological changes and granularity, 0=all worms dead, severe granularity) (Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007). For example, phenotypic changes following praziquantel incubation (100 μg/ml) for 72 h can be observed by means of light microscopy (Fig. 3c) and SEM (Fig. 3d). Blebbing, furrows, swelling, loss of structure and the lack of spines were visible following incubation with praziquantel. Since the assessment of morphological and behavioural responses of schistosomula to treatment is quite labour intensive and time consuming, the use of a drug response predictor (e.g. colourimetric assays, such as MTT formazan (3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium formazan), which measures the metabolic activity as a marker of viability in mammalian cell culture systems) should be explored. The latter assay technology might offer distinct advantages such as standardization, reproducibility and accuracy and can be performed more rapidly and easily, when compared to phenotypic evaluated drug screens.

Fig. 3.

Schistosoma mansoni – (a): light microscopic (LM) image of newly transformed control schistosomula. (b) SEM image of newly transformed control schistosomula. (c) LM image of newly transformed schistosomula incubated for 72 h in the presence of 100 μg/ml praziquantel (d) SEM image of newly transformed schistosomula incubated for 72 h in the presence of 100 μg/ml praziquantel.

Adult S. mansoni in vitro assay

In parallel to the development of schistosomula culture systems, culture methods for adult worms have been established (Clegg, Reference Clegg1965; Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007). Today, in vitro chemotherapeutic studies mainly rely on S. mansoni adults (Table 1, Fig. 2). For example, standard operating procedures (SOPs) for compound screening of the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) are based on adult worms. Since the adult worm supply is somewhat restricted, the in vitro throughput capacity of this assay is limited (∼2,000 compounds are screened each year at TDR screening centers) (Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007). For this assay, mice are infected with a high number (up to 600) of S. mansoni cercariae. Adult S. mansoni are obtained by portal perfusion of mice after 40 days post-infection (Yolles et al. Reference Yolles, Yolles, Moore, De Giusti, Ripsom and Meleney1947; Smithers and Terry, Reference Smithers and Terry1965). 3–4 week old worms might also be used for in vitro studies (Hobbs et al. Reference Hobbs, Fryer, Duimstra, Hedstrom, Brodie, Collodi, Menino, Bayne and Barnes1993) including drug sensitivity assays, as juvenile worms might show differences with regard to drug sensitivity when compared to larvae and adult worms. Following several washing steps, worms are incubated for example in DMEM, RPMI 1640 or HBSS supplemented with foetal calf serum, penicillin and streptomycin (Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007; Xiao et al. Reference Xiao, Keiser, Chollet, Utzinger, Dong, Vennerstrom and Tanner2007). Schistosomes survive in a range of supplemented tissue culture media for periods ranging from 3–120 days (Smyth and Halton, Reference Smyth and Halton1983). Test drugs at various concentrations (5 μg/ml is the standard concentration in the primary screen at WHO-TDR screening centers (Ramirez et al. Reference Ramirez, Bickle, Yousif, Fakorede, Mouries and Nwaka2007)) are added to each culture well containing 6–10 worms. Worms are examined under a dissecting microscope at different time points (e.g. 24, 48 and 72 h). Control schistosomes are incubated in the presence of the highest concentration of solvent used. Drug effects are commonly evidenced by phenotypic changes (motility and morphological changes and death) using an inverted microscope as described above. Markers such as MTT formazan have also been used to evaluate the viability of adult schistosomes (Oliveira et al. Reference Oliveira, d'Avila, Tempone, Soares, Rumjanek, Ferreira-Pereira, Ferreira and Oliveira2004). For more thorough studies on the effects of antischistosomal drugs, detailed surface features of treated and untreated specimens might be investigated by means of SEM as the tegument of trematodes is a highly susceptible site of damage following drug treatment (Manneck et al. Reference Manneck, Haggenmüller and Keiser2009). For example, in Fig. 4b the male tegument is depicted. Fig. 4c shows the tegument of a male S. mansoni following drug treatment: 72 h post incubation with 100 μg/ml praziquantel blebbing was observed.

Fig. 4.

Schistosoma mansoni – SEM observations. (a) dorsal male tegument and (b) blebbing (b) observed on the dorsal male tegument following incubation with 100 μg/ml praziquantel for 72 h.

S. mansoni in vivo model

The mouse is the most commonly used mammalian host for experimental studies with S. mansoni (Table 2, Fig. 2). Though a difference has been observed regarding the percentage of cercariae that mature in different mouse strains, most mice strains tested were susceptible to an infection with S. mansoni (Lewis, Reference Lewis2001). 129/Ola and WEHI 129J mice are two examples of non-permissive hosts (Elsaghier et al. Reference Elsaghier, Knopf, Mitchell and McLaren1989). Hamsters (Xiao, et al. Reference Xiao, Keiser, Chollet, Utzinger, Dong, Vennerstrom and Tanner2007), gerbils (Chisty et al. Reference Chisty, Nargis, Sato, Inaba, Takahashi and Kamiya2002), rabbits (Kasilima et al. Reference Kasilima, Wango, Kigondu, Mutayoba and Nyindo2004) or primates (Webbe et al. Reference Webbe, James, Nelson and Sturrock1981) have also been used to study S. mansoni in vivo. The rat is only a semi-permissive host (Cioli et al. Reference Cioli, Knopf and Senft1977) and therefore unlikely to be suitable for the present purpose.

Table 2.

In vivo drug screening: rodent host, infection and evaluation procedures of juvenile and adult S. mansoni, F. hepatica and E. caproni assays

NA Not applicable

Animals are infected either by skin penetration, usually through the tail or shaved abdominal skin or via subcutaneous or intraperitonal injection of cercariae (Lewis, Reference Lewis2001). For chemotherapeutic studies it is important to compare the number of schistosomes in treated and untreated control animals, which is usually done after schistosomes have matured (6 to 8 weeks after infection).

Schistosomes are obtained and counted either via perfusion of the portal venous system (Yolles et al. Reference Yolles, Yolles, Moore, De Giusti, Ripsom and Meleney1947; Lewis, Reference Lewis2001) or examining the mesenteric veins and the liver for the presence of schistosomes (Xiao et al. Reference Xiao, Keiser, Chollet, Utzinger, Dong, Vennerstrom and Tanner2007). In the latter case, the liver and the small and large intestines are removed from the animal. The liver is placed into a 20×20 cm transparent plastic folder, compressed between 2 glass plates and the parenchyma is examined under a stereoscopic microscope. In addition, the mesenteric veins are systematically examined using a stereoscopic microscope. All schisosomes present in the liver and mesenteric veins are sexed and counted (Xiao et al. Reference Xiao, Keiser, Chollet, Utzinger, Dong, Vennerstrom and Tanner2007).

To revisit our example of praziquantel, the current drug of choice, the in vivo antischistosomal properties of this drug have been well studied in the S. mansoni-mouse model. For example, a single 300 mg/kg oral dose of praziquantel resulted in a worm burden reduction of 81% (Xiao et al. Reference Xiao, Chollet, Booth, Weiss and Tanner1999).

F. hepatica in vitro assays

Newly encysted juveniles, young and adult worms can be cultured in vitro (Fig. 5, Table 1). An assay based on newly excysted juveniles offers the advantage that, as for the schistosomula assay described above, a mammalian host is not required. Metacarcariae excyst when exposed to appropriate stimuli (Dixon, Reference Dixon1966). Commonly used SOPs include exposure of metacercariae to an activation medium followed by incubation in an emergence medium. For example, the following protocol has been described by McGonigle and colleagues: metacercariae are excysted by incubation in 0·5% sodium hypochlorite for 20 min prior to centrifugation (2000 g for 2 min), washing in distilled water and incubation in 0·5% sodium bicarbonate, 0·4% sodium chloride, 0·2% sodium tauroglycholate, 0·07% concentrated HCl and 0·006% l-cysteine for up to 3 h at 37°C (McGonigle et al. Reference McGonigle, Mousley, Marks, Brennan, Dalton, Spithill, Day and Maule2008). Excysted juveniles are maintained in nutritive culture medium, such as RPMI 1640, supplemented with penicillin and streptomycin and possibly serum or red blood cells (Ibarra and Jenkins, Reference Ibarra and Jenkins1984). In vitro assays based on newly excysted flukes have been used to study the fasciolicidal activities of investigational compounds; for example, 20 plant extracts were recently evaluated using this parasite stage (Vera-Montenegro et al. Reference Vera-Montenegro, Ibarra-Velarde, Ramirez-Avila and Munguia-Xochihua2008). Nonetheless, newly excysted flukes might have different drug sensitivities when compared to juvenile and adult flukes: a study which compared the fasciolicidal activity of known fasciolicides demonstrated that newly excysted flukes were unaffected by most of these compounds (Ibarra and Jenkins, Reference Ibarra and Jenkins1984).

Fig. 5.

Life cycle of F. hepatica highlighting collection points for in vitro and in vivo chemotherapeutic studies. Maturation of parasite occurs within final host (red arrows).

Assays based on juvenile or adult Fasciola are more often used to study fasciolicidal properties of investigational drugs in vitro. The main disadvantages of these assays are that only few flukes can be recovered from the laboratory rat, which is the main rodent host. Rats tolerate an infective dose of 20–30 metacercariae, yielding between 2 and 13 mature flukes (Chapman and Mitchell, Reference Chapman and Mitchell1982). In addition, as mentioned above, Fasciola spp. grow for 8–12 weeks until they are fully mature. Hence, these assays are expensive and time consuming. Another possibility to obtain parasite material in a more efficient and less costly manner in endemic countries is to collect worms from animals slaughtered in abattoirs (Shalaby et al. Reference Shalaby, El Namaky and Kamel2009). Juvenile or adult Fasciola are incubated in the presence of RPMI 1640 or NTCT 135, supplemented with antibiotics and the investigational drug (Anderson and Fairweather, Reference Anderson and Fairweather1995; Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006). Though supplementation of the medium with serum is recommended for long term culture of Fasciola spp. (longer than 4 days), serum substitution may not be advised for Fasciola drug activity studies, as many drugs bind strongly to serum proteins (Jenkins et al. Reference Jenkins, Topley and Rapson1987). Fasciolicidal drugs such as rafoxanide or bithionol showed a decreased activity in vitro in the presence of serum compared to serumless medium (Jenkins et al. Reference Jenkins, Topley and Rapson1987). The presence of bile in the incubation medium has also been shown to influence the uptake pattern of fasciolicidal drugs, such as triclabendazole or albendazole (Alvarez et al. Reference Alvarez, Mottier and Lanusse2004). As described above for the schistosomes, fasciolicidal properties are examined documenting motility, morphological changes and death. To my knowledge metabolic markers have not been used in Fasciola drug sensitivity assays. Drug effects have also been well documented using SEM and transmission electron microscopy (TEM) (Anderson and Fairweather, Reference Anderson and Fairweather1995; McConville et al. Reference McConville, Brennan, McCoy, Castillo, Hernandez-Campos, Ibarra and Fairweather2006; Keiser and Morson, Reference Keiser and Morson2008). For example, Fig. 6a shows a SEM image of the anterior region of a F. hepatica. We have recently described that the semi-synthetic artemisinin derivatives artemether and artesunate, which are essential components of malaria therapy (White, Reference White2008) and synthetic derivatives possess a broad range of trematodicidal activities (Keiser and Utzinger, Reference Keiser and Utzinger2007; Utzinger et al. Reference Utzinger, Xiao, Tanner and Keiser2007). Fig. 6b depicts the head region of a Fasciola fluke 48 h post-treatment with a single 200 mg/kg oral dose of artemether, revealing blebbing, sloughing and roughening of the tegumental surface (Keiser and Morson, Reference Keiser and Morson2008).

Fig. 6.

Fasciola hepatica – SEM observations. (a) anterior region of an adult F. hepatica recovered from a rat (OS: oral sucker, VS: ventral sucker). (b) Blebbing (b) and sloughing (s) visible near the ventral sucker (VS) on a F. hepatica recovered from a rat 48 h post-treatment with a single oral dose of 200 mg/kg artemether.

F. hepatica in vivo assays

F. hepatica can be grown in different laboratory animals but, as mentioned above, the rat is the most commonly used rodent to study F. hepatica in vivo (Fig. 5, Table 2). No significant difference was observed between different rats strains infected with F. hepatica with regard to worm burden (Chapman and Mitchell, Reference Chapman and Mitchell1982). Rats, commonly infected with 20–30 metacercariae, harbour between 2–13 adult flukes (Chapman and Mitchell, Reference Chapman and Mitchell1982). The laboratory mouse is not a suitable model to grow adult F. hepatica, as mortality of mice is high and the flukes do not fully develop in this model (Chapman and Mitchell,Reference Chapman and Mitchell1982; Smyth and Halton, Reference Smyth and Halton1983). The infectivity of F. gigantica was found to be low in Wistar rats (Itagaki et al. Reference Itagaki, Sakamoto, Tsutsumi and Itagaki1994). Rabbits, the rat-like hamster, Tscherskia triton, and sheep have also been used for studies of experimental fascioliasis (Terasaki et al. Reference Terasaki, Noda, Shibahara, Itagaki, Fukuda and Tsuchiya2003; McConville et al. Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernandez-Campos and Fairweather2009).

In chemotherapeutic studies, groups of rats or sheep, which had been experimentally infected with F. hepatica for 3–5 weeks (juvenile infection) and 8–12 weeks (adult infection) or sheep harbouring a natural infection are treated with the investigational drugs and the numbers of flukes present in the livers and bile ducts are counted several weeks post treatment. Groups of untreated animals serve as controls (Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006, Reference Keiser, Rinaldi, Veneziano, Mezzino, Tanner, Utzinger and Cringoli2008; McConville et al. Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernandez-Campos and Fairweather2009). Besides the effect of the drug on the Fasciola worm burden, the egg count reduction and the effect of the drug on the morphology, tegument or intestinal structures are studied (McKinstry et al. Reference McKinstry, Fairweather, Brennan and Forbes2003; Keiser and Morson, Reference Keiser and Morson2008; McConville et al. Reference McConville, Brennan, Flanagan, Edgar, Hanna, McCoy, Gordon, Castillo, Hernandez-Campos and Fairweather2009).

Revisiting the artemisinins, the fasciolicidal properties of artemether were thoroughly studied in F. hepatica-infected rats (both a triclabendazole-resistant and -sensitive isolate) and sheep harbouring a natural F. hepatica infection: single oral artemether at 200 mg/kg achieved a complete cure in rats (Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006, Reference Keiser, Utzinger, Vennerstrom, Dong, Brennan and Fairweather2007), while a worm burden reduction of 91% was recorded in sheep following a single intramuscular administration of 160 mg/kg artemether (Keiser et al. Reference Keiser, Rinaldi, Veneziano, Mezzino, Tanner, Utzinger and Cringoli2008).

E. caproni in vitro assay

Encysted metacercariae of E. caproni can be excysted in a trypsin-bile salts-cysteine medium (Table 1) (Saxton et al. Reference Saxton, Fried and Peoples2008; Fried and Peoples, Reference Fried, Peoples, Fried and Toledo2009). Excysted larvae have been mainly used in in vitro culture studies and might also prove useful for chemotherapeutic, biological or physiological studies as reviewed elsewhere (Fried, Reference Fried1994). Excysted larvae are cultured in a defined medium such as NCTC 135 supplemented with antibiotics and different types of natural products such as serum, egg yolk or mucus (Fried, Reference Fried, Fried and Graczyk2000).

Several studies have examined the echinostomicidal properties of investigational drugs using adult echinostomes in vitro (Leger et al. Reference Leger, Notteghem and Cavier1973; Leger and Notteghem, Reference Leger and Notteghem1975; Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006). Though adult echinostomes maintained in vitro deteriorate quickly, possibly due to microbial contamination and/or worm autolysis (Fried, Reference Fried, Fried and Graczyk2000), echinostomes can be maintained in nutritive media such as RPMI 1640 ideally supplemented with additional glucose (e.g. 1%) for several days (Leger et al. Reference Leger, Notteghem and Cavier1973). As described above worms incubated in the presence of drugs are analyzed for occurrence of death, motility disturbances or morphological changes.

E. caproni in vivo assay

While there is a high degree of compatibility between E. caproni and mice and hamsters, rats show a low degree of compatibility with E. caproni (Toledo and Fried, Reference Toledo and Fried2005). E. caproni can survive for more than 20 weeks in mice and hamsters (Toledo and Fried, Reference Toledo and Fried2005). The laboratory mouse is most commonly used to study E. caproni in vivo (Table 2). As adult infections are established already by 10–14 d after infection, this model allows for quicker patent infections in the definitive hosts than the F. hepatica or S. mansoni assays described above. Hence, results are obtained rapidly and cost-effectively in the E. caproni-mouse model. In vivo studies in Echinostoma spp. infected mice, with various test compounds such as the benzimidazoles, praziquantel, the artemisinins or tribendimidine against echinostomes in mice have been summarized recently (Saric et al. Reference Saric, Li, Wang, Holmes, Utzinger, Keiser, Fried and Toledo2009). For example, E. caproni-infected mice were cured following a single oral dose of 700 mg/kg artesunate (Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006). In Fig. 8a the head region of an untreated E. caproni is depicted. Four h after treatment with a single 700 mg/kg oral dose of artesunate eruption and sloughing was observed on the ventral surface of an E. caproni fluke (Fig. 8b) (Keiser et al. Reference Keiser, Brun, Fried and Utzinger2006).

Fig 8.

Echinostoma caproni – SEM observations. (a) anterior region of an adult E. caproni recovered from a mouse. (b) eruption (e) and sloughing (s) visible on the ventral surface of an E. caproni 4 h post-treatment with a single oral dose of 700 mg/kg artesunate.