**4. Parasite culture system**

340 Current Topics in Tropical Medicine

al., 1997). In addition to the wide geographical distribution and their use in folk medicine, the interest in these compounds and plant extracts is based on the fact that it is easy to isolate secondary metabolites and to propagate the plant, which has a short reproductive cycle. Thus, considering the *in vitro* schistosomicidal activity of the amide piplartine, the importance of more research on the biological activity of the natural compounds isolated

Schistosome species are dioecious (having male and female reproductive organs in separate individuals) platyhelminthes and have complex life cycles comprising multiple morphologically distinct phenotypes in definitive mammalian and intermediate snail hosts. *S. mansoni* is one of the most common etiological agents of human schistosomiasis and is the most widely used schistosome model for chemotherapeutic studies.Schistosome infection of humans (or another definitive host) occurs by direct contact with freshwater containing freeswimming larval forms of the parasite, known as cercariae. Cercariae penetrate the intact human skin and transform into schistosomula, which reside in the skin for up to 72 hours before entering a blood vessel. Within the vascular system, schistosomula migrate via complex routes to their nal venous destination, where they mature into male and female adults. The mature flukes dwell in the human portal vasculature, depositing eggs in the intestinal wall that either pass to the gut lumen and are expelled in the faeces or travel to the liver and trigger immune-mediated granuloma formation and peri-portal fibrosis. Egg production commences 5 to 6 weeks after infection and continues for the life of the worm. The life cycle is completed when the eggs passed in the faeces hatch in the water, releasing the larval form miracidia, which then infect freshwater snails of the *Biomphalaria* spp. The infected snails, bearing schistosomal sporocysts, release cercariae into the water, which in

To complete a life cycle in the laboratory, *S. mansoni* is commonly maintained using rodents, ranging from hamsters to mice, as the definitive hosts and *Biomphalaria glabrata* as the intermediate host snail species (Figure 1). Infections of rodents of the same gender, 3 to 4 weeks of age and weighing 18 to 25 g, with *S. mansoni* are commonly initiated by subcutaneous injection of 100 to 150 cercariae (infective larvae). At 42, 49 or 56 days postinfection, animals are sacrificed with CO2, dissected, and miracidia are hatched from *S. mansoni* eggs taken from animal livers. Each intermediate host snail is exposed to approximately 10 miracidia. All animals should be handled in strict accordance with good animal practice adhering to the institutional guidelines for animal husbandry. Thus, all studies should have a statement from their ethics committee or institutional review board

The use of *in vivo* animal models in drug discovery and the techniques used for these studies in the laboratory have recently been described in detail elsewhere by Keiser (2010) and Ramirez et al. (2007). This chapter will focus on the techniques used for *in vitro* studies. Many methods have been described that aim to determine the antischistosomal activity of drugs *in vitro*. The assessment of the viability of different stages of schistosome, tegumental changes, oviposition, toxicity in mammalian cells and other parameters are important in the search for antischistosomal substances. The techniques detailed here will be the key to better

**3.** *Schistosoma mansoni* **life cycle and maintenance in the laboratory** 

from the family Piperaceae and other plants is apparent.

turn penetrate the skin of their denitive host (Gryseels et al., 2006).

indicating the approval of the research.

assess the methodology employed during screening tests.

*In vitro* studies with schistosomula, juvenile and adult worms of *S. mansoni* are frequently used in screening strategies for the discovery of new antischistosomal drugs (Abdulla et al., 2009; Keiser, 2010; Mølgaard et al., 2001; Peak et al., 2010; Ramirez et al., 2007; Smout et al., 2010; Yousif et al., 2007). Parasites at different stages might show differences with regard to drug sensitivity. The *in vitro* methods currently utilised have recently been reviewed, and following the establishment of the *S. mansoni* life cycle in the laboratory, *in vitro* parasite culture techniques were developed (Keiser, 2010; Ramirez et al., 2007). For *in vitro* trials, parasites of different ages are used, such as 3-h-old and 1-, 3-, 5- and 7-day-old schistosomula, 21 day-old juveniles, and 42- to 56-day-old adults. Figure 1 shows the life cycle of *S. mansoni* in the laboratory, illustrating the collection points for *in vitro* chemotherapeutic studies.

Fig. 1. Life cycle of *S. mansoni*, illustrating the collection points for *in vitro* chemotherapeutic studies. Black arrow: maturation of parasite within final host. Blue arrow: aquatic phase

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 343

Obtaining sufficient quantities of schistosomula directly from skin or lung tissue for most research purposes is time consuming and involves working with mammalian hosts. These difficulties led to the development of techniques for transforming cercariae and maintaining schistosomula. In fact, schistosomula can be obtained by transforming cercariae using simple techniques, such as centrifugation, vortexing, repeated aspiration through a syringe needle, or chemical stimulation, and these schistosomula are easily maintained *in vitro* for several days (Basch, 1981). These *in vitro* strategies are advantageous because they confer uniformity in parasite maturation, which cannot be achieved *in vivo* due to the variation in the time required for individual parasites to penetrate the host skin and enter the vasculature. In addition, the use of mechanically obtained schistosomula is an alternative method that reduces, refines and replaces the use of animals in laboratory research in accordance with animal protection principles

Among the techniques available for the production of schistosomula, the current methods most commonly used for the removal of tails from cercariae are the repeated aspiration through a syringe needle, based on Colley and Wikel (1974), and the use of a Vortex mixer, based on Ramalho-Pinto et al. (1974). In our laboratory, for example, schistosomula are mechanically transformed according to Ramalho-Pinto et al. (1974) and cultured *in vitro* in 169 medium, as described by Bash (1981). This method is recommended over the syringepassage technique, which severely stresses and damages the parasites. In addition, the mechanical transformation procedure in a Vortex mixer is simpler than the passage of the parasites under pressure many times through a needle. Briey, to obtain cercariae, *B. glabrata* snails infected with miracidia are exposed to incandescent light for 2 h, and then cercariae are collected, concentrated in glass conical centrifuge tubes and cooled in a ice bath for 10 minutes to reduce the motility of the worms. The ice-cold cercarial suspension is centrifuged, resuspended in RPMI 1640 medium with 25 mM HEPES, 200 UI/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, and vortexed for 2 minutes to trigger tail loss. The resulting cercarial bodies are isolated from free tails by centrifugation through a 60% Percoll gradient (Lazdins et el., 1982) or decantation (Ramalho-Pinto et al., 1974). Microscope examination is used to assess the quantity and quality of purified schistosomula. Finally, schistosomula are cultivated in 169 medium containing antibiotics and supplemented with 10% bovine foetal serum at 37 ºC in a 5% CO2 atmosphere. Schistosomula are cultured until day 7, which corresponds to the lung-stage worm. For *in vitro* drug screening assays, schistosomula are transferred into 96-well culture microplates, with approximately 50 parasites per well, and maintained in 200 µl 169 medium under the

After penetrating the definitive host, significant morphological, physiological, and biochemical changes occur in the developing schistosomula (Gobert et al., 2007; Skelly & Alan Wilson, 2006). Although mechanically transformed schistosomula are different from the schistosomula that have penetrated the skin, these larval cultures have been maintained in *vitro* for different amounts of time to produce all the mammalian host stages, including skin- and lung-stage schistosomula and paired, mature adult males and females (Basch, 1981). Mechanically transformed schistosomula are now commonly used in studies of behaviour, development, metabolic activity, biochemistry, molecular biology, immunology,

**4.2 Schistosomula** 

(Broadhead & Bottrill, 1997).

conditions described above.

#### **4.1 Juvenile and adult schistosomes**

Adult worms have been more commonly used for antischistosome drug discovery. Today, *in vitro* chemotherapeutic studies using juvenile worms are also highly recommended. For these assays, each rodent is commonly infected with either 100-150 or 400-500 cercariae. Rodents exposed to 400-500 cercariae are sacriced 21 days after infection for juvenile recovery, while mice exposed to 100-150 cercariae are sacriced 42 to 56 days after infection for adult recovery. Juveniles or adults are collected using a perfusion technique with Hanks' balanced salt solution (HBSS), Dulbecco's Modified Eagle's Medium (DMEM), or Roswell Memorial Park Institute (RPMI) 1640 medium, containing an anticoagulant such as heparin at a concentration range of 5-20 U/ml (Smithers & Terry, 1965). The worms are washed in RPMI 1640 medium, kept at pH 7.5 with 25 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES) containing 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. After washing, ten to fifteen juveniles or one pair of adult worms (male and female coupled) are transferred to each well of a 24-well culture plate containing 2 ml of the same medium supplemented with 10% bovine foetal serum and incubated at 37 ºC in a humid atmosphere containing 5% CO2. Only viable, contractile worms showing total tegument integrity as assessed by light microscopy should be included in the different investigations (Figure 2).

Fig. 2. *In vitro* assay models with *Schistosoma mansoni* adult worms.

#### **4.2 Schistosomula**

342 Current Topics in Tropical Medicine

Adult worms have been more commonly used for antischistosome drug discovery. Today, *in vitro* chemotherapeutic studies using juvenile worms are also highly recommended. For these assays, each rodent is commonly infected with either 100-150 or 400-500 cercariae. Rodents exposed to 400-500 cercariae are sacriced 21 days after infection for juvenile recovery, while mice exposed to 100-150 cercariae are sacriced 42 to 56 days after infection for adult recovery. Juveniles or adults are collected using a perfusion technique with Hanks' balanced salt solution (HBSS), Dulbecco's Modified Eagle's Medium (DMEM), or Roswell Memorial Park Institute (RPMI) 1640 medium, containing an anticoagulant such as heparin at a concentration range of 5-20 U/ml (Smithers & Terry, 1965). The worms are washed in RPMI 1640 medium, kept at pH 7.5 with 25 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES) containing 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. After washing, ten to fifteen juveniles or one pair of adult worms (male and female coupled) are transferred to each well of a 24-well culture plate containing 2 ml of the same medium supplemented with 10% bovine foetal serum and incubated at 37 ºC in a humid atmosphere containing 5% CO2. Only viable, contractile worms showing total tegument integrity as assessed by light microscopy should be included

**4.1 Juvenile and adult schistosomes** 

in the different investigations (Figure 2).

Fig. 2. *In vitro* assay models with *Schistosoma mansoni* adult worms.

Obtaining sufficient quantities of schistosomula directly from skin or lung tissue for most research purposes is time consuming and involves working with mammalian hosts. These difficulties led to the development of techniques for transforming cercariae and maintaining schistosomula. In fact, schistosomula can be obtained by transforming cercariae using simple techniques, such as centrifugation, vortexing, repeated aspiration through a syringe needle, or chemical stimulation, and these schistosomula are easily maintained *in vitro* for several days (Basch, 1981). These *in vitro* strategies are advantageous because they confer uniformity in parasite maturation, which cannot be achieved *in vivo* due to the variation in the time required for individual parasites to penetrate the host skin and enter the vasculature. In addition, the use of mechanically obtained schistosomula is an alternative method that reduces, refines and replaces the use of animals in laboratory research in accordance with animal protection principles (Broadhead & Bottrill, 1997).

Among the techniques available for the production of schistosomula, the current methods most commonly used for the removal of tails from cercariae are the repeated aspiration through a syringe needle, based on Colley and Wikel (1974), and the use of a Vortex mixer, based on Ramalho-Pinto et al. (1974). In our laboratory, for example, schistosomula are mechanically transformed according to Ramalho-Pinto et al. (1974) and cultured *in vitro* in 169 medium, as described by Bash (1981). This method is recommended over the syringepassage technique, which severely stresses and damages the parasites. In addition, the mechanical transformation procedure in a Vortex mixer is simpler than the passage of the parasites under pressure many times through a needle. Briey, to obtain cercariae, *B. glabrata* snails infected with miracidia are exposed to incandescent light for 2 h, and then cercariae are collected, concentrated in glass conical centrifuge tubes and cooled in a ice bath for 10 minutes to reduce the motility of the worms. The ice-cold cercarial suspension is centrifuged, resuspended in RPMI 1640 medium with 25 mM HEPES, 200 UI/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, and vortexed for 2 minutes to trigger tail loss. The resulting cercarial bodies are isolated from free tails by centrifugation through a 60% Percoll gradient (Lazdins et el., 1982) or decantation (Ramalho-Pinto et al., 1974). Microscope examination is used to assess the quantity and quality of purified schistosomula. Finally, schistosomula are cultivated in 169 medium containing antibiotics and supplemented with 10% bovine foetal serum at 37 ºC in a 5% CO2 atmosphere. Schistosomula are cultured until day 7, which corresponds to the lung-stage worm. For *in vitro* drug screening assays, schistosomula are transferred into 96-well culture microplates, with approximately 50 parasites per well, and maintained in 200 µl 169 medium under the conditions described above.

After penetrating the definitive host, significant morphological, physiological, and biochemical changes occur in the developing schistosomula (Gobert et al., 2007; Skelly & Alan Wilson, 2006). Although mechanically transformed schistosomula are different from the schistosomula that have penetrated the skin, these larval cultures have been maintained in *vitro* for different amounts of time to produce all the mammalian host stages, including skin- and lung-stage schistosomula and paired, mature adult males and females (Basch, 1981). Mechanically transformed schistosomula are now commonly used in studies of behaviour, development, metabolic activity, biochemistry, molecular biology, immunology,

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 345

Current methods utilised to assess schistosomal viability have recently been reviewed, and most of these methods involve microscopic techniques (Keiser, 2010; Ramirez et al., 2007). The phenotypic changes are scored by using a viability scale. For example, a scale of 0 – 4, where 4= normally active, 3= slowed activity, 2= minimal activity, 1= absence of motility apart from gut movements, and 0= total absence of mobility, is based on standard procedures for compound screening at the Special Programme for Research and Training in Tropical Diseases, World Health Organization, WHO-TDR (Ramirez et al., 2007). Alternatively, as described by Manneck et al. (2010, 2011), drug activity is defined as 3= totally vital, normally active, and no morphological changes; 2= slowed activity, primary morphological changes and visible granularity; 1= minimal activity, severe morphological changes and granularity; 0= all worms dead, severe morphological changes and granularity; the granularity is characterised only for schistosomula. The regular movement of both larval and adult schistosomes has proven to be a valuable trait in assessing schistosome viability *in vitro* because lack of movement is a good indicator of death. Worm death is usually dened as no movement observed for at least 2 min of examination (Manneck et al., 2010). In this context, the viability of worm during the culture period is also assessed by motor activity reduction, and it is defined as "slight " or "significant". This subjective criterion is commonly used by several research groups (Braguine et al., 2009; de Melo et al., 2011; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011, Parreira et al., 2010; Pereira et al., 2011; Xiao et al., 2007). Size measurements of parasites are also employed to study phenotypic

In addition to the phenotypic approaches, another *in vitro* drug-screening assay method is based on microcalorimetry. Manneck et al. (2011) analysed the effects of drugs on the metabolic activity of schistosomula and adult *S. mansoni* by comparing their heat ow. In this study, a multi-channel isothermal microcalorimeter equipped with 48 measuring channels was used to monitor the heat production by schistosomes as a result of their metabolism over time. The results show that microcalorimetry can be a valuable tool to study antischistosomal drugs, and the microcalorimetric measurements conrmed, in part, the results of the phenotypic evaluation. However, the level of agreement between microscopy and microcalorimetry data requires further investigation (Manneck et al., 2011). In the following section, other methods are described that are used to determine the effect of

Phenotypic changes are determined as mentioned above. However, because of the lack of standardisation between laboratories, the replication of results obtained by microscopic means is not always possible. In an effort to avoid the subjective nature of quantifying schistosome viability from the microscopic examination of phenotype alone, further adaptations have been developed and are based on the differentiating potential of some colorimetric vital dyes. Diamidinophenylindole (DAPI) has been used as a differential stain of dead schistosomula during microscopy; in addition, the low DAPI concentration (1 µg/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence (Van Der Linden & Deelder, 1984). Trypan blue has also been shown to be a reliable dye for differentially staining dead schistosomula (Harrop & Wilson, 1993) and by means of a methylene blue dye exclusion test (Gold, 1997). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is also a vital dye that has been successfully used to assess the viability of worms. The use of this assay on helminths was pioneered by Comley et al. (1989), and several nematode

changes.

drugs on schistosomula and adult *S. mansoni.* 

and in vaccine development and drug screening protocols (Abdulla et al., 2009; Gobert et al., 2007; Harrop & Wilson, 1993; Peak et al., 2010). There is evidence that mechanically transformed schistosomula are structurally similar to their lung schistosomula counterparts (Chai et al., 2006), and after 7 days in culture, the larvae have the morphological features of lung worms and are capable of maturation when introduced into the portal vein of mice (Harrop & Wilson, 1993). Furthermore, mechanically transformed schistosomula are able to develop steadily until adult worm pairing (Basch, 1981). Because of these reasons, drugscreening assays in our laboratory are based on mechanically transformed schistosomula of different ages *in vitro* (3-h-, 1-, 3-, 5- and 7-day-olds). It takes roughly 3 h for the cercariae to secrete the contents of their acetabular glands; the 1- to 7-day-olds correspond to the skinand lung-stage schistosomula.
