**Tegument of** *Schistosoma mansoni* **as a Therapeutic Target**

Claudineide Nascimento Fernandes de Oliveira, Rosimeire Nunes de Oliveira, Tarsila Ferraz Frezza, Vera Lúcia Garcia Rehder and Silmara Marques Allegretti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53653

**1. Introduction**

Schistosomiasis is a parasitic disease with great social impact, being regarded as a relevant public health issue in 76 countries in Africa, Asia, and South and Central Americas [1, 2]. It is one of the main water-borne parasitic diseases in the world and it continues to be a major cause of morbidity and mortality, disabling and killing thousands of people every year. Considering that, both public health bodies and pharmaceutical companies need to more diligent regarding that issue [3, 4].

The parasite that causes schistosomiasis mansoni is the *Schistosoma mansoni,* an intravascular digenetic trematode from the family Schistosomatidae*.* In Brazil, where only that particular schistosome can be found, there are 25 million people living in endemic areas, from which 4 to 6 million are infected, which makes the country the most affected by intestinal schistoso‐ miasis in all Americas. Popularly known as barriga-d'água (water belly) in Brazil, the dis‐ ease is transmitted by planorbides from genus *Biomphalaria* [5, 6]*.*

The transmission occurs when an infected definitive host eliminates viable eggs of the para‐ site through stool, getting in contact with bodies of fresh water and contaminating them. Therefore, the disease is directly related to fast urban growth and lack of resources such as safe water supply and adequate sewage system in peri-urban areas [7]. The pathology of the disease is characterized by having two phases, acute and chronic, which are dependent on the life stage of the parasite, as shown in Figure 1.

© 2013 de Oliveira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tion of cases are associated with human activities that interfere with both landscape structure and human behavior, so there is a relationship between land occupation and health decline. In reference [9], the authors have recently confirmed this hypothesis by re‐ porting the rise of the disease in the state of Bahia (Brazil), particularly in the town of Lauro de Freitas, which full ongoing economic growth is attracting intense human migration, which results in disordered urban occupation and environmental disturbances, increasing the risk of expanding the endemic area. Because of that, schistosomiasis mansoni is consid‐ ered one of the most critical health issues in Brazil, occurring in 19 states (out of 26 states

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**2. Therapeutics of schistosomiasis mansoni: Searching for new**

control, safe water supplies, and environmental education.

Several intervention measures can be taken to reduce the morbidity of the disease, as well as, to prevent or interrupt the transmission of the parasite from the mollusk intermediate host to humans. Such measures include chemotherapy, environmental sanitation, mollusk

Chemotherapy provides a double benefit: it reduces both the morbidity caused by the presence of adult worms in the human host and the number of eggs eliminated to the

There are only two drugs available for the treatment of schistosomiasis mansoni, i.e., oxam‐ niquine and praziquantel. However, since the former has side effects on the human organ‐ ism (mutagenic and carcinogenic effects, as well as effects on the central nervous system), its production and commercialization are controlled and reduced. Therefore, praziquantel has been practically the only drug available for that treatment since the 1970s [12-14]. To exacer‐ bate the situation, cases of tolerance and resistance of *S. mansoni* to the treatment with both drugs have been recently reported, which raises the need to develop new drugs and forms

In this context, research with medicinal plants becomes a viable alternative, especially in countries with large biodiversity and rich cultural and ethnic diversity, like Brazil, because of the resulting accumulation of local traditional knowledge, which is passed from genera‐ tion to generation and includes the use and management of medicinal plants as home rem‐ edies [18]. Furthermore, there are about 100,000 catalogued plant species in Brazil, and their active ingredients are mostly unknown, as only 8% of those species were studied regarding their chemical composition and therapeutic properties [19]. In recent years, the scientific community has been conducting *in vitro* and *in vivo* tests to examine a variety of essential oils, extracts and isolated compounds from different species with respect to their schistoso‐

In the last few years, our research group carried out *in vitro* and *in vivo* tests using three species of Brazilian medicinal plants – *Baccharis trimera* (Less) DC.*, Cordia verbenacea* DC

and one Capitol) [9, 10].

**alternatives**

environment [11].

micidal potential.

of controlling the disease [15-17].

**Figure 1.** Cycle and pathology of schistosomiasis mansoni.

According to [8], environmental degradation is a determining factor to the dissemination of the disease, even more than poverty and underdevelopment. The authors also consider that some factors contribute to both development and maintenance of schistosomiasis mansoni breeding sites, such as subsistence cultivation; perennial cultivation; flooded areas; mean‐ ders and natural channels; springs and taps; environments likely to be polluted by human waste; fish-breeding ponds; ponds or watercourses used for sport fishing, washing of uten‐ sils, and bathing; sand deposits on river banks with no vegetation; large debris and garbage deposits and activities along watercourse banks. As a result, the authors believe that the dis‐ tribution of the disease is not as random as it seems, and that the localities with concentra‐ tion of cases are associated with human activities that interfere with both landscape structure and human behavior, so there is a relationship between land occupation and health decline. In reference [9], the authors have recently confirmed this hypothesis by re‐ porting the rise of the disease in the state of Bahia (Brazil), particularly in the town of Lauro de Freitas, which full ongoing economic growth is attracting intense human migration, which results in disordered urban occupation and environmental disturbances, increasing the risk of expanding the endemic area. Because of that, schistosomiasis mansoni is consid‐ ered one of the most critical health issues in Brazil, occurring in 19 states (out of 26 states and one Capitol) [9, 10].

## **2. Therapeutics of schistosomiasis mansoni: Searching for new alternatives**

Several intervention measures can be taken to reduce the morbidity of the disease, as well as, to prevent or interrupt the transmission of the parasite from the mollusk intermediate host to humans. Such measures include chemotherapy, environmental sanitation, mollusk control, safe water supplies, and environmental education.

Chemotherapy provides a double benefit: it reduces both the morbidity caused by the presence of adult worms in the human host and the number of eggs eliminated to the environment [11].

There are only two drugs available for the treatment of schistosomiasis mansoni, i.e., oxam‐ niquine and praziquantel. However, since the former has side effects on the human organ‐ ism (mutagenic and carcinogenic effects, as well as effects on the central nervous system), its production and commercialization are controlled and reduced. Therefore, praziquantel has been practically the only drug available for that treatment since the 1970s [12-14]. To exacer‐ bate the situation, cases of tolerance and resistance of *S. mansoni* to the treatment with both drugs have been recently reported, which raises the need to develop new drugs and forms of controlling the disease [15-17].

In this context, research with medicinal plants becomes a viable alternative, especially in countries with large biodiversity and rich cultural and ethnic diversity, like Brazil, because of the resulting accumulation of local traditional knowledge, which is passed from genera‐ tion to generation and includes the use and management of medicinal plants as home rem‐ edies [18]. Furthermore, there are about 100,000 catalogued plant species in Brazil, and their active ingredients are mostly unknown, as only 8% of those species were studied regarding their chemical composition and therapeutic properties [19]. In recent years, the scientific community has been conducting *in vitro* and *in vivo* tests to examine a variety of essential oils, extracts and isolated compounds from different species with respect to their schistoso‐ micidal potential.

**Figure 1.** Cycle and pathology of schistosomiasis mansoni.

152 Parasitic Diseases - Schistosomiasis

According to [8], environmental degradation is a determining factor to the dissemination of the disease, even more than poverty and underdevelopment. The authors also consider that some factors contribute to both development and maintenance of schistosomiasis mansoni breeding sites, such as subsistence cultivation; perennial cultivation; flooded areas; mean‐ ders and natural channels; springs and taps; environments likely to be polluted by human waste; fish-breeding ponds; ponds or watercourses used for sport fishing, washing of uten‐ sils, and bathing; sand deposits on river banks with no vegetation; large debris and garbage deposits and activities along watercourse banks. As a result, the authors believe that the dis‐ tribution of the disease is not as random as it seems, and that the localities with concentra‐

In the last few years, our research group carried out *in vitro* and *in vivo* tests using three species of Brazilian medicinal plants – *Baccharis trimera* (Less) DC.*, Cordia verbenacea* DC and *Phyllanthus amarus* – on adult *S. mansoni* worms. The three species are widely used in Brazilian folk medicine in the forms of infusion and tea on account of their anti-in‐ flammatory properties.

opening placed posteriorly to the ventral sucker. The author also reported the presence of tubercles on the dorsal surface of male worms starting from the posterior portion of the ven‐ tral sucker, both sides being covered by thorns. The number of such tubercles starts to de‐

Tegument of *Schistosoma mansoni* as a Therapeutic Target

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155

Furthermore, Hockley [31] observed that in the areas between the tubercles the parasite's surface is rugged, with several grooves, as well as a few isolated thorns. On the male worm lateral edge, which bends to form the gynaecophoric canal, the author noticed the presence of large thorns whose function is to capture the female in the canal. Finally, he noticed that females also presents more thorns at the final portion of their surface, but in less quantity

Using TEM, Hockley and McLaren [33] concluded that the surface of *S. mansoni* consists of two opposite lipid bilayers very close to each other and having the form of a cell membrane. Since the tegument does not have lateral membranes, its cytoplasm extends as a continuous unity, or syncytium, around the body of the worm. According to the authors, that syncytial complex is the main route of nutrient absorption (glucose, amino acids, among others), me‐ tabolite excretion (lactic acid and others) and protection against attacks by the host's im‐

mune system, whereupon it is a crucial target of drugs with schistosomicidal activity.

the drugs used in the treatment for the disease, i.e., oxamniquine and praziquantel.

was noticed that male worms showed more damage in the tegument than females.

drug is the rupture of the surface of the worm, leading to its death [40].

search to solve that puzzle.

Actually, the action mechanism of praziquantel on *S. mansoni* has yet to be fully understood. On the other hand, there is no doubt that studies using SEM and TEM were important in the

The morphological changes that praziquantel causes to the tegument and in the sarcoplas‐ mic membranes of the parasite are thought to be followed by an increase of antigen expo‐ sure on its surface. The antigens are identified and connected with the host's immune response required to complement the activity of the drug [38, 39]. Therefore, praziquantel is believed to interact with the host's immune system to kill the parasite. The last effect of the

From the 1980s on, after the establishment of the therapeutics of schistosomiasis mansoni, TEM and especially SEM were also used in an attempt to clarify the action mechanisms of

Becker et al*.* [34], by means of SEM and TEM, realized that worms subjected to oxamniquine showed tegumental vacuolization. Using SEM, Kohn et al*.* [35] noticed that the drug was produc‐ ing changes in the structure of the worm on tegumental, muscular and parenchymal levels, caus‐ ing bubble-like lesions. Magalhães-Filho et al*.* [36] also noticed vacuolization, destruction of tubercles in male worms, and surface erosion. Recently, praziquantel has been subjected to fur‐ ther studies because it still is the most used drug in the treatment of all types of schistosomiasis. Using SEM and TEM, Shaw and Erasmus [37] observed extensive damage to the structure of *S. mansoni* specimens subjected to praziquantel, including vacuolization of the tegument and subtegument of females, and destruction of the tegument and musculature. In males, in ad‐ dition to vacuolization and destruction of the parenchymal tissue, mainly in the dorsal re‐ gion, loss of cytoplasm, and structural damages to the musculature could be observed. It

crease from the posterior lateral edges of the dorsal surface.

than males. Senft and Gibler [32] termed such thorns sensory papillae.

Amongst the several criteria analyzed by our research group to evaluate the therapeutic effi‐ ciency of the tested plants, the morphological changes in the tegument of the worms (both males and females) were considered essential, due to the fact that literature reports the dam‐ age caused to that structure as cardinal in causing the parasite's death, and studies for the development of new schistosomicidal drugs have been setting it as a target [20].

## **3. The importance of the tegument of** *Schistosoma mansoni*

The tegument of *S. mansoni* plays a key role in its protection against the action of the host's immune system, as it is renovated every six hours [20]. In addition to that, it is capable of absorbing nutrients and molecules and synthesizing some proteins [21-24]. That structure is also extremely important for the success of the infection and the survival of the worm in the host [25-27]. For all those reasons, it has been vastly studied since the end of the 1960s.

## **3.1. The analysis evolution of the tegument of** *S. mansoni***: From optical to electron microscopy**

Before microscopy techniques were available for the evaluation of the worm ultrastructure, little was known about the importance of the tegument of *S. mansoni*. The first detailed stud‐ ies on that structure were carried out in the 1940s, when Gönnert [28], using light microsco‐ py, described the differences between *S. mansoni* males and females, including the fact that males have more and larger thorns.

With the emergence of transmission electron microscopy (TEM) and scanning electron mi‐ croscopy (SEM) there was a revolution regarding the 'appearance' of the worm and the de‐ scription of its sexual dimorphism. By using electron beams that either pass through (in the case of TEM) or scan the specimen under analysis (in the case of SEM), electron microscopy, which was developed in the 1930s, had a significantly higher resolving power than optical microscopy, allowing for a detailed observation of samples.

According to [29, 30], *S. mansoni* was the first digenetic trematode to be examined under electron microscopy. For that reason, the ultrastructure of that helminth has been more often studied than the ultrastructure of any other digenetic trematode. By the end of the 1960s and beginning of the 1970s, electron microscopy was used in studies on schistosomiasis mansoni to confirm details of the parasite's tegument and thus allowed for interpretation of the func‐ tions of that structure.

In 1968, Hockley [31] described the surface of the worm using SEM, pointing out, for in‐ stance, the presence of thorns in the more internal portion of the oral sucker in both sexes, and the fact that the ventral sucker (acetabulum) is longer and more conspicuous in males. Still according to that author, the genital pore was detected in both sexes in the form of an opening placed posteriorly to the ventral sucker. The author also reported the presence of tubercles on the dorsal surface of male worms starting from the posterior portion of the ven‐ tral sucker, both sides being covered by thorns. The number of such tubercles starts to de‐ crease from the posterior lateral edges of the dorsal surface.

and *Phyllanthus amarus* – on adult *S. mansoni* worms. The three species are widely used in Brazilian folk medicine in the forms of infusion and tea on account of their anti-in‐

Amongst the several criteria analyzed by our research group to evaluate the therapeutic effi‐ ciency of the tested plants, the morphological changes in the tegument of the worms (both males and females) were considered essential, due to the fact that literature reports the dam‐ age caused to that structure as cardinal in causing the parasite's death, and studies for the

The tegument of *S. mansoni* plays a key role in its protection against the action of the host's immune system, as it is renovated every six hours [20]. In addition to that, it is capable of absorbing nutrients and molecules and synthesizing some proteins [21-24]. That structure is also extremely important for the success of the infection and the survival of the worm in the host [25-27]. For all those reasons, it has been vastly studied since the end of the 1960s.

Before microscopy techniques were available for the evaluation of the worm ultrastructure, little was known about the importance of the tegument of *S. mansoni*. The first detailed stud‐ ies on that structure were carried out in the 1940s, when Gönnert [28], using light microsco‐ py, described the differences between *S. mansoni* males and females, including the fact that

With the emergence of transmission electron microscopy (TEM) and scanning electron mi‐ croscopy (SEM) there was a revolution regarding the 'appearance' of the worm and the de‐ scription of its sexual dimorphism. By using electron beams that either pass through (in the case of TEM) or scan the specimen under analysis (in the case of SEM), electron microscopy, which was developed in the 1930s, had a significantly higher resolving power than optical

According to [29, 30], *S. mansoni* was the first digenetic trematode to be examined under electron microscopy. For that reason, the ultrastructure of that helminth has been more often studied than the ultrastructure of any other digenetic trematode. By the end of the 1960s and beginning of the 1970s, electron microscopy was used in studies on schistosomiasis mansoni to confirm details of the parasite's tegument and thus allowed for interpretation of the func‐

In 1968, Hockley [31] described the surface of the worm using SEM, pointing out, for in‐ stance, the presence of thorns in the more internal portion of the oral sucker in both sexes, and the fact that the ventral sucker (acetabulum) is longer and more conspicuous in males. Still according to that author, the genital pore was detected in both sexes in the form of an

**3.1. The analysis evolution of the tegument of** *S. mansoni***: From optical to electron**

development of new schistosomicidal drugs have been setting it as a target [20].

**3. The importance of the tegument of** *Schistosoma mansoni*

flammatory properties.

154 Parasitic Diseases - Schistosomiasis

**microscopy**

males have more and larger thorns.

tions of that structure.

microscopy, allowing for a detailed observation of samples.

Furthermore, Hockley [31] observed that in the areas between the tubercles the parasite's surface is rugged, with several grooves, as well as a few isolated thorns. On the male worm lateral edge, which bends to form the gynaecophoric canal, the author noticed the presence of large thorns whose function is to capture the female in the canal. Finally, he noticed that females also presents more thorns at the final portion of their surface, but in less quantity than males. Senft and Gibler [32] termed such thorns sensory papillae.

Using TEM, Hockley and McLaren [33] concluded that the surface of *S. mansoni* consists of two opposite lipid bilayers very close to each other and having the form of a cell membrane. Since the tegument does not have lateral membranes, its cytoplasm extends as a continuous unity, or syncytium, around the body of the worm. According to the authors, that syncytial complex is the main route of nutrient absorption (glucose, amino acids, among others), me‐ tabolite excretion (lactic acid and others) and protection against attacks by the host's im‐ mune system, whereupon it is a crucial target of drugs with schistosomicidal activity.

From the 1980s on, after the establishment of the therapeutics of schistosomiasis mansoni, TEM and especially SEM were also used in an attempt to clarify the action mechanisms of the drugs used in the treatment for the disease, i.e., oxamniquine and praziquantel.

Becker et al*.* [34], by means of SEM and TEM, realized that worms subjected to oxamniquine showed tegumental vacuolization. Using SEM, Kohn et al*.* [35] noticed that the drug was produc‐ ing changes in the structure of the worm on tegumental, muscular and parenchymal levels, caus‐ ing bubble-like lesions. Magalhães-Filho et al*.* [36] also noticed vacuolization, destruction of tubercles in male worms, and surface erosion. Recently, praziquantel has been subjected to fur‐ ther studies because it still is the most used drug in the treatment of all types of schistosomiasis.

Using SEM and TEM, Shaw and Erasmus [37] observed extensive damage to the structure of *S. mansoni* specimens subjected to praziquantel, including vacuolization of the tegument and subtegument of females, and destruction of the tegument and musculature. In males, in ad‐ dition to vacuolization and destruction of the parenchymal tissue, mainly in the dorsal re‐ gion, loss of cytoplasm, and structural damages to the musculature could be observed. It was noticed that male worms showed more damage in the tegument than females.

Actually, the action mechanism of praziquantel on *S. mansoni* has yet to be fully understood. On the other hand, there is no doubt that studies using SEM and TEM were important in the search to solve that puzzle.

The morphological changes that praziquantel causes to the tegument and in the sarcoplas‐ mic membranes of the parasite are thought to be followed by an increase of antigen expo‐ sure on its surface. The antigens are identified and connected with the host's immune response required to complement the activity of the drug [38, 39]. Therefore, praziquantel is believed to interact with the host's immune system to kill the parasite. The last effect of the drug is the rupture of the surface of the worm, leading to its death [40].

In view of the fact that certain strains of the parasite have proved to be tolerant and resistant to the treatment with oxamniquine and praziquantel, it has become necessary to test new drugs. Electron microscopy has been used since the 1990s to know if such drugs are active against the worm by observing the damages caused to its tegument. Many studies using SEM have shown that drugs active against *S. mansoni* are responsible for severe damage to its tegument.

reported damage to the tegument of *S. mansoni* specimens subjected to *in vitro* assays with *Dry‐ opteris* sp. and *Piper tuberculatum*, respectively, considering them either moderate or severe.

Therefore, there are other methods besides electron microscopy to evaluate the activity of a drug or candidate drug on the tegument of *S. mansoni.* Such methods can be classified as qualitative (i.e., it is only possible to notice changes, not to measure them), quantitative (changes can be quantified, i.e., it is possible to count and compare, for instance, the number of tubercles in male worms subjected to the tested sample with the number found in the control group), and qualitative-quantitative (in this case, changes can be both visualized and

**Method Classification Advantages Disadvantages References**


Tegument of *Schistosoma mansoni* as a Therapeutic Target

http://dx.doi.org/10.5772/53653

157

[51, 52,53-57]

[51]

[43, 58, 59]

[24, 46,58]

intermediate, moderate, or even





and difficult work



absent




three-dimensional images, especially on the tegument of male worms, where the number of changed or damaged tubercles is counted -Provides images of great contrast even with


caused to the tegument of the worm but also of changes in its musculature and internal




**Table 1.** Methods to analyze damage to the tegument of *S. mansoni* specimens subjected to either *in vitro* or *in vivo*

quantified). Table 1 presents that classification.

Qualitative - No special preparation is required to visualize the sample

Quantitative - Allows damages to be quantified through

weakly fluorescent specimens

of the surface of the worm

Qualitative - Allows for the analysis not only of damages


the surface of the worm

surface of the worm

organs

quantified

tests *for* evaluation of candidate drugs*:* classification. Sources: [60-63].

out

time

Inverted Optical Microscope (used during *in vitro* tests)

Confocal Laser Scanning Fluorescence Microscopy

Transmission Electron Microscopy

Scanning Electron Microscopy

Qualitative/ Quantitative

Some drugs, which had their schistosomicidal activity studied, have proved capable of pro‐ ducing tegumental changes. Albuquerque et al. [41] noticed that imidazole derivatives caus‐ es damage to the oral sucker in males, in addition to reducing and disorganizing the tubercles. In females the authors noticed erosion and peeling of the tegument, rupture of the surface membrane, and the complete disappearing of sensory structures.

Manneck et al. [42] observed that mefloquine causes higher degree of changes to the tegument of schistosomula and adult females, including peeling and bubble formation. Tests with that drug have also been carried out with other *Schistosoma* sp. (e.g., *S. japonicum*, which causes in‐ testinal schistosomiasis), and these were the effects described: peeling in males and females, fu‐ sion of thorns in males, collapse of the sensory papillae, and erosion on the suckers [43].

In assays carried out with arachidonic acid, El Ridi et al. [44] showed extensive changes in the aspect of tubercles in males, including reduction in size and loss of thorns. The suckers also underwent changes such as oedemas and loss of thorns.

Other authors used SEM to attest the activity of natural compounds on the tegument of the worm. Shuhua et al. [24] reported that artemether, a derivative compound of artemisinin, which is extracted from *Artemisia annua,* causes damage to the tegument of both males and females, including peeling, which was more intense in females – not surprisingly, as artemi‐ sinin itself is most effective against females [45]. Oliveira et al*.* [46] noticed extensive peeling on the tegument of male and female worms, destruction of tubercles, thorns and sensory papillae, and changes in the suckers (oral and ventral) after *in vitro* exposure to essential oil of *Baccharis trimera* over an incubation period of 24 hours.

#### **3.2.** *S. mansoni* **tegument analysis method**

Nowadays, other techniques are being used in an attempt to evaluate the activity of candidate drugs on the tegument of the worm. One of them is confocal laser scanning microscopy (CLSM), which, like SEM, provides three-dimensional images. The confocal microscope was developed in 1950, but it only became popular for analysis of biological samples in the 1970s [47-50].

Moraes et al. [27-51] used confocal laser scanning microscopy to analyze the activity of pi‐ plartine (isolated from *Piper tuberculatum*) on the tegument of *S. mansoni* adults and schisto‐ somula, and reported reduction in the quantity of tubercles in males and damage to the surface membrane of schistosomula. Moraes [51] proposed the use of both CLSM and SEM to perform a quantitative analysis (by counting the tubercles in a specific area) of the dam‐ age caused to the tegument of worms used in drug testing.

Another way to evaluate tegument damage caused by candidate drugs is to use an inverted op‐ tical microscope during *in vitro* assays. Magalhães et al. [52] and Moraes et al. [51], for example, reported damage to the tegument of *S. mansoni* specimens subjected to *in vitro* assays with *Dry‐ opteris* sp. and *Piper tuberculatum*, respectively, considering them either moderate or severe.

In view of the fact that certain strains of the parasite have proved to be tolerant and resistant to the treatment with oxamniquine and praziquantel, it has become necessary to test new drugs. Electron microscopy has been used since the 1990s to know if such drugs are active against the worm by observing the damages caused to its tegument. Many studies using SEM have shown

Some drugs, which had their schistosomicidal activity studied, have proved capable of pro‐ ducing tegumental changes. Albuquerque et al. [41] noticed that imidazole derivatives caus‐ es damage to the oral sucker in males, in addition to reducing and disorganizing the tubercles. In females the authors noticed erosion and peeling of the tegument, rupture of the

Manneck et al. [42] observed that mefloquine causes higher degree of changes to the tegument of schistosomula and adult females, including peeling and bubble formation. Tests with that drug have also been carried out with other *Schistosoma* sp. (e.g., *S. japonicum*, which causes in‐ testinal schistosomiasis), and these were the effects described: peeling in males and females, fu‐

In assays carried out with arachidonic acid, El Ridi et al. [44] showed extensive changes in the aspect of tubercles in males, including reduction in size and loss of thorns. The suckers

Other authors used SEM to attest the activity of natural compounds on the tegument of the worm. Shuhua et al. [24] reported that artemether, a derivative compound of artemisinin, which is extracted from *Artemisia annua,* causes damage to the tegument of both males and females, including peeling, which was more intense in females – not surprisingly, as artemi‐ sinin itself is most effective against females [45]. Oliveira et al*.* [46] noticed extensive peeling on the tegument of male and female worms, destruction of tubercles, thorns and sensory papillae, and changes in the suckers (oral and ventral) after *in vitro* exposure to essential oil

Nowadays, other techniques are being used in an attempt to evaluate the activity of candidate drugs on the tegument of the worm. One of them is confocal laser scanning microscopy (CLSM), which, like SEM, provides three-dimensional images. The confocal microscope was developed

Moraes et al. [27-51] used confocal laser scanning microscopy to analyze the activity of pi‐ plartine (isolated from *Piper tuberculatum*) on the tegument of *S. mansoni* adults and schisto‐ somula, and reported reduction in the quantity of tubercles in males and damage to the surface membrane of schistosomula. Moraes [51] proposed the use of both CLSM and SEM to perform a quantitative analysis (by counting the tubercles in a specific area) of the dam‐

Another way to evaluate tegument damage caused by candidate drugs is to use an inverted op‐ tical microscope during *in vitro* assays. Magalhães et al. [52] and Moraes et al. [51], for example,

in 1950, but it only became popular for analysis of biological samples in the 1970s [47-50].

sion of thorns in males, collapse of the sensory papillae, and erosion on the suckers [43].

that drugs active against *S. mansoni* are responsible for severe damage to its tegument.

surface membrane, and the complete disappearing of sensory structures.

also underwent changes such as oedemas and loss of thorns.

of *Baccharis trimera* over an incubation period of 24 hours.

age caused to the tegument of worms used in drug testing.

**3.2.** *S. mansoni* **tegument analysis method**

156 Parasitic Diseases - Schistosomiasis

Therefore, there are other methods besides electron microscopy to evaluate the activity of a drug or candidate drug on the tegument of *S. mansoni.* Such methods can be classified as qualitative (i.e., it is only possible to notice changes, not to measure them), quantitative (changes can be quantified, i.e., it is possible to count and compare, for instance, the number of tubercles in male worms subjected to the tested sample with the number found in the control group), and qualitative-quantitative (in this case, changes can be both visualized and quantified). Table 1 presents that classification.


**Table 1.** Methods to analyze damage to the tegument of *S. mansoni* specimens subjected to either *in vitro* or *in vivo* tests *for* evaluation of candidate drugs*:* classification. Sources: [60-63].

Our research group has evaluated the activity of different fractions of *B. trimera, C. verbena‐ cea* and *P. amarus* on the tegument of adult *S. mansoni* males and females using SEM. We un‐ derstand that this method provides important data regarding tegumentary changes because its high angular resolution provides high-quality images that allow us to analyse the para‐ site's surface in detail.

The use of 2.5% glutaraldehyde as fixative for a shorter incubation period (24 hours) was,

Figure 8 shows the standardized protocol that was applied to all SEM assays with the stud‐ ied plant species, i.e., *B. trimera, C. verbenacea* and *P. amarus.* The samples were mounted on aluminum stubs, placed in a Balzers critical point dryer, model CPD 030, and a Bal-Tec/Balz‐ ers sputtering system (Sputter Coater), model SCD 50, and then were analyzed under a Jeol

**Methodology Type Advantages Disadvantages References**





**Table 2.** Methodological differences for the preparation of *S. mansoni* specimens for SEM. Sources: [73, 74].

Formalin - Low cost


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toxic substances [70-72]

[65-67]

[68,69]

[65, 67, 70,71]

[24,41]

thus, adopted by our group.

Pre-fixatives Glutaraldehyde (AG)

Karnovsky (2.5% glutaraldehyde and 4% paraformaldehyde)

Phosphate

Buffer solution Sodium cacodylate

scanning electron microscope, model JSM 5800LV.

## **4. Methodologies used in scanning electron microscopy for studies of** *S. mansoni*

On account of their naturally hydrated condition, biological samples are relatively complex to process, and only hard objects (e.g., seeds) can be observed through SEM with minimum preliminary treatment. Therefore, the preparation of a biological sample for SEM includes various stages [60, 64].

From the first observations using SEM to the present ones, it can be noticed that the method‐ ology for *S. mansoni* worms preparation has significantly varied from one author to the oth‐ er. Nevertheless, the steps for preparing the worms have always been respected: fixation, washing to remove the excess of fixatives, post-fixation in osmium tetroxide, dehydration at growing concentrations of ethanol, critical point drying, mounting on aluminum stubs, gold sputtering, and observation under a scanning electron microscope. Table 2 shows differen‐ ces in the methodologies for preparing *S. mansoni* specimens for SEM analysis.

Although some methodologies for preparation of *S. mansoni* samples for SEM use phosphate buffer, some authors use sodium cacodylate buffer. Considering that, an experiment was carried out in order to evaluate possible differences between the use of such biological buf‐ fers. A protocol was used, and only the biological buffer was changed:

1. Fixation in Karnovsky (2.5% glutaraldehyde and 4% paraformaldehyde) with buffering pH between 7.0 and 7.3 (adjusted with 0.2 M HCL) with 0.1 M sodium cacodylate buffer sol‐ ution or 0.1 M phosphate during 48 hours; 2. Washing in 0.1 M sodium cacodylate buffer or 0.1 M phosphate for one hour, changing the solution every 15 minutes; 3. Post-fixation in 1% osmium tetroxide for one hour; 4. Washing in 0.1 M sodium cacodylate buffer or 0.1 M phosphate for 30 minutes, changing the solution every 10 minutes; 5. Dehydration at grow‐ ing concentrations of ethanol (50% and 70% during 30 minutes, changing the solution after 15 minutes; 90% and 100% during 30 minutes, changing the solution every 10 minutes); 6. Drying of worms in a critical point dryer; 7. Mounting of the samples on aluminum stubs; 8. Gold Sputtering; 9. Observation under a scanning electron microscope

Figures 2 to 7 present images obtained with different buffers. It could be noticed that the samples subjected to phosphate buffer showed inferior fixation as compared to sodium ca‐ codylate, since the worms became malleable and, especially males, seemed to dehydrate. For that reason, sodium cacodylate buffer was used in the subsequent assays. However, it could also be noticed that the use of Karnovsky solution in fixation, as well as the long period in which the samples stayed therein, rendered the worms stiff, friable and hard to manipulate. The use of 2.5% glutaraldehyde as fixative for a shorter incubation period (24 hours) was, thus, adopted by our group.

Our research group has evaluated the activity of different fractions of *B. trimera, C. verbena‐ cea* and *P. amarus* on the tegument of adult *S. mansoni* males and females using SEM. We un‐ derstand that this method provides important data regarding tegumentary changes because its high angular resolution provides high-quality images that allow us to analyse the para‐

**4. Methodologies used in scanning electron microscopy for studies of** *S.*

On account of their naturally hydrated condition, biological samples are relatively complex to process, and only hard objects (e.g., seeds) can be observed through SEM with minimum preliminary treatment. Therefore, the preparation of a biological sample for SEM includes

From the first observations using SEM to the present ones, it can be noticed that the method‐ ology for *S. mansoni* worms preparation has significantly varied from one author to the oth‐ er. Nevertheless, the steps for preparing the worms have always been respected: fixation, washing to remove the excess of fixatives, post-fixation in osmium tetroxide, dehydration at growing concentrations of ethanol, critical point drying, mounting on aluminum stubs, gold sputtering, and observation under a scanning electron microscope. Table 2 shows differen‐

Although some methodologies for preparation of *S. mansoni* samples for SEM use phosphate buffer, some authors use sodium cacodylate buffer. Considering that, an experiment was carried out in order to evaluate possible differences between the use of such biological buf‐

1. Fixation in Karnovsky (2.5% glutaraldehyde and 4% paraformaldehyde) with buffering pH between 7.0 and 7.3 (adjusted with 0.2 M HCL) with 0.1 M sodium cacodylate buffer sol‐ ution or 0.1 M phosphate during 48 hours; 2. Washing in 0.1 M sodium cacodylate buffer or 0.1 M phosphate for one hour, changing the solution every 15 minutes; 3. Post-fixation in 1% osmium tetroxide for one hour; 4. Washing in 0.1 M sodium cacodylate buffer or 0.1 M phosphate for 30 minutes, changing the solution every 10 minutes; 5. Dehydration at grow‐ ing concentrations of ethanol (50% and 70% during 30 minutes, changing the solution after 15 minutes; 90% and 100% during 30 minutes, changing the solution every 10 minutes); 6. Drying of worms in a critical point dryer; 7. Mounting of the samples on aluminum stubs; 8.

Figures 2 to 7 present images obtained with different buffers. It could be noticed that the samples subjected to phosphate buffer showed inferior fixation as compared to sodium ca‐ codylate, since the worms became malleable and, especially males, seemed to dehydrate. For that reason, sodium cacodylate buffer was used in the subsequent assays. However, it could also be noticed that the use of Karnovsky solution in fixation, as well as the long period in which the samples stayed therein, rendered the worms stiff, friable and hard to manipulate.

ces in the methodologies for preparing *S. mansoni* specimens for SEM analysis.

fers. A protocol was used, and only the biological buffer was changed:

Gold Sputtering; 9. Observation under a scanning electron microscope

site's surface in detail.

158 Parasitic Diseases - Schistosomiasis

various stages [60, 64].

*mansoni*

Figure 8 shows the standardized protocol that was applied to all SEM assays with the stud‐ ied plant species, i.e., *B. trimera, C. verbenacea* and *P. amarus.* The samples were mounted on aluminum stubs, placed in a Balzers critical point dryer, model CPD 030, and a Bal-Tec/Balz‐ ers sputtering system (Sputter Coater), model SCD 50, and then were analyzed under a Jeol scanning electron microscope, model JSM 5800LV.


**Table 2.** Methodological differences for the preparation of *S. mansoni* specimens for SEM. Sources: [73, 74].

#### **Sodium Cacodylate Group – mated worms**

**Sodium Cacodylate Group – Suckers**

sucker; va. ventral sucker; f. female worm.

**Phosphate Group – Mated Worms**

**Figure 4.** Scanning electron microscopy of *S. mansoni* male worm suckers using sodium cacodylate buffer. vo. oral

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**Figure 5.** SEM of adult *S. mansoni* worms using sodium phosphate buffer. A- male worm; **f-** female worm; B- male

worm; **vo-** oral sucker; **va**- ventral sucker. **cg**- gynaecophoric canal.

**Figure 2.** Scanning electron microscopy of adult *S. mansoni* using sodium cacodylate buffer. A-B – mated *S. mansoni* worms; m: male worm; f: female worm; cg: gynaecophoric canal, vo: oral sucker, Va: ventral sucker.

#### **Sodium Cacodylate Group – Tegument**

**Figure 3.** Scanning electron microscopy of the tegument of adult *S. mansoni* worms using sodium cacodylate buffer. **A** – tegument of male worm; **B** – tegument of female worm. **t**. tubercles. **ep** – excretory pore.

#### **Sodium Cacodylate Group – Suckers**

**Sodium Cacodylate Group – mated worms**

160 Parasitic Diseases - Schistosomiasis

**Sodium Cacodylate Group – Tegument**

**Figure 2.** Scanning electron microscopy of adult *S. mansoni* using sodium cacodylate buffer. A-B – mated *S. mansoni*

**Figure 3.** Scanning electron microscopy of the tegument of adult *S. mansoni* worms using sodium cacodylate buffer.

**A** – tegument of male worm; **B** – tegument of female worm. **t**. tubercles. **ep** – excretory pore.

worms; m: male worm; f: female worm; cg: gynaecophoric canal, vo: oral sucker, Va: ventral sucker.

**Figure 4.** Scanning electron microscopy of *S. mansoni* male worm suckers using sodium cacodylate buffer. vo. oral sucker; va. ventral sucker; f. female worm.

#### **Phosphate Group – Mated Worms**

**Figure 5.** SEM of adult *S. mansoni* worms using sodium phosphate buffer. A- male worm; **f-** female worm; B- male worm; **vo-** oral sucker; **va**- ventral sucker. **cg**- gynaecophoric canal.

**Phosphate Group – Tegument**

**Figure 6.** SEM of the tegument of *adult S. mansoni* worms using sodium phosphate buffer. A – tegument of male worm; B – tegument of female worm. **t**: tubercles.

**Figure 8.** Protocol used by our research group to prepare *S. mansoni* specimens for scanning electron microscopy.

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The species *B. trimera, C. verbenacea* and *P. amarus,* whose activity on the tegument of *S. mansoni* was evaluated*,* are widely used in Brazilian folk medicine. The studied plants were obtained from the Experimental Field of the Chemical, Biological and Agricultural Pluridisciplinary Re‐ search Center (CPQBA), Paulínia (22º45'40" S – 47º09'15" W), São Paulo, Brazil. The following fractions were tested, all of them coming from the aerial parts (flowers and/or inflorescences).

**A -** *Baccharis trimera* **B-** *Cordia verbenacea* **C-** *Phyllanthus amarus*

**Figure 9.** *B. trimera, C. verbenacea* and *P. amarus* specimens*.* Source: CPQBA, Unicamp, 2011.

**5. Study of the activity of medicinal plants on the tegument of** *S. mansoni: Baccharis trimera* **(Less) DC,** *Cordia verbenacea* **DC and**

*Phyllanthus amarus*

#### **Phosphate Group – Suckers**

**Figure 7.** SEM of adult *S. mansoni* worms suckers using sodium phosphate buffer*.* **A**- male worm, genital pore high‐ lighted (**pg**).

**Phosphate Group – Tegument**

162 Parasitic Diseases - Schistosomiasis

A B

worm; B – tegument of female worm. **t**: tubercles.

**Phosphate Group – Suckers**

lighted (**pg**).

**Figure 6.** SEM of the tegument of *adult S. mansoni* worms using sodium phosphate buffer. A – tegument of male

**Figure 7.** SEM of adult *S. mansoni* worms suckers using sodium phosphate buffer*.* **A**- male worm, genital pore high‐

**Figure 8.** Protocol used by our research group to prepare *S. mansoni* specimens for scanning electron microscopy.

## **5. Study of the activity of medicinal plants on the tegument of** *S. mansoni: Baccharis trimera* **(Less) DC,** *Cordia verbenacea* **DC and** *Phyllanthus amarus*

The species *B. trimera, C. verbenacea* and *P. amarus,* whose activity on the tegument of *S. mansoni* was evaluated*,* are widely used in Brazilian folk medicine. The studied plants were obtained from the Experimental Field of the Chemical, Biological and Agricultural Pluridisciplinary Re‐ search Center (CPQBA), Paulínia (22º45'40" S – 47º09'15" W), São Paulo, Brazil. The following fractions were tested, all of them coming from the aerial parts (flowers and/or inflorescences).

**Figure 9.** *B. trimera, C. verbenacea* and *P. amarus* specimens*.* Source: CPQBA, Unicamp, 2011.

## **•** *Baccharis trimera* (Less) DC

The species *B. trimera* (Figure 9-A), known in Brazil as "*carqueja-amarga"*, belongs to the fam‐ ily Asteraceae and is used in folk medicine for the treatment of many diseases, in particular hepatic ones. The plant allegedly has tonic, mouth-healing, antipyretic, analgesic, anti-dia‐ betic, and anti-inflammatory properties [75-81]. It is native to the South and Southeast re‐ gions of Brazil, also being found in Argentina, Bolivia, Paraguay and Uruguay [82].

*B. trimera* was used in *in vitro* assays in which mating worms were kept in RPMI-1640 medi‐ um with penicillin/streptomycin, incubated in a controlled environment (5% CO2 and 37°C) [53], and exposed to a fraction hexane fraction, obtained from the fractionation of dichloro‐ methane extract, in lethal concentration of 130 μg/mL for 24 hours. After that period, the worms were prepared for SEM, also according to the protocol shown in Figure 8*.*

The hexane fraction of *B. trimera* caused changes to the tegument of both males and females and on the oral and ventral suckers. The tegumental peeling was particularly worth noting (Figures 10 to 12).

**Figure 11.** SEM of adult *S. mansoni* specimens after *in vitro* exposure to the hexane fraction obtained from the crude dichloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **B.** Male worm, destruction of tubercles and thorns on its tegumental surface. **C.** Female worm with extensive tegumental

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**Figure 12.** SEM of adult *S. mansoni* specimens after *in vitro* exposure to the hexane fraction obtained from the crude dichloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **D.** Male worm with changes in the oral (**vo**) and ventral (**va**) suckers. **E.** Female worm with changes in the suckers and tegu‐

*C. verbenacea* (Figure 9-B), also referred to as *C. salicina, C. curassavica, C. cylindristachia, Litho‐ cardium fresenii, L. salicinum and L. verbaceum*, is popularly known as *erva-baleeira* or salicina in Brazil. It belongs to the family Boraginaceae and is widely distributed along the Brazilian coast, being found mainly in the littoral zone extending from São Paulo to Santa Catarina [83]. Species of the genus *Cordia* are present in tropical and subtropical areas of Asia, South‐ ern Africa, Australia, Guyana, and South America [84]. Several compounds are found in their aerial parts, including tanins, flavonoids, mucilage, and essential oils. Such parts, along with leaves and inflorescences, have been used in folk medicine in the form of infusions and alcohol extracts because of their antiulcer, antimicrobial and antirheumatic activities, and

peeling on its dorsal surface.

mental wrinkling and erosion.

**•** *Cordia verbenacea* DC

**Figure 10.** SEM of a *S. mansoni* adult couple after *in vitro* exposure to hexane fraction obtained from the crude di‐ chloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **m**- male worm showing changes in its oral **(vo)** and ventral **(va)** suckers, as well as destruction of its tegument; **f –** female worm with tegumental peeling on its body surface.

**Figure 11.** SEM of adult *S. mansoni* specimens after *in vitro* exposure to the hexane fraction obtained from the crude dichloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **B.** Male worm, destruction of tubercles and thorns on its tegumental surface. **C.** Female worm with extensive tegumental peeling on its dorsal surface.

**Figure 12.** SEM of adult *S. mansoni* specimens after *in vitro* exposure to the hexane fraction obtained from the crude dichloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **D.** Male worm with changes in the oral (**vo**) and ventral (**va**) suckers. **E.** Female worm with changes in the suckers and tegu‐ mental wrinkling and erosion.

#### **•** *Cordia verbenacea* DC

**•** *Baccharis trimera* (Less) DC

164 Parasitic Diseases - Schistosomiasis

(Figures 10 to 12).

The species *B. trimera* (Figure 9-A), known in Brazil as "*carqueja-amarga"*, belongs to the fam‐ ily Asteraceae and is used in folk medicine for the treatment of many diseases, in particular hepatic ones. The plant allegedly has tonic, mouth-healing, antipyretic, analgesic, anti-dia‐ betic, and anti-inflammatory properties [75-81]. It is native to the South and Southeast re‐

*B. trimera* was used in *in vitro* assays in which mating worms were kept in RPMI-1640 medi‐ um with penicillin/streptomycin, incubated in a controlled environment (5% CO2 and 37°C) [53], and exposed to a fraction hexane fraction, obtained from the fractionation of dichloro‐ methane extract, in lethal concentration of 130 μg/mL for 24 hours. After that period, the

The hexane fraction of *B. trimera* caused changes to the tegument of both males and females and on the oral and ventral suckers. The tegumental peeling was particularly worth noting

**Figure 10.** SEM of a *S. mansoni* adult couple after *in vitro* exposure to hexane fraction obtained from the crude di‐ chloromethane extract at the lethal concentration of 130 μg/mL, over an incubation period of 24 hours. **m**- male worm showing changes in its oral **(vo)** and ventral **(va)** suckers, as well as destruction of its tegument; **f –** female

worm with tegumental peeling on its body surface.

gions of Brazil, also being found in Argentina, Bolivia, Paraguay and Uruguay [82].

worms were prepared for SEM, also according to the protocol shown in Figure 8*.*

*C. verbenacea* (Figure 9-B), also referred to as *C. salicina, C. curassavica, C. cylindristachia, Litho‐ cardium fresenii, L. salicinum and L. verbaceum*, is popularly known as *erva-baleeira* or salicina in Brazil. It belongs to the family Boraginaceae and is widely distributed along the Brazilian coast, being found mainly in the littoral zone extending from São Paulo to Santa Catarina [83]. Species of the genus *Cordia* are present in tropical and subtropical areas of Asia, South‐ ern Africa, Australia, Guyana, and South America [84]. Several compounds are found in their aerial parts, including tanins, flavonoids, mucilage, and essential oils. Such parts, along with leaves and inflorescences, have been used in folk medicine in the form of infusions and alcohol extracts because of their antiulcer, antimicrobial and antirheumatic activities, and tonic, analgesic and anti-inflammatory properties [83,85,86]. In view of the variety of chemi‐ cal groups found in extracts of *C. verbenacea* and their alleged biological properties, that plant is an important material for pharmaceutical research [87].

tion of coupled worms, thus female worms remained in the gynaecophoric canal protected

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**Figure 13.** SEM of adult *S. mansoni* specimens after *in vivo* assay with fraction 3 obtained from the organic fraction of *C. verbenacea* at the concentration of 300 mg/kg. **A-B.** Male and female worms, respectively, showing peeling of the tegument. **C-D.** Male worms showing adhesion of host's cells to its tegument and formation of vesicles. **et** – erosion of the tegument; **ch** – host's cells adhered to the surface of *S. mansoni*; **l** – host's leukocytes; **dt** – destruction of the tegu‐

from the action of the butanolic fraction 2.

ment; **v** – vesicle.

*C. verbenacea* were used in *in vivo* assays with mice Balb/c (*Mus musculus*), infected with 70 cercariae of *S. mansoni* (BH strain) by tail immersion [98], and kept in an isolated environ‐ ment. Forty-five days following infection, the animals were treated orally by esophageal in‐ tubation with 300 mg/kg, administered in a single dose, of fraction 3, obtained from the fractionation of the organic fraction, originated from the ethanol extract [99], with a specific concentration of the tested fraction. Fifteen days after treatment, the animals were euthan‐ ized by cervical dislocation. The worms were collected by perfusion of the hepatic portal system [100], and washed in 0.9% NaCl solution and subjected to the protocol shown in Fig‐ ure 8 in order to be analyzed by scanning electron microscopy.

Fraction 3 of *C. verbenacea* caused an erosion on the tegument of both male and female worms. Formation of vesicles and adhesion of host's cells to the surface of worms (Figure 13) were also observed*.* No damages to the sucker were found.

*• Phyllanthus amarus*

The plants belonging to the genus *Phyllanthus* are widely distributed in most of the tropical and subtropical countries (in both hemispheres) and include between 550 and 750 species. It is be‐ lieved that around 200 species of that genus are distributed in the Americas, chiefly in the Car‐ ibbean and in Brazil [88, 89]. In Brazil, the plants of that genus are popularly known as stonebreaker ("*quebra-pedra*" in Brazilian Portuguese) because they are recognized by their diuretic properties in Brazilian and other countries' folk medicine, being used in the treatment for kid‐ ney and bladder disorders. In addition to helping the elimination of kidney stones, they com‐ bat intestinal infections, diabetes and hepatitis B [88-90]. The interest in plants of the genus *Phyllanthus* has been considerably increasing, especially for the species *P. amarus* (family Eu‐ phorbiaceae) (Figure 9-C), which is scientifically one of the most studied, many of its com‐ pounds having already been isolated and chemically identified. *P. amarus* has a long history of usage in folk medicine because of its rich medicinal effects, being reported to possess potent hepatoprotective [91, 92], anti-inflammatory, analgesic [93-94], hypoglycaemic [95], antiplas‐ modial (against *Plasmodium berghei*) [96], and antioxidant [97] properties.

*P. amarus* were used in *in vivo* assays with mice Balb/c (*Mus musculus*), infected with 70 cercar‐ iae of *S. mansoni* (BH strain) by tail immersion [98], and kept in an isolated environment. Fortyfive days following infection, the animals were treated orally by esophageal intubation [99], with the butanolic fraction 2 in the concentration of the 100 mg/kg for three days. Fifteen days after treatment, the animals were euthanized by cervical dislocation. The worms were collect‐ ed by perfusion of the hepatic portal system [100], washed in 0.9% NaCl solution and subjected to the protocol shown in Figure 8 in order to be analyzed by scanning electron microscopy.

The butanolic fraction 2 of *P. amarus* caused damage to the male worms' tegument, includ‐ ing perforations, changes in the tubercles, peeling, and formation of vesicles and protuber‐ ances. Contraction and swelling were noticed in the region around the suckers (Figure 14). No damage were found in the tegument of females. This fraction did not cause the separa‐ tion of coupled worms, thus female worms remained in the gynaecophoric canal protected from the action of the butanolic fraction 2.

tonic, analgesic and anti-inflammatory properties [83,85,86]. In view of the variety of chemi‐ cal groups found in extracts of *C. verbenacea* and their alleged biological properties, that

*C. verbenacea* were used in *in vivo* assays with mice Balb/c (*Mus musculus*), infected with 70 cercariae of *S. mansoni* (BH strain) by tail immersion [98], and kept in an isolated environ‐ ment. Forty-five days following infection, the animals were treated orally by esophageal in‐ tubation with 300 mg/kg, administered in a single dose, of fraction 3, obtained from the fractionation of the organic fraction, originated from the ethanol extract [99], with a specific concentration of the tested fraction. Fifteen days after treatment, the animals were euthan‐ ized by cervical dislocation. The worms were collected by perfusion of the hepatic portal system [100], and washed in 0.9% NaCl solution and subjected to the protocol shown in Fig‐

Fraction 3 of *C. verbenacea* caused an erosion on the tegument of both male and female worms. Formation of vesicles and adhesion of host's cells to the surface of worms (Figure

The plants belonging to the genus *Phyllanthus* are widely distributed in most of the tropical and subtropical countries (in both hemispheres) and include between 550 and 750 species. It is be‐ lieved that around 200 species of that genus are distributed in the Americas, chiefly in the Car‐ ibbean and in Brazil [88, 89]. In Brazil, the plants of that genus are popularly known as stonebreaker ("*quebra-pedra*" in Brazilian Portuguese) because they are recognized by their diuretic properties in Brazilian and other countries' folk medicine, being used in the treatment for kid‐ ney and bladder disorders. In addition to helping the elimination of kidney stones, they com‐ bat intestinal infections, diabetes and hepatitis B [88-90]. The interest in plants of the genus *Phyllanthus* has been considerably increasing, especially for the species *P. amarus* (family Eu‐ phorbiaceae) (Figure 9-C), which is scientifically one of the most studied, many of its com‐ pounds having already been isolated and chemically identified. *P. amarus* has a long history of usage in folk medicine because of its rich medicinal effects, being reported to possess potent hepatoprotective [91, 92], anti-inflammatory, analgesic [93-94], hypoglycaemic [95], antiplas‐

*P. amarus* were used in *in vivo* assays with mice Balb/c (*Mus musculus*), infected with 70 cercar‐ iae of *S. mansoni* (BH strain) by tail immersion [98], and kept in an isolated environment. Fortyfive days following infection, the animals were treated orally by esophageal intubation [99], with the butanolic fraction 2 in the concentration of the 100 mg/kg for three days. Fifteen days after treatment, the animals were euthanized by cervical dislocation. The worms were collect‐ ed by perfusion of the hepatic portal system [100], washed in 0.9% NaCl solution and subjected to the protocol shown in Figure 8 in order to be analyzed by scanning electron microscopy.

The butanolic fraction 2 of *P. amarus* caused damage to the male worms' tegument, includ‐ ing perforations, changes in the tubercles, peeling, and formation of vesicles and protuber‐ ances. Contraction and swelling were noticed in the region around the suckers (Figure 14). No damage were found in the tegument of females. This fraction did not cause the separa‐

plant is an important material for pharmaceutical research [87].

ure 8 in order to be analyzed by scanning electron microscopy.

13) were also observed*.* No damages to the sucker were found.

modial (against *Plasmodium berghei*) [96], and antioxidant [97] properties.

*• Phyllanthus amarus*

166 Parasitic Diseases - Schistosomiasis

**Figure 13.** SEM of adult *S. mansoni* specimens after *in vivo* assay with fraction 3 obtained from the organic fraction of *C. verbenacea* at the concentration of 300 mg/kg. **A-B.** Male and female worms, respectively, showing peeling of the tegument. **C-D.** Male worms showing adhesion of host's cells to its tegument and formation of vesicles. **et** – erosion of the tegument; **ch** – host's cells adhered to the surface of *S. mansoni*; **l** – host's leukocytes; **dt** – destruction of the tegu‐ ment; **v** – vesicle.

**•** The tested fractions of *B. trimera, C. verbenacea* and *P. amarus* caused tegumental changes in adult *S. mansoni* specimens, including peeling or erosion of the surface membrane, for‐ mation of vesicles, destruction of tubercles, and modifications in the suckers. It is worth

**•** Since the tegument of *S. mansoni* is a major chemotherapeutic target, we can infer that the fractions of *B. trimera, C. verbenacea* and *P. amarus* have a promising schistosomicidal ac‐ tivity, more studies being needed in order to isolate and identify their compounds active

against the worm and to understand their mechanism of action on the tegument.

noting that such changes were more intense in male worms.

The authors are thankful to CAPES and FAPESP for financial support.

, Vera Lúcia Garcia Rehder2

Pluridisciplinary Research Center (CPQBA), Paulínia, São Paulo, Brazil

The Lancet Infectious Diseases 2006; 6: 411-425.

na Tropical 2003; 36: 211-6.

\*Address all correspondence to: claudineide@gmail.com

Claudineide Nascimento Fernandes de Oliveira1\*, Rosimeire Nunes de Oliveira1

1 Department of Animal Biology, State University of Campinas (Unicamp), Campinas,

2 Division of Organic Chemistry and Pharmaceuticals, Chemical, Biological and Agricultural

[1] Engels D, Chitsulo L, Montresor A, Savioli L. The global epidemiological situation of schistosomiasis and new approaches to control and research. Acta Tropica 2002; 82:

[2] Steinmann P, Keiser J, Bos R, Tanner M, Utzinger J. Schistosomiais and water resour‐ ces development: systematic review, meta-analysis, and estimates of people at risk.

[3] Bina JC, Prata A. Esquistossomose na área hiperendêmica de Taquarendi. I – Infecção pelo *Schistosoma mansoni* e formas graves. Revista da Sociedade Brasileira de Medici‐

,

and Silmara Marques Allegretti1

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169

**Acknowledgments**

**Author details**

Tarsila Ferraz Frezza1

São Paulo, Brazil

**References**

139-146.

**Figure 14.** SEM of adult *S. mansoni* specimens after *in vivo* assay with butane fraction 2 from butane extract at the concentration of 100 mg/kg/3 consecutive days. **A.** Area of the gynaecophoric canal showing perforations (**p**). **B.** Rup‐ ture of tubercles (**rtb**) and peeling (**d**) on the tegument of a male worm. **C.** Peeling (**d**) and destruction of tubercles (**dtb**). **D.** Formation of several vesicles (**v**). **E.** Formation of protuberances (**pt**) on the tegument. **F.** Contraction of the ventral sucker (**cvv**) and swelling (**i**) of the region around the suckers.

## **6. Conclusions**

**•** The combined use of 0.1 M sodium cacodylate buffer and 2.5% glutaraldehyde and the reduction in fixation time provided more distinct images with no artefacts, in contrary to the combination of Karnovsky solution with 0.1 M phosphate buffer.


## **Acknowledgments**

The authors are thankful to CAPES and FAPESP for financial support.

## **Author details**

Claudineide Nascimento Fernandes de Oliveira1\*, Rosimeire Nunes de Oliveira1 , Tarsila Ferraz Frezza1 , Vera Lúcia Garcia Rehder2 and Silmara Marques Allegretti1

\*Address all correspondence to: claudineide@gmail.com

1 Department of Animal Biology, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil

2 Division of Organic Chemistry and Pharmaceuticals, Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA), Paulínia, São Paulo, Brazil

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**Figure 14.** SEM of adult *S. mansoni* specimens after *in vivo* assay with butane fraction 2 from butane extract at the concentration of 100 mg/kg/3 consecutive days. **A.** Area of the gynaecophoric canal showing perforations (**p**). **B.** Rup‐ ture of tubercles (**rtb**) and peeling (**d**) on the tegument of a male worm. **C.** Peeling (**d**) and destruction of tubercles (**dtb**). **D.** Formation of several vesicles (**v**). **E.** Formation of protuberances (**pt**) on the tegument. **F.** Contraction of the

**•** The combined use of 0.1 M sodium cacodylate buffer and 2.5% glutaraldehyde and the reduction in fixation time provided more distinct images with no artefacts, in contrary to

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**6. Conclusions**

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**Chapter 9**

**Solving the Riddle of the Lung-Stage Schistosomula**

**Vaccine for Schistosomiasis**

Rashika El Ridi and Hatem Tallima

http://dx.doi.org/10.5772/52922

**1. Introduction**

have delayed progress.

tioned or referred to.

able to interact with the parasite.

Additional information is available at the end of the chapter

**Paved the Way to a Novel Remedy and an Efficacious**

The field of schistosomiasis vaccine has suffered from several entrenched dogmas, which

The first dogma states that the main mechanism of innate and acquired immunity-related parasite attrition is antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC has been shown to effectively mediate killing of 3-, 18- or 24 hr-old schistosomula in human, mouse, and rat models. However, this phenomenon is of no in vivo relevance as larvae of this age are still in the epidermis, impervious to host immune attacks. Intact, healthy older larvae, pre-adults and adult schistosomes are entirely invisible to the immune system, and thus, are never threatened by ADCC in vitro or in vivo. Concurrently, the immune effectors "hunt" for larvae in the pulmonary capillaries, proposed by von Lichtenberg et al. in 1977 [1] as a plausible mechanism for resistance to infection, was entirely neglected, and never men‐

Second dogma is to consider parasite surface membrane antigens of great importance as vaccine antigens, because they reside at the host-parasite interface, and were shown to in‐ duce robust immune responses. However, schistosome surface membrane molecules are hidden, inaccessible to host antibodies, and accordingly, induced immune effectors are un‐

Third, stressing that Th1 immune responses are the pillars for acquired immunity to larval infection in mice. This dogma is entrenched notwithstanding the numerous re‐ ports documenting the importance of exclusive type 2 immune responses in rat, monkey, and human schistosomiasis, and despite that larval antigens induce principally Th1 relat‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 El Ridi and Tallima; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
