Abstract: Light Microscopy (LM) and Scanning Electron Microscopy (SEM) were used to study the egg and miracidium of Fasciola gigantica and redia, cercaria and metacercaria from field infected Lymnaea (cailliaudi) natalensis snails, to analyze the differences between eggs and intra-molluscan stages of F. hepatica and F. gigantica in a zone of sympatry. The egg of F. gigantica has an umbilicus-like invagination at the posterior end of the egg shell. The emerged miracidium has an elongated conical body that has a broad anterior end and tapering posterior end. The surface was found to be covered with varied lengths of cilia except regions of lateral connection of epidermal plates. The redia of Fasciola sp. has a caudal papilliform process. Tail of cercaria was found to be provided with two fin folds. Steps of encystation of the cercaria were described as a variable morphological change in cercarial body and cyst wall.
INTRODUCTION
Fascioliasis is a worldwide problem caused by the liver fluke (Dalton, 1999; Hurtrez et al., 2001). In Egypt, this re-emerging disease is caused mainly by Fasciola hepatica and F. gigantica (Lotfy et al., 2002). It is a serious disease of herbivorous animals (Torgerson and Claxton, 1999), leading to huge economic losses in live-stock production, while human infection has long been seemed to be accidentally. Fascioliasis has recently appeared as an outstanding health problem in Egypt, causing severe illness of human liver (Farag et al., 1979; Farag, 1998), which is unfortunately suffering also from schistosomiasis (Esteban et al., 2003). Studies on fasciolosis are therefore required to understand the epidemiology of this re-emerging disease (Hurtrez et al., 2001).
Many studies have been undertaken in medical and veterinary parasitology, as well as in molecular biology (Dalton, 1999). Surprisingly, there is still much to be discovered in morphology of the larval stages for a better understanding. The larval stages of F. hepatica had been widely studied (Andrews, 1999), while those of F. gigantica have been only partly described. However, it is extremely important to have reliable criteria to distinguish the two species in zones of sympatry, like in Egypt (Lotfy et al., 2002). A lot of studies deal with the measurements and shape of the eggs of Fasciola sp. Eggs of Fasciola consist of a fertilized ovum with vitelline cells surrounded with proteinous shell. Eggs of F. hepatica are operculated with characteristic yellowish brown colour (Andrews, 1999). They are not readily differentiated from eggs of F. gigantica (Chen et al., 1990). The miracidium of F. hepatica (130x28 μm) comes out of the egg and swims at great speed. It has a conical shaped body covered with cilia (Malek, 1980). Formation of sporocyst, the different generation of rediae, morphologically and the conditions to produce cercariae, the structure and development of cercariae and steps of encystation had been studied (Koie et al., 1976; Malek, 1980; Spithill et al., 1999). Metacercariae have a big importance for parasitologists so the shape, structure, the size, the life spane, the encystation and excystation steps and infectivity were studied (Robert, 1950; Dinnik and Dinnik, 1956; Dawes, 1959; Kendall, 1965; Wilson and Draskau, 1976; Koie et al., 1976, 1977; Malek, 1980; Beaver et al., 1984; Yadav and Gupta, 1988; Chen et al., 1990; Rakotondravao et al., 1992; Abrous et al., 1998; Srimuzipo et al., 2000). Koie et al. (1977) have studied the redia of F. hepatica but they had not recorded any process at the posterior end, although Krejci and Fried (1994) reported papilliform process at the posterior end of the rediae of Echinostoma caproni and E. trivolvis. Belding (1965) described the cercariae of Fasciola, but they have not recorded any fin folds like structure. Koie et al. (1977) reported that the tail of F. hepatica cercaria is provided with dorsolateral folds. Krejci and Fried (1994) reported fin folds but they are two in dorsal position in both Echinostoma caproni and E. trivolvis, although they have one ventral fold and two ventral folds, respectively.
So, the aim of this study is to analyze the differences between eggs and intra-molluscan stages of F. hepatica and F. gigantica in a zone of sympatry.
MATERIALS AND METHODS
The sexually mature Fasciola were collected from different slaughterhouses in Qena governorate in Egypt. The study has been done within a period between 1993-1997. The collected flukes were washed several times in saline solution to be free from the debris; dissecting needle was used in extraction of intrauterine eggs. 50-100. According to Drury and Wallingten (1980), the eggs were fixed in 10% neutral formalin and mounted in Kaiser’s glycerol-jelly (aqueous media). A considerable amount of eggs (more than 50) were put in small Petri dish in an incubator at 28°C for hatching. The obtained miracidia were studied with light microscopy.
L. (cailliaudi) natalensis snails were collected, according to Frandsen (1983) from suitable places and carried in plastic containers to the laboratory, identified and counted. Emergence of cercariae was induced by placing groups of snails under indirect sunlight or artificial illumination in glass containers with dechlorinated tap water for half an hour, according to Frandsen and Christensen (1984). In case of emergence of cercariae, the snails were subsequently transferred individually to small beakers and the above scheduled procedures were repeated. According to Khalifa (1972) and Hassan (1987), biological characters and structure were studied for identification of these cercariae. According to Abdel-Ghani (1958), Willomitzer (1974) and Schillhorn-Van-Veen (1980), the shell of infected snails was crushed gently and the fleshy part was dissected. The different intramolluscan stages were transferred to a glass slide for detailed examination. For light microscopic studies, miracidiae were fixed in 5% neutral formalin, cercariae, rediae and metacercariae were fixed in 10% neutral formalin for 2 h. Staining was done according to Drury and Wallington (1980) using Kirkpatrick’s carmalum staining method. Differentiation, dehydration, clearing and mounting were carried out. Preparation of samples for scanning electron microscope was done as follows: eggs or larval stages of Fasciola spp. were fixed in 5% glutaraldehyde and dropped in sodium cacodylate buffer (pH 7.3) for 48 h. The samples were washed 3 changes in the same buffer. Postfixation was done by adding 1% osmium tetroxide for 2 h. The samples were washed again in the same buffer 3 times. Dehydration was done in ascending concentrations of ethanol. The excess alcohol was withdrawn after passing from water to amylacetate. The specimen was placed in a chamber where the liquid carbon dioxide is used to substitute for amylacetate, then heated to 35°C to separate carbon dioxide from the specimens and then mounted on holders. Sputter coating (using gold) was done immediately after critical point. It took about 2-3 min and then the specimens were used for examination with scanning electron microscope.
RESULTS
F. gigantica Eggs
Eggs are large yellowish and operculated with thin shell. It has a distinct,
flat operculum and an umbilicus-like invagination at the posterior end of the
shell (Fig. 1). Using SEM in studying the eggs has shown the
outer surface of the egg shell to be smooth and devoid of any microspines and
highly conspicuous umbilicus-like invagination on the shell in the opposite
side of the operculated end (Fig. 2 and 3).
This umbilicus is sometimes containing some fine debris taking different shapes
in SEM photos. This debris may give a false impression of a knob in some eggs
seen by light microscopy.
F. gigantica Miracidium
The emerged miracidium swims rapidly in aimless directions. It has an elongated
conical body that has a broad anterior end and tapering posterior end. The surface
is covered with numerous cilia, except in lateral connection regions of epidermal
plates. These cilia are found to be characteristically longer on the apical
part of the anterior end and the posterior extremity than the cilia on the rest
of the body.
| Fig. 1: | Light micrograph of egg of F. gigantica showing flat small operculum (short arrow) and an umbilicus-like invagination (arrowhead). X 400 |
| Fig. 2: | Scanning electron micrograph of the egg showing an umbilicus-like invagination (arrowhead). X 5.000 |
| Fig. 3: | Scanning electron micrograph of the egg showing another shape of the umblicus-like invagination (arrowhead). X 5.000 |
| Fig. 4: | Light micrograph of miracidium of F. gigantica showing apical papilla (ap. p.) and varied length of cilia; anterior and posterior long cilia (l. c.), eye spot (e. s.), short cilia (s. c.) and cilia free region (c. f. r.). X 400 |
There is an apical or boring papilla, on which open one apical gland and two pair of penetration glands. There is one pair of eye spots at the right side of midline of anterior part (Fig. 4). The miracidium has one pair of flame cells situated at the end of second third of the body. Each flame cell leads to a tubule which goes down to the lateral side to end in an excretory pore. Germ cells are scattered in the posterior part. Studying the miracidium with SEM has shown the typical pyriform shape of the body. The apical papilla is shown in the middle of the anterior broad part, while the whole surface of the body was illustrating dark pits with variable size (Fig. 5).
| Fig. 5: | Scanning electron micrograph of the miracidium showing the conical body shaped, apical papilla (ap. p.) and many pits on the surface. X 1,000 |
| Fig. 6: | Light micrograph of mother redia of Fasciola sp. showing the muscular pharynx (ph.), collar (c.), germ cells (arrow heads) and a caudal papilliform process (p. p.). X 40l |
Description of Larval Stages of Fasciola sp. Obtained from Field
Infected Snails
Redia
Mother redia has an elongated, flat body with an anterior projecting circular
ridge or collar and ended with caudal papilliform process (Fig.
6). It has a muscular pharynx followed by a simple sac-like intestine (gut).
The mother redia contains undifferentiated structures and germ cells. Mother
redia measures 1.47 - 1.86 (av 1.65) mm in length and 0.39-0.59 (av 0.47) mm
in width. The gut length varied between 0.68-0.95 (av 0.81) mm in length, (it
occupy about two thirds of the total body length). Daughter redia (Fig.
7) has a long, cylindrical body but hasn't collar and the caudal papilliform
process is inconspicuous. It has two posterior processes (lappet) at the beginning
of the posterior third of the body. It contains developing cercariae and germ
cells. The alimentary canal begins with mouth, which leads to suctorial pharynx,
followed by a short and simple gut. There is a birth pore at the anterior end
through it the developed cercariae emerge. Daughter redia measured 1.26-3.01
(av 1.65) mm in length by 0.16-0.37 (av 0.29) mm in width. It has a short gut
that measures 0.42-1.02 (av 0.66) mm in length (occupy about one-third of the
total body length). SEM study of the daughter redia revealed the conspicuous
suctorial pharynex with rounded muscular walls (Fig. 8, 9).
Cercaria
The emerged cercaria in water swims actively in aimless direction. It has
a large heart shaped body and simple long tail. The body has a characteristically
thick wall (Fig. 10) and is surrounded by minute spines all
over its surface. The body length ranged between 0.15-0.21 mm (mean 0.18 mm)
in length and 0.18-0.24 mm (mean 0.20 mm) in width. The tail length is nearly
three times as the body, as its length ranges between 0.56-0.7 mm (mean 0.61
mm) its width was 0.35-0.056 mm (mean 0.048 mm). The ventral sucker is larger
than the oral sucker. The former varied from 0.028-0.039 mm (mean 0.033 mm),
while the latter 0.037-0.048 mm (mean 0.041 mm). The rudiment of the alimentary
canal consists of a mouth followed by pharynx surrounding the oesophagus that
leads to intestine. The latter bifurcates into two simple branches that extend
around the ventral sucker to a level below the posterior border of the ventral
sucker. The genital primordium is dumble-shaped with two unequal rounded or
oval masses connected together with a longitudinal bar.
| Fig. 7: | Light micrograph of daughter redia of Fasciola sp. showing developing cercariae (arrowheads) and germ cells (short arrow). X 40 |
| Fig. 8: | Scanning electron micrograph of daughter redia showing pharynx (ph.), collar (c.) and a posterior process (post. p.). X 150 |
| Fig. 9: | Scanning electron micrograph of the anterior end of daughter redia showing the muscular pharynx (ph.) and collar (c.). X 750 |
It is located between the upper surface of the excretory vesicle and the upper border of the ventral sucker. The body is full of numerous cystogenous glands. Due to the thick body wall and the densely deposited cytogenous glands, it was difficult to visualize the excretory system and flame cell formula. Study of the cercaria with SEM has shown that the body is concave ventrally (Fig. 11) and the tail is provided with two fin-like processes at its lateral sides (Fig. 12).
| Fig. 10: | Light micrograph of cercaria of Fasciola sp. showing the general characters. X 100 |
| Fig. 11: | Scanning electron micrograph of the cercaria, ventral view showing the general shape and ventral concavity of the body. X 200 |
| Fig. 12: | Scanning electron micrograph of tail of cercaria showing two fin folds (arrowheads). X 500 |
Cercarial Encystation
Encystation of cercariae occurred in the following steps:
| Fig. 13: | Light micrograph of recently formed metacercaria of Fasciola sp. X 400 |
| Fig. 14: | Scanning electron micrograph of the metacercaria, showing the general shape and the felt-work filaments. X 350 |
| • | Just after attachments to a suitable object, or even in water (if this was not available) and while the tail is still lashing vigorously from side to side, a thin irregular wall starts to be secreted around the body from the cystogenous glands |
| • | Tail becomes separated from the body while the cystogenous glands secretions become more accumulated in several layers usually four around the more or less rounding-up body. At that stage, many of the internal structures of the cercarial body were easily seen through the cyst wall particularly the suckers and the genital primordium (Fig. 13) |
| • | The wall becomes more and more thick and the cercarial body becomes curled on itself in the cyst, which become slightly smaller in size but with a well developed cyst wall. At that stage it is difficult to differentiate any of the internal structures of the body |
Metacercaria
Metacercaria has a double thick cyst wall. The recently formed metacercaria
has larger diameter than the mature one where the former diameter ranging between
0.224-0.272 mm (mean 0.257 mm) and the latter's diameter ranging between 0.215-0.256
mm (mean 0.235 mm). Non-infected L. natalensis snail swallowed a few
number of metacrecariae, which were later on passed out in a linear shape mixed
with the snail faeces (Fig. 13). Study of the metacercaria
with SEM has shown that the outer layer consists of felt work filaments (Fig.
14).
DISCUSSION
Koie et al. (1976) had not reported the umbilicus-like invagination in the eggs of F. hepatica when they study these eggs with stereoscan microscope, but Allam (1992) had been differentiate between the eggs of F. gigantica and F. hepatica according to the measurements. Krejci and Fried (1994) mentioned the presence of abopercular knobs of the eggs of Echinostoma caproni and E. trivolvis. The measurements of F. hepatica eggs depend on the host (Abrous et al., 1998; Srimuzipo et al., 2000). The present work included light and scanning electron microscopic studies of eggs and larval stages of Fasciola. This study showed that eggs of both species have an umbilicus-like invagination at the posterior rounded end of the shell. Miracidia and all intramolluscan stages and encysted metaceracrial formation. This was shown by SEM pictures (also with light microscope) may be for the first time during the present study. Most of the previous work on miracidia was done on that of F. hepatica. It has been also shown that cilia on anterior and posterior borders of the miracidium are particularly larger than on the rest of the body. Also, disconnections of the miracidial cilia were clearly seen. The SEM also illustrated the miracidium. Koie et al. (1977) have studied the redia of F. hepatica but they had not recorded any process at the posterior end of the rediae, although Krejci and Fried (1994) reported papilliform process at the posterior end of the rediae of Echinostoma caproni and E. trivolvis. In the present study, mother and daughter redia of Fasciola sp. were described by light microscopy and SEM, which recorded presence of caudal papilliform process (conspicuous in mother redia and inconspicuous in daughter redia) in the caudal end of the egg shell. Belding (1965) described the cercariae of Fasciola, but they have not recorded any fin folds like structure. Koie et al. (1977) reported that the tail of F. hepatica is provided with dorso-lateral folds. Krejci and Fried (1994) reported fin folds but they are two in dorsal position in both Echinostoma caproni and E. trivolvis, although they have one ventral fold and two ventral folds, respectively. During the present study cercariae of Fasciola sp. seemed to be not different from previously described forms. However, detail structure of the genital premordium is given. Moreover, SEM has shown a characteristic sochet at the posterior border of the body in which the tail is usually attached. The tail was also shown for the first time in F. gigantica cercariae to be provided with two lateral fin folds, which might to have a great help in swimming of the cercariae. Boray and Enigk (1964) and Dixon (1966) described the encystations process, most of their studies were concentrated on the wall of the metacercariae. During the present study the steps of metacercarial encystations and the variable morphological changes in cercarial body and cyst wall during the steps of encystation were described. Moreover, SEM has shown that the outer layer consisted of felt work filaments. Yadav and Gupta (1988) reported no affection on the viability of F. gigantica metacercariae ingested by non-infected L. natalensisi snails. The present data confirmed this work that the metacercariae were found to be passed unchanged with faeces of the snail.