ISSN (Print) 0023-4001

ISSN (Online) 1738-0006

Korean J Parasitol Vol. 53, No. 1: 59-64, February 2015

▣ ORIGINAL ARTICLE http://dx.doi.org/10.3347/kjp.2015.53.1.59

Experimental Life History and Biological Characteristics of

Fasciola gigantica (Digenea: Fasciolidae)

Anawat Phalee1,2, Chalobol Wongsawad1,*, Amnat Rojanapaibul1, Jong-Yil Chai3

1
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; 2Fisheries Program, Faculty of Agriculture and
Technology, Nakhon Phanom University, Nakhon Phanom 48000, Thailand; 3Department of Parasitology and Tropical Medicine, Seoul National
University College of Medicine, Seoul 110-799, Korea

Abstract: This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult
worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and
Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for mat-
uration study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched
out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and
gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cer-
cariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae
were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice
plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from
day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the av-
erage recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that ex-
perimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study re-
ports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained informa-
tion can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.

Key words: Fasciola gigantica, life history, biological characteristic, Digenea, Fasciolidae

INTRODUCTION conducted on F. gigantica. F. hepatica is widely distributed in

temperate zones, whereas F. gigantica is typically found in tropi-
The giant liver fluke, Fasciola gigantica (Digenea: Fasciolidae), cal zones around the world [5,6].
is important as a plant-borne trematode together with the The general life cycle of fasciolids is described in the follow-
sheep liver fluke, Fasciola hepatica. These parasites are the main ing passage. Adults expel eggs and these are evacuated in the fe-
cause of fascioliasis in ruminants and humans. They live in the ces of the definitive host, usually cows or buffaloes. Miracidia
liver of cattle, buffaloes, sheep, goats, and swines, which have a are hatched from the eggs in water, and they penetrate into sev-
significant impact on growth rate, development, and productiv- eral lymnaeid snails (intermediate hosts), such as Lymnaea viri-
ity of ruminants, and therefore, are considered economically dis, L. columella, L. cousin, L. ollula, L. natalensis, and L. auricularia
significant [1]. In some occasions, these flukes can be infected rubiginosa [7-11], and then develop into the sporocysts, rediae,
in humans, and it has been rarely reported that adult flukes and cercariae. Cercariae separate from the snails and are encyst-
have been recovered from the bile duct of people in Japan, ed to become metacercariae on vegetation (infective stage). The
northern Iran, and Thailand [2-4]. Most of the previous reports metacercariae can then be found on vegetation, such as the rice
have focused on F. hepatica, whereas fewer studies have been plant, stubble, Japanese parsley, and water lilies [8,12]. These
plants are important sources of fodder for definitive hosts. The
• Received 31 May 2014, revised 25 October 2014, accepted 24 December 2014 life cycle completes itself when the definitive hosts eat vegeta-
* Corresponding author ([email protected]) tion containing the metacercariae.
© 2015, Korean Society for Parasitology and Tropical Medicine
Although studies on the life history of F. gigantica have been
This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) sometimes reported in several countries, those studies did not
which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited. cover all life cycle stages. Dreyfuss and Rondeland [13] com-

59
60  Korean J Parasitol Vol. 53, No. 1: 59-64, February 2015

pared the productivity of infected F. gigantica and F. hepatica in cidia. Notes on differentiation of the eggs were recorded daily
Lymnaea tomentosa and found that the number of rediae of F. under a stereomicroscope until miracidia hatched from the
gigantica were substantially greater than that of F. hepatica. The eggs.
patent period and the number of cercariae of both fasciolids
were closely correlated with particular Lymnaea truncatula pop- Larval development in snail hosts
ulation and trematode species [14]. Moreover, Yadav and Gup- One-month-old non-parasitized snails, L. auricularia rubigi-
ta [15] reported that after infection of 2 rabbits with 50 F. gigan- nosa, were used for the experimental infection. One hundred
tica metacercariae derived from L. truncatula, both rabbits died snails were placed in 5 clay pots (20 snails/pot) with a holding
at days 83 and 87 post-infection (PI), and 8 and 10 immature capacity of 2 L of dechlorinated tap water. After then, 200 mi-
adults were recovered from the liver, respectively. This aspect racidia were placed in each clay pot. The exposed snails in the
has not been studied in Thailand, and most reports were fo- clay pots were supplied with fresh lettuce leaves for feeding the
cused on the epidemiology and molecular detection of F. gigan- snails. One exposed snail was crushed daily to observe the lar-
tica. val stages until cercarial-shedding occurred. The cercarial shed-
Fascioliasis in domestic ruminants of Thailand is mainly ding was investigated with the aid of a stereomicroscope.
passed on by F. gigantica, and the adult stage has commonly One-month old cultured rice plants (Oryza sativa) were used
recorded in cows and water buffaloes from different parts of for encystation of the cercariae to develop into the metacercar-
the country. The highest prevalence of F. gigantica in cows and iae. Mature cercariae were placed in rice plant pots containing
water buffaloes was found in the north and northeast, and the dechlorinated tap water for the cercarial encystment. Rice
lowest incidence in the south [16,17]. However, there is sparse plants were examined for the presence of metacercariae both
information on the life history of F. gigantica, especially with by naked eyes and with a stereomicroscope.
regard to the dynamics of the larval stage in Thailand. The lar-
vae are transmitted to the intermediate hosts, which in many Growth and development of worms in mice
cases are vegetation, as well as to the definitive hosts. Under- Sixteen albino mice (Mus musculus domesticus) were used as
standing of these stages is needed to understand the epidemi- the experimental definitive host. Thirty experimentally encyst-
ology of fascioliasis in Thailand. ed metacercariae were fed to each mouse, and then they were
Thus, the present study was designed to investigate the com- sacrificed every 3 days post-infection (PI). Adult flukes were
plete life history of F. gigantica in Thailand. Previous studies observed in the intestine and liver, and worm recovery and
have been only on 1 or 2 stages of the life history. Moreover, adult maturity were examined.
the previous studies focused on F. hepatica, although F. hepatica
and F. gigantica are closely related species. The results can be Ethical statements
applied for treatment, management, and control of these para- All experimental hosts were managed according to the
sitic infections. guidelines approved by the Animal Ethics Committee of the
Faculty of Science, Chiang Mai University, and this document
MATERIALS AND METHODS (no. RE 002/13) was approved by the committee. The guide-
lines for animal care were used according to the International
Collection and culture of eggs Guiding Principles of Biomedical Research Involving Animals
Fresh eggs of F. gigantica were recovered from bile of the gall of Council for International Organizations of Medical Sciences
bladder of water buffaloes (Bubalus bubalis) in the abattoir of (CIOMS). The gall bladders of the water buffaloes that were
Chiang Mai Province, Thailand. The eggs were washed several used for this study were processed as part of the work of the
times with dechlorinated tap water and collected under a ste- abattoir.
reomicroscope.
A total of 1,000 F. gigantica eggs were placed in each well of RESULTS
multiple-well plates (6 wells) containing dechlorinated tap wa-
ter, and were then incubated at room temperature (27-31˚C; av. Experimental life cycle and biological characteristics
29˚C) under natural light to allow the development of mira- After incubation, the eggs developed from the unembryo-
 Phalee et al.: Experimental life history of Fasciola gigantica  61

A B C D

Fig. 1. Photographs demonstrating the different stages of Fasciola gigantica eggs during the incubation period. (A) Unembryonated egg.
(B) Embryonated egg. (C) Egg with fully matured miracidium. (D) Egg with escaping miracidium. Scale bar= 0.05 mm.

nate stage (Fig. 1A) to the embryonated stage (Fig. 1B) on day
3 post incubation, and then they fully developed into eggs
containing miracidia (Fig. 1C). Hatching began to occur on
day 11, while most eggs hatched on day 12. Fully-developed
miracidia then protruded from the eggs by pushing through
the operculum of the eggs (Fig. 1D). 2
5 4 3
Free-swimming miracidia encountered and penetrated the
appropriate snail intermediate host (L. auricularia rubiginosa).
6 1
Miracidia then attached themselves to the snails’ body via the
apical papillae and lost its ciliated covering and transformed
into the next stage. The miracidia that failed to find a snail 7

host died within 24 hr. In infected snails, the miracidia trans-

formed to the next 3 larval stages, referred to as the sporocyst,
redia, and cercaria. The developmental stages in the snails are
depicted in Fig. 2.
The miracidium lost its ciliated covering and became a young
sporocyst on day 3. The young sporocyst had packed germinal Fig. 2. Illustration demonstrating the development of the larval
cells and eyespots (no. 1 in Fig. 2). On day 7 post incubation, stages of F. gigantica, as found in the experimental snail host,
the young sporocyst transformed to become a mature sporocyst, Lymnaea auricularia rubiginosa.

redia-like in shape, but with no pharynx or primitive gut (no. 2).

On day 10 post incubation, the mature sporocyst transformed the plant or substrate, released the outer layer of the cyst, and
to become a young redia, with the visible pharynx and primi- produced capsules to cover their bodies. At this point, the cer-
tive gut, the unique characteristics of the redia (no. 3). Each ger- cariae lost their tails and developed into the metacercarial
minal cell in the sporocyst developed and formed into the ger- stage (Fig. 3). The life cycle was completed when the metacer-
minal ball on day 14 post incubation (no. 4). Young daughter cariae were eaten by a definitive host, such as a mammal.
rediae and cercariae were present on day 21 (no. 5). On day 24,
daughter rediae and young cercariae became fully developed Morphology of larval stages
(no. 6), and then the fully developed cercariae were separated Eggs: The eggs are large, oval, yellowish-brown with a thin
from the rediae through a birth pore to the snail’s tissue and shell, and are flat and operculated. They are 0.12-0.18 (0.15)
then shed to the water on day 39 post incubation (no. 7). mm in length and 0.08-0.11 (0.09) mm in width. The outer
Active cercariae emerged from the snail and swam freely to surface of the eggs was smooth (Fig. 4A).
search for the substrate for encystment. The cercariae came Miracidia: The body is elongated and conical and has a
into contact with the rice plant or other substrate, adhered to broad anterior part and a posterior part that tapers to a blunt
62  Korean J Parasitol Vol. 53, No. 1: 59-64, February 2015

A B C

A C

Fig. 3. Photographs demonstrating the metacercariae of F. gigan-

tica. (A) Metacercariae (arrowhead) adhered to the stem of the
rice plant. (B) A metacercaria. (C) Capsule of a metacercaria. D
Scale bar= 0.05 mm.

E F
end. It is 0.13-0.17 (0.15) mm long and 0.05-0.09 (0.07) mm
wide. The surface is completely occupied with cilia. An apical
papilla is seen in the middle of the anterior part, and there is a
pair of darkly stained eyespots that are visible near the anterior
part of the body. Germinal cells are scattered at the posterior
segment (Fig. 4B).
Sporocysts: The young sporocyst is oval, 0.11-0.15 (0.13)
G H I
mm in length and 0.10-0.11 (0.10) mm in width. The sporocyst
consists initially of a minute ball of tightly packed germinal
Fig. 4. Illustration demonstrating the larval morphology of F. gi-
cells. At this stage, the eyespots can be seen (Fig. 4C). Each ger- gantica. (A) Egg. (B) Miracidium. (C) Young sporocyst. (D) Mature
minal cell gives rise to new germinal cells and these then multi- sporocyst. (E) Mother redia. (F) Daughter redia. (G) Cercaria. (H)
Encapsulated metacercaria. (I) Metacercaria. Scale bars (A-
plies to become germinal balls. After that, the bodies of mature
D)= 0.03 mm; (E-G)= 0.1 mm; (H-I)= 0.05 mm.
sporocysts are elongated and formed to rediae (Fig. 4D).
Rediae: The redia is roughly cylindrical in shape, and the
birth pore is located at the anterior end. The unique character- pacts. The diameters of the capsules range from 0.26 to 0.30
istics of this stage include 2 lateral projections at the posterior (0.28) mm (Fig. 4H). The metacercariae have a double thick
end. The redia stage consists of a mother redia and a daughter wall that consists of an outer and inner cyst, which is 0.19-0.23
redia. The mother redia contain many daughter rediae and (0.20) mm in diameter. The cyst is white when laid and is al-
germinal balls (Fig. 4E), while the daughter redia contain most immediately infective to the definitive host. After 1 or 2
many cercariae and germinal balls (Fig. 4F). days, the cyst gradually becomes yellow and darkens in color
Cercariae: The cercaria is tadpole-like with a discoidal body (Fig. 4I).
and a long tail. They possess an oral sucker and a ventral suck-
er in the center of their bodies and have very conspicuous cys- Growth and development of worms in mice
togenous glands and a forked intestine. A pharynx and preph- The morphological changes of the adult worms are shown
arynx are present (Fig. 4G). in Fig. 5. The metacercariae excysted to become young adult
Metacercariae: The metacercariae of F. gigantica are covered worms and were then recovered in the intestine on days 3 and
with capsules, which serve to protect from environmental im- 6 PI, until day 9 PI when they were found in the liver of the
 Phalee et al.: Experimental life history of Fasciola gigantica  63

Fig. 5. Different stages of F. gigantica recovered from albino mice. Scale bar= 1 mm.

host. The rate of parasitic incidence was 100%, and the average of the embryo in the eggs [18]. After the miracidia hatched,
worm recovery rate was 35.8%. The genital pore was initially they actively swam to search for lymnaeid snail species, such as
revealed on day 9 PI, while ceca were found on day 18 PI. L. columella, L. cousin, L. natalensis, and L. truncatula [9,20]. The
However, the testes and ovary were discovered on day 27 PI, most frequently involved intermeadiate hosts were L. auricularia
and they developed to maturity on day 39 PI. Immature eggs rubiginosa and L. natalensis [21]. In our study, L. auricularia rubig-
were discovered on day 42 PI, and these eggs were developed inosa were used as the experimental snail host, and miracidia
fully on day 48 PI, which indicated parasite maturation. successfully penetrated this snail host and developed to be-
come sporocysts, rediae, and cercariae. When the miracidia (F.
DISCUSSION gigantica) failed to find a snail host, they died within 24 hr. This
was also true for the miracidia of F. hepatica [19].
The present study is the first to report the complete life his- In infected snails, the larval stages of F. gigantica were com-
tory of F. gigantica in Thailand. Our findings show that L. au- pletely developed, and the cercariae were separated from the
ricularia rubiginosa is an intermediate host of F. gigantica, which snails within 42 days post incubation. This was different from
is distributed in all regions of Thailand. Moreover, the ob- the reports of Dreyfuss and Rondelaud [13] in which the cer-
tained metacercariae in this study developed to adults in mice, carial shedding occurred at day 54 post incubation at 23˚C.
other than ruminants and humans. The pattern of the life his- The free-swimming cercariae contracted and successfully ad-
tory consisted of 7 stages involving the egg, miracidium, spo- hered to the rice plant or other substrate and suddenly formed
rocyst, redia, cercaria, metacercaria, and adult stage, which is the metacercarial cysts. The metacercarial encystment was suit-
closely similar to the related species, F. hepatica [18]. able at high temperatures of above 24˚C [22]. The life cycle
In this study, the suitable hatching temperature ranged from was completed when the metacercariae were eaten by a mam-
27-31˚C, at which the eggs successfully developed and hatched malian definitive host.
after 12 days, whereas the eggs of F. hepatica hatched within 2-4 In this study, eggs of F. gigantica were found to be larger than
weeks at the temperature range of 23-26˚C [18,19]. This indi- those reported for F. hepatica [23]. The miracidia of F. gigantica
cated that the development of F. gigantica eggs in this study was were also bigger than those of F. hepatica [18]. The sporocyst
more rapid than that of F. hepatica, and the high temperature was oval, the redia was roughly cylindrical, and the cercaria
may have affected the development and hatching of the eggs. A was tadpole-like with a long tail. The metacercarial cysts were
lower temperature resulted in a slower rate of development, protected with capsules, which had a double thick wall that
while higher temperature results in a faster rate of development consisted of an outer cyst and an inner cyst, all of which were
64  Korean J Parasitol Vol. 53, No. 1: 59-64, February 2015

of equal size to the cyst of F. hepatica [21]. 1995; 58: 313-318.

The present study dealt with the completion of the full life 8. Yoshihara S, Ueno H. Ingestion of Fasciola gigantica metacercari-
ae by the intermediate host snail, Lymnaea ollula, and infectivity
history of F. gigantica using experimental hosts in the laborato-
of discharged metacercariae. Southeast Asian J Trop Med Public
ry. Our findings indicate that F. gigantica can be completely de- Health 2004; 35: 535-539.
veloped in nature, and ruminants and humans living in Thai- 9. Salazar L, Estrada VE, Velásquez LE. Effect of the exposure to Fas-
land may be at the risk of infection with F. gigantica. ciola hepatica (Trematoda: Digenea) on life history traits of Lym-
naea cousini and Lymnaea columella (Gastropoda: Lymnaeidae).
Exp Parasitol 2006; 114: 77-83.
ACKNOWLEDGMENTS
10. Magalhães KG, Jannotti-Passos LK, Caldeira RL, Berne ME,
Muller G, Carvalho OS, Lenzi HL. Isolation and detection of Fas-
We would like to thank Ms. Waraporn Noikong and Mr. ciola hepatica DNA in Lymnaea viatrix from formalin-fixed and
Suksan Chuboon for their laboratory assistance. We also thank paraffin-embedded tissues through multiplex-PCR. Vet Parasitol
Mr. Russell Kirk Hollis for approving English grammar. Grate- 2008; 152: 333-338.
ful thanks are extended to the Department of Biology, Faculty 11. Hussein AN, Khalifa RM. Experimental infections with Fasciola
in snails, mice and rabbits. Parasitol Res 2008; 102: 1165-1170.
of Science, Chiang Mai University, Applied Technology for
12. Suhardono, Roberts JA, Copeman DB. Distribution of metacer-
Biodiversity Research Unit, Institute for Science and Technolo- cariae of Fasciola gigantica on rice straw. Trop Anim Health Prod
gy, and the Graduate School, Chiang Mai University, Thailand 2006; 38: 117-119.
for provision of the facilities. 13. Dreyfuss G, Rondelaud D. Comparative studies on the produc-
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CONFLICT OF INTEREST
Vet Res 1995; 81: 531-536.
14. Dreyfuss G, Rondelaud D. Fasciola gigantica and F. hepatica: a
We have no conflict of interest related to this study. comparative study of some characteristics of Fasciola infection in
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