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vertebrates, was defined as a potential candidate antigen to develop vaccines against some
helminthiases, i.e. schistosomiasis and filariasis. Several vaccination trials are under way, or
have been conducted, using either the native or recombinant protein. Different levels of
response and protection have been achieved in animals (Cancela et al., 2004).
This work aims to evaluate an early immunodiagnosis protocol for fascioliasis using sandwich
ELISA to Antigen. On the other hand, study the humoral and cellular immune response for
developing a vaccine using F. gigantica Antigen which is isolated from adult worm
homogenate during parasite invasion to give protection against this disease.
6
Review of Literature
Fascioliasis may lead to economic losses of weight gain, fertility and milk production, in case
of infection with F. hepatica, ovine fascioliasis can result in significant blood loss, representing
a loss of metabolizable energy. This together with impaired appetite can have an adverse effect
on weight gain, the greatest reduction in weight gain occurred in first 16 week (wk) of infection
and there is still a significant decrease in weight gain during chronic stage of the disease
(Torgerson and Claxton, 1999).
Goat fascioliasis is considered less frequent and less important than sheep and cattle infection;
however, fascioliasis occurs as a major constraint for goat production in many areas of the
world. It is known that the course of fascioliasis in the host varies according to the ruminant
species; for example, fascioliasis is often chronic and subclinical in cattle, but acute and
accompanied by high mortality in sheep (Soulsby, 1982). Furthermore, the response against the
parasite differs in different host species. Humans are usually infected by ingestion of leafy
plants that contain infective metacercariae (Mas-Coma et al., 1999).
Epidemiology of the infection with F. gigantica is similar to that with F. hepatica, because the
two parasites infect the liver of herbivorous animals and have similar forms of life cycle. Social
and agricultural differences between tropical and temperate regions where F. gigantica and F.
hepatica are endemic are considered as important differences that separate between the
epidemiology of the two parasites (Spithill et al., 1997).
In Egypt, Haridy et al. (1999) reported that, during the years 1994 to 1997 the overall
slaughtered animals in Egyptian abattoirs was 2,003,200 sheep and goats, 2,624,239 cattle and
3,536,744 buffaloes. The overall rates of fascioliasis were 2.02 % for sheep and goats, 3.54 %
for cattle and 1.58 % for buffaloes. Macroscopic examination of sheep liver showed up to 100
flukes per liver inside largely dilated thick walled bile ducts. Cattle liver showed up to 275
flukes per liver inside thickened dilated and calcareous bile ducts with offensive yellowish
brown bile. Buffalo's liver showed up to 330 flukes per liver. Microscopic examination showed
mainly thickened wall, hyperplasia and marked fibrosis.
Eggs of Fasciola passed from bile duct into duodenum and later into feces. Eggs are
composed of large number of yolk granules surrounding the fertilized ovum (140 µm long).
When, these eggs are passed out into feces. They undergo embryonation, operculate and consist
of two types of cells, somatic cell, which develops into the succeeding stage (miracidia), and
germ or propagative cell responsible for the formation of the following stage outside the host,
where they are exposed to several physio-chemical factors as temperature, humidity, and
oxygen tension (Andrews, 1999).
7
Miracidium escapes the egg within 1-2 wk in water, where it infects the snail as an
intermediate host, Lymnaea spp. The penetration process involves a mechanical boring to the
snail, which is facilitated by secretion of proteolytic enzymes, the time required for the
development of miracidia in the eggs varies with temperature, 10-11 days at 37-38
o
C, 21-24
days at 25
o
C and 33 days at 17-22
o
C (Garcia, 2001). Eggs do not survive at temperatures
higher than 43-44
o
C (Spithill et al., 1999a). Eggs of F. gigantica do not all develop at the same
rate so that, from the same batch, miracidia may hatch over a period up to 14 wk, thus
enhancing their opportunity to infect a snail (Guralp et al., 1964). Once miracidia released
from the egg, they survive in water for 18-26 hour (hr) (Asanji, 1988). When the miracidium
reaches its proper host, it adheres to its body by the apical papilla and penetrates its tissues,
finding its way to the blood spaces in the roof of the pulmonary cavity (Soliman, 1996).
Within the intermediate host, it develops into sporocyst (Agarwal, 2003). Each sporocyst gives
rise to 5-8 rediae that developed later into cercariae. The later leave the snail in 4-7 wk from the
time of infection (Soulsby, 1973), The cercariae has an oval body and a long unforked tail
(leptocercous); the body possesses a small anterior sucker surrounding the mouth, a large
ventral sucker, a pharynx, a short esophagus and a two branched intestine in addition to
rudiments of the genital organs (Soliman, 1996). Cercariae are shed in up to 15 waves (usually
three or fewer), 1-8 days apart over a period of about 7-50 days, in each wave, 50-70 cercariae
are released (Da-Costa et al., 1994). Shedding commences as early as 20 days post infection
(PI) of snails (Sharma et al., 1989). About 80% of cercariae are shed at night (Da-Costa et al.,
1994). Cercariae swim for some days and eventually attach to aquatic grass blades. After losing
the tail, they become encysted within a chitinous shell secreted by the cystogenous glands, thus
changing into metacercariae (the infective stage). They can withstand adverse conditions for a
wk (Agarwal, 2003). About two-thirds attach to objects within 6.4 cm of the surface of the
water (Ueno and Yoshihara, 1974), and the remainders do not attach but become floating
cysts (Spithill et al., 1999a).
The proportion of floating cysts is higher for F. gigantica than for F. hepatica
(Dreyfuss and Rondelaud, 1997). Newly encysted metacercariae requires at least 24 hr to
become infective (Boray, 1969), and when swallowed by the definitive host, the metacercariae
excysts in the small intestine (Dixon, 1966), where the cyst wall dissolves by the effect of the
intestinal enzymes. The newly encysted juveniles (NEJs) penetrate intestinal mucosa, and then
they develop into young flukes, bore their way through the wall of the host's intestine and
migrate to the peritoneal cavity. After 3-5 days of wandering, they enter the liver causing
serious damage to the hepatic tissue, or they may enter the bile ducts directly from the intestine
8
or through the blood circulation. They become sexually mature within 2-3 months (Soliman,
1996).
Human infection with fascioliasis can result from ingestion of encysted metacercariae
attached to aquatic vegetation and plants (Ragab and Farag, 1978). Eating raw or
incompletely cooked crab or Cray fish, or by accidental transfer of cyst to mouth after handling
raw vegetables, crabs or cray fish during preparation of food (Yokogawa, 1982). Drinking of
contaminated water with encysted metacercariae might be another way of infection, on the
other hand, consuming raw liver dishes prepared from fresh livers infected with immature F.
gigantica that lead to acute and chronic disease characterized by clinical symptoms as anorexia,
fever and weight loss (Taira et al., 1997).
The metacercarial larvae of Fasciola escape from cysts in duodenum and cause no
significant damage as they migrate through duodenum wall into peritoneal cavity. When young
migrating flukes reached the liver parenchyma, they cause traumatic and necrotic lesions due to
heavily infiltration with eosinophils (Eo) (Arora, 2005). The major pathological changes are
seen during migration of these young flukes through liver parenchyma before they enter biliary
tract. They digest the hepatic tissue and cause intensive hemorrhagic lesions, inflammatory
reactions, and destruction of liver tissue and inflammation of bile ducts (Marsden and
Warren, 1984). While, less pathogenic effect to liver can be found when small numbers of
flukes reach the bile duct, leading to inflammation and fibrosis of the bile duct (Smithers and
Doenhoff, 1982). When larvae reach the bile duct, it develops into adult and causes
gastrointestinal symptoms. Some patients develop chronic cholecystitis, cholangitis and
cholilithiasis, which may be accompanied by biliary colic, epigastric pain, jaundice, nausea,
pruritis and upper right quadrant pain. In heavy infections, young worms may wonder back into
liver parenchyma producing abscesses. Larvae migrating through peritoneal cavity may
become lodged in ectopic foci, where abscesses or fibrotic lesions may develop. These sites
include blood vessels, lungs, subcutaneous tissue, ventricles of brain (Arora, 2005).
Consequences of liver damage resulting from the migrating flukes compromises liver
function which is reflected in changes of plasma protein concentration (albumin and globulin).
Changes of levels of hepatic enzymes released into the blood as a result of liver tissue damage
are used as monitor to progress of infection and sensitive diagnostic aid in field infection
(Gajewska et al., 2005). A decrease in hemoglobin, packed cell volume, total erythrocyte
counts and appearance of reticulocytes in blood of F. gigantica infected buffaloes to
hypothyroidism (Ganga et al., 2007). Most patients with fascioliasis develop cell-mediated
immune response and specific antibodies against Fasciola worms. This specific humoral
immune response could be followed by formation of localized or circulating immune complex
9
(CIC), which could be involved in the immunological mechanism of host-fluke relationship
(Shaker et al., 1994). Human fascioliasis is considered as a serious hepatic pathological threat
to livers of Egyptian population, where it is diagnosed from some diseases as acute hepatitis,
initial symptoms most frequently include severe headache, chills and fever. Enlarged tender or
cirrhotic liver accompanied by diarrhea and anemia indicates advanced infection (Keyyu et al.,
2006).
1.
Pathology
A.
In human
The clinical picture of fascioliasis is usually divided into three major forms:
- Acute Fascioliasis: It corresponds to the migratory phase of the NEJs in the liver and occurs
within 2-3 months after acquiring the infection. Fever, abdominal pain, headache, pruritis,
urticaria, weight loss, and Eosinophilia. Transaminase levels are in normal range or are only
minimally elevated, and bilirubin levels are typically in normal range (Patil et al., 2009).
- Chronic Fascioliasis: It corresponds to the presence of mature adult parasites in bile ducts
inside the liver and symptoms begin about 1 month after the exposure to metacercariae, are
fever, general malaise, fatigue, hepatomegaly, anorexia, weight loss, urticaria with
dermatographism and peripheral blood Eosinophilia. The symptoms may be absent in cases of
light infection. The biliary phase may be asymptomatic or there may be symptoms related to
cholangitis and obstruction of the biliary tract due to the enlarging fluke(s). The biliary phase
may last for months or years (Kanoksil et al., 2006).
- Ectopic Fascioliasis: It is found in organs and subcutaneous tissues in the abdominal region.
They were also found in lungs, heart, eyes, brain, and lymph nodes. This is due to secondary
infection in the liver by clostridium species occurring in necrotic hemorrhagic lesions produced
by young parasites migrating within the liver (Cho et al., 1994). Also, rarely, a condition
known as "black disease" occurs as a complication of fascioliasis (Boray, 1999).
B.
In animals
The clinical picture of fascioliasis is usually divided into three major stages:
- Pre-hepatic stage: It is associated with the least pathology but sheep may experience ascites,
pneumonia and fibrous pleuritis. Other signs include edema "bottle jaw" (Behm and Sangster,
1999).
- Hepatic or parenchymal stage: It is associated with immature flukes burrowing through the
liver. Clinical signs are preferable to hepatitis and include signs referable to derangement of
amino acid metabolism, carbohydrate and lipid balance, urea synthesis, detoxification
metabolism, ketogenesis and albumin and glutathione synthesis. Clinical signs include
inappetance, ill-thrift, abdominal pain, jaundice, anemia, weakness, respiratory distress and
10
collapse. Hepatic fibrosis and calcification may occur as a result of chronic infection,
particularly in less permissible hosts like cattle and humans (Behm and Sangster, 1999).
- Post-hepatic stage: It is associated with the establishment of adult flukes in the bile ducts
where they become patent. This occurs approximately 7 to 8 wk PI. Little inflammatory
response occurs once flukes are established in the bile duct, however bile duct hypertrophy and
calcification is not uncommon in cattle or humans. The presence of a large number of flukes
can mechanically obstruct bile ducts (Smyth, 1994; Andrews, 1999; Behm and Sangster,
1999).
Economical effect of fascioliasis in sheep is due to sudden deaths of animals as well as
reduction of weight gain and wool production. In goats and cattle, the clinical manifestation is
similar to sheep. However, acquired resistance to F. hepatica infection is well-known in adult
cattle (Behm and Sangster, 1999; Boray, 1999).
2.
Immune Response Against Fasciola
Whether, the immune responses which develop in humans in response to liver and lung fluke
infections confer protective immunity against the currently held parasites or NEJs remains
unclear. Little direct progress has been made towards vaccine development for humans;
however, a great deal of work has been carried out to characterize certain enzymes shared
by these fluke (Cox et al., 2005). Human and experimental animals develop a complex array
of humoral and cellular immune responses during the course of infection (Osman et al., 1992;
Shaker et al., 1994). Both cellular and humoral immune responses were induced in the liver of
cattle and buffaloes during infection with F. gigantica probably by antigens released by the
developing flukes and by damage caused by the flukes during their migration in the liver
(Molina and Skerratt, 2005).
Most workers investigating the mechanisms of immunity of Fasciola have used the rat as their
experimental animal because of its superior ability to develop resistance to reinfection.
Rajasekariah and Howell (1977) showed that, previously infected rats resist an oral challenge,
but develop no immunity to a similar challenge given intraperitonealy (i.p.). In contrast, Kelly
et al. (1980) claimed that, acquired resistance is expressed as effectively against i.p. challenge
against oral challenge. A recent observation suggests that at least two distinct mechanisms may
be involved. Resistance at the intestinal barrier might be non-specific and thymus-independent,
whereas specific acquired immunity operates beyond the intestine (Smithers, 1982).
Several lines of evidence support the role of T cells in protective immunity. In vaccine trials
with F. hepatica, antibody titers were not generally associated with protection (Dalton
et al., 1996). Protection in F. gigantica vaccine trials were not antibody mediated (Estuningsih
et al., 1997). Passive transfer of protective immunity required volumes of immune serum too
11
large to be consistent with humoral based protection (Armour and Dargie, 1974). In contrast,
lymphoid cells have proven to be more efficient at transferring immunity (Armour and
Dargie, 1974; Chapman and Mitchell, 1982b). While the studies support a role for T cells,
little work has been conducted characterizing specific T cell responses in F. hepatica
infection. Better elucidation of host immunity is critical to the logical development of
vaccines against F. hepatica and related trematodes. In previous work, Shoda et al. (1999)
have characterized Th cell lines and CD4
+
T cell clones specific for F. hepatica soluble worm
antigen (SWA). The T helper (Th) cell lines co-expressed the cytokines, interleukin-4 (IL-4),
and interferon-gamma (IFN-); identifying these lines as having unrestricted T helper type 0
cell (Th0)-like phenotypes. Similarly, most T cell clones expressed a Th0-like cytokine profile;
a few expressed an IL-4 dominant Th2like profile, but no clones were identified with IFN-
dominant Th1 type profile. Th2-like responses has also been implicated by the predominant
immunoglobulin G1 (IgG1) response upon re-exposure of previously infected cattle (Clery
et al., 1996; Brown et al., 1999). These IgG1-based responses are consistent with recent data
on Schistosoma-infected human suggesting that Th2 responses are inversely related to disease
in the natural host (Mwatha et al., 1998).
In the study of cattle fascioliasis, lymphocyte proliferation assays showed a positive correlation
between the cumulative lymphocyte response to fluke antigen and the mature fluke burden. The
antibody responses, moreover, was dominated by the IgG1 isotype. Lastly, blood cultures
stimulated with adult fluke antigen failed to produce IFN-, a Th1type cytokine, in keeping
with previous work showing a low level if IFN- transcripts in F. hepatica-specific bovine Th-
cell clones (Brown et al., 1994).
In primary
infected cattle, however, an early production of
IFN- has been recorded (Clery and Mulcahy, 1998), suggesting differing immune response in
chronically infected and naïve infected cattle. Recently, Brown et al. (1999) demonstrated that,
most CD4
+
T-clones co-express IFN- and IL-4, underscoring the heterogeneous nature of the
cytokine response by CD4
+
T-cells. Little is known about how the rate at which the infection is
demonstrated affects the development of resistance.
During the migration of F. hepatica to the bile duct of the mammalian host, numerous antigens
that are potentially capable of inducing a specific Th
2
-like response are expressed (Reddington
et al., 1984). Of particular interest is the tegument and outer glycocalyx, which are maintained
through constitutive granule secretion by developmentally regulated secretory cells (Shoda et
al., 1999). The antigenicity of the glycocalyx has been demonstrated by incubation of worms
with immune sera which resulted in extensive Ig surface coating, although rapid turnover
mediated by granule secretion apparently permitted antibody shedding (Hanna, 1980). Both
rapid turnover and developmentally regulated expression of distinct secretory cells may
12
contribute to immune evasion by the parasite (Tkalcevic et al., 1996). They have focused on
tegument antigens that are common to the juvenile and adult worm stages as potential targets of
the immune response directed against both life stages. One candidate protein is the 12 kDa
F. hepatica thioredoxin molecules that has been previously identified by immunoscreening
using F. hepatica immune sera and has been characterized as being present on both NEJs and
adult worm stages (Richardson, 1994).
Increased levels of eosinophils are found in the lamina propria of the rat small intestine 3 wk
PI with F. hepatica and there is a marked increase in intestinal Eosinophilia immediately after
challenge (Doy et al., 1978). Adult flukes introduced into peritoneal cavity of immune rats
show palliating attachment of cells, mostly Eo, around there tegument within 1.5 hr, other Eo
degranulate at a distance from the parasite. The destruction of the fluke's tegument however,
appears to be also affected by neutrophils and macrophages (Bennett et al., 1980).
Recently, some aspects of immune response against F. hepatica in the goat have been
described, such as antibody production and lymphocyte proliferative response to
excretory/secretory (E/S) products (Martinez-Moreno et al., 1997). But the nature of that
responses and its relationship with the pathogenesis of the disease have not been considered. In
sheep and rats, there are some work described how NEJs induce a granulomatous lesion in the
hepatic parenchyma, with numerous macrophages, lymphocytes and Eo (Chauvin and
Boulard, 1996). This reaction is dependent on cell-mediated responses against parasite, but no
such studies have been carried out in goat fascioliasis.
By using peripheral blood mononuclear cells (PBMNCs), proliferation assays assess the cell
mediated immune response involved in fascioliasis under the same experimental conditions.
Bossaert et al. (2000) have paid a special attention to the type of cells involved in the immune
response. Eo have been reported as the main leukocyte-type helping to fight parasitic infection
(Hansen et al., 1999). In rats, Eo may have a role in the development of resistance to
reinfection (Davies and Goose, 1981; Milbourne and Howell, 1990). In sheep, skin Eo counts
following an intradermal reaction to mitogens appeared to correlate with the level of resistance
to a Trichostronglus colubriformis infection (Rothwel et al., 1991). A possible involvement of
Eo in antibody-dependant cell-mediated cytotoxicity (ADCC) mechanisms (Burden et al.,
1983; Van Milligen et al., 1998) and in IgE-dependant hypersensitivity reaction was reported
(Doy et al., 1981).
In vitro, Doy and Hughes (1980) had shown that, Eo from a mixed population of rat peritoneal
cell selectively adhere to the tegument of F. hepatica NEJs in the presence of serum from
previously infected rats. An artificially raised antiserum to dead fluke antigens failed to induce
Eo adherence. Neutrophils and Eo from cattle were also shown to adhere in large numbers to
13
NEJs coated with antibody from infected cattle, attachment was dependent on fraction
crystalline (Fc) receptor, although the adherence of Eo was more prolonged than that of the
neutrophils. Despite this cell-parasite interaction, damage to the fluke was measured by
chromium release could not be demonstrated, although it was subsequently shown that a major
basic protein isolated from cattle Eo caused damage and death of NEJs (Duffus and Frankes,
1980).
Th1, Th2, B cells and macrophages are all activated during Fasciola infection (Osman and
Abo El-Nazar, 1999). They cooperate in overcoming the parasite and work in benefit of the
host. The immunological response is carefully regulated by the aid of a complex network of
immunoregulatory mediators (cytokines) (Khalil et al., 1999).
Interleukin-1 (IL-1) is well known as a pro-inflammatory macrophage/monocyte driven
cytokine, secrete by B cells as well as other cell types. It achieves alone or with other cytokines
namely tumor necrosis factor (TNF), IL-6 and IL8, as a set of systematic events modulating
the inflammatory reaction. It co-stimulates the cell activation and promotes B-cell maturation
(Chensue et al., 1993; Ruth et al., 1996). IL-4 is released predominantly by Th2 cells; it has
important effects in relation to T and especially B cell function. It is primarily an anti-
inflammatory cytokine that causes coordinate down-regulation of macrophage derived IL-1 and
TNF. It has inhibitory effects on macrophage function. It provides a potent stimulus for B cell
switching to production of IgE antibody. As such it is important from clinical point of view in
relation to parasitic infections in which IgE antibody-mediated response plays an important role
(Medhat et al., 1998; Schof et al., 1998). IgE is present in the serum of healthy individuals at
extremely low levels. Its level rises in response to parasitic infections. The induction of IgE
synthesis by parasitic infection has been shown to be T cell dependent and these cells appears
to exert their effect through the production of soluble factors that enhance the production of IgE
by B cells. It activates mast cells and it is important in defense against parasitic
infections, e.g. worms (Yamaguchi et al., 1997).
- Resistance to infection
The relative susceptibility or resistance of the host to Fasciola spp. infection is associated with
the biochemical characteristics of parasites and the host immune response during fascioliasis
(Spithill et al., 1999b; Meeusen and Piedrafita, 2003). Sheep are susceptible to F. hepatica
infection but less susceptible to F. gigantica infection (Boyce et al., 1987; Roberts et al.,
1997).
Acquired resistance to a secondary F. gigantica infection following a primary infection or
vaccination has been demonstrated in cattle, goats and sheep (Haroun and Hillyer, 1986).
Low or no resistance is seen hamsters and mice and the infection is highly pathologic in both
14
the acute and chronic phases. In heavy infection death is the usual sequel (Smithers and
Doenhoff, 1982).
It is well established that sheep do not acquire resistance to F. hepatica as determined from the
observed yields of mature parasites after primary and secondary infections with F. hepatica
(Haroun and Hillyer, 1986; Boyce et al., 1987). In European sheep, yields of F. hepatica
ranged from 16 to 38 % and from 13 to 31 % after primary and secondary infection,
respectively, indicating that resistance to F. hepatica does not develop in these sheep breeds
(Boyce et al., 1987). In contrast, acquired resistance to F. gigantica has been observed in
sheep. Flynn et al. (2007) reported that, infection with F. hepatica results in polarization of the
host's immune response and generation of Th2 immune responses, which are known to be
inhibitory to Th1 responses.
Buffaloes were highly susceptible to F. gigantica infection, and this susceptibility could be
associated with the late and weak cellular immune response in the early phase of infection
(Zhang et al., 2006). There was a trend toward higher parasite-specific IgG2 titers in
sheep infected with lower worm burdens, suggesting that higher F. gigantica or F.
hepatica burdens suppress IgG2 responses. The findings of this study suggested that, in early
infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica
because of its rapid increase in size and the speed of its progression through the migratory
phases of its life cycle (Raadsma et al., 2007). Cervi et al. (1999) reported that, The E/S
antigen of F. hepatica is involved in the suppressive phenomena of cellular immune responses
in rats.
Most workers investigating the mechanism of immunity to Fasciola have used the rat as their
experimental animal because of its superior ability to develop resistance to reinfection.
Smithers (1982) suggested that, resistance at the intestinal barrier might be non-specific and
thymus-independent, whereas specific acquired immunity operates beyond the intestine.
Armour and Deargie (1974) noted that, when high levels of immunity are transferred with
serum, the worms of the challenge appear to be destroyed before they enter the liver. On the
other hand, a similar level of protection against a challenge after adoptive transfer of cells is
invariably accompanied by marked liver lesions associated with cellular infiltrates and dead
immature parasites.
Bossaert et al. (2000) have paid special attention to the type of cells involved in the immune
response. Eo have been reported as the main leukocyte-type helping to fight parasitic infection
(Butterworth, 1977). The cellular immune response to F. hepatica infection in different
animal species has been widely studied. Especially, in F. hepatica-infected sheep, a significant
transient proliferation in vitro of PBMNCs stimulated by parasite antigens appears during the
15
first wk of infection (Moreau et al., 1998; Mulcahy et al., 1999). PBMNCs proliferation
induced by F. gigantica E/S products increased from 2 wk PI with a peak at 5 wk PI (Zhang et
al., 2006). Peripheral blood Eo counts increased as previously described in F. hepatica
and in F. gigantica infected sheep (Chauvin et al., 1995; Hansen et al., 1999; Zhang et
al., 2005). Eo numbers increased significantly from 3 wk PI in F. gigantica-infected
buffaloes and displayed a peak at 8 wk PI (Zhang et al., 2006).
Neutrophils and Eo from cattle were also shown to adhere in large numbers to NEJs coated
with antibody from infected cattle (Duffus and Frankes, 1980). The total number of hepatic
mononuclear cells increased significantly following infection, but the proportion of Natural
killer (NK) cells did not change. After infection, these cells were found around the portal space,
around the centrolobular vein, in the periportal fibrosis and in the band of collagen. However,
no NK cells could be detected in or around the granuloma during infection (Tliba et al., 2002).
The cell mediated immunity to F. hepatica antigens in cattle is investigated by establishing the
lymphocyte proliferation and tests for quantitative determination of IL2 production (Oldham
and William, 1985). Some aspects of the immune response against F. hepatica in the goat have
been described, such as antibody production and lymphocyte proliferative response to E/S
products (Martinez-Moreno et al., 1997). In sheep and rats, there are various data describing
how the NEJs induce a granulomatous lesion in the hepatic parenchyma, depending on cell-
mediated response against parasite with numerous macrophages, lymphocytes and Eo
(Chauvin and Boulard, 1996).
Bovine peripheral blood lymphocytes isolated 2-5 wk PI undergo strong proliferative responses
upon co-culture with F. hepatica specific antigen, although this response is transient and
lymphocytes gradually lose the ability to respond to F. hepatica antigens as the infection
matures (Clery and Mulcahy, 1998; McCole et al., 1999). The local cellular response may
not, however, always reflect that found in the periphery: in mice and rats, both the specificity
and isotype of antibody (Meeusen and Brandon, 1994), together with PBMNCs cytokine
profiles (Tliba et al., 2002) have been found to differ in the different body and immune
compartments examined.
Waldvogel et al. (2004) investigated F. hepatica specific IL-4 and IFN- messenger
ribonucleic acid (mRNA) expression in PBMNCs from calves experimentally infected
with F. hepatica. Cells were collected prior to infection and on days 10, 28 and 70 PI.
Interestingly, PBMNCs responded to stimulation with F. hepatica E/S products and expressed
high amounts of IL-4 but not of IFN- mRNA suggesting that F. hepatica induced a Th2 biased
early immune response which was not restricted to the site of infection. Later in infection, IL-4
mRNA expression decreased whereas IFN- mRNA expression increased slightly.
16
In vitro, it is established that the cytokine production of monocytes from patients with acute or
chronic fascioliasis is increased (Khalil et al., 1999). IL-8 and IL-6 are produced in higher
amounts from the monocytes of patients with acute fascioliasis compared to the healthy
controls. The production of these lymphokins is decreased during the chronic stage of the
disease in comparison to the acute stage of the disease.
The production of IL-1 from mononuclear phagocytes, IL-4 from Th2 lymphocytes and IgE
level are measured in patients with fascioliasis during the acute and chronic stage of the disease
and after triclabendazole (TCBZ) treatment (Allam et al., 2000) The levels of IL-1 and IL-4 are
significantly decreased and the IgE level is increased both in acute and chronic stage of
fascioliasis. After the drug treatment, the cytokine values restore to the control.
Osman et al. (1989) reported that, antibodies, cytotoxic T-cells, activated macrophages, NK
cells and many other cells, mediators of the ADCC, and modulator of the immune system as
cytokines, are involved in the immune mechanisms, which are efficient against parasites.
Clery and Mulcahy (1998), established early stimulation of IFN- production in the peripheral
lymphocytes of F. hepatica infected cattle. Others also established that the IFN- level was
increased only during the first 2 wk PI the F. hepatica in sheep, IL-10 is secreted mainly during
the first 6 wk PI and monocytes were inhibited till the 35
th
wk PI (Moreau et al., 1998).
3.
Treatment
A number of drugs have been used to control fascioliasis in animals. Drugs differ in their
efficacy, mode of action, price, and viability. Fasciolicides (drugs against Fasciola spp.) fall
into five main chemical grs:
Halogenated phenols: bithionol (Bitin), hexachlorophene (Bilevon) and nitroxynil (Trodax).
Salicylanilides: closantel (Flukiver, Supaverm) and rafoxanide (Flukanide, Ranizole).
Benzimidazoles: triclabendazole (Fasinex), albendazol (Vermitan, Valbazen), mebendazol
(Telmin) and luxabendazole (Fluxacur).
Sulphonamides: clorsulon (Ivomec Plus).
Phenoxyalkanes: diamphenetide (Coriban).
TCBZ is considered as the most common drug due to its high efficacy against adult as well as
NEJ flukes. It is used in control of fascioliasis of livestock in many countries. Nevertheless,
long-term veterinary use of TCBZ has caused appearance of resistance to F. hepatica in sheep
(Overend and Bowen, 1995).
The cost of the treatment with TCBZ prohibits its wide adoption by rural producers in
developing countries. Scientists have started to work on the development of new drug.
Recently, a new Fasciolicides was successfully tested in naturally and experimentally infected
17
cattle in Mexico. This new drug is called compound Alpha and is chemically very much
closed to TCBZ (Ibarra et al., 2004).
Treatment is too late to decrease or avoid the side effects of the infection, also the extensive use
of these drugs increase the risk of developing resistance to them. So, diagnosis is very
important specifically early one to control the effect and spreading of infection.
4.
Antigens-derived Fasciola
- Tegumental antigens (TA)
The tegument of F. hepatica provides the interface at which the host immune system interacts
with the parasite. Its structure has been determined by microscopy to be a syncytium formed by
the fusion of specialized tegumental cells located beneath the longitudinal and lateral muscle
layers (Bennett and Threadgold, 1975). It is speculated that protein expression in the
tegument regulates and plays an important part in the fluke's defense against the host's immune
system (Bennett et al., 1980).
Three types of nucleated cells are found within the nucleated layer of the tegument, which are
active during various stages of the fluke's development within the mammalian host (Hanna,
1980).
Initially type o (To) bodies are produced but during the migratory stage, they are the first to
become active and then only within the metacercariae and the NEJs stage. These cells are
known to produce granules that are then released at the apical surface of the NEJs fluke
allowing for the continual turnover of the outer tegument in response to host antibody
attachment (Hanna, 1980) and there is a switch to type 1 (T1) bodies and on reaching the bile
duct type 2 (T2) bodies become predominant (Bennett and Threadgold, 1975).
- Somatic antigens
Somatic antigens were fractionated by column chromatography for diagnosis of human
fascioliasis. Somatic extracts of adult F. hepatica have been used both as a vaccine to induce
resistance to challenge and to monitor the host immune response to infection. Sinclair and
Joyner (1974) reported a 54% decrease in the fluke burden following challenge with the
worms extract when compared to untreated control rabbits.
The humoral response to adult somatic antigens has also been examined by indirect fluorescent
antibody-labeling of plastic embedded sections of NEJs and adult flukes. Estimates of the
levels of specific IgG and IgA antibodies in the serum and bile were determined during the
course of infection (Hughes et al., 1981).
The immune response of sheep to somatic components of adult F. hepatica was studied during
an experimental infection. Antibodies against adult fluke somatic antigens were detected by
thin layer immunoassay from the 2
nd
wk PI. Similarly, the results of western blotting (WB)
18
analysis showed a specific recognition of several components as early as 2 wk PI. However, an
increase in the number and intensity of bands with time of infection was observed in the
patterns of antigenic recognition towards components of 20-23 kDa in the somatic
preparation of F. hepatica, especially noticeable after the 6
th
wk PI. Since these polypeptides
were recognized by all infected animals, they could play an important role in the diagnosis of
sheep fascioliasis (Ruiz-Navarrete et al., 1993). Somatic antigens of F. gigantica, G.
explanatum, S. spindale and hydatid cyst ingredients were analyzed to identify the cross-
reactive antigens among them using WB technique (Yokananth et al., 2005).
- Excretory/Secretory antigens (E/S)
Several F. gigantica antigens with immunodiagnostic potential have been identified in
preparations of the flukes and their E/S products. These E/S antigens are probably composed of
molecules released from the continuous turnover of the glycocalyx coating the tegument
surface membrane as well as some enzymes released from the caecum. E/S antigens contained
several enzymes such as glutathione-S-transferase (GST), CP and CL (Espino and Finaly,
1994). It was found that CP of F. hepatica is very important candidates for a vaccine antigen
because of their role in fluke biology and in the host-parasite relationship (Wedrychowicz
et al., 2007).
Subproteomics has been used to compare E/S products produced by adult F. hepatica in vivo,
within ovine host bile, with classical exhaust in vitro E/S methods. Only CL proteases
from F. hepatica were identified in our ovine host bile preparations. Several host proteins were
also identified including albumin and enolase with host trypsin inhibitor complex identified as
a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed CL
proteases as the major constituents of the in vitro E/S proteome. In addition, detoxification
proteins, actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate
dehydrogenase were all identified in vitro. WB of in vitro and in vivo E/S proteins showed
only CL proteases were recognized by serum pooled from F. hepatica-infected animals. Other
liver fluke proteins released during in vitro culture may be released into the host bile
environment via natural shedding of the adult fluke tegument. These proteins may not have
been detected during our in vivo analysis because of an increased bile turnover rate and may not
be recognized by pooled liver fluke infection sera as they are only produced in adults. This
study highlights the difficulties identifying authentic E/S proteins ex host, and further confirms
the potential of the CL proteases as therapy candidates (Morphew et al., 2007).
Serradell et al. (2007) observed that, E/S products induced an early apoptosis of rat peritoneal
Eo and that this phenomenon was time- and concentration-dependent. Furthermore, activation
of protein tyrosine kinases (TyrK) and caspases were necessary to mediate the Eo apoptosis
19
induced by the E/S products, and that carbohydrate components present in these antigens were
involved in this effect. They described for the first time the ability of E/S products from F.
hepatica to modify the viability of Eo by apoptosis induction. Besides that, we have observed
Eo apoptosis in the liver of rats 21 days after F. hepatica infection. The diminution in Eo
survival in early infection could be a parasite strategy in order to prevent a host immune
response.
Ortiz et al. (2000) used E/S, somatic and surface antigens of adult F. hepatica for antibody
response determination in dairy cattle naturally infected with F. hepatica. They reported that,
antibody responses were developed against 60-66 kDa in E/S and surface antigens and 17 kDa
in somatic antigen. Qureschi et al. (1995) used
E/S
antigens
and
reported
that,
at
approximately 15 kDa F. hepatica E/S antigens can be used for species specific diagnosis in
cattle. Hillyer and Soler De Galanes (1991), obtained sera from human patients, calves, sheep,
and rabbits infected with F. hepatica and tested the WB techniques with F. hepatica E/S
antigens in order to evaluate their immunodiagnostic potential. Researchers reported that the
serum samples from humans, rabbits, cattle, and sheep infected with fascioliasis recognized
two antigenic polypeptides of 17 and 63 kDa in the form of sharp bands.
The E/S antigens of Fasciola spp. or their partially purified components are the most common
sources of antigens for use in ELISA methods and antibodies to these antigens can be detected
as early as 2 wk and peak concentrations are reached at 8-10 wk PI (Fagbemi and Guobadia,
1995).
Evaluation of efficacy of E/S antigen by ELISA (Osman et al., 1995) revealed that, the crude
preparation had 100% sensitivity. 94% specificity and 98% accuracy at cut off level of 0.3 in
acute cases and positive results in 77% of chronic cases. Cross reactivity with Schistosoma (S.)
and Toxoplasma was reported. So E/S is recommended for diagnosis of fascioliasis in acute
cases. However, low positive results with chronic case may limit its application in routine
investigation adding to the cross reaction with sera of other parasites.
The E/S antigen of F. gigantica was tested by ELISA against sera from fascioliasis,
schistosomiasis and hydatidosis cases to determine its sensitivity and specificity in the
detection of specific IgG antibodies (Ortiz et al., 2000). The sensitivity of E/S antigen was
found to reach up to 100% and the specificity of E/S was 89.47% and 86.8% for IgG and IgM,
respectively (Mousa, 1994). Cattle naturally exposed to F. hepatica, in Cajamarac, Peru;
develop a significant IgG response to this parasite E/S antigen (Ortiz et al., 2000).
- The E/S products of adult F. hepatica released during the course of the infection have been
investigated. Cuperlovic and Movsesijan (1972), using a fluorescent antibody test, found the
caecal contents of adult flukes gave the strongest specific fluorescence during the course of
20
infection. Fluorescent labeling also occurred on the epithelial cells lining the uterus and
excretory ducts as well as the spermatogenic cells. Ericksen and Flagsted (1974) proposed
that, host resistance induced by subcutaneous (s.c) implantation of adult worms might be
directed against the fluke's metabolic or E/S products. The finding of Goose (1978), in which
the E/S products of adults were found to be toxic to the lymphocytes from infected rats,
suggested that some parasite products released many services to protect the parasite. E/S
products have served as effective immunogens when mature flukes, contained within diffusion
chambers, were implanted i.p. or s.c in rats. Resistance was imported by these implanted flukes
whether they were present at the time of challenge or if they had been removed 2 wk earlier
(Haroun et al., 1980). Proteases
The ability of flukes to secrete proteolytic enzymes is critical for their survival in the definitive
host, facilitating migration, and utilization of host tissue as nutrient and cleaving of host
Igs (Mulcahy and Dalton, 1998). A major component of the enzymes secreted by F.
gigantica consists of cysteine proteases (CP), of which two major forms have been
characterized, cathepsin L1 (CL1) and cathepsin L2 (CL2) (Fagbemi and Hillyer,
19911992; Smith et al., 1993; Dowd et al., 1994).
Cells of the fluke gut produce these enzymes, which are homologous to mammalian lysosomal
enzymes, CL1 and CL2 proteinases which act in the gut to breakdown ingested blood and other
tissue, and are also regurgitated by the flukes. CL1 has been shown to cleave Igs in the hinge
region and then prevent antibody-mediated attachment of Eo to NEJ flukes (Carmona et al.,
1993; Smith et al., 1993). The fluke CL1 and CL2 can also cleave host collagen and other
extra cellular matrix proteins (Beresain et al., 1997). Several reports have demonstrated that,
proteases from parasites may be useful as protective vaccine (Knox, 1994). It was found that,
the secreted proteases were able to cleave Ig, a finding that strengthened the case for targeting
these proteases as potential vaccine antigens for controlling infection with F. gigantica
(Chapman and Mitchell, 1982a). Yamasaki and Aoki (1993), Heussler and Dobbelaere
(1994) and Wijffels et al. (1994a) revealed that, the secreted adult proteases are of the CL
class.
CL of Fasciola, formulated in Freund's adjuvant, has been shown to protect cattle against F.
gigantica (Dalton et al., 1996) and to induce high reduction (70%) in the output of eggs by
the parasites in vaccinated sheep (Wijffels et al., 1994b) and cattle (Dalton et al., 1996). These
results suggested a critical role for Freund's adjuvant in vaccine efficacy using CL that may
relate to the induction of a certain arm of the immune responses.
21
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