J. Parasitol., 95(2), 2009, pp. 450–455
? American Society of Parasitologists 2009
DEVELOPMENT AND APPLICATION OF A FECAL ANTIGEN DIAGNOSTIC SANDWICH
ELISA FOR ESTIMATING PREVALENCE OF FASCIOLA GIGANTICA IN CATTLE IN
CENTRAL JAVA, INDONESIA
Endah Estuningsih, Terry Spithill*, Herman Raadsma†, Ruby Law‡, G. Adiwinata, Els Meeusen§, and David Piedrafita?
Indonesian Research Institute for Veterinary Science, Bogor, West Java, Indonesia. e-mail: email@example.com
antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti–F. gigantica antibodies in
serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood,
feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen
detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The
results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than
the anti–F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The
coproantigen ELISA was able to detect 100% of the cattle with ?15 flukes. A survey of 305 cattle in central Java over a 10-
mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful
diagnostic method to determine patent F. gigantica infections in cattle.
The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica
Most of the 589 million cattle and buffaloes in Asia are kept
in villages, where they play a central role in the economies of
households by providing draft power and cash from the sale of
milk and meat, and by contributing to the recycling of nutrients
from crop residues. Infection by the liver fluke (Fasciola gi-
gantica) is regarded as the single most important parasitic dis-
ease in large ruminants in Asia, causing ill thrift, lower pro-
ductivity, and reduced draft power (Fabiyi, 1987; Spithill et al.,
1999). In Indonesia the prevalence of fascioliasis in cattle is
estimated to be 60–100% (Roberts and Suhardono, 1996), and
the annual economic loss due to fascioliasis in cattle is esti-
mated at US$107 million, representing an annual loss per ani-
mal of US$42, or about 10% of the meat value of each animal
(Spithill et al., 1999). Control of fascioliasis currently relies on
selective treatment of individual animals with anthelmintic. Im-
plementation of an anthelmintic control program would bring
significant economic benefit to subsistence farmers in Indonesia
and other endemic areas in Asia and Africa. However, the ef-
fectiveness of such a control program would be enhanced by
methods to accurately diagnose fascioliasis, so that delivery of
treatment could be targeted to areas where disease is prevalent.
The current diagnosis of liver fluke infection is performed
using the identification of eggs in the feces, or detection of
serum antibodies to specific antigens of Fasciola species (Hil-
lyer, 1999). However, during the 12–14 wk prepatent period of
the disease, the presence of the parasite cannot be determined
because of the lack of egg output (Spithill et al., 1999; Almazan
et al., 2001). Antibodies to ES antigens and cathepsin L are
detected in chronically infected cattle and humans, which sug-
gests that the continued release of ES antigens and cathepsin L
by adult flukes induces an ongoing antibody response to these
antigens (Fagbemi and Guobadia, 1995; O’Neill et al., 1998;
Received 30 April 2008; revised 30 July 2008, 14 August 2008; ac-
cepted 18 August 2008.
* School of Animal and Veterinary Sciences, Charles Stuart University,
Wagga Wagga, NSW, Australia 2678.
† Faculty of Veterinary Science University of Sydney, Camden NSW,
‡ Department of Biochemistry, Monash University, Victoria, Australia
§ Department of Physiology, Monash University, Victoria, Australia
? To whom correspondence should be addressed.
Cornelissen et al., 1999; Hillyer, 1999). However, serodiagnosis
does not differentiate between patent and past infections in cat-
tle because circulating anti-Fasciola antibodies can persist in
the serum for many months after worm expulsion (Cornelissen
et al., 1999; Hillyer, 1999) Thus, cattle with resolved parasite
infection will still be scored as infected because of the presence
of residual anti–F. gigantica serum antibodies; these ‘‘false-
positives’’ are a real impediment to detecting accurately cattle
An alternative to the detection of F. gigantica eggs or serum
antibodies is the detection of circulating antigens using defined
antibodies (Hillyer, 1999). The most abundant immunodomi-
nant antigens released by F. gigantica parasites are the excre-
tory/secretory (ES) antigens, of which a family of cathepsin L
proteases is the major component (Wijffels et al., 1994; O’Neill
et al., 1998). ES antigens of Fasciola hepatica could be de-
tected in cattle serum from the first week post-infection and in
fecal supernatant from the fourth week post-infection (Alama-
zan et al., 2001). A monoclonal antibody has been shown to
detect F. hepatica antigens in human sera and feces as well as
in sheep and cattle samples (Espino et al., 1990; Espino and
Finlay, 1994; Dumenigo, 1996, 2000; Abdel-Rahman et al.,
1998). These observations suggest that detection of fecal ES or
cathepsin L antigens shed by liver flukes in infected ruminants
may provide a useful test for detection of F. gigantica. Here
we apply this assay for the detection of F. gigantica cathepsin
L in the feces of infected cattle and assess the prevalence of
fasciolosis in 2 regions of central Java.
MATERIALS AND METHODS
Production of ES antigens of F. gigantica
ES antigens were obtained from adult F. gigantica as described by
Wijffels et al. (1994) with slight modifications. Briefly, adult flukes were
removed from bile ducts of naturally infected cattle at a local abattoir,
and about 50 flukes were put into 100 ml PBS at 37 C for 15 min.
Initial regurgitant containing blood, bile, and debris was removed by
washing the parasites in RPMI culture media containing 50 U/ml strep-
tomycin, and subsequently live flukes were removed and incubated in
fresh RPMI medium for 4–6 hr at 37 C (2 flukes/ml RPMI). The culture
medium was then removed, centrifuged at 2,500 rpm at 4 C for 10 min,
and stored at ?20 C until required. Protein concentration was deter-
mined using the Bio-Rad DC colorimetric assay following detergent
solubilization (Lowry et al., 1951).
ESTUNINGSIH ET AL.—FECAL ANTIGEN TO DIAGNOSE F. GIGANTICA451
Generation of monoclonal antibodies
The IgG1 monoclonal antibodies were generated commercially at the
Walter and Eliza Hall Institute of Medical Research, Melbourne, Aus-
tralia, by fusion of myeloma cells with spleen cells from BALB/c mice
hyperimmunized with F. gigantica ES antigens. The immunization of
BALB/c mice consisted of 5 intraperitoneal injections, 3 wk apart, of
50 ?g of ES antigens. The first injection was emulsified in Freund’s
complete adjuvant, and subsequent immunizations used Freund’s in-
complete adjuvant. Putative monoclonal antibodies, expressed from suc-
cessful hybridoma fusions, were screened for the IgG isotype and tested
for recognition of F. gigantica antigens in a standard ELISA using
plates coated with ES antigens (2 ?g/ml). Positive hybridoma cells were
cloned and injected intraperitoneally into pristan-primed BALB/c mice,
and the anti–F. gigantica IgG1 antibodies were purified from the ascites
fluid by affinity chromatography on a protein G column (5 ml HiTrap
Protein G, Amersham Biosciences, Sydney, Australia) according to the
manufacturer’s protocol. To allow a more rapid and sensitive test, mono-
clonal antibodies with high end-point titers were also covalently labeled
with biotin using a succinimide ester of biotin using a standard protocol
(Harlow and Lane, 1988).
Purification of cathepsin L from ES antigens of F. gigantica
Native cathepsin L was enriched from F. gigantica ES by size ex-
clusion chromatography. The cysteine protease inhibitor E64 was added
to prevent protein degradation, and the ES proteins were concentrated
down to 10% of the original volume by dialysis (MW cutoff 2,000,
Serva, Sydney, Australia) in polyethylene glycol (mw of 15,000–
20,000, Sigma, St. Louis, Missouri) at 4 C on a rocker. The concentrated
ES was then dialyzed against PBS overnight. Cathepsin L in ES was
purified on a Superdex G75 10/30 column using PBS as the running
buffer and fractions collected subsequently analyzed by SDS-PAGE and
Western blot using rabbit anti-cathepsin L (1:1,000 dilution) anti-sera.
Those fractions containing cathepsin L were pooled, and the protein
concentration determined and stored at ?20 C in the presence of 30%
glycerol for later use.
ES antigens (10 ?g) or purified cathepsin L (2 ?g) were separated
by 12% SDS-PAGE and transferred to a nitrocellulose filter. The filter
was blocked with 5% skim milk for 1 hr at room temperature and
incubated for 1 hr with the generated mAB1 monoclonal antibody. After
washing 3 times with 0.1% Tween 20 in PBS, bound monoclonal an-
tibodies on the membranes were detected by incubation with donkey
anti-mouse immunoglobulin conjugated to alkaline phosphatase (Sile-
nus, Melbourne, Australia). Bands were developed with p-nitro blue
tetrazolium chloride and 5-bromo-4-chloro-3-indoyl phosphate p-tolui-
dene salt (Bio Rad Laboratories, Sydney, Australia) following the man-
ufacturer’s instructions. The reaction was stopped with distilled water.
Cattle were killed according to the Veterinary Law of Indonesia by
severing the jugular vein with a sharp knife. The liver was removed,
and all the flukes from the major bile ducts and in the gall bladder were
collected into a small jar for subsequent counting. The liver was cut
into slices of about 1 cm in thickness, and the sliced liver was thor-
oughly squeezed and washed in saline to recover parasites within the
liver tissue. The total number of parasites were counted and measured
as previously described (Roberts et al., 1997).
Fecal and serum sample analysis
Fasciola egg counts: Fecal egg counts were performed by the sedi-
mentation method as described by Parfitt and Banks (1977) with slight
modification. Briefly, 3 g of feces were put into a conical flask and tap
water added to 250 ml. The fecal suspension was allowed to sediment
for 5 min, then the supernatant decanted and the sediment re-suspended
in 15 ml of tap water. This process was repeated 3 times, and after the
addition of 1–2 drops of methylene blue, 1 ml of the suspension was
counted in a McMaster Egg Counting Slide (J. A. Whitlock, Sydney,
Australia) at 40? magnification.
Detection of F. gigantica ES antigens in feces: Two monoclonal an-
tibodies, found to give the highest sensitivities for the detection of F.
gigantica ES antigens, designated mAB1 and biotinylated mAB2, were
tested for their ability to detect F. gigantica antigens in cattle feces.
One gram wet weight of feces extract was suspended in 2 ml of PBS
containing 0.1% Tween 20 (PBST), then centrifuged at 13,000 rpm for
10 min, and the supernatant was collected and stored at ?20 C until
use (Espin et al., 1990). Microtitre plates (Nunc, Roskilde, Denmark)
were coated with 2.5 ?g/ml of mAB1 in 100 ?l/well of carbonate buffer,
pH 9.6, and incubated overnight at 4 C. The wells were washed 3 times
with PBST and blocked with 200 ?l of 5% skim milk in PBST for 1
hr at 37 C, then washed and 100 ?l of fecal supernatant added in
duplicate and incubated for 1 hr at 37 C. The wells were then washed
4 times with PBST and 100 ?l of biotinylated mAB2 (0.63 ?g/ml)
added to each well before incubation for 1 hr at 37 C. After washing
again, 100 ?l of extravidin (peroxidase conjugate; Sigma) diluted
1:1,000 was added to the wells and incubated for 45 min at 37 C. The
substrate was prepared by dissolving 1 tablet of Tetramethylbenzidine
(TMB) in 1 ml of dimethylsulfoxide and mixed with 9 ml of citrate
phosphate buffer and 2 ?l of H2O2(1 tablet of TMB for 1 plate). After
washing again, 100 ?l of substrate was added to each well, and after
10 min the reaction was stopped with 25 ?l of 2 M H2SO4. The optical
density (OD) was measured at 450 nm in an ELISA reader. The cutoff
value between negative and positive samples was calculated as the av-
erage of the OD450nmof fecal supernatant from F. gigantica naive cattle
(n ? 32) plus 3 times the SD of these fecal supernatants.
Detection of anti–F. gigantica ES antibodies in serum: ELISA plates
were coated with 2 ?g/ml of ES of F. gigantica antigen (100 ?l well)
in 0.1 M sodium carbonate buffer, pH 9.6, and incubated overnight at
4 C. IgG antibodies were detected with HRP conjugated anti-bovine
IgG (H ? L; Silenus) peroxidase conjugate (1/1,000).
Calculation of sensitivity and specificity of ELISA and fecal egg
counts: The sensitivity and specificity were calculated using the for-
Specificity ? TN/(TN ? FP) ? 100% and
Sensitivity ? TP/(TP ? FN) ? 100%,
where TN stands for true negative, TP for true positive, FN for false
negative, and FP for false positive. Animals were considered TP if F.
gigantica were found in their liver, otherwise they were classified as
Abattoir survey: Serum, feces, and livers were collected from 150
cattle from an abattoir in Jakarta. The total fluke number and length of
each recovered parasite was recorded. Fecal samples were assayed for
coproantigens using the capture ELISA and F. gigantica eggs counts,
and serum IgG anti–F. gigantica ES antibody titers were determined.
Small numbers of paramphistomes, Gigantocotyle sp. and Eurytrema
sp., were recovered from all cattle, but no correlation between these
burdens and a positive or negative signal was seen (data not shown).
Field survey: Repeated blood and fecal samples were collected
monthly for 10 mo from 305 cattle in 2 Yogyakarta regions. Serum was
assayed for anti–F. giganticaa antibody, and the fecal samples were
analyzed for the presence of coproantigens and F. gigantica eggs.
Development of the 2-site capture ELISA using
monoclonal antibodies to ES antigens
Fasciola gigantica excretory/secretory antigens were a com-
plex mixture of proteins, with molecular masses ranging from
5 to ?70 kDa (Fig. 1a). Immunizing mice with this material
yielded 4 monoclonal antibodies of the IgG1 subclass, which
recognized 2 predominant bands about 28 and 60 kDa (Fig. 1b,
lane 1), as well as cathepsin L migrating at 28 kDa (Fig. 1b,
lane 2). It is possible that the 60 kDa ES was a dimer of the
28 kDa cathepsin L proteins because the PAGE was run under
non-reducing conditions. Each monoclonal antibody recognized
the same bands (data not shown), which suggests that either
they distinguished the same or different epitopes on a single
452 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 2, APRIL 2009
SDS-PAGE (12%) Coomassie Blue–stained polyacrylamide gel. (b)
Western blot analysis of the immunoreactivity of ES antigens and native
cathepsin L. Samples separated by SDS-PAGE (12%) under nonreduc-
ing conditions were transferred to nitrocellulose and probed with the
monoclonal antibody mAB1.
(a) Profile of ES antigens and native cathepsin L on an
pernatants of cattle with, or without, the addition of decreasing amounts
of F. gigantica ES antigens.
Detection of coproantigens in Fasciola-naive fecal su-
cathepsin L or they reacted with different cathepsin L isoforms,
at least 8 of which have been identified (Wijffels et al., 1994).
Several combinations and dilutions of capture and biotiny-
lated detector antibodies were evaluated to determine the great-
est sensitivity for detection of ES antigens. The best combina-
tion was to use the mAB1 for capture (2.5 ?g/ml) and bioti-
nylated mAB2 (0.63 ?g/ml) as detector. Initially feces from F.
gigantica–naive animals (defined as seronegative with no flukes
or eggs in the liver or feces) were spiked with ES antigens and
then tested. The ELISA detected as little as 200 ng ES/ml of
feces in a dose-dependent manner (Fig. 2). The assay was sub-
sequently validated in naturally infected cattle and compared
for sensitivity and specificity with egg counting and anti–F.
Determination of the cutoff value in the ELISAs for feces
This was obtained from 32 serum and fecal samples of F.
gigantica–naive cattle (as defined above). The ‘‘cutoff’’ OD
value for a negative sample, i.e., background of the assay, was
defined as the average of the OD absorbance ? 3 SD. These
came to 0.21 and 0.40 for the capture and serum antibody
Application of the coproantigen sandwich-ELISA in cattle
Abattoir survey: Ninety-two of the 150 cattle (61%) con-
tained F. gigantica flukes (mean 32, range 1–426), the majority
of which were adults 28–46 mm in length. Eighty-seven of the
fecal samples from these cattle contained F. gigantica coproan-
tigens detected by the capture ELISA, indicating a sensitivity
of 95% (87/92). The 5 cattle negative by ELISA contained less
than 15 flukes each. The specificity of the coproantigen ELISA
assay was 91% (53/58). The sensitivity and specificity of the
serum anti–F.gigantica ES ELISA was 91% (84/92) and 88%
(51/58), whereas fecal egg counting gave figures of 87% (80/
92) and 100%. Thus, the coproantigen ELISA was the only test
to score higher than 90% in both parameters. There was a better
correlation between the capture ELISA and worm burden (Fig.
3a, r2? 0.243) than between egg counts and worm burden (Fig.
3b, r2? 0.205), while the serum ES antibodies ELISA did not
correlate with worm numbers (Fig. 3c). The coproantigen
ELISA could detect 95% of cattle with 1–15 flukes and 100%
of cattle with ?15 flukes (Fig. 4).
Field survey: Three-hundred and five cattle from 2 regencies
in Yogyakarta province were monitored monthly over 10 mo.
The first regency, Kulon Progo, is situated in mountains, 1,000
ESTUNINGSIH ET AL.—FECAL ANTIGEN TO DIAGNOSE F. GIGANTICA453
of infected cattle with (a) coproantigen ELISA absorbance value; (b)
Fasciola eggs per gram of feces; and (c) serum antibody ELISA ab-
The relationship between the number of flukes in the liver
value (OD) and the number of flukes in the liver recovered from abattoir
killed cattle. Horizontal line represents cutoff OD value at ?0.21.
The relationship between the coproantigen absorbance
2 regions of Yogyakarta based on detection by (a) coproantigen ELISA
or (b) eggs per gram in feces.
Percentage of cattle positive for F. gigantica infection in
m above sea level, whereas Bantul is coastal and only 55 m
above sea level. Trends of infection were determined by testing
serial blood and fecal samples by ELISA and egg counting. The
coproantigen ELISA showed that the incidence of F. gigantica
infection was about 50% but increased as the study progressed,
peaking at 90% in Bantul, although egg counting did not reveal
this trend (Figs. 5a, 5b). The coproantigen ELISA suggested
that Ongole cattle were more susceptible to F. gigantica than
Simental and Limosin breeds, particularly in Kulon Progo,
where the prevalence in Ongole cattle reached 90% compared
with 50–60% in the other 2 breeds (Figs. 6a, 6b).
The problem of accurately diagnosing liver fluke infection
using the current conventional tests, i.e., identification of eggs
454 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 2, APRIL 2009
ferent breeds of cattle in 2 regions of Yogyakarta based on detection
by (a) coproantigen ELISA or (b) eggs per gram in feces.
Percentage positive for F. gigantica infection of 3 dif-
in the feces or detection of serum antibodies, has driven at-
tempts to develop a more accurate diagnostic tool. In the pres-
ent study we evaluated whether monoclonal antibodies raised
against F. gigantica ES antigens would be able to detect the
presence of ES antigens in the feces of animals infected with
We developed a capture ELISA that was capable of detecting
coproantigens of F. gigantica in infected cattle. The monoclo-
nal antibodies reacted with a predominant band at 28 kDa and
with the 28 kDa purified cathepsin L antigen, which suggests
that these monoclonal antibodies most likely recognize cathep-
sin L. The detection of F. gigantica antigens in the feces of
infected animals has several important advantages over the se-
rological assay or detection of eggs in the feces, which are
commonly utilized for F. gigantica diagnosis. First, the co-
proantigen ELISA was the only test to score greater than 90%
in sensitivity and specificity when compared to the detection of
anti–F. gigantica antibodies in serum or fecal egg detection.
Second, although fecal egg detection has the advantages of be-
ing 100% specific, is simple to perform, and can detect patent
infections, the detection of eggs in feces has low sensitivity
when compared to coproantigen ELISAs. Third, although serum
anti–F. gigantica antibodies can be often detected very early
following infection, this test does not discriminate between ac-
tive infection and prior exposure in animals recovered from
infection. Our previous work (Estuningsih et al., 2004) has
shown the coproantigen ELISA detects patent infection.
Our interest in a diagnostic assay for the detection of patent
F. gigantica infection was to facilitate strategic anthelmintic
treatment in developing countries, that is, to allow targeting of
treatment to infected animals whose production traits are af-
fected by fascioliasis. A shotgun approach of treating all ani-
mals with anthelmintics several times a year is unaffordable and
impracticable for subsistence farmers in developing countries
(Roberts and Suhardono, 1996). Intensity of infection is an im-
portant parameter in fascioliasis because losses due to fluke
infection in cattle are greater with increasing burdens (Spithill
et al., 1999). The sandwich ELISA for coproantigens described
in this study suggests control agencies and farmers will be able
to specifically target anthelmintic treatment to ruminants with
burdens above 15 flukes, increasing the effectiveness of treat-
ment and reducing overall costs.
There are virtually no published reports of the prevalence of
F. gigantica infections in Indonesia. The limited surveys carried
out to date indicated prevalence levels between 60 and 100%
(Suhardono et al., 1991; Roberts and Suhardono, 1996); in gen-
eral, the prevalence in cattle and buffaloes is considered to be
high in Indonesia. In the present study, we found a high oc-
currence of fascioliasis in the Yogyakarta region of Indonesia
with prevalence in cattle up to 80–90% in both regions studied.
The percentage of animals infected increased during the study
from August to July and was higher in Bantul relative to the
Kulon Progo region. This was expected because the Bantul re-
gion is near a coastal area where there is more rice cultivation,
one of the major predisposing risk factors for liver fluke infec-
tion (Spithill et al., 1999). Our study also suggests that there
are between-breed variations in susceptibility to F. gigantica
infection. Ongole cattle showed the highest mean F. gigantica
intensity of infection overall in both regions, but it was partic-
ularly higher in the Kulon Progo region. These finding are sim-
ilar to those of Wiedosari et al. (1999), who reported that On-
gole cattle were more susceptible to F. gigantica infection than
other breeds, e.g., Bali cattle and buffalo.
In summary, ELISA for coproantigens evaluated in this study
was able to detect 100% of cattle infected with greater than 15
liver flukes, suggesting that this test may be used as a detection
system to aid strategic targeted anthelmintic treatment of those
infected animals at highest risk of suffering economic loss. The
coproantigen ELISA capture assay has the advantage over the
conventional indirect ELISA for the detection of anti–F. gigan-
tica antibodies in serum or the detection of fecal egg counts
currently available in Indonesia in that patent migrating and
adult infections in the majority of cattle will be identified. Col-
lection of feces, rather than serum, will also allow a more cost-
effective, field-adaptable, and socially acceptable assay.
This study was supported by the Department for International De-
velopment (DFID), Edinburgh, United Kingdom, and the Australian
Centre for International Agricultural Research, Canberra, Australia. The
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