Download Immunodiagnosis of fasciolosis using recombinant

Diagnostic Microbiology and Infectious Disease
41 (2001) 43– 49
www.elsevier.com/locate/diagmicrobio
Immunodiagnosis of fasciolosis using recombinant procathepsin L
cystein proteinase
Silvana Carnevalea,*, Mónica I. Rodrı́gueza, Eduardo A. Guarneraa, Carlos Carmonab,
Tamara Tanosa, Sergio O. Angela
a
Departamento de Parasitologı́a, ANLIS “Dr. Carlos G. Malbrán,” Av. Vélez Sársfield 563, (1281) Buenos Aires, Argentina
b
Unidad de Biologı́a Parasitaria, Facultad de Ciencias, Instituto de Higiene, Montevideo, Uruguay
Received March 29, 2001; accepted August 10, 2001
Abstract
Cathepsin L1, a cystein protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli
as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1).
In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent
assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other
parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect
antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post
infection in all the studied animals.
Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis. © 2001 Elsevier
Science Inc. All rights reserved.
1. Introduction
Fasciola hepatica, the common bile duct fluke, is a
prevalent and economically important parasite in the husbandry industry. Although fasciolosis is predominantly a
disease of domestic animals such as sheep and cattle, it is
now emerging as an important chronic disease in humans
with 2.5 million people at risk in the highly endemic areas
of Bolivia and Perú (Chen & Mott, 1990; Arjona et al.,
1995; Hillyer & Apt, 1997; Mas-Coma, 1999).
Definitive diagnosis of fasciolosis in humans is achieved
parasitologically by finding the fluke eggs in feces. However, the flukes start releasing eggs in feces after 8 weeks of
infection, and that is why coprological methods cannot
detect infection during previous weeks when the immature
worms are migrating through the liver parenchyma.
The detection of anti-fluke antibodies in serum by
ELISA is considered a sensitive method and can be used in
conjunction with fecal examination (Hillyer et al., 1992). In
the past, ELISA techniques have used crude somatic ex* Corresponding author. Tel.: ⫹54-11-4301-7437; fax: ⫹54-11-43017437.
E-mail address: [email protected] (S. Carnevale).
tracts of parasites or a preparation of excretory-secretory
products as antigen for the detection of serum antibodies
(Knobloch, 1985; Espino et al., 1987; Khalil et al., 1990;
Espino Hernandez et al., 1991; Hillyer et al., 1992; Sampaio
Silva et al., 1996; Carnevale et al., 2001), but the employment of complex antigens reduce the specificity of the test
due to cross-reactivity with parasites sharing similar immunogens (Hillyer & Serrano, 1983).
Recently, an IgG4-ELISA test has been employed to
detect infected humans using as antigens purified cathepsin
L1 cystein proteinase or recombinant protein expressed in
yeast (O’Neill et al., 1998, 1999). The results of these
reports showed an excellent potential for the development
of a standardized assay for the diagnosis of human fasciolosis.
Cathepsin L1 is secreted by all stages of the developing
parasite; it is capable of cleaving host immunoglobulins and
can prevent in vitro attachment of eosinophils to newly
excysted juveniles (Carmona et al., 1993; Smith et al.,
1993a, b). Additionally, CL1 can degrade extracellular matrix and basal membrane components thus collaborating in
the migration of the parasite (Berasaı́n et al., 1997). This
protease, a vaccine candidate (Spithill & Dalton, 1998), is
one of the major molecules in the excretory-secretory prod-
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 2 8 8 - 7
44
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
ucts of F. hepatica and has shown to be highly immunogenic in infected animals.
A total of seven complete nucleotide sequences of F.
hepatica cathepsin L1 are deposited in the database from the
National Center for Biotechnology Information Bank (Yamasaki & Aoki, 1993, Accession number AB009306; Heussler & Dobbelaere, 1994, Accession number Z22765; Wijffels et al., 1994a, Accession number L33771; Roche et al.,
unpublished data, Accession number U62288; Panaccio et
al., unpublished data, Accession number L33772; Smooker
et al. unpublished data, Accession number AF271385; Cornelissen et al., unpublished data, Accession number
AJ279092).
In order to obtain a specific serologic tool for the diagnosis of F. hepatica infections, a recombinant protein
(rproCL1) was designed and expressed in Escherichia coli.
The rproCL1 protein is codified by nucleotides 82 to 1019
of the cDNA (derived from the data of Heussler & Dobbelaere, 1994), encoding the 90 aminoacid proregion and
the 219 aminoacid mature protease. This construction contains six histydine residues at he N-terminal end. The fusion
protein was purified and tested with experimentally infected
sheep and human sera to determine its antigenic value based
on the detection of IgG anti F. hepatica during infection.
2. Materials and methods
2.1. Sera
All human sera belonged to the sera library at the Departamento de Parasitologı́a Sanitaria, ANLIS “Dr. Carlos
G. Malbrán,” with the exception of Schistosoma mansonipositive sera that were kindly provided by Dr. José Mauro
Peralta, from the Instituto de Microbiologia, CCS-UFRJ,
Rio de Janeiro, Brazil.
Positive control sera (n ⫽ 16) had been obtained from
patients with fasciolosis that had been coprologically or
surgically confirmed.
Negative control sera (n ⫽ 99) belonged to persons
without clinical symptoms of fasciolosis from a non-endemic area in the country and who did not recall watercress
consumption.
One hundred eleven sera from patients with other parasitic and non-parasitic diseases were also employed: schistosomiasis (Sm; n ⫽ 10), toxoplasmosis (Tg; n ⫽ 5), toxocariasis (Tc; n ⫽ 4), hydatid disease (Hd; n ⫽ 15),
cysticercosis (Cy; n ⫽ 9), trichinosis (Ts; n ⫽ 10), Plasmodium vivax malaria (Pv; n ⫽ 2), strongyloidiasis (Ss; n ⫽
3), ascariasis (Al; n ⫽ 6), syphilis (Sf; n ⫽ 5), hepatitis A
(HA; n ⫽ 10), hepatitis B (HB; n ⫽ 20), hepatitis C (HC;
n ⫽ 10), and cyrrosis (Cr; n ⫽ 2).
Sera from ten experimentally infected sheep were also
employed. Ten Corriedale sheep (1 year old) were housed in
a paddock with an artificial water supply. The animals were
purchased from a fluke-free area and shown to be free of
infection by fecal analysis. Serum samples were collected
from each animal weekly between week 0 and 19 of the
experiment. At week 8 all the animals were orally infected
with 300 viable F. hepatica metacercariae obtained in the
laboratory by passage through the intermediary host Lymnaea viatrix.
2.2. Parasite RNA purification
Live adults of F. hepatica were obtained from naturally
infected bovine livers at a local abattoir. Flukes were
washed three times in 0.01 M phosphate-buffered saline
(PBS) pH 7.2 and stored at –70°C until used.
Two parasites were disrupted in 4 M guanidine isothiocyanate by vigorous homogenization using a tissue grinder.
Total RNA was extracted following Chomczynski protocol
(Chomczynski & Sacchi, 1987) with the exception that the
RNA pellet was disolved in diethylpyrocarbonate-treated
water instead of 0.5% sodium dodecyl sulfate (SDS). RNA
was quantified by absorbance at 260 nm.
2.3. RT-PCR and cloning of cystein protease specific
cDNA
Total RNA (1.5 ␮g) was employed to obtain cathepsin
L1 cDNA by RT-PCR using the Access RT-PCR System
(Promega) according to the manufacturer’s instructions.
Two primers were designed, CL1F and CL1R, corresponding to positions 82 to 102 and 998 to 1019 respectively of
the cDNA sequence of F. hepatica cathepsin L1 (Heussler
& Dobbelaere, 1994; GenBank Accession number Z22765).
The RT-PCR protocol included: 1 cycle at 48°C during
45 min for reverse transcription; 1 cycle at 94°C 2 min for
AMV reverse transcriptase inactivation and denaturation;
40 cycles of a denaturation step at 94°C for 30 s, an annealing step at 49°C for 1 min, and an extension step at
68°C for 2 min, with a final extension cycle of 68°C for 7
min.
The 938-bp cDNA fragment was recovered from agarose
gels by elution from DEAE-cellulose membranes and quantified by comparison with DNA standards (Gibco) by UV
fluorescence.
The cDNA (50 ng) was cloned in pGEM T-vector (Promega) and the recombinant plasmid was transformed into
competent DH5␣ E. coli cells.
2.4. DNA sequencing
The nucleotide sequence of the plasmid insert was determined by automatic DNA sequencing employing the ABI
PRISM Big DyeTM Terminator Cycle Sequencing Ready
Reaction Kit (Perkin Elmer Applied Biosystems). Gel electrophoresis was carried out on an ABI 377 Automated DNA
Sequencer (Perkin Elmer).
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
45
Fig. 1. Comparison of the predicted aminoacid sequence of rproCL1 with the seven available sequences of cathepsin L1 from F. hepatica. Alignment between
rproCL1 and cathepsin L1 sequences corresponding to Accesion numbers AJ279092, AF271385, L33772, U62288, L33771, AB00306 and Z22765 showed
identities of 96, 87, 88, 93, 95, 92 and 78%, respectively.
2.5. Construction of expression plasmid
The pGEM T-vector containing the procathepsin L1
cDNA was digested with Bam HI and Sal I endonucleases.
The resulting DNA fragment was purified from agarose
(Qiagen) and cloned into Bam HI-Sal I-digested plasmid
pQE-30 (Qiagen) in order to create the plasmid pQErproCL1. E. coli M15 competent cells were transformed
with this plasmid.
2.6. Expression and purification of recombinant protein
E. coli M15 bacteria containing pQE-rproCL1 from an
overnight culture, diluted 1:20, were grown in Luria broth
supplemented with ampicillin (100 ␮g/mL) and kanamycin
(10 ␮g/mL) for 4 h at 37°C with the addition of IPTG
(isopropyl-␤-D-thiogalactopyranoside) at a final concentration of 2 mM to induce the fusion protein rproCL1. The
bacteria were harvested by centrifugation. The pellet was
resuspended in sample solution (20 mM Tris-HCl pH 8, 1
mM EDTA pH 8, 5% ␤-mercaptoethanol, 2.5% SDS) and
boiled for 5 min. The sample was centrifuged for 30 min at
4°C at 15,000⫻ g and the supernatant was recovered. The
SDS present in the sample was precipitated by the addition
of 1 volume of 100 mM KCl, incubation for 10 min at room
temperature and centrifugation at 15,000⫻ g for 3 min. The
supernatant was equilibrated in lysis buffer (8 M urea, 0.1
M NaH2PO4, 10 mM Tris-HCl, pH 8) by dialysis in two
changes of 10 volumes of lysis buffer during 48 h at 4°C.
The fusion protein was passed through a Ni2⫹-nitrilotriacetic acid resin (Qiagen), and bacterial proteins were eluted
from the column with washing solution (lysis buffer at pH
6.3). Finally, the rproCL1 protein was eluted from the column with purification solution (lysis buffer at pH 4.5).
Protein concentration in the purified sample was measured
by Bradford method (Bradford, 1976). rproCL1 was stored
at –20°C in aliquots containing 40 ␮g of protein.
2.7. PAGE and Western blot
Proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 15% acrylamide gels (Laemmli, 1970) in the MiniProtean System (BioRad). After
electrophoresis proteins were stained with Coomassie blue
or transferred to nitrocellulose membranes (BioRad). For
immunoblot analysis, filters were blocked with blocking
46
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
solution (PBS, 5% skim milk) for 1 h and washed three
times in PBS, 0.05% Tween 20. Positive and negative
control human sera for F. hepatica infection were probed at
1:100 dilution in blocking solution for 1 h. A peroxidase
immunoconjugate (Sigma) diluted 1:1,500 was used as the
second antibody, and specific binding was developed with
diaminobenzidine as a chromogenic substrate.
2.8. rproCL1 ELISA
Microtiter plates (PolySorp, Nunc) were coated with the
rproCL1 antigen (4 ␮g/mL) in 0.05 M carbonate buffer, pH
9.6 (100 ␮L/well), and incubated for 3 h at 37°C and
overnight at 4°C. After three washes with PBS, 0.05%
Tween 20, the plates were blocked with 100 ␮L of blocking
reagent (3% bovine serum albumin (BSA) diluted in
PBS,0.05% Tween 20) by incubation for 30 min at 37°C.
The plates were then washed as described above and 100
␮L of serum were applied to each well. To test human sera,
they were diluted 1:250 in blocking reagent. In the case of
sheep sera, the optimal dilution was 1:100. The plates were
incubated for 30 min at 37°C and washed as described
above. Goat anti-human IgG (Sigma) or donkey anti-sheep
IgG (The Binding Site) horseradish peroxidase-labeled antibodies, diluted 1:3,000 and 1:1,000 respectively, were
used as the secondary antibody. After incubation for 30 min
at 37°C and washes, the reaction was developed with 2–2⬘azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) as
the chromogenic substrate. Plates were read after 10 min on
a Dynatech MR4000 plate reader at an absorbance of 410
nm (A410). All samples were run in duplicate. The cutoff
points were established as the mean value of reactivity plus
3.09 standard deviations of the negative controls.
The data collected were compared by analysis of variance. P values lower than 0.05 were considered significant.
3. Results
Fig. 2. Purified fusion protein rproCl1 resolved by SDS-PAGE on 15%
polyacrylamide gel. (A) Coomasie blue staining of the gel. Lanes: 1, first
fraction from the Ni2⫹-nitrilotriacetic acid resin; 2 and 3, washing steps; 4
to 7, elution fractions of the specific protein. (B) Immunoblots of 3 ␮g of
fusion protein rproCL1 with an anti-F. hepatica positive (lane 1) and
negative (lane 2) human serum. Molecular masses are shown on the left.
The predicted proenzyme (36 kDa) is marked with arrows.
coli, and the recombinant protein (rproCL1) was purified by
affinity chromatography and analyzed by SDS-PAGE (Fig.
2A). Coomassie blue staining of the purified rproCL1
showed a strong signal visible at 36 kDa, which corresponded to the expected mass of the proenzyme according
to the aminoacid sequence.
After Western blotting, purified rproCL1 was probed
with human control sera. Serum from a patient with surgically proven fasciolosis was the only one that reacted
against the fusion protein (Fig. 2B).
After purification, the rproCl1 was recovered in the order
of 800 –1,000 ␮g/100 mL of induced culture.
3.3. ELISA with human sera
3.1. Sequence analysis
The predicted aminoacid sequence of rproCL1 was compared with the seven F. hepatica cathepsin L sequences
deposited in the database (Fig. 1). The overall aminoacid
identity varied from 96% to 78%. Residues at positions
106 –107 (positions are given on the basis of the complete
predicted aminoacid sequence of cathepsin L1), corresponding to the cleavage site to form the mature protein are
conserved. Residues 126 to 136 contain similarity to the
thiol cathepsin consensus pattern (Gln-Xaa(3)-[Gly Glu]Xaa-Cys-Trp-Xaa(2)-[Ser-Thr-Ala-Gly]).
3.2. Expression of Fasciola hepatica recombinant
procathepsin L1 protein
The cDNA of the procathepsin L1 from F. hepatica was
cloned into the expression vector pQE-30, expressed in E.
The rproCL1 was used to sensitize ELISA plates which
were probed with human sera. Frequency distribution of
positive and negative control sera was plotted as histogram
(Fig. 3A). The mean A410 of the negative control group was
0.26 with a standard deviation of 0.05 and a range between
0.15 and 0.36. The cutoff point resulted in 0.41 (dashed line
in Fig. 3A). Sera from patients with fasciolosis showed a
mean value of 0.63 ⫾ 0.11 with a range between 0.49 and
0.90. Using the calculated cutoff point, the sensitivity of the
assay was 100%.
The specificity of the test was analyzed by measuring the
reactivities of sera from patients with other diseases (Fig.
3B). No cross-reactivity was detected in any of the 111 sera,
since none of them showed A410 values above the cutoff
point. The mean absorbances obtained for all these sera
were not significantly different from those obtained for the
negative control sera (p ⬎ 0.05). Moreover, all samples
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
47
Fig. 3. Immunoreactivity of the rproCL1 with a collection of sera from humans infected and non infected with F. hepatica. (A) Analysis by ELISA of sera
obtained from 99 negative control patients and 16 individuals with parasitologically positive F. hepatica infection. The y axis shows the frequency of
absorbance measurements. The vertical dashed line indicates the calculated cutoff point at 3.09 SD from the mean of the seronegative group. (B) ELISA
absorbances of 111 sera from individuals with parasitic and non parasitic diseases other than fasciolosis. Sm: schistosomiasis, Tg: toxoplasmosis, Tc:
toxocariasis, Hd: hydatid disease, Cy: cysticercosis, Ts: trichinosis, Pv: Plasmodium vivax malaria, Ss: strongyloidiasis, Al: ascariasis, Sf: syphilis, HA:
hepatitis A, HB: hepatitis B, HC: hepatitis C, and Cr: cyrrosis. Each individual is indicated by a closed square. The dashed horizontal line depicts the cutoff
value.
from the F.hepatica-infected patients had significantly
higher absorbance readings than those obtained from individuals with other diseases (p ⬍ 0.05). According to these
results, the specificity of the assay was 100%.
and by three weeks post-infection (week 11) showed values
higher than the cutoff. There was a steady increase, reaching
a peak at weeks 6 –7 post-infection (weeks 14 –15 of the
experiment).
3.4. Antibody response to Fasciola hepatica rproCL1 in
sheep
4. Discussion
To determine the antigenic value of rproCL1 in farm
animals, serum samples from experimentally infected sheep
obtained at different time points were probed by rproCL1ELISA. Development of anti-F. hepatica rproCL1 IgG levels in all animals is shown in Fig. 4. The absorbance values
at weeks 0 to 8 were used to calculate the cutoff point
resulting in 0.25. The dynamics of IgG response was similar
in all infected animals. Specific IgG levels ascended quickly
Fig. 4. Development of anti-F. hepatica rproCL1 IgG levels in 10 sheep
experimentally infected with 300 metacercariae at Week 8 of the experiment, tested by rproCL1-ELISA. Each curve corresponds to one animal.
Human fasciolosis is becoming increasingly recognized
as a serious public health problem. Clinical manifestations
include fever, pain in the right hypochondrium, anorexia,
weight loss, persistent diarrhea and vomiting, but many
individuals are asymptomatic or present vague symptoms
(Bunnag et al., 1991; Arjona et al., 1995; Bryan & Michelson, 1995).
To overcome the clinical diagnostic problems, there is a
need for a reliable, simple, cost-effective diagnostic tool to
determine F. hepatica infections. Here, we report that a
recombinant procathepsin L1 employed as antigen, ensures
the sensitivity and specificity of the indirect ELISA for the
immunodiagnosis of F. hepatica.
The antigenicity of rproCL1 was evaluated with human
sera by ELISA and its specificity was demonstrated by the
low reactivity shown by sera from patients without fasciolosis and no-cross reactions with other parasitic and non
parasitic infections. As the validation of an ELISA is a
balance between sensitivity and specificity, our results are
promising as both parameters reached values of 100%.
However, a larger number of positive control sera should be
analyzed to confirm the sensitivity value. Additionaly, a
large scale validation of specificity of the test should include
sera from other helminthiasis, particularly paragonimiasis
which is a major cause of cross reactivity (Ikeda et al.,
1996).
48
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
Our results are in agree with previous studies that
showed that cathepsin L1 purified from the excretory-secretory products of F. hepatica as single antigen in ELISA
provided a more conclusive diagnosis compared with excretory-secretory extract, discriminating more clearly between seropositive and seronegative populations (O’Neill et
al., 1998). However, production of sufficient quantities of
pure cathepsin L1 is a complex, time-consuming and expensive process. To overcome this problem, recombinant
cathepsin L1, produced in yeast (Roche et al., 1997) was
used in an IgG4-ELISA as antigen and showed a similar
performance to the native antigen (O’Neill et al., 1999).
The important development in this study is the production of a recombinant antigen in an economical and rapid
expression system, providing a sensitive, specific and standardized test for the serologic diagnosis of human fasciolosis. The expression of the recombinant protein in its immature form in bacteria culture has the advantages of an
easy and low cost-system, an standardized protocol to produce similar batches of antigen and a reduced toxicity for
bacteria.
Although we centered our attention on the diagnosis of
fasciolosis in humans, we tested the recombinant antigen
with sera from experimentally infected sheep. F. hepatica is
a common parasite of domestic livestock (cattle, sheep,
goats), causing severe disease and economic losses (Froyd,
1975; Haroun & Hillyer, 1986). Several antigens and test
systems have been described for the detection of F. hepatica
in ruminants, including indirect hemagglutination and
ELISA with crude and excretory-secretory antigen (Zimmerman et al., 1982; Santiago & Hillyer, 1988; Cornelissen
et al., 1992).
Attempts using cathepsin L proteases, as well as peptides
selecting epitopes of cathepsin L1, in order to develop
reliable tests for the diagnosis of F. hepatica infections have
been performed in sheep and cattle (Wijffels et al., 1994b;
Cornelissen et al., 1999). The results of our study, although
preliminary, are promising: sera from sheep experimentally
infected with F. hepatica gave a seroconversion in the
ELISA as soon as three weeks post infection, in concordance with the pattern of antibody response to F. hepatica
excretory-secretory products in goats (Martı́nez-Moreno et
al., 1999). Therefore, this first result encourages us to study
the potential use of this recombinant proenzyme in the
serologic diagnosis of F. hepatica in sheep and cattle.
The results shown here demonstrate that the recombinant
protein procathepsin L1 from F. hepatica can be employed
as a powerful antigen in a human and veterinarian serologic
diagnostic system.
Acknowledgments
The authors would like to thank Dr. José Mauro Peralta,
from the Instituto de Microbiologia, UFRJ, Rio de Janeiro,
Brasil, for providing Schistosoma mansoni serum samples.
S. O. Angel (Researcher) is member of National Council
Research (CONICET). This work was partially supported
by an ANPCyT grant (BID802/OC-AR-PICT 05– 04831).
REFERENCES
Arjona, R., Riancho, J., Aguado, J., Salesa, R., & Gonzalez-Macias, J.
(1995). Fasciolosis in developed countries: a review of classic and
aberrant forms of the disease. Medicine (Baltimore), 74, 13–23.
Berasaı́n, P., Goñı́, F., McGonicle, S., Dowd, A., Dalton, J., Frangione, B.,
& Carmona, C. (1997). Proteinases secreted by Fasciola hepatica
degrade extracelullar matrix and basement membrane components. J
Parasitol, 83, 1–5.
Bradford, M. M. (1976). A rapid sensitive method for the quantitation of
protein utilizing the principle of protein dye binding. Anal Biochem, 72,
248 –256.
Bryan, R. T., & Michelson, M. K. (1995). Parasitic infections of the liver
and biliary tree. In Gastrointestinal and Hepatic Infections. Eds, C
Surawicz and RL Owen. Philadephia: WB Saunders, pp 405– 454.
Bunnag, D., Thanongsak, B., & Goldsmith, R. (1991). Fasciolosis. In
Hunter’s Tropical Medicine. Ed, GT Strickland. Philadelphia: WB
Saunders, pp 823– 826.
Carmona, C., Smith, A., Dowd, A., & Dalton, J. (1993). Cathepsin L
proteinase secreted by Fasciola hepatica in vitro prevents antibodymediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol, 62, 9.
Carnevale, S., Rodrı́guez, M. I., Santillan, G., Labbé, J. H., Cabrera, M. G.,
Bellegarde, E. J., Velásquez, J. N., Trgovcic, J. E., & Guarnera, E. A.
(2001). Immunodiagnosis of human fasciolosis by enzyme-linked immunosorbent assay (ELISA) and Micro-ELISA. Clin Diagn Lab Immunol, 8, 174 –177.
Cornelissen, J. B. W. J., de Leeuw, W. A., & van der Heijden, P. J. (1992).
Comparisson of the indirect haemagglutination assay and the ELISA
for diagnosing Fasciola hepatica in experimentally and naturally infected sheep. Vet Q, 14, 152–156.
Cornelissen, J. B. W. J., Gaasenbeek, C. P. H., Boersma, W., Borgsteede,
F. H. M., & van Milligen, F. J. (1999). Use of a pre-selected epitope of
cathepsin-L1 in a highly specific peptide-based immunoassay for the
diagnosis of Fasciola hepatica infections in cattle. Int J Parasitol, 29,
685– 696.
Chen, M. G., & Mott, K. E. (1990). Progress in morbidity due to Fasciola
hepatica infection. Trop Dis Bull, 87, 1–37.
Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem, 162, 156 –159.
Espino, A. M., Dumenigo, B. E., Fernandez, R., & Finlay, C. M. (1987).
Immunodiagnosis of human fasciolosis by enzyme-linked immunosorbent assay using excretory-secretory products. Am J Trop Med Hyg, 37,
605– 608.
Espino Hernandez, A. M., Seuret, N., Morlote, C., & Dumenigo Ripoll, B.
(1991). Antı́genos de Fasciola hepatica. Su utilidad en el diagnóstico
de la fasciolosis humana. Rev Cubana Med Trop, 43, 151–155.
Froyd, G. (1975). Liver flukes in Great Britain: a survey of afected livers.
Vet Rec, 97, 492– 495.
Haroun, E. M., & Hillyer, G. V. (1986). Resistenace to fasciolosis: a
review. Vet Parasitol, 20, 63–93.
Heussler, V. T., & Dobbelaere, D. A. (1994). Cloning of a protease gene
family of Fasciola hepatica by the polymerase chain reaction. Mol
Biochem Parasitol, 64, 11–23.
Hillyer, G. V., & Apt, W. (1997). Food borne trematode infections in the
Americas. Parasitol Today, 13, 87:88.
Hillyer, G. V., & Serrano, E. A. (1983). The antigens of Paragonimus
westermani, Schistosoma mansoni and Fasciola hepatica adult worms.
Evidence for the presence of cross-reactive antigens and for cross-
S. Carnevale et al. / Diagnostic Microbiology and Infectious Disease 41 (2001) 43– 49
protection to Schistosoma mansoni infection using antigens of Paragonimus westermani. Am J Trop Med Hyg, 32, 350:358.
Hillyer, G. V., Soler de Galanes, M., Rodriguez-Perez, J., Bjorland, J., De
Lagrava, M. S., Ramirez Guzman, S., & Bryan, R. T. (1992). Use of the
Falcon assayTM screening test-enzyme linked immunosorbent assay
(FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot
(EITB) to determine the prevalence of human fasciolosis in the Bolivian Altiplano. Am J Trop Med Hyg, 46, 603– 609.
Ikeda, T., Oikawa, Y., & Nishiyama, T. (1996). Enzyme-linked immunosorbent assay using cysteine proteinase antigens for immunodiagnosis of human paragonimiasis. Am J Trop Med Hyg, 55, 435– 437.
Khalil, H. M., Abdel, T. M., Maklad, M. K., Abdallah, H. M., Fahmy, I. A.,
& El Zayyat, E. A. (1990). Specificity of crude and purified Fasciola
antigens in immunodiagnosis of human fasciolosis. J Egipt Soc Parasitol, 20, 87:94.
Knobloch, J. (1985). Human fasciolosis in Cajamarca/Peru: humoral antibody response and antigenaemia. Trop Med Parasitol, 36, 91–93.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680 – 685.
Martı́nez-Moreno, A., Jiménez-Luque, V., Moreno, T., Rodondo, E. S. H.,
Martı́n de las Mulas, J., & Pérez, J. (1999) Liver pathology and immune
response in experimental Fasciola hepatica infections in goats. Vet
Parasitol, 82, 19 –33.
Mas-Coma, M. S., Esteban, J. G., & Bargas, M. D. (1999). Epidemiology
of human fasciolosis: a review and proposed new clasiffication. Bull
World Health Organ, 77, 340 –346.
O’Neill, S. M., Parkinson, M., Dowd, A. J., Strauss, W., Angles, R., &
Dalton, J. P. (1999). Short report: immunodiagnosis of human fasciolosis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg, 60, 749 –751.
O’Neill, S. M., Parkinson, M., Strauss, W., Angles, R., & Dalton, J. P.
(1998). Immunodiagnosis of Fasciola hepatica infection (fasciolosis)
in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg, 58, 417– 423.
Roche, L., Dowd, A. J., Tort, J., McGonigle, S. M., McSweeney, A.,
Curley, P., Ryan, T., & Dalton, J. P. (1997). Functional expression of
49
Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae. Eur
J Biochem, 245, 373–380.
Sampaio Silva, M. L., Correia da Costa, J. M., Viana da Costa, A. M.,
Pires, M. A., Lopes, S. A., Castro, A. M., & Monjour, L. (1996).
Antigenic components of excretory-secretory products of adult Fasciola hepatica recognized in human infections. Am J Trop Med Hyg, 54,
146 –148.
Santiago, N., & Hillyer, G. V. (1988). Antibody profiles by EITB and
ELISA of cattle and sheep infected with Fasciola hepatica. J Parasitol,
74, 810 – 818.
Smith, A., Dowd, A., McGonigle, S. M., Keegan, P., Brennan, G., Trudgett, A., & Dalton, J. P. (1993a). Purification of a cathepsin L-like
proteinase secreted by adult Fasciola hepatica. Mol Biochem Parasitol,
62, 1– 8.
Smith, A., Dowd, A. J., Hefferman, M., Robertson, C., & Dalton, J. P.
(1993b). Fasciola hepatica: a secreted cathepsin L-like proteinase
cleaves host immunoglobulins. Int J Parasitol, 23, 977–983.
Spithill, T. W., & Dalton, J. P. (1998). Progress in development of liver
fluke vaccines. Parasitol Today, 14, 224 –228.
Wijffels, G. L., Panaccio, M., Salvatore, L., Wilson, L., Walker, I. D., &
Spithill, T. W. (1994a). The secreted cathepsin L-like proteinase of the
trematode. Fasciola hepatica, contains, 3-hydroxy-proline residues.
Biochem J, 299, 781–790.
Wijffels, G. L., Salvatore, L., Dosen, M., Waddington, J., Wilson, L.,
Thompson, C., Campbell, N., Sexton, J., Wicker, J., Bowen, F., Friedel,
T., & Spithill, T. W. (1994b). Vaccination of sheep with purified
cysteine proteinases of Fasciola hepatica decreases worm fecundity.
Exp Parasitol, 78, 132–148.
Yamasaki, H., & Aoki, T. (1993). Cloning and sequences analysis of the
major cysteine proteases expressed in the trematode parasite Fasciola
spp. Biochem Mol Biol Int, 31, 537–542.
Zimmerman, G. L., Jen, L. W., Cerro, J. E., Farnsworth, K. L., & Wescott,
R. B. (1982). Diagnosis of Fasciola hepatica in sheep by an enzymelinked immunosorbent asssay. Am J Vet Res, 43, 2097–2100.