Coprological diagnosis have been routinely applied for
diagnosis of F. hepatica eggs in stools, but have several drawbacks. Antibodies
detection based on specific antigens of Fasciola spp. may be helpful in
diagnosis of fascioliasis after 2–4 weeks post-infection (Dixit et al., 2002; Dumenigo
and Espini, 2003). Over the past decades, serological tests have been increasingly developed for detecting antibodies
in the sera of human and ruminant fascioliasis. Different kinds of parasite
products such as crude extracts of adult parasites, purified proteins from
excretory-secretory products (ESP) have been used for early detection of
fascioliasis (O’Neill et al., 1980).

Nevertheless, problems with specificity and previous
infections have limited specific antibody detection, especially in animal
because of false positive result with a loss of diagnostic specificity. Therefore,
antibody detection by specific
Fasciola antigen may play an important complementary role in serodiagnosis of fascioliasis
(Attalah et al., 2013; Kuerpick et al., 2013 Gottstein et al., 2014).

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Among various antigens, proteases have been considered as
valuable diagnostic antigen (Robinson et al., 2011). It
has been described that cysteine proteinases released from adult and juvenile
fluke in the host circulation (Ruiz et al., 2003), of which the CL1 is
an appropriate antigen for detection of Fasciola spp (Robinson et al.,
2008; Jefferies et al., 2001). This study was conducted to investigate the
potential influence of surface epitopes of CL1 in the serodiagnosis of animal fascioliasis.
A complete
source of protein
cannot be considered as an effective diagnostic antigen due to its expression
in different helminthes. Therefore, to overcome potential cross-reactivity
issues, the specific epitopes of CL1 can be employed for a specific antibody
response. A previous study indicated potential of CL-based synthetic peptides for
discriminating between early and late infection with F.  gigantica in experimentally
infected sheep (Jezek et al., 2008). In
the current study, all infected sera with Fasciola
spp. were positive in the peptide ELISA that the sensitivity of the ELISA test was determined as
100% here for the both synthetic peptides. The test reported 100% negative
predictive value for both peptides. Our findings demonstrated that the tests

using both peptide did not reach the expected specificities. However,
the first peptide showed a higher specificity (87.3%) as
compared to peptide 2 (79%). Therefore, it is likely that if experimentally
infected sera was available, this peptide may well detect positive and negative
cases with a very high specificity, indicating
a need for evaluation of the
cross-reaction
with parasitic sera in experimental infection of the host. Recombinant CL1 with 220 amino acid showed a high level
of cross-reactivity with both, sera from cancer patients and sera from patients
with other parasitic disease such as alveolar echinococcosis and visceral
leishmaniosis, whereas the test did not revealed the expected performance in
ELISA (Gottstein et al, 2014). Conversely, Gonzales Santana et al., (2013) produced
recombinant CL1 based upon Pichia pastoris, that the peptide revealed
excellent sensitivity and specificity for diagnosis of F. hepatica in human.

Of course, the expressive conditions
have been mutually different in the two studies mentioned above, metazoan gene
expression in Pichia pastoris may lead to carboxylation of the peptide,
and thus to a favorable conformation of epitopes. Favorable conformation of
peptide are very important, whereas the three-dimensional
structure could have resulted in the loss of some conformational epitopes
needed for antibody binding. The differential expression pattern of
protease have been accordingly, which this condition can be linked to major changes
in the active site and substrate specificity (Cwiklinski et al., 2015). Therefore,
this factors cab be involved in performance of ELISA upon use of the protease.

Previously, an epitope of CL1 showed high sensitivity and
specificity in ELISA for detection of antibodies in the sera
from cattle experimentally infected with F. hepatica (Cornelissen et
al., 1999). It has been
reported that the ELISA with mentioned peptide had a sensitivity of 98.9% and
specificity of 99.8%. The synthetic peptide 2 was selected as previously
described by Cornelissen et al. (1999). Specificity (79%) of peptide 2 was lower than the
specificity obtained with CL1 by mentioned study (99.8%). It is worth noting
that mentioned
study performed using
sera from calves experimentally infected with pure isolates of parasitic
infection and naturally infection.

Cornelissen et al. (2001) used a recombinant F.
hepatica CL1 for diagnosis of bovine fascioliasis using
sera from naturally infected cattle,
which its specificity was calculated as 75.3%. Our findings indicated relatively better specificity
for the first and second peptides in the ELISA than study conducted by Cornelissen et al., (2001). However, O’Neill et al., (1998) did not find any cross-reactivity between
CL1 and numerous human parasitic disease such as Schistosoma
mansooni, hydatidosis, cysticercosis and chagas in the ELISA test using
IgG4. They indicated the role of IgG4 secondary antibodies in
improving discrimination between positive and negative sera than anti-total
IgG.

Dixit et al., (2002 and 2004) reported a sensitivity 100% for CL1
using ELISA, Dipstick-ELISA and Western blotting. CL
cysteine proteinase has been reported as suitable
antigen for immunodiagnosis
of fascioliasis in experimentally
infected calves with F. gigantica which
demonstrated a sensitivity rate of 100%,
4
weeks post-infection without cross-reactivity
with hydatid cysts, Gigantocotyle
explanatum, and Paramphistomum epiclitum antigens (Sriveny et
al., 2006).

In a study by Varghese et al., (2012), Native CL1 based
Dot-ELISA with a molecular
weight of 28 kDa was used for detection of F. gigantica infection in
buffaloes, the findings showed that this antigen reached ?90% sensitivity and 100% specificity.

Rokni and Gharavi, (2002) used a preliminary epitope of CL1 for diagnosis
of human fascioliasis, which showed a sensitivity of 77% without sufficient
efficiency for antibody detection. Rokni et al., (2002) extracted cathepsin L from execratory and secretory products of F.
hepatica for diagnosis of human fascioliasis using ELISA method. They
reported high sensitivity and specificity (100%), while the rate of specificity
was reported to be 98% using execratory and secretory products.

Many studies have used CL1 as antigen for serodiagnosis of F. hepatica and F. gigantica
with a different molecular nature and different expression system, such as
production of recombinant, synthetic, and fusion peptide in Saccharomyces
cerevisiae, P. pastoris, Escherichia coli,
etc. with various molecular weights
(Dixit et al., 2002; Cornelissen et al., 2001; Sriveny et al., 2006; Raina et
al., 2006; Gonzales Santana et al., 2013; Gottstein et al.,
2014). Contradictory results are
achieved where some of produced peptide did not have the desired performance (Gharavi, 2002; Cornelissen et al., 2001 Gottstein et al., 2014).

 Jezek et al., (2008) evaluated F. gigantica cathepsin L
proteinase-based synthetic peptide for
sheep immunization. This peptide has been
synthesized in three different forms,
and could
be valuable for distinguishing between
early and late, and low and high burden of F. gigantica infection in
sheep (Jezek et al., 2008).

Yamasaki et
al., (1989) used cathepsin L proteinase-based Elisa (Japanese isolates) for
immunodiagnosis of human fascioliasis.
They recorded a high sensitivity, whereas cross-reactivity
was detected in Japanese Schistosoma in a case. Fagbemi and Goubadia, (1995) applied a cysteine
protease with a molecular weight of 28 kDa of F. gigantica
for detection of bovine fascioliasis using ELISA test, and consequently reported cross-reactivity with
different  helminth infections.  The cross-reactivity of the peptide
2 increased remarkably than that of the peptide 1
using serum from animal cystic echinococcosis and dicrocoeliasis.

Regarding
the specificity of our both peptide, an overview of
the findings did not indicate the favorable performance of both antigenic
peptides derived from CL1 for the
serodiagnosis of animal fascioliasis. This can be a result of host
infection with an immature fasciola infection,or very low burden which has not
been detected during slaughter inspection, or because of the presence of
antibodies in the previous host exposure with the parasite or even receiving previous anthelmintic treatments. In other word, sera from naturally infected animal with other infections
might be obtained at an initial stage of infection or might have had previous
antibody response and or the burden of fascioliasis be very low. It is worth noting that several previous studies have not mentioned this effective factors in cross-reactivity that are involved
in performance of ELISA for evaluating animal and human sera. As matter of fact
ccriteria for the fascioliasis sera were not described by many studies,
especially in naturally infected animal. However, this cannot be ascertained because the exact
level of infection was not available for us in naturally infected animals with
other parasitic in the panel. Moreover, the
probability of different results can be related to the population
of subjects evaluated.
The differential expression pattern of protease have been shown which this
issue can be associated with the performance of diagnostic assays base upon protease
application (Cwiklinski et al., 2015).

However, as other
studies indicated the favorable diagnostic value of recombinant CL1, if its
production be carried out by a various expression method (O’Neill
et al., 1999) or as a chemically synthesized peptides polypeptide (Tantrawatpan
et al., 2007). Therefore, it will be necessary for future
studies to, examine other systems of
expression / synthesis at the same time, using the same sera and ELISA protocols. There is a need for standardized test and
performance of ELISA needs to be assessed by a large panel of sera from animal infected with F.
hepatica and other parasitic infections.