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Acta Tropica 118 (2011) 217222
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Short communication
Lack of protective efcacy in buffaloes vaccinated with Fasciola gigantica leucine
aminopeptidase and peroxiredoxin recombinant proteins
O.K. Raina
a,
, Gaurav Nagar
a
, Anju Varghese
a
, G. Prajitha
a
, Asha Alex
a
, B.R. Maharana
a
, P. Joshi
b
a
Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P. 243122, India
b
Division of Biochemistry, IVRI, Izatnagar, India
a r t i c l e i n f o
Article history:
Received 25 December 2010
Received in revised form 10 February 2011
Accepted 12 February 2011
Available online 3 March 2011
Keywords:
Fasciola gigantica
Leucine aminopeptidase
Peroxiredoxin
Vaccination
Protection
a b s t r a c t
Gene codingfor leucine aminopeptidase (LAP), a metalloprotease, was identiedinthe tropical liver uke,
Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopepti-
dase of the temperate liver uke, Fasciola hepatica. The recombinant leucine aminopeptidase protein
was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an
immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes
was conducted with these two recombinant proteins, with 150 and 300g of leucine aminopeptidase
and a cocktail of 150g each of recombinant leucine aminopeptidase and peroxiredoxin in three groups,
respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization
with these antigens. A challenge study with 400 metacercariae did not show a signicant protection in
terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized
groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are
contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate
vaccine molecule. Identication of leucine aminopeptidase gene and evaluation of the protein for its
protective efcacy in buffaloes is the rst scientic report on this protein in F. gigantica.
2011 Elsevier B.V. All rights reserved.
1. Introduction
Fasciola gigantica (tropical liver uke) inicts substantial pro-
ductivity losses on the livestock industry in the tropical countries
and adversely affects the health, weight gain, feed conversion ef-
ciency and reproduction of buffaloes (FAO, 1994; Mehra et al.,
1999). The pathogenesis of tropical fasciolosis in buffaloes has
been remarkably complex, involving a liver migratory phase of
the parasite causing traumatic hepatitis and a phase of matura-
tion and establishment of the adult parasite in the bile ducts,
causing hyperplastic obstructive cholangitis (Radostits et al., 1994;
Aiello and Mays, 1998). India, being an agrarian society, has the
largest buffalo population in the world, which constitutes a very
signicant component of the Indian livestock sector (Agricultural
Research Data Book, 2002). There are endemic pockets of fas-
ciolosis in India (Sanyal, 2001), a major constraint in buffalo
productivity. Despite intensive research efforts, progress in the
development of effective vaccine for Fasciola hepatica and F. gigan-
tica control has been relatively slow (Mendes et al., 2010). Several
parasite molecules have been identied that have the potential to
induce a protective immune response in the laboratory and large

Corresponding author. Tel.: +91 9458269041; fax: +91 5812302368.

E-mail address: rainaok@rediffmail.com (O.K. Raina).
animals but the protection conferred to the host has been shown
to vary.
Leucyl aminopeptidases (LAPs), members of the M1 or M17
peptidase families, constitute a group of diverse and ubiquitous
Zn-dependent metallopeptidases and play a crucial role in the par-
asite biology of protozoa and helminths. LAPs present broader
amidolytic activity beyond leucine hydrolysis and participate
in processing/maturation/activation or degradation of substrates
(Rawlings et al., 2006; Matsui et al., 2006). The homo-hexameric
M17 LAPs are being revealed as novel drug targets and vaccine can-
didates against parasitic infections. In helminths, LAPs have been
poorly characterized although there is evidence that support their
participation in vital processes in the parasite life cycle. LAP has
been detected, puried and characterized in Ascaris suum, Schis-
tosoma mansoni, Schistosoma japonicum and Haemonchus contortus
(Rogers and Brooks, 1978; Rhoads and Fetterer, 1998; McCarthy
et al., 2004; Abouel-Nour et al., 2005). The F. hepatica leucine
aminopeptidase was identied, characterized (Acosta et al., 1998,
2008) andevaluatedina single vaccinationtrial insheep, where the
proteinwas shownas a relevant candidate for vaccine development
(Piacenza et al., 1999). In F. gigantica, the pathogenic species of the
tropical countries, the protein has not been identied.
The F. hepatica peroxiredoxin (Prx) plays an important role in
the parasites defense against host generated oxidants (McGonigle
et al., 1997, 1998). The proteinalso acts as animmunomodulator by
0001-706X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2011.02.008
Author's personal copy
218 O.K. Raina et al. / Acta Tropica 118 (2011) 217222
inducing the recruitment of alternatively activated macrophages
to the peritoneum (Donnelly et al., 2005, 2008). Alternatively
activated macrophages are key to promoting Th2 responses and
suppression of Th1 inammatory pathology during helminth infec-
tions. Using murine models for the infections caused by F. hepatica
and S. mansoni, it has been shown that parasite peroxiredoxin
induces alternative activation of macrophages by way of expres-
sion of surface markers, Ym1, Fizz1, arginase on macrophages, a
key to promoting Th2 response in the host (Donnelly et al., 2005,
2008).
In the present study, gene coding for leucine aminopeptidase
was identied in F. gigantica and recombinant protein expressed
in Escherichia coli. Another protein of the parasite, peroxiredoxin,
was alsoclonedandexpressed. The tworecombinant proteins were
evaluated for vaccine potential in buffaloes.
2. Materials and methods
2.1. Parasite collection
F. gigantica ukes were collected from a local buffalo abattoir,
Bareilly, U.P., India, in physiological saline and transported to the
laboratory in chilled saline.
2.2. Cloning and expression of recombinant leucine
aminopeptidase protein
The ukes were washed extensively in normal saline and total
RNA was isolated from 30 to 50mg of the parasite tissue, using
Trizol reagent (Invitrogen, USA) following manufacturers proto-
col. The RNA was used in the synthesis of cDNA using oligo-dT
primer and M-MLV reverse transcriptase enzyme (MBI Fermen-
tas, USA). The cDNA was subjected to polymerase chain reaction
(PCR) using primers specic to the temperate liver uke F. hepat-
ica leucine amino-peptidase (GenBank accession no: AY644459),
with sequences forward primer 5

-atggcggcgttggctgtgggcgt-3

and
reverse primer 5

-ctatttgaatcccagtcgtgg-3

. The full length F. gigan-

tica leucine aminopeptidase open reading frame was amplied,
cloned in a TA cloning vector (pDRIVE, Qiagen, Germany) and
sequenced. The gene was sub-cloned in a prokaryotic expression
vector (pPROEXHT-b, Life Technologies, USA) and the recombinant
leucine aminopeptidase expressed in the E. coli DH5 at 28

C for
68h post-induction with 1mM IPTG. The recombinant protein
was puried by NiNTA afnity chromatography in its native con-
formation. Briey, the bacterial cells were exposed to 34 cycles
of alternate freezing and thawing at 40

C and 37

C. Bacterial
cells were sonicated with 5 bursts of 30s each at 10 amplitude
(Soniprep, USA) and cell lysate centrifuged at 10,000g. Soluble
recombinant leucine aminopeptidase was puried from the bacte-
rial lysate by binding to NiNTA resin (Qiagen, GmbH, Germany)
in tris-phosphate buffer (10mM Tris, 100mM NaH
2
PO
4
, pH 8.0;
supplemented with 20mM imidazole). A step elution protocol
was developed to remove the contaminating bacterial proteins by
washing the recombinant protein bound resin with 10ml each of
tris-phosphate buffer (pH 8.0), supplemented with imidazole con-
centrations of 25, 50 and 80mM, respectively. The recombinant
protein was eluted in the same buffer, containing 250mM imi-
dazole. The tris-phosphate buffer used in the above purication
protocol was supplemented with 300mM NaCl.
2.3. Expression of peroxiredoxin protein
F. gigantica cDNA was used for the PCR amplication of
peroxiredoxin gene using primers specic to F. gigantica perox-
iredoxin (Prx) gene (GenBank accession no: GQ 845012), with
primer sequences forward 5

-atgttgcagcctaatatgccc-3

and reverse
5

-ctagttggctgaggagaaata-3

. The ampliedPCRproduct was cloned

in a TA cloning vector (pDRIVE, Qiagen, Germany) and sequence
information determined. Peroxiredoxin gene was sub-cloned in
a prokaryotic expression vector (pPROEXHT-b, Life Technologies,
USA) and protein expressed in E. coli DH5. Expression and puri-
cation protocol for the recombinant peroxiredoxin protein was
identical to the one followed for recombinant leucine aminopepti-
dase, except for the resin washing being carried out in wash buffer,
supplemented with 25, 35 and 50mM imidazole, respectively. The
recombinant protein was eluted at 250mM imidazole concentra-
tion in the wash buffer.
2.4. Immunization protocol
Twenty nine buffalo calves, 810 months old were purchased
through a local animal contractor from F. gigantica non-endemic
areas of North India. Serological and faecal screening was carried
out to rule out previous exposure to F. gigantica. Calves were main-
tained under normal husbandry conditions on a concrete oor in
the experimental shed of the Institute. Experimental procedures
were carried out as per the guidelines of the Institute Animal
Ethics Committee. These calves were randomly distributed in Gr.I
and Gr.II with 7 calves each, Gr.III with 6 calves and Gr.IV with 9
calves as non-immunized control group. Animals in Gr.I and II were
immunized with 150 and 300g recombinant leucine aminopep-
tidase, respectively, and Gr.III calves received a cocktail of leucine
aminopeptidase and peroxiredoxin proteins at 150g of each pro-
tein. Both the antigens were delivered to the host with montanide
M-70 VG (Seppic, France) at 70:30 adjuvantantigen ratios. The
antigenadjuvant emulsion was prepared in a glass homogenizer
with a Teon coated piston at a speed of 4000rpm on ice for ini-
tial 2min, followed by another 2min homogenization at 4000rpm
on ice after a 2min resting period. Antigen emulsion was injected
to the calves via intramuscular route in the neck and glutial mus-
cles. Three immunizations were given at 3 week interval. A gap of
one month was maintained after the last immunizing dose before
challenging these animals along with the un-immunized control
calves. Animals were challenged per os with a dose of 400 viable
metacercariae prepared in a wheat our bolus. The metacercariae
were harvested from F. gigantica infected Lymnaea auricularia,
collected from the natural water bodies (ponds). Snails positive
for F. gigantica metacercariae were maintained in the laboratory.
Metacercariae were harvested on polyethylene strips and stored in
distilled water at 4

C before infection of the experimental animals.

Metacercariae were in vitro hatched to newly excysted juveniles
and conrmed for F. gigantica species by PCR amplication and
sequencingof ITS-2sequence of the ribosomal DNAinthe juveniles.
Buffalo calves were infected within one month of the harvesting
of metacercariae. Experimental animals were euthanized by intra-
venous injection of 50100ml of saturated magnesium sulphate
at 1820weeks post-challenge infection. Liver was cut into slices
approximately 1cm thick and squeezed in warm saline for uke
recovery.
2.5. Enzyme linked immunosorbent assay
Serum samples collected periodically from each animal under
experiment were probed by ELISA (Wijffels et al., 1994). Checker-
board titration was initially performed to determine the optimum
quantum of antigen, sera dilution and antiglobulin enzyme conju-
gate concentration. Polystyrine microtitre plates (Nunc, Denmark)
were sensitized with 100l of 0.05M carbonatebicarbonate
buffer, pH9.6, containing 2g/ml of rLAP or peroxiredoxinantigen,
followed by overnight incubation at 4

C. The wells were washed

with PBS containing 0.05% Tween-20 (PBS-T), thrice for 5min each.
Subsequently, blocking was performed using 3% skimmed milk in
Author's personal copy
O.K. Raina et al. / Acta Tropica 118 (2011) 217222 219
PBS-T at 37

C for 2h. After 3 washings with PBS-T, 100l of 1%

skimmed milk in PBS containing test sera (1:100) was added in
each well and incubated at 37

C for 1h. Plates were washed thrice

with PBS-T and incubated for 1h at 37

C after adding 100l of

1% skimmed milk in PBS containing 1:8000 dilution of rabbit anti-
bovine IgG-HRP-conjugate (Sigma Chemicals, USA). Finally, after
3 washes with PBS-T, 100l substrate buffer (OPD-8mg, 10ml of
citrate buffer, pH 5.0, 10l of H
2
O
2
) was added to the wells and
kept in dark for 510min. The absorbance was read at 492nm on
microplate ELISA reader (Bio-Rad, USA). The results, expressed as
the mean of the optical density (OD) were obtained from dupli-
cate samples. Sera fromuninfected and F. gigantica experimentally
infected animals were used as negative and positive controls,
respectively.
The protocol followed for assessment of IgG response was also
usedfor measuringtheIgG1andIgG2, responses. Sheepanti-bovine
IgG1 and IgG2HRP conjugate (Serotech Inc., USA) were used at
1:2000 dilutions.
2.6. Statistical analysis
Data were subjected to one-way analysis of variance using SPSS
softwarefor determiningthesignicanceof differenceintheexper-
imental data. A p value less than 0.05 was considered as signicant.
3. Results
3.1. Sequence analysis of F. gigantica LAP gene
The complete leucine aminopeptidase open reading frame of
1572bp in F. gigantica was amplied using primer sequences tar-
geting F. hepatica LAP gene. Sequence analysis of F. gigantica LAP
gene showed a close homology of the two parasite species, with an
identity of 98.6% at the nucleotide level. A change of 22 nucleotides
at 78, 145, 261, 391, 457, 486, 489, 498, 624, 628, 634, 639, 801, 922,
1194, 1210, 1239, 1337, 1383, 1452, 1489 and 1504th bp caused an
overall change of 7 amino acids fromasparagine to histidine (145th
bp), glycine to serine (628th bp), methionine to leucine (634th bp),
glutamic acid to glutamine (922th bp), proline to alanine (1210th
bp), threonine to isoleucine (1337th bp) and threonine to alanine
(1489th bp) in the leucine aminopeptidase protein of F. gigantica,
(GenBank accession no: GQ214329).
3.2. Generation of LAP and Prx recombinant proteins
The recombinant leucine aminopeptidase and peroxiredoxin
proteins were puried by step elution protocol, with wash buffer
supplemented with 25mM, 50mM and 80mM for recombinant
LAP and 25mM, 35mM and 50mM imidazole for the recombinant
Prx purication, respectively. The yield of two proteins was 23
and 56mg/l of bacterial culture, respectively. The recombinant
LAP resolved at 56kDa and peroxiredoxin at 24kDa in SDSPAGE
(Fig. 1). The recombinant proteins were stable at 4

C for more than

a month but aggregated/precipitated on storage at 20

C and at
a range of temperatures (room temperature, 37

C and 50

C for
24h). The peroxiredoxin protein was more stable than recombi-
nant leucine aminopeptidase, as it did not aggregate on storage at
room temperature and at 37

C for 24h, respectively.

3.3. Humoral immune response
Humoral immune response in immunized and infected control
calves was probed with recombinant LAP and Prx antigens, respec-
tively. Humoral response was measured in groups of buffaloes that
received 150 and 300g of the recombinant LAP and a cocktail
Fig. 1. F. gigantica recombinant leucine aminopeptidase (56kDa) (a) and peroxire-
doxin (24kDa) (b) puried by NiNTA afnity chromatography and resolved on 12%
SDSPAGE.
0
0.5
1
1.5
2
O
D
4
9
2
IgG LAP Imm
Un-imm cont
Pre
vac
1w
ist bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
16w
ch
Fig. 2. Mean IgG response in buffalo calves immunized each with LAP-150g (Gr.I)
(abbreviations: vac, vaccination; bt, booster; ch, challenge, w, weeks).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Pre
vac
1w
Ist bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
16w
ch
O
D
4
9
2
IgG1
IgG2
un-imm cont
Fig. 3. Mean IgG1 and IgG2 responses in buffalo calves immunized each with LAP-
150g (Gr.I).
0
0.5
1
1.5
2
2.5
Pre
vac
1w
Ist bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
16w
ch
O
D

4
9
2
IgG LAP imm
un-imm cont
Fig. 4. MeanIgGresponse inbuffalocalves immunizedeachwithLAP-300g (Gr.II).
Author's personal copy
220 O.K. Raina et al. / Acta Tropica 118 (2011) 217222
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Pre
vac
1w Ist
bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
16w
ch
O
D
4
9
2
IgG1
IgG2
un-imm
cont
Fig. 5. Mean IgG1 and IgG2 responses in buffalo calves immunized each with LAP-
300g (Gr.II).
of LAP and Prx antigens, respectively. It was measured in indi-
vidual animal and data expressed as mean of the group. The IgG
response in the animals of Gr.I (LAP 150g immunized) showed
a steep increase up to 1st wk of 1st booster, with steady increase
up to 2nd wk of 2nd booster, followed by a plateau till 16th wk
post challenge. A steady increase in the IgG1 titre up to 4th wk
of 2nd booster, followed by a plateau was observed in the group.
Same pattern of IgG2 response was detected in these experimen-
tal animals but with a lower antibody level. The difference in the
IgG1 and IgG2 responses was statistically insignicant (p>0.05).
The IgG response in Gr.II animals (LAP 300g immunized) showed
a steep increase in its titre when probed on 1st wk of 1st booster,
thereafter; a plateau was maintained up to 4th wk of 2nd booster
(mean OD
492
ranging between 1.52 and 1.92) and the antibody
titre showed no further increase up to 16th wk post-challenge. A
steady increase in IgG1 titre up to 4th wk of 2nd booster, with a
further increase on 4th wk of challenge and a plateau in the anti-
body level upto16thwk of challenge were observed. IgG2response
showed a steady increase up to 4th wk of 2nd booster. Thereafter, a
plateau was maintained till the termination of experiment on 16th
wk post-challenge (Figs. 25). Results indicated that both 150 and
300g LAP immunization induced a dominant response of IgG1,
with lower IgG2 titre.
The groupof calves immunizedwiththe cocktail of twoantigens
(Gr.III) when probed with LAP and Prx antigens for anti-LAP and
anti-Prx IgGantibody responses, showed a steep increase up to 2nd
wk of 2nd booster, followed by a consistent plateau up to 16th wk
of probing for these antibodies (Fig. 6). Like wise, a steep increase
in IgG1 response up to 1st wk of 1st booster, followed by a plateau
of the antibody titre till 2nd booster, thereafter a steady decline
in the antibody level till 12th wk of post-challenge was observed
in this group probed with the recombinant LAP. A sharp increase
in the antibody level up to 1st wk of 1st booster, followed by its
decline up to 4th wk of 2nd booster and then a plateau till 12th wk
of probing were observed with respect to anti-LAP IgG2 response
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Pre
vac
1w Ist
bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
16w
ch
O
D
4
9
2
IgG-LAP
IgG-Prx
Un-imm cont
Fig. 6. Mean IgG response in buffalo calves immunized with LAP and Prx anti-
gens (cocktail group, Gr.III). Antibody response probed with LAP and Prx antigens,
respectively.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Pre
vac
1w Ist
bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
O
D
4
9
2
IgG1
IgG2
un-imm cont
Fig. 7. Mean IgG1 and IgG2 responses in buffalo calves immunized with LAP and
Prx antigens (cocktail group, Gr.III). Antibody response probed with LAP antigen.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Pre
vac
1w Ist
bt
2w
2bt
4w
2bt
4w
ch
6w
ch
8w
ch
12w
ch
O
D
4
9
2
IgG1
IgG2
un-imm cont
Fig. 8. Mean IgG1 and IgG2 responses in buffalo calves immunized each with LAP
and Prx antigens (cocktail group, Gr.III). Antibody response probed withPrx antigen.
(Fig. 7). Anti-Prx IgG1 antibody showed a steep increase up to 2nd
wk of 2nd booster but a steady decline in its titre up to 12th wk of
probingtheresponse. Anti-PrxIgG2titrealsoincreasedupto1st wk
of 1st booster, thereafter aplateauof antibodylevel was maintained
till 12th wk of probing (Fig. 8). There was a predominance of IgG1
response over IgG2.
Challenge infection of the LAP and LAP+Prx immunized and
non-immunized infected calves with the F. gigantica metacercariae
did not cause any increase in the titre of IgG, IgG1 or IgG2 isotypes.
3.4. Fluke recovery
The mean number of adult ukes recovered at necropsy from
the hepatic parenchyma, bile duct and gall bladder of the ani-
mals that received LAP 150g, LAP 300g, cocktail of these
antigens and challenge control animals was 249.4, 182.0, 198.8
and 198.7, respectively. Difference in the mean adult uke recov-
ery in the LAP-300g and LAP-150g groups was statistically
signicant (p<0.05). A mean uke reduction of 8.4% observed
in the buffalo calves that received 300g of the LAP antigen
was statistically insignicant (p>0.05). Immunization with the
Table 1
Fluke recovery data of buffalo immunization with F. gigantica LAP and PRx antigens.
Antigen Fluke recovery Mean uke
recovery
% Protection
LAP 167 281 325 237 322 249.4 0.0
150g 242 172
LAP 170 162 103 305 171 182.0 8.4
300g 135 228
LAP+Prx 167 206 193 193 156 198.8 0.0
150g each 278
Challenge
control
160 234 202 214 205 198.7
193 181 163 237
% Protection: (1mean number of ukes in vaccinated animals/mean number of
ukes in challenged control animals) 100.
Author's personal copy
O.K. Raina et al. / Acta Tropica 118 (2011) 217222 221
two antigens (recombinant LAP+Prx) in combination did not
evoke a signicant protection (p>0.05) vis a vis animals receiv-
ing LAP antigen alone (Table 1). There was no signicant (p>0.05)
difference in the mean wet weight of 55.9, 52.4, 56.4 and 51.3mg
of the ukes recovered from Gr.I, Gr.II, Gr.III and Gr.IV calves,
respectively. Also, mean length and width (3.5cm0.39cm)
of the ukes recovered from the two immunized groups was
not signicantly (p>0.05) different from the size of the ukes
(3.3cm0.38cm) recovered from the non-immunized challenge
controls.
There was no reduction in the mean bile egg count (47,251,
73,000 and 68,341) in the group I, II and III, respectively, when
compared to the group IV non-immunized control calves (59,270),
depicting no anti-fecundity effect of the immunization on the
ukes. Also, when the eggs released by the adult ukes in the bile
of the animals immunized with either LAP or a cocktail of the LAP
and Prx, were allowed to hatch to miracidia, there was no statis-
tically signicant (p>0.05) difference in the hatching percentage
of the miracidia from the eggs recovered from immunized and un-
immunized groups. This indicated no anti-embryonation effect of
the vaccine.
4. Discussion
Despite intensive research efforts, progress in the development
of effective vaccine for F. hepatica and F. gigantica control has been
relatively slow (Mendes et al., 2010). Several molecules with a
potential to induce protective responses in laboratory and large
animals have been identied in the parasite but the protection
conferred by these molecules has been shown to vary. Peptidases
accomplish tasks associated critically with successful parasitism
such as invasion, migration, acquisition of nutrients and evasion
of inammatory and immune responses. Although endopeptidases
have been extensively studied due to their prominent participa-
tion in these processes, exopeptidases have been largely neglected
in parasites (Williamson et al., 2003; Acosta et al., 2008). F. hep-
atica leucine aminopeptidase was identied, characterized and
expressed by Acosta et al. (1998, 2008). A single vaccination trial
withthe native F. hepatica LAPinovine fasciolosis, usedeither alone
or in combination with native cathepsin (CL1 and CL2), gave a very
high level of protection (89% and 76%, respectively) against chal-
lenge infection (Piacenza et al., 1999). These results represent the
highest protectionlevel achievedinruminants by a natural antigen,
therefore, positioning FhLAP as one of the leading vaccine candi-
dates in ruminant fasciolosis (Hillyer, 2005). However, no studies
have been reported on the leucine aminopeptidases of F. gigan-
tica, except for a recent report on the characterization of a novel
aminopeptidase of F. gigantica named leucine aminopeptidase-2
(LAP-2) (Mohamed et al., 2009). F. gigantica LAP reported here is
different from F. gigantica LAP-2 on account of its size, activity
maxima at 37

C and being unstable at higher temperature. Lim-

ited vaccination trials with different antigens of Fasciola have been
conducted in buffaloes (Nambi et al., 2005; Kumar, unpublished
data) which showed a moderate level of protection (35%) achieved
with different antigens. Present vaccination trial in buffaloes with
the recombinant leucine aminopeptidase in different doses, using
montanide as a commercially acceptable adjuvant, did not evoke a
signicant protection, with a mean reduction in the worm burden
of 8.4% in the group receiving 300g of leucine aminopeptidase.
Vaccination also showed no anti-fecundity/embryonation effect of
the immunization on the ukes. These results are contrary to the
earlier report of a high level of protection (89%) achieved with F.
hepatica leucine aminopeptidase in sheep. However, sheep vac-
cination trial was carried out with the native LAP antigen with
Freunds adjuvant, whereas the present trial was conducted with
the recombinant antigen and a different adjuvant, permitted for
use in food animals. These factors, including a change (if any) in
the post-translational modications of the E. coli expressed recom-
binant LAP could be responsible for the poor protection achieved
in buffaloes. Also, variations in the host susceptibility to F. gigantica
infectionhave beenreportedandbuffaloes are highlysusceptible to
fasciolosis (Zhang et al., 2006) and this susceptibility could be asso-
ciatedwiththe late andweak cellular immune response inthe early
phase of F. gigantica infection and the Th0-like response through-
out the infection (Zhang et al., 2006). Identication of leucine
aminopeptidase gene and evaluation of the protein for its protec-
tive efcacy in buffaloes is the rst scientic report on this protein
in F. gigantica. The LAP gene of F. gigantica showed a sequence vari-
ation of 22 nucleotides with its homologue in F. hepatica, causing
a change of 7 amino acids, with an overall nucleotide identity of
98.6%. Whether this change of 7 amino acids does alter the bio-
logical and immunological activity of the enzyme, needs further
characterization.
Immunogenic potential of the recombinant F. hepatica perox-
iredoxin in goats, when used with quil A adjuvant, showed strong
antibody response and 33.04% protection, suggesting FhPrx as a
potential candidate for vaccination against ruminant fasciolosis
(Mendes et al., 2010). No studies have been reported on the per-
oxiredoxins of F. gigantica except for a recent identication of
two F. gigantica peroxiredoxin genes (Chaithirayanon and Sobhon,
2010). The F. hepatica peroxiredoxin plays an important role in
the parasites defense against host-generated oxidants (McGonigle
et al., 1997, 1998) and in immuno-modulating the host response
(Donnelly et al., 2005, 2008). It has been postulated that success-
ful vaccination against F. hepatica and S. mansoni is dependent on
preventing the Th2 responses and the induction of Th1 responses
(Mulcahy et al., 2004; Pearce et al., 2004). Therefore, in the present
preliminary study it was reasoned that evoking anti-peroxiredoxin
antibodies by recombinant peroxiredoxin immunization in buf-
faloes will block the parasite secreted peroxiredoxin antigen on
challenge infection, thereby modulation of the immune response
to Th2 type caused by the peroxiredoxin antigen, will be altered.
In this context, a group of calves was immunized with parasite
peroxiredoxin and LAP in combination to determine the effect of
peroxiredoxin on Th1/Th2 responses and also an overall effect on
the level of protection, if any. Our results indicated no change in the
expression of the IgG1 or IgG2 antibodies in this group of animals
vis a vis other two groups immunized with leucine aminopeptidase
alone, withco-dominance of boththe antibody isotypes. Also, there
was no difference in the protection achieved in this experimental
animal groupvis a vis other groups of animals receiving LAP antigen
alone. Cell mediated immune responses evoked to immunization
with leucine aminopeptidase and peroxiredoxin for determining
the Th1/Th2 response were not measured in the present study.
This was only a pilot study to determine an overall effect on the
reduction in wormburden caused by peroxiredoxin protein immu-
nization. However, detailedstudies onthe expressionof the surface
markers of alternative activation on macrophages and cell medi-
ated immune response, with cytokine prole, in the vaccinated
buffaloes need to be carried out to understand the vaccine effect
of this protein. Further, vaccination trials with novel target anti-
gens are essential to develop control measures against fasciolosis
in buffaloes and above all, not many trials have been conducted in
these ruminants.
Acknowledgments
The authors are thankful to the Department of Biotechnology,
Govt. of India, NewDelhi, for the grants provided to the rst author.
We are also thankful to the Director, Indian Veterinary Research
Author's personal copy
222 O.K. Raina et al. / Acta Tropica 118 (2011) 217222
Institute, Izatnagar for providing necessary facilities for completion
of this research work.
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