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Identification of a 66 kDa Haemonchus contortus
excretory/secretory antigen that inhibits host monocytes
D.K. Rathore a, S. Suchitra a, M. Saini a, B.P. Singh b, P. Joshi a,*
a Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, UP, Indiab Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, UP, India
Received 5 May 2005; received in revised form 27 December 2005; accepted 26 January 2006
Abstract
A 66 kDa adult Haemonchus contortus excretory/secretory (E/S) antigen was identified in Western blot by reaction with sera
from the infected goats. The protein was purified from the adult worm extract and E/S products by anion exchange and ConA-
Sepharose chromatography. The purified protein inhibited monocyte function in vitro as judged by decreased production of
hydrogen peroxide and nitric oxide in the culture medium. The protein also caused proliferation of peripheral blood
mononuclear cells. The absence of protein in the free living L3 larvae suggests that the expression of this protein coincides
with the adaptation to the parasitic life. A correlation of antibody titre and worm burden was observed in the infected goats with
higher antibody levels in high worm burdened animals. Anti-protein antibody caused loss of adult worm motility in vitro
resulting in the death of the parasite. The fact that the protein is recognized by the host together with in vitro killing of adult
parasites by antibodies makes this protein a promising candidate for vaccination trial.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Haemonchus contortus; Excretory/secretory products; Immunomodulatory protein; Monocytes
www.elsevier.com/locate/vetpar
Veterinary Parasitology 138 (2006) 291–300
1. Introduction
Haemonchus contortus is a gastrointestinal parasite
of small ruminants and feeds on the blood of its host
causing anaemia that may be fatal particularly for the
young animals (Newton and Munn, 1999). Anthel-
mintics are commonly used for the control of this
parasite. However, the development of anthelmintic
* Corresponding author. Tel.: +91 581 2301 638.
E-mail addresses: pj@ivri.up.nic.in,
parit_joshi@rediffmail.com (P. Joshi).
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved
doi:10.1016/j.vetpar.2006.01.055
resistance in many Haemonchus strains together with
the concern for chemical residues in the animal tissues
for human consumption as well as the contamination
of the environment has necessitated the search for
alternate strategies for the control of this parasite
(Newton and Munn, 1999). During the past decade,
efforts were made to identify the protective antigenic
molecules of this parasite in order to develop an
effective vaccine. Two types of antigens have been
recognized in H. contortus. The first category includes
natural antigens which are recognized by the host
during infection with the generation of an immune
.
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300292
response and the second type are hidden. Though the
hidden antigens are not exposed to the host immune
system during natural infection, some of these have
shown promising results in the protection trials (Knox,
2000; Schallig, 2000).
Excretory/secretory (E/S) products are the sub-
stances released by the parasite during in vitro
cultivation and are presumed to be released in vivo
as evidenced by the presence of antibodies in the
infected animals against many E/S proteins (Schallig
et al., 1994). These molecules may perform diverse
functions such as aid in tissue penetration and
degradation of host proteins for nourishment (Cox
et al., 1990; Karanu et al., 1993), modulation of host
immune response and prevention of blood clotting,
etc. (Joshi and Singh, 2000; Suchitra and Joshi, 2005).
Two low molecular weight natural E/S antigens (15/
24 kDa) caused significant reduction in worm popula-
tion in the vaccinated animals during protection trials
(Schallig and van Leeuwen, 1997). Subsequently,
recombinant forms of these proteins were generated
due to limitations in the E/S products and the
engineered proteins gave protection though to a lesser
extent compared to native proteins. However, the
protection results could not be reproduced with a new
batch of recombinant proteins probably due to
defective folding (Vervelde et al., 2002). Recently, a
comprehensive analysis was made which suggests the
presence of more than 100 proteins including some yet
unidentified proteins in H. contortus E/S products
(Yatsuda et al., 2003).
Here we report the identification and partial
characterization of a 66 kDa E/S antigen (p66) and
discuss its significance in host–parasite relationship.
2. Materials and methods
2.1. Materials
RPMI 1640 tissue culture medium, Histopaque
1077, concanavalin A (Con A), lipopolysaccharide
(LPS), phytohemaglutinin (PHA), phorbol myristate
acetate (PMA), 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide (MTT dye), DEAE-
Sepharose, Sephadex G-100, acrylamide, bis-acryla-
mide, 3,30-diaminobenzidine (DAB) were purchased
from Sigma chemical co., U.S.A., ELISA plates were
from Nunc (Denmark). All other reagents were of the
highest purity available.
2.2. Collection of adult worms, excretory/
secretory products and larvae culture
H. contortus were harvested from the abomasum of
goats procured from the local abattoir and the goats
sacrificed in the institute. Worms were washed twice
with saline and those with high motility were
transferred to sterilized normal saline in sterile
condition and finally to RPMI-1640 medium contain-
ing penicillin and streptomycin (Joshi and Singh,
2000; Suchitra and Joshi, 2005) and kept in a candle
jar at 37 8C for 8 h. Approximately 1 ml medium was
used for 10 parasites. After the incubation, culture
medium was collected by decantation, centrifuged and
the supernatant was labeled as E/S product and stored
at �85 8C. The recovered worms were also stored at
�85 8C.
H. contortus (L3) larvae were obtained as described
previously with modifications (Gamble et al., 1989).
In brief, adult worms were crushed gently in PBS in a
pestle and mortar and the suspension was layered over
a mixture of autoclaved goat faecal matter and
charcoal kept on a moist filter paper in a petri dish.
The petri dish was placed in a glass container with
distilled water at the bottom and kept at 30–35 8C.
Larvae were recovered from water by centrifugation at
10,000 � g for 20 min and stored at 4 8C in fresh
distilled water for about 2–3 weeks.
2.3. Purification of 66 kDa protein (p66)
Adult parasites were homogenized in a pestle and
mortar with 20 mM sodium phosphate buffer (pH 7.4)
containing 1 mM EDTA and PMSF. The suspension
was centrifuged at 10,000 rpm in a SS 34 rotor
(Sorvall RC 5C plus) for 30 min at 4 8C. The
supernatant was passed over a DEAE-Sepharose
column equilibrated with 20 mM phosphate buffer
(pH 7.4) and eluted with stepwise increase in sodium
chloride. Fractions were checked for the presence of
p66 by Western blot using serum from H. contortus-
infected animals. The positive fractions (50 and
75 mM NaCl elutes) were pooled, dialyzed against
Con A-Sepharose equilibrating buffer (10 mM Tris–
HCl pH 7.4/150 mM NaCl and 0.025% sodium azide)
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300 293
and loaded to a Con A-Sepharose column prepared as
described earlier (Mohan et al., 1995). The column
buffer contained 1 mM each of CaCl2 and MnCl2. The
unbound fraction was re-chromatographed on a
DEAE-Sepharose column and eluted with smaller
cuts of sodium chloride.
p66 was also purified from E/S products first by
concentrating the products by lyophilization followed
by equilibration against the column buffer. The
fractionation protocol included DEAE-Sepharose fol-
lowed by ConA-Sepharose chromatography as above.
2.4. Lymphocyte proliferation assay
Peripheral blood mononuclear cells (PBMCs) were
separated from the blood of three healthy adult goats,
maintained by the Division of Pharmacology, by
density gradient centrifugation using Histopaque 1077
(Baker and Knoblock, 1982). The cells were
suspended in RPMI 1640 medium containing 10%
fetal calf serum, 20 mM HEPES and 20 mM sodium
bicarbonate and the viability was checked by trypan
blue dye exclusion.
One hundred microlitre of cells at a concentration
of 5 � 106 cells/ml were placed in wells of a flat-
bottomed microtitre plate. p66 was added in varying
amounts and the plate was incubated at 37 8C for 72 h
in a humidified atmosphere with 5% CO2. The protein
was dialyzed against normal saline and then filter
sterilized to rule out interference by impurities. Wells
containing ConA (10 mg/ml), PHA (5 mg/ml) and LPS
(10 mg/ml) served as positive controls. At the end of
the incubation, 20 ml of MTT dye (5 mg/ml stock) was
added and further incubated at 37 8C for 4 h.
Subsequently, 150 ml/well of DMSO was added, to
dissolve the formazan crystals, and the absorbance
was read at 540 nm with reference reduction at
650 nm (Bounous et al., 1992). Stimulation index (SI)
was calculated as:
SI ¼ Absorbance of stimulated cells
Absorbance of unstimulated cells
2.5. Quantitation of nitric oxide and hydrogen
peroxide
Nitric oxide production was measured in mono-
cytes. These cells were isolated by their adherence to
plastic surface (Kaleab et al., 1990). Approximately
5 � 104 cells/well (of a 96 well plate) were used in the
assay in a medium consisting of RPMI 1640 without
phenol red and antibiotic but contained 5 mM L-
arginine. Varying dilutions of p66 was added to wells
in triplicate. ConA (2.5 mg) and LPS (2.5 mg) served
as controls. The total assay volume was 100 ul/well
and the plate was incubated at 37 8C in a humidified
chamber with 5% CO2. Nitric oxide was measured as a
stable end product nitrite (NO2�) (Green et al., 1982).
Culture supernatant (50 ml) was collected after 24, 48
and 72 h and transferred to a new plate containing
100 ml/well of Griess reagent. The absorbance was
measured at 545 nm in a Bio-Rad microplate reader
(Model 680). The standard curve was prepared with a
range of 5–50 mM sodium nitrite.
Hydrogen peroxide released by the monocytes was
also measured (Pick and Mizel, 1981). This method is
based on the horseradish peroxidase (HRPO) depen-
dent oxidation of phenol red by H2O2 into a yellow
compound. Briefly, 5 � 105 monocytes/well of a 24-
well plate were activated with mitogens namely LPS
(10 mg/ml), PMA (200 ng/ml) or p66 (2.5, 5 and
10 mg/well) in a total volume of 1 ml and incubated at
37 8C. After 4 h, 950 ml of the supernatant was
collected and 10 ml of 1N NaOH was added. The
absorbance of the coloured product was read at
610 nm. The standard curve was prepared with a range
of 12.5–50 mM hydrogen peroxide.
2.6. Antibody production
Purified p66 was subjected to preparative SDS-
PAGE. After electrophoresis, a vertical gel strip was
cut from the middle of the gel and negatively stained
(Ferreras et al., 1993). The area corresponding to p66
band in the unstained gel was cut into small pieces
and incubated overnight with small volume of PBS-
EDTA at 4 8C. It was then centrifuged and the
presence of p66 in the extract was confirmed by
Western blot and Coomassie blue staining. Two
healthy male rabbits were immunized with 50–60 mg
of purified p66 emulsified in Freund’s complete
adjuvant intramuscularly. Rabbits were boosted
every 3rd week with the same amount of protein
mixed with incomplete adjuvant. Presence of anti-
body in rabbits was checked a week after the third
booster by Western blot.
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300294
2.7. Effect of anti-p66 antibody on parasite
Effect of anti-p66 antibody on adult worms and
infective stage larvae (L3) was investigated. The assay
was carried out in a 24 well microtitre plate containing
500 ml RPMI-1640 medium with 4 adult worms and
varying dilutions of antiserum. The plate was kept at
37 8C in a humidified atmosphere of 5% CO2. The
worm motility was observed under a light microscope
at different time intervals and differentiated as ++++
for highly motile parasite followed by +++, ++, + for
moderate to slow moving worms. Parasites with no
motility were given a negative (�) score. Sera from
normal rabbit and goat, de-complemented anti-p66
antiserum (56 8C/30 min) and sera from low and high
burdened H. contortus-infected animals were also
tested.
2.8. SDS-PAGE, Western blotting and protein
determination
SDS-PAGE was performed in 5–15% linear
gradient gels in a discontinuous buffer system
(Laemmli, 1970). Protein bands were identified by
staining with Coomassie brilliant blue.
Western blot was performed according to Towbin
et al. (1979). Protein bands were transferred from gel
onto a nitrocellulose paper at 200 mA for 3 h and then
blocked with 5% skimmed milk powder. Incubation
with H. contortus-infected sera (1:100 dilution) was
done at room temperature for 4 h. After several
washes, the paper was incubated with rabbit anti-goat
IgG-peroxidase conjugate for 2 h followed by
washings and development with DAB.
Protein content in samples was determined by
Lowry’s method (1951).
2.9. Enzyme linked immunosorbent assay (ELISA)
ELISA was developed to quantitate the amount of
p66 in E/S products as well as to measure p66
antibody levels in the infected goats. For protein
quantitation, direct ELISA was performed using
purified p66 (1–20 ng) as a reference. The primary
antibody (anti-p66 antiserum) was used at 1:1500
dilution and the secondary antibody conjugated
to peroxidase at 1:3000 dilution. A standard curve,
based on the absorbance of reference, was prepared
and used for the quantitation of p66 in E/S products.
For measurement of antibody levels in the infected
animals, wells were coated overnight at 4 8C with
100 ml/well of p66 solution at a concentration of
12 mg/ml in 0.06 M carbonate buffer (pH 9.6). This
was followed by blocking the wells with gelatin
(0.2%) for an hour. H. contortus-infected goat sera
(100 ml/well) was added in varying dilutions from
1:50 to 1:6400 in PBS-Tween 20 (PBS-T) and the plate
was incubated at room temperature for 4 h. Wells were
washed thrice with PBS-T and 100 ml of rabbit anti-
goat IgG-peroxidase (conjugate) was added at 1:1000
dilution and further incubated for 2 h. After several
washings, colour was developed with OPD as a
substrate and the absorbance was read at 490 nm.
Wells coated with gelatin (0.2%) served as control and
the absorbance of these wells was subtracted from the
experimental values.
3. Results
3.1. Identification and characterization of p66
Fractionation of adult H.contortus E/S products on
SDS-polyacrylamide gel showed at least 20 polypep-
tides after staining the gel with Coomassie blue
(Fig. 1A). The size of these polypeptides ranged from
15 to 150 kDa. A 66 kDa protein reacted with sera
from H. contortus-infected goats in Western blot
(Fig. 1B). All batches of E/S products collected during
many months contained this protein which reacted
with infected sera. No reaction was observed with sera
from uninfected animals. Extracts of infective L3
larvae did not react with the antiserum raised against
p66 (not shown). Quantitation of E/S products by
ELISA suggests a value of 20–30 ng of p66 released
per parasite in an hour.
A simplified purification protocol was developed
which involved anion-exchange chromatography as a
first step. This step resulted in an enriched protein
preparation with a little bit of contaminants. Further
purification on a ConA-Sepharose column and re-
chromatography on anion-exchanger yielded a homo-
geneous p66 preparation that showed a single band in
SDS-gel with or without 2-mercaptoethanol (Fig. 1C).
The fact that p66 did not bind to ConA-Sepharose
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300 295
Fig. 1. (A) SDS-PAGE analysis of H. contortus E/S products stained with coomassie brilliant blue R 250. Lane 1, E/S proteins. Lane 2, marker
proteins. (B) Western blot analysis of E/S products with pre-infected goat serum (lane 1) and pooled sera from H. contortus-infected animals
(lane 2). (C) Purification of p66. SDS-PAGE analysis showing molecular weight markers (1), adult worm extract (2), E/S products (3), ConA-
Sepharose unbound fraction (4) and eluted fractions from the second cycle of DEAE-Sepharose (5,6). (D) Reaction of DEAE-Sepharose
fractionated E/S products with pooled infected sera in Western blot. Total E/S products (Lane 1), 50 mM NaCl eluted fraction (2), ConA unbound
fraction (3). (E) Reaction of concentrated E/S products with rabbit anti-adult worm p66 antiserum in Western blot. Lanes 1–3 represents different
E/S batches.
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300296
Fig. 2. Measurement of serum antibody levels to p66 in H. contortus-infected goats by ELISA. The protein concentration for coating the wells
was 12 mg/ml. Values represent mean absorbance � standard deviation of three experiments.
suggests that it lacked at least mannose and glucose
moieties. Because of limitations in E/S products and
comparatively less complexity of protein profile of E/
S products, the second DEAE-Sepharose cycle was
omitted and the protein obtained had >90% purity.
The elution profile of E/S p66 was similar to that of
adult worm extract p66 and the fractionated protein
reacted with the infected animal sera (Fig. 1D). To
confirm that the E/S p66 was similar to adult worm
protein, reaction of E/S protein with rabbit anti-adult
worm p66 antiserum was studied by Western blot. A
single band of 66 kDa reacted with the antiserum
(Fig. 1E).
p66 was differentiated from albumin, the major
host blood protein, by comparing the chymotrypsin
digestion products of these two proteins. No
similarity was observed between the two proteins
as judged by gel electrophoresis (not shown). Also,
anti-p66 antibody did not react with albumin (not
shown). Studies were performed to assess if p66
possesses proteolytic activity as many E/S proteins do
show this property (Cox et al., 1990; Karanu et al.,
1993). No degradation of albumin was observed at
different pH after incubation with p66 for many hours.
Similarly, no breakdown of gelatin was observed in
gelatin containing gels (not shown; Karanu et al.,
1993).
3.2. Antibody levels in the infected animals
Antibody levels in H. contortus-infected animals
was measured by ELISA using p66 as an antigen. A
positive correlation of antibody levels and worm
burden was observed. Animals with higher worm
burden had higher antibody titres (Fig. 2).
3.3. Effect of p66 on blood cells
Lymphocytes from uninfected normal goats pro-
liferated in the presence of p66 (Fig. 3). Though the
effect was of lower degree, it was significant and
concentration dependent. These results are in accor-
dance with the immune response generated during H.
contortus infection. ConA, used as a positive control,
also elicited low degree of cell stimulation. The
reason(s) for this are not clear and may be due to
genetic make up of goats which were of local breed
(Rohilkhand). p66 also potentiated the mitogen
induced proliferation of PBMCs.
Functional activity of monocytes was assessed by
measuring the hydrogen peroxide and nitric oxide
released into the culture medium. Peroxide production
decreased in presence of p66 and was concent-
ration dependent with �50% reduction at 10 mg
p66. Moreover, PMA and LPS induced production of
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300 297
Fig. 3. Proliferation (Mean � S.D.; n = 9) of normal goat lymphocytes in the presence of p66. The values represent mean stimulation index of
three independent experiments each in triplicate.
peroxide was also inhibited by the protein (Fig. 4A).
Likewise, production of NO was reduced by these
cells in the presence of p66 as compared to respective
cell control at 24, 48 and 72 h intervals (Fig. 4B).
3.4. Effect of anti-p66 antibody on parasites
Antibodies raised against purified p66 inhibited
adult worm motility in a concentration dependent
manner (Table 1). At 1:500 dilution of antiserum,
motility decreased gradually with time but at higher
antibody concentration (1:100 dilution), it decreased
faster and by 2 h all the adult worms were immotile.
Serum recovered from H. contortus-infected goats
also inhibited worm motility. Sera from animals with
higher number of worms were more effective
compared to sera from low worm burdened animals
which took longer time to immobilize the worms.
Decomplementation of anti-p66 antiserum at 56 8Cdid not alter inhibitory properties. Sera from normal
uninfected goats or rabbits had no effect on worm
motility. The motility of L3 larvae was unaffected in
the presence of infected-animal sera or anti-p66
antiserum and were motile for at least up to 24 h.
Taken together, these results suggest that the reduction
in worm motility was mediated by anti-p66 antibody.
4. Discussion
In this study it was demonstrated that adult H.
contortus E/S p66 is a natural antigen. Furthermore,
the authenticity of this protein as a true E/S
protein was evident as higher antibody levels were
observed in animals with increased worm numbers
suggesting enhanced secretion of the protein in these
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300298
Fig. 4. Evaluation of goat monocyte function in the presence of p66
(A) hydrogen peroxide (mM) production (B) nitric oxide production
measured as nitrite (mM). The values represent mean � standard
deviation of three separate observations.
animals. A protein of this size was earlier identified
in E/S products and was recognised by sera from H.
contortus-infected animals (Schallig et al., 1994).
More recently, multiple spots were observed in the
66 kDa region in 2-D gel electrophoresis of H.
contortus E/S products. Four of these spots have been
identified; three as proteases and one appear to be of
host origin. There remain as yet unidentified spots in
that region (Yatsuda et al., 2003). Results presented
here establishes a separate identity for p66 and its
origin from the parasite in spite of size similarity to
host albumin that is taken up during blood meal. The
variation in the amount of p66 in different batches of
E/S products probably reflects heterogeneous popu-
lation of male and female parasites and or variation
in their metabolic activities.
The inhibition of monocytes in vitro is an important
functional attribute of this protein and seems an
important defense strategy devised by the parasite to
protect itself from the oxidative damage of peroxide
and NO generated by these cells (Jungi et al., 1996).
This may also explain the absence of protein in the L3
larvae which being free living do not come in contact
with the host monocytes. At this juncture, it is not clear
if the expression of this protein starts immediately
after conversion to L4 larvae inside the host. The L4
stage larvae, like the adult worms, also feeds on the
host blood (Clark et al., 1962). Thus, it is likely that
the expression and release of this protein may start at
the L4 stage with the commencement of blood feeding
to prevent generation of free radicals by host
monocytes. The mechanism of monocyte inhibition
is not clear and no cell damage occurred as suggested
by trypan blue dye exclusion test. The protein had no
inhibitory effect on neutrophils which are also
involved in immunity.
The precise mechanism of adult worm death by p66
antibody is not clear. The antibody in all probability
would bind to the exterior of the active parasite
suggesting surface or near surface localization of p66
which would also facilitate protein secretion. The
antibody could bring about worm damage by
complement proteins. Since worm damage occurred
even after inactivation of complement by heat suggest
other mechanism(s) by which antibody exerts its
effect. It is, thus, possible that p66 in addition to
inhibiting host monocytes may have an additional and
important function for the parasite and blocking by
D.K. Rathore et al. / Veterinary Parasitology 138 (2006) 291–300 299
Table 1
Inhibition of adult H. contortus motility by anti-66 kDa protein antibody
Serum (dilution) Worm motility (time)
15 min 30 min 60 min 2 h 3 h 6 h 8 h
RPMI-1640 medium only ++++ ++++ ++++ ++++ ++++ +++ ++
Normal rabbit (1:100) ++++ ++++ ++++ ++++ ++++ +++ ++
Normal goat (1:100) ++++ ++++ ++++ ++++ ++++ +++ ++
Rabbit anti-66 kDa antiserum
1:100 ++++ ++ + � � � �1:250 ++++ +++ ++ +� + � �1:500 ++++ ++++ +++ +++ ++ + �
H. contortus-infected (600 worms infection)
1:100 ++++ ++ + � � � �1:250 ++++ +++ ++ + � � �1:500 ++++ ++++ ++++ +++ +++ ++ �
H. contortus-infected (50 worms infection)
1:100 ++++ ++++ +++ ++ ++ � �1:250 ++++ ++++ ++++ ++++ +++ ++ �1:500 ++++ ++++ ++++ ++++ ++++ +++ �
Note: The degree of worm motility is expressed as ++++ (very fast) to + (slow); � indicates worm movement observed after 30–40 s and �means no motility observed up to 3 min. indicating death. The results shown above for H. contortus-infected sera represent two extreme cases,
one with high worm counts and the other with low numbers. In an initial experiment, worm motility was inhibited by all the seven sera obtained
from H. contortus-infected goats.
antibody would make the protein unavailable for that
function. Many proteins including H. contortus
calreticulin are multifunctional proteins (Suchitra
and Joshi, 2005). Further work on p66 and its
expression pattern would throw light on its func-
tion(s).
It is important to address the reasons for the
survival and establishment of the parasite in the
infected animals in spite of antibody formation.
Foremost is the fact that the formation of antibodies
would take at least a few weeks, by that time the
parasite would have established itself. Second, the
antibodies are probably neutralized by the high output
of the protein. E/S products appear to contain a good
amount of the protein. Third, the proteases released by
the parasite (Cox et al., 1990; Karanu et al., 1993) may
degrade antibody. Lastly, the binding of the antibody
as well as the immune effecter cells to its target may
not be easily achievable given the fact that the parasite
attaches itself to the surface of the stomach wall.
These arguments may also hold true for many other
natural antigens including E/S proteins which have
shown promising results in challenge studies (Jacobs
et al., 1999; Schallig and van Leeuwen, 1997). Thus, it
would be of interest to assess the potential of p66 as an
immunogen as prior activation of the immune system
may prove protective.
Acknowledgements
We thank Director, IVRI for providing the
necessary facilities and our colleagues for encourage-
ment. The laboratory assistance of Ram Kishore is
duly acknowledged. This work was supported by a
grant from the Department of Biotechnology, New
Delhi to PJ. DKR is a DBT research associate.
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