Identification of a 66 kDa Haemonchus contortus excretory/secretory antigen that inhibits host...

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: [email protected],

[email protected] (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

.

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mailto:[email protected]

http://dx.doi.org/10.1016/j.vetpar.2006.01.055

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.


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


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.


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


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


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


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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Identification of a 66 kDa Haemonchus contortus excretory/secretory antigen that inhibits host...

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