Immune responses in chickens against Eimeria tenella sporozoite antigen

Immune responses in chickens against

Eimeria tenella sporozoite antigen

Rajat Garg, D.P. Banerjee*, S.K. Gupta

Department of Veterinary Parasitology, CCS Haryana Agricultural University, Hisar 125004, India

Received 4 February 1998; accepted 14 September 1998

Abstract

Two-day old broiler chicks were subcutaneously immunized with Eimeria tenella sporozoite

antigen (25 mg per chick) with or without adjuvants on 2 and 18 days of age and the effect of

induced immunity was determined by challenging the chickens with 104 homologous sporulated

oocysts at 32 days of age. Chicks immunized with sporozoite antigen emulsified in Freund's

Complete Adjuvant (FCA) showed protection in terms of oocyst production, mortality and mean

lesion scores. Antigen emulsified in FCA produced significant cell mediated immune responses (as

assessed by lymphocyte migration inhibition test) from 12 to 30 days post-immunization. Antibody

responses as assessed by enzyme linked immunosorbent assay were significant from 12 days post-

immunization when the antigen was administered with or without adjuvants by subcutaneous route.

# 1999 Elsevier Science B.V. All rights reserved.

Keywords: Chicken; Immune response-protozoa; Eimeria tenella

1. Introduction

Cecal coccidiosis has tremendous impact on poultry throughout the world because of

consequent mortality, morbidity and weight loss in affected birds. Control of Eimeria

tenella in poultry is presently accomplished by prophylactic chemotherapy, but the rapid

emergence of drug resistant parasites, coupled with difficulty and expense of developing

new drugs, has led to a search for new approaches to coccidiosis control involving

immunological, biotechnological and genetical methods. Of these, immunological

approach is assuming more importance. The problems of controlling pathogenicity,

coupled with limited shelf life of live, virulent and avirulent vaccines has prompted work

towards the development of subunit vaccines against poultry coccidiosis (Chapman,

Veterinary Parasitology 81 (1999) 1±10

* Corresponding author. Tel.: +91-1662-31171/4297; fax: +91-1662-34952; e-mail: hau@ hau.ren.nic.in

0304-4017/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 4 0 1 7 ( 9 8 ) 0 0 2 3 1 - 3

1988). Various types of E. tenella derived antigens viz. sporulated oocyst, sporozoite,

merozoite antigens etc. have been tried as vaccinal candidates with various degrees of

success (Wisher, 1986; Sutton et al., 1989; Karkhanis et al., 1991; Rhalem et al., 1993).

Of these, sporozoites are potential candidates as vaccinal antigens as they are the first

developmental stage which comes in direct contact with the host immune system.

Keeping this in view, the present study was planned with an aim to immunize chicks

against E. tenella using sporozoite antigen and to monitor the antibody and cellular

immune responses produced by this antigen.

2. Materials and methods

2.1. Experimental chicks

One-day old broiler chicks (Ross breed) of both sexes were reared under coccidia-free

environment in horizontal battery brooders with raised wire netting floor inside a well

ventilated room. Commercial chick mash, free of any coccidiostat additive was fed ad

libidum to all the chickens.

2.2. The parasite

Field isolate of E. tenella was obtained from a post-mortem case brought to the Disease

Investigation Laboratory, Hisar. The infected cecum was examined grossly and

microscopically to identify the species of coccidia. Identification was based on the

appearance of characteristic schizonts and gametocytes in fresh smear of cecal mucosa

(Davies et al., 1963). Further, its monospecific character was ascertained by experimental

infection of 3-weeks old susceptible broiler chickens. For propagation of E. tenella

oocysts, 3-weeks old broiler chickens (Ross breed) were infected with 5000 sporulated

oocysts and feces collected from days 5 and 9 post-inoculation. The oocysts were purified

by salt flotation technique, sporulated and sterilized by hypochlorite treatment as

described previously (Davies et al., 1963). Purified oocysts were suspended in 0.1 M PBS

(pH 7.6) at the concentration of 5 � 107 oocysts mlÿ1.

For collection of sporozoites 2 ml suspension of sporulated oocysts (5 � 107 mlÿ1) was

first ground at 500 rpm for 10 min at 48C in a tissue homogenizer with a loose fitting

pestle. Following centrifugation (1000 rpm for 10 min) the supernatant was discarded and

pellet was further processed. Excystation of sporozoites from the released sporocysts in

the pellet was carried out as described previously (Davies et al., 1963). The freshly obtained

sporozoites were purified from the excystation debris by using a DE-52 cellulose anion ex-

changer (Sisco Research Laboratories, Bombay, India) as described by Schmatz et al. (1984).

2.3. Preparation of sporozoite antigen

Sporozoites (109) were washed three times in 0.1 M PBS (pH 7.6) containing l mM

phenyl methyl sulfonyl fluoride (E. Merck, Germany). They were then sonicated with a

Branson Sonifier 250 (B. Braun Instruments, U.S.A.) at a power setting of 3� and 30%

2 R. Garg et al. / Veterinary Parasitology 81 (1999) 1±10

duty cycle for 10 min. Sonication was performed on ice and sample was examined by

light microscopy for disruption. The sonicated material was then centrifuged at 10 000�g

for 1 h at 48C and the supernatant used as soluble antigen for further experiments. Protein

content of the antigen was determined by the method of Lowry et al. (1951).

2.4. Experimental design

A total of 125 one-day old broiler chicks (Ross breed) were divided into five groups

and immunized as follows:

Group A ± 25 chicks were vaccinated subcutaneously (s/c) on the breast region at 2

days of age with 25 mg antigen emulsified in FCA (Sigma, St. Louis, U.S.A.) and a

booster dose of 25 mg antigen alone was given at 18 days of age.

Group B ± 25 chicks were vaccinated subcutaneously at 2 days of age with 25 mg

antigen absorbed on aluminium hydroxide gel (1 ml antigen added to 3 ml aluminium

hydroxide gel, pH 7.6, and incubated at 208C for 48 h) and a booster dose of 25 mg

antigen alone was given at 18 days of age.

Group C ± 25 chicks were vaccinated s/c with 25 mg antigen alone on 2 and 18 days of

age.

Group D ± 25 chicks were inoculated s/c with FCA alone on 2 and 18 days of age.

Group E ± 25 chicks were kept as unimmunized controls.

Extreme care was taken to avoid adventitious exposure of chicks to coccidia during

immunization period and feces from each group were periodically examined for the

absence of oocysts.

All the chicks were challenged at 32 days of age with 104 E. tenella sporulated oocysts

per bird and the surveillance for coccidial oocysts was continued until the day 5 post-

challenge.

2.5. Lesion scoring, fecal oocyst counts

On day 6 post-challenge, 10 birds from each group were killed and lesion scores

assessed by the method of Johnson and Reid (1970). Also, the feces from each group

were collected separately from day 5 to 9 post-challenge and oocysts counted from 1 gm

of faeces from each group using McMaster's counting technique and protection

percentage was calculated (Rose and Mockett, 1983) as follows:

100ÿ 100� number of oocysts from vaccinated birds

number of oocysts from control birds

2.6. Immunological studies

Whole blood and serum were collected from three chicks selected randomly from each

group on 4, 8, 12, 16, 23, 30 and 37 days post-immunization. After collecting blood from

chicks, they were marked with leg bands so that they are not reused for blood collection.

However, all the chicks were challenged on 32 days of age.

R. Garg et al. / Veterinary Parasitology 81 (1999) 1±10 3

2.6.1. Lymphocyte migration inhibition test

This was performed after the method of Chhabra and Goel (1981) with some

modifications. 5 ml of heparinized blood was layered over 3 ml lymphoprep (E.

Nyegaard, Oslo, p � 1.077) in 15 ml centrifuge tubes and centrifuged at 400�g for

25 min. The leucocytes at the interface of plasma and lymphoprep were collected with

Pasteur pipette and washed twice with culture medium (RPMI-1640 containing foetal calf

serum; pH 7.1). The pellet was finally suspended in culture medium at a concentration of

2 � 107 to 5 � 107 cells per ml. Microcapillaries were filled with this cell suspension,

plugged at one end with plasticin and centrifuged at 200�g for 3 min. The capillaries

were cut at the liquid cell interface and the stubs were anchored over migration surface of

the chamber wells with the help of silicon grease applied at the bottom. A dose of 45 mg

antigen per ml of RPMI-1640 (minimum toxic dose) was used to charge the wells of LMI

plate (Laxbro, Pune, India) and area of lymphocyte migration was noted after incubating

the plates for at least 18 h at 378C. Lymphocyte migration (LM) index was calculated as

below:

LM index � Average area of lymphocyte migration in presence of antigen

Average area of lymphocyte migration in absence of antigen

2.6.2. Enzyme linked immunosorbent assay

This was performed after the method of Onaga et al. (1986) with some modifications.

Distilled water rinsed wells of microtitre plates were coated for 3 h at 378C with 50 ml of

1 : 20 dilution of E.tenella sporozoite antigen (80 mg mlÿ1). The coated plates were

further incubated at 48C for 18 h. The coated plates were washed once with 0.1 M PBS

containing 0.05% (v/v) Tween-80 (pH 7.6) and air dried. Sera were diluted 1/20 in PBS

containing 1% bovine serum albumin (BSA). The diluted sera were incubated on E.

tenella sporozoite antigen coated microtitre wells (4 mg antigen/well) of the microtitre

plate (Dynatek micro ELISA plates, Virginia, U.S.A.) at 378C for 1 h. Serum was

removed and wells were washed three times in washing solution. 50 ml of Anti-chicken

IgG, diluted 1/1000 and conjugated with horse-radish peroxidase were then added and

incubated at 378C for 1 h. Once again the plate wells were washed four times with

washing solution. Amounts of bound isotypes were quantified by the 405 nm absorbance

values after 1 h and 30 min enzymatic reaction with 2, 20-Azino-di-(3-ethyl-benzyl-

thiazoline sulphonic acid) i.e., ABTS as substrate at room temperature.

3. Results

The immunizing efficacy of E. tenella sporozoite antigen in terms of mean oocyst

production, mortality and mean lesion scores after homologous oocyst challenge is

depicted in Table 1. The chicks immunized with the sporozoite antigen emulsified in

FCA showed 57.4% reduction in oocyst output as compared with controls

(1.41 � 0.07 � 107 oocysts per gram feces (OPG) in immunized chicks vs.

3.31 � 0.35 � 107 for the controls) and had mean lesion scores of �1.9. Similarly, the

chickens immunized with antigen absorbed on aluminium hydroxide gel resulted in


Tab

le1

Oo

cyst

pro

du

ctio

nan

dce

cal

lesi

on

sco

res

ob

serv

edin

chic

ken

sim

muniz

edw

ith

E.

tenel

lasp

oro

zoit

ean

tigen

and

chal

lenged

wit

hhom

olo

gous

sporu

late

doocy

sts

Gro

ups

Tre

atm

ent

Num

ber

of

bir

ds

chal

len

ged

Num

ber

test

edfo

r

each

par

amet

er

E.

tenel

laoocy

st

chal

lenge

dose

Oocy

stpro

duct

ion

(�10

7)

Per

cent

reduct

ion

inoocy

stoutp

ut

Mort

alit

yM

ean

ceca

l

lesi

on

score

s

AA

nti

gen�

FC

A2

010

10

000

1.4

1�

0.0

757.4

±�1

.90

BA

nti

gen�

Alu

min

ium

hy

dro

xid

eg

el

20

10

10

000

2.1

3�

0.1

835.6

2�2

.30

CA

nti

gen

alo

ne

20

10

10

000

3.2

3�

0.2

523.5

3�2

.60

DF

CA

alo

ne

20

10

10

000

3.2

3�

0.2

6±

5�3

.30

EC

ontr

ols

20

10

10

000

3.3

1�

0.3

5±

6�3

.40

FC

A±

Fre

und's

Com

ple

teA

dju

van

t.


2.13 � 0.18 � 107 OPG after challenge which represented 35.6% reduction in oocyst

output as compared with controls. They had mean lesion scores of �2.3. Birds

immunized with antigen alone showed 23.5% reduction in oocyst output as compared

with controls and had mean lesion scores of �2.6. The unimmunized controls had a mean

lesion score of �3.4.

The cell mediated immunity patterns as indicated by LM index in the immunized and

control chickens is depicted in Fig. 1. In controls the LM index ranged between

0.971 � 0.018 (4 days PI) and 0.930 � 0.051 (16 days PI). In group A, a significant cell

mediated immune response (CMIR) was noted at 12 days PI (LM index of 0.589 � 0.086;

Fig. 1. Lymphocyte migration indices of chicken in different groups immunized with Eimeria tenella sporozoite

antigen.


p � 0.05) which peaked on 23 days PI (0.570 � 0.086; p � 0.05) and then declined by

day 30 PI (0.871 � 0.037) below significant levels, but was again found significant on 37

days PI. In group B, a significant cellular response was noted on day 12 PI (0.631 � 0.14;

p � 0.05) which declined by day 23 PI below the significant levels. In group C, peak

cellular responses were noted on day 16 PI (0.668 � 0.72; p � 0.05) after which they

declined and became insignificant on day 23 PI.

The results of ELISA performed on sera samples from E. tenella sporozoite antigen

immunized and control chickens are shown in Fig. 2 at mean absorbance levels (405 nm).

In the chickens immunized with antigen given with or without adjuvants, a significant

increase in mean absorbance values were noted from 12 day PI (0.164 � 0.005 in group

A, 0.175 � 0.006 in group B, 0.145 � 0.003 in group C, p � 0.05) and were still at

Fig. 2. ELISA mean absorbance levels of sera from Eimeria tenella sporozoite antigen immunized and control

chickens.


significantly high levels on the day of challenge i.e. 1 month PI (0.245 � 0.028 in group

A, 0.274 � 0.028 in group B, 0.235 � 0.028 in group C, p � 0.05). In the unimmunized

controls the mean absorbance values (405 nm) ranged from 0.106 � 0.003 on day 16 PI

to 0.157 � 0.016 on day 37 PI.

4. Discussion

In the present study we have compared the effect of two different types of adjuvants

(aluminium hydroxide gel and Freund's complete adjuvant) when given along with the

sporozoite antigen. It can be inferred that the chickens immunized with the antigen

emulsified in FCA showed best protection against homologous oocyst challenge as

evidenced by a reduced number of oocyst production (57.4% reduction in oocyst count),

no mortality and lower mean lesion scores (�1.9). Protection against challenge with E.

tenella have been obtained in the past with killed sporozoites (Rose, 1982) and even with

recombinant antigens (Miller et al., 1988; Danforth et al., 1989; Crane et al., 1991).

Cecal lesions evaluated by the method of Johnson and Reid (1970) were rated on a

scale of 0 (no pathological change) to �4 (most severe pathological changes) on the basis

of subjective observations. Lesion scores of less than �2.0 represented cecal coccidiosis

which would not have serious effect on the broiler performance. Thus, in our study the

reduction in mean lesion scores from �3.4 in controls to a mean of �1.9 in chickens

immunized with 25 mg antigen emulsified in FCA represents a substantial degree of

protection. However, Karkhanis et al. (1991) were able to induce significant reduction in

mean cecal lesion scores (less than �2.0) with less than 10 mg alum precipitated

sporozoite antigen given intramuscularly in the thigh on 2nd, 9th and 16th day of age of

chickens. The difference could be due to the repeated inoculations done in the later case.

CMIR was assessed by the lymphocyte migration inhibition test using peripheral blood

lymphocytes. Chickens immunized with the antigen emulsified in FCA showed peak

CMIR on 23rd day PI, whereas those immunized with the antigen absorbed on aluminium

hydroxide had a peak on 16th day PI. These results are in close conformity with those

reported by Bumstead et al. (1995) who recorded peak CMIR between 14 and 21 days

post-infection by performing lymphocyte proliferation assay after oral immunization of

bird with 100 sporulated E. tenella oocysts at 4 weeks of age and challenging the birds

with 10 000 sporulated E. tenella oocysts at 35 days post-immunization. A longer

duration of CMIR in chickens immunized with the antigen emulsified in FCA could be

attributed to the fact that the FCA is considered to be an adjuvant for both humoral and

CMIR whereas aluminium hydroxide gel is an adjuvant for humoral immune response

only.

ELISA has proved to be the most convenient and sensitive technique, with regard to

ease of preparation, evaluation time and quantities of antigen and test samples required

for detection of antibody responses to Eimeria species (Rose and Mockett, 1983). Very

few studies on the time course of circulating antibody responses of Eimeria have been

conducted. However, it is known that specific serum ELISA antibodies are detectable

shortly after 1 week of infection (Rose and Mockett, 1983; Lillehoj and Ruff, 1987). In

the present study, significant increase in antibody levels could be detected on 12 days PI.


It is also seen that ELISA antibody are produced for �25 days and protection was

conferred on most of the chickens by 37th day PI. These results further substantiate

the findings of Onaga et al. (1989) who have stressed that antibodies against E. tenella

are produced for �22 days and protection was conferred on most of the chickens by

38 days PI.

Antigen given alone induced limited protection (Table 1) in chickens; and both CMIR

and HIR seemed to operate together only for a shorter duration (Figs. 1 and 2) in our

study. Therefore, it seems logical to recommend that further studies should be aimed at

achieving a better protection in chickens against coccidiosis using purified sporozoite

antigens along with adjuvants. Schedule of immunization can also be modified to obtain

more accurate and specific results.

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Immune responses in chickens against Eimeria tenella sporozoite antigen