Immunological Evaluation of Two Local Isolates of Eimeria tenella Gametocytes against Coccidiosis in Poultry

Abstract

Two local isolates of Eimeria tenella gametocytes against coccidiosis were immunologically evaluated in chickens. Cell mediated immune response were detected by modified splenic cell migration inhibition assay (MSCMIA) and data was expressed in terms of per cent migration index. No significant difference in per cent migration index was detected for the chickens immunized either with Vaccine-I (local isolate-I) or with Vaccine-II (local isolate-II); however per cent migration index was comparatively lower in chickens immunized with Vaccine-II as compared to Vaccine-I; indicating a somewhat higher cell mediated immune (CMI) response. Humoral immune response was monitored by ELISA in vaccinated and control chickens. Significantly elevated (P<0.05) antibody titer (IgG) in chickens immunized with Vaccine-II as compared to Vaccine-I was detected. Significantly higher protection (67%) in chickens immunized with Vaccine-II followed by vaccine-I (49%) was recorded. Further, oocyst count was significantly lower (P<0.05) in chickens immunized with Vaccine-II as compared to those immunized with Vaccine-I. It was concluded that vaccinal strain (Vaccine-II) contained additional protein of high molecular weight (49.23 kDa) in its gametocytes provided cross protection and can be used to prepare commercial vaccine against coccidiosis in poultry.

Full text

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Pakistan Veterinary Journal
ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE)
Accessible at: www.pvj.com.pk
RESEARCH ARTICLE


Immunological Evaluation of Two Local Isolates of Eimeria tenella Gametocytes against
Coccidiosis in Poultry

Masood Akhtar*, M. Irfan Anwar, Zafar Iqbal, Faqir Muhammad1, Mian Muhammad Awais, Ahsan ul Haq2 and Elzbieta
Hiszczynska-Sawicka3

Immunoparasitology Laboratory, Department of Parasitology; 1Department of Physiology & Pharmacology; 2Department
of Poultry Science, University of Agriculture, Faisalabad-38040, Pakistan; 3School of Biological Sciences,
University of Canterbury, Private Bag 4800, Christchurch, New Zealand.
*Corresponding author: drakhtar@brain.net.pk


ARTICLE H I STOR Y

Received:
Revised:
Accepted: August 14, 2011
Key words:
Coccidiosis
Eimeria tenella
Gametocytes vaccine
Local isolates






(P<0.05) in chickens immunized with Vaccine-II as compared to those immunized
with Vaccine-I. It was concluded that vaccinal strain (Vaccine-II) contained
additional protein of high molecular weight (49.23 kDa) in its gametocytes provided
cross protection and can be used to prepare commercial vaccine against coccidiosis
in poultry.



AB STRACT
June 25, 2011
August 12, 2011


Two local isolates of Eimeria tenella gametocytes against coccidiosis were
immunologically evaluated in chickens. Cell mediated immune response was
detected by modified splenic cell migration inhibition assay (MSCMIA) and data
were expressed in terms of per cent migration index. No significant difference in per
cent migration index was detected for the chickens immunized either with Vaccine-I
(local isolate-I) or with Vaccine-II (local isolate-II); however per cent migration
index was comparatively lower in chickens immunized with Vaccine-II as
compared to Vaccine-I; indicating a somewhat higher cell mediated immune (CMI)
response. Humoral immune response was monitored by ELISA in vaccinated and
control chickens. Significantly elevated (P<0.05) antibody titer (IgG) in chickens
immunized with Vaccine-II as compared to Vaccine-I was detected. Significantly
higher protection (67%) in chickens immunized with Vaccine-II followed by
vaccine-I (49%) was recorded. Further, oocyst count was significantly lower
©2011 PVJ. All rights reserved
To Cite This Article: Akhtar M, Anwar MI, Z Iqbal, F Muhammad, MM Awais, AU Haq and EH Sawicka, 2012.
Immunological evaluation of two local isolates of Eimeria tenella gametocytes against coccidiosis in poultry. Pak Vet
J, 32(1): 77-80.

INTRODUCTION

Coccidiosis remains one of the major menaces for
poultry industry throughout the world (Hafez, 2011). The
disease is traditionally controlled by the use of chemopro-
phylactic measures including anti-coccidials in feed that
inhibit the developmental stages of Eimeria (Calnek et al.,
1997). No doubt, the continuous use of anti-coccidials has
proved to be quite helpful in controlling the disease in
commercial poultry production systems (Allen and
Fetterer, 2002). But there could be several shortcomings
related to this strategy. These include cost, variable
withdrawal times, emergence of drugs resistant strains
(Dalloul and Lillehoj, 2005) and the possible constraints
by the regulatory authorities on medication of feed in
animals (McEvoy, 2001) that are used for human
consumption. Thus, vaccination of birds seems to be a
safer and promising tool to control avian coccidiosis.
Vaccines available in market such as Immucox,
Coccivac, Livacox, Paracox and Viracox (Chapman et al.,
2002; Williams, 2002) contained live virulent or
attenuated lines of Eimerian parasites (Bedrnik et al.,
1995) except CoxAbic that contained native gametocytes
(Wallach, 2002). Many of the commercial vaccines are
being used in different parts of the world with variable
results due to the fact that coccidial strains from different
geographical regions exhibit different antigenicity (Martin
et al., 1997). Laboratory and field trials conducted
previously by using gametocyte vaccines from local
isolate of Eimeria tenella were found to be effective in
mixed spp. coccidial infection in chickens (Hafeez et al.,
2006; Anwar et al., 2008). Present study reports the

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Pak Vet J, 2012, 32(1): 77-80.

immunological evaluation of the two isolates of Eimeria
against coccidiosis in chickens.

MATERIALS AND METHODS

Parasite Stages: Sporulated oocysts of 2 local isolates of
E. tenella maintained in Immunoparasitology Laboratory,
Department of Parasitology, University of Agriculture,
Faisalabad-Pakistan were processed for excystation to get
sporozoites following the methodology of Speer et al.
(1973). The sporozoites were washed twice with
phosphate buffered saline (PBS) and their concentration
was adjusted to 1.8x103- 2x103 per 100 µL and stored at
4°C until use.
Nine days old chicken embryos (2 batches of 200 each)
were purchased from a hatchery at Faisalabad, Pakistan and
maintained in an incubator at 39°C with 70% humidity. The
viability of the embryos was confirmed by candling. On day
12th of age, 100 µl from sporozoites suspensions of each
isolate, containing penicillin (2000 IU) and streptomycin
(0.05mg), was inoculated into the embryos through chorio-
allantoic membranes (2 batches). The embryos were
maintained for 5-7 days at 39°C temperatures and 70%
relative humidity. On day 5th-7th post-inoculation,
gametocytes were harvested and washed according to the
methodology described by Hafeez et al. (2006).

Vaccine formulation: Gametocyte antigens of both the
isolates were prepared
methodology described previously (Hafeez et al., 2006).
In brief, gametocytes were homogenized by sonication
(1x3 minutes; Nissei, Model US 330, Japan) at 4-8oC.
Supernatant obtained after centrifugation (9500 g/15
minutes) was used as antigen and their protein contents
measured by using the methodology of Bradford (1976)
were adjusted to 500µg/0.2ml with PBS. Each vaccine
dose contained 500 µg proteins. Both the vaccines were
designated as Vaccine-I and Vaccine-II, respectively.

Experiment: A total of 180 chicks (Hubbard) procured
from a hatchery at Faisalabad, Pakistan were reared under
standard management conditions (specific pathogen free
environment) at Experimental shed, Department of
Parasitology, University of Agriculture, Faisalabad-
Pakistan. On day 5th of age, chickens were separated into
three equal groups A, B and C (n= 60 in each group). On
day 10th of age, chickens in groups A and B were
administered with experimental gametocyte vaccines
(Vaccine-I and Vaccine- II, respectively) with the help of
an oral gavage; whereas group C served as control was
administered with PBS. Chickens were confirmed for
their coccidia free status by coprological examination.
On day 14th post vaccination (PV), blood and spleens
were collected from fifteen chickens in each group. On
day 15th PV, the remaining chickens of groups A and B
(n=45) were administered with the booster dose of the
respective vaccine and those in group C with PBS. On day
21st PV (primary dose), again blood and spleen samples
were collected from fifteen chickens in each group for
detection of cellular and humoral immune responses.
Spleens were used immediately just after collection to
demonstrate the cell mediated immunity by splenic cell
migration inhibition assay (Akhtar et al., 1999). On the
78
separately following the
other hand, to detect the humoral immunity, serum was
collected from blood samples and subjected to ELISA
(Hafeez et al., 2006).
On day 21st, the remaining chicken in each group
(n=30) were orally challenged with 6.5 X 104 sporulated
oocysts of mixed species of Eimeria [local isolates;
mainly E. maxima, E. tenella and E. acervulina (1:1:1)].
Fecal examination was performed on daily basis up to day
10th post challenge and oocysts per gram of droppings
(OPG) were calculated by McMaster counting technique.
Clinical symptoms and mortality during the experiment in
each group was monitored up to 10 days post challenge.
Per cent protection and mortality was also calculated in
each group post challenge. Dead and survived chickens in
all the groups were monitored for lesion scoring upto day
10th post challenge (Johnson and Reid, 1970).

RESULTS AND DISCUSSION

Numerous commercial vaccines are being used in
many parts of the world to control coccidiosis in chickens.
A generalized limitation regarding their use in broiler and
high roster birds is marked reduction in weight gain and
feed conversion ratios (FCR) as compared to birds
receiving some anticoccidial agents in feed as a
prophylactic measures (Shapiro, 2001; Crouch et al.,
2003). In addition, there is a threat of the introduction of
unwanted Eimeria (E.) species into the environment due
to variant antigenicity of coccidial strains in different
constituencies of the world (Martin et al., 1997). Further,
some strains of Eimeria showed immunological variation
(Lee, 1993) and such strains could impair the efficacy of
vaccines. In such circumstances, vaccines prepared from
local isolates of parasite have been reported to give
promising results (Hafeez et al., 2006; Anwar et al.,
2008). This study reports the immunological evaluation of
the two isolates of Eimeria in terms of cellular, humoral
and challenge responses.
In the present study, CMI in vaccinated and control
chicken, was demonstrated by MSCMIA; and the results
were expressed in terms of per cent migration index. In
vaccinated chickens, sensitized splenic T-cells become re-
sensitized with the test antigen in vitro and therefore the
migration of T-cells inhibited more with antigen. It can be
speculated that these sensitized splenic Tdth cells release
lymphokines including interleukins (IL-1, IL-2, IL-4) and
other cytokines such as migration inhibition factor, which
inhibit the migration (Oldham, 2009). This reflects that
the test antigen used for vaccination activated the T-cells
to initiate the cellular immune response. In majority of the
parasitic infections, protection
experimentally in normal chickens by the transfer of
splenocytes, especially the T-cells, from the immunized
birds. This is due to the fact that T-cells secrete IL-10,
which hinders the release and activity of the INF-γ needed
to activate macrophages and rule out the parasitic
infection, possibly by the enhanced production of
cytotoxic T-cells and NK cells (Roitt et al., 1998). There
was no significant difference (p>0.05) in per cent
migration index in the chickens immunized either with
Vaccine-I or with Vaccine-II; however it was
comparatively lower in chickens immunized with
Vaccine-II as compared to Vaccine-I (Table 1); indicated
can be obtained

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Pak Vet J, 2012, 32(1): 77-80.

to somewhat higher CMI response. Moreover, significant
difference in CMI response in terms of per cent migration
index was recorded on days 14th and 21st post vaccination,
irrespective of the vaccine administered. Further,
significantly lower (P<0.05) migration index in vaccinated
groups as compared to control; indicated the higher CMI
response in vaccinated chickens as compared to the control.

Table 1: Migration distance with and without antigen on days 14th and
21st post vaccination
Migration Distance (µm±SE)
Days Post
Vacci-
nation
Antigen
14 Vaccine-I 24.9±1.18b
Vaccine-II 26.7±1.60b
Control 36.0±1.27a
21 Vaccine-I 49.8±2.25b
Vaccine-II 46.2±1.27b
Control 70.1±2.87a
Means having different letters in a column are statistically significant
(P<0.05).

Humoral response in vaccinated and control chickens
were monitored through ELISA and results were
demonstrated in terms of optical density (OD) values.
ELISA results showed a significantly higher (P<0.05)
antibody titer (IgG) in chickens immunized with Vaccine-
II as compared to Vaccine-I. Further, irrespective of
vaccine, the antibody titer was significantly higher
(P<0.05) in vaccinated as compared to control chickens
(Table 2). The role of antibodies in providing protective
immunity against coccidiosis is controversial. It has been
assumed that antibodies probably develop in parallel with
the cellular immunity (Gilbert et al., 1988). In the preset
study, vaccines contained gametocyte proteins that are
present in the wall forming bodies of the macro-gametes
(WFBs) and play a pivotal role in the development of
Eimerian parasites (Mello et al., 2006).

Table 2: ELISA results on days 14th and 21st Post Vaccination (PV)
14th day pv Vaccine
OD (Mean) SD (±)
Vaccine-I 0.189aB 0.041
Vaccine-II 0.302bB 0.037
Control 0.048cB 0.017
Means having different small letters in a column are statistically
significant (P<0.05); Mean having different capital letters in rows
represents significant difference (P<0.05) within the days.

Results of the challenge experiment on 21 day PV
revealed significantly higher protection (67 %) in
chickens immunized with Vaccine-II followed by
vaccine-I (49%). Significantly lower (P<0.05) oocyst
count in group of chickens immunized with Vaccine-II as
compared to Vaccine-I was recorded (Figure-I). This
reduced oocyst count could be caused by immunity
produced that may reduce the development of disease
through inhibition of the growth, development and/or
fertilization of gametes (Anwar et al., 2008). During the
course of experiment, vaccinated chickens of both the
groups were active and alert with normal feed and water
intake contrary to chickens in control group that were
dull, depressed with ruffled feathers and decreased feed
and water intake.
Survived and dead chickens (during experiment after
challenge) in experimental and control groups were
monitored for lesion scoring to assess the biological
79
Vaccine
With Without
Antigen
32.5±1.36b
36.2±1.07b
38.3±1.33a
62.0±1.87b
59.0±2.24b
74.8±2.80a
Migration
Index (%)
76
73
93
80
78
93
21st day PV
OD (Mean)
0.279aA
0.424bA
0.057cA
SD (±)
0.049
0.044
0.019
protection. In control group, majority of the chickens (66-
70%) demonstrated severe lesions (3.0-4.0) while 25-27
per cent chickens showed moderate lesions (2.0). On the
other hand, chickens immunized with Vaccine-I and
Vaccine-II developed 74 and 69 per cent mild to
moderated lesions (1.0-2.0), respectively.

25
Oocysts per gram of droppings
b
b
b
b
b
b
c
c
c
c
c
c
a
a
a
a
a
a
a
0
5
10
15
20
45678910
Days post challenge
(X10000)
A
B
C


Fig. 1: Oocysts per gram of droppings post challenge with Eimeria
species (local isolates). A = Vaccine-I; B= Vaccine-II; C= Non-vaccinated
infected Control. Bars sharing different letters on a particular day are
statistically significant (P<0.05).

Gametocytes of the two isolates used in Vaccine-I
and Vaccine-II subject to SDS-PAGE contained soluble
proteins had molecular weights 27.65, 24.90, 22.75, 13.20
kDa and 49.23, 27.65, 24.90, 22.75, 13.20 kDa,
respectively (details not given). Chickens immunized with
vaccine-II contained additional gametocyte protein (49.23
kDa) produced high level of antibodies that somehow
prevent the formation of dityrosine bonds and their
associated protein matrices (Wallach, 2002; Mello et al.,
2006). It can be speculated that these antibodies might
hamper with the oocyst’s wall formation (Belli et al.,
2009). Further, the maternal antibodies produced due to
these gametocyte proteins have the ability to protect
hatchlings against homologous infection with Eimeria
species (Hafeez et al., 2007; Belli et al., 2009).
On the whole, these results demonstrated that
vaccinal isolates (Vaccine-II) contained additional
gametocyte protein of 49.23 kDa provided maximum
protection to chickens in challenge experiment. It can be
assumed that the significantly higher cross species
protection in our previous (Hafeez et al., 2006; Ayaz et
al., 2008) and present studies may be due to this
additional molecular weight gametocyte protein (49.23
kDa), which may contain the major portion of conserved
epitopes common to the gametocytes of other Eimerian
species (Wallach et al., 1995). In some other studies, all
most similar size gametocyte proteins (56 kDa) purified
from E. maxima protected the chickens in challenge
experiment (Wallach et al., 1989). From these results it
was concluded that vaccinal strain contained additional
gametocyte proteins of high molecular weight provided
cross protection against mixed species of genus Eimeria
(local isolates) and has practical implications to prepare
commercial vaccine against avian coccidiosis. Further
studies are underway on the role of high molecular weight
gametocyte proteins to be used in vaccine preparation
against coccidiosis.

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Pak Vet J, 2012, 32(1): 77-80.

Acknowledgements
The funds for this project were sponsored by Pakistan
Science Foundation, Islamabad, Pakistan (Project No. P-
AU/Bio/347).

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