Veterinary Parasitology 186 (2012) 170–177

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Immunostimulatory and protective effects of Aloe vera against

coccidiosis in industrial broiler chickens
Masood Akhtar a,∗ , Abdul Hai a , Mian Muhammad Awais a , Zafar Iqbal a , Faqir Muhammad b ,
Ahsan ul Haq c , Muhammad Irfan Anwar d
a
IImmunoparasitology Laboratory, Department of Parasitology, University of Agriculture, Faisalabad 38040, Pakistan
b
Department of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan
c
Department of Poultry Science, University of Agriculture, Faisalabad 38040, Pakistan
d
Poultry Research Institute, Office of Deputy District Livestock Officer (Poultry), Faisalabad, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: This paper reports the immunostimulatory and protective effects of Aloe vera extracts
Received 7 July 2011 (aqueous and ethanolic) against coccidiosis in industrial broiler chickens. The study was
Received in revised form 9 November 2011 divided into two experiments. Experiment-I was conducted for the evaluation of immunos-
Accepted 16 November 2011
timulatory activity of A. vera and experiment-II demonstrated the protective efficacy of
A. vera extracts against coccidiosis in chickens. Results of the experiment-I revealed sig-
Keywords:
nificantly higher (p < 0.05) lymphoproliferative responses in chickens administered with
Coccidiosis
Immunostimulation
ethanolic extract of A. vera as compared to those administered with aqueous extract and
Aloe vera control group. Microplate haemagglutination assay for humoral response on day 7th and
Broilers 14th post primary and secondary injections of sheep red blood cells (SRBCs) revealed sig-
nificantly higher (p < 0.05) anti SRBC antibody (total Igs, IgG and IgM) titers in chickens
of experimental groups as compared to the control group. None of the extracts, how-
ever, demonstrated significant effects on the development of lymphoid organs. Results
of experiment-II revealed maximum protection (60%) in chickens administered with aque-
ous Aloe extract as compared to the ethanolic extract administered chickens (45%). Mean
oocysts per gram of droppings in the control group was significantly higher (p < 0.05) as
compared to the chickens in both the experimental groups. Chickens administered with
aqueous Aloe extract showed a minimal mean lesion score (2.3) followed by those admin-
istered with ethanolic Aloe extract (2.6) and control chickens (3.05) for caeca, and a similar
pattern was observed for intestinal lesion scoring. Further, significantly higher weight gains
and antibody titers (p < 0.05) were observed in chickens administered with A. vera extracts
as compared to those in the control group. It was concluded that A. vera may be a potential
and valuable candidate to stimulate the immune responses and can be used successfully as
an immunotherapeutic agent against coccidiosis in industrial broiler chickens.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
Coccidiosis, caused by parasitic protozoa of genus Eime-
ria, is an important disease in poultry production, leading
∗ Corresponding author. Tel.: +92 419201094; fax: +92 419201094.
E-mail address: drakhtar@brain.net.pk (M. Akhtar).
to poor growth rate and high mortality with significant eco-
nomic losses up to 3 billion US dollars annually worldwide
(Williams, 1999; Dalloul and Lillehoj, 2006). Generally,
Eimeria (E.) species responsible for coccidiosis in chick-
ens include E. tenella, E. necatrix, E. acervulina, E. maxima,
E. brunetti and E. mitis but the first four are economically
important and prevalent worldwide including Pakistan
(Ayaz et al., 2003; Shah et al., 2009). Eimeria species infect

0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2011.11.059
 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177
epithelial cells of the bird’s intestine causing enteritis and
bloody diarrhoea. Severity of disease, however, depends
on the specie(s) of Eimeria involved. On the other hand,
in a sub-clinical form, it may cause immunosuppression in
chickens that paves the way to secondary disease condi-
tions. So, management of disease and maintenance of the
immune functions for maximum performance, growth and
production in poultry industry are fundamental require-
ments for profitable farming. To meet these requirements,
approaches adopted by the poultry farmers are the uses of
chemotherapeutic and biological agents including vaccines
(Lillehoj and Trout, 1996; Mehala and Moorthy, 2008). Nev-
ertheless, development of resistant pathogenic strains and
drug residues in animal products used by the human beings
are the major constraints in this regard (Delespaux and
Koning, 2007; Reig and Toldra, 2008).
Current scenario demands for the introduction of
some alternative and effective strategies that may con-
fer the poultry birds a strong immune status to fight
against the pathogenic organisms. In such circumstances,
synthetic and native immunomodulators seem to be suit-
able candidates to maintain the immune status of the
birds to combat different infections. Several synthetic and
native immunomodulators have been reported to regu-
late the natural immune responses and thus increased
production performance of the birds (Yamamoto, 1996;
Nundkumar and Ojewole, 2002; Awais et al., 2011). Botan-
ical immunomodulators are considered ideal because of
their abundant availability, easy processing, potent efficacy
and minimal or no chances of residues in animal products,
which are used for human consumption (Patwardhan and
Gautam, 2005).
In this regard, Aloe vera, also known as the medicinal
Aloe, has been shown to have diverse biological activities.
It is frequently cited as being used in herbal medicines for
its curative and therapeutic properties and over 75 bio-
logically active compounds have been identified from A.
vera (Reynolds and Dweck, 1999). It has been used ther-
apeutically for centuries and is of particular interest due
to its historic reputation as a curative agent and dietary
supplement (Mehala and Moorthy, 2008).
In addition, A. vera components also exhibit
immunomodulatory activities that have been demon-
strated in numerous animal models (Talmadge et al., 2004;
Kil, 2006) other than poultry except for some preliminary
studies on its therapeutic efficacy (Choi and Chung, 2003).
Keeping in view its diverse biological activities, present
study was conducted to investigate the immunostimula-
tory effects of A. vera extracts in industrial broiler chickens
and their subsequent protection against avian coccidiosis.
2. Materials and methods
2.1. Preparation of A. vera extracts
Leaves of A. vera used in the present study were obtained
from Botanical Garden, University of Agriculture Faisal-
abad (UAF), Pakistan and its authenticity was confirmed
by the concerned botanist of UAF, and a voucher sam-
ple was kept there. Immediately after harvesting, leaves
were washed with chlorinated water (chlorine 5–10 ppm)
171
Table 1
Composition of aqueous and ethanolic extracts of Aloe vera based on prox-
imate analysis.
Constituent (%) Aqueous extract Ethanolic extract
Crude protein 4.85 3.89
Crude fat 0.25 54.15
Ash 18.18 21.10
Nitrogen free extract 76.72 20.86
followed by distilled water (Femenia et al., 1999). There-
after, pulp was collected from the cleaned leaves with the
help of a wooden spatula within 3–4 h post-harvesting to
minimize any deterioration. A. vera pulp was processed
for aqueous and ethanolic extracts following Madan et al.
(2008) with minor modifications briefly described as fol-
lows.
2.1.1. Aqueous extract
Pulp was blended in an electric blender. The blended
material (100 g in 1 l double distilled H2 O) was homoge-
nized for 10 min at 4 ◦ C in a homogenizer (Ultra-Turrax,
Janke & Kunkel UK). The homogenous suspension was
boiled for 10–12 h in a water bath at 80 ◦ C. The gelatinous
material obtained (750 ml) was filtered through Buckner
funnel followed by Whatman No. 1 filter paper to remove
the floating material in the suspension. The filtrate thus
obtained was subjected to lyophilization at a temperature
of −65 ◦ C using freeze drying system (CHRIST, alpha 1-4
LD, Gfiertrocknugsanlagen Freeze dryers, Germany) that
yielded 28 g dried extract. Dried extract was subjected
to proximate analysis (A.O.A.C., 1980) (Table 1) and the
final concentration was reconstituted in sterile phosphate
buffered saline (PBS; pH 7.2) at a dose rate of 100 mg of
dried extract per ml of PBS.
2.1.2. Ethanolic extract
The blended A. vera pulp was homogenized in a homog-
enizer by taking 100 g pulp in 1 l absolute ethanol (95%;
Merck® , Germany). The homogenized suspension was
shaken vigorously on a magnetic stirrer (Fisher Scien-
tific Co., USA) for 6–8 h at room temperature (25 ◦ C). The
suspension thus obtained was subjected to filtration and
lyophilized as described above. The yield of dried ethanolic
extract was 37 g. Dried extract was subjected to proximate
analysis (A.O.A.C., 1980) (Table 1) and the final concentra-
tion was reconstituted in sterile PBS at a dose rate of 100 mg
of dried extract per ml of PBS.
2.2. Infective material
Chicken guts suspected to be naturally infected with
Eimeria species were collected from different poultry sale
points and outbreak cases of poultry farms in and around
Faisalabad, Pakistan. The guts were processed for col-
lection and sporulation of oocysts as described earlier
(Reid and Long, 1979) in the Immunoparasitology Labora-
tory, Department of Parasitology, University of Agriculture,
Faisalabad (UAF), Pakistan. Briefly, contents of the posi-
tive guts were placed in potassium dichromate solution
(2.5%) and incubated for 60–72 h (37 ◦ C temperature and
60–80% humidity) for sporulation of the oocysts. After
172 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177
Table 2
Chemical composition of withdrawal feed used in the experiment.
Chemical analysis g/kg
Protein 200.0
Fat 45.0
Carbohydrate 420.0
Fiber 50.0
Lysine 12.0
Ash 55.0
Calcium 10.0
Phosphorus 5.0
Sodium 1.5
Methionine + cystine 7.0
Methionine 4.0
ME: 11.995 MJ/kg.
confirmation of sporulation, the suspension of sporulated
oocysts was subjected to zinc sulphate (ZnSO4 ) floata-
tion (Levine, 1961). Sporulated oocysts were washed thrice
with PBS and subjected to McMaster counting technique
to count the number of sporulated oocysts per ml of sus-
pension (Ryley et al., 1976). The final concentration of
suspension was adjusted to 6.5 × 104 –7.0 × 104 sporulated
oocysts per 2 ml of PBS and stored in a sterilized screw
capped bottle at 4 ◦ C until further use.
2.3. Morphometric analysis of sporulated oocysts for
identification of different species
Final suspension of sporulated oocysts was subjected to
morphometric analysis and different species of genus Eime-
ria were identified based upon their predilection sites from
where they were collected, morphology and size (Reid and
Long, 1979). Morphometric analysis of sporulated oocysts
revealed the presence of three species of Eimeria (E. tenella,
E. acervulina, and E. necatrix) in the final suspension pro-
cessed for challenge experiment.
2.4. Experimental design
A total of 180-day-old industrial broiler chicks (Hub-
bard) were procured from local hatchery and reared on
the floor system under standard management conditions at
Animal House, Institute of Microbiology, UAF. All the chicks
were provided withdrawal feed (Table 2) and water ad libi-
tum. All the chickens were acclimatized for 5 days before
the initiation of experimental procedures and were inoc-
ulated with the routine vaccination (Anwar et al., 2008).
At 5th day of age, chickens were randomly divided into
two main groups namely A (n = 60) and B (n = 120) and
housed in two separate sheds. Group A was assigned to
experiment-I (evaluation of immunostimulatory effect of
A. vera) and group B to experiment-II (protective efficacy
of A. vera against coccidiosis).
2.5. Experiment-I: effect of A. vera extracts on humoral
and cellular immune responses
To evaluate the immunostimulatory effects of A. vera
extracts, chicks of group A (n = 60) were randomly divided
into three sub-groups (A1 : aqueous Aloe extract, A2 :
ethanolic Aloe extract, and A3 : untreated control). Each
group consisted of 20 chicks, which were administered
orally with the assigned extracts at a dose rate of 300 mg/kg
of body weight/day for three consecutive days, i.e., 5th, 6th
and 7th days of age. The dose rates of the Aloe extracts used
in the present study were optimized in a preliminary dose
titration pilot project (data not shown).
2.5.1. Evaluation of cellular immune response
In vivo lymphoproliferative response to
phytohaemagglutinin-P (PHA-P; Sigma® , USA) by using the
classic toe-web assay was used to demonstrate the cellular
immune response as described by Corrier (1990). Briefly,
half of the chickens of both the experimental and control
groups were injected PHA-P (100 ␮g/100 ml/chicken;
intradermally) between the third and fourth digits of
the right foot on day 14th post administration of A. vera
extracts. The left foot injected with PBS (100 ␮l) served as
control. The thickness of the interdigital skin was mea-
sured with a pressure-sensitive micrometer screw gauge
at 24 and 72 h post PHA-P injection. Lymphoproliferative
response to PHA-P was calculated by using the following
formula:
Lymphoproliferative response =
(PHA-P response, right foot) − (PBS response, left foot)
2.5.2. Evaluation for humoral immune response
To assess the humoral response, anti-SRBC anti-
body (total Igs, IgM and IgG) titers were detected by
using microplate haemagglutination test according to the
methodology described by Yamamoto and Glick (1982)
with minor modifications suggested by Qureshi and
Havenstein (1994). Briefly, on day 14th post administra-
tion of A. vera extracts, chickens were injected SRBCs
(5%) via intramuscular route (1 ml/chicken) followed by
a booster at day 14th post primary injection. Blood was
collected at day 7th and 14th post primary and sec-
ondary injections to separate the sera. All the samples were
analyzed for total Igs, IgM (mercaptoethanol-sensitive)
and IgG (mercaptoethanol-resistant) anti-SRBCs antibod-
ies. For this purpose, two fold serial dilutions (50 ␮l volume
in each well) of the sera samples were made by using PBS
as a diluent. In each well of the microtitration plate, 50 ␮l
suspension of 5% (v/v) SRBCs was added and mixed gently.
The plates were incubated at room temperature (25 ◦ C) for
30 min. The titer of the well containing 50% agglutination
and 50% reticulum settling (clumping) was considered as
the total anti-SRBC antibody titer of the test sera. To detect
IgG titer, 0.01 M mercaptoethanol (50 ␮l) in PBS was added
instead of using PBS alone, followed by the previously men-
tioned procedure. IgM titers were calculated by subtracting
the IgG titers from total antibody titers of the respective
sera samples.
2.6. Relative weight of the lymphoid organs
Chickens from all the groups were individually weighed
and slaughtered at day 35th post administration of A. vera
extracts. Lymphoid organs including bursa of fabricius,
 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177
thymus, spleen and cecal tonsils were removed and
weighed. The data thus collected were expressed as organ
weight ratio relative to the live body weight (Giamborne
and Closser, 1990).
2.7. Experiment-II: protective effects of A. vera extracts
against coccidiosis
To evaluate the protective effects of A. vera extracts
against coccidiosis, chicks of group B (n = 120) were ran-
domly assigned to three sub-groups (B1 : aqueous Aloe
extract, B2 : ethanolic Aloe extract and B3 : untreated con-
trol). Each group consisted of 40 chicks, which were
administered orally with the assigned extracts at a dose
rate of 300 mg/kg body weight/day for three consecutive
days, i.e., 5th, 6th and 7th days of age. The dose rates of the
Aloe extracts used in the present study were optimized in
a preliminary dose titration pilot project (data not shown).
2.7.1. Therapeutic evaluation
Therapeutic efficacy of A. vera was determined
by inoculation of mixed species of genus Eimeria
(6.5 × 104 –7.0 × 104 sporulated oocysts/chicken) in all the
groups on day 14th post administration of A. vera extracts.
Chickens of all the groups were monitored for body weight
gain per day from day 3rd to 12th post challenge. Fecal
examination was conducted daily up to day 12th post
challenge and numbers of oocysts per gram of droppings
were calculated by using the McMaster counting technique
(Ryley et al., 1976). Clinical symptoms and mortality dur-
ing the experiment in each group were also recorded. The
lesions on the intestine and caeca of dead and survived
chickens were enumerated from day 6th to 9th post chal-
lenge and were scored on a scale from 0 to 4 (Johnson and
Reid, 1970). Per cent mortality was calculated in each group
post challenge by using the following formula:
No. of birds died due to coccidiosis post challenge
× 100
No. of total birds challenged
The cause of death of all the birds died post challenge was
confirmed by autopsy findings. Per cent protection was cal-
culated by subtracting the per cent mortality from 100. On
day 12th post challenge, lymphoid organs from the sur-
vived chickens of all the groups were removed and organ
to body weight ratios of lymphoid organs were calculated.
2.7.2. Evaluation of elicited humoral response against
Eimeria spp. in chickens administered with A. vera
extracts
Elicited humoral response against coccidial species used
in the challenge experiment was assessed by enzyme
linked immunosorbent assay (ELISA). Briefly, on day 7th
and 14th post infection, blood was collected from the chick-
ens of each group (both treated and control) to get sera.
ELISA was performed according to Garcia et al. (2006) with
minor modifications (Awais et al., 2011). For this assay,
2 ml complete fraction of sporulated oocysts was sonicated
for 15 (5 × 3) min in a water-jacketed processing vessel
with cold water circulation and centrifuged for 10 min at
10,000 × g to get the soluble antigen (supernatant) and
used for coating the plates. Flat bottomed microtitration
173
plates (96-wells; medium binding, polystyrene, Flow Lab.,
UK) were coated with 0.1 ml of the antigen (10 ␮g/ml)
diluted in 0.1 M carbonate buffer (pH 9.6) and incubated
overnight at 4 ◦ C. The plates were washed thrice with wash-
ing buffer (0.05% PBS–Tween 20; pH 7.4); whereas, non
specific protein binding sites were blocked by adding car-
bonate buffer containing 8% non-fat dry milk for 2 h at
37 ◦ C. The control and test sera were diluted (1:10) in
PBS–Tween 20 and added to each well in the microti-
tration plate in duplicate, having 0.1 ml in each well and
then incubated for 2 h at 37 ◦ C. After washing with the
washing buffer, horseradish peroxidase conjugated rab-
bit anti-chickens IgG (1:400) in PBS–Tween 20 was added
(100 ␮l/well) and incubated for 1 h at 37 ◦ C. After wash-
ing, the peroxidase activity was observed by adding 0.1 ml
of ortho-phenylenediamine (OPD) solution (20 mg ortho-
phenylenediamine/50 ml of 0.1 M phosphate citrate buffer,
pH 5.0, and 20 ␮l of 30% H2 O2 ). The reaction was blocked by
adding 0.05 ml of 1.0 N HCL. The optical density (OD) was
read at 492 nm in an ELISA reader. The mean absorbance
values were recorded and the OD value was calculated. Pos-
itive and negative control sera were run in each plate and
the corrected OD value was determined as follows:
ODSample − ODNegative control of plate
ODcorrected =
ODPositive control of plate − ODNegative control of plate
2.8. Statistical analysis
One way analysis of variance (ANOVA) and Duncan’s
multiple-range tests were used for the determination of
statistical significance using statistical analysis software
(SAS® , 2004). Data on per cent mortality were analyzed
using the Chi-square test. Value of p < 0.05 was consid-
ered to be statistical significant for lymphoproliferative
response, antibody titers, daily weight gain and oocysts per
gram of droppings; whereas, for per cent mortality, pro-
tection and relative organ weight ratio of lymphoid organs
value of p was considered to be <0.01.
3. Results
3.1. Experiment-I
Cell mediated immunity in terms of lymphoprolifer-
ative response was assessed by measuring amplitude of
toe-web swelling at 24 and 72 h post PHA-P injection, and
the results revealed significantly higher (p < 0.05) lympho-
proliferative responses in chickens administered with A.
vera extracts (either ethanolic or aqueous) as compared to
those in the control group. Furthermore, at 24 h post PHA-P
injection significantly higher (p < 0.05) lymphoprolifera-
tive response was recorded in chickens administered with
ethanolic extract as compared to those administered with
aqueous extract (Fig. 1). These results indicated the high-
est cellular immune response against PHA-P injection in
chickens administered with ethanolic extract of A. vera fol-
lowed by the chickens administered with aqueous extract
as compared to control group.
To demonstrate the humoral immune response, sheep
red blood cells (SRBCs) were used as non-pathogenic
174 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177

Fig. 2. Oocysts per gram of droppings post-challenge in experimental and

Fig. 1. Lymphoproliferative response to phytohemagglutinin-P in exper- control chickens. Bars sharing similar letters on each particular day are
imental and control chickens. Bars sharing similar letters at 24 and 72 h statistically non-significant (p > 0.05). B1 : aqeuous extract of Aloe vera;
are statistically non-significant (p > 0.05). A1 : aqeuous extract of Aloe vera; B2 : ethanolic extract of Aloe vera; B3 : control.
A2 : ethanolic extract of Aloe vera; A3 : control.

3.2. Experiment-II

antigens for stimulating T-cell dependent response (Saxena

In therapeutic evaluation, fecal oocyst shedding, intesti-
et al., 1997; Kundu et al., 1999). Results revealed that
nal lesion scores, body weight gain and per cent protection
oral administration of A. vera extracts resulted in sig-
were used to evaluate the anticoccidial efficacy (Johnson
nificantly higher (p < 0.05) total immunoglobulins (Igs),
and Reid, 1970; Dalloul et al., 2003) of A. vera extracts. Fecal
immunoglobulin-G (IgG) and immunoglobulin-M (IgM)
oocyst shedding was significantly lower (p < 0.05) in chick-
titers against SRBCs at day 7th and 14th post primary and
ens administered with A. vera extracts when compared to
secondary injections of SRBCs as compared to the control
the infected control group, whereas, among the experimen-
group (Table 3). However, difference among the experi-
tal groups, the difference was statistically non-significant
mental groups (administered with A. vera extracts) at day
(Fig. 2).
7th and 14th post primary injection was statistically non-
Protection was maximum (60%) in the group admin-
significant (p > 0.05); whereas, at day 7th and 14th post
istered with aqueous Aloe extract followed by group of
secondary injection chickens administered with ethanolic
chickens administered with ethanolic Aloe extract (45%).
extract showed statistically higher (p < 0.05) response as
On the other hand, significantly lower (p < 0.01; 20%)
compared to those administered with aqueous extract.
protection in the control group as compared to both exper-
Effects of the A. vera extracts on the development of
imental groups was recorded. Lesion scoring (scale 0–4)
lymphoid organs including bursa of fabricius, spleen, thy-
of the survived and dead chickens was performed on day
mus and caecal tonsils were also calculated, and results
6th to 9th post challenge with mixed species of Eimeria.
were expressed in terms of organ–body weight ratio. The
Chickens administered with aqueous Aloe extract showed
results revealed apparently higher per cent organ–body
a minimal mean lesion score (2.3) followed by those
weight ratio in both the experimental groups as compared
administered with ethanolic Aloe extract (2.6) and con-
to control group; but the difference was statistically non-
trol chickens (3.05) for caeca, and a similar pattern was
significant (p > 0.01) (data not shown).
observed for intestinal lesion scoring (Table 4). Signifi-
cantly higher (p < 0.05) body weight gain was recorded
in chickens of experimental groups administered with A.
Table 3 vera extracts as compared to those in the control group;
Antibody response to sheep red blood cells in experimental and control
whereas, chickens administered with aqueous extract
chickens.
showed significantly higher (p < 0.05) body weight gains,
Group Day 7 PPI Day 14 PPI Day 7 PSI Day 14 PSI compared with those administered with ethanolic extract
Total anti-SRBCs antibody titer (Fig. 3). Organ–body weight ratio of all the lymphoid organs
A1 55.46a 55.97a 64b 63.97b was higher in chickens of experimental groups as compared
A2 55.97a 55.46a 73.52a 64.56a
A3 27.66b 23.98b 32c 27.54c
Immunoglobulin-M Table 4
A1 34.57a 28.31a 32.38b 8.51a Per cent mortality and Lesion scores in experimental and control chickens.
A2 35.08a 23.84b 38.44a 8.59a
A3 19.66b 10.16c 16c 6.65b Group Mortality (%) Mean lesion score
Immunoglobulin-G Intestine Caeca
A1 20.89a 27.66b 31.62b 55.46a
b
A2 20.89a 31.62a 35.08a 55.97a B1 40 2.0 2.3
A3 8b 13.82c 16c 20.89b B2 55a 2.1 2.6
B3 80c 3.4 3.05
Means sharing similar letters in a column are statistically non-significant
(p > 0.05). A1: aqeuous extract of Aloe vera; A2: ethanolic extract of Aloe For mortality, per cent values sharing similar letters in column are sta-
vera; A3: control. PPI: post-primary injection; PSI: post-secondary injec- tistically non-significant (p > 0.05). B1 : aqueous extract of Aloe vera; B2 :
tion. ethanolic extract of Aloe vera; B3 : control.
 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177
Fig. 3. Weight gains in chickens from day 3rd to 12th post challenge
in experimental and control chickens. Bars sharing similar letters on
each particular day are statistically non-significant (p > 0.05). B1 : aqeuous
extract of Aloe vera; B2 : ethanolic extract of Aloe vera; B3 : control.
to control group; although the difference was statisti-
cally non-significant (p > 0.01). The antibody titers obtained
from ELISA are shown in Fig. 4. In the chickens administered
with A. vera extracts, either aqueous or ethanolic, a signifi-
cant increase in mean absorbance values was noted on day
7th post challenge (0.27 ± 0.02 in group B1 and 0.31 ± 0.02
in group B2 ) as compared to control group (0.14 ± 0.02)
(p < 0.05). A similar trend was observed on day 14th post
challenge; however, on day 14th, higher OD values were
observed as compared to those obtained on day 7th post
challenge.
4. Discussion
Coccidiosis is primarily controlled by medication under
field conditions in spite of limitations like drug resis-
tance, and other concerns apprehended about food chain
contamination (Martin et al., 1997). As a substitute, the
use of plants and their products as immunodulators and
therapeutics have a traditional history. According to a
report, more than 64% of the world’s population use
botanical drugs to combat health problems (Farnsworth,
1999). In this regard, therapeutic properties of A. vera
have been studied in different animal models and human
beings. These include anti-inflammatory, immunomodu-
latory, wound healing, promotion of radiation damage
repair, antibacterial, antiviral, antifungal, antidiabetic and
Fig. 4. Serum antibody titers on day 7th and 14th post infection with
Eimeria species (local isolates). Bars sharing similar letters on each partic-
ular day are statistically non-significant (p > 0.05). B1 : aqeuous extract of
Aloe vera; B2 : ethanolic extract of Aloe vera; B3 : control.
175
antineoplastic activities (Stanic, 2007; Pandey and Mishra,
2010). As far as could be ascertained, except Yim et al.
(2010), therapeutic efficacy of A. vera in poultry has not
been explored.
In the present study, aqueous and ethanolic extracts
from the A. vera pulp exerted stimulatory effects on
humoral and cellular immune responses in chickens. The
higher cellular immune responses in A. vera administered
chickens may be due to the Aloe polysaccharides, espe-
cially acemannan (ACM), which activated the macrophages
to produce inflammatory cytokines such as IL-1, IL-6, and
TNF-␣ (Womble and Helderman, 1988, 1992; Zhang and
Tizard, 1996; Tan and Vanitha, 2004; Chow et al., 2005).
Further, ACM-induced nitric oxide synthesis, mediated
through macrophage mannose receptors and macrophage
activation, may also be responsible for immunomodula-
tory effects in chickens (Karaca et al., 1995). Ethanolic
extract administered chickens showed significantly higher
(p < 0.05) response as compared to aqueous extract at 24 h
post-PHA-P injection. The higher response may be due
to the lipid components from the cell membranes of A.
vera, solubilized by alcoholic treatment (Reynolds, 2004)
which enhanced the phagocytic activity of macrophages
and release of cytokines (Zhang and Tizard, 1996).
Oral administration of A. vera extracts resulted in
higher anti-SRBC antibody (total Igs, IgM and IgG) titers
as compared to control, indicating their stimulatory effects
on humoral immunity. The activity of the A. vera to
stimulate humoral response may be due to aloeride (a
polysaccharide from A. vera) that induces the activity
of IL-6, a potent B-cell stimulant, to produce antibodies
(Tan and Vanitha, 2004).
In the challenge experiment, mean OPG in the con-
trol group was significantly higher (p < 0.05) as compared
to chickens in both the experimental groups. Chickens
administered with Aloe extracts showed lower mean lesion
scores as compared to control chickens both for ceacal and
intestinal lesions. A. vera contains anthraquinones, sterol,
sterol type 5 , saponins and carbohydrates (Vazquez
et al., 1996) that might have inhibited the multiplica-
tion of the Eimerian parasites leading to low OPG. In vitro
inhibitory effects of A. vera against coccidiosis in chick-
ens have also been reported previously (Mwale et al.,
2006). Maximum protection (60%) was recorded in chick-
ens administered with aqueous Aloe extract that may be
due to the high concentration of mucilagus components
and pectins with the potential to reduce inflammation
due to their inhibitory action on the arachidonic acid
via cyclooxygenase pathways (Vazquez et al., 1996). On
the other hand, 20% protection was also observed in
the control group that might be due to the self-limiting
mechanism(s) of coccidiosis in chickens (Sharma, 1991).
Further, experimental chickens irrespective of the extract
they received were comparatively active with normal feed
and water intake and showed the least abnormal signs.
On the other hand, chickens in control groups were dull
and depressed with ruffled feather and took less feed
and water that may be due to altered gut homeosta-
sis (Kettunen et al., 2001) leading to poor feed intake,
metabolism and thus decreased weight gains (Adams et al.,
1996).
176 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170–177
Results of the present study revealed no significant dif-
ference (p > 0.05) in organ–body weight ratio of all the
lymphoid organs indicating that Aloe extracts have no effect
on the development of lymphoid organs.
In the current study, elicited humoral response against
challenge species of Eimeria was noted in chickens admin-
istered with A. vera extracts as compared to those in the
control group. Recently, it has been reported that anti-
bodies have a key role in protective immunity against
coccidial infection (Wallach, 2010). Moreover, antibodies
can efficiently hinder the development of Eimeria in the
intestine (Rose, 1974). A positive correlation between anti-
body titer and protection against coccidiosis has also been
reported by Smith et al. (1994). In similar studies, anti-
bodies have been described for inducing partial protective
passive immunity by blocking the growth, development
and replication of parasite (Crane et al., 1988; Hafeez et al.,
2007; Anwar et al., 2008). In the present study, the thera-
peutic efficacy of Aloe extracts may be attributed to their
stimulatory effects on the production of antibodies against
experimentally induced coccidiosis and thus leading to
higher weight gains and lower fecal egg count.
In conclusion, the results of the present study demon-
strated that A. vera may be a potential and valuable
candidate to stimulate the immune responses in chick-
ens and can be used successfully as an immunotherapeutic
agent against coccidiosis. Further, it can also be used as
a low cost alternative to allopathic drugs to control coc-
cidiosis in chickens. Further studies on the isolation and
identification of its bioactive components responsible for
such activities are underway in our lab.
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