Professional Documents
Culture Documents
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
a r t i c l e
i n f o
Article history:
Received 7 July 2011
Received in revised form 9 November 2011
Accepted 16 November 2011
Keywords:
Coccidiosis
Immunostimulation
Aloe vera
Broilers
a b s t r a c t
This paper reports the immunostimulatory and protective effects of Aloe vera extracts
(aqueous and ethanolic) against coccidiosis in industrial broiler chickens. The study was
divided into two experiments. Experiment-I was conducted for the evaluation of immunostimulatory activity of A. vera and experiment-II demonstrated the protective efcacy of
A. vera extracts against coccidiosis in chickens. Results of the experiment-I revealed signicantly higher (p < 0.05) lymphoproliferative responses in chickens administered with
ethanolic extract of A. vera as compared to those administered with aqueous extract and
control group. Microplate haemagglutination assay for humoral response on day 7th and
14th post primary and secondary injections of sheep red blood cells (SRBCs) revealed signicantly 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, however, demonstrated signicant effects on the development of lymphoid organs. Results
of experiment-II revealed maximum protection (60%) in chickens administered with aqueous Aloe extract as compared to the ethanolic extract administered chickens (45%). Mean
oocysts per gram of droppings in the control group was signicantly 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 administered with ethanolic Aloe extract (2.6) and control chickens (3.05) for caeca, and a similar
pattern was observed for intestinal lesion scoring. Further, signicantly 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 Eimeria, is an important disease in poultry production, leading
to poor growth rate and high mortality with signicant economic losses up to 3 billion US dollars annually worldwide
(Williams, 1999; Dalloul and Lillehoj, 2006). Generally,
Eimeria (E.) species responsible for coccidiosis in chickens include E. tenella, E. necatrix, E. acervulina, E. maxima,
E. brunetti and E. mitis but the rst four are economically
important and prevalent worldwide including Pakistan
(Ayaz et al., 2003; Shah et al., 2009). Eimeria species infect
171
Table 1
Composition of aqueous and ethanolic extracts of Aloe vera based on proximate analysis.
Constituent (%)
Aqueous extract
Ethanolic extract
Crude protein
Crude fat
Ash
Nitrogen free extract
4.85
0.25
18.18
76.72
3.89
54.15
21.10
20.86
followed by distilled water (Femenia et al., 1999). Thereafter, pulp was collected from the cleaned leaves with the
help of a wooden spatula within 34 h post-harvesting to
minimize any deterioration. A. vera pulp was processed
for aqueous and ethanolic extracts following Madan et al.
(2008) with minor modications briey described as follows.
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 homogenized for 10 min at 4 C in a homogenizer (Ultra-Turrax,
Janke & Kunkel UK). The homogenous suspension was
boiled for 1012 h in a water bath at 80 C. The gelatinous
material obtained (750 ml) was ltered through Buckner
funnel followed by Whatman No. 1 lter paper to remove
the oating material in the suspension. The ltrate thus
obtained was subjected to lyophilization at a temperature
of 65 C using freeze drying system (CHRIST, alpha 1-4
LD, Gertrocknugsanlagen 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
nal 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 homogenizer by taking 100 g pulp in 1 l absolute ethanol (95%;
Merck , Germany). The homogenized suspension was
shaken vigorously on a magnetic stirrer (Fisher Scientic Co., USA) for 68 h at room temperature (25 C). The
suspension thus obtained was subjected to ltration 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 nal concentration 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 collection and sporulation of oocysts as described earlier
(Reid and Long, 1979) in the Immunoparasitology Laboratory, Department of Parasitology, University of Agriculture,
Faisalabad (UAF), Pakistan. Briey, contents of the positive guts were placed in potassium dichromate solution
(2.5%) and incubated for 6072 h (37 C temperature and
6080% humidity) for sporulation of the oocysts. After
172
Table 2
Chemical composition of withdrawal feed used in the experiment.
Chemical analysis
g/kg
Protein
Fat
Carbohydrate
Fiber
Lysine
Ash
Calcium
Phosphorus
Sodium
Methionine + cystine
Methionine
200.0
45.0
420.0
50.0
12.0
55.0
10.0
5.0
1.5
7.0
4.0
173
174
Fig. 1. Lymphoproliferative response to phytohemagglutinin-P in experimental and control chickens. Bars sharing similar letters at 24 and 72 h
are statistically non-signicant (p > 0.05). A1 : aqeuous extract of Aloe vera;
A2 : ethanolic extract of Aloe vera; A3 : control.
3.2. Experiment-II
antigens for stimulating T-cell dependent response (Saxena
et al., 1997; Kundu et al., 1999). Results revealed that
oral administration of A. vera extracts resulted in signicantly higher (p < 0.05) total immunoglobulins (Igs),
immunoglobulin-G (IgG) and immunoglobulin-M (IgM)
titers against SRBCs at day 7th and 14th post primary and
secondary injections of SRBCs as compared to the control
group (Table 3). However, difference among the experimental groups (administered with A. vera extracts) at day
7th and 14th post primary injection was statistically nonsignicant (p > 0.05); whereas, at day 7th and 14th post
secondary injection chickens administered with ethanolic
extract showed statistically higher (p < 0.05) response as
compared to those administered with aqueous extract.
Effects of the A. vera extracts on the development of
lymphoid organs including bursa of fabricius, spleen, thymus and caecal tonsils were also calculated, and results
were expressed in terms of organbody weight ratio. The
results revealed apparently higher per cent organbody
weight ratio in both the experimental groups as compared
to control group; but the difference was statistically nonsignicant (p > 0.01) (data not shown).
Table 3
Antibody response to sheep red blood cells in experimental and control
chickens.
Group
Day 7 PPI
Day 14 PPI
Day 7 PSI
Day 14 PSI
64b
73.52a
32c
63.97b
64.56a
27.54c
32.38b
38.44a
16c
8.51a
8.59a
6.65b
31.62b
35.08a
16c
55.46a
55.97a
20.89b
In therapeutic evaluation, fecal oocyst shedding, intestinal lesion scores, body weight gain and per cent protection
were used to evaluate the anticoccidial efcacy (Johnson
and Reid, 1970; Dalloul et al., 2003) of A. vera extracts. Fecal
oocyst shedding was signicantly lower (p < 0.05) in chickens administered with A. vera extracts when compared to
the infected control group, whereas, among the experimental groups, the difference was statistically non-signicant
(Fig. 2).
Protection was maximum (60%) in the group administered with aqueous Aloe extract followed by group of
chickens administered with ethanolic Aloe extract (45%).
On the other hand, signicantly lower (p < 0.01; 20%)
protection in the control group as compared to both experimental groups was recorded. Lesion scoring (scale 04)
of the survived and dead chickens was performed on day
6th to 9th post challenge with mixed species of Eimeria.
Chickens administered with aqueous Aloe extract showed
a minimal mean lesion score (2.3) followed by those
administered with ethanolic Aloe extract (2.6) and control chickens (3.05) for caeca, and a similar pattern was
observed for intestinal lesion scoring (Table 4). Signicantly higher (p < 0.05) body weight gain was recorded
in chickens of experimental groups administered with A.
vera extracts as compared to those in the control group;
whereas, chickens administered with aqueous extract
showed signicantly higher (p < 0.05) body weight gains,
compared with those administered with ethanolic extract
(Fig. 3). Organbody weight ratio of all the lymphoid organs
was higher in chickens of experimental groups as compared
Table 4
Per cent mortality and Lesion scores in experimental and control chickens.
Group
B1
B2
B3
Mortality (%)
40
55a
80c
Caeca
2.0
2.1
3.4
2.3
2.6
3.05
For mortality, per cent values sharing similar letters in column are statistically non-signicant (p > 0.05). B1 : aqueous extract of Aloe vera; B2 :
ethanolic extract of Aloe vera; B3 : control.
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-signicant (p > 0.05). B1 : aqeuous
extract of Aloe vera; B2 : ethanolic extract of Aloe vera; B3 : control.
to control group; although the difference was statistically non-signicant (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 signicant 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
eld conditions in spite of limitations like drug resistance, 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 worlds 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-inammatory, immunomodulatory, 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 particular day are statistically non-signicant (p > 0.05). B1 : aqeuous extract of
Aloe vera; B2 : ethanolic extract of Aloe vera; B3 : control.
175
176
Results of the present study revealed no signicant difference (p > 0.05) in organbody 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 administered with A. vera extracts as compared to those in the
control group. Recently, it has been reported that antibodies have a key role in protective immunity against
coccidial infection (Wallach, 2010). Moreover, antibodies
can efciently hinder the development of Eimeria in the
intestine (Rose, 1974). A positive correlation between antibody titer and protection against coccidiosis has also been
reported by Smith et al. (1994). In similar studies, antibodies 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 therapeutic efcacy 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 demonstrated that A. vera may be a potential and valuable
candidate to stimulate the immune responses in chickens 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 coccidiosis in chickens. Further studies on the isolation and
identication of its bioactive components responsible for
such activities are underway in our lab.
References
Adams, C., Vahl, H., Veldman, A., 1996. Interaction between nutrition and
Eimeria acervulina infection in broiler chickens: development of an
experimental infection model. Brit. J. Nutr. 75, 867873.
Anwar, M.I., Akhtar, M., Hussain, I., Haq, A.U., Muhammad, F., Hafeez, M.A.,
Mahmood, M.S., Bashir, S., 2008. Field evaluation of Eimeria tenella
(local isolates) gametocytes vaccine and its comparative efcacy with
imported live vaccine LivaCox. Parasitol. Res. 104, 135142.
A.O.A.C., 1980. Ofcial Methods of Analysis, 13th ed. Association of Ofcial
Analytical Chemists, Washington, DC, USA.
Awais, M.M., Akhtar, M., Muhammad, F., Haq, A.U., Anwar, M.I., 2011.
Immunotherapeutic effects of some sugar cane (Saccharum ofcinarum L.) extracts against coccidiosis in industrial broiler chickens.
Exp. Parasitol. 128, 104110.
Ayaz, M.M., Akhtar, M., Hayat, C.S., Hafeez, M.A., Haq, A.U., 2003. Prevalence of coccidiosis in broiler chickens in Faisalabad, Pakistan. Pak.
Vet. J. 23, 5152.
Choi, S., Chung, M., 2003. A review on the relationship between Aloe vera
components and their biologic effects. Sem. Integ. Med. 1, 5362.
Chow, J.T.N., Williamson, D.A., Yates, K.M., Goux, W.J., 2005. Chemical
characterization of the immunomodulating polysaccharide of Aloe
vera L. Carbohydr. Res. 340, 11311142.
Corrier, D.E., 1990. Comparison of phytohemagglutinin-induced cutaneous hypersensitivity reactions in the interdigital skin of broiler and
layer chicks. Avian Dis. 34, 369373.
Crane, M.S., Goggin, B., Pellegrino, R.M., Ravino, O.J., Lange, C., Karkhanis,
Y.D., Kirk, K.E., Chakraborty, P.R., 1988. Passive protection of chickens against Eimeria tenella infection by monoclonal antibody. Infect.
Immun. 56, 972976.
Dalloul, R.A., Lillehoj, H.S., Shellem, T.A., Doerr, J.A., 2003. Intestinal
immunomodulation by vitamin A deciency and Lactobacillus-based
probiotic in Eimeria acervulina-infected broiler chickens. Avian Dis. 47,
13131320.
Dalloul, R.A., Lillehoj, H.S., 2006. Poultry coccidiosis: recent advancements
in control measures and vaccine development. Expert Rev. Vacc. 5,
143163.
Delespaux, V., Koning, H.D., 2007. Drugs and drug resistance in African
trypanosomiasis. Drug Resist. Updates 10, 3050.
Farnsworth, N.R., 1999. The role of ethnopharmacology in drug development. In: Anonymous (Ed.), Bioactive Compounds from Plants. Ciba
Foundation Symposium, Wiley Intersci., New York.
Femenia, A., Sanchez, E.S., Simal, S., Rossello, C., 1999. Compositional features of polysaccharides from Aloe vera (Aloe barbadensis Miller) plant
tissues. Carbohydr. Polym. 39, 109117.
Garcia, J.L., Navarro, I.T., Vidotto, O., Gennari, S.M., Machado, R.Z., Pereira,
A.B.L., Sinhorini, I.L., 2006. Toxoplasma gondii: comparison of a
rhoptry-ELISA with IFAT and MAT for antibody detection in sera of
experimentally infected pigs. Exp. Parasitol. 113, 100105.
Giamborne, J.J., Closser, J., 1990. Efcacy of live vaccine against serologic
subtypes of infectious bursal disease. Avian Dis. 34, 711.
Hafeez, M.A., Akhtar, M., Javed, M.T., Haq, A.U., 2007. Maternal immunization by egg propagated gametocyte vaccine to control Eimeria tenella
infections in newly hatched chicks. Parasitol. Res. 100, 11391141.
Johnson, J., Reid, W.M., 1970. Anticoccidial drugs: lesion scoring techniques in battery and oor pen experiments with chickens. Exp.
Parasitol. 28, 3036.
Karaca, K., Sharma, J.M., Nordgren, R., 1995. Nitric oxide production by
chicken macrophages activated by Acemannan, a complex carbohydrate extracted from Aloe vera. Int. J. Immunopharmacol. 17, 183188.
Kettunen, H., Tiihonen, K., Peuranen, S., Saarinen, M.T., Remus, J.C., 2001.
Dietary betaine accumulates in the liver and intestinal tissue and
stabilizes the intestinal epithelial structure in healthy and coccidiainfected broiler chicks. Comp. Biochem. Physiol. Part A: Mol. Integ.
Physiol. 130, 759769.
Kil, L.C., 2006. New Perspectives on Aloe. College of Pharmacy, Chungbuk
National University Press, South Korea.
Kundu, A., Singh, D.P., Mohapatra, S.C., Dash, B.B., Moudgal, R.P., Bisht,
G.S., 1999. Antibody response to sheep erythrocytes in Indian native
vis--vis imported breeds of chickens. Brit. Poult. Sci. 40, 4043.
Levine, N.D., 1961. The Protozoan Parasite of Domestic Animals and Man.
Burgess Publication Company, Minnesota, USA.
Lillehoj, H.S., Trout, J.M., 1996. Avian gut-associated lymphoid tissues and
intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev.
9, 349360.
Madan, J., Sharma, A.K., Inamdar, N., Rao, H.S., Singh, R., 2008.
Immunomodulatory properties of Aloe vera gel in mice. Int. J. Green
Pharm. 2, 152154.
Martin, A., Danforth, H., Barta, J., Fernando, M., 1997. Analysis of immunological cross-protection and sensitivities to anticoccidial drugs among
ve geographical and temporal strains of Eimeria maxima. Int. J. Parasitol. 27, 527533.
Mehala, C., Moorthy, M., 2008. Effect of Aloe vera and Curcuma longa
(Turmeric) on carcass characteristics and biochemical parameters of
broilers. Int. J. Poult. Sci. 7, 857861.
Mwale, M., Bhebhe, E., Chimonyo, M., Halimani, T.E., 2006. The in vitro
studies on the effect of Aloe vera ((L.) webb. and berth.) and Aloe spicata
(L.f.) on the control of coccidiosis in chickens. Int. J. Appl. Res. Vet. Med.
4, 25.
Nundkumar, N., Ojewole, J.A.O., 2002. Studies on the antiplasmodial properties of some South African medicinal plants used as antimalarial
remedies in Zulu folk medicine. Clin. Pharmacol. 24, 397401.
Pandey, R., Mishra, A., 2010. Antibacterial activities of crude extract of Aloe
barbadensis to clinically isolated bacterial pathogens. Appl. Biochem.
Biotechnol. 160, 13561361.
Patwardhan, B., Gautam, M., 2005. Botanical immunodrugs: scope and
opportunities. Drug Discov. Ther. 10, 495501.
Qureshi, M.A., Havenstein, G.B., 1994. A comparison of the immune performance of a 1991 commercial broiler with a 1957 randombred
strain when fed typical 1957 and 1991 broiler diets. Poult. Sci. 73,
18051812.
Reid, W.M., Long, P.L., 1979. A diagnostic chart for nine species of fowl
coccidia. Research Report. University of Georgia.
Reig, M., Toldra, F., 2008. Veterinary drug residues in meat: concerns and
rapid methods for detection. Meat Sci. 78, 6067.
Reynolds, T., 2004. Aloe chemistry. In: Reynolds, T. (Ed.), Aloes: The Genus
Aloe. CRC Press, Boca Raton, pp. 3974.
Reynolds, T., Dweck, A.C., 1999. Aloe vera leaf gel: a review update. J.
Ethnopharmacol. 68, 3337.
Rose, M.E., 1974. Protective antibodies in infections with Eimeria maxima:
the reduction of pathogenic effects in vivo and a comparison between
oral and subcutaneous administration of antiserum. Parasitology 68,
285292.
Ryley, J.F., Meade, R., Ifazulburst, J., Robinson, T.E., 1976. Methods in coccidiosis research: separation of oocyst from faeces. Parasitology 73,
311326.
177
Wallach, M., 2010. Role of antibody in immunity and control of chickens coccidiosis: a review. Trends Parasitol. 26,
382387.
Williams, R.B., 1999. A compartmentalised model for the estimation o f
the cost of coccidiosis to the worlds chicken production industry. Int.
J. Parasitol. 29, 12091229.
Womble, D., Helderman, J.H., 1988. Enhancement of allo-responsiveness
of human lymphocytes by acemannan. Int. J. Immunopharmacol. 10,
967973.
Womble, D., Helderman, J.H., 1992. The impact of acemannan on the generation and function of cytotoxic T-lymphocytes. Immunopharmacol.
Immunotoxicol. 14, 6377.
Yamamoto, K., 1996. Elimination of minimal residual leukemic
cells by biological response modiers. Rinsho Ketsueki 37,
666670.
Yamamoto, Y., Glick, B., 1982. A comparison of the immune
response between two lines of chickens selected for differences in the weight of the bursa of fabricius. Poult. Sci. 61,
21292132.
Yim, D., Kang, S.S., Kim, D.W., Kim, S.H., Lillehoj, H.S., Min, W.,
2010. Protective effects of Aloe vera-based diets in Eimeria maxima-infected broiler chickens. Exp. Parasitol. 127,
322325.
Zhang, L., Tizard, I.R., 1996. Activation of a mouse macrophage cell line
by acemannan: the major carbohydrate fraction from Aloe vera gel.
Immunopharmacology 35, 119128.