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Veterinary Parasitology 186 (2012) 170– 177

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

jou rn al h om epa ge: www.elsev ier .com/ locate /vetpar

mmunostimulatory and protective effects of Aloe vera againstoccidiosis in industrial broiler chickens

asood Akhtara,∗, Abdul Haia, Mian Muhammad Awaisa, Zafar Iqbala, Faqir Muhammadb,hsan ul Haqc, Muhammad Irfan Anward

IImmunoparasitology Laboratory, Department of Parasitology, University of Agriculture, Faisalabad 38040, PakistanDepartment of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, PakistanDepartment of Poultry Science, University of Agriculture, Faisalabad 38040, PakistanPoultry Research Institute, Office of Deputy District Livestock Officer (Poultry), Faisalabad, Pakistan

r t i c l e i n f o

rticle history:eceived 7 July 2011eceived in revised form 9 November 2011ccepted 16 November 2011

eywords:occidiosis

mmunostimulationloe veraroilers

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 wasdivided into two experiments. Experiment-I was conducted for the evaluation of immunos-timulatory activity of A. vera and experiment-II demonstrated the protective efficacy ofA. vera extracts against coccidiosis in chickens. Results of the experiment-I revealed sig-nificantly higher (p < 0.05) lymphoproliferative responses in chickens administered withethanolic extract of A. vera as compared to those administered with aqueous extract andcontrol group. Microplate haemagglutination assay for humoral response on day 7th and14th 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 chickensof experimental groups as compared to the control group. None of the extracts, how-ever, demonstrated significant effects on the development of lymphoid organs. Resultsof experiment-II revealed maximum protection (60%) in chickens administered with aque-ous Aloe extract as compared to the ethanolic extract administered chickens (45%). Meanoocysts per gram of droppings in the control group was significantly higher (p < 0.05) ascompared to the chickens in both the experimental groups. Chickens administered withaqueous 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 gainsand antibody titers (p < 0.05) were observed in chickens administered with A. vera extractsas compared to those in the control group. It was concluded that A. vera may be a potentialand valuable candidate to stimulate the immune responses and can be used successfully asan immunotherapeutic agent against coccidiosis in industrial broiler chickens.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Coccidiosis, caused by parasitic protozoa of genus Eime-ia, 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).

304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.11.059

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 economicallyimportant and prevalent worldwide including Pakistan(Ayaz et al., 2003; Shah et al., 2009). Eimeria species infect

Parasitology 186 (2012) 170– 177 171

Table 1Composition of aqueous and ethanolic extracts of Aloe vera based on prox-imate analysis.

Constituent (%) Aqueous extract Ethanolic extract

Crude protein 4.85 3.89

M. Akhtar et al. / Veterinary

epithelial cells of the bird’s intestine causing enteritis andbloody diarrhoea. Severity of disease, however, dependson the specie(s) of Eimeria involved. On the other hand,in a sub-clinical form, it may cause immunosuppression inchickens that paves the way to secondary disease condi-tions. So, management of disease and maintenance of theimmune functions for maximum performance, growth andproduction in poultry industry are fundamental require-ments for profitable farming. To meet these requirements,approaches adopted by the poultry farmers are the uses ofchemotherapeutic and biological agents including vaccines(Lillehoj and Trout, 1996; Mehala and Moorthy, 2008). Nev-ertheless, development of resistant pathogenic strains anddrug residues in animal products used by the human beingsare the major constraints in this regard (Delespaux andKoning, 2007; Reig and Toldra, 2008).

Current scenario demands for the introduction ofsome alternative and effective strategies that may con-fer the poultry birds a strong immune status to fightagainst the pathogenic organisms. In such circumstances,synthetic and native immunomodulators seem to be suit-able candidates to maintain the immune status of thebirds to combat different infections. Several synthetic andnative immunomodulators have been reported to regu-late the natural immune responses and thus increasedproduction performance of the birds (Yamamoto, 1996;Nundkumar and Ojewole, 2002; Awais et al., 2011). Botan-ical immunomodulators are considered ideal because oftheir abundant availability, easy processing, potent efficacyand minimal or no chances of residues in animal products,which are used for human consumption (Patwardhan andGautam, 2005).

In this regard, Aloe vera, also known as the medicinalAloe, has been shown to have diverse biological activities.It is frequently cited as being used in herbal medicines forits 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 dueto its historic reputation as a curative agent and dietarysupplement (Mehala and Moorthy, 2008).

In addition, A. vera components also exhibitimmunomodulatory activities that have been demon-strated in numerous animal models (Talmadge et al., 2004;Kil, 2006) other than poultry except for some preliminarystudies on its therapeutic efficacy (Choi and Chung, 2003).Keeping in view its diverse biological activities, presentstudy was conducted to investigate the immunostimula-tory effects of A. vera extracts in industrial broiler chickensand 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 obtainedfrom Botanical Garden, University of Agriculture Faisal-

abad (UAF), Pakistan and its authenticity was confirmedby the concerned botanist of UAF, and a voucher sam-ple was kept there. Immediately after harvesting, leaveswere washed with chlorinated water (chlorine 5–10 ppm)

Crude fat 0.25 54.15Ash 18.18 21.10Nitrogen free extract 76.72 20.86

followed by distilled water (Femenia et al., 1999). There-after, pulp was collected from the cleaned leaves with thehelp of a wooden spatula within 3–4 h post-harvesting tominimize any deterioration. A. vera pulp was processedfor aqueous and ethanolic extracts following Madan et al.(2008) with minor modifications briefly described as fol-lows.

2.1.1. Aqueous extractPulp was blended in an electric blender. The blended

material (100 g in 1 l double distilled H2O) was homoge-nized for 10 min at 4 ◦C in a homogenizer (Ultra-Turrax,Janke & Kunkel UK). The homogenous suspension wasboiled for 10–12 h in a water bath at 80 ◦C. The gelatinousmaterial obtained (750 ml) was filtered through Bucknerfunnel followed by Whatman No. 1 filter paper to removethe floating material in the suspension. The filtrate thusobtained was subjected to lyophilization at a temperatureof −65 ◦C using freeze drying system (CHRIST, alpha 1-4LD, Gfiertrocknugsanlagen Freeze dryers, Germany) thatyielded 28 g dried extract. Dried extract was subjectedto proximate analysis (A.O.A.C., 1980) (Table 1) and thefinal concentration was reconstituted in sterile phosphatebuffered saline (PBS; pH 7.2) at a dose rate of 100 mg ofdried extract per ml of PBS.

2.1.2. Ethanolic extractThe 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 wasshaken vigorously on a magnetic stirrer (Fisher Scien-tific Co., USA) for 6–8 h at room temperature (25 ◦C). Thesuspension thus obtained was subjected to filtration andlyophilized as described above. The yield of dried ethanolicextract was 37 g. Dried extract was subjected to proximateanalysis (A.O.A.C., 1980) (Table 1) and the final concentra-tion was reconstituted in sterile PBS at a dose rate of 100 mgof dried extract per ml of PBS.

2.2. Infective material

Chicken guts suspected to be naturally infected withEimeria species were collected from different poultry salepoints and outbreak cases of poultry farms in and aroundFaisalabad, 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 and60–80% humidity) for sporulation of the oocysts. After

172 M. Akhtar et al. / Veterinary Parasit

Table 2Chemical composition of withdrawal feed used in the experiment.

Chemical analysis g/kg

Protein 200.0Fat 45.0Carbohydrate 420.0Fiber 50.0Lysine 12.0Ash 55.0Calcium 10.0Phosphorus 5.0Sodium 1.5

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Methionine + cystine 7.0Methionine 4.0

E: 11.995 MJ/kg.

onfirmation of sporulation, the suspension of sporulatedocysts was subjected to zinc sulphate (ZnSO4) floata-ion (Levine, 1961). Sporulated oocysts were washed thriceith PBS and subjected to McMaster counting technique

o count the number of sporulated oocysts per ml of sus-ension (Ryley et al., 1976). The final concentration ofuspension was adjusted to 6.5 × 104–7.0 × 104 sporulatedocysts per 2 ml of PBS and stored in a sterilized screwapped bottle at 4 ◦C until further use.

.3. Morphometric analysis of sporulated oocysts fordentification of different species

Final suspension of sporulated oocysts was subjected toorphometric analysis and different species of genus Eime-

ia were identified based upon their predilection sites fromhere they were collected, morphology and size (Reid and

ong, 1979). Morphometric analysis of sporulated oocystsevealed the presence of three species of Eimeria (E. tenella,. acervulina, and E. necatrix) in the final suspension pro-essed for challenge experiment.

.4. Experimental design

A total of 180-day-old industrial broiler chicks (Hub-ard) were procured from local hatchery and reared onhe floor system under standard management conditions atnimal House, Institute of Microbiology, UAF. All the chicksere provided withdrawal feed (Table 2) and water ad libi-

um. All the chickens were acclimatized for 5 days beforehe initiation of experimental procedures and were inoc-lated with the routine vaccination (Anwar et al., 2008).t 5th day of age, chickens were randomly divided into

wo main groups namely A (n = 60) and B (n = 120) andoused in two separate sheds. Group A was assigned toxperiment-I (evaluation of immunostimulatory effect of. vera) and group B to experiment-II (protective efficacyf A. vera against coccidiosis).

.5. Experiment-I: effect of A. vera extracts on humoralnd cellular immune responses

To evaluate the immunostimulatory effects of A. veraxtracts, chicks of group A (n = 60) were randomly dividednto three sub-groups (A1: aqueous Aloe extract, A2:thanolic Aloe extract, and A3: untreated control). Each

ology 186 (2012) 170– 177

group consisted of 20 chicks, which were administeredorally with the assigned extracts at a dose rate of 300 mg/kgof body weight/day for three consecutive days, i.e., 5th, 6thand 7th days of age. The dose rates of the Aloe extracts usedin the present study were optimized in a preliminary dosetitration pilot project (data not shown).

2.5.1. Evaluation of cellular immune responseIn vivo lymphoproliferative response to

phytohaemagglutinin-P (PHA-P; Sigma®, USA) by using theclassic toe-web assay was used to demonstrate the cellularimmune response as described by Corrier (1990). Briefly,half of the chickens of both the experimental and controlgroups were injected PHA-P (100 �g/100 ml/chicken;intradermally) between the third and fourth digits ofthe right foot on day 14th post administration of A. veraextracts. The left foot injected with PBS (100 �l) served ascontrol. The thickness of the interdigital skin was mea-sured with a pressure-sensitive micrometer screw gaugeat 24 and 72 h post PHA-P injection. Lymphoproliferativeresponse to PHA-P was calculated by using the followingformula:

Lymphoproliferative response =(PHA-P response, right foot) − (PBS response, left foot)

2.5.2. Evaluation for humoral immune responseTo assess the humoral response, anti-SRBC anti-

body (total Igs, IgM and IgG) titers were detected byusing microplate haemagglutination test according to themethodology described by Yamamoto and Glick (1982)with minor modifications suggested by Qureshi andHavenstein (1994). Briefly, on day 14th post administra-tion of A. vera extracts, chickens were injected SRBCs(5%) via intramuscular route (1 ml/chicken) followed bya booster at day 14th post primary injection. Blood wascollected at day 7th and 14th post primary and sec-ondary injections to separate the sera. All the samples wereanalyzed for total Igs, IgM (mercaptoethanol-sensitive)and IgG (mercaptoethanol-resistant) anti-SRBCs antibod-ies. For this purpose, two fold serial dilutions (50 �l volumein each well) of the sera samples were made by using PBSas a diluent. In each well of the microtitration plate, 50 �lsuspension of 5% (v/v) SRBCs was added and mixed gently.The plates were incubated at room temperature (25 ◦C) for30 min. The titer of the well containing 50% agglutinationand 50% reticulum settling (clumping) was considered asthe total anti-SRBC antibody titer of the test sera. To detectIgG titer, 0.01 M mercaptoethanol (50 �l) in PBS was addedinstead of using PBS alone, followed by the previously men-tioned procedure. IgM titers were calculated by subtractingthe IgG titers from total antibody titers of the respectivesera samples.

2.6. Relative weight of the lymphoid organs

Chickens from all the groups were individually weighedand slaughtered at day 35th post administration of A. veraextracts. Lymphoid organs including bursa of fabricius,

Parasit

M. Akhtar et al. / Veterinary

thymus, spleen and cecal tonsils were removed andweighed. The data thus collected were expressed as organweight ratio relative to the live body weight (Giamborneand Closser, 1990).

2.7. Experiment-II: protective effects of A. vera extractsagainst coccidiosis

To evaluate the protective effects of A. vera extractsagainst coccidiosis, chicks of group B (n = 120) were ran-domly assigned to three sub-groups (B1: aqueous Aloeextract, B2: ethanolic Aloe extract and B3: untreated con-trol). Each group consisted of 40 chicks, which wereadministered orally with the assigned extracts at a doserate of 300 mg/kg body weight/day for three consecutivedays, i.e., 5th, 6th and 7th days of age. The dose rates of theAloe extracts used in the present study were optimized ina preliminary dose titration pilot project (data not shown).

2.7.1. Therapeutic evaluationTherapeutic 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 thegroups on day 14th post administration of A. vera extracts.Chickens of all the groups were monitored for body weightgain per day from day 3rd to 12th post challenge. Fecalexamination was conducted daily up to day 12th postchallenge and numbers of oocysts per gram of droppingswere 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. Thelesions on the intestine and caeca of dead and survivedchickens were enumerated from day 6th to 9th post chal-lenge and were scored on a scale from 0 to 4 (Johnson andReid, 1970). Per cent mortality was calculated in each grouppost challenge by using the following formula:

No. of birds died due to coccidiosis post challengeNo. of total birds challenged

× 100

The cause of death of all the birds died post challenge wasconfirmed by autopsy findings. Per cent protection was cal-culated by subtracting the per cent mortality from 100. Onday 12th post challenge, lymphoid organs from the sur-vived chickens of all the groups were removed and organto body weight ratios of lymphoid organs were calculated.

2.7.2. Evaluation of elicited humoral response againstEimeria spp. in chickens administered with A. veraextracts

Elicited humoral response against coccidial species usedin the challenge experiment was assessed by enzymelinked immunosorbent assay (ELISA). Briefly, on day 7thand 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) withminor 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 vesselwith cold water circulation and centrifuged for 10 min at10,000 × g to get the soluble antigen (supernatant) andused for coating the plates. Flat bottomed microtitration

ology 186 (2012) 170– 177 173

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 incubatedovernight at 4 ◦C. The plates were washed thrice with wash-ing buffer (0.05% PBS–Tween 20; pH 7.4); whereas, nonspecific protein binding sites were blocked by adding car-bonate buffer containing 8% non-fat dry milk for 2 h at37 ◦C. The control and test sera were diluted (1:10) inPBS–Tween 20 and added to each well in the microti-tration plate in duplicate, having 0.1 ml in each well andthen incubated for 2 h at 37 ◦C. After washing with thewashing 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 mlof 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% H2O2). The reaction was blocked byadding 0.05 ml of 1.0 N HCL. The optical density (OD) wasread at 492 nm in an ELISA reader. The mean absorbancevalues were recorded and the OD value was calculated. Pos-itive and negative control sera were run in each plate andthe corrected OD value was determined as follows:

ODcorrected = ODSample − ODNegative control of plate

ODPositive control of plate − ODNegative control of plate

2.8. Statistical analysis

One way analysis of variance (ANOVA) and Duncan’smultiple-range tests were used for the determination ofstatistical significance using statistical analysis software(SAS®, 2004). Data on per cent mortality were analyzedusing the Chi-square test. Value of p < 0.05 was consid-ered to be statistical significant for lymphoproliferativeresponse, antibody titers, daily weight gain and oocysts pergram of droppings; whereas, for per cent mortality, pro-tection and relative organ weight ratio of lymphoid organsvalue 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 oftoe-web swelling at 24 and 72 h post PHA-P injection, andthe results revealed significantly higher (p < 0.05) lympho-proliferative responses in chickens administered with A.vera extracts (either ethanolic or aqueous) as compared tothose in the control group. Furthermore, at 24 h post PHA-Pinjection significantly higher (p < 0.05) lymphoprolifera-tive response was recorded in chickens administered withethanolic extract as compared to those administered withaqueous extract (Fig. 1). These results indicated the high-est cellular immune response against PHA-P injection inchickens administered with ethanolic extract of A. vera fol-

lowed by the chickens administered with aqueous extractas compared to control group.

To demonstrate the humoral immune response, sheepred blood cells (SRBCs) were used as non-pathogenic

174 M. Akhtar et al. / Veterinary Parasitology 186 (2012) 170– 177

Fig. 1. Lymphoproliferative response to phytohemagglutinin-P in exper-imental and control chickens. Bars sharing similar letters at 24 and 72 haA

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

re statistically non-significant (p > 0.05). A1: aqeuous extract of Aloe vera;2: ethanolic extract of Aloe vera; A3: control.

ntigens for stimulating T-cell dependent response (Saxenat al., 1997; Kundu et al., 1999). Results revealed thatral administration of A. vera extracts resulted in sig-ificantly higher (p < 0.05) total immunoglobulins (Igs),

mmunoglobulin-G (IgG) and immunoglobulin-M (IgM)iters against SRBCs at day 7th and 14th post primary andecondary injections of SRBCs as compared to the controlroup (Table 3). However, difference among the experi-ental groups (administered with A. vera extracts) at day

th and 14th post primary injection was statistically non-ignificant (p > 0.05); whereas, at day 7th and 14th postecondary injection chickens administered with ethanolicxtract showed statistically higher (p < 0.05) response asompared to those administered with aqueous extract.

Effects of the A. vera extracts on the development ofymphoid organs including bursa of fabricius, spleen, thy-

us and caecal tonsils were also calculated, and resultsere expressed in terms of organ–body weight ratio. The

esults revealed apparently higher per cent organ–bodyeight ratio in both the experimental groups as compared

o control group; but the difference was statistically non-

ignificant (p > 0.01) (data not shown).

able 3ntibody response to sheep red blood cells in experimental and controlhickens.

Group Day 7 PPI Day 14 PPI Day 7 PSI Day 14 PSI

Total anti-SRBCs antibody titerA1 55.46a 55.97a 64b 63.97b

A2 55.97a 55.46a 73.52a 64.56a

A3 27.66b 23.98b 32c 27.54c

Immunoglobulin-MA1 34.57a 28.31a 32.38b 8.51a

A2 35.08a 23.84b 38.44a 8.59a

A3 19.66b 10.16c 16c 6.65b

Immunoglobulin-GA1 20.89a 27.66b 31.62b 55.46a

A2 20.89a 31.62a 35.08a 55.97a

A3 8b 13.82c 16c 20.89b

eans sharing similar letters in a column are statistically non-significantp > 0.05). A1: aqeuous extract of Aloe vera; A2: ethanolic extract of Aloeera; A3: control. PPI: post-primary injection; PSI: post-secondary injec-ion.

control chickens. Bars sharing similar letters on each particular day arestatistically non-significant (p > 0.05). B1: aqeuous extract of Aloe vera;B2: ethanolic extract of Aloe vera; B3: control.

3.2. Experiment-II

In therapeutic evaluation, fecal oocyst shedding, intesti-nal lesion scores, body weight gain and per cent protectionwere used to evaluate the anticoccidial efficacy (Johnsonand Reid, 1970; Dalloul et al., 2003) of A. vera extracts. Fecaloocyst shedding was significantly lower (p < 0.05) in chick-ens administered with A. vera extracts when compared tothe infected control group, whereas, among the experimen-tal groups, the difference was statistically non-significant(Fig. 2).

Protection was maximum (60%) in the group admin-istered with aqueous Aloe extract followed by group ofchickens administered with ethanolic Aloe extract (45%).On the other hand, significantly lower (p < 0.01; 20%)protection in the control group as compared to both exper-imental groups was recorded. Lesion scoring (scale 0–4)of the survived and dead chickens was performed on day6th to 9th post challenge with mixed species of Eimeria.Chickens administered with aqueous Aloe extract showeda minimal mean lesion score (2.3) followed by thoseadministered with ethanolic Aloe extract (2.6) and con-trol chickens (3.05) for caeca, and a similar pattern wasobserved for intestinal lesion scoring (Table 4). Signifi-cantly higher (p < 0.05) body weight gain was recordedin chickens of experimental groups administered with A.vera extracts as compared to those in the control group;whereas, chickens administered with aqueous extractshowed significantly higher (p < 0.05) body weight gains,

compared with those administered with ethanolic extract(Fig. 3). Organ–body weight ratio of all the lymphoid organswas higher in chickens of experimental groups as compared

Table 4Per cent mortality and Lesion scores in experimental and control chickens.

Group Mortality (%) Mean lesion score

Intestine Caeca

B1 40b 2.0 2.3B2 55a 2.1 2.6B3 80c 3.4 3.05

For mortality, per cent values sharing similar letters in column are sta-tistically non-significant (p > 0.05). B1: aqueous extract of Aloe vera; B2:ethanolic extract of Aloe vera; B3: control.

M. Akhtar et al. / Veterinary Parasit

Fig. 3. Weight gains in chickens from day 3rd to 12th post challenge

in experimental and control chickens. Bars sharing similar letters oneach particular day are statistically non-significant (p > 0.05). B1: aqeuousextract 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 obtainedfrom ELISA are shown in Fig. 4. In the chickens administeredwith A. vera extracts, either aqueous or ethanolic, a signifi-cant increase in mean absorbance values was noted on day7th post challenge (0.27 ± 0.02 in group B1 and 0.31 ± 0.02in group B2) as compared to control group (0.14 ± 0.02)(p < 0.05). A similar trend was observed on day 14th postchallenge; however, on day 14th, higher OD values wereobserved as compared to those obtained on day 7th postchallenge.

4. Discussion

Coccidiosis is primarily controlled by medication underfield conditions in spite of limitations like drug resis-tance, and other concerns apprehended about food chaincontamination (Martin et al., 1997). As a substitute, theuse of plants and their products as immunodulators andtherapeutics have a traditional history. According to areport, more than 64% of the world’s population usebotanical drugs to combat health problems (Farnsworth,1999). In this regard, therapeutic properties of A. verahave been studied in different animal models and human

beings. These include anti-inflammatory, immunomodu-latory, wound healing, promotion of radiation damagerepair, antibacterial, antiviral, antifungal, antidiabetic and

Fig. 4. Serum antibody titers on day 7th and 14th post infection withEimeria species (local isolates). Bars sharing similar letters on each partic-ular day are statistically non-significant (p > 0.05). B1: aqeuous extract ofAloe vera; B2: ethanolic extract of Aloe vera; B3: control.

ology 186 (2012) 170– 177 175

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 notbeen explored.

In the present study, aqueous and ethanolic extractsfrom the A. vera pulp exerted stimulatory effects onhumoral and cellular immune responses in chickens. Thehigher cellular immune responses in A. vera administeredchickens may be due to the Aloe polysaccharides, espe-cially acemannan (ACM), which activated the macrophagesto produce inflammatory cytokines such as IL-1, IL-6, andTNF-� (Womble and Helderman, 1988, 1992; Zhang andTizard, 1996; Tan and Vanitha, 2004; Chow et al., 2005).Further, ACM-induced nitric oxide synthesis, mediatedthrough macrophage mannose receptors and macrophageactivation, may also be responsible for immunomodula-tory effects in chickens (Karaca et al., 1995). Ethanolicextract administered chickens showed significantly higher(p < 0.05) response as compared to aqueous extract at 24 hpost-PHA-P injection. The higher response may be dueto the lipid components from the cell membranes of A.vera, solubilized by alcoholic treatment (Reynolds, 2004)which enhanced the phagocytic activity of macrophagesand release of cytokines (Zhang and Tizard, 1996).

Oral administration of A. vera extracts resulted inhigher anti-SRBC antibody (total Igs, IgM and IgG) titersas compared to control, indicating their stimulatory effectson humoral immunity. The activity of the A. vera tostimulate humoral response may be due to aloeride (apolysaccharide from A. vera) that induces the activityof 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 comparedto chickens in both the experimental groups. Chickensadministered with Aloe extracts showed lower mean lesionscores as compared to control chickens both for ceacal andintestinal lesions. A. vera contains anthraquinones, sterol,sterol type �5, saponins and carbohydrates (Vazquezet al., 1996) that might have inhibited the multiplica-tion of the Eimerian parasites leading to low OPG. In vitroinhibitory 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 bedue to the high concentration of mucilagus componentsand pectins with the potential to reduce inflammationdue to their inhibitory action on the arachidonic acidvia cyclooxygenase pathways (Vazquez et al., 1996). Onthe other hand, 20% protection was also observed inthe control group that might be due to the self-limitingmechanism(s) of coccidiosis in chickens (Sharma, 1991).Further, experimental chickens irrespective of the extractthey received were comparatively active with normal feedand water intake and showed the least abnormal signs.On the other hand, chickens in control groups were dulland 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).

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76 M. Akhtar et al. / Veterinary

Results of the present study revealed no significant dif-erence (p > 0.05) in organ–body weight ratio of all theymphoid organs indicating that Aloe extracts have no effectn the development of lymphoid organs.

In the current study, elicited humoral response againsthallenge species of Eimeria was noted in chickens admin-stered with A. vera extracts as compared to those in theontrol group. Recently, it has been reported that anti-odies have a key role in protective immunity againstoccidial infection (Wallach, 2010). Moreover, antibodiesan efficiently hinder the development of Eimeria in thentestine (Rose, 1974). A positive correlation between anti-ody titer and protection against coccidiosis has also beeneported by Smith et al. (1994). In similar studies, anti-odies have been described for inducing partial protectiveassive immunity by blocking the growth, developmentnd replication of parasite (Crane et al., 1988; Hafeez et al.,007; Anwar et al., 2008). In the present study, the thera-eutic efficacy of Aloe extracts may be attributed to theirtimulatory effects on the production of antibodies againstxperimentally induced coccidiosis and thus leading toigher weight gains and lower fecal egg count.

In conclusion, the results of the present study demon-trated that A. vera may be a potential and valuableandidate to stimulate the immune responses in chick-ns and can be used successfully as an immunotherapeuticgent against coccidiosis. Further, it can also be used as

low cost alternative to allopathic drugs to control coc-idiosis in chickens. Further studies on the isolation anddentification of its bioactive components responsible foruch activities are underway in our lab.

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