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Experimental Parasitology 128 (2011) 104–110

Contents lists available at ScienceDirect

Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Immunotherapeutic effects of some sugar cane (Saccharum officinarum L.)

extracts against coccidiosis in industrial broiler chickens
Mian Muhammad Awais a,⇑, Masood Akhtar a, Faqir Muhammad b, Ahsan ul Haq c, M. Irfan Anwar a
a
Immunoparasitology 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

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

Article history: Present paper reports the effects of aqueous and ethanolic extracts of sugar cane (Saccharum officinarum L.)
Received 15 December 2010 juice and bagasse, respectively on protective immune responses in industrial broiler chickens against
Received in revised form 11 February 2011 coccidiosis. Immunotherapeutic efficacies of the extracts were measured by evaluating their effect on
Accepted 21 February 2011
body weight gain, oocyst shedding, lesion score, anti-coccidial indices, per cent protection and elicited
Available online 24 February 2011
serum antibody responses against coccidiosis. Results revealed a significantly lower (P < 0.05) oocyst
shedding and mortality in chickens administered with sugar cane extracts as compared to control. Further,
Keywords:
significantly higher (P < 0.05) body weight gains and antibody responses were detected in chickens
Saccharum officinarum
Chicken
administered with sugar cane extracts as compared to chickens of control group. Moreover, ethanolic
Immunotherapeutic extract showed higher anti-coccidia index (227.61) as compared to aqueous extract (192.32). The organ
Coccidiosis body weight ratio of the lymphoid organs of experimental and control groups were statistically non-
significant (P > 0.01). These results demonstrated that both ethanolic and aqueous extracts of sugar cane
possess immune enhancing properties and their administration in chickens augments the protective
immunity against coccidiosis.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction
Coccidiosis is one of the most important protozoal diseases of
poultry causing heavy economic losses around the globe in terms
of mortality, morbidity and weight loss in affected birds (Garg
et al., 1999). Morbidity is exhibited through higher feed conver-
sions and depressed average daily weight gains leading to high cost
of production. Although, vaccines and anticoccidials are contribut-
ing a lot for the control of coccidiosis but the efficacies of conven-
tional medications against coccidiosis have been reported with
variable success (Basu and Aaldar, 1994). Moreover, toxic effects
of chemotherapeutic agents on humans in the from of drug resi-
dues in meat (Kaemmerer and Butenkotter, 1973; Murray et al.,
1992), the development of resistance to these drugs by target par-
asites (Maingi et al., 1996) as well as high cost of drugs (Cheema
and Ward, 1990) pave way for the introduction and development
of some alternative remedies. Additionally, the disadvantages of
conventional vaccines have also stimulated the research for some
new alternatives (Talebi and Mulcahy, 2006). Therefore, efforts
⇑ Corresponding author. Fax: +92 419201094.
E-mail addresses: awais009966@hotmail.com, drawaisuaf@gmail.com (M.M.
Awais).
are being made to enhance the host immunity against coccidial
infection.
One of the practical methods to protect the immunobalance is
by the use of pharmacological immunomodulators. In this regard,
herbal immunomodulators are considered to be promising candi-
dates to influence the host inflammatory responses and to boost
up the natural immunity (Patwardhan and Gautam, 2005). There
are number of natural biological response modifiers from plant ori-
gin such as Aloe vera (Zhang and Tizard, 1996; Choi and Chung,
2003), Astragalus membranaceous (Wang et al., 2002; Shabbir
et al., 2008), Azadirachta indica (Ray et al., 1996; SaiRam et al.,
2000), Allium sativum (Morioka et al., 1993; Chang et al., 2005)
and Andrographis peniculata (Chiou et al., 2000); which have immu-
nostimulatory effects in animals making them less susceptible to
certain infectious insults by boosting their intrinsic potential to
perform better immunogenically (Gore and Qureshi, 1997).
In this regard, sugar cane has also been reported to have biolog-
ical as well as immunological properties including adjuvant effects
on the immune responses (El-Abasy et al., 2002, 2003a); therapeu-
tic effects against Eimeria infection (El-Abasy et al., 2003b; Akhtar
et al., 2008) and endotoxic shock in mice (Motobu et al., 2006);
radioprotective effects (Amer et al., 2004, 2005); reconstituting
effects on the B-cells in cyclophosphamide induced immunosup-
pression in chickens (El-Abasy et al., 2004); immunostimulatory

0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2011.02.024
 Author's personal copy
M.M. Awais et al. / Experimental Parasitology 128 (2011) 104–110
and immunomodulatory effects in chickens (Hikosaka et al., 2007;
Akhtar et al., 2008).
Keeping in view, the present study was conducted to investigate
the efficacy of aqueous and ethanolic extracts of sugar cane juice
and bagasse, respectively as natural alternatives to chemothera-
peutic agents for the prevention of coccidiosis in industrial broiler
chickens.
2. Materials and methods
2.1. The plant material
Fresh sugar cane (Saccharum officinarum) plants were procured
from the Sugar Cane Section, Ayub Agricultural Research Institute
(AARI), Faisalabad, Pakistan and its authenticity was confirmed
by the concerned botanist of AARI and a voucher sample was kept
there.
2.1.1. Aqueous extract
The stalks from sugar cane plants were mechanically crushed in
an electric cane crusher immediately after cutting to get fresh su-
gar cane juice and bagasse.
Aqueous extract was separated following the methodology de-
scribed previously (Awais, 2010). Briefly, sugar cane juice (SCJ);
(1.5 L) was centrifuged at 5000g for 15 min at 4 °C. The sediment
was discarded and supernatant was subjected to filtration
(0.45 lm followed by 0.22 lm). The filtrate (1.3 L) thus obtained
was subjected to rotary evaporator (R-210, BÜCHI Laboratories,
UK) at 80 °C to remove the solvent from the juice. The concentrate
obtained (200 mL) was subjected to lypholization (65 °C) using
freeze drying system (CHRIST, alpha 1-4 LD, Gfiertrocknugsanlagen
Freeze dryers, Germany) that yielded 80 g of dried extract. Dried
extract was subjected to proximate analysis (AOAC, 1980) (Table
1) and final dose was prepared with 0.1 M phosphate buffered sal-
ine (PBS) at a dose rate of 100 mg of dried extract per mL of PBS.
2.1.2. Ethanolic extract
Sugar cane bagasse (SCB) was used to extract the ethanolic ex-
tract as described earlier (Awais, 2010). Briefly, SCB was air dried
for 15–20 days at room temperature (25 °C) and ground in an elec-
tric grinder to get powder that was subjected to ethanolic extrac-
tion (20 g powder/500 ml of ethanol) at 80 °C using Soxhlet
apparatus that yielded 5 ml concentrated ethanolic extract. Extract
was lyophilized at 65 °C using freeze drying system and final ex-
tract obtained was 3.8 g. Dried extract was subjected to proximate
analysis (Table 1) and final dose was prepared with 0.1 M phos-
phate buffered saline (PBS) at a dose rate of 100 mg of dried extract
per mL of PBS.
2.2. Experimental design
A total of 120 (1-day-old hubbard) industrial broiler chicks
were purchased from local hatchery and reared under standard
management conditions at Animal House, Faculty of Veterinary
Table 1
Composition of aqueous and ethanolic extracts based on proximate analysis.
Constituent (%) Aqueous extract of Ethanolic extract of
sugar cane juice sugar cane bagasse
Crude protein 2.50 3.45
Crude fat 0.4 49.15
Ash 16.08 27.1
Nitrogen free extract 81.02 20.29
105
Science, University of Agriculture, Faisalabad, Pakistan. All the
chickens were fed commercial withdrawal feed. Chickens were al-
lowed to acclimate for 5 days before the initiation of any experi-
mental procedures. All the chickens of this experimental study
were inoculated with the routine vaccination (Anwar, 2008). At
5th day of age, chickens were randomly divided into three groups
namely A, B and C, each comprising of 40 chickens and were
administered orally with sugar cane extracts for three consecutive
days (5th, 6th and 7th days of age) with the help of an oral gavage
as per schedule.
Group A: Aqueous extract, 4 mL/kg body weight.
Group B: Ethanolic extract, 4 mL/kg body weight.
Group C: Phosphate buffered saline, 4 mL/kg body weight and
served as a control.
2.3. Preparation of infective material
Chickens guts suspected to be naturally infected with coccidia
were collected from different poultry sale points and outbreak
cases of poultry farms of Faisalabad, Pakistan. Positive guts were
processed for the collection and sporulation of oocysts as described
earlier (Reid and Long, 1979) with minor modification. Briefly, con-
tents of the positive guts were placed in 2.5 per cent potassium
dichromate (K2Cr2O7) solution and incubated at 37 °C temperature
and 60–80% humidity for 60–72 h. The sporulation of the oocysts
was confirmed by taking a drop of suspension on glass slide and
examining the sporocysts containing sporozoites under the
microscope (40X). Suspension having the sporulated oocysts was
subjected to zinc sulfate (ZnSO4) floatation (Levine, 1961). Sporu-
lated oocysts were washed thrice with phosphate buffered saline
and subjected to McMaster counting technique to quantify the
number of sporulated oocysts per ml of suspension (Ryley et al.,
1976). The final concentration of suspension was adjusted to
65,000–70,000 sporulated oocysts per 2 mL of PBS and placed in
a sterilized screw capped bottle and stored at 4 °C till further use.
2.4. Microscopy of sporulated oocysts for identification of different
species
Final suspension of sporulated oocysts was subjected to mor-
phometric analysis by microscopic examination and different spe-
cies of genus Eimeria were identified by their predilection site,
morphology and size (Reid and Long, 1979). Morphometric analy-
sis of sporulated oocysts revealed the presence of three species of
Eimeria (Eimeria tenella, Eimeria acervulina, and Eimeria necatrix) in
the final suspension processed for challenge experiment.
2.5. Challenge experiment
Chickens of all the groups were challenged with 65,000–70,000
sporulated oocysts/2 mL of mixed species of genus Eimeria with
the help of an oral gavage on day 14th post administration of sugar
cane extracts. Chickens of each group were monitored for body
weight gain per day from day 3rd to 12th post challenge. Drop-
pings from each group were collected in triplicate and examined
daily from day 3rd to 12th post challenge for the determination
of oocysts per gram (OPG) of droppings by McMaster counting
technique (Ryley et al., 1976). During challenge experiment, chick-
ens of each group were monitored for mortality; dead and survived
chickens in all the groups were also slaughtered and scored for le-
sion scoring (Johnson and Reid, 1970). Per cent protection against
lesions was also determined by using the formula described by
Singh and Gill (1975) as: (Average lesion score (IUG)  Average le-
sion score (IMG)/Average lesion score (IUG)  100).where IUG = In-
fected untreated group and IMG = Infected medicated group.
 Author's personal copy
106 M.M. Awais et al. / Experimental Parasitology 128 (2011) 104–110
2.6. Evaluation of anti-coccidial index (ACI)
Anti-coccidial index was calculated by using the formula as de-
scribed by (Shah et al., 2009).
ACI = (relative rate of weight gain + survival rate)  (lesion va-
lue + oocyst value), whereas, survival rate was estimated by the
number of surviving chickens divided by the number of initial
chickens. Relative rate of weight gain of the chickens in each group
was determined by subtracting the body weight of the chickens at
the time of challenge from the body weight at the end of the
experiments.
Lesion score of the chickens from each group was investigated
according to the method of Johnson and Reid (1970) and oocyst
value was calculated using the formula described by Peek and
Landman (2003) as: (the number of oocysts from positive control
chickens  Treated chickens)/positive control chickens  100%).
2.7. Estimation of elicited humoral response against Eimeria spp. in
chickens administered with sugarcane extracts
Enzyme linked immunosorbent assay (ELISA) was used to de-
tect the elicited humoral response, in experimental chickens as
compared to control group, against coccidial species used in exper-
iment (Section 2.4). For this purpose, blood was collected from the
chickens of each group on day 5th and 10th post infection to get
sera. ELISA was performed as previously described by Garcia
et al. (2006) with minor modifications. Briefly, the complete frac-
tion (2 ml) of sporulated oocysts (obtained in Section 2.3), was son-
icated for 15 (5  3) minutes in a water-jacketed processing vessel
with cold water circulation and centrifuged for 10 min at 10,000g.
The supernatant (soluble antigen) was used as antigen to coat the
wells of microtitration plates. Flat bottomed microtitration plates
(96-wells; Medium binding, polystyrene, Flow Lab. UK) were
coated with 0.1 ml of the antigen (10 lg/ml) diluted in 0.1 M car-
bonate buffer (pH 9.6) and incubated overnight at 4 °C. The plates
were washed thrice with washing buffer (0.05% PBS-Tween 20; pH
7.4); whereas non specific protein binding sites were blocked by
adding carbonate 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 microtitration plate in duplicates;
having 0.1 ml in each well, and then incubated for 2 h at 37 °C.
After washing twice with washing buffer, horseradish peroxidase
conjugated rabbit anti-chickens IgG (1:400) in PBS-Tween 20 was
added (100 lL/well) and incubated for 1 h at 37 °C. After washing,
the peroxidase activity was revealed by adding 0.1 ml of ortho-
phenylenediamine (OPD) solution (20 mg ortho-phenylenedi-
amine/ 50 ml of 0.1 M phosphate citrate buffer, pH 5.0, and 20 lL
of 30% H2O2); the reaction was blocked by the addition of
0.05 mL of 1.0 N HCl. The optical density (OD) was observed at
492 nm in an ELISA microplate reader. The mean absorbance val-
ues were recorded and the OD value was calculated. Positive and
negative control sera were run in every plate and the corrected
OD value was determined as follows:
ODcorrected ¼ ðODSample  ODNegative control of plate =ODPositive control of plate
 ODNegative control of plate Þ
2.8. Effect on relative weight of lymphoid organs
Chickens from each group were individually weighed and
slaughtered on day 12th post challenge. Lymphoid organs includ-
ing bursa of fabricius, thymus, spleen and caecal tonsils were re-
moved surgically and weighed. The data was expressed as per
cent organ weights ratio relative to the live body weight by using
the formula: (Organ weight (gm)/Body weight (gm)  100).
2.9. Statistical analysis
One way analysis of variance (ANOVA) and Duncan’s multiple-
range tests were used for the determination of statistical signifi-
cance. Data on percent mortality and protection against lesions
were analyzed using the Chi square test. Values of P < 0.05 were
considered to be statistical significant for daily weight gain,
oocysts per gram of droppings and antibody titers, whereas for
percent mortality, protection against lesions and relative organ
weight ratio of lymphoid organs value of P was considered to be
<0.01.
3. Results
3.1. Oocyst Count
Immunotherapy with sugar cane extracts resulted in consider-
ably lower oocyst production in chickens challenged with local
isolates of Eimeria (E. tenella, E. acervulina, and E. necatrix) as com-
pared to challenged control group. OPG was at peak on day 9th
post challenge and mean OPG of control group was significantly
higher (P < 0.05) as compared to chickens in both the experimental
groups (A and B) (Fig. 1). However, there was apparently lower
OPG in group B as compared to group A; although the difference
was statistically non-significant (P > 0.05).
3.2. Daily weight gains post challenge
The average daily weight gain (gm ± SE) of all the groups is
shown in Fig. 2. Body weight gain was significantly higher
(P < 0.05) in chickens administered with aqueous and ethanolic ex-
tracts of sugar cane juice and bagasse, respectively as compared to
chickens of control group. Further, weight gains in chickens admin-
istered with ethanolic extract were significantly higher (P < 0.05)
as compared to those administered with aqueous extract.
3.3. Percent mortality and protection
Per cent protection was maximum in chickens administered
with ethanolic extract of sugar cane bagasse followed by chickens
administered with aqueous extract of sugar cane juice; although
the difference between both the groups was nonsignificant,
whereas significantly lower protection (20%) in control group as
compared to experimental groups was observed. On the other
hand, chickens administered with sugar cane extracts showed
significantly lower mortality as compared to control (Table 2).
Oocysts per gram of droppings (x1000)
60
A
50 B * *
*
C
40
*
30 *
*
20
*
10 *
* *
0
3 4 5 6 7 8 9 10 11 12
Days post challenge
Fig. 1. Oocysts per gram of droppings post challenge with Eimeria species (local
isolates). A = aqueous extract of sugar cane juice; B = ethanolic extract of sugar cane
bagasse; C = non-treated infected control. (⁄Significantly higher values)
 Author's personal copy

M.M. Awais et al. / Experimental Parasitology 128 (2011) 104–110 107

Table 3
35 A B C D
Estimation of anti-coccidial index (ACI).
Daily weight gain (gm)

w
30 x x x wx w w x x x
x x x y x x x y Group Relative rate of Survival Lesion Oocyst decrease ACI
y x y x
25 y y y w y wt. gain rate value ratio (%)
y x
z
20 z z z y z A 201 0.65 2.35 93.02 192.32
z z
15 z z B 234 0.70 1.95 94.86 227.61
z
10 C 137 0.20 3.05 0.00 34.15

5 A = aqueous extract of sugar cane juice; B = ethanolic extract of sugar cane bagasse;
0 C = control.
3 4 5 6 7 8 9 10 11 12
Days post challenge

Fig. 2. Weight gains in chickens post challenge with Eimeria species (local isolates). 0.4 x
x
A = aqueous extract of sugar cane juice; B = ethanolic extract of sugar cane bagasse; 0.35 A B C
C = non-treated infected control; D = non infected non-treated control group {data 0.3

Mean OD
adopted from study site (Awais, 2010)}. Bars sharing similar letters on a particular 0.25
x x
day are statistically non-significant (P > 0.05). 0.2
0.15
0.1 y
y
0.05
Table 2 0
Per cent mortality, lesion scores and per cent protection against lesions in chickens 5th Day 10th Day
after challenge with Eimeria species (local isolates). Days post challenge
Group (N) Mortality Lesion scoring of birds Protection against
Fig. 3. Serum antibody titers on day 5th and 10th post challenge with Eimeria
lesions
species (local isolates). A = aqueous extract of sugar cane juice; B = ethanolic extract
(%) 0 1 2 3 4 (%)
of sugar cane bagasse; C = non-treated infected control. Bars sharing similar letters
A (40) 35b 4 8 10 6 12 41.25b on a particular day are statistically non-significant (P > 0.05).
B (40) 30b 8 10 8 4 10 51.25a
C (40) 80a 2 0 8 14 16 23.74c

Values sharing similar letter in a column are statistically non-significant (P > 0.05). pared to those in control group after challenge with local isolates
A = aqueous extract of sugar cane juice; B = ethanolic extract of sugar cane bagasse; of Eimera (mainly E. tenella, E. acervulina, and E. necatrix) (Table 3).
C = control. (N) = No. of birds.

3.6. Antibody response

Further, chickens of both the experimental groups were active, The results of ELISA performed on sera samples from sugar cane
with normal feed and water intake as compared to the chickens extracts administered and control chickens are shown in Fig. 3 at
in control group that were dull/depressed, with ruffled feathers mean absorbance level (492 nm). In the chickens administered
and decreased feed and water intake. with sugar cane extracts, either ethanolic or aqueous, a significant
increase in mean absorbance values were noted on day 5th post
challenge (0.1708 ± 0.018 in group A and 0.1699 ± 0.015 in group
3.4. Lesion scoring and per cent protection against lesions B) as compared to control group (0.0419 ± 0.008) (P < 0.01). A sim-
ilar trend was observed on day 10th post challenge; however all
Postmortem findings revealed extensive hemorrhagic lesions on the groups showed higher OD values on day 10th as compared to
the intestine and caeca filled with blood in control chickens. No or those on day 5th post challenge.
minimal lesions were recorded in chickens of both the experimen-
tal groups. Up to day 12th post challenge with mixed species of
3.7. Organ-body weight ratios in experimental and control chickens
Eimeria, survived and dead chickens (during experiment after chal-
lenge) were monitored for lesion scoring on a scale 0–4. Maximum
On day 12th post challenge, the organ body weight ratio of the
chickens (75%) in control group showed severe lesions (3.0–4.0);
lymphoid organs including bursa of fabricius, spleen, thymus and
moderate lesions (2.0) were also observed in 20 per cent chickens,
caecal tonsils were calculated. Results showed that all the lymphoid
whereas in group A, 45% chickens developed mild to moderate le-
organs had higher values in chickens administered with ethanolic
sions (1.0–2.0) and 45% chickens showed severe lesions (3.0–4.0)
extract of sugar cane bagasse as compared to group administered
and 10% showed no lesions. In group B (administered with etha-
with aqueous extract of sugar cane juice (group A); although the
nolic extract of sugar cane bagasse), 35% chickens developed severe
difference was statistically non-significant (P > 0.01). Further, high-
lesions, 45% chickens of this group had mild to moderate lesions
er per cent organ body weight ratio were recorded in experimental
(1.0–2.0), whereas 20 % chickens of this group showed no signs
groups (A and B) as compared to control group (C) but the differ-
and symptoms (Table 2).
ence was statistically non-significant (P > 0.01).
To access the protective efficacy of sugar cane extracts against
Eimeria induced lesions, per cent protection against lesions was
also calculated in experimental and control groups. Per cent pro- 4. Discussion
tection against lesions was maximum in group B followed by group
A and control group C (Table 2). Coccidiosis is an important parasitic disease in intensive poultry
production, resulting in reduced growth rate and mortality in broi-
ler chickens with huge economic losses up to three billion dollars
3.5. Anti-coccidial index (ACI) annually throughout the world (Williams, 1999; Dalloul et al.,
2003). The severity of Eimeria infection is directly correlated with
A similar trend was observed with respect to anti-coccidial in- reduction in weight gain and shedding of oocysts in droppings
dex. ACIs of sugar cane extracts, either aqueous or ethanolic, were (Idris et al., 1997). Efficient protective effects in terms of increased
higher in chickens administered with sugar cane extracts as com- weight gain and reduced oocyst shedding are considered to be the
 Author's personal copy
108 M.M. Awais et al. / Experimental Parasitology 128 (2011) 104–110
indicators of host resistance to coccidiosis (Dalloul et al., 2005; Lee
et al., 2007) and are therefore highly desired. Keeping in view, the
present study was designed to evaluate the immunotherapeutic ef-
fects of aqueous and ethanolic extracts of sugar cane juice and ba-
gasse, respectively against coccidiosis in chickens.
The results revealed highest protection (70%) in chickens
administered with ethanolic extract of sugar cane bagasse followed
by chickens administered with aqueous extract of sugar cane juice,
whereas only 20% protection was observed in control group. It may
be assumed that natural antibodies (Bo) against a sugar cane poly-
saccharide mediate its complement-activating effect that prevents
the invasion and development of the coccidian parasites and thus
protected the chickens from infection (Li et al., 1983; Li and Vogt,
1982). On the other hand, 20% protection in the control group
might be due to the self-limiting mechanisms of coccidiosis in
chickens (Sharma, 1991). Therapeutic effects of sugar cane extracts
against Eimeria species had also been reported previously (El-
Abasy et al., 2003; Akhtar et al., 2008). In the current study, signif-
icantly higher (P < 0.05) oocyst count was recorded in control
group as compared to chickens of experimental groups adminis-
tered either with the ethanolic or aqueous extract of sugar cane.
Further, experimental chickens of both the groups irrespective of
the extract administered were comparatively active with normal
feed and water intake and showed least abnormal signs. On the
other hand, chickens in control groups were dull and depressed
with ruffled feather and took less feed and water intake that may
be due to altered gut homeostasis (Fernando and McCraw, 1973;
Stephens et al., 1974; McKenzie et al., 1987; Kettunen et al.,
2001a,b) leading to poor feed intake, metabolism and thus de-
creased weight gains (Adams et al., 1996).
Decreased damage to the caecal mucosa in chickens adminis-
tered with sugar cane extracts suggested the involvement of im-
mune effector components in the sugar cane that may cause
inhibition of development of the parasite’s life cycle (Lillehoj,
1989; Trout and Lillehoj, 1996) and thus decreased oocyst count.
During coccidiosis, the cytokine metabolite milieu produced with-
in the microenvironment of intestine, may lead to physiological
alterations including vasodilation that cause increased hemor-
rhagic lesions (Allen, 1997). In the present study, less lesion scores
in chickens administered with ethanolic extract of sugar cane ba-
gasse may be correlated with anti-inflammatory effects of fatty
acids from sugar cane wax oil present in sugar cane bagasse extract
(Ledon et al., 2007).
Significantly reduced (P < 0.05) body weight gain per day in con-
trol group during coccidial infection was due to the inflammatory
immune responses occurred during the disease that divert energy
from growth that may affect the weight gain (Klasing et al.,
1987). Mean body weight gains in chickens administered with eth-
anolic extract of sugar cane bagasse were significantly higher
(P < 0.05) as compared to chickens administered with aqueous ex-
tract of sugar cane juice and control group; but were comparable
to the weight gains of non-treated non infected chickens (historic
data adopted from the same study site; Awais, 2010) except on
day 7, 8, and 9 post challenge that are the days of peaks of infection.
At the same time, weight gains in chickens administered with aque-
ous extract were also significantly higher (P < 0.05) as compared to
control group. Growth promoting effects of sugar cane in healthy as
well as Eimeria infected chickens had been reported previously
(El-Abasy et al., 2002, 2003a,b; Akhtar et al., 2008). In the current
study, improved body weight gain and less mortality in chickens
administered with sugar cane extracts may be associated with su-
gar cane factor that has been reported to induce protective effects
against bacterial infection (Pryce et al., 1990). The protective effects
of sugar cane may also be correlated with local immune responses
(El-Abasy et al., 2002, 2003, 2004; Amer et al., 2004) that may
correspond with the onset of specific immunity to coccidial infec-
tion. Parmentier et al. (2001) also reported the immune responses
and resistance to E. acervulina in chickens divergently selected for
anti-sheep red blood cells antibody response.
In the present study, elicited humoral response against Eimeria
species (used for challenge) was observed in chickens administered
with sugar cane extracts as compared to control chickens. It has
been reported earlier that antibodies do play a role in protective
immunity against Eimeria (Wallach, 2010). Moreover, antibodies
can effectively block the development of Eimeria in intestine as sug-
gested by Rose (1974). Smith et al. (1994) also reported a positive
correlation between antibody titer and protection against coccidio-
sis. In other studies, antibodies have also been reported to induce
partial protective passive immunity by blocking the growth, devel-
opment and replication of parasite (Crane et al., 1988; Hafeez et al.,
2007; Anwar et al., 2008). In the present study, elicited humoral re-
sponse in chickens administered with sugar cane extracts can be
correlated with their therapeutic efficacy against coccidiosis and
thus leading to higher anti-coccidial indices; a synthetic criterion
for assessing the protective effect of any therapeutic agent.
Previously, similar findings have been observed that showed
that oral administration of sugar cane extract resulted in higher
antibody (Total, IgM and IgG) response to sheep red blood cells
in chickens initially infected with oocysts of E. tenella (El-Abasy
et al., 2003; Awais, 2010); enhanced humoral response to sheep
red blood cells in radiation induced immunosuppressed chickens
(Amer et al., 2004); increased serum antibody responses against
sheep red blood cells and Brucella abortus and the number of
antibody-producing cells in peripheral blood leukocytes, intestinal
leukocytes and splenocytes of chickens administered sugar cane
extract and polyphenol rich fraction of sugar cane extract
(Hikosaka et al., 2007); stimulatory effect of sugar cane juice on
antibody production (Akhtar et al., 2008).
In chickens, coccidiosis have been reported to have adverse ef-
fects on the immune status of chickens by somehow having a di-
rect or indirect effect on the development of lymphoid organs
including bursa of fabricius, spleen, thymus and caecal tonsils
(Akhtar and Zafar, 2003; Rashid, 2004). Although, there are reports
that coccidiosis had either no effect on the lymphoid organs
(Augustine and Thomas, 1979) or an increase in their weight
(Venkatratnam et al., 1985) that may be due to the hypertrophy
and cellular infiltration of the lymphoid organs occurred during
coccidiosis (Bettsille, 1986). Results of the current study revealed
non-significant difference in per cent organ-body weight ratio in
experimental chickens administered with sugar cane extracts as
compared to control.
From the results of the present study, it was concluded that su-
gar cane extracts (both aqueous and ethanolic) had immunothera-
peutic efficacy against coccidiosis in broiler chickens as these
significantly enhanced the body resistance against coccidiosis in
terms of elicited humoral response; relatively higher weight gain,
anti-coccidial indices and protection; and reduced oocyst shedding
and lesion score.
Acknowledgment
The funds for this project were sponsored by Pakistan Science
Foundation, Islamabad, Pakistan (Project No. P-AU/Bio (412)).
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