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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
Marizvikuru Mwale, BSc, MSc1
Evison Bhebhe, BSc, MS, PhD2
Michael Chimonyo, BSc, MSc, PhD3
Tinyiko Edward Halimani, BSc, MSc4
1
Department of Agriculture
Bindura University of Science Education
Bindura, Zimbabwe
2
Department of Paraclinical Veterinary Studies
University of Zimbabwe
Harare, Zimbabwe
3
Department of Livestock and Pasture Science
Faculty of Science and Agriculture
University of Fort Hare
Eastern Cape, Republic of South Africa
4
Department of Animal Science
University of Zimbabwe
Harare, Zimbabwe

KEY WORDS: Aloe vera, Aloe spicata, oocysts at 45% treatment concentration.
coccidia oocysts, sporulation Aloes can, therefore, be used to control
coccidiosis in chickens, especially among
ABSTRACT the resource-poor smallholder farmers.
An in vitro trial was undertaken to deter-
mine the effect of Aloe vera and Aloe spi- INTRODUCTION
cata on the inhibition of the sporulation of Aloe vera and Aloe spicata belong to the
avian coccidia oocysts. Petri dishes contain- lily family. They are related to garlic, onion,
ing coccidia oocysts isolated from drop- and asparagus. They have been reported to
pings of coccidiosis-infected chickens were have medicinal properties.1 Aloe vera, for
randomly assigned to 0%, 15%, 30%, and example, is thought to help in the external
45% aloe extract, and the control, sul- healing of many kinds of wounds, to be
phachlopyrazine sodium monohydrate excellent in soothing minor burns, scrapes,
(Esb3®, Novartis AG, Basel, Switzerland).
ulcers, and to alleviate arthritis, constipa-
The Petri dishes were incubated at 25°C for
tion, and piles. The leaf and juice may be
48 hours. Aloe spicata inhibited sporulation
better than A vera (P < 0.05). Increasing used in animals internally or externally. It is
aloe concentration reduced the number of believed to possess pharmacological anti-
coccidia oocysts that sporulated (P < 0.05). bacterial, antivenin, and immunological
Aloe spicata had the least number of sporu- properties. The potential for using A vera
lated oocysts at 30% concentration while A and A spicata in livestock health manage-
vera had the least number of sporulated ment is, however, not documented.

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There is little documentation on broiler MATERIALS AND METHODS


production in the smallholder sector. Most
Study Site
studies are on village chicken production2 of
which farmers are engaged in both village The trial was carried out in the Parasitology
chicken and broiler chicken production. Of Laboratory, Department of Paraclinical
the several constraints to chicken produc- Veterinary Studies, University of
tion, the major chicken disease problems are Zimbabwe.
coccidiosis and Newcastle disease.3 Experimental Design and Experimental
Incidences of coccidiosis, among other dis- Procedure
eases, were reported to be high in chickens
Extraction of aloe juice
age 3-4 weeks.3,4
Fresh aloe leaf extract of the broad leaf vari-
In developing countries, poultry produc-
ety was used. The aloe leaves were bought
tion is largely based on traditional extensive
at Greendale in Harare, in March 2004. The
poultry production systems.5 Village chick-
leaves were thoroughly washed using water
ens represent an appropriate system to pro- before being minced in preparation for
vide high-quality protein to the fast growing extracting juice. Thorns on the leaves were
human population. They also provide removed using a scalpel blade after which
income to resource-poor smallholder farm- the leaves were cut into small cubes for
ers, especially women. However, high mor- easy mincing. Aloe juice was collected in a
tality rates, mainly due to diseases, bucket from the mincer and then stored in
including coccidiosis, constitute one of the sample bottles. Before use, the juice was
greatest constraints on chicken develop- thoroughly mixed to obtain a uniform solu-
ment. Resource-poor smallholder farmers tion, using a kitchen blender. The aloe
usually use various aloe species to reduce extracts were stored for 6 days in a refriger-
chicken mortalities. Common examples of ator at 4°C pending use to avoid oxidation,
aloe used in Southern Africa include A vera, thereby maintaining the active ingredients in
A excelsa, A christiana, and A spicata. They the liquid extracts.
are easy to use and are available all year Isolation of Coccidia From Poultry
round.6 There is, thus, a need to investigate Droppings
and document the therapeutic importance of
Preparation of saturated salt solution
the aloes, which may in turn result in the
About 400 g of sodium chloride were mixed
preservation and protection of the natural
with 1000 mL of distilled water in a 10-L
plant resource-base. Smallholder farmers are
plastic container at ambient temperature.8
able to contribute in the conservation efforts
The prepared saturated salt solution was
if they use the herb for ethno-veterinary and used in the isolation of coccidia from coc-
medicinal purposes. cidiosis-infected chicken feces.
Although ethno-veterinary medicines are
Isolation of coccidia
widely used, the optimum dosage and the
dose response characteristic are not known.7 The flotation method was used to test for the
Dose response curves are difficult to deter- presence of coccidia oocysts in poultry drop-
pings. Fresh chicken feces (2 g) of birds sus-
mine when using ethno-veterinary medi-
pected of being infected with coccidia
cines. The objective of the current study
parasites were thoroughly mixed with a satu-
was, therefore, to determine the optimum
rated salt solution using a pestle and mortar.
concentration of the commonly used aloes
The mixture was filtered through a strainer
in Zimbabwe, A vera and A spicata, to into a 250-mL plastic beaker. The saturated
inhibit sporulation of coccidia oocysts. salt solution makes the oocysts float. The
oocysts in suspension were withdrawn using

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a 5-mL Pasteur pipette and mounted onto a Sample preparation for incubation and
slide for counting of oocysts under a light counting
microscope. Oocysts were counted using the The prepared oocysts-potassium dichro-
McMaster technique.8 mate-water mixture was centrifuged for 5
Fresh chicken feces of birds suspected minutes at a gravity of 2000 rpm so as to
of coccidia oocysts infection were used. The increase oocysts counts in 1 mL oocysts-
oocysts were washed and separated from the water-potassium dichromate mixture/solu-
litter using a standard 63 μm Tyler mesh tion. The supernatant was discarded until 15
sieve. Initially, the droppings were mechan-
9 mL of solution was left. Fifteen petri dishes,
ically mixed with water using a pestle and 3 for each treatment, were labeled and pre-
mortar. The oocysts were then washed with pared for incubation, and 1-mL aliquots of
water and filtered first through the 180 μm oocyst solution were withdrawn using a 5-
mL Pasteur pipette and randomly allocated
Tyler mesh sieve. A small hand-held
to each of the prepared petri dishes into
sprayer and flexible hose attached directly
which various A vera and sulphachlopy-
to the water faucet at a sink, ideally suited
razine sodium monohydrate (Esb3®,
for this cleaning process, was used. The
Novartis AG, Basel, Switzerland) concen-
coarse residual material retained in the sieve trations were added. The oocyst solution
was discarded. The filtrate containing was thoroughly mixed mechanically each
oocysts was collected in a 5-L beaker and time 1 mL of oocysts solution was with-
allowed to stand for 30 minutes in a refrig- drawn. The A vera extract concentrations
erator at 4°C until the oocysts settled to the used were 0%, 15%, 30%, and 45%. The
bottom. Esb3® (0.02 g) per petri dish was introduced
After the oocysts had settled, the super- into 3 petri dishes as a control. The different
natant was siphoned off and the sediment dilutions of A vera consisted of 100% A
containing oocysts were transferred to a 106 vera extract. To make a total volume of 20
μm Tyler mesh sieve through which the mL for each treatment, the A vera, water,
oocysts were flushed with water into a 5-L and oocysts volumes were prepared as
beaker. The filtrate was once more collected shown in Table 1. All the petri dishes were
and allowed to settle in a refrigerator (4°C) incubated at 25°C for 48 hours. The same
for 30 minutes. Thereafter, the supernatant preparation procedure for incubation was
done for A spicata.
was discarded and the sediment flushed
with water using a 63 μm Tyler mesh sieve. The McMaster oocyst counting technique
The process was repeated using the same One gram of feces was suspended in 30 mL
sieve. The last sediment was flushed, this of saturated salt solution. Dilutions of the
time into a smaller container (250-mL plas- saturated salt solution were calculated, as
tic beaker). The water was then siphoned different weights were obtained for oocysts
out and a potassium dichromate solution in each petri dish. The suspension was
(2.5%) was mixed with the concentrated stirred well to obtain a completely homoge-
oocysts to a depth of 40 mm Table 1. Preparations for the Aloe Extract Concentrations.
above the oocysts. One part
Volume (mL)
oocysts were diluted with 4
Concentration (%) Aloe Water Oocysts Total Volume
parts 2.5% potassium dichro-
0 0 19 1 20
mate. Potassium dichromate
15 3 16 1 20
was added to inhibit bacterial
30 6 13 1 20
growth that may hinder the
45 9 10 1 20
sporulation of oocysts. Esb3®
0 19 1 20

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neous distribution of the oocysts in the liq- centration of the ith species, μ = overall mean,
uid. All 3 counting chambers of the count- Si = effect due to species (i = 1 and 2), Cj =
ing cell were filled with the aid of a Pasteur effect due to concentration (j = 1, 2, 3, 4, and
pipette. After 5 minutes, the oocysts floated 5), (SC)ij = species and concentration interac-
on the surface of the concentration solution tion, and εijk = residual error.
and stuck to the cover slip. The oocysts The data were converted to percentages
were then counted under a microscope at a and tested for normality using a univariate
low magnification (×10 μm). The counting procedure.10 The data was not normally dis-
cells had 3 compartments, each compart- tributed, and arcsine transformations were
ment having a surface of 10 × 10 mm2; the performed. The collected data was then ana-
space between objective glass and cover slip lyzed using the General Linear Model pro-
was 1.5 mm. Each compartment contained cedures.10 Tukey's test was used to compare
0.15 mL liquid, and at least 4 compartments differences among means.
were counted. The following formula was
used to obtain the actual number of oocysts: RESULTS
EPG = X × 200 Aloe spicata and A vera had significant
where EPG = eggs (oocysts) per gram of effects on the sporulation of coccidia
feces, and X = the number of eggs/oocysts oocysts (P < 0.05). Increase in aloe extract
counted in 1 counting cell. concentration had an effect on the sporula-
tion of coccidia oocysts (P < 0.05): the
If 2 or more counting cells were filled
number of sporulated oocysts decreased
and counted, then X changed as per the for-
with increasing aloe treatment concentra-
mula below.
tions (Table 2). The 45% concentration had
X = Total number of eggs ÷ Total number of
the least number of sporulated oocysts for
counting cells
both aloes. The 15% A vera concentration
The coccidia oocysts were then counted and the 30% A vera concentrations were not
under a light microscope using the significantly different from the 0% A vera
McMaster technique (quantitative method).8 concentration in inhibiting coccidia oocysts
The counts were converted to a percentage sporulation, while the 15% A spicata con-
of both the sporulated and unsporulated coc- centration was not different from the 0%
cidia oocysts. and 30% A spicata concentrations (Table 2).
Experimental design Conversely, the 15% and 30% A vera treat-
Oocysts were randomly allocated to 4 con- ments were not significantly different from
centrations (v/v; 0%, 15%, 30%, and 45%) the Esb3® treatment, whereas for A spicata,
of either A vera or A spicata extracts. Three only the 0% concentration was not different
replications were made for each concentra- from the Esb3® treatment.
tion. Esb3® was applied to 6 Table 2. Arcsine Oocyst Mean Values (±SE) Following Aloe vera
petri dishes at a recommend- and Aloe spicata Treatment on Sporulation.
ed application rate of 0.1%
Unsporulated
(w/v). Oocysts were incubat- Sporulated Oocysts Oocysts
ed for 48 hours at 25°C. (counts/g feces) (counts/g feces)

Statistical Analyses Concentration (%) A vera A spicata A vera A spicata

The following model was 0 0.7ab 1.2a 0.6 0.07c


used to analyze the effect of 15 0.5ab 1.0ab 0.5 0.14bc
each aloe on sporulation of 30 0.5ab 0.7b 0.6 0.37a
coccidia oocysts: 45 0.3b 0.8b 0.8 0.27ab
Yijk = µ + Si + Cj + (SC)ij + εijk Esb3® 1.3a 1.3a 0.1 0.04c
SEM 0.18 0.08 0.21 0.038
where Yijk = the jth con-
Lsmeans with the same superscript within a column are not different (P > 0.05).

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The Esb3® treatment had the highest concentrations were significantly different
number of sporulated oocysts in comparison in inhibiting sporulation (P < 0.05). There
to petri dishes with aloe extracts, while the was no interaction between the aloe species
45% concentration had the least number of and treatment concentrations (P > 0.05).
sporulated oocysts for A vera and the 30%
concentration had the least number of DISCUSSION
sporulated oocysts for A spicata (Table 2). The observation that aloe extract concentra-
The 45% concentration was significantly tions had an effect on the sporulation of
different from the Esb3® treatment (P < coccidia oocysts indicates that aloe extracts
0.05). There was no significant difference are able to kill or inhibit growth and devel-
in the number of unsporulated oocysts in all opment of oocysts. The finding that A spica-
the A vera treatments (P > 0.05). However, ta had the least sporulation at 30% while
for A spicata, there was a significant differ- A vera was most effective at 45% could
ence (P < 0.05), with the 30% concentra- suggest that the former is more effective in
tion having the highest number of treating coccidiosis. The observation that
unsporulated oocysts and the 0% and Esb3® Esb3® could not inhibit sporulation could be
having the least number of unsporulated explained by the fact that, because it is a
coccidia oocysts. bactericidal drug as well, it killed the bacte-
The 2 aloe species A vera and A spicata ria present, thereby enhancing sporulation of
were significantly different in inhibiting oocysts. The effect of bacteria on sporula-
sporulation of coccidia parasites (P < 0.05). tion of coccidia oocysts has been document-
Table 3 illustrates that A spicata inhibited ed earlier.9 Potassium dichromate killed
sporulation better than A vera. As shown in bacteria in a sample containing coccidia
Table 4, Esb3® was not effective in inhibit- oocysts, thereby enhancing sporulation of
ing sporulation. The formulated treatment coccidia oocysts.9 It could be that bacteria,
Table 3. Arcsine Oocyst Mean Values (±SE) Following if present, could interfere with the
Aloe vera and Aloe spicata Treatment. sporulation of oocysts, possibly by
Sporulated Unsporulated competing for nutrients and/or feed-
Oocysts Oocysts ing on the oocysts. This also explains
Species (counts/g feces) (counts/g feces) the differences in the 0% treatment
A vera 1.0a
0.5
a
concentration values of the aloes that
A spicata 0.7b
0.2
b was expected to be equal. It is not
known whether the formed sporo-
SEM 0.06 0.07
zoites were capable of infecting
Lsmeans with the same superscript within a column are not different chickens or not, depending on the
(P > 0.05).
coccidia species. It could be that
Table 4. Arcsine Oocyst Mean Values (±SE) Following although the coccidia sporulated, they
Different Aloe Concentrations.
were not in the infective state. Thus,
Sporulated Unsporulated more studies are required to deter-
Oocysts Oocysts
mine the effect of the aloes on infec-
Concentration (%) (counts/g feces) (counts/g feces)
tivity of the sporulated coccidia
0 0.9ab
0.4
ab
oocysts.
15 0.8b
0.3
ab
The differences between the 2
30 0.6b
0.5
ab
aloe species in inhibiting sporulation
45 0.5b
0.6
a of the coccidia oocysts may be due to
Esb3 ®
1.3a
0.1
b differences in chemical compositions.
The inability of the aloe species to
SEM 0.10 0.11 completely inhibit sporulation of the
Lsmeans with the same superscript within a column are not different coccidia oocysts is an indication that
(P > 0.05).

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ACKNOWLEDGEMENTS
The authors are grateful to the University of
Zimbabwe Research Board for financial
assistance, the Department of Paraclinical
Veterinary Studies University of Zimbabwe,
and Mr. Taruvinga for all the technical
assistance.

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Related Interests