STUDENTS NAMES REG NUMBERS

NYASHA NEDZIWE H200410A

SARAH TINAZVO H200781M
ZONDA MUDENDA H200633Q
PRECIOUS GARIRAI H200315M
TADIWANAHSE J MANDIGONA H200471Z
PASCAL CHINHAMO H200540P

DEGREE PROGRAMME : B TECH IN BIOTECHNOLOGY

COURSE : HIT 2001

SUPERVISOR. : MR NCUBE

LECTURER : MRS. CHIDZIYA

PROJECT PROPOSAL

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TOPIC: Formulation and Development of an Avian Colibacillosis Anticoccidial
using acemannan from Aloe Vera for Broiler Chickens.

CHAPTER 1

INTRODUCTION
1.1 BACKGROUND
Colibacillosis is an infectious disease caused by the bacterium Escherichia coli (E.
coli) and is seen in poultry flocks worldwide. E. coli is a gram negative facultative
anaerobic bacilli that are part of the normal intestinal microflora of poultry. Although
most E. coli are non-pathogenic, some strains are able to establish themselves outside
of the intestines and cause disease. These strains are known as Avian Pathogenic E.
coli (APEC). APEC is the major cause of colibacillosis in poultry production (Dahbi
et al, 2015). It is a common disease in poultry flocks worldwide especially in the
intensive farming system (Chansiripornchai et al, 2009). It affects birds of all ages.
This disease has an important economic impact on poultry production worldwide
(Blanco et al, 2012). It is one of the most common causes of mortality in commercial
layer and breeder chickens (Nolan et al, 2013). Signs in birds affected with
colibacillosis vary from sudden death to birds being off-colour with their necks pulled
into their bodies, respiratory distress, reduced appetite and poor growth. Lesions seen
at post mortem are air-sacculitis, pericarditis, perihepatitis and peritonitis. APEC
strains are the etiologic agents of colibacillosis in birds (Know et al, 2008). Treatment
of food-producing animals without conducting sensitivity test is a common practice
by non-veterinarians and because there are no strict regulations guiding the use of
antibiotics in food-producing animals in Masvingo, farmers often use different
antibiotics as growth promoters or prophylaxis in brooding their birds. This scenario
has led to selection pressure and development of multidrug resistance among E. coli
isolates. This, in turn, has resulted to the increased virulence of Escherichia coli and
consequently to high rate of treatment failures and great economic losses often
experienced by farmers (Oladele et al, 2013). Avian colibacillosis in chicken is
endemic in Masvingo. Intensification of poultry production and the quick expansion
of free-range production systems will increase the incidence of colibacillosis through
greater exposure of birds to pathogens and stress (Hossain et al, 2004). Environmental

2
influences, such as temperature, humidity, and high concentration of ammonia and
dust in poultry houses, also contribute to the respiratory stress of birds.

In recent years, pharmaceutical companies have spent a lot of time and money in
developing natural products extracted from plants, to produce more cost effective
remedies that are affordable to the population. The rising incidence in multidrug
resistance amongst pathogenic microbes has further necessitated the need to search
for newer antibiotic sources. This gave rise to the study of Etho Veterinary Medicines
(EMV). EMV also known as folklore medicine (Akinpelu and Onakoya, 2006),
encompasses the knowledge, skills, methods, practices and beliefs about animal
health care found among the members of a community. The increasing resistance to
common antibiotics by microorganisms has prompted the ongoing search for new
antimicrobial agents either by the design or synthesis of new agents or through the
search of natural sources for as yet undiscovered antimicrobial agents (Cock, 2008).
Much recent attention has focused on extracts and biologically active compounds
isolated from plants used in herbal medicine (Essawi and Srour, 2000; Alemda and
Agaoglu, 2009).

The inclusion of medicinal plants in alternative medicine has been increasing in our
society because of the therapeutic properties of plants. Aloe vera (A.barbadensis
Miller) is a cactus-like plant that grows in dry climates. Currently, because of
demand, it is cultivated in large quantities (Chantarawaratit et al 2014). Aloe vera is
used to produce two different products, each of which has a completely different
composition and therapeutic properties, A. vera gel and the bitter yellow llatex.
According to the International Aloe Scientific Council, aloe leaf can be processed into
two types of juices for commercial use: the inner leaf juice and decolorized whole leaf
juice. A. vera juice is approximately 99% water and the rest contains potentially 75
active constituents.(Reynolds and Dweck, 1999). Aloes have been used
therapeutically for centuries. Different properties are ascribed to the inner, colorless,
leaf gel and to the exudates from the outer layers. The reasons for aloe gel efficacy are
varied. Polysaccharides are one of the gel constituents to which activity has been
ascribed. These polysaccharides can be acetylated, partially acetylated or not
acetylated. Acemannan, β-(1, 4)-acetylated polymannose is the major polysaccharide
of A. vera gel. It has significant beneficial therapeutic effects such as immune-

3
stimulating, antineoplasic and wound-healing actions (Sehgal et al 2013). Acemannan
has been used for the management of wounds using protocols approved by the US
Food and Drug Administration (FDA) for the past decade. In addition, FDA approval
was recently obtained for the treatment of alveolar osteitis. This mini review gives an
overview of the present knowledge about the pharmacological potential of acemannan
and related problems.(King et al 1995).

There is considerable discrepancy in the literature as to the structure of acemannan.

Acemannan, a β-(1, 4)-linked polydispersed, highly acetylated mannan is found in
the inner leaf gel of the aloe plant, where it is produced by a specialized cells called
leucoplasts.

Figure 1: Structure of the mucopolysaccharide acemannan, extracted from Aloe vera.

Lobo et al (2010), called acemannan aloeverose. As a polysaccharide, acemannan is

composed of mannose, glucose and galactose monomers. The approximate
monosaccharide composition of acemannan is 31 β-(1, 4)-linked mannoses, 1 β-(1, 4)-
linked glucose, (Manna S, McAnalley BH. (1993)

The Aloe is an entirely African genus, occurring naturally in Africa and on some
islands off the coast (Baker and Linley, 1983). Over 150 Aloe species are found in
Southern Africa and over 30 species are known to occur in Zimbabwe (West, 1974).
The Aloe is one of the most commonly used herbs in Zimbabwean concoctions for the
treatment of both human and livestock diseases (Matekaire and Bwakura, 2004). Aloe
preparations are used for a wide range of applications in both human and animal ethno

4
medicine. In fact according to recent studies, the Aloe plant was found to be the most
commonly used herb in rural poultry management in Zimbabwe (Mwale et al, 2005).
In Zimbabwe, Aloes are used to treat such conditions as diarrhoea, burns and septic
wounds. In all cases, fresh leaves are used as a water infusion taken orally in the first
case or as a water extract that is used to folklore medicine where it is used as a basis
for medicinal washing of the wounds (Mwale et al, 2005).

Different Aloe sp. are used for different applications and differences on this aspect are
minor within communities and wider across communities. According to a study
carried out in western Kenya, Aloes were found to be the most commonly used herb
in communal poultry management, mostly in the treatment of a diarrhoeal infection
(Okitoi et al, 2007). In Trinidad and Tobago, Aloe sap is used to enhance productivity
in poultry (Lans, 2001). Aloe arborescens is used extensively in livestock ethno
medicine in South Africa. The sap is used to prevent illness in poultry (Jaarsvelcl,
2002). In the Westem world, Aloe preparations are used to treat wounds (including
ordinary and x ray burn wounds), flea bites, acne, constipation and to treat herpes
lesions (Atherton, 1997). In folklore veterinary medicine, Aloe chabaudii is used for
poultry which may be dipped in an infusion of the leaves to kill external parasites and
the leaf juice may be mixed with drinking water as a cure for poultry with blood in
their faeces. The aim of this study was to evaluate the antimicrobial activities or
effectiveness of acemannan from Aloe vera leaf extracts against E. coli causing Avian
colibacillosis.

1.2 PROBLEM STATEMENT

During avian post mortems carried out in most Zimbabwean poultry companies, a
relatively large proportion of birds examined are often found to be affected by
colibacillosis. This probably indicates that colibacillosis could be one of the leading
causes of avian mortalities in Masvingo. Farmers in Zimbabwe are experiencing great
losses due to death of broilers caused by Avian colibacillosis. Infected broilers
consume more feeds; stunted growth and extended growth period for example broilers
take approximately six weeks to be fully matured while infected broilers can take
more than six weeks therefore more feeds are required which may result in lot of

5
expenses.Aloe vera is one of the traditional medicines used to treat sick birds without
any specification on the disease being treated against and without a known
concentration of the medicine to be administered. The commercial drugs such
florouquine which is banned in some other countries including United States of
America (USA), Saponin, cephradine, chloramphenicol, sulphonamides, amino-
glycosides, B-lactam and tetracycline used to treat colibacillosis are much more
expensive and the bacteria E. coli is becoming more and more resistance to these
drugs thereby propose a research to study the anticoccidial activity of acemannan
from Aloe vera on avian colibacillosis treatment.

1.3 JUSTIFICATION

Commercial drugs to treat colibacillosis are much more expensive due to the current
economic hardships in Zimbabwe so there is a need to resort to a cheaper, less risks of
adverse effects, less resistance rates, readily available and environmentally friendly
medicine to treat colibacillosis. This study is an initial step looking into the potential
of acemannan from Aloe vera to treat avian colibacillosis. The use of acemannan to
treat colibacillosis might help farmers to maximise their profits through the
production of healthy birds, increased egg production, reduced mortality of birds. The
Aloe vera plant is readily available, can be grown with minimum maintenance
required and is easily prepared. Due to E. coli becoming increasingly resistant to
frequently used antibiotics such as third-generation cephalosporins and
fluoroquinolones (Dierikx, 2013), the use of acemannan could potentially help to
limit the increasing rate of drug resistance in the pathogens.

CHAPTER 2
LITERATURE REVIEW

Escherichia coli are a bacterium widespread in the intestine of animals and humans,

and a pathogen that can induce enteric and extra intestinal infections. In particular,
avian pathogenic E. coli (APEC) is the main cause of colibacillosis in the poultry
industry; a syndrome associated to airsacculitis, perihepatitis, pericarditis, and
sometimes fatal septicemia. APEC strains are responsible for the mortality of 3–4% of
the animals in a specific farm, and for the reduction of 2–3% of egg production

6
(Morley et al, 1984), resulting in an economic burden to the poultry industry.
Treatment of food-producing animals without conducting sensitivity test is a common
practice by non-veterinarians. Since there are no strict regulations guiding the use of
antibiotics in food-producing animals, farmers often use different type’s antibiotics as
growth promoters/prophylaxis in brooding their birds. This scenario has led to
selection pressure and development of equivalently high drug-resistance amongst the
E. coli isolates. This has resulted to the increased virulence of E. coli and
consequently to high rate of treatment failures and heavy economic losses often
experienced by farmers (Oladele et al, 2013). Intensification of poultry production
and the quick expansion of free-range production systems will increase the incidence
of colibacillosis through greater exposure of birds to pathogens and stress (Hossain et
al, 2004).

The successful treatment of avian colibacillosis caused by APEC strains mainly

depends on the use of antimicrobials. However, increasing resistance to critically
important antimicrobials, such as third-generation cephalosporins and
fluoroquinolones, is nowadays common in E. coli from poultry origin (Dierikx, 2013).
These resistances can be transmitted to humans via the food supply so there is a need
to use locally available and environmentally friendly earthomedicine to treat
Colibacillosis.

Plants remain the most common source of antimicrobial agents. Their usage as
traditional health remedies is most popular for 80% of world population in Asia and
all over the world and is reported to have minimal side effects (Bibitha et al.2002).
Ethno Veterinary Medicine (EMV) also known as folklore medicine (Akinpelu and
Onakoya, 2006), encompasses the knowledge, skills, methods, practices and beliefs
about animal health care found among the members of a community. The increasing
resistance to common antibiotics by microorganisms has prompted the ongoing search
for new antimicrobial agents either by the design or synthesis of new agents or
through the search of natural sources for as yet undiscovered antimicrobial agents
(Cock, 2008). Much recent attention has focused on extracts and biologically active
compounds isolated from plants used in herbal medicine (Essawi and Srour, 2000;
Alemda and Agaoglu, 2009).

7
Aloe vera belongs to Liliacea family which is a cactus-like plant that grows readily in
hot, dry climates as the gel consists of water. Aloe barbadensis miller and Aloe
aborescens are grown commercially as they are among the four of 250 species of Aloe
vera which are cultivated for their health properties. The parenchymatous cells, in the
fresh leaves of Aloe vera, secrete a colorless mucilaginous gel that contains 98-99%
water and 1-2% active compounds like Aloesin, Aloin, Aloe-mannan, Flavonoids,
Saponin, Sterols, Amino acids and Vitamins. Free Anthroquinones and their
derivatives like Barbaloin-IO-Aloe emodin-9 anthrone, Isobarbaloin and chromones in
Aloe vera leaves exert a strong purgative effect and are potent anti-microbial
agents (Vogler, 1994).

Aloe preparations are used for a wide range of applications in both human and animal
earthomedicine. In fact according to recent studies, the Aloe plant was found to be the
most commonly used herb in rural poultry management in Zimbabwe (Mwale et al,
2005). In Zimbabwe, Aloes has been used to treat such conditions as diarrhoea, burns
and septic wounds. In all cases, fresh leaves are used as a water infusion taken orally
in the first case or as a water extract that is used to folklore medicine where it is used
as a basis for medicinal washing of the wounds (Mwale et al., 2005). In Trinidad and
Tobago, Aloe sap was used to enhance productivity in poultry (Lans, 2001). Aloe
arborescens is used extensively in livestock ethno medicine in South Africa. The sap
was used to prevent illness in poultry (Jaarsvelcl, 2002). In folklore veterinary
medicine, Aloe chabaudii is used for poultry which may be dipped in an infusion of
the leaves to kill external parasites and the leaf juice may be mixed with drinking
water as a cure for poultry with blood in their faeces.

Aloe vera has potent antibacterial, antifungal, and antiviral properties. The
antibacterial activity of the extract was tested using Agar well diffusion technique. A
clear zone of inhibition was obtained against Escherichia coli, Enterococcus
faecalis and Staphylococcus aureus (Ramasubramanian et al, 2010). Antibacterial
activity of Aloe barbadensis Miller (Aloe Vera) was tested against bacterial
strains; Escherichia coli, Bacillus subtilius, Salmonella typhi, Pseudomonas,
Klebsiella pneumonia and Staphylococcus epidermidis. The methanolic extract of
Aloe vera resulted in the greatest antibacterial effect as other solvent extracts. Some

8
fractions were tested in which fraction 8 possessed greatest antibacterial actions
against all aforementioned bacterial strains (Saba et al. 2011) .

The antimicrobial activity of Aloe barbadensis, A. chabaudii and A. arborescens leaf

(methanol and water) extracts was investigated using the agar disc diffusion method
against a panel of bacteria and yeast. For A. barbadensis methanol extracts, C.
albicans and E. coli gave the largest inhibition zones thus were the most susceptible.
Cold water was the second best solvent after methanol, judging by the sizes of the
inhibition zones (Joshua et al, 2010). On this study cold water, hot water and
methanol extracts were used and zones of inhibition were observed and measured on
all the extracts for E. coli. Such activity against some Enterobacteriae e.g., E. coli is
probably behind the successful use of Aloe sap or extract in the treatment of livestock
gastrointestinal disorders. It was demonstrated in one study that Aloe preparations
added to the drinking water of chickens were useful in treating a diarrhoeal condition
with bloody droppings, thought to be colibacillosis (Okitoi et al, 2007). Colibacillosis
is an important disease of chickens caused by E. coli (Bames et al, 2003).

Aqueous, ethanol and acetone were used to extract the bioactive compounds from the
leaves of Aloe vera to screen the antimicrobial action selected human clinical
pathogens by agar diffusion technique. The greatest antibacterial actions were
observed in acetone extracts other than ethanol extracts and aqueous extracts (Kumar
et al, 2009). Antibacterial action of A. Vera was tested against Staphylococcus aureus,
Streptococcus pyogenes, Pseudomonas aeruginosa and E. coli. A. vera leaf gel can
restrain the development of two gram positive microscopic organisms Shigella
flexneri and Streptococcus pyogenes. Clear zones of inhibition were clearly seen in
the gram positive bacteria rather than gram negative bacteria (Mariappan et al, 2012).

With respect to all studies that have been done on Aloe vera by other researchers, this
study is more focused on finding the potential of acemannan in treating Avian
colibacillosis in poultry as farmers in Masvingo uses the basics and nature of the
plants properties, and to identify the MIC of the acemannan to be administered to
birds affected by colibacillosis.

CHAPTER 3

9
AIM AND OBJECTIVES
AIM
To formulate, develop and validate an avian colibacillosis anticoccidial from
acemannan for broiler chickens

OBJECTIVES
 To extract acemannan from Aloe vera
 To formulate Anticoccidial using acemannan extracted from Aloe vera
 To determine the effectiveness of the antibacterial activity of the Anticoccidial
against Escherichia coli.

CHAPTER 4

RESEARCH HYPOTHESIS

H0: Anticoccidial is not effective in treating Avian colibacillosis

H1: Anticoccidial is effective in treating Avian colibacillosis

CHAPTER 5

METHODOLOGY

Collection of samples
A total of 50 samples were collected, randomly selected from dead birds that were
brought to Masvingo Provincial laboratory for post mortems

Isolation of E. coli from affected Birds

The whitish material resembling colibacillosis infection from the upper surface of the
liver, lung and heart of different affected birds is going to be swabbed with sterile
swabs. The swabbed bacteria will then cultured in MacConkey and Blood agar and
incubated overnight at 37°C. Gram staining, citrate testing, motility testing and indole
testing will be performed on the resulting colonies.

10
Preparation of Acemannan
Fresh Aloe vera leaves will be collected from Mbare, Harare, Zimbabwe. Mabel
Alonso et al.(2012) studied the application of four processes for obtaining acemannan;
size exclusion chromatography (SEC) using Sepharose CL-4B matrix followed by
ethanolic precipitation; size exclusion chromatography, ultrafiltration using hollow-
fiber cartridges and ethanol precipitation and size exclusion chromatography,
precipitation with cetyltrimethylammonium bromide (CTAB) and ethanolic
precipitation, and direct precipitation with CTAB and ethanolic precipitation. For the
extraction of the active substance a collection of 100 plant leaves will be used,
washed, decontaminated, and the pulp gel will be homogenized in a domestic blender.
Ethanol will be added slowly, and the suspension will be left with slow agitation and
then centrifuged. Acemannan is extracted from fresh leaves. First, the skin is washed
and removed from the leaves and the parenchyma is soaked for 30 minutes. Then, the
parenchyma gels are blended using a homogenizer and centrifuged. The supernatant is
collected and mixed with absolute ethanol at a 1:3ratio and the precipitated white
opaque particles are collected before further centrifugation and lyophilization.

Acemannan disk preparation

100 of 6mm diameter filter paper disks will be cut using a puncher and each of the 25
disks will be impregnated with 20 microlitres of different concentration of acemannan
. The impregnated filter paper disks will be left to dry at room temperature for 3
hours.

Control disk preparation

50 filter paper disks with a diameter of 6mm will be cut using a puncher. A pipet will
be used to inject 20 microlitres of distilled water into a filter paper disk to make a
negative control disks. 20 micro litres of Aliseryl diluted as per manufacturer’s
instructions will be injected in each of the 25 filter paper disks to make positive
controls. The filter paper disks will be left to dry at room temperature for 3 hours in
petri dishes.

Procedure

11
The pathogenic E. coli from the infected birds from post mortem findings will be
isolated. The isolated Pathogenic E. coli will then cultured in Blood and MacConkey
Agar. Gram staining, motility testing, citrate testing and indole testing will be
performed on the resulting colonies for confirmatory reasons. Spread plate streaking
of the required pure E. coli will be performed on blood agar plates. A marker will be
used to divide the petri dish into six compartments. Sterile forceps is then used to
place control disks and different concentration of acemannan disks into the marked
compartments in the streaked blood agar and incubated the inverted culture plates at
37oC for 18 to 24 hours. After Incubation the zones of inhibition on each and every
disk is going to be checked, measured and recorded in millimetres.

Determination of the Minimum inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) is the concentration giving the least
inhibitory activity and below which there is no further inhibition. It is therefore
regarded as the concentration giving the lowest possible zones of inhibition. The MIC
acemannan from Aloe vera will be obtained by dissolving a constant volume of the
extracts in various volume of distilled water. 0.2g of acemannan extract will be
dissolved in 2ml, 4ml, 16ml, 32ml of distilled water to obtain 1mg/ml, 0.5mg/ml,
0.25mg/ml, 0.125mg/ml and 0.0625mg/ml respectively. 0.2ml of the dilutions
obtained 1mg/ml-0.0625mg/ml will be introduced into different wells made. After 24
hours of incubation, the results of each concentration is going to be measured .

Sample size
50 samples of E. coli will be collected from avian birds brought for post mortems.
The E. coli is going to be isolated and differentiated by first swabbing the infected
area, culturing the sample, performing gram staining, indole test, motility testing and
citrate test following the Laboratory Standard Operating procedures at Masvingo
Provincial Veterinary services. Sensitivity testing will be done using the Kirby disk
diffusion method and Aliseryl imbedded in a filter paper disk will be a positive
control and distilled water imbedded in a filter paper disk will be the negative control
whilst the acemannan different concentrated disks will be the test disks.

Study type and Variables

12
A simple random design where a sample will be chosen randomly and entirely by
chance will be used in this study for accurate and efficient analysis. All the
experiments and tests will be carried out in a laboratory where all other factors such
as temperature, humidity will be taken care of. The independent variable will be the
acemannan used whilst the dependent variables will be the results obtained due to the
effectiveness of acemannan on avian colibacillosis.

Data Analysis
Microsoft excel is going to be used to calculate the average diameter of the zones of
inhibition for each Aloe vera concentrate. The results will be categorised as sensitive
or resistant which is either effective (ZOI ≥ 7mm) or not effective (ZOI < 7mm)
according to their average diameter of zone of inhibition.

CHAPTER 6

EXPECTED RESULTS
-Successful extraction of acemannan from aloe vera
-Successful formulation of anticoccidial using the extracted acemannan
-Successful determination of the effectiveness of the antibacterial activity of the
anticoccidial against Escherichia coli.

CHAPTER 7

BUDGET
Objective Materials Quantity Cost ($)
1`
 Fresh Aloe Vera leaves 100 10

2 Normal saline 2litre 5

Micropipette 2 2
Sodium hypochlorite 1litre 3
3
4  Swabs 1 roll 5

13
 1 litre
 Mackonkey broth 100g 5
 Agar 6 7
 Agar plates 6

CHAPTER 8

TIMEFRAME

DECEMBE JANUAR FEBRUAR MARC APRI MA

R Y Y H L Y
EXTRACTION
OF
ACEMANNAN

FORMULATIO
N OF THE
ANTICOCCIDI
AL
TESTING THE
EFFECTIVENE

14
SS
DATA
ANALYSIS

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