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Isolation of Aerobic Bacteria Associated with
Fistulous Withers in Donkeys
By
Shereen Farog Mostafa
University of Khartoum
B.V.M. 2004
Superviser
Dr. Awad Elkarim Abd Elghaffar Ibrahim
A thesis submitted to the University of Khartoum in partial
fulfillment of the requirements For the Degree of M. Sc.
Department of Microbiology
Faculty of Veterinary Medicine
University of Khartoum
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INTRODUCTION
The domestic donkey traces its ancestry to the wild asses found
in Egypt, the Sudan, Somalia and Ethiopia (Dreyfus, 1976). Packing is
one of the most ancient forms of transport which preceded even the
invention of the wheel. That it has survived to the present day serves to
emphasize its value. The major advantage of pack transport is its
effectiveness in the absence of roads. It is particularly effective where
access is limited ,this applies not only to remote mountainous areas but
also to areas of high population density as in city centres. The steady
speed of a donkey’s walk is what makes it so popular as a pack animal or
for pulling small carts. Donkeys are usually cheap compared to other
draught animals (Clark , 1974). Donkeys are widely used in both rural
and urban areas of the Sudan for transporting a wide variety of items
such as grain, construction materials, goods and animal feed (Wylam ,
1991). Such transport is an important stimulus to trading and also
represents a source of income for the donkey owners. A large part of the
people and of the economy of Omdurman depends on donkey transport
for the movement of goods from wholesale centers to retail outlets and
households. The service is cheap, flexible and readily accessible in most
parts of the city. It is also an essential source of livelihood and income
to many households. Housing for donkeys is rarely constructed as, in
most cases, they are simply tied to a pole inside the owner’s compound.
Most of the donkeys used in the city are imported from other places. In
most countries breast band harnesses are used for donkeys pulling carts
or doing field work. The most common causes of saddle sores on
donkeys are by and large incorrectly fitting saddles and harness
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(Pritchard, 2005). This material causes friction, rubs, sweat and
overheating problems. This leaves the animal more prone to withers
galls, open back sores and rubs. Conformation faults are often at the root
of the problem. If your horse has low withers, or in the other extreme is
high withered, or perhaps is a narrowly built horse fitting the saddle can
be difficult. A poorly fitted saddle will either put pressure on the donkey
where it shouldn't be, or it will slide around, causing friction .
Fistulous withers (supraspinous bursitis) is an inflammatory
conditions of supraspinous bursa of equine . The bursa is located over
the second to fifth thoracic spine of equine horses and donkeys. In the
early stage of the disease, a fistula is not present . In more chronic,
advanced cases, the ligament and the dorsal vertebral spines are affected,
and occasionally these structures undergo necroses . When the bursal sac
ruptures or when it is opened for surgical drainage, and secondary
infection with pyogenic bacteria occurs, it usually assumes a true
fistulous character.
The number of donkeys in Sudan is about 7522690 given by
(Statistical bulletin for animal resources , 2011). In Sudan, donkeys are
mostly used for transport. They pull carts and carry goods and
characterized by their body conformation and working speed into three
breeds: Mekadi, Atbawi and Reef. Little information is available about
donkey type and distribution in Sudan. Their origins are Eddamer and
Atbara in the north and Jalabi from Gazira in central Sudan. The scope
of the present study is limited to the donkey pack-transport of goods
within the city boundary .The main objectives were as follows:
1-To identify and quantify factors associated with the presence of pack
wounds in donkeys working in the transport of goods.
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2-To isolate and identify aerobic bacteria associated with fistulous
wither and other saddle and harness wounds.
3-Assessment of the welfare of working donkeys, using health and
behavior parameters.
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CHAPTER ONE
1.
LITERATURE REVIEW
1.1 Donkey
1.1.1 Donkey population and history
Donkeys Equus asinus are domestic animals falling under the
equine family, which includes horses, zebras and the mules (Wilson,
1990). There are 44.3 million donkeys or Ass (Equus asinus) worldwide.
This number has increased by 15.6 per cent since 1981 (FAO, 1992). A
small number are kept in the western world as pets. However over 95 per
cent live in the developing world where they are kept mainly for work.
The precise wild progenitor of the domestic donkey is disputed. Bökönyi
(1991) argued that domestication took place in Egypt and Clutton-Brock
(1992) notes that the skeletons of three domestic donkeys have been
found in an Egyptian tomb dated to 4500–4000 BC. There are
comparably early skeletons in the Near East but, whether these are
domestic remains uncertain (Eisenmann, 1995).The two recognised races
of the ass are: Equus asinus africanus and Equus asinus somaliensis.
The Nubian wild ass is one subspecies of the wild ass, Equus africanus
which cited in many textbooks as the progenitor of the donkey, although
other races may also have contributed to the gene-pool (Epstein, 1984).
The dominant color is the mousy grey, although the whole range from
black to white are generally rare. The average weight of donkeys (both
males and females),was approximately 105 kg (Wilson,1991). Donkeys
reach maturity around four years of age .Breeding age for female
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donkeys is 4–5 years (Feseha and Yoseph, 1996). Both males (intact
males are called jacks and castrated males are geldings) and females
(jennies) can be used for work. With good care, donkeys can have a
working life of 12-15 years, and they can live even longer. Castration
will help to improve the temperament and reliability of males. However,
good jacks are important for breeding, and farmers may be able to obtain
fees for allowing their jacks to breed. Donkeys are more common in
Africa than horses with an exception of Ethiopia and Morocco. Egypt,
Nigeria, Mali, Niger and Sudan provide examples of the numerical
dominance of donkeys over horses (Payne and Wilson, 1999).
Equids used to transport people by cart, to carry goods by pack,
or to work in bricks kilns. Rural equids had more problems than urban
ones, but urban equids had more lesions. Equids were significantly
thinner when climates were warmer. These results should aid the
development and targeting of specific welfare interventions.
1.1.2. The place of donkeys in society
A number of factors help to explain why donkeys have low status.
They are usually the cheapest, often the only affordable, work animal
and therefore tend to be associated with the poor. If donkeys are not
available, women often have to do the same work (Mohammed, 1991).
In contrast with cattle, buffalo and camels which are usually kept for
their milk and meat as well as work, whose hides are cured for leather,
and whose dung even has a number of uses (Pearson, 1992), donkey
bye-products are not generally used.
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Map 1: Estimates of donkey populations in Africa and the Arabian
peninsula in 1996. The figures show the estimated donkey population
of each country in thousands. The intensity of shading gives an
approximate visual indication of the numbers of donkeys in the
different countries. Authors’ estimates are shown in italics. (FAO,
1997).
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1.1.3. General donkey features and Behavior
Donkeys are often very inexpensive and have little, or no,
disposal value. Although they have sometimes been considered as
animals of ridicule or low status, they have excellent reputations as
easily trainable and very dependable work animals.
Draught animals were used as pack animals for goods and people.
In some cases they are the only source of transport. Animals driven
vehicles are an important part of the transport network and were prime
source of transport used by farmers to bring their agro-products to
market and take back their necessities from cities. Such types of vehicles
are not only being used in villages but are still on the roads in big cities
and are contributing a lot in the saving of petrol energy. donkeys are
used as singles or in teams depending on the load and the operations
performed.
Cattle are hardly used at all for functions other than cultivation
except for threshing which is done simply by walking round on top of a
heap of crop or by dragging a heavy stone or wooden baulk. Donkeys
are the only other species used in this function either loosely attached to
each other in pairs or in multiples of up to six animals.
Cart transport is much less common than in the past but the
importance of this function in urban areas does vary. It is more common
in the rural areas of the country . Although the market is the main centre
for the donkey pack-transport service most donkey operators reside in
the peripheral parts of the city. The majority of the donkey operators are
able-bodied males who perform the loading and unloading tasks by
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themselves . The donkey pack-transport business, like many other
informal activities, has little restriction to entry.
Table 1: Main advantages and disadvantages of using donkeys
(Jones,1997).
Advantages
Disadvantages
Friendly towards humans
Willing to work
Can turn in small space
Easy to train
Need little supervision in work
Can utilize poor feed well
Not affected much by external
parasites
Need little water
Can survive droughts better than
cattle
Can survive well in tsetse-infested
areas
Comparatively cheap to buy
Strong relative to size
Live and work many years in good
care
Useful for calming and guarding other
kinds of animal
Fast walking speed
Suffer from being alone
Noisy when frustrated or lonely
Friends not easily separated
Uncastrated males aggressive
towards other donkeys
Skin easily wounded
Wander long distance if not
supervised
Do not move out of the way of
traffic
Need shelter from cold and
damp
Meat not generally eaten
Comparatively small in size
Mature slowly
Breed slowly
Manure more fibrous than
nutrient-rich
1.1.4. Selection characteristics of a working donkey
When
selecting
an
animal
for
work
certain
physical
characteristics should be observed. These include: a large frame with
wide shoulders and a deep chest, a straight back and well-muscled
straight legs which have a 90° angle to the ground . The donkey should
have good eyesight and agility and an attractive hair coat, without skin
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diseases or an abundance of ticks. It is important to observe an animal
while it is working, to detect whether it has a physical disability, such as
coughing, poor breathing, lameness, sores or wounds.
1.1.5. Economic importance
The donkey is one of the man oldest domesticated animals.
Donkeys therefore contribute to economic development as they are used
in every-day life. They are even used more frequently than cattle and
horses and have taken the work that was in the past done by cattle,
horses and machinery such as tractors (Starkey, 1994). Most farmers are
able to keep donkeys because they do not require technical husbandry
practices. They are tolerant to some tropical diseases and parasites.
Donkeys are therefore easy to manage and not too demanding in terms
of feeding. They can almost survive on poor quality feeds and they can
tolerate a considerable heat and dehydration. (Aganga et al., 2000). Most
traditional households use their donkeys for transport, fetching water and
for gathering firewood . This is shown by the wide spread use of
donkeys in urban and rural areas in Africa, as well as parts of the Central
America and Asia (Aganga et al., 1994). The major cost of starting a
donkey pack-transport business is investment in donkeys. Other start-up
costs include straps , packsaddle and whip .The donkeys are usually
overloaded and suffer from wounds related to overwork.
Therefore it would be a good idea for the country to try to
improve the poor animals social standing or social position. Donkeys are
multipurpose, as they not only provide transport, but also milk to
children as a medicine against whooping cough.
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1.1.6. Fitting Equipment
Harnesses and saddles are usually not fitted properly on the
donkeys. This causes terrible discomfort and deep wounds that are
usually left untreated, since the donkeys are forced to continue working.
The face, back, neck, hind legs and under the legs are the areas that are
most subjected to deep-seated wounds that can often lead to infection.
Breast band harnesses this kind of harness is cheap, and easy to
manufacture locally were, however, originally constructed for horses
which have a wide and muscular chest, providing enough space for a
breast band. This is not the case with donkeys. The breast band on the
donkey rests on the shoulder joint and will either have the tendency to
glide up, pressing onto the trachea (windpipe), or down getting onto the
animal’s forearm. In addition, the donkeys chest being less muscular
than the horse’s, increases the risk of bruises. Mostly donkeys were
harnessed with homemade breast band harnesses of poor quality .This is
not an unusual sight in many parts of Omdurman market places . Donkey
users preferred the local round collar harness (traditional) to the threepad collar (improved). The general perception of most farmers on
improving harnessing systems underscored the use of quality materials
to ensure efficient draught power supply.
Saddles sores caused by poor saddle fit usually show up around
the withers, shoulder, or back under the cantle. Sometimes saddle sores
are caused by a dirty saddle pad or a saddle cinched up with the hair not
laying flat underneath. Both of these problems can be big enough
irritants to cause open sores or wounds within the space of a short time
of work. Saddle sores can be caused by girths and cinches being too
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tight, too loose, too rough, too dirty, or skin being bunched underneath
them- also called girth galls.
Fig 1. The arrangement off the breast band, breech strap and saddle for
harnessing a donkey to the shafts of a two-wheeled cart (Pritchard, 2005).
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1.1.7. General causes of injuries
Donkey welfare will be best if donkeys pull well-balanced carts with good,
lubricated wheels. They also need good, simple harnesses. This makes pulling easier and
the animals stay in good condition. Skin injuries to the back, shoulders or other parts of
the body due to saddles, harnesses or collars are brought about by combinations of friction
and pressure. If a donkey with a saddle sore must be worked the pressure and friction on
the sore must be reduced or preferably eliminated. The ordinary neck collar oscillates
from side to side while the animal walks. The breast collar doesn’t oscillate, but has a
sawing action. Sawing is obviously a greater source of friction than oscillation, and in
consequence the make and fit of the breast collar must be such as to present a perfectly
smooth surface next to the donkey’s skin. Neglect of this precaution is soon evident as any
small projection in the harness is capable of doing great harm. Harness materials should
be from the local environment.
Injuries often occur when insufficient attention has been paid to equipment. Many
owners consider that the weight of a donkey’s body is equally distributed over its limbs.
This is not true, the fore limbs carry more of the body weight than the hind, and the
amount which they carry is influenced by the position of the head, which, if held high,
relieves the forelegs from weight, and if depressed increases the weight. In regular use the
saddle requires attention every day. Saddles should be inspected almost as regularly as the
feet. Each weak point in the harness should be known and repaired before trouble occurs
(Fahmy, 1992).
1.1.8. Wounds
Wounds are often a result of a direct injury, ill fitting tack, harness or hobbles or
being attacked by other animals. Some wounds or injuries may need veterinary attention.
Others may simply need to be cleaned. If a wound or injury affects the donkey’s ability to
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work, it will ultimately affect the livelihood of the owner as well as the welfare of the
donkey. If treatment is administered quickly and flies are kept away from the wound, the
risk of infection is minimized.
Fig 2. Donkey was repeatedly whipped on an open wound, that led
to deep infection (Omdurman Market).
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1.2. Fistulous withers
1.2.1. Definition of fistulous withers
Fistulous withers is a chronic inflammatory disease of the
supraspinotus bursa and associated tissues (Gaughan et al., 1988;
Rashmir-Raven et al., 1990;Cohen et al., 1992). The supraspinous bursa
is located between the funicular portion of the nuchal ligament and the
dorsal spinous processes of the second to fifth thoracic vertebrae
(Hawkins and Fessler 2000). The bursa is approximately 5 cm wide and
ranges in length from 5–11 cm and has a capacity in the normal horse of
30–90 ml. Although infection by Brucella abortus has been associated
with the condition (Duff, 1937). Other infectious organisms and trauma
can also cause the disease. Indeed organisms commonly isolated from
clinical cases in geographical areas with a low prevalence of brucellosis
in cattle, B. abortus is rarely isolated from fistulous withers cases
(Gaughan et al., 1988; Cohen et al., 1992). Infection by multiple bacteria
is often Present. Other than B. abortus, include Streptococcus
zooepidemicus
,Streptococcus
equi,
Staphylococcus
aureus,
Staphylococcus epidermidis, Proteus mirablis, Actinomyces bovis,
Bacteroides
fragilis,
Escherichia
coli,
Pasteurella
spp.
and
Corynebacterium spp. (Guard 1932; Gaughan et al., 1988; Cohen et al.,
1992; Hawkins and Fessler 2000). Infection by Onchocerca cervicalis
has also been incriminated in some cases (Lyons et al., 1988; RashmirRaven et al., 1990; Hawkins and Fessler 2000).
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Fig. 3: Names of the different body parts of a donkey.
1.2.2. Clinical signs
Clinical signs of supraspinous bursitis (fistulous withers) include
singular or multiple draining tracts or diffuse swelling of the withers
without drainage. The onset of clinical signs may be abrupt or insidious.
Early signs include localized heat, pain and swelling of the bursa. There
may be lethargy and general stiffness. In most cases the bursa ruptures
and purulent exudates is discharged from one or more fistulae. These
fistulae may heal over, but may subsequently reform .Extensive fibrosis
may occur. However, horses with fistulous withers that are seropositive
to B. abortus are significantly more likely than seronegative horses with
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fistulous withers to have radiographic evidence of osteomyelitis of the
underlying dorsal spinous processes (Cohen et al., 1992).
Poll evil (septic supra-atlantal bursitis) causes similar clinical
signs to fistulous withers in the poll region. There is frequently pain and
neck stiffness. Swelling of the region occurs which may be followed by
discharge of purulent material.
1.2.3. Etiological agent of fistulous withers
1.2.3.1. Brucella abortus
Bacteria of the genus Brucella are nonmotile, aerobic,
intracellular Gram-negative cocci, cocco-bacilli or short rods. Brucella
spp. are transmissible to a wide range of species and among the
domesticated animals (Godfroid et al., 2004). B. bortus infections in
domestic animals have been reported worldwide, There is no apparent
age, gender or breed predisposition to infection in horses, although most
cases have been reported in horses aged >3 years and equine infections
usually involve the cattle pathogen B. abortus (Nicoletti, 2007). Horses
usually become infected by the ingestion of B. abortus-contaminated
feed, and most reported cases indicate a history of contact with cattle
(Duff 1937; McCaughey and Kerr 1967; Denny 1973; O’Sullivan 1981;
Ocholi et al. 2004).
Serological surveys in endemic areas indicate that many horses
can be exposed and infected by B. abortus without any clinical signs of
disease (Denny 1973; Dawson and Durrant 1975; Nicoletti et al., 1982;
MacMillan and Cockrem 1986). The commonest clinical diseases
associated with Brucella spp. infection in horses are septic supraspinotus
bursitis (fistulous withers) and septic supra atlantal bursitis (poll evil).
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Duff (1937) reported 85 cases of fistulous withers and identified B.
abortus infection in 80%.
1.2.3.2. Staphylococcus
Bacteria of the genus Staphylococcus are strongly Gram-positive
cocci (0.5– 1.5 min diameter) that occur singly, in pairs, tetrads, short
chains ,spherical bacteria that occur in microscopic clusters resembling
grapes (Todar, 2011). Staphylococci grow in clusters because of the
special division way of Staphylococci, the cells dividing in both three
dimensional axis and the new cells remaining attached to each other
followed by each division successively. Since there is no exact point of
division, the result is to form an irregular cells cluster (Pinho and
Errington, 2003). Colonies are usually large (6-8 mm in diameter),
smooth, and translucent. The colonies of most strains are pigmented,
ranging from cream-yellow to orange. Staphylococci are nonmotile,
non–spore-forming, catalase-positive and oxidase-negative bacteria.
Staphylococci are facultative anaerobes that grow by aerobic respiration
or by fermentation that yields principally lactic acid.
Typical animal strains of S. aureus are important pathogens in
veterinary medicine, causing disease in horses, the bacterium may cause
dermatitis and cellulitis. Staphylococcus aureus is the most common
species of staphylococci to cause Staph. infections. One of the reasons
for this is a carotenoid pigment staphyloxanthin that is responsible for
the characteristic golden color of S. aureus colonies. This pigment acts
as a virulence factor, with an antioxidant action that helps the microbe
evade death by reactive oxygen species used by the host immune
system(Clauditz et al ., 2006; Liu GY et al., 2005 ). On the basis of the
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coagulase test, the genus Staphylococcus was originally divided into the
coagulase-positive
species
S.
aureus
and
coagulase-negative
staphylococci.
1.2.3.2.1. Bacterial virulence factor
Several potential virulence factors have been described as
important in staphylococcal infections. Most of these factors have been
studied in S. aureus ,but some of them have also been found in other
Staphylococcus species. The virulence factors can be divided into cell associated components, exoenzymes
and exotoxins. The known
virulence factors of staphylococci are described as follows :-
1.2.3.2.2. Cell - Associated Components
Which include:
- Protein A: is a surface protein of S. aureus that binds IgG
molecules by their Fc region. In serum, the bacteria bind IgG molecules
in the wrong orientation, which disrupts opsonization and phagocytosis.
- Capsular Polysaccharide: These capsular polysaccharides have
been proposed to interfere with host defense mechanisms by inhibiting
the attachment of antibodies. They have also been described to bind to
epithelial and endothelial cells and to monocytes, and they induce the
release of cytokines.
- Adhesins : Staphylococcal bacteria may express proteins on
their surface that promote attachment to host proteins ,such as
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fibronectin, laminin and collagen which important in promoting bacterial
attachment to damaged tissue .
1.2.3.2.3. Exoenzymes
Coagulase is an extracellular protein that binds to prothrombin in
the host to form a complex called staphylothrombin. The protease
activity is activated in the complex, leading to conversion of fibrinogen
to fibrin . Probably, the bacteria protect themselves from phagocytic and
immune defenses by causing localized clotting.
1.2.3.2.4. Exotoxins
Staphylococcus aureus can express several different types of
protein toxins which are probably responsible for symptoms during
infections:
- Superantigens: S. aureus secretes two different toxins that have
Superantigens activity. The secreted Superantigens stimulate the nonspecific activity of T cell without a normal antigenic recognition.
Cytokines are released in a large amount and lead to disease symptoms
(Lowy, 1998). One is enterotoxin which can cause food poisoning. A
Second superantigens that S. aureus produced is toxic shock syndronme
toxin (TSST-1), which is the cause of toxic shock syndrome (TSS)
(Johnson et al., 1991).
1.2.3.2.5. Membrane-damaging toxins
Which include: Alpha toxin (alpha-hemolysin), Beta – toxin,
Delta – toxin and Gamma – toxin.
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1.2.3.3. Streptococcus
Streptococci are Gram-positive spherical bacteria less than 2mm
that typically grow by cell division in one plane, so that nascent cells
form a linear array. Most are facultatively anaerobic and catalase
negative. Identification is based on colony characteristics; hemolytic
properties; fermentation and other biochemical reactions. The majority
of pathogenic streptococci possess a serologically active carbohydrate
antigenically different from one species or group of species to another.
Some pathogenic streptococci, are identified by features such as
fermentation behavior, ability to grow at different temperatures, salt
tolerance, optochin sensitivity, bile solubility.
1.2.3.3.1 Virulence factors
In general ,streptococcal virulence is based on surface and
secreted proteins and on structures that directly or indirectly impede
phagocytosis, are involved in adhesion and carbohydrate metabolism, or
induce release of pro - inflammatory cytokines. The best understood
streptococcal virulence factors are the hyaluronic acid capsule, the
antiphagocytic M proteins ,and the pyrogenic exotoxins. However, other
molecules
including
streptolysins,
proteases,
leukocidal
toxins,
plasminogen activators (streptokinase), and possibly plasmin receptors
found on the surface or secreted, also contribute to pathogenicity. In
addition, most pathogenic streptococci have the ability to bind
components of the host’s plasma, such as albumin, immunoglobulins,
fibrinogen, and to bind to fibronectin, lamini and other components of
the host cell. Organisms coated with one or more of these plasma
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components may be able to evade host defenses either by escaping
detection or by blocking deposition of opsonic components of
complements .
1.2.3.3.2. Streptococcal toxins
Few of the cellular components of group A streptococci appear to
be directly toxic for animals or humans. Cell wall fragments produce a
chronic Multi nodular inflammatory lesion of dermal connective tissue.
The peptidoglycan component of cell walls has many of the biologic
features of endotoxins. The exotoxins of group A streptococci include
the erythrogenic toxins (pyrogenic exotoxins) and the cytolytic toxins
(streptolysins S and O). The pyrogenic exotoxins are associated with the
enhancement of endotoxin shock and a wide variety of other biologic
properties. Streptolysin S is a non antigenic polypeptide associated with
various stabilizing carrier molecules. It lyses a wide range of mammalian
cells, influences T lymphocyte functions, and is probably responsible for
the leukotoxic property of group A streptococci. Streptolysin O is an
oxygen-labile (thiol-activated) cytolysin. It is inhibited by non esterified
cholesterol and binds to cholesterol in the membranes of mammalian
cells and organelles, an interaction producing ring-like and C-shaped
structures demonstrable by electron microscopy. Streptolysin O affects a
number
of
leukocyte
functions.
It
produces
profound
electrocardiographic changes in experimental animals and toxic effects
on pulsating heart cells in tissue culture (Wannamaker,1983).
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1.2.3.3.3. Streptococcus equi
Strangles is an acute disease of horses caused by infection with
Streptococcus equi. It is characterized by inflammation of the upper
respiratory tract and abscessation in the adjacent lymph nodes. The
source of infection of strangles is the nasal discharge from infected
animals which contaminate the feed and water. The disease is worldwide in distribution. In Sudan it is reported in the list of animal diseases
in Statistical Bulletin for Animal Recourses (2000).
1.2.3.4. Corynebacterium
Corynebacterium species are non motile, non sporulating, short,
pleomorphic, gram - positive rods with a high G + C content in DNA.
They belong to the class Actinobacteria and are part of the larger
Corynebacterium , Mycobacterium , Nocardia (CMN) grouping.
Bacteria of this group are characterized by cell walls containing long –
chain fatty acids called mycolic acids; corynebacterial mycolic acids
have relatively short chains, typically of 28 – 40 carbons.
Corynebacterium species are found in a wide range of ecological niches,
such as soil ,sewage, and plant surfaces, and some are important
pathogens of humans or animals. Use of partial gene sequences from the
RNA polymerase subunit - encoding gene has also proved useful in
identification and classification of Corynebacterium species.
1.2.3.4.1. Virulence factor
Clear understanding of virulence of this organism, leading to
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effective, easily delivered means of prevention, will have major impact
on many nations but especially on the developing world.
Cell wall lipids of C. pseudotuberculosis have been considered
virulence factors since their cytotoxicity was demonstrated more than 50
years ago (Carne etal.,1956). They protect the bacteria from intra
phagocytic destruction but also induce hemorrhagic necrosis and chronic
abscessation (Jolly, 1966; Hard, 1975; Tashjian and Campbell, 1983).
An extracellular protein, renalin production by most isolates of
C. renale This protein lyses erythrocytes in synergy with S. aureus
toxin. The staphylococcal toxin apparently hydrolyzes sphingomyelin,
producing ceramide, and then renalin interacts non enzymatically with
ceramide to lyse cells (Bernheimer and Avigad, 1982).
Others virulence factors, including tissue - damaging toxins and
enzymes and attachment and colonization factors. It can cause a variety
of diseases in multiple species ,but there is no indication that particular
virulence factors are associated with specific disease.
1.2.3.5. Actinomycetes
Actinomycetes
appear
as
Gram-positive
bacilli
(Hall,
2006),which comprise a group of branching unicellular microorganisms.
They produce branching mycelium which may be of two kinds substrate
mycelium
and
aerial
mycelium.
Among
actinomycetes,
the
streptomycetes are the dominant. The non-streptomycetes are called
actinomycetes, comprising approximately 100 genera .Members of the
actinomycetes, which live in marine environment, are poorly understood
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and only few reports are available (Siva, 2001;Vikineswari et al., 1997;
Rathana
and
Chandrika,
1993;
Lakshmanaperumalsamy,
1978).
actinomyces species require enriched culture and growth is enhanced by
an atmosphere with 6-10% added carbon dioxide. The optimum growth
temperature is 37°C. Colonies may appear after 3 - 7 days of incubation.
Colonies are described often as ‘breadcrumb’ colonies. The reason for
different sensitivity between gram-positive and gram-negative bacteria
could
be
described
to
the
morphological
differences
these
microorganisms, gram-negative bacteria having an outer polysaccharide
membrane carrying the structural lipopolysaccharide components. This
makes the cell wall impermeable to lipophilic solutes, the gram positive
should more susceptible having only an outer peptidoglycan layer which
is not an effective permeability barrier.
1.2.3.5.1 Virulence factor
Actinomyces pyogenes, a gram-positive, normally commensal
bacterium, resides on the mucous membranes of cattle, sheep, swine, and
other economically important animals (Carter et al., 1991).Despite the
versatility of A. pyogenes as an agent of disease in domestic animals .
Actinomycetes pyogenes produces virulence factors (pyolysin [PLO],
collagen binding protein, neuraminidase, and DNase) are ubiquitous
among isolates . The hemolytic exotoxin pyolysin (PLO) (Ding and
La¨mmler, 1996), which is cytolytic for the erythrocytes of a number of
animal species (Funk et al., 1996) , as well as dermonecrotic and lethal
for laboratory animals (Lovell, 1944). The role of this toxin in
pathogenesisis unclear. However, it is expressed in vivo and is
immunogenic, as anti hemolysin antibodies have been found in the sera
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of naturally (Lovell, 1939) and experimentally ( Matthews et al., 1963) .
Cytolysin produced by a number of gram-positive bacteria (Alouf ,
1980; Boulnois et al., 1991; Cossart et al., 1989) and its potential role
in virulence. Also Actinomyces pyogenes produces neuraminidase which
cleave
terminal
sialic
acid
residues
from
carbohydrates
and
glycoproteins. In some bacteria, this allows bacterial adhesion.
1.2.3.6. Bacillus
Bacillus species are Gram-positive rods often arranged in pairs or
chains with rounded or square ends and usually have a single endospore.
The endospores are generally oval and are very resistant to adverse
condition sporulation is not repressed by exposure to air (Holt et al.,
1994). Bacillus species can be broadly divided in three groups based on
the morphology of the spore and sporangium (Berkeley et al., 1997 ) .
The genus Bacillus currently comprises in excess of 60 species,
commonly found in the environment and as laboratory contaminants
(Konemann et al., 1997). They are aerobic or facultatively anaerobic and
most species are motile by peritrichous flagella. Most species are
oxidase- negative and catalase-positive.
1.2.3.6.1. Virulence factor
The many species of the genus exhibit a wide range of
physiologic abilities that allow them to live in every natural environment
enable them to survive or thrive in harsh environments, ranging from
desert sands and hot springs to Arctic soils and from fresh waters to
marine sediments. Spores formed under different conditions have
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different stabilities and degrees of resistance to heat, radiation,
chemicals, desiccation, and other hostile conditions. The genus includes
thermophilic, acidophilic, alkaliphilic, pH values and salt concentrations
at which few other organisms could survive. Because the spores of many
Bacillus species are resistant to heat, radiation, disinfectants, and
desiccation, they are exacerbate preexisting infections by producing
either tissue-damaging toxins or metabolites such as penicillinase that
interfere with treatment.
1.2.4. Epidemiology
Very Little data about the epidemiology of fistulous withers in
donkeys. They are given less consideration than other species of
livestock and their welfare is often neglected. The data concerning the
casual agent , transmission and significance of the disease . It is
important to look for donkeys , due to their hard working ability and
their tough nature.
1.2.5. Disease of equine similar to fistulous withers
1.2.5.1. Granulomatus lesion of the skin
A wide range of etiologies have been reported to cause
granulomatous inflammatory lesions localized to the deep layers of the
skin in equine. Habronema and Onchocerca species of nematode have
been shown (Jones et al., 1997). Fungal infections, including
sporotrichosis, and histoplasmosis, can produce prolific and often deep
granulomatous
inflammation
(Scott
et
al.,
2003).
Similarly,
botryomycosis, which has been associated mainly with Staphylococcus
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spp., is characterized by a chronic pyogranulomatous dermatitis and
formation of micro abscesses and bacterial pseudomycetoma (Pascoe et
al., 1999 ; Scott et al., 2003). In addition, a generalized idiopathic
granulomatous disease syndrome has been reported in horses(Axon et
al., 2004; Heath et al .,1990 ; Sellers et al., 2001) . Although generally
thought of as a disseminated disease affecting many organ systems,
idiopathic granulomatous disease has been described to produce only
cutaneous lesions in some cases (Heath et al.,1990).
1.2.5.2. Saddle sores
A saddle sore is an inflammation of the hair follicles of the horse at
pressure points where tack comes in contact with the skin. The most
common location is the withers. Girth sores are similar friction wounds
found in the girth area. Sores can also occur on the horse's face where
the bridle contacts the skin. In addition, riding a horse that has not been
properly groomed or using dirty tack can result in saddle sores. The
pressure of tack grinding against grit or sand on the skin can have to
same effect as rubbing the skin with sand paper. Clean tack after every
ride, and always groom your horse before every ride. Another cause of
saddle sores is poor equitation. An off-balance, bouncing rider can cause
the horse a great deal of muscle pain, as well as sores. All that excessive
movement causes friction against the horse's skin. The first symptom of
a saddle sore is inflammation and sometimes blistering. The condition
starts as an acute inflammation of the hair follicles and progresses to a
purulent folliculitis. Affected areas show hair loss and are swollen,
warm, and painful. The serous or purulent exudates dries and forms
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crusts and in the most severe cases infection and necrosis will occur. The
best treatment for saddle sores is complete rest until it has healed. Cold
water or ice packs can help reduce the swelling. Your veterinary may
recommend giving the horse an anti-inflammatory drug. If there are open
wounds he/she will probably prescribe antibiotics to ward off infection.
A topical antibacterial ointment should be applied to the raw area. In
severe cases, where there is a blood blister or necrotic tissue, surgery
might be required to remove that tissue. As usual, prevention is easier
than the cure. Groom your horse thoroughly before riding, paying
particular attention to areas that come in contact with the tack. Make
sure your tack is clean, has no rough places, and fits properly. Lift the
pad up slightly at the withers before tightening the girth, so that it does
not bind in that area. Learn to ride correctly, in balance with your horse,
and avoid excessive up and down hill riding. If a donkey has one of the
conformation faults it might need extra or specially designed pads under
its saddle and/or a breast collar to stabilize the saddle.
1.2.5.3. Capped hocks
Capped hock is inflammatory swellings of the subcutaneous bursae
(acquired bursitis) located over the olecranon process and tuber
calcaneus, respectively, of horses. Trauma from lying on poorly bedded
hard floors, kicks, falls , iron shoes projecting beyond the heels, and
prolonged recumbency are frequent causes. Edematous swelling
develops over and around the affected bursa. Lameness is rare in either
case. The affected bursa may be fluctuating and soft at first but, in a
short time, a firm fibrous capsule forms, especially if there is a
recurrence of an old injury. Initial bursal swellings may be hardly
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noticeable or quite sizable. Chronic cases may progress to abscessation.
Acute early cases may respond well to applications of cold water,
followed in a few days by aseptic aspiration and injection of a
corticosteroid. The bursa may also be reduced in size by application of a
counterirritant or by ultrasonic or radiation therapy. Older encapsulated
bursae are more refractory. Surgical treatment is recommended for
advanced chronic cases or for that become infected.
1.2.6. Prevention
Since horses usually acquire B. abortus infection from cattle (Denny,
1973; Cramlet and Bernhanu, 1979), they should not be housed or
pastured with seropositive cattle.
Since trauma is considered to be a predisposing factor for the
development of fistulous withers, properly-fitting saddles and tack
should always be used. Parasite control measures to reduce the incidence
of Onchocerca spp.
1.2.7. Treatment
Treatment of fistulous withers and poll evil can be difficult. Medical
treatment is directed towards controlling infection and inflammation.
Broad spectrum antibiotics are recommended. The wounds generally
resulted from poorly fitting pack equipment. Treatment regimens are
divided into two categories:
1) conservative topical therapy plus padding .
2) surgical debridement and primary wound closure.
Surgical treatment is used primarily in cases refractory to conservative
therapy. As a result of surgical intervention, clients tend to comply with
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recommendations to stop working the animal. The surgery may be
performed in the standing horse or under general anaesthesia.
The
convalescent time was shorter, and the recurrence rate was lower
following surgery than for conservative therapy ( Miguel et al., 1997).
Treatment of brucellosis in donkey generally involves a combination of
systemic antimicrobials and local surgical drainage of infected tissue. In
a survey of veterinarians in Florida concerning the treatment of fistulous
withers, treatment with antibiotics was reported to be effective in 37 of
46 horses (80.4%) (Gardner et al., 1983). Lavage of draining tracts with
antiseptic solutions and dimethyl sulphoxide may be helpful (Cohen et
al., 1992).
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CHAPTER TWO
2.
MATERIALS AND METHODS
2.1. Sterilization
a-Flaming
It was used to sterilize glass slides, cover slip, needles, scalpels points of
scissors and mouth of culture tubes by passing them through Bunsen burner
flame without allowing becoming red hot .
b-Red heat
It was used to sterilize loop wires, point of forceps and searing spatulas by
holding them over Bunsen burner flame until become red-hot .
c-Hot air oven
It was used to sterilize glassware such as bottles, flasks, test tubes, Petri
dishes, Pasteur pipettes, graduated pipettes and forceps. They were sterilized
in hot air oven at 180°C for one hours .
d-Steaming at 100°C
It was used for sterilization of sugars and media that could not be autoclaved
without determent effect to their constituents .It was carried out as described
by Cruickshank et al., (1975).
e-Moist heat (Autoclave)
Autoclaving at 121°C for 15 minutes was used for sterilization of media and
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plastic wares . Autoclaving at 115°C for 10 minutes was used for
sterilization of some media .
2.2. Reagent
Most reagents were obtained from British Drug House (BDH).
2.2.1. Sodium Chloride/ normal saline
Normal, physiological or isotopic saline was prepared as described in
Oxoide manual by dissolving 8.5 g of sodium chloride in 1 liter of distilled
water to obtain 0.85% concentration.
2.2.2. Alpha-naphthol solution
Alpha-naphthol solution was obtained from BDH Ltd . This solution was
prepared as 5% aqueous solution for Voges-Proskaur (VP) test and prepared
according to (Hopkins and Williams) .
2.2.3. Potassium hydroxide (Hopkins and Williams):
This reagent was prepared as 40% aqueous solution for Voges-Proskaur
(VP) test and prepared according to (Hopkins and Williams) .
2.2.4. Hydrogen peroxide (H2O2)
This reagent was prepared as 3% aqueous solution and stored in dark and
cool place. It was used for catalase test and obtained from BDH Ltd .
2.2.5. Kovac's reagent
This reagent composed of 5 g para- dimethyl amino benzaldehyde, 75ml
amyl alcohol and 25ml concentrated hydrochloride acid. It was prepared
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as described by Barrow and Feltham (1993). The aldehyde was dissolved in
alcohol gently in water bath at temperature of 50-55C, then cooled and the
acid was added with care. The reagent was protected from light and stored
at 4°C for indole test .
2.2.6. Ttetramethyl-p-phenylene diamine dihydrochloride
This reagent was prepared as 1% aqueous solution according to (Hopkins
and Williams) and it was used for oxidase test.
2.2.7. Nitrate test reagent
Nitrate test reagent was consisted of two solution and they were prepared
according to Barrow and Feltham (1993). Solution A was composed of
0.33% sulphanilic acid dissolved by gentle heating in 5N- acetic acid.
Solution B was composed of 0.6% dimethyl amine-alpha-naphthylamine
dissolved by gentle heating in 5N- acetic acid.
2.3 Indicators
2.3.1. Andrade's indicator
This was prepared according to Barrow and Feltham (1993) . It was
composed of 5g of acid fuchsin, 1 liter of distilled water and 150ml of NNaOH. The acid fuchsin was dissolved in distilled water, then the alkali
solution was added, mixed and was allowed to stand at room temperature
for 24 hour with frequent shaking until the color changed from red to
brown. It was used in sugar fermentation .
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2.3.2. Bromothymol blue
It was obtained from BDH Ltd . The solution was prepared by dissolving
0.2g of the bromothymol blue powder in 100ml distilled water. It was used
for Oxidation fermentation test .
2.3.3. Plasma
The plasma used for coagulase test was human plasma , prepared by
centrifugation of citrated human blood .
2.3.4. KOH and HCL
KOH and HCL were used for adjusting the pH .
2.4. Collection of blood for enriched media
Blood for enriched media was collected aseptically into sterile flask
containing glass beads venipuncture of jugular vein of a healthy sheep kept
for this purpose. The blood was defibrinated by shaking the sterile flask that
containing glass beads while and after collection .
2.5. Preparation of Culture Media
Different type of media including solid ,semisolid and liquid media were
used for isolation and identification of bacteria in this study .
2. 5.1. Liquid media
2.5.1.1. Nutrient broth
This medium contained peptic digest of animal tissue 5g , beef
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B274 . Thirteen grams of nutrient broth (Oxoid) were added to one litre of
distilled water, mixed well and pH adjusted to 7.2 -7.4 ,distributed in 3ml
amount into clean test tubes, then sterilized by autoclaving at 121°C for 15
minutes.
2.5.1.2. Peptone water
This medium contained peptic digest of animal tissue 10 g and sodium
Chloride 5g with code No. M 028 according to (HIMEDIA). To rehydrate
the medium, 15g were suspended in 1000 ml distilled water, mixed well
and pH adjusted to 7.2 -7.4 , distributed in 3ml amount into clean test
tubes and Sterilized by autoclaving at 121°C for 15 minutes.
2.5.1.3. Peptone water sugar
This media was prepared according to Barrow and Feltham (1993). 900ml
of peptone water were prepared and andrade's indicator 10 ml . Sugar
solution was prepared by dissolving 10 grams of appropriate sugar in 90 ml
of peptone water . The pH was adjusted to 7.1- 7.3 before the addition of
andrade's indicator. The complete medium was mixed, distributed in to 2
ml volumes into test tubes containing inverted Derham's tube (for gas
trapping in case of glucose) andsterilized by autoclaving at 115°C for 10
minutes and kept at 4°C until used .
2.5.1.4. Glucose-phosphate (MR-VP test) medium
This medium contained peptone (Oxoid L49) 5g , dextrose 5g and
phosphate buffer (k2HPO4) 5g with code NO. 43 according to (Oxoid).
The powder of 15 g were added to 1 liter of distilled water, dissolved by
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steaming , mixed well ,the pH was adjusted to 7.5 , distributed into clean
test tubes and sterilized by autoclaving at 121°C for 15 minutes.
2.5.1.5. Nitrate broth medium
This media was prepared according to Barrow and Feltham (1993). It
contained potassium nitrate 0.2g and peptone 5g . Solid ingredients were
dissolved into one liter of distilled water. The medium mixture was
distributed in 5 ml amount in clean test tubes and then autoclaved for 15
minutes at 121°C.
2.5.1.6. Serum water sugars
The medium was consisted of peptone
4g
,sodium phosphate
0.8g
,distilled water 800ml ,sterile serum 200ml and bromcresol purple(0.2%)
10ml. dissolved the peptone and phosphate in the water, steamed at 100°C
for 15 minutes. Add the serum ,checked pH to 7.6_7.8 , add the indicator
and sterilized at 115°C for 10 minutes. Then aseptically add 1% of the
appropriate sugar as sterile solution and distributed into sterile tubes.
2.5.2. Semi solid media
2.5.2.1. Motility medium
Motility medium was prepared as described by Barrow and Feltham (1993).
It consisted of nutrient broth (Oxoid) 13g and agar (Oxoid agar No.1) 5g.
The dehydrated of nutrient broth were added to agar in one liter of distilled
water , steamed to dissolved . The pH was adjusted to 7.4 . The medium was
dispended in
5 ml volume into 20 ml capacity test tubes containing
appropriate Cragie tubes. The medium in test tubes were sterilized by
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autoclaving at 121°C for 15 minutes. Then the medium was allowed to cool
to become semi solid before using.
2.5.2.2. Hugh and Leifson's (O/F) medium
This medium was composed of dipotassium hydrogen phosphate ,sodium
chloride, peptone, agar and 0.2 % aqueous solution of bromocersol purple.
The medium was prepared as described by Barrow and Feltham (1993) .
Two grams of peptone powder, 5g of sodium chloride, 0.3g of potassium
hypophosphate and 3g of agar were add to 1 liter of distilled water then
heated in water path at 55°C to dissolve the solid . The pH was adjusted to
7.1 . Then the indicator bromothymol blue (0.2 % aqueous solution ) was
added and mixture was sterilized by autoclaving at 115 °C for 15 minutes.
Then sterile glucose solution was added aseptically to give final
concentration 1% . Then the medium was mixed and distributed aseptically
in 10 ml amount into sterile test tubes .
2.5.3. Solid media
2.5.3.1. Blood agar
This medium was composed of dehyderated blood agar base obtained
from(Scharlau) and defibrinated sheep blood . The blood agar base with
code NO. 01-352 contained meat extract 10g , tryptone 10g , Sodium
chloride 5g and agar 15g . It was prepared according to manufacturer’s
instruction by dissolved 40g of blood agar base in one liter of distilled
water by boiling, mixed and sterilized by autoclaving at 121°C for 15
minutes. Then cooled to about 50°C and defibrinated sheep blood was added
aseptically to give final concentration 10% , mixed gently and 20ml of
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complete medium was poured into each sterile Petri dishes, the plates were
allowed to solidify at room temperature on leveled surface .
2.5.3.2. MacConkey agar
The dehydrated form obtained from (Scharlau) with code No. 01-118 and it
consist of peptone 20g , lactose 10g , bile salt 5g , sodium chloride 5g ,
neutral red 0.03g , crystal violet 0.001g and agar 15g . Then 51.5 g of the
powder were suspended in 1 liter of distilled water, boiling until dissolved
the ingredient completely then sterilized by autoclaving at 121°C for 15
minutes. The medium was then cooled to about 50 °C and poured into sterile
Petri dishes in 20 ml amount and left to solidify at room temperature on
leveled surface .
2.5.3.3. Nutrient agar
The medium was obtained from (Scharlau) with code No. 01-144 and it was
composed of peptone 5g , meat extract 3g and agar 15g .To rehydrate the
medium , 23g of nutrient agar powder and 5 g of sodium chloride were
suspended in 1 liter of distilled water, brought to boiling until dissolved
completely then sterilizing by autoclaving at 121°C for 15 minutes. The
medium was then cooled to about 50°C and distributed in 20 ml amount
sterile Petri dishes.
2.5.3.4. Ammonium salt sugars (ASS)
The medium was prepared as described by Barrow and Feltham (1993) .
One gram of (NH4)2HPO4 , KCL (0.2g) , MgSO4.7H2O (0.2g) and yeast
extract (0.2g) were added to 1 liter distilled water .Then indicator
bromothymol blue (0.04ml) was added. The solids were dissolved by
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steaming
and sterilized by autoclaving at 115°C for 20 minutes. The
mixture was allowed to cool to about 60°C, then appropriate carbohydrate
was added as sterilized solution to give final concentration 1% , mixed and
distributed aseptically into sterile test tubes and allowed to solidify in
slope position.
2.5.3.5. Aesculin – bile azide agar
The medium was prepared as described by Barrow and Feltham (1993) .
The medium was composed of ox bile (dehydrated) 40g , asculin 1g ,
ferric citrate 0.5g , sodium azide 0.2g , agar 15g and nutrient broth
1000g . All of the ingredients except aesculin were dissolved in water by
heating . Allowed to cool and then aesculin was added . The medium was
then suspended in screw capped bottles, sterilized by autoclaving 115°C
for 20 minutes and allowed to set in slope position .
2.5.3.6. Milk agar
The medium was prepared as described by Barrow and Feltham (1993) .
This medium was composed of milk ( skim ) 500ml and nutrient agar
(double- strength) 5ooml . Prepared the skim-milk and sterilized by heating
at 115°C for 10 minutes. Cool to about 50°C and add to the nutrient agar
double- strength , melted and cooled to 50- 55°C. Mixed and distributed in
Petri dishes.
2.5.3.7. Nutrient gelatin medium
This medium was composed of beef extract 3 g , peptone 15 g , gelatin
120g and Distilled water 1000 ml . One hundred and twenty eight
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grams of nutrient gelatin (Oxoid) ,were added to one liter of distilled water ,
steam to dissolved, mixed well, pH was adjusted to 6.8 , the medium
distributed in sterile screw capped ( Bijou) bottles in 2ml amount and
sterilized by autoclaving at 121°C for 15 minutes.
2.5.3.8. Simmons citrate agar
This medium was composed of magnesium sulfate o.2g , manoammonium
dihydrogen phosphate 1g , dipotassium phosphate 1g , sodium citrate 2g ,
sodium chloride 5g , bacto- agar 15g , brom thymol blue 0.08 g . To
rehydrate the medium, 24.2g (Scharlau) were suspended in 1000ml distilled
water , dissolved by steaming completely and the pH was adjusted to 7.0 .
The medium sterilized by autoclaving for 15 minutes at 121°C . Then
dispended into Bijou bottles in portions of 5ml and allowed to solidify in
slope position .
2.5.3.9. Starch agar
It consisted of potato starch 10g , nutrient agar 50g, there were suspended
in 1000ml distilled water (Barrow and Feltham, 1993). Triturated the starch
with the water to a smooth cream and added to the molten nutrient agar.
Mixed and sterilized at 115°C for 10 minutes. Distribute in 20 ml amount
sterile Petri dishes .
2.5.3.10. Urea agar Base
The base medium contained peptone (Oxoid L37) 1g , dextrose 1g ,
sodium chloride 5g ,disodium phosphate 1.2g , potassium dihydrogen
phosphate 0.8g , phenol red 0.012g and agar No.3 (Oxoid L13) 15g with
code No. CM53 . The medium was prepared according to manufacturer’s
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(Oxoid) by dissolving 2.4g of powder in 95ml distilled water by boiling ,
pH was adjusted to 6.8 , sterilized at 121°C for 15 minutes and cooled to
50-55°C . Five ml of sterile urea solution was added aseptically .The
medium was distributed in 5 ml amount in sterile test tubes and allowed to
set in slope position .
2.6. Clinical investigations
2.6.1. Study area
A protocol was developed to assess the welfare of a cross-sectional study of
randomly selected, male donkeys used for pack and ridden work found in
Khartoum State in urban and peri-urban areas namely Elsagana, Elremila,
Mayo in Khartoum, Halaib, Elsouk Elshabi Omdurman in Omdurman and
shambat in Bahri. clinical examination and a questionnaire survey were both
carried out simultaneously between December 2010 and May 2011.
2.6.2. Animals
One hundred and fifty of indigenous breeds from regions of selected areas
were investigated using direct observation of health and behavior
parameters and to isolate and identify aerobic bacteria associated with
infection by fistulous wither in donkeys in Khartoum state. Parameters
examined were general condition , body temperature and pulse rate.
2.6.3. Clinical signs
Clinical signs of fistulous wither include localized heat, pain and swelling of
the bursa. The early lesion is a simple thin-walled sac with serous exudates.
This exudates arises in the cause of the inflammatory change in inner layer
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of the bursal sac. The latter stages involve considerable necrosis.
2.6.4. Collection of Samples
In this study, sixty out of 150 donkeys, which showed fistulous wither were
considered for bacteriological examination. The lesion were open (fistulae)
and samples collected after cleaned with cotton pieces soaked in 70% ethyl
alcohol. All donkey samples were collected by a sterile cotton swabs ,
which were then labeled and carried in container with ice to the laboratory
and kept at -10°C.
2.7. Bacteriological examination
2.7.1. Primary isolation
All samples were cultured onto blood agar and MacConkey agar.
a-Blood agar
It was used as an enriched , non- inhibiting medium for primary isolation of
bacteria and for determination of colonial morphology and hemolytic
activity of Streptococci, Staphylococci and other organisms.
b-MacConkey agar
This medium was used in this study for isolation of enterobacteria .
2.7.2. Isolation
Isolation attempts were made for all samples at medical laboratory,
University of Khartoum. The swabs were inoculated onto 10% defibrinated
sheep blood agar and MacConkey agar .The inoculated plates were then
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incubated aerobically at 37°C for 24 hours as described by Barrow and
Feltham (1993). Further incubation was continued for another 24 h and if no
growth was evident ,then the plates were discarded as negative .
2.7.3. Cultural characteristics
All cultures on solid media were examined with nacked eye for growth and
colonial morphology and any change in medium.The liquid media were also
examined with eye for turbidity , color change ,formation of sediments and
accumulation of gas in the Durham’s tube in case of carbohydrates media .
2.8. Purification
Purification was done by several subculturing from single well separated
colony on separate nutrient agar plates or blood agar plates for those which
do not grow on nutrient agar. Then examined for purity microscopically as
described later for identification.
2.9. Preservation
The isolates were preserved for further studies to determine their cultural
and biochemical characterizations. Preservation was made by subculturing
the purify isolates and store at 4°C.
2.10. Microscopic Examination
Smears were made from purified colonies, fixed by heating and stained by
Gram stain method of
Barrow and Feltham(1993). Then examined
microscopically for cell purity , morphology , arrangement
characterization of bacteria and staining reaction .
ϰϯ
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2.11. Biochemical tests
2.11.1. Sugar fermentation test
The test was carried out as described by Barrow and Feltham (1993). The
ped isolates on fresh sheep blood agar plates weakly. Cultures were kept.
peptone water sugar was inoculated with organism under the test, incubated
at 37°C and then examined daily for several days. Acid production was
indicated by the appearance of pink color, while gas production was
indicated by presence of an empty space in the inverted Durham's tubes.
2.11.2. Oxidase test
The method of Barrow and Feltham (1993) was followed. Strips of filter
paper was soaked in 1% solution of tetramethyl- p-phenylenediamine
dihydrochloride and dried in hot air oven and then placed on a clean glass
slide by a sterile forceps. A fresh young tested culture on nutrient agar was
picked off with sterile glass rod and rubbed on the filter paper strips. If a
purple color developed within 5-10 seconds, the reaction was considered
positive.
2.11.3. Catalase test
The test was carried out as described by Barrow and Feltham (1993). A drop
of 3% aqueous solution of hydrogen peroxide was placed in a clean glass
slide. A colony of tested culture on nutrient agar was picked off and put on
the drop of hydrogen peroxide. Evolution of gas and appearance of bubbles
indicated positive test.
2.11.4. Coagulase test
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2.11.4.1. Slide coagulase test
The test performed on slide by added drops of physiological saline were
placed in each end of slide .Two colonies of tested culture were emulsified
in each of drops to make two thick suspensions . A drop of human plasma
was added to one of the suspension and mixed gently . Clumping of the
organisms within 10 seconds was recorded as positive reaction .
2.11.4.2. Tube coagulase test
The test was performed as described by Barrow and Feltham (1993). To
0.5ml of 1:10 dilution of human plasma in physiological saline, 0.5ml of an
18-24h old broth culture of the tested organism was added in asterile
agglutination test tube then incubated at 37°C and examined after 2-24 h for
coagulation. Definite clot formation indicated positive result.
2.11.5. The oxidation- fermentation (o/F) test
The test was carried out as described by Barrow and Feltham (1993).
Duplicate test tubes of Hugh and Leifeson's medium were inoculated by the
tested organism with straight wire. To one of the test tube a layer of sterile
melted soft paraffin oil was added to a depth of 3 cm above the medium to
seal it from air. The inoculated tubes were incubated at 37°C and examined
daily for fourteen days. Yellow color in the open tube only indicated
oxidation of glucose, yellow color in both tubes showed fermentation
reaction and blue or green color in open tube and green color in the sealed
tube indicated production of alkali.
2.11.6. Indole production test
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Indole production test was carried out as described by Barrow and Feltham
(1993). The tested organism was inoculated into peptone water and
incubated at 37°C for 48h . One milliliter of the Kovac's reagent was added
. Production of red color on the upper layer of the reagent indicated positive
reaction.
2.11.7. Voges- Proskauer (VP) test
The test was performed as described by Barrow and Feltham (1993). The
tested culture was inoculated into glucose phosphate medium (MR-VP
medium) then incubated at 37°C for 48h. Then 0.6ml of 5% alphanaphthol solution followed by a 0.2ml of 4% potassium hydroxide were
added to one ml of the culture, shaked well and examined after 15-30
minutes . When bright pink color developed the reaction was regarded as
positive.
2. 11.8. Nitrate reduction
The nitrate test was carried out as described by Barrow and Feltham (1993).
The tested culture was lightly inoculated into nitrate broth and incubated at
37°C for up to 5 days. Then 0.5ml of solution A followed by 0.5ml of
solution B of nitrate test reagent were added. Red color indicated positive
reaction that showed nitrate had been reduced. If red did not develop,
powdered zinc was added to see whether there was residual nitrate or not.
Red color development indicated that nitrate in medium had been reduced to
nitrite by zinc but not by organism, whereas unchanged color indicated
nitrate in original medium had been reduced completely and nitrite was
further broken down by the organism.
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2.11.9. Urease activity test
The test was carried out as described by Barrow and Feltham (1993). The
tested organism was inoculated heavily onto slope urea agar medium and
then incubated at 37°C for 7 days. Appearance of pink color indicated
positive reaction.
2.11.10. Citrate utilization test
The test was performed as described by Barrow and Feltham (1993). The
tested culture was inoculated as a single streak over the surface of a slope of
Simmon’s citrate medium at 37°C up to 48 hours . Examined daily for 7
days for growth of the organism and changed color to blue indicated
positive test.
2.11.11. Aesculin hydrolysis
The procedure of Barrow and Feltham (1993) was followed. The tested
organism was inoculated
into aesculin agar
, incubated at 37°C and
examined daily for up to 7 days. Black color indicated positive reaction .
2.11.12. Hydrogen sulphide (H2S) production
The method of
Barrow and Feltham (1993) was followed. The tested
culture was inoculated into peptone water, filter paper impregnated with
10% lead acetate solution was inserted
between the plug and the tube .
The tubes were examined daily for 7 days for black color of paper due to
hydrogen sulphide production as positive reaction .
2.11.13. Ammonium salt sugar test
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The test was performed as described by Barrow and Feltham (1993). The
tested organism was inoculated onto a slope of ASS medium and incubated
at 37°C for up to 7 days. The medium was examined on alternative days for
growth and acid production.
2.11.14. Gelatin hydrolysis
Gelatin hydrolysis test was carried out as described by Barrow and Feltham
(1993). The tested culture was stabbed into nutrient gelatin and was
incubated at 37°C for up to 14 days. The inoculated tube was placed in
refrigerator for 2 hours every 2-3 days and was examined. The liquefaction
of gelatin indicated positive test.
2.11.15. Motility test
The semi-solid motility medium containing Craigi tube prepared as
described by Cruickshank et al. (1975) was used to detect motility . A small
piece of the colony of the bacterium under the test was picked by the end of
the straight wire loop and stabbed in the center of semi-solid agar in the
Craigi tube ,then incubated at 37°C.The was considered motile if there was
turbidity in the medium in/outside the Craigi tube .
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CHAPTER THREE
3.
RESULTS
3.1. Clinical findings
The data showed that donkeys demonstrate avoidance or aggressive
behavior towards an observer. Fewer than 60% of working donkeys had
abnormal mucous membranes, ectoparasites or poor coat condition. Over
30% of donkeys demonstrated limb deformities and abnormalities of gait ,
overgrown hooves and wounds caused by incorrect tethering or hobbling .
In cases of injury seen the animals were usually thinner and the pads under
the pack saddle were of a synthetic fiber material such as polypropylene.
The results of this study might be used as the initial stage of a long-term
strategy to inform priorities for welfare interventions in working donkeys
and to establish a welfare benchmark. Therefore, the status and dynamics of
donkeys husbandry, as well as a general assessment of the needs of donkeys
owners in this area were researched. The prevalence of external injuries in
donkeys was observed in different ages with different status from mild,
moderate and severe and different causes. The survey which targeted to
detect fistulous wither among donkeys, its investigation was focused on
etiological agent. Almost all donkeys were under hard working conditions
treated by owners. Donkeys were used always exclusively for transport
purposes , abused and poorly managed . feeding is mainly green fodder as
well as Sorghum grains (Fetarita) (tables 2,3 and 4).
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Table2: Distribution of External Injuries on Various Body Parts in donkeys
Location of Injures
Donkeys (n)
%
Wither
60
40
Flank
4
2.7
Back / Shoulder
71
47.3
Under Tail
3
2
Front Leg
5
3.3
Abdomen
3
2
Head / Neck
-
-
Hind Leg
4
2.7
150
100
Total
Table 3: Causes of External Injuries in Donkeys in Khartoum State
Cause
Donkeys
n(%)
Improper harness and saddle
38 (25.3)
Overloading and overworking
55 (36.7)
Biting
20 (13.3)
Infection disease
14 (9.3)
Un known
5 (3.3)
Maltifactorial causes
18 (12)
Total
150
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Table 4: Owner’s Responses to the Management of External Injuries in donkeys
Owner’s Responses
Donkeys
(%)
(n)
Take to nearly health center
32
21. 3
Treat with medication purchased
20
31. 3
Treat with medical plant
28
18. 7
Do nothing
70
46. 7
Total
150
100
from local market
3.2. Bacterial isolation
From 150 donkeys, only 60 samples which collected from donkeys
infected with fistulous wither and cultured onto blood agar and MacConkey
agar media and incubated aerobically at 37°C. Fifty-four (90%) showed
bacterial growth, while six samples (10%) did not demonstrated any growth
of bacteria.
3.3. Characterization and identification of isolates
The bacterial isolates obtained in this investigation were identified to
species level on the basis of their cultural Characteristics, cell morphology,
Gram stain reaction and their biochemical properties as described by
Barrow and Feltham (1993) as showen in (table 5). The isolate consisted of
142 Gram positive isolate (236.7%) as showen in (Table 4).
3.4. Gram- positive Bacteria
Total of 142 (236.7%) organism were characterized as Grampositive bacteria. They were further characterized as Staphylococcus spp 55
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(91.7%), Corynebacterium spp 8 (13.3%), Streptococcus spp 6 (10%),
Actinomycetes 4 (6.6%) and Bacillus spp 69 (115%) as showed in (Table 4).
Bacillus spp and Staphylococcus spp represented the highest percentage of
the total Gram-positive bacteria isolated (Fig.1).
3.4.1. Staphylococcus
The Staphylococcus spp percentage (38.7%) of the total Grampositive isolated. Staphylococcus species were identified according to the
scheme of Staphylococcus species identification of Barrow and Feltham
(1993). They were identified as twenty-five coagulase –positive
staphylococci, this represent (45.5%) of the total Staphylococci species.
They were twenty-two (40%) S. aureus and three (5.5%) S. intermedius.
Thirty isolates were coagulase –negative Staphylococci, this represent
(54.5%) of the total Staphylococci species. They were eighteen (32.7%) S.
chromogen, eight (14.5%) S. haemolyticus and four (7.3%) S. epidermidis.
3.4.2. Streptococcus
The Streptococcus spp isolated in this study were identified
according to their morphology and Gram-stain reaction. They were Grampositive cocci, non-motile, non-sporing and arranged in short or long chain.
They were also identified on the basis of their biochemical properties. They
were
(4.3%)
of
the
total
Gram-positive
bacterial
isolation.
3.4.3. Actinomycetes
Four isolates were identified as Actinomycetes spp and these
represent (2.8%) of the total Gram-positive isolates. The different species
isolated were Actinomycetes bovis and Actinomycetes pyogenes.
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3.4.4. Corynebacterium
Eight isolates were identified as Corynebacterium spp and these represent
(5.6%) of the total Gram-positive isolates. The species isolated were
Corynebacterium pseudotuberclosis, Corynebacterium aminoniagenes,
Corynebacterium kutscheri, Corynebacterium ulcerans.
3.4.5. Bacillus
Sixty-nine isolates were identified as Bacillus spp and these represent
(48.6%) of the total Gram-positive isolates. The different species isolated
were Bacillus mycoides, Bacillussubtilius, Bacillus circulans, Bacillus
licheniformis, Bacillus firmus , Bacillus alvei.
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Table 5: Isolation frequency of Gram-positive bacteria isolated from donkeys
infected with fistulous withers .
No. of
Sources of samples
Infected donkeys with
No. of
samples
Species of bacteria
examined
isolated
ϲϬ
fistulous withers
Total
isolated
Staphylococcus spp
ϱϱ
Corynebacterium spp
ϴ
Streptococcus spp
ϲ
Actinomycetes spp
ϰ
Bacillus spp
ϲϵ
ϭϰϮ
Table 6: Biochemical properties of some Grame+ve bacteria isolated from
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donkeys infected with fistulous withers in Khartoum State
Staphylococcs
Streptococcus
Actinomycetes
Test
spp
spp
spp
Gram reaction
+
+
+
Growth
+
+
+
Catalase
+
-
+
Oxidase
-
-
-
Motility
-
-
-
Growth
+/-
+
+
F
F
F
VP
+/-
-
-
Nitrate
+
-
+/-
+
-
-
at37°C.
anaerobically
Oxidation
fermentation
reduction
Coagulase
Key:
(-) = Negative
(+) = Positive
F = fermentative
Table 7: Gram stain reaction and biochemical properties of Staphylococcus spp
isolated from donkeys infected with fistulous withers
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Test
Staphylococcus
Staphylococcs
Staphylococcus
Staphylococcus
aureus
intermedius
chromogens
haemolyticus
St
ep
Gram-reaction
+
+
+
+
+
Oxidase
-
-
-
-
-
Catalase
+
+
+
+
+
Oxidation
F
F
F
F
F
Coagulase
+
+
-
-
-
VP
+
-
-
+
+
Nitrate
+
+
+
+
+
Urease
+
+
+
-
+
Mannitol
+
+
-
+
-
Maltose
+
-
+
+
+
Sucrose
+
+
+
+
+
fructose
+
+
+
+
+
Xylose
-
-
-
-
-
fermentation
Key:
(-) = Negative
(+) = Positive
F =fermentative
Table 8: Gram stain reaction and some biochemical properties of Bacillus spp
isolated from donkeys infected with fistulous wither
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Test
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
mycoides
subtilius
circulans
licheniformis
firmus
B
l
a
Gram-reaction
+
+
+
+
+
+
Oxidase
-
-
-
-
-
-
Catalase
+
+
+
+
+
+
Oxidation
F
F
F
F
F
F
Motility
-
+
+
+
+
+
VP
+
+
-
-
-
+
Nitrate
+
+
-
+
+
+
Urease
+
-
-
-
-
-
Glucose
+
+
+
+
+
+
Gluctose
+
+
+
+
-
+
Salicin
+
-
+
+
-
+
Xylose
-
-
+
+
-
-
Citrate
+
+
-
+
-
-
fermentation
Key:
(-) = Negative
(+) = Positive
F = fermentative
Table 9: Gram stain reaction and some biochemical properties of
Corynebacterium spp isolated from donkeys infected with fistulous withers
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Test
Corynebacterium
Corynebacterium
Corynebacterium
Pseudotuberclosis
aminoniagenes
Gram-reaction
+
+
+
Oxidase
-
-
-
Catalase
+
+
+
Oxidation
F
F
F
VP
-
-
-
Nitrate
-
+
+
Urease
+
+
+
Mannitol
-
-
-
Maltose
+
-
+
Sucrose
+
-
+
Xylose
-
-
-
kutscheri
fermentation
Key:
(-) = Negative
(+)= Positive
F = fermentative
Table 10: Gram stain reaction and some biochemical properties of
Streptococcus spp and Actinomycetes spp isolated from donkeys infected with
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fistulous withers
Test
Streptococcus
Streptococcus
Actinomycetes
equi
zooepidemicus
bovis
Actinomycetes
pyogene
Gram-reaction
+
+
+
+
Oxidase
-
-
-
-
Catalase
-
-
-
-
Motility
-
-
-
-
Oxidation
F
F
F
F
VP
-
-
-
-
Growth
+
+
+
+
Mannitol
+
+
-
-
Sucrose
-
-
+
-
Lactose
+
+
+
+
Starch
ND
ND
+
+
ND
ND
-
+
fermentation
anaerobically
hydrolysis
Gelatin
liquefaction
Key:
(-) = Negative
(+) = Positive
F = fermentative
ϱϵ
ND=NOT done
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ϳϬ
ϲϬ
ϱϬ
ϰϬ
No. of isolate
ϯϬ
ϮϬ
ϭϬ
Ϭ
Type of bacteria
Figure 4 : Frecquency of Gram
Gram-positive
positive bacterial species isolated from
donkeys with fistulous withers
ϲϬ
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Figure 5 : Staphylococcus aureus isolated from donkey with fistulous
withers on blood agar medium
Figure 6 : Actinomyces spp bacteria isolated from donkey with
fistulous withers on blood agar medium
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Figure 7: Smear from blood agar culture of Staphylococcus aureus
Figure 8: Smear from blood agar culture of Actinomyces spp
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CHAPTER FOUR
Discussion
In underdeveloped countries donkeys play an essential role in the
agricultural economies, but these animals have not yet been given sufficient
care and they are subjected to many diseases which affect their viability and
lower their work ability (Khalifa et al., 1988). The use of donkeys in doorto-door transport service also provides urban dwellers with the opportunity
of income generation (Kathy et al., 2004). In the Sudan the use of donkeys
for transportation will continue for years to come because of the rugged
terrain characteristics inaccessible for modern road transportation facilities
as well as the absence of well-developed modern transport networks and the
prevailing low economic status of the community.
This study was carried out in Khartoum state that is heavily populated with
donkeys which are considered to be the most important draught animals, in
addition to other towns and rural areas of the Sudan.
Despite their invaluable contributions, donkeys are the most neglected
animals, accorded low social status, particularly the male working donkeys
often are involved in more multipurpose activities than horses. They
transport goods to and from markets, farms, and shops, traveling long
distances. They also pull carts carrying heavy loads 3 to 4 times their body
weight. They work from 8 to 12 hours/day, depending on the season and
type of work. Unlike horses, donkeys are not provided with feed
supplements. They are brutally treated, made to work overtime without
adequate feed or health care. The increasing human population,
demands
for transport of goods to and from far, remote areas, and construction
activities around the town are making equines highly demanded animals.
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Though equines provide several advantages, health and welfare is a visible
problem. Studies to elucidate the magnitude of this problem are lacking.
Such information would be useful for designing strategies that would help
improve donkeys health and welfare. This study describes causes and
influencing factors of external injuries in a working population of donkeys
in Khartoum State. The results of this investigation demonstrated that
external injuries were highly prevalent among the working donkeys in the
region, showing their inhumane suffering due to inappropriate management
and neglect. The study also revealed that donkeys were much more affected
than horses contrary to the widely prevailing opinion that they are tolerant
to hardship conditions. Donkeys were involved in a wide array of activities,
yet very little management was accorded to them. They were made to carry
heavy loads over long distances and hours. They travel as far as 70 km/day
while carrying an average weight load of 150 kg. This was evident in the
present findings as more cases of injuries in donkeys (36.7%) were due to
overloading and overweight as reported by Demelash et al. (2006) , a
similar situation in Ethiopia where over-weight and type of load/work
contributed to high cases of back sores in donkeys. In agreement with the
present report, improper harness and saddle were major causes of injuries in
equines, these were demonstrated to be commonly distributed on wither and
back coinciding with poorly designed and ill-fitted harnesses and saddles.
Manufactured by unskilled artisans, equine-drawn carts are often designed
unbalanced and too heavy and do not consider load distribution in relation
to the body balance and style of movement. Wooden- or iron-made saddles
are constantly put on the back/shoulder and strongly tied to the body by
plastic rope, which causes persistent irritation and injuries. Donkeys are also
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rarely bathed by their owners. Sand, dirt and debris from the transported
goods and pollution find their way between the equipment and the donkeys
skin. The friction caused by these elements rubbing together against the skin
cause chaffing and wounds that are left untreated and that can progress to
serious injuries and infections: if the owner is actually riding the donkey,
there is more weight on the saddle, predisposing for heavier rubbing against
the wound.
In most cases, harnesses were made of car tire strips, which cut in to the
skin of the donkeys and form large open wounds. It can be concluded that
whenever an animal works, there is a potential for injuries caused by either
absence of or inappropriate equipment and harnessing. This substantiates
the report of Pearson, (1992).
The high prevalence of infection-related injuries in donkeys suggests the
microbial pathogens as either primary or secondary causes, contributing to a
decrease in sale value and work performance. A higher number of donkeys
with lacerated wounds due to bites was reported in the present study
(13.3%) . Damages caused by barbed wire, sharp objects and trauma due to
fighting among donkeys were also common causes of lesions in donkeys in
Khartoum State.
Adequate feeding and proper health care should thus be an integral part of
injury prevention. In many areas it is often virtually impossible for the
donkey owner to provide fencing around the area in which the donkey lives.
In these situations, although far from ideal, a neck tether, leg hobbles, or a
combination of both is used to ensure the donkey does not stray. Unsuitable
materials, such as wire, and incorrect fitting cause the majority of injuries
which can often be very serious. One of the main problems facing donkeys
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is fistulous withers that being the major cause of ill-health donkeys and
owner should have knowledge of these problems for control and prevention.
Occurrence of
fistulous withers in working donkey and horses were
reported previously (Pearson et a1., 1999). In addition , fistulous withers
needed surgical removal of necrotic tissue and local systemic prophylactic
treatment with antibiotic. This study showed that wound due to fistulous
withers which might be due to trauma, inhumane care which constitutes the
main problem or continuous irritation of the animal’s back; this is justified
by the fact that most of the donkeys in the area examined are used for
riding or for pulling carts. Moreover, treatment for most of the infected
wounds may prove costly.
The isolated bacteria in the present study included Bacillus spp (69) ,
Staphylococcus spp (55) , Corynebacterium spp (8) , Streptococcus spp (6)
and Actinomyces spp (4). In this study Staphylococci isolated from fistulous
withers
were
Staphylococcus
Staphylococcus
Staphylococcus
chromogen,
aureus,
Staphylococcus
Staphylococcus
intermedius,
haemolyticus
epidermidis. Staphylococcus aureus
and
is the potential
pathogen isolated from wounds which in agreement with Ellis et al., (1998).
While the isolation of commensal microflora such as Staphylococcus
epidermidis is in line with the findings of El Abdeen (2001), who isolated
them from contusions of sheep skins. Isolated of Staphylococcus aureus
and Staphylococcus epidermidis , substantiated the result of Guard (1932)
and Gaughan et al., (1988). The commonest Staphylococcus were
Staphylococcus aureus, Staphylococcus chromogen and Staphylococcus
haemolyticus. The Staphylococci associated with wound infections were
also previously reported by Beers and Berkow (1999) as in the present
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study, the higher rate of isolation of Staphylococci indicated that these
organism were more active in causing putrefaction of the skins as they were
isolated alone or in mixed infection with other organisms. The isolation of
Staphylococci from putrefied skin was reported by other authors(Unsworth
1946; Esuruoso 1977 and Ruhmann 1987).
The species of Corynebacteria were normal flora of the skins (Barrow and
Feltham1993). In this study species of Corynebacterium isolated were
Corynebacterium Pseudotuberclosis , Corynebacterium aminoniagenes ,
Corynebacterium kutscheri and Corynebacterium ulcerous.,0 which similar
to the bacterial species isolated by Cohen et al., (1992); Hawkins and
Fessler (2000). Isolation of Corynebacterium ulcerans confirms what was
reported by Asha et al., (2006), whereas isolation of Corynebacterium
pseudotuberculosis and Staphylococcus spp. were in agreement with the
findings of Stephen (2005). Corynebacterium was isolated from putrefied
skins , this confirms what was reported by Ibrahim (1989).
The Isolates of Streptococcus, were identified as Streptococcus equi and
Streptococcus zooepidemicus. The same organisms were isolated by
Gaughan et al., (1988), but Streptococcus salivarius and Streptococcus
pyogenes were reported by Asha et al., (2006). The previous report by
Woolcock (1975), substantiated the present finding, who isolated
Streptococcus zooepidemicus from infected equines. Streptococci spp.
caused a wide variety of local and generalized disease conditions in equines
such as pneumonia and sinusitis and as commonest secondary invaders in
wounds such as fistulous withers and poll evil. Streptococcus spp. was also
isolated from damaged skins as reported by Holt et al., (1994).
Actinomycetes spp. isolated were Actinomycetes bovis and Actinomycetes
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pyogenes. Isolation of Actinomycetes bovis, is in agreement with Kimball
and Frank, (1945) and Actinomycetes pyogenes is in agreement with Asha et
al., (2006). Actinomycetes bovis causes Actinomycosis which is a sub-acute
or
chronic
progressive
disease
characterized
by development
of
granulomatus, supperative lesion involving bone and soft tissue. The present
result demonsterated abscesses discharge through fistulous and sinuses. In
cattle the disease involves the mandible or other bony tissue of the head
(lumpy jow).
The species of Bacillus isolated: Bacillus mycoides, Bacillus subtilius,
Bacillus circulans, Bacillus licheniformis, Bacillus firmus and Bacillus
alvei. Isolation of
Bacillus
licheniformis and Bacillus firmus,
is in
agreement of the previous report by Asha et al., (2006). Bacillus causes
putrefaction of skins due to gelatinolytic activity of
bacteria (Cooper
,1973). Bacillus spp. were contaminating microorganism and were found in
bad hygiene and were main source of contamination. In contrast to Guard
(1932) and Asha et al., (2006) in this study Escherichia coli, Pasteurella
spp. Enterococcus faecalis, Listeria species and Kurthia species were not
isolated. This difference might be due to difference geographical areas .
Hawkins and Fessler (2000) , were frequently isolated ß- hemolytic
Streptococcus spp.
from horses, whereas Staphylococcus aureus was
frequently isolated from fistulous withers samples collected from donkeys in
the present study, which in agreement with the report of Cohen et al.,
(1992).
Lastly, Equines are important animals to the resource-poor communities in
rural and urban areas of Sudan, providing traction power and transport
services at low cost. Veterinary clinic service should be upgraded and made
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available to donkey owners, especially where private and state veterinarians
are unavailable or too expensive in such resource-limited community. Bad
hygiene and poor general condition are the factor which help the
multiplication of bacteria, that lead to offensive skin, hair slopping and
putrefaction of skins (Knew, 1952).
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CONCLUSION
The present study can be concluded in the followings :
1. The major problems of donkey identified were:
• Injuries to the animal.
• Inadequate harness materials.
• Lack of knowledge of correct adjustment and use.
• Different hitching systems for different implements.
2.The major pathogenic bacteria isolated were Staphylococcus spp.,
Corynebacterium spp., Streptococcus spp. and Actinomycetes spp.
3. The most common health problems of donkeys arise from overuse,
misuse and under-nutrition.
4. Donkeys remain to play an important economic and social roles and are
still important in transportation and some families depend on donkey as
financial source.
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RECOMMENDATIONS
From the findings of the present study it is recommended that :
1. There is a need for greater awareness of donkeys' welfare issues, with
improved facilities and services for donkey management, feeding, watering
and Veterinary care, supported by possible legislation.
2. There is a need for training and extension programs targeting on donkey
owners. Extension staff needs more training related to donkeys .
3. Appropriate padding materials should be used to allow sufficient
ventilation on the skin surface hence preventing infections.
4. Local artisans should be given basic training on harnessing theories and
working principles of carts to prevent the manufacture of shoddy carts and
donkey owners should be adequately trained on how to adjust the improved
harnesses.
5. It is important that, with the influx of synthetic material, owners are
educated to the benefits of natural materials over synthetics with respect to
the long-term health and efficiency of their animals.
6. Bacteriological laboratories should be consulted to determine the
organism and most effective treatment to be used.
7. When antimicrobial drugs are administered they should be given in full
therapeutic dose for adequate period.
8. Donkeys with fistulous withers or other type of wounds should be
prohibited from working by the authorities .
9. Further extensive work should be carried out, to survey the prevalence of
fistulous withers in donkeys.
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10. Possible improvements by development of a new harness for donkeys .
11. Good management, treatment of wounds in addition to use of suitable
saddles may decrease chance of bacterial infection of the affected skins in the
area of damage.
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Appendix
Age :
Sex :
Type of breed :
Feeding :
Activity :
Behavior :
Posture and gait :
Coat condition :
Infected by injury :
Site of injuries :
Site of injuries :
Causes of injuries :
Treat of injuries :
Fate of injuries :
Clinical sign :
Specimen collection :
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DEDICATION
To the soul of my father, to my lovely mother, supportive
Husband, unique sweet son and daughters, wonderful sisters
and brother and Friends for their unlimited assistance.
With my deep love.
i
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PREFACE
This work was carried out at the
Department of Microbiology, Faculty of
Veterinary Medicine, University of Khartoum,
Under supervision of Dr. Awad Elkarim Abd Elghaffar Ibrahim.
ii
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ACKNOWLEDGEMENTS
First and foremost I would like to thank my Merciful Allah for giving me strength and
health to do this work.
I would like to express my sincere thankfulness, indebtedness and appreciation to
my Supervisor Dr. Awad Elkarim Abd Elghaffar Ibrahim for his guidance, advice, help,
encouragement and patience throughout the period of this work.
My gratitude is also extended to all staff of the Bacteriology laboratory for the
technical assistance during the laboratory work.
My thanks also extended to my friends, and collages who help me.
iii
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ABSTRACT
Working animals provide an essential transport resource in
developing countries worldwide. Many of these animals are owned by poor
people and work in harsh environments, so their welfare is a cause for
concern. The present study was carried out to assess the welfare of working
donkeys in Khartoum State, using clinical examination and a questionnaire
survey , to identify and quantify factors associated with the presence of
wounds in donkeys working in the transport of goods and to isolate and
identify aerobic bacteria associated with fistulous withers. One hundred fifty
male donkey were randomly chosen. They were used for work and found in
markets and along the roads in Elsagana, Elremila, Mayo, Halaib, Elsouk
Elshabi Omdurman and Shambat. The study was carried out between
December 2010 and May 2011.The results showed that improper harness
and saddle were the major causes of injuries in donkeys. The prevalence of
injuries was influenced by the type of work carried out, and the body lesions
occurred predominantly in the areas of the breast/shoulder, withers and girth,
while Lesions on the head, neck,
flank and tail base were seen in less than
10% of the animals. From 150 donkeys examined, only 60 of them were had
fistulous withers, and their collected samples were cultured on blood agar ,
nutrient agar and MacConkey’s agar. Bacterial isolates were identified to the
species level, according to their cultural characteristics and biochemical
tests. A total of one hundred and forty two isolates were obtained and
identified as Gram positive bacteria. These isolates were belonging to
Bacillus spp. (48.6%), Staphylococcus spp. (38.7%), Corynebacterium spp.
(5.6%), Streptococcus spp.(4.3%) and Actinomyces spp. (2.8%),which
represents potential pathogens that induced fistulous withers. The following
iv
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species
were
isolated:
pseudotuberclosis,
Staphylococcus
aureus,
Corynebacterium
Streptococcus zooepidemicus, Actinomyces bovis and
Actinomyces pyogenes, which might lead to the chronicity of the disease.
v
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Staphylococcus spp. (38.7%), Bacillus spp
(48.6%), Corynebacterium spp.
(5.6%), Streptococcus spp. (4.3%) and Actinomyces spp. (2.8%).
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Staphylococcus aureus, Corynebacterium pseudotuberclosis, Streptococcus
zooepidemicus, Actinomyces bovis and Actinomyces pyogenes.
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vi
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TABLE OF CONTENTS
DEDICATION…………………………………………………………………i
PREFACE……………………………………………………………………...ii
ACKNOWLEDGEMENTS……………………………………………………iii
ABSTRACT……………………………………………………………………iv
ARABIC ABSTRACT…………………………………………………….......vi
LIST OF CONTENTS…………………………………………………………vii
LIST OF TABLES…………………………………………………………….xv
INTRODUCTION…………………………………………………………......1
1. CHAPTER ONE: LITERATURE REVIEW ……………………………….4
1.1. Donkey…………………………………………………………………….4
1.1.1. Donkey population and history …………………………………………4
1.1.2. The place of donkeys in society………………………………………...5
1.1.3. General donkey features and behavior…………………………………7
1.1.4. Selection characteristics of a working donkey………………………….8
1.1.5. Economic importance………………………………………………
9
1.1.6. Fitting equipment…………………………………………………
10
1.1.7. General causes of injuries …………………………………………
12
1.1.8. Wounds……………………………………………………………
12
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1.2. Fistulous withers……………………………………………………
14
1.2.1. Definition of fistulous withers……………………………………
14
1.2.2. Clinical signs……………………………………………………..
15
1.2.3. Etiological agent of fistulous withers……………….....................
16
1.2.3.1. Bruccella abortus………………………………….....................
16
1.2.3.2.Staphylococcus spp……………………………………………….
17
1.2.3.2.1. Bacterial virulence factor……………………………………
18
1.2.3.2.2. Cell - Associated Components………………………………
18
1.2.3.2.3. Exoenzymes…………………………………………………
19
1.2.3.2.4. Exotoxins……………………………………….......................
19
1.2.3.2.5. Membrane-damaging toxins …………………………………
19
1.2.3.3. Streptococcus…………………………………………………
20
1.2.3.3.1.Virulence factors……………………………………………
20
1.2.3.3.2. Streptococcal toxins…………………………………………
21
1.2.3.3.3. Streptococcus equi………………………………………….
22
1.2.3.4. Corynebacterium………………………………………………
22
1.2.3.4.1.Virulence factor……………………………………………..
22
1.2.3.5.Actinomycetes……………………………………………........
23
1.2.3.5.1. Virulence factor…………………………………………….
24
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1.2.3.6. Bacillus……………………………………………….............
25
1.2.3.6.1. Virulence factor ……………………………………………
25
1.2.4. Epidemiology……………………………………………………
26
1.2.5. Disease of equine similar to fistulous withers…………………
26
1.2.5.1. Granulomatus lesion of the skin………………………………
26
1.2.5.2. Saddle sores…………………………………………………...
27
1.2.5.3. Capped hocks………………………………………….............
28
1.2.6. Prevention……………………………………………………….
29
1.2.7. Treatment ……………………………………………………….
29
2. CHAPTER TWO: MATERIALS AND METHODS………………
31
2.1. Sterilization………………………………………………………
31
a-Flaming………………………………………………………………
31
b-Red heat……………………………………………………………...
31
c-Hot air oven…………………………………………………………
31
d-Steaming at 100°C…………………………………………………..
31
e-Moist heat (Autoclave)……………………………………………..
31
2.2. Reagent…………………………………………………………..
32
2.2.1. Sodium Chloride/ normal saline………………………………
32
2.2.2. Alpha-naphthol solution ……………………………………..
32
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2.2.3. Potassium hydroxide …………………………………………
32
2.2.4. Hydrogen peroxide …………………………………………
32
2.2.5. Kovac's reagent……………………………………………….
32
2.2.6. Ttetramethyl-p-phenylene diamine dihydrochloride …………
33
2.2.7.Nitrate test reagent……………………………………………
33
2.3. Indicators ………………………………………………………
33
2.3.1. Andrade's indicator……………………………………………
33
2.3.2. Bromothymol blue…………………………………………….
34
2.3.3. Plasma…………………………………………………………
34
2.3.4. KOH and HCL……………………………………………….
34
2.4. Collection of blood for enriched media………………………….
34
2.5. Preparation of Culture Media……………………………………
34
2. 5.1. Liquid media…………………………………………………
34
2.5.1.1. Nutrient broth …………………………………………….
34
2.5.1.2. Peptone water…………………………………………….
35
2.5.1.3. Peptone water sugar………………………………………
35
2.5.1.4. Glucose-phosphate (MR-VP test) medium……………….
35
2.5.1.5. Nitrate broth medium……………………………………..
36
2.5.1.6. Serum water sugars……………………………………….
36
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2.5.2. Semi solid media……………………………………………
36
2.5.2.1. Motility medium………………………………………….
36
2.5.2.2. Hugh and Leifson's (O/F) medium……………………….
37
2.5.3. Solid media…………………………………………………
37
2.5.3.1. Blood agar ………………………………………………
37
2.5.3.2. MacConkey agar…………………………………………
38
2.5.3.3. Nutrient agar……………………………………………..
38
2.5.3.4. Ammonium salt sugars (ASS) …………………………..
38
2.5.3.5. Aesculin – bile azide agar ……………………………….
39
2.5.3.6. Milk agar ………………………………………………..
39
2.5.3.7. Nutrient gelatin medium …………………………………
39
2.5.3.8. Simmons citrate agar …………………………………..
40
2.5.3.9. Starch agar……………………………………………….
40
2.5.3.10. Urea agar Base …………………………………………
40
2.6. Clinical investigations……………………………………….
41
2.6.1. Study area…………………………………………………
41
2.6.2. Animals……………………………………………………
41
2.6.3. Clinical signs………………………………………………
41
2.6.4. Collection of Samples ……………………………………
42
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2.7. Bacteriological examination…………………………………
42
2.7.1. Primary isolation…………………………………………..
42
a- Blood agar …………………………………………………….
42
b- MacConkey agar………………………………………………
42
2.7.2. Isolation……………………………………………………
43
2.7.3. Cultural characteristics ……………………………………
43
2.8. Purification…………………………………………………..
43
2.9. Preservation……………………………………………….....
43
2.10. Microscopic Examination………………………………….
44
2.11. Biochemical tests…………………………………………...
44
2.11.1. Sugar fermentation test……………………………………
44
2.11.2. Oxidase test ……………………………………………...
44
2.11.3. Catalase test ………………………………………………
45
2.11.4. Coagulase test……………………………………………
45
2.11.4.1. Slide coagulase test ………………………………….....
45
2.11.4.2. Tube coagulase test …………………………………….
45
2.11.5. The oxidation- fermentation (o/F) test……………………
45
2.11.6. Indole production test…………………………………….
46
2.11.7. Voges- Proskauer (VP) test ……………………………..
46
xii
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2. 11.8. Nitrate reduction…………………………………………
46
2.11.9. Urease activity test ………………………………………
47
2.11.10. Citrate utilization test……………………………………
47
2.11.11. Aesculin hydrolysis………………………………………
47
2.11.12. Hydrogen sulphide (H2S) production……………………
48
2.11.13. Ammonium salt sugar test ………………………………
48
2.11.14. Gelatin hydrolysis………………………………………..
48
2.11.15. Motility test………………………………………………
48
3. CHAPTER THREE: RESULTS……………………………….
50
3.1. Clinical findings……………………………………………...
50
3.2. Bacterial isolation ……………………………………………
52
3.3. Characterization and identification of isolates………………
52
3.4. Gram- positive Bacteria……………………………………..
52
3.4.1. Staphylococcus …………………………………………..
53
3.4.2 Streptococcus ……………………………………………..
53
3.4.3. Actinomycetes ……………………………………………
53
3.4.4. Corynebacterium …………………………………………
54
3.4.5. Bacillus ……………………………………………………
54
xiii
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CHAPTER FOUR: DISCUSSION…………………………........
64
CONCLUSION…………………………………………………..
71
RECOMMENDATIONS………………………………………...
72
REFERENCES…………………………………………………..
74
APPENDIX……………………………………………………..
84
xiv
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LIST OF TABLES
Page
Table
1.
Main advantages and disadvantages of using donkeys.............
2.
Distribution of External Injuries on Various Body Parts in
8
donkeys…………………………………………………………
51
3.
Causes of External Injuries in donkeys in Khartoum State……
51
4.
Owner’s Responses to the Management of External Injuries
in donkeys…………………………………………………
52
5.
Isolation frequency of Gram-positive bacteria isolated from
donkeys infected with fistulous withers ………………………….
6.
Biochemical properties of some bacteria isolated from donkeys
infected with fistulous withers in Khartoum State………………
7.
55
56
Gram stain reaction and biochemical properties of Staphylococcus
spp isolated from donkeys infected with fistulous withers……… 57
8.
Gram stain reaction and biochemical properties of Bacillus
spp isolated from donkeys infected with fistulous withers……… 58
9.
Gram stain reaction and biochemical properties of Bacillus
spp isolated from donkeys infected with fistulous withers………. 59
10.
Gram stain reaction and some biochemical properties of
Streptococcus spp and Actinomycetes spp isolated from donkeys
infected with fistulous withers……………………………………. 60
xv
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LIST OF FIGURES
Figure
1.
Page
The arrangement of the breast band, breech strap and saddle
for harnessing a donkey to the shafts of a two-wheeled cart …. 11
2.
Donkey repeatedly whipped on an open wound, that
led to deep infection (Omdurman Market)……………………
13
3.
Names of the different body parts of a donkey……….
15
4.
Frecquency of Gram-positive bacterial species isolated
From donkeys with fistulous withers……………………….
5.
Staphylococcus aureus isolated from donkey with
Fistulous withers on blood agar medium……………………..
6.
61
62
Actinomyces spp bacteria isolated from donkey with
fistulous withers on blood agar medium…………………… .
62
7.
Smear from blood agar culture of Staphylococcus aureus…… 63
8.
Smear from blood agar culture of Actinomycetes spp………… 63
xvi